Biocorrosion and Aseptic Loosening of Metal Implants:

Novel Pathophysiological Mechanisms

Dr. Dieter Cadosch, MD

This thesis is presented for the degree of

Doctor of Philosophy At

The University of Western Australia

School of Anatomy and Human Biology

2011

This thesis is dedicated to my loving and wonderful parents,

My loved daughters Lia Charleen and Zoë Alina

&

Anne

“By seeking and blundering we learn”

Johann Wolfgang von Goethe

II

Abstract Biocorrosion and Aseptic Loosening of Metal Implants: Novel Pathophysiological Mechanisms

Metal orthopaedic devices exposed to a physiological environment remain prone to

corrosion by several mechanisms, leading to the production of significant amounts of

non-biocompatible wear particles and metal ions. Beside the well-known mechanical

wear and electrochemical redox reactions, metal implants undergo corrosion through

biological activities. The results presented in this thesis suggest that mature

osteoclasts are able to enhance corrosion of titanium and stainless steel implants

and contribute to the release of relevant amounts of corresponding metal ions into

the peri-implant tissues and the systemic blood circulation. In addition to the

increased osteolytic activity caused by wear particles, the results detailed in this

thesis strongly suggests that released titanium ions in the 4+ oxidation state (Ti(IV))

contribute to the pathophysiological mechanism of aseptic loosening by stimulating

both the bone metabolism and immune system through a series of direct and indirect

pathways. Aseptic loosening is believed to be caused by an increased osteolytic

activity at the bone-implant interface leading to loss of stability and ultimately implant

failure. Titanium (IV) ions are able to directly induce the differentiation of osteoclast

precursors toward mature and functional osteoclasts in ~20% of individuals.

Additionally, Ti(IV) ions stimulate the secretion of pro-inflammatory cytokines that are

known to enhance osteoclast recruitment, differentiation, activation and survival.

More evidence is linking the immune system to the bone and to its involvement in the

pathophysiology of aseptic loosening. Titanium (IV) ions influence phenotype and

function of T-lymphocytes, resulting in activation of a CD69+ and CCR4+ T-

lymphocyte and secretion of receptor activator of NF-κB ligand (RANK-L).

To date, revision surgery remains the only option for the treatment of a failed

orthopaedic implant caused by aseptic loosening. These results represent an

important step towards a better understanding of the complex pathophysiological

mechanism of aseptic loosening in patients with titanium implants, and may open

new research perspectives in this field which could offer innovative therapeutic

options and reduce the rate of aseptic loosening.

III

Table of Contents

Abstract III

Table of Contents IV

Publications Arising VI

Talks VII

Posters VIII

Prizes IX

Acknowledgments X

Statement of Candidate Contribution XI

Abbreviations XII

Preface XIII

Chapter 01 | Background and Literature Review 1

Introduction

Biocorrosion

Osteoimmunology: Links between Immunology and Bone System

Chapter 02 | Research Questions 5

Aims

Chapter 03 9

Uptake and intracellular distribution of various metal ions in monocyte-derived

dendritic cells detected by Newport Green DCF diacetate ester

Chapter 04 16

Bio-Corrosion of Stainless Steel by Osteoclasts - In vitro Evidence

Chapter 05 23

Biocorrosion and uptake of titanium by human osteoclasts

Chapter 06 31

Titanium IV ions induced human osteoclast differentiation and

enhanced bone resorption in vitro

IV

Chapter 07 40

Titanium induced production of chemokines CCL17/TARC and CCL22/MDC

in human osteoclasts and osteoblasts

Chapter 08 50

Titanium uptake, induction of RANK-L expression, and enhanced proliferation of

human T-lymphocytes

Chapter 09 | Discussion 58

Cellular Mechanisms of Corrosion

Effects of Metal Ions on Bone Metabolisms

Effects of Titanium Ions on Osteoimmunology

Summary and Outlook

Bibliography 63

Appendix 67

Poster 1

Poster 2

V

Publications Arising The following publications originated from this research project and have been published in peer-reviewed journals. *This manuscript is still under review at the time of thesis submission. Chapter 3: Uptake and intracellular distribution of various metal ions in

human monocyte-derived dendritic cells detected by Newport Green DCF diacetate ester. Cadosch D, Meagher J, Gautschi OP, Filgueira L (2009). J Neurosci Methods. Mar 30;178(1):182-7.

Chapter 4: Biocorrosion of stainless steel by osteoclasts –

in vitro evidence. Cadosch D, Chan E, Gautschi OP, Simmen HP, Filgueira L (2009). J Orthop Res. Jul;27(7):841-6.

Chapter 5: Biocorrosion and uptake of titanium by human osteoclasts.

Cadosch D, Al-Mushaiqri MS, Gautschi OP, Meagher J, Simmen HP, Filgueira L (2010). J Biomed Mater Res A. Dec 15;95(4):1004-10.

Chapter 6: Titanium IV ions induced human osteoclast differentiation and

enhanced bone resorption in vitro. Cadosch D, Chan E, Gautschi OP, Meagher J, Filgueira L (2009). J Biomed Mater Res A. Oct;91(1):29-36.

Chapter 7: Titanium induced production of chemokines CCL17/TARC and

CCL22/MDC in human osteoclasts and osteoblasts. Cadosch D, Gautschi OP, Chan E, Simmen HP, Filgueira L (2010). J Biomed Mater Res A. Feb;92(2):475-83.

Chapter 8: Titanium uptake, induction of RANK-L expression, and

enhanced proliferation of human T-lymphocytes. Cadosch D, Sutanto M, Chan E, Mhawi A, Gautschi OP, von Katterfeld B, Simmen HP, Filgueira L (2010). J Orthop Res. Mar;28(3):341-7.

Chapter 1 & 9: Metal is not inert: role of metal ions released by biocorrosion in

aseptic loosening-current concepts. Cadosch D, Chan E, Gautschi OP, Filgueira L (2009). J Biomed Mater Res A. Dec 15;91(4):1252-62.

Influence of metal ions on human lymphocytes and the generation of titanium specificT-lymphocytes. Chan E, Cadosch D, Gautschi OP, Sprengel K, Filgueira L (2011). J Appl Biomater Biomech. Accepted April 2011.

*Pharmacological blocking of osteoclastic biocorrosion of surgical stainless steel. Lionetto S, Little JA, Heymann D, Filgueira L, Cadosch D (2011).

VI

Talks The thesis results have been presented at the following national and international meetings: 6/2008 Immune reactivity to metal implants; an in vitro investigation.

Australian Society for Medical Research: WA Scientific Symposium

Perth, WA, Australia

8/2008 Biocorrosion of implants and metal sensitivity reactions.

Australian Orthopaedic Association: Annual Scientific Meeting

Crawley, WA, Australia

10/2008 Titanium induced osteoclast recruitment and activation resulting

in enhanced bone resorption: a human in vitro Study.

American College of Surgeons: 94th Annual Clinical Congress

San Francisco, CA, USA

6/2009 Titanium IV Ions Induced Human Osteoclast Differentiation and

Enhanced Bone Resorption in vitro.

96. Jahreskongress der Schweizerischen Gesellschaft für Chirurgie

Montreaux, VD, Switzerland

9/2009 Osteoclastic Corrosion of Stainless Steel and Titanium Implants

22nd European Conference on Biomaterials.

The annual conference of the European Society for Biomaterials

Lausanne, VD, Switzerland

10/2009 Titanium uptake and induction of TGF-beta and RANK-L

expression in human T-lymphocytes.

American College of Surgeons: 95th Annual Clinical Congress

Chicago, IL, USA

10/2009 Increased expression of pro-inflammatory and osteogenic

cytokines by osteoclasts cultured on surgical stainless steel.

American College of Surgeons: 95th Annual Clinical Congress

Chicago, IL, USA

VII

Posters The thesis results have been presented as posters at the following national and international meetings. Selected posters are listed in the appendix. 9/2008 Titanium (IV) ions induced human osteoclast differentiation and

enhanced bone resorption in vitro.

American Association for the Surgery of Trauma

67th Annual Meeting, Maui, Hawaii, USA

10/2008 Titanium ions induced bone resorption due to osteoclast

recruitment and activation: a human in vitro study.

Symposium on Biotechnology in Musculoskeletal Repair

2nd International Symposium, Lausanne, VD, Switzerland

1/2009 Immune reactivity to metal implants: an in vitro investigation.

2nd Singapore Symposium for Immunology, Singapore

5/2010 Titanium IV Uptake, Induction of RANK-L Expression and

Enhanced Proliferation of Human T-Lymphocytes

97. Jahreskongress der Schweizerischen Gesellschaft für Chirurgie

Interlaken, BE, Switzerland

VIII

Prizes The results presented in this thesis have been awarded with the following prices: Scholarship for International Research Fees (SIRF)

University of Western Australia, Crawley, Australia, 2008 - 2011

Excellence in Research Award in Orthopaedic Surgery 2008 American College of

Surgeons

American College of Surgeons, San Francisco, CA, USA, 2008

Travel Award University of Western Australia

University of Western Australia, Crawley, Australia, 2009

IX

X

Acknowledgements Though my name is printed on the cover of this thesis, this work would not have been

possible without the support of many people. It is a great pleasure to thank those

who made this thesis possible. To my loving and wonderful parents: Thank you for

always believing in me and giving me the freedom and the opportunity to pursue my

interests and dreams. I am heartily thankful to my supervisor and mentor Professor

Luis Filgueira. Thank you for your encouragement, guidance and the opportunity to

work at the School of Anatomy and Human Biology at the University of Western

Australia, but especially for your friendship. The friendly and supportive atmosphere

inherent to the whole School of Anatomy and Human Biology contributed essentially

to the final outcome of my studies. I would like to make a special reference to the

entire School of Anatomy and Human Biology. I want to thank my external

supervisor, Professor Hans-Peter Simmen for his generous support, and Professor

Marco Decurtins for his encouragement to undertake a PhD study in Western

Australia. I am particularly grateful to Tricia Knox, who was always so kind to edit all

my manuscripts. I am sincerely thankful to Dr. Oliver P. Gautschi, who has always

been of great support and a dear friend. Finally, I want to thank all of my friends who

made life in Perth so enjoyable, especially Urban M. and his lovely family; you made

me feel at home. For their hospitality and advice I would like to thank Professor Allan

P. and Carole Skirving.

State of Candidate Contribution The work presented in this thesis results from an external PhD study at the School of

Anatomy and Human Biology, University of Western Australia and the Clinic of

Trauma Surgery, University of Zurich. The experiments detailed in this thesis have

been performed at the School of Anatomy and Human Biology, University of Western

Australia under the supervision of Professor Luis Filgueira, and at the Clinic of

Trauma Surgery at the University Hospital of Zurich under the supervision of

Professor Hans-Peter Simmen. Contributions and collaborations with other

colleagues are mentioned accordingly and listed as co-authorships in the published

papers.

Dr. Dieter Cadosch

MD, PhD Candidate

Prof. Hans-Peter Simmen Prof. Luis Filgueira

MD, External Supervisor MD, Supervisor

XI

Abbreviations

Al Aluminium AL Aseptic loosening CCL22 Macrophage- derived chemokine CCL17 Thymus and activation- regulated chemokine Co Cobalt Cr Chromium EFTEM Energy-filtered electron microscopy ELISA Enzyme-linked Immunosorbent Assay Fe Iron IFN-γ Interferon-gamma IL Interleukin M-CSF Macrophage colony- stimulating factor Mn Manganese

Mo Molybdenum Ni Nickel OC Osteoclast/s PCR Polymerase chain

reactions PHA Phytohemagglutinin RANK-L Receptor activator of NF-κB ligand Ti Titanium Ti(IV) Titanium ions in 4+

oxidation state TNF-α Tumour necrosis

factor-alpha TRAP Tartrate-resistant

acid phosphatise V Vanadium Zr Zirconium

XII

Preface

To date, the pathophysiological mechanisms of biocorrosion and aseptic loosening of

metal implants remain not fully understood. Whether cellular mechanisms are able to

enhance biocorrosion of metal implants remains controversial. Furthermore, the

effects of metal ions released by biocorrosion on the immune system and bone

metabolisms remain under-studied. This thesis presents research into understanding

the effects of mature osteoclasts on biocorrosion as well as titanium ions on the

recruitment, differentiation and activation of human osteoclasts.

Chapter 02 gives an overview on the current knowledge on biocorrosion and aseptic

loosening of orthopaedic implants. From this, the hypothesis of enhanced

biocorrosion by osteoclasts and the effects of metal ions on bone metabolisms and

immune system are postulated. A brief outline of the methodology employed to

investigate the hypothesis is also described.

With the aim of understanding cellular biocorrosion and the effects of titanium ions on

osteoclastogenesis, a range of investigations were carried out. Six papers (published

in peer-reviews journals) investigating cellular biocorrosion of stainless steel and

titanium alloys (Chapter 04 and 05), the effects of titanium ions on bone metabolism

(Chapter 06 and 07), and on the immune system (Chapter 08) are presented. Each

paper is self-contained and presented in its entirety in the published format. A short

introduction highlighting the aims and the key results of the paper are provided at the

beginning of each chapter. The discussion attempts to unify the results presented by

the individual papers and to incorporate the possible pathways involving titanium ions

into the pathophysiological mechanism of aseptic loosening.

This journey of discovery inflamed my desire to uncover more of the complex

pathophysiology of aseptic loosening and metal ions. I hope that this thesis will allow

you to re-live the exciting journey of scientific research.

Dr. Dieter Cadosch, MD, PhD Candidate

The University of Western Australia

XIII

Chapter 01

Background Introduction

Metal implants are essential therapeutic tools for the treatment of bone fractures and

joint replacements. The first metal hip prosthesis was implanted in 1956 under great

engineering and medical efforts. In the meantime, total joint arthroplasty has been

performed routinely in over a million patients worldwide every year. The constant

aging of our population will contribute to an increasing number of patients with

implanted metal devices (1). As long as tissue engineering and biodegradable bone

substitutes do not lead to products that will be applicable in clinical routine, prosthetic

and osteosynthetic devices, made of metal and metal alloys, will remain

indispensable in orthopaedic and trauma surgery. Most patients tolerate metal

implants well; however, complications resulting from inflammatory and immune

reactions to metals have been well documented in up to 10% of patients, with the

incidence continuing to increase (2-8).

Many different metals and metal alloys have been used for joint replacement, internal

fixation of bone fractures, and after osteotomy. The metal implants predominately

used in contemporary surgery are made of commercially pure titanium, titanium

alloys (e.g., Ti6Al7Nb, TiAl6V4), and medical grade stainless steel (SS316L). The

employed metallic biomaterials are composed of a variety of metals including

aluminium (Al), chromium (Cr), cobalt (Co), nickel (Ni), molybdenum (Mo), vanadium

(V), titanium (Ti), and iron (Fe). Stainless steel SS316L, for example, contains Cr

(16–18%), Ni (10–14%), and various trace metals (5%) balanced with Fe (9,10). Over

recent decades, significant developments have taken place to provide suitable

metallic biomaterials with optimal biofunctionality, such as stability and a surface

texture allowing cellular adhesion, combined with excellent biocompatibility of low

intrinsic toxicity, inflammatory activation, and immunogenicity (11). However, their

permanent tendency to corrode when implanted into a physiological environment

remains a serious concern (12-14). Several case reports and studies have indicated

that metal implants undergo corrosion inside the human body by various

mechanisms including mechanical wear, biological activity, and mechanically

accelerated electrochemical processes (15-16). Thus, significant amounts of metallic

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particles are released into the tissues surrounding metal implants and elevated

concentrations of metal ions have been measured in clinically retrieved capsular and

peri-prosthetic tissues, as well as in distant organs (liver, spleen, and lymph nodes)

and body fluids (serum and urine) in total hip arthroplasty patients (17-20). Implants

used for osteosynthesis have to withstand completely different forces than implants

used for joint replacements. Thus, large implant-derived particles (in the nanometer

range) are produced exclusively by the mechanical wear process in articular coupling

of prostheses and are not present in patients with osteosynthesis implants. However,

both osteosynthesis and joint replacement implants are exposed to biological activity

and electrochemical processes.

A wide range of molecular and cellular interplays have been demonstrated between

the released biocorrosion products and the human organism, involving the immune

and skeletal system. There is increasing clinical and research-based evidence that a

relevant percentage of patients with metal implants may develop metal

hypersensitivity and severe inflammatory side effects, leading to aseptic loosening

(AL) of the implant and even systemic reactions (21-24). The phagocytosis of metal

wear particles by tissue macrophages induces production of pro-inflammatory

cytokines that enhance osteolytic activity at the implant-bone interface (22, 25-27).

Although many studies have investigated the role of metal wear particles in

osteoclastogenesis and AL, little is known about the direct effects of metal ions

released by biocorrosion. At the bone metal interface, metal ions may directly interact

with bone cells contributing to AL by accelerating osteoclastic bone resorption and/or

inhibiting the function of osteoblasts. Various studies have demonstrated that

nontoxic concentrations of metal ions affect the differentiation and function of

osteoblastic cells in vitro (28,29). Furthermore, once released into the systemic blood

circulation, the metal ions bind to serum proteins and form haptens or hapten-like

complexes, which are considered to be relevant antigens recognized by T-

lymphocytes and candidates for eliciting hypersensitivity reactions (18,30). Exposed

to metal–protein complexes, T-lymphocytes proliferate and differentiate to produce

soluble factors such as interleukin (IL)-6, IL-1a/b, and tumour necrosis factor-alpha

(TNF-α) that trigger a particular immune response and activate osteoclastogenesis

(31–33). The extent of this immune response upon implantation of metallic devices

depends predominantly on the individual immune reactivity and on material

characteristics (34).

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Biocorrosion

To date, it is well recognized that corrosion of metallic materials implanted in the

human body is an inevitable deteriorating reaction leading to the release of

undesirable metal ions/corrosion products, which are not biocompatible. In the

chemist’s view, corrosion is the visible destruction of a metal caused by interactions

with its environment, which may cause rupture of a structure or loss of function, for

example, breakage of an orthopaedic implant. This aspect is no longer important for

modern metals/alloys in surgery as material loss due to any corrosive attack is

minimal and does not usually compromise the stability of the implant (16,35).

However, despite the great progress in providing corrosion resistant metallic

biomaterials, in a physiological environment corrosion remains a slow and continuous

process, which leads to the release of significant amounts of metal ions and other

corrosion products. For instance, the corrosion of a stainless steel implant releases

Fe, Cr, and Ni ions; corrosion of Ti and Ti alloys release Ti (mostly at the 4+ oxidation

state; Ti(IV)), V and Al ions (36-48). Dissolved metal ions can accumulate in the

tissue, surrounding metal implants or can be released into the systemic blood

circulation and transported to distal organs (17–20). Indeed, significantly higher

concentrations of metal ions have been observed in the body fluids of patients with

stainless steel implants 10–13 years after primary hip arthroplasty when compared

with individuals without implants. This included concentrations of Ni in blood of ~0.51

μg/L, in plasma of ~0.26 μg/L, and in urine of ~2.26 μg/L, and plasma Cr levels of

~0.19 μg/L (14). Similarly, Ti concentrations of ~135.57 μg/L were measured in

patients with Ti-6Al-4V total knee replacements after 57 months (49). The amount of

metal ions released depends on the quality of the surgical procedure and on the

function of the implant. Leopold et al. measured a threefold increase in Ti levels in

patients with well-functioning Ti-alloy joint replacements, while patients with failed

implants showed as much as a 50-fold increase in serum concentrations when

compared with individuals without implants (49). A further study reported ~1 μg/L of

Ti in serum of patients with stable prosthesis; however, the concentrations increased

up to ~4 μg/L in the case of failed implants (51). In a physiological environment,

metallic biomaterials are known to undergo corrosion through a variety of

mechanisms. The most commonly observed forms of corrosion include mechanical

wear processes (e.g., in the articular coupling of prostheses leading to the formation

of wear particles) and electrochemical corrosion (11,22,35). As tissue fluids enter into

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contact with the metal surface, corrosion occurs via an electrochemical redox

reaction, in which oxidation (electron loss of the metal) is coupled with reduction

(electron gain of electrolyte components) (35). In terms of how a biological

environment can affect corrosion, it is believed that proteins can influence the

electrochemical behaviour of implant metals/alloys. However, overall results are not

conclusive and the exact effect of proteins on corrosion is still the topic of much

debate (52). Some authors demonstrate that proteins can enhance the propagation

of charges between implant and surrounding fluids while others suggest a reduced

charge transfer by proteins (53–56).

Osteoimmunology: Links between Immunology and Bone System

Recently, published evidence has shown that the immune system is closely

connected to the development of bone cells. This might not be surprising,

considering that the production of immune cells of hematopoietic origin comes from

the bone marrow and that improper bone development leads to a deficient bone

marrow size and function (84). In addition, OC, the only and essential bone resorbing

cells, derive from the same precursor cells as macrophages and dendritic cells,

professional antigen-presenting cells (70). This linkage plays also a major role in

rheumatoid arthritis, cancerous bone metastasis, and other destructive bone

disorders as indicated by an increasing amount of published studies (69,85–87).

There is increasing evidence indicating that inflammatory immune responses

influence bone metabolism. A variety of T-lymphocyte derived factors and cytokines,

including RANK-L and TNF-α, have been shown to directly and indirectly promote

OC activity and inhibit osteoblast function (72,88–93). Other T-lymphocyte-derived

cytokines, including interferon-gamma (IFN-γ) and IL-4, have been shown to

decrease OC activity and thereby decrease bone remodelling and probably delay

bone fracture healing (94,95). Increased levels of IL-10, a cytokine produced by

regulatory T-lymphocytes, have been measured in patients with orthopaedic implants

(96). However, despite increasing evidence supporting the linkage between the

immune system and bone cells, the role of the immune system in the systemic and

peri-implant tissue responses, characterized by increased osteolysis and implant

failure (AL), remains poorly understood with several studies reporting conflicting

results regarding their actual involvement.

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Chapter 02 Research Questions and Aims

The project described in this thesis was based on the following hypothesis:

1: Mature Osteoclasts are a Major Cause of Biocorrosion of Metal Implants

To date, it is well recognized that corrosion of metallic materials implanted in the

human body is an inevitable deteriorating reaction leading to the release of

undesirable metal ions and other corrosion products, which are not bio-compatible. In

the chemist’s view, corrosion is the visible destruction of a metal caused by

interactions with its environment, which may cause rupture of a structure or loss of

function, e.g. breakage of an orthopaedic implant. This aspect is no longer important

for modern metals/alloys in surgery as material loss due to any corrosive attack is

minimal and does not usually compromise the stability of the implant (17,37). Despite

the great progress in providing corrosion resistant metallic biomaterials, they remain

prone to corrosion in a physiological environment through a variety of mechanisms.

The most commonly observed include mechanical wear processes (e.g. in the

articular coupling of prostheses leading to the formation of wear particles) and

physiochemical corrosion (electrochemical redox reaction) (14,21,37). Beside the

mechanical and electrochemical aspect of corrosion, we hypothesise that mature

human OC are able to directly corrode the metal surface (e.g. pure Ti) and release

corresponding metal ions. We propose that this process may take place at the bone-

implant interface, representing an additional mechanism of metal corrosion in vivo

and contributing to the levels of metal ions measured in the periprosthetic tissues and

serum from total arthroplasty patients. Previous investigations have demonstrated

that Ti is relatively inert and resistant to corrosion with low water solubility (in the

range of 1 M concentration) at physiologic pH and low reactivity with biomolecules

in an aqueous environment (11). However, Ti concentrations up to ~136 μg/L were

measured in patients with Ti-6Al-4V total knee replacements (15). These levels are

much higher than would be expected given the “corrosion resistant” quality of Ti

demonstrated by in vitro analysis, and leads to our hypothesis that additional

mechanisms must be involved. Additionally, the amount of metal ions released

depends on the function of the implant. Leopold et al. measured a three-fold increase

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in Ti levels in patients with well-functioning Ti-alloy joint replacements, while patients

with failed implants showed as much as a 50-fold increase in serum concentrations

when compared with individuals without implants (52). A further study reported ~1

μg/L of Ti in serum of patients with stable prosthesis; however the concentrations

increased up to ~4 μg/L in the case of failed implants surrounded by osteolytic

lesions (53). Given that the most frequent cause of implant failure is AL, it is

reasonable to assume that the OC responsible for the osteolytic lesions may also be

responsible for the increased levels of metal ions measured in patients with failed

implants.

2: Titanium Ions released by Biocorrosion Enhance Osteolysis

Recently great effort has been directed towards understanding the

pathophysiological cascade of events initiated by biocorrosion products, resulting in

periprosthetic bone loss and ultimately aseptic loosening. The mechanical wear

process in articular coupling of prostheses is responsible for severe inflammatory

reactions and bone resorption, which has been recognized as one of the primary

biological mechanism leading to periprosthetic osteolysis (23). Phagocytosis of Ti

wear particles by macrophages induces their activation, producing mediators that

enhance OC formation (27). However, the potential role of Ti ions released by

biocorrosion from the implant surface must also be considered.

Normal bone maintenance relies on the balance between bone formation and bone

resorption (77). Thus the net bone loss at the metal-tissue interface occurs because

of an increased bone resorption or reduced bone formation (77). Previous studies

have demonstrated that non-toxic concentrations of metal ions affect the

differentiation and function of osteoblastic cells in vitro (30). Although there have

been several studies investigating the effect of metal ions on bone formation, very

little is known about their effects on bone resorption (29,30). Increased bone

resorption can be due to an increase of one or more of the following events:

recruitment of OC precursors from the blood circulation at the bone-implant interface,

their differentiation into mature multi-nucleated cells, their functional activation, and

finally their survival. Our hypothesis was that Ti ions may increase recruitment of OC

precursors (due to increased production of chemotactic cytokines) from the systemic

blood circulation to the peri-implant tissues and enhance their subsequent

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differentiation into mature OC. This hypothesis was supported by several animal

studies demonstrating that OC precursors in tissues surrounding subcutaneously

implanted wear particle differentiate into mature OC (65-67). Additionally, more

recently, particulate wear debris has been shown to induce chemokine expression in

macrophages, fibroblasts and osteoblasts, including IL-8, monocyte chemoattractant

protein-1, macrophage inflammatory protein 1, and eotaxin (68-71).

3: Titanium Ions affect the Phenotype and Function of human T lymphocytes

Dermal hypersensitivity to metal is common, with up to 20% of Caucasians being

sensitive to Ni (107). Immune reactions to dermal contact and ingestion of metals,

manifested as skin conditions such as eczema, urticaria, erythema and pruritis, are

believed to be of a type IV cell mediated hypersensitivity (12). The earliest case of an

allergic manifestation towards an orthopaedic implant was reported by Foussereau et

al., when a patient presented with an eczematous rash over a stainless steel fracture

plate (108). Beside the clinical observations of metal hypersensitivity observed in

some patients with orthopaedic implants, strengthened by the alleviation of

symptoms after the removal of the causative metal implant, evidence for the

involvement of the immune system comes from several histological studies of

retrieved peri-implant tissues (109). These studies have shown the infiltration of

lymphocytes, monocytes, dendritic cells, macrophages and mast cells into the peri-

implant tissues at various time points after metal implantation. Furthermore, there is

increasing evidence indicating that inflammatory immune responses influence bone

metabolism. A variety of T-lymphocyte derived factors and cytokines, including

RANK-L and TNF-α, have been shown to directly and indirectly promote OC activity

and inhibit osteoblast function (97,98). However, little is known about the metabolic

effects of Ti ions on human T-lymphocytes and its antigenicity, since the majority of

studies have focused on other cells (such as macrophages) and other metal ions

(such as Ni) (142). This study tested the hypothesis that Ti ions released by

biocorrosion, will be taken up by immune cells and cause alterations in T-

lymphocytes phenotype and function including: expression of surface markers,

proliferation, activation and regulation of cytokine expression and secretion.

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Aims

The aims of this study were first, to investigate whether human monocytes are able

to grow on metal implants (such as Ti and stainless steel) and differentiate into

mature and functional OC. Second, to test the hypothesis that mature OC are able to

directly corrode the metal surface and release corresponding metal ions into their

environment. Third, to investigate the effects of the released Ti ions on bone

resorption, including OC recruitment, differentiation and activation, and finally, to

investigate their effects on T-lymphocytes, including expression of surface markers,

proliferation, activation and regulation of cytokine expression.

Methodology

The methods used in the presented studies for qualitative and quantitative analysis

have already been well documented and described for use in areas of toxicology,

immunology and morphological studies. They include: (1) Isolation of peripheral

blood cells, (2) in vitro cell cultures of human monocytes, OC and lymphocytes from

blood of healthy donors, (3) Functional assays on dentine slices, (4) Electron

Microscopy, Scanning Electron Microscopy and Energy-Filtered Electron Microscopy

(EFTEM), (5) Flow Cytometry, (6) Proliferation Assays, (7) Cytometric Bead Assays

for cytokines measurements, (8) Enzyme-linked Immunosorbent Assay (ELISA) for

Chemokines Measurements, (9) quantitative reverse transcription polymerase chain

reactions (PCR), (10) Atomic emission spectrometry. All these methods are

described in the corresponding papers (Chapters 4-8). Additionally, a novel method

using Newport GreenTM DCF diacetate ester to fluorescently label intracellular

protein metal complexes was developed (Chapter 3).

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- 9 -

Chapter 03 Uptake and intracellular distribution of various metal ions in human monocyte-derived dendritic cells detected by Newport Green DCF diacetate ester The attempt to visualise intracellular protein metal complexes in biomedical research

has currently been limited due to the lack of adequate metal detection methods and

the unavailability of probes for such molecular structures. This first project aimed to

develop a novel detection method to overcome these limitations and enable the

planned research in the field of metals and metal ions. Newport GreenTM DCF

diacetate ester is a cell permeant acetate ester, which becomes fluorescent after

hydrolysis. This molecule is initially uncharged, allowing it to pass through cell

membranes. Once in the cell, it is hydrolysed and becomes charged, hindering its

escape from the cell and allowing it to bind charged protein metal complexes, which

then become fluorescent. In this study, we exposed cultured human monocyte-

derived dendritic cells to a variety of metal ions (including Ti with different oxidation

state: Ti(III) and Ti(IV)) with the aim of having the cells take up and process protein

metal complexes. Newport GreenTM DCF diacetate ester was used to fluorescently

label intracellular protein metal complexes. Flow cytometry analysis and confocal

imaging showed specific staining for monocyte-derived dendritic cells exposed to Al,

Cr, Ni, Ti and Zr ions. The intensity of staining varied between ion types, whereby

Ti(III) resulted in the brightest fluorescence signal. Aluminium, Cr(III), Ni, Ti(IV) and

Zr(IV) were also clearly detectable. Incorporating a new method of fluorescence

metal detection using both, flow cytometry and confocal laser microscopy, this first

study outlined the capacity of Newport GreenTM DCF diacetate ester to fluorescently

stain a variety of intracellular metal ion protein complexes (including Ti) as well as

unveiling the morphology of intracellular protein-bound metals in a monocyte-derived

dendritic cells culture setting. Additional experiments clearly demonstrated that this

protocol is suitable for investigating other cell lines including T-lymphocytes and OC.

Journal of Neuroscience Methods 178 (2009) 182–187

Contents lists available at ScienceDirect

Journal of Neuroscience Methods

journa l homepage: www.e lsev ier .com/ locate / jneumeth

Uptake and intracellular distribution of various metal ions inhuman monocyte-derived dendritic cells detected byNewport GreenTM DCF diacetate ester

Dieter Cadosch ∗, James Meagher, Oliver P. Gautschi, Luis FilgueiraSchool of Anatomy and Human Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

a r t i c l e i n f o

Article history:Received 29 May 2008Received in revised form 4 December 2008Accepted 6 December 2008

Keywords:Newport GreenMetal protein complexMetal ionsTitaniumDendritic cellFlow cytometryConfocal microscopy

a b s t r a c t

Background: The attempt to visualise intracellular protein metal complexes has currently been difficultdue to the unavailability of probes for such molecular structures. Newport GreenTM DCF diacetate esteris a cell permeant acetate ester, which becomes fluorescent after hydrolysis. This molecule is initiallyuncharged, allowing it to pass through cell membranes. Once in the cell, it is hydrolysed and becomescharged, hindering its escape from the cell and allowing it to bind charged protein metal complexes,which then become fluorescent.Methods: In this study, we exposed cultured human monocyte-derived dendritic cells (mDC) to a varietyof metal ions with the aim of having the cells take up and process protein metal complexes. NewportGreenTM DCF diacetate ester was used to fluorescently label intracellular protein metal complexes.Results: Flow cytometry analysis and confocal imaging showed specific staining for mDC exposed toaluminium, chromium, nickel, titanium and zirconium ions. The intensity of staining varied between ion

types, whereby Ti(III) resulted in the brightest fluorescence signal. Aluminium, Cr(III), Ni, Ti(IV) and Zr(IV)were also clearly detectable.Conclusion: For the first time, intracellular metal ion protein complexes undergoing cellular processing

ed in

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. Introduction

Humans are exposed to metals on a daily basis due to anthro-ogenic activities in their metals containing natural environment.s metals are used for the production of daily used commodi-

ies, potentially everyone is exposed to metals in Western societiesNestle et al., 2002). Metals enter the human body through theigestive and the respiratory system or through the skin (Hostynek,003; Kusaka et al., 2001; Sunderman, 2001). Additionally, nearlyll medical disciplines apply an increasing number of metalmplants, most notably in orthopaedic and trauma surgery. There isncreasing evidence that all metals in contact with biological sys-ems corrode and release metal ions into surrounding tissues andhe systemic blood circulation (Jacobs et al., 1998; Steens et al.,006; Tezer et al., 2005).

It is known that certain metals are essential for the structurend function of specific proteins in most cells and tissues, includ-ng the nervous system, e.g. iron (Fe) and zinc (Zn) (Insel et al., 2008;

urakami and Hirano, 2008). Those metals at low concentrations,

∗ Corresponding author. Tel.: +61 8 6488 3647; fax: +61 8 6488 1051.E-mail address: dcadosch@gmx.net (D. Cadosch).

165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.jneumeth.2008.12.008

human mDC using flow cytometry and confocal microscopy.© 2008 Elsevier B.V. All rights reserved.

of determined oxidation state and of specific bioorganic formula-tion are needed for adequate function of the human body and fora healthy life. However, the same metals at higher concentrations,of different oxidation states and formulations may be very toxic(Burger et al., 2003; Bush, 2008; Rensing and Maier, 2003; Thomasand Jankovic, 2004). The oxidation state of metal ions may deter-mine whether the metal acts as a physiologically required element,as an allergen or as a toxic molecule. Chromium (Cr) is probably thebest-known example beside nickel (Ni), where Cr(VI) acts as a toxicion, and Cr(III) is an antigenic ion causing inflammatory and allergicreactions (Artik et al., 1999; Shrivastava et al., 2002). To date, it isnot well understood why differences in the oxidation state result insuch diverse responses, and whether or how metal ions may changetheir oxidation state in vivo.

Metals may influence the nervous system by directly interferingwith biochemical and physiological activities, or by inducing andsustaining inflammatory and damaging reactions (Bush, 2008; Inselet al., 2008; Murakami and Hirano, 2008; Thomas and Jankovic,

2004). Sudden, acute intoxication with high metal concentrationscauses usually significantly correlated, unequivocal neurotoxicsymptoms, as described for Cr, cobalt (Co) and lead (Pb) (Gobba,2003; Halatek et al., 2008; Kumar, 2001; Pecze et al., 2005; Songet al., 2008). Little is known about long-term exposure to lower

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D. Cadosch et al. / Journal of Neur

etal concentrations, however, there is increasing evidence thatransitional and heavy metals, including aluminium (Al), Cr, cop-er (Cu), Pb, manganese (Mn) and titanium (Ti), may be involved

n chronic nervous diseases, such as Alzheimer’s and Parkinson’sisease (Armstrong et al., 1995; Campbell et al., 2004; Rogers andahiri, 2004; Smorgon et al., 2004; Wu et al., 2008).

The main reason there is limited knowledge about the role ofetals in nervous functions and diseases has been due to the lack

f adequate methods for the detection of metals in a biologicalontext (Domaille et al., 2008). Newport Green has been showno bind to Zn, cadmium (Cd), mercury (Hg), Pb, Ni, Fe and CuThompson et al., 2002) in salt solution and to form fluorescentomplexes. Newport GreenTM DCF diacetate ester (Newport GreenCF) is a molecule that is uncharged, making it highly cell perme-nt and therefore very beneficial for use in cellular experiments.nce inside the cell the probe is rapidly hydrolysed, rendering

he probe charged and cell impermeable, thus preventing the dyerom leaving the cell. Hydrolysis also results in the probe becominglightly fluorescent (Thierse et al., 2004). Any subsequent bindingo metal ions results in a significant increase in fluorescence inten-ity with no change in wavelength, allowing for the detection ofabelled intracellular metal ions. Exploiting this quality, our studyxtended the use of Newport Green DCF beyond the metals previ-usly investigated to include Al, Co, Cr, molybdenum (Mo), Ti andanadium (V).

We applied this fluorescent probe in an in vitro model usinguman monocyte-derived dendritic cells (mDC). Dendritic cellslay a role in immune reactions and are closely related toicroglia, which are also monocyte-derived cells (Filgueira et

l., 1996). Although microglia are thought to be a separateyeloid cell population unique to the central nervous system,

here is increasing evidence that additional monocyte-derivedlia-like cells may migrate into the brain under certain condi-ions (Bulloch et al., 2008; Mildner et al., 2007; Rodriguez etl., 2007). With this in mind, human mDC seem to be goodepresentative cellular model to investigate the influence of met-ls on monocytic cells, and as a result mDC were used for thistudy.

. Materials and methods

.1. Dendritic cells

Human mDC were obtained from adherent peripheral bloodononuclear cells (PBMCs). Briefly, PBMCs were isolated from buffy

oats of 10 healthy blood donors (Australian Red Cross Blood ServiceARCBS), Perth, Western Australia) using a Ficoll-gradient den-ity separation protocol as previously described (Filgueira et al.,996; Meagher et al., 2005). Freshly isolated PBMCs were culturedor 2 h (25 cm2 culture flasks; Sarstedt, Germany) in RPMI 1640lutamax medium (RPMI, Invitrogen, NZ) containing 10% humanerum (ARCBS) and antibiotics (Invitrogen). Subsequently, the non-dherent PBMCs were discarded and the adherent cells wereashed thoroughly with phosphate buffered saline (PBS) (Invit-

ogen). The remaining adherent cells were then cultured in RPMIedium supplemented with 5% human serum, antibiotics, recom-

inant human granulocyte macrophage colony-stimulating factor50 ng/mL; Leucomax, Schering-Plough, Australia), and interleukin-(10 ng/mL; R and D Systems, MN) (Filgueira et al., 1996) for 1 week

o obtain immature mDC.

.2. Cell culture conditions

Cells were incubated with one of 10 different metal ion solutions,omprising of Al(I), Co(III), Cr(II), Cr(III), Mo(II), Ni(II), Ti(III), Ti(IV),

ce Methods 178 (2009) 182–187 183

V(II), or Zr(IV) at 50 �M concentration. After overnight incubationthe cells were fixed with 1% paraformaldehyde in PBS. Untreatedcell cultures were used as negative controls.

2.3. Newport GreenTM DCF diacetate ester staining

Fixed cells (106/mL) were incubated for 1 h at room tem-perature with Newport GreenTM DCF diacetate ester at 1 �Mconcentration and washed with PBS solution. Cells for flowcytometry were resuspended in 2 mL PBS. Dendritic cells forconfocal microscopy were spun onto glass slides, stained withDAPI nuclear stain (Roche Diagnostics, Germany) and mounted inDako Fluorescent Mounting Medium (DakoCytomation, Carpinte-ria, CA).

2.4. Flow cytometry (FACS)

Fluorescence quantification was carried out using flow cytom-etry (FACScan, 488 nm laser; BD Biosciences, San Jose, CA).Approximately 20,000 cells were acquired per condition and gatedfor granular large cells according to their forward and side scatterprofile.

2.5. Confocal microscopy

Cell morphology was analysed using confocal fluorescencemicroscopy, including the UV-laser for the detection of DAPI, MPlaser (488 nm) for Newport Green DCF detection and the cor-responding software for image analysis (Leica TCS SP2 AOBS,Germany).

3. Results

3.1. Flow cytometry

3.1.1. Titration of Newport GreenTM DCF diacetate ester in thecontext of Ti(III)

To assess the full working range of Newport Green DCF as aprobe for the detection of intracellular metal ion complexes, New-port Green fluorescence was titrated against Ti(III) (Fig. 1). Thiswas determined by measuring intracellular fluorescence throughflow cytometry of cell populations incubated with increasing con-centration (0, 0.06, 0.08, 0.125, 0.25, 0.5 �M) of Newport GreenDCF with constant Ti(III) concentration (50 �M), giving fluores-cence of cells to the point at which adequate fluorescence wasachieved (Fig. 1a). Furthermore, Ti(III) itself was titrated (0, 2, 10,20, 40, 60, 80, 120 �M) from control levels to the point whereno further change in fluorescence was observed in combina-tion with a constant 0.1 �M Newport Green DCF concentration(Fig. 1b). These first results indicate that Newport Green DCF isan effective probe between the range of 0.08–0.125 �M and thatTi(III) can be detected in mDC at concentrations greater than20 �M using this staining protocol, a comparable range to thatdescribed for other metals and fluorescent probes (Domaille et al.,2008).

3.1.2. Detection of different intracellular metal ions in dendriticcells using flow cytometry

Fig. 2 summarises the results of quantitative analyses per-formed using flow cytometry to measure metal uptake by mDC.

Titanium (III) displayed the highest fluorescence signal, indicat-ing significant metal protein complex binding to the fluorescentNewport Green DCF molecule. Dendritic cells exposed to 50 �MAl(III), Cr(III), Mo(II), Ni(II), Ti(IV), and Zr(IV) also exhibited signif-icant increases in fluorescence signal in comparison to controls.

184 D. Cadosch et al. / Journal of Neuroscience Methods 178 (2009) 182–187

F wporto n of 1l ions t

IicbHmb

FC

ig. 1. Titration of Newport Green (a) and Ti(III) (b). Increasing concentration of Nef 50 �M; and Ti(III) (0, 2, 10, 20, 40, 60, 80, 120 �M) using a constant concentratioine corresponds to 0 �M. The curves line up according to the increasing concentrat

n contrast, cells exposed to Cr(II), Co(III), and V(II) showed min-mal or no increase in fluorescence signal when compared toontrol cells. These results indicate that Newport Green DCF can

e reliably used to detect and measure metal ion uptake by cells.owever, not surprisingly, different metals, oxidation states andetal ion concentrations all affect the fluorescence signal that can

e detected.

ig. 2. Detection of various metal ions in dendritic cells using Newport Green DCF stain ao (b), Cr(II) (c), Cr(III) (d), Mo (e), Ni (f), Ti(III) (g), Ti(IV) (h), V (i) or Zr (k) before stained

Green DCF (0, 0.06, 0.08, 0.125, 0.25, 0.5 �M) using a constant Ti(III) concentration�M Newport Green DCF results in an increasing fluorescence intensity. The black

o the right.

3.2. Microscopy

3.2.1. Detection of intracellular metal ions in dendritic cells using

confocal microscopy

Flow cytometry data were further confirmed with confocalmicroscopy. The microscopy analysis provided additional relevantinformation about the subcellular distribution of the metal ions

nd flow cytometry. Cells were incubated with no metal (faint line) or 50 �M Al (a),with Newport Green DCF (bold line).

D. Cadosch et al. / Journal of Neuroscience Methods 178 (2009) 182–187 185

F ence oG etatiov

(acflintgm

ig. 3. Confocal fluorescence microscopy. Dendritic cells were cultured in the presreen DCF. A blue nuclear stain (DAPI) was used in images a, b, c and e. (For interprersion of the article.)

Fig. 3). For that purpose, mDC were incubated with Cr(III), Ti(III)nd Ti(IV), stained with Newport Green DCF and prepared foronfocal microscopic analysis. Confocal images showed intenseuorescence throughout the cytoplasm and nucleolus of mDC

ncubated with Ti(III). Comparable fluorescence intensities wereot observed in control cells. Similar results were confirmed byransmission electron microscopy (data not shown). This sug-ests that Ti(III) ions may bind to proteins of the surface cellembrane, cytoskeletal proteins and ribonucleoproteins found

f Ti(III) (a and b), Ti(IV) (c), Cr(III) (d) or no metal (e) before stained with Newportn of the references to colour in this figure legend, the reader is referred to the web

in the nucleolus. Distinct staining of different regions for DAPIand Newport Green DCF indicates that Ti(III) does not bind tothe same nuclear DNA compounds labelled by DAPI. In addi-tion to Ti(III), confocal imaging also showed specific staining for

Cr(III) and Ti(IV) however neither of these fluorescent signalswere as intense as Ti(III), indicating that these metal ions do notbind to cellular proteins with greater affinity as that of Ti(III).There was no observed nucleolus staining for metals other thanTi(III).

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86 D. Cadosch et al. / Journal of Neur

. Discussion

Biomedical research in the context of metals and metal ions haseen limited due to a lack of adequate metal detection methods.nly recently, there has been an increasing panel of fluorescent

eagents available that can be used effectively for the detection of aariety of metals (Domaille et al., 2008). Newport Green, a fluores-ent chelator molecule, is able to bind specifically to intracellularetal protein complexes (Thompson et al., 2002). Incorporatingnew method of fluorescence metal detection using both flow

ytometry and confocal laser microscopy, this study has outlinedhe capacity of Newport GreenTM DCF diacetate ester to fluores-ently stain a variety of metal ion protein complexes as well asnveiling the morphology of intracellular protein-bound metals inn in vitro mDC culture setting.

Chelator molecules with affinities for a variety of metal ionsay interact with metals native to the cell, thus resulting in flu-

rescence information, which does not correspond to the metal ofnterest. Cellular Zn(II) and Cu(II) are the main metals that could bexpected to interfere with cellular fluorescence levels when usingewport Green DCF (Thierse et al., 2004). Consequently, some low

evel background staining of untreated control cells was observed.owever, incubation of the cells in the presence of increasing metal

on concentrations and Newport Green DCF concentrations resultedn significant fluorescence onset values from titration data. The fluo-escence signal of metal ion treated cells, particularly in the contextf Ti(III), sufficiently increased to a level where we could be surehat any background fluorescence from native metal ion complexesas essentially insignificant. In addition, a significant increase of

he fluorescent signal was seen for Al, Cr(III), Mo, Ni and Ti(IV), alletals that have been reported to be either neurotoxic or nomi-

ated to play a role in chronic nervous diseases (Campbell et al.,004; Halatek et al., 2008; Smorgon et al., 2004; Song et al., 2008;ang et al., 2008; Wu et al., 2008).It is also important to consider the toxicity of the various metal

ons when interpreting flow cytometric results of this nature. Pub-ished studies have shown that concentrations of 1% titaniumarticles, eluting Ti ions, kill immune cells (Pioletti et al., 1999) andhat some aqueous Ti, such as TiCl4, is cytotoxic (Yamamoto et al.,998). Our flow cytometry observations suggest that the morphol-gy of the mDC changed due to exposure to Ti(III) and other metallicons, whereby the cells became larger and more mature (data nothown). Cell gating was adjusted to compensate for this changen morphology where possible. However, cell death due to toxicityid not appear to significantly affect the results in this experimentaletting.

Wendt-Larsen et al. (2001) have shown that cells concen-rate metal ions into granules when extracellular metal ion levelsncrease. Our study confirms that observation, as with increasedevels of Ti(III), Newport Green DCF staining displayed a granular

orphology. It is possible that the metal ions are taken up by theell and then concentrated in the endo-lysosomal compartment.owever, the Newport Green DCF stain not only shows granularreas of higher fluorescence intensity but also a general dispersionf fluorescence throughout the cell cytoplasm. This indicates pos-ible diffusion of Ti(III) ions throughout the cytoplasm, cell matrixnd nuclear structures. However, the fact that Ti(III) does not bindnywhere within the nucleus other than the nucleolus confirmseliefs that Ti(III) will readily complex with certain proteins (Hallabt al., 2000, 2001), but also suggests that Ti(III) does not bind to DNAolecules.

The confocal images produced by using this new stain show dif-

erences in the distribution and intensity of fluorescence betweenifferent metal types as well as between different oxidationtates of the same metal. Consequently, one has to postulatehat mDC may process metal ions of different oxidation states in

ce Methods 178 (2009) 182–187

different ways resulting in a diverse metal-dependent immuneresponse.

By combining flow cytometry with confocal microscopy, ourstudy demonstrates that Newport GreenTM DCF diacetate ester canbe used to fluorescently label metal ion protein complexes for avariety of different metal ions. Furthermore, our study highlightsthat cellular proteins demonstrate different binding affinities fordifferent metal ion types of the same metal, with for example Ti(III)appearing to have the higher binding affinity with this particularacetate ester compared with Ti(IV). It remains unclear whether thebinding affinity of the protein metal complex to the fluorescentdiacetate molecule is responsible for the difference or whether itis an indication of binding affinity between the metal ion and thecellular proteins alone. Nevertheless, this study will help to furtherunderstand how the mDC process and present metal ions.

Finally, Newport Green DCF is an excellent fluorescent probe forAl(III), Cr(III), Mo(II), Ni(II), Ti(III), Ti(IV) and Zr(IV) in the context ofcells and can be incorporated into procedures involving both flowcytometry and confocal fluorescence microscopy. Ongoing experi-ments have clearly demonstrated that this protocol is suitable forinvestigating tissue sections and other cell lines (T-lymphocytes,neuronal PC12 cells and osteoclastic cells), and that it will help theunderstanding of further pathophysiological mechanisms involvingmetal ions.

Acknowledgements

We thank John Kuo, John Murphy, Paul Rigby and Kathryn Heel(Centre for Microscopy, Characterisation and Analysis, University ofWestern Australia), and Guy Ben-Ary (Image Acquisition and Anal-ysis Facility, School of Anatomy and Human Biology) for excellenttechnical support and advice. We would also like to acknowledgeTrish Knox for her editorial support. This study was supported bythe NIH grant 1 R01 GM072726-01A1.

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Chapter 04 Biocorrosion of stainless steel by osteoclasts - In vitro evidence Most metals in contact with biological systems undergo corrosion by an

electrochemical process. This study investigated whether human OC are able to

grow on stainless steel and enhance corrosion of the metal alloy leading to an

increased release of corresponding metal ions, which may cause inflammatory

reactions and activate the immune system. Scanning electron microscopy analysis

demonstrated long-term viable OC cultures and evident resorption features on the

surface of stainless steel discs on which OC were cultured for 21 days. The findings

were confirmed by atomic emission spectrometry investigations showing significantly

increased levels of Cr, Ni, and Mn in the supernatant of OC cultures. Furthermore,

significant levels of pro-inflammatory cytokines IL-1b, IL-6, and TNF-α, which are

considered to be major mediators of osteolysis, were revealed in the same cultures

by cytometric bead array analysis. Within the present study, it was shown that human

OC precursors are able to grow and differentiate towards mature OC on stainless

steel. The mature cells are able to directly corrode the metal surface and release

corresponding metal ions, which induce the secretion of pro-inflammatory cytokines

that are known to enhance OC differentiation, activation, and survival. Enhanced

corrosion and the subsequently released metal ions may therefore result in enhanced

osteolytic lesions in the peri-prosthetic bone, contributing to the AP of the implant.

- 16 -

Bio-Corrosion of Stainless Steel by Osteoclasts—In Vitro Evidence

Dieter Cadosch,1,2 Erwin Chan,1 Oliver P. Gautschi,1,2 Hans-Peter Simmen,3 Luis Filgueira1

1School of Anatomy and Human Biology, University of Western Australia, Crawley, Australia, 2Department of Orthopaedic and Trauma Surgery,Royal Perth Hospital, Perth, Australia, 3Division of Trauma Surgery, University Hospital Zurich, Zurich, Switzerland

Received 22 August 2008; accepted 18 November 2008

Published online 22 December 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20831

ABSTRACT: Most metals in contact with biological systems undergo corrosion by an electrochemical process. This study investigatedwhether human osteoclasts (OC) are able to grow on stainless steel (SS) and directly corrode the metal alloy leading to the formation ofcorresponding metal ions, which may cause inflammatory reactions and activate the immune system. Scanning electron microscopy analysisdemonstrated long-term viable OC cultures and evident resorption features on the surface of SS discs on which OC were cultured for 21 days.The findings were confirmed by atomic emission spectrometry investigations showing significantly increased levels of chromium, nickel, andmanganese in the supernatant of OC cultures. Furthermore, significant levels of pro-inflammatory cytokines IL-1b, IL-6, and TNF-a, whichare considered to be major mediators of osteolysis, were revealed in the same cultures by cytometric bead array analysis. Within the presentstudy, it was shown that human osteoclast precursors are able to grow and differentiate towards mature OC on SS. The mature cells are ableto directly corrode the metal surface and release corresponding metal ions, which induce the secretion of pro-inflammatory cytokines thatare known to enhance osteoclast differentiation, activation, and survival. Enhanced corrosion and the subsequently released metal ionsmay therefore result in enhanced osteolytic lesions in the peri-prosthetic bone, contributing to the aseptic loosening of the implant.

� 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:841–846, 2009

Keywords: osteoclast; bio-corrosion; stainless steel; osteolysis; TNF-a

Over the last decades, significant developments havebeen taking place to provide suitable metal alloys forimplants with optimal mechanical characteristics andgood biocompatibility with minimal adverse host tissuereactions.1 However, their permanent tendency tocorrode when exposed to a physiological environmentremains a serious concern.2–4 Elevated concentrationsof metal ions have been measured in clinically retrievedperi-prosthetic tissues, distal organs, and body fluids intotal hip arthroplasty patients.5–7 The biology behindcorrosion inside the human body and the distribution ofthe metal ions throughout the organism is not fullyunderstood. It has been suggested that extracellularbody fluids have corrosive proprieties and contain metalbinding proteins.2,3 Furthermore, it has been debatedwhether osteoclasts (OC) are able to corrode pure metalwhen entering into contact with metal surfaces. How-ever, direct evidence of metal corrosion by OC has beenmissing to date.

Osteoclasts are highly specialized multinucleatedcells, which are capable of bone resorption. They areformed by the fusion of marrow-derived mononuclearprecursors, which circulate in the peripheral blood.Osteoclastic differentiation from hematopoietic andcirculating monocytes occurs in the presence of macro-phage-colony stimulating factor (M-CSF) and the recep-tor activator of NF-kB ligand (RANK-L) which areexpressed on osteoblastic cells.8 Mature OC displaydistinct morphological characteristics, such as multiplenuclei and a basal ruffled border surrounded by amembrane stabilizing the extent ring of actin filaments,and express cathepsin K and tartate-resistant acidphosphatase.9–11

The presence of implant-derived wear debris areknown to be responsible for severe inflammatory andhypersensitivity responses due to the release of pro-inflammatory cytokines that lead to increased osteolyticactivity at the bone–implant interface.12–15 We havepreviously demonstrated that metal ions (such astitanium) directly induce the differentiation of osteoclastprecursors towards mature OC, able of effective boneresorption.16 Furthermore, once released into the sys-temic blood circulation, the metal ions bind to serumproteins and form haptens or hapten-like complexes,which are considered to be relevant antigens recognizedby T lymphocytes and candidates for eliciting hyper-sensitivity reactions.5,17 Exposed to metal–protein com-plexes, T lymphocytes proliferate and differentiate toproduce effectors such as interleukin (IL)-6, IL-1a/b,interferon-gamma (IFN-g) and tumor necrosis factor-alpha (TNF-a) that trigger a particular immuneresponse.18–20

To date, surgical stainless steel 316L (SS) is one of themost frequently used biomaterials because of a favorablecombination of strength, good fabrication properties, lowinherent toxicity, minimal reactivity with biomolecules,and cost efficiency when compared with other metallicimplant materials.1,21 The aims of this study were firstly,to investigate whether human monocytes are able togrow on SS implants and differentiate into mature OC.Secondly, to test the hypothesis that mature OC areable to directly corrode SS and release correspondingmetal ions into the peri-implant environment, whichmay subsequently induce inflammatory reactions.

METHODSMetal DiscsSurgical SS [316L medical grade (UNS S31673) 17–19 Cr, 13-15 Ni, 2.25–3.0 Mo, max. 2.0 Mn, max 0.030 C, max 0.75 Si,max 0.025 P, max 0.010 S, max 0.10 N, max 0.50 Cu] discs were

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Correspondence to: Dieter Cadosch (T: þ61 8 6488 3647; F: þ61 86488 1051; E-mail: dcadosch@gmx.net)

� 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

kindly provided by the RMS Foundation, Bettlach, Switzer-land. The polished test discs were 13 mm in diameter, 1 mmthick, and fitted into the wells of 24-well plates (Sarstedt,Nuernbrecht, Germany). Before use, the discs were washed inethanol, rinsed in distilled water, and steam sterilized.

Isolation of Peripheral Blood Monocytic CellsHuman monocytic cells (MC) were obtained from adherentperipheral blood mononuclear cells (PBMCs) as previouslydescribed.7 Briefly, PBMCs were isolated from buffy coats ofsix healthy blood donors [Australian Red Cross Blood Service(ARCBS), Perth, Australia] through Ficoll-gradient centri-fugation (Amersham Biosciences, Uppsala, Sweden). ThePBMCs were cultured (378C, humidified, 5% CO2) in 25 cm2

tissue culture flasks (Sarstedt) in RPMI-1640 glutamaxmedium (RPMI) (Gibco/Invitrogen, Auckland, New Zealand),supplemented with 10% human serum (ARCBS) and 1%antibiotics (10,000 units/mL penicillin G sodium, 10,000 mg/mL streptomycin sulfate, and 25 mg/mL Amphotericin B,Gibco) (standard medium). After 1 h in culture, the non-adherent PBMCs were discarded and the adherent PBMCsconsisting of 95% of MC cells were washed twice with 0.1 Mphosphate buffered saline (PBS) pH 7.2 (Gibco). The collectedcells were resuspended in RPMI medium and counted in ahemocytometer. Ethical approval for using human blood cellsfor this study was granted by the Ethics Committee of theUniversity of Western Australia and the ARCBS.

Cell Culture Conditions on Stainless Steel Discsand CoverslipsThe isolated MC were seeded either onto the SS discs (106 cells/disc) or glass coverslips (106 cells/coverslips) (12 mm diame-ter), resulting in a nonconfluent cell layer, placed into 24-welltissue culture plates (Sarstedt). After overnight incubation(378C, humidified, 5% CO2), the cultures were replenishedwith 1,500 mL of standard medium. In order to generate OC,half of the cell cultures were supplemented with osteoclastdifferentiation cytokines (10 ng/mL recombinant humanRANK-L and 10 ng/mL recombinant human M-CSF; R&D,Minneapolis, MN).10 Half of the cultures were left as MC. Asnegative controls, SS discs and glass coverslips were incubatedin standard culture medium only. After every incubation week,the conditioned supernatant was collected, pooled, frozen at�208C, and replaced with fresh standard medium. After21 days, the supernatant was collected and the cells fixedwith 2.5% EM grade glutaraldehyde (Electron MicroscopyScience, Washington, PA) in culture medium before processedfurther as described below. Six specimens were cultured pereach condition, and all experiments were carried out intriplicate.

Scanning Electron MicroscopyScanning electron microscopy (SEM) analysis were performedin order to assess whether MC were able to grow anddifferentiate on SS. The fixed cells adherent on the metaldiscs and glass coverslips (one/condition) were dehydratedthrough graded ethanol, critical point dried (BAL-TEC,Balzers, Liechtenstein) and mounted on SEM stubs (AgarScientific Ltd., Stansted, United Kingdom) for 4 nm platinumcoating before examination by SEM. Additionally, functionalevidence of osteoclastic corrosion was determined. For thatpurpose, after 3 weeks, incubation discs and coverslips onwhich MC and OC had been cultured (one/experiment), as well

as negative controls incubated without cells in standardmedium, were washed vigorously with distilled water and leftovernight in 0.25% ammonium hydroxide to remove theadherent cells. After rinsing and ultrasonication for 10 min,the discs and coverslips were air dried and mounted on SEMstubs. Subsequently, the entire surface was investigated bySEM. Documentation and analysis of the specimens wasperformed with a high resolution field emission SEM [Zeiss1555 VP-FESEM, 0.1-30 kV, variable pressure (up to 133 Pa,dry only), with BSE, CL, Oxford Instruments EDS and In LensSE detector capabilities, Oberkochen, Germany; available atthe Centre for Microscopy, Characterization and Analysis(CMCA), University of Western Australia, Perth, Australia].

Atomic Emission SpectrometryThe amount of bio-corrosion was quantified by measuring theconcentrations of chromium (Cr), nickel (Ni), manganese (Mn),and molybdenum (Mo) released into the culture supernatantcollected every 7 days as described above. Ion level detectionwas carried out using atomic emission spectrometry [Induc-tively Coupled Plasma-Optical (Atomic) Emission Spectrom-eter, Varian VISTA Axial, Mulgrave, Australia; available atMurdoch University, Perth, Australia]. The detection limitswere 0.01 mg/L for Cr, 0.002 mg/L for Mn, and 0.04 mg/L for Niand Mo.

Cytometric Bead Array AnalysisThe release of IL-1b, IL-6, and TNF-a from MC and OCcultured on metal discs and glass into the culture medium(collected as described above) was evaluated using a com-mercially available Cytometric Bead Array (CBA) kit (BDBiosciences, San Jose, CA). Fluorescence intensity wasmeasured with FACSCanto II Flow Cytometer (BD Bioscien-ces; available at CMCA) and quantified from a calibrationcurve using FCAP Array v1.0 Software (Softflow Technologies,New Brighton, MN). All assays were performed according tothe manufacturers’ instructions.

Calculations and Statistical AnalysisData were analyzed using SPSS for Windows (version 15.0;SPSS Inc., Chicago, IL). Independent-samples t-tests wereconducted in order to determine if mean ion concentrationswere significantly greater than minimal detection limits. Aone-way ANOVA was used to test mean differences in secretedcytokines in the various cell cultures. A p-value of <0.05 wasconsidered statistically significant.

RESULTSThe aim of this study was to investigate whether humanOC are able to directly corrode SS and release metal ionswhen entering in contact with metal surfaces. The veryfirst step was to evaluate the ability of MC and in vitrogenerated OC to grow on medical grade SS. For thatpurpose, the cells were cultured for 21 days either onglass coverslips or SS discs before being processed forSEM. The experiments demonstrated that human MCand OC grow as well on polished SS surfaces as theygrow on glass and culture dish (Fig. 1A,B). Nomorphological differences were detected in the cellscultured on metal discs when compared to the cultureson glass.

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Evidence and Quantification of Bio-CorrosionThe ability of human OC to directly corrode SS wasinvestigated by documenting erosion lacunae on metaldiscs and by measuring the concentration of metal ionsreleased into the culture supernatant.

Detection of Bio-Corrosion Features Using ScanningElectron MicroscopyAfter 21-days incubation, the cell layers were removedfrom the metal discs and coverslips, and the bio-corrosion features documented by SEM. Figure 1Cshows representative images of resorption pits on SSdiscs on which OC had been cultured. As expected,no corrosion features were detected on metal discs onwhich MC had been cultured and on control discsincubated in standard culture medium only (Fig. 1D).No signs of erosion were detected on glass coverslips onwhich osteoclasts were cultured (data not shown).

Measurement of Metal Ions in Culture Supernatant byAtomic Emission SpectrometryThe amount of bio-corrosion was quantified by measur-ing the levels of metal ions released into the culturesupernatant after every incubation week. Significantlyincreased concentrations of Cr, Ni, and Mn weredetected in the supernatant of OC cultured on SS discswhen compared to the levels measured in the super-natant of MC cultured on SS discs and control discs leftin culture medium alone ( p< 0.01) (Fig. 2). Spectrom-etry analysis revealed concentrations of Mo belowdetection limits (0.04 mg/L) in OC supernatant. How-ever, no differences in ion concentrations were meas-ured between the different collection points in thesupernatant of OC cultures on SS ( p> 0.05) (data notshown). The levels of the different metal released by OCinto the supernatant were congruent with the percent-

age of the corresponding metal contained in the metalalloy. All measurements were below detection limits inthe culture supernatant of MC cultures on SS discs.Furthermore, our analysis indicates that the discswere corrosion resistant when left in standard culturemedium over 3 weeks, as no metal ions were detected inthe medium.

Cytokine MeasurementsIn order to assess the effects of the metal ions releasedinto the supernatant on the MC and OC cultures, theamount of IL-1b, IL-6, and TNF-a secreted into theculture medium were measured. Analysis revealedsignificantly increased concentrations of IL-1b, IL-6,and TNF-a in the supernatant of OC cultured on metaldiscs when compared with MC cultures on SS and OCcultured on glass coverslips ( p< 0.05) (Fig. 3). Analysisrevealed no significant differences in all cytokinemeasurements in the supernatant of MC and OCcultured on glass coverslips ( p>0.05).

DISCUSSIONDespite the enormous progress in providing suitablemetallic biomaterials with minimal bio-reactivity andrejection by the body, metal alloys in contact withbiological systems remain prone to corrosion. Thedetection of resorption pits on the surface of SS discs,on which OC were cultured and the significant levels ofcorresponding ions measured in culture supernatants,deliver first evidence for our hypothesis that maturehuman OC are able to directly corrode metal andsubsequently release metal ions into the cellularenvironment.

In tissue fluids, corrosion occurs at the metal surfaceas an electrochemical redox reaction, in which oxidationis coupled with reduction. In terms of how a biologicalenvironment can affect corrosion, it is believed thatproteins can influence the electrochemical behavior ofimplant metals/alloys. However, the overall resultsare not conclusive and the exact effect of proteins on

Figure 1. SEM images showing human in vitro generatedosteoclasts growing on glass coverslips (A) and on stainless steeldiscs (B). (C) Representative image of resorption pits after 21-daysincubation of human osteoclasts on stainless steel discs (inset showsmagnified resorption pits). (D) Surface detail of metal disc left for21 days in standard culture medium (note that the entire discsurface was investigated and no resorption pits were detected).

Figure 2. Histogram showing the significantly higher meanconcentrations (n¼27, �SD) of Ni, Cr, and Mn released into thesupernatantof human osteoclasts cultured over 21 days on stainlesssteel discs when compared to the levels measured in the super-natant of monocytes cultured on metal discs (below detection limits)and control discs (below detection limits). Mo concentrations inosteoclast cultures were below detection limits. *p<0.01.

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corrosion is still the topic of much debate.22 Some authorsdemonstrate that proteins can enhance the propagationof charges between implant and surrounding fluids whileother publications suggest a reduced charge transfer byproteins.23–25 In addition, it has been debated whethermature OC are able to corrode the metal surface, butevidence has yet been missing. Our results clearlyindicate that osteoclast precursors are able to grow anddifferentiate on SS, as well as to directly corrode themetal surface as demonstrated by resorption pits on themetal discs. Moreover, the corresponding significantlevels of metal ions measured in the supernatant of OCcultures support the observed resorption features andsuggest that OC release the metal ions into theirenvironment. It is reasonable to assume that this processmay take place at the bone–implant interface, repre-senting an additional mechanism of metal corrosionin vivo and contributing to the levels of metal ionsmeasured in the peri-prosthetic tissues and in the serumfrom total arthroplasty patients.

The longevity of prosthetic implants depends on anumber of independent factors that are determined bythe surgical techniques, the implant, and the host’sreaction to the implant. To date, the formation of

osteolytic lesions, impairing the maintenance of a stableinterface between the implant and the bone remains theleading cause of failure of total joint arthroplasty.26 Peri-prosthetic osteolysis and subsequent aseptic looseningultimately develops in approximately 20% of patientswith reported failure rates of 13% for the femoralcomponent and 34% for the acetabular component.27,28

Several studies have shown the role of metal wearparticles in the initiation and development of asepticloosening by inducing an inflammatory response thatleads to an increased osteolytic activity at the implant–bone interface.12, 29–31 It is well recognized that activatedmacrophages, in response to titanium wear particles,produce pro-inflammatory cytokines, such as TNF-a,IL-6, and IL-1a/b.32–34 TNF-a acts directly on osteoclastprecursors, whereas IL-6 and IL-1a/b act indirectly byincreasing the expression of RANK-L and M-CSFby osteoblasts.35 In the present study, significantlyincreased levels of IL-1b, IL-6, and TNF-a were meas-ured in the supernatant of OC cultured on SS discs.These results are in line with several previous studiesshowing an increased release of bone resorptive cyto-kines IL-1b, IL-6, and TNF-a by cells of the monocyte/macrophage lineages in aseptic loosening.20,34,36 Theenhanced secretion of pro-inflammatory cytokines bythe metal ions (Cr, Ni, Mn) described here indicatesa possible additional mechanism to the macrophage-mediated foreign body reaction induced by the phagocy-tosis of small wear debris, which may contribute to theinflammatory reactions leading to an increased osteo-lytic activity reported in patients with aseptic loosening.

In addition to the interactions mentioned above,metals may also influence the immune system by directlyinterfering with biochemical and physiological activ-ities.37 More importantly, once released into the systemiccirculation, most metal ions have the ability to formcomplexes with native proteins; such metallo-organiccomplexes can induce allergy or may act as allergens inthe body.5,38–40 Metals known as sensitizers are nickelfollowed by cobalt and chromium.2 Such sensitizers arepresent in SS and potentially released by bio-corrosion.They are presented by dendritic cells, the most potentantigen-presenting cells, and subsequently recognizedby specific T-lymphocytes, thereby resulting in metal-specific immune responses including systemic auto-immune-like diseases and hypersensitivity reactions.These immune responses most typically manifest asskin lesions such as hives, eczema, redness, itching, andasthma-like symptoms.2

A metal in living tissue is prone to corrosion dueto electrochemical reactions.4 In addition to the well-investigated electrochemical aspect of corrosion in aphysiological system, this study demonstrates thathuman OC grow as well on SS as they grow on glass,and are able to directly corrode the metal surface andsubsequently release metal ions. Furthermore, theseresults indicate that the released metal ions may causeinflammatory reactions by inducing the secretion of pro-inflammatory cytokines IL-1b, IL-6, and TNF-a, which in

Figure 3. Bar chart showing the expression levels (�SD) of TNF-a, IL-1b, and IL-6 in the supernatant of monocytes and in vitrogenerated osteoclasts cultured on stainless steel discs at 3 weeks ofincubation. The amount of cytokines released was normalized to theconcentrations secreted by the cells (monocytes and osteoclasts)from the same individual incubated during the same period of timeon glass coverslips and averaged (n¼32). *p<0.05.

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turn may enhance osteoclastic activity at the bone–metal interface.

Prosthetic and osteosynthetic devices made of metalalloys will remain indispensable in orthopedic andtrauma surgery. Therefore, the elucidation of theinteractions between metal surface and the surroundingtissues remains of clinical relevance. Implant topogra-phy modulates osteoclastic corrosion thus inducing therelease of metal ions which may mediate aseptic loosen-ing. Activation of cells from the macrophage lineage hasbeen reported to be dependent on the topography and, toa certain extent, to the chemistry of the metal implant.31

Our results may help developing new metal alloys and/orimplant surface topographies, which may reduce osteo-clastic corrosion without compromising the bonding ofthe peri-implant tissue with the metal indispensable forthe implant stability. Further studies are required inorder to outline a complete picture of the interactionbetween metal implant and bone cells, and the molecularpathways, which are involved in their activation throughreleased metal ions.

CONCLUSIONSWithin the present study, it was shown that humanosteoclast precursors were able to grow and differ-entiate towards mature OC on surgical SS. The maturecells were able to directly corrode the surface of SSand released corresponding metal ions into theirenvironment. The released ions induced the secretionof pro-inflammatory cytokines, which may lead to theformation of osteolytic lesions in the peri-prostheticbone, contributing to the loosening of the implant.

ACKNOWLEDGMENTSThis study was supported by the RMS Foundation, Bettlach,Switzerland. The authors gratefully acknowledge the Centrefor Microscopy, Characterization and Analysis (CMCA) for theuse of their facilities, as well as S. Parkinson for his excellentassistance with the SEM analysis. We thank T. Knox fromPathWest Laboratory Medicine WA for her excellent assis-tance, and B. von Katterfeld for the statistical analysis. AtomicEmission Spectrometry Analysis was done at the Marine andFreshwater Research Laboratory, Murdoch University, Perth,Australia.

REFERENCES1. Long M, Rack HJ. 1998. Titanium alloys in total joint

replacement—a materials science perspective. Biomaterials19:1621–1639.

2. Hallab NJ, Anderson S, Stafford T, et al. 2005. Lymphocyteresponses in patients with total hip arthroplasty. J OrthopRes 23:384–391.

3. Jacobs JJ, Hallab NJ, Skipor AK, et al. 2003. Metaldegradation products: a cause for concern in metal-metalbearings? Clin Orthop Relat Res 417:139–147.

4. Tezer M, Kuzgun U, Hamzaoglu A, et al. 2005. Intraspinalmetalloma resulting in late paraparesis. Arch Orthop TraumaSurg 125:417–421.

5. Jacobs JJ, Skipor AK, Patterson LM, et al. 1998. Metal releasein patients who have had a primary total hip arthroplasty. A

prospective, controlled, longitudinal study. J Bone Joint Surg[Am] 80:1447–1458.

6. Savarino L, Granchi D, Ciapetti G, et al. 2002. Ion release inpatients with metal-on-metal hip bearings in total jointreplacement: a comparison with metal-on-polyethylene bear-ings. J Biomed Mater Res 63:467–474.

7. Urban RM, Jacobs JJ, Tomlinson MJ, et al. 2000. Dissem-ination of wear particles to the liver, spleen, and abdominallymph nodes of patients with hip or knee replacement. J BoneJoint Surg [Am] 82:457–476.

8. Lacey DL, Timms E, Tan HL, et al. 1998. Osteoprotegerinligand is a cytokine that regulates osteoclast differentiationand activation. Cell 93:165–176.

9. Akisaka T, Yoshida H, Inoue S, et al. 2001. Organization ofcytoskeletal F-actin, G-actin, and gelsolin in the adhesionstructures in cultured osteoclast. J Bone Miner Res 16:1248–1255.

10. Filgueira L. 2004. Fluorescence-based staining for tartrate-resistant acidic phosphatase (TRAP) in osteoclasts combinedwith other fluorescent dyes and protocols. J HistochemCytochem 52:411–414.

11. Walsh NC, Cahill M, Carninci P, et al. 2003. Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene. Gene 307:111–123.

12. Bauer TW. 2002. Particles and periimplant bone resorption.Clin Orthop Relat Res 405:138–143.

13. Graves SE, Davidson D, Ingerson L, et al. 2004. TheAustralian Orthopaedic Association National Joint Replace-ment Registry. Med J Aust 180:S31–S34.

14. Harris WH. 1995. The problem is osteolysis. Clin Orthop RelatRes 311:46–53.

15. Voggenreiter G, Leiting S, Brauer H, et al. 2003. Immuno-inflammatory tissue reaction to stainless-steel and titaniumplates used for internal fixation of long bones. Biomaterials24:247–254.

16. Cadosch D, Chan E, Gautschi OP, et al. 2008. Titanium IVions induced human osteoclast differentiation and enhancedbone resorption in vitro. J Biomed Mater Res A (in press).

17. Martin SF. 2004. T lymphocyte-mediated immune responsesto chemical haptens and metal ions: implications for allergicand autoimmune disease. Int Arch Allergy Immunol 134:186–198.

18. Hallab NJ, Caicedo M, Finnegan A, et al. 2008. Th1 typelymphocyte reactivity to metals in patients with total hiparthroplasty. J Orthop Surg 3:6.

19. Streich NA, Breusch SJ, Schneider U. 2003. Serum levels ofinterleukin 6 (IL-6), granulocyte-macrophage colony-stimu-lating factor (GM-CSF) and elastase in aseptic prostheticloosening. Int Orthop 27:267–271.

20. Taki N, Tatro JM, Lowe R, et al. 2007. Comparison of the rolesof IL-1, IL-6, and TNFalpha in cell culture and murine modelsof aseptic loosening. Bone 40:1276–1283.

21. Singh R, Dahotre NB. 2007. Corrosion degradation andprevention by surface modification of biometallic materials.J Mater Sci Mater Med 18:725–751.

22. Yan Y, Neville A, Dowson D. 2006. Understanding the role ofcorrosion in the degradation of metal-on-metal implants. ProcInst Mech Eng [H] 220:173–181.

23. Khan MA, Williams RL, Williams DF. 1999. Conjointcorrosion and wear in titanium alloys. Biomaterials 20:765–772.

24. Williams RL, Brown SA, Merritt K. 1988. Electrochemicalstudies on the influence of proteins on the corrosion of implantalloys. Biomaterials 9:181–186.

25. Zhu J, Xu N, Zhang C. 1999. Characteristics of coppercorrosion in simulated uterine fluid in the presence of protein.Adv Contracept 15:179–190.

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26. Harris WH. 1994. Osteolysis and particle disease in hipreplacement. A. review. Acta Orthop Scand 65:113–123.

27. Aspenberg P, Van der Vis H. 1998. Migration, particles, andfluid pressure. A. discussion of causes of prosthetic loosening.Clin Orthop Relat Res 352:75–80.

28. Keener JD, Callaghan JJ, Goetz DD, et al. 2003. Twenty-five-year results after Charnley total hip arthroplasty in patientsless than fifty years old: a concise follow-up of a previousreport. J Bone Joint Surg [Am] 85-A:1066–1072.

29. Bi Y, Van De Motter RR, Ragab AA, et al. 2001. Titaniumparticles stimulate bone resorption by inducing differentiationof murine osteoclasts. J Bone Joint Surg [Am] 83-A:501–508.

30. Matthews JB, Green TR, Stone MH, et al. 2001. Comparisonof the response of three human monocytic cell lines tochallenge with polyethylene particles of known size and dose.J Mater Sci Mater Med 12:249–258.

31. Sommer B, Felix R, Sprecher C, et al. 2005. Wear particles andsurface topographies are modulators of osteoclastogenesisin vitro. J Biomed Mater Res A 72:67–76.

32. Jiranek WA, Machado M, Jasty M, et al. 1993. Production ofcytokines around loosened cemented acetabular components.Analysis with immunohistochemical techniques and in situhybridization. J Bone Joint Surg [Am] 75:863–879.

33. Kim KJ, Rubash HE, Wilson SC, et al. 1993. A histologic andbiochemical comparison of the interface tissues in cementless

and cemented hip prostheses. Clin Orthop Relat Res 287:142–152.

34. Stea S, Visentin M, Granchi D, et al. 2000. Cytokines andosteolysis around total hip prostheses. Cytokine 12:1575–1579.

35. Ragab AA, Nalepka JL, Bi Y, et al. 2002. Cytokinessynergistically induce osteoclast differentiation: support byimmortalized or normal calvarial cells. Am J Physiol CellPhysiol 283:C679–C687.

36. Goodman SB, Huie P, Song Y, et al. 1998. Cellular profile andcytokine production at prosthetic interfaces. Study of tissuesretrieved from revised hip and knee replacements. J BoneJoint Surg [Br] 80:531–539.

37. Shrivastava R, Upreti RK, Seth PK, et al. 2002. Effects ofchromium on the immune system. FEMS Immunol MedMicrobiol 34:1–7.

38. Budinger L, Hertl M. 2000. Immunologic mechanisms inhypersensitivity reactions to metal ions: an overview. Allergy55:108–115.

39. Swiontkowski MF, Agel J, Schwappach J, et al. 2001.Cutaneous metal sensitivity in patients with orthopaedicinjuries. J Orthop Trauma 15:86–89.

40. Valentine-Thon E, Schiwara HW. 2003. Validity of MELISAfor metal sensitivity testing. Neuro Endocrinol Lett 24:57–64.

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Chapter 05 Biocorrosion and uptake of titanium by human osteoclasts As tissue fluids enter into contact with the metal surface, corrosion occurs via an

electrochemical redox reaction. In terms of how a biological environment can affect

corrosion, it is believed that proteins can influence the electrochemical behaviour of

implant metals/alloys. Besides the electrochemical aspect of corrosion, it has been

debated whether cellular mechanisms are able to corrode the metal surface. This

study investigated whether human OC are able to grow on Ti and Al, and have

corrosive potency leading to an increased release of corresponding metal ions.

Scanning electron microscopy analysis demonstrated long-term viable OC cultures

on the surface of Ti and Al foils. Atomic emission spectrometry investigations showed

significantly increased levels of Al ions in the supernatant of OC cultured on Al;

however, all measurements in the supernatants of cell cultures on Ti were below

detection limits. Despite this, confocal microscopy analysis with Newport Green DCF

diacetate ester staining depicted intense fluorescence throughout the cytoplasm and

nucleolus of OC cultured on Ti foils. Comparable fluorescence intensities were not

observed in monocytes and control cells cultured on glass. It must be assumed that

Ti ions bind with a strong affinity to phosphorylated intracellular proteins.

Consequently, Ti was not released into the culture supernatant, explaining the no

detectable Ti levels. Titanium–protein complexes may be eventually released into the

extracellular space once the OC die and break down. This study demonstrated that

human OC precursors are able to grow and differentiate toward mature cells on Ti.

Furthermore, it suggests that mature OC enhance corrosion of Ti implants, most

likely by removing the protecting metal oxide layer from the metal surface, and take

up corresponding Ti ions.

- 23 -

Biocorrosion and uptake of titanium by human osteoclasts

Dieter Cadosch,1,2† Mohamed S. Al-Mushaiqri,2* Oliver P. Gautschi,2* James Meagher,2

Hans-Peter Simmen,1 Luis Filgueira2

1Division of Trauma Surgery, University Hospital Zurich, Zurich, Switzerland2School of Anatomy and Human Biology, University of Western Australia, Crawley, Australia

Received 11 September 2009; revised 6 May 2010; accepted 7 June 2010

Published online 24 September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32914

Abstract: All metals in contact with a biological system

undergo corrosion through an electrochemical redox reac-

tion. This study investigated whether human osteoclasts (OC)

are able to grow on titanium and aluminum, and directly cor-

rode the metals leading to the release of corresponding

metal ions, which are believed to cause inflammatory reac-

tions and activate osteoclastic differentiation. Scanning elec-

tron microscopy analysis demonstrated long-term viable OC

cultures on the surface of titanium and aluminum foils.

Atomic emission spectrometry investigations showed signifi-

cantly increased levels of aluminum in the supernatant of OC

cultured on aluminum; however, all measurements in the

supernatants of cell cultures on titanium were below detec-

tion limits. Despite this, confocal microscopy analysis with

Newport Green DCF diacetate ester staining depicted intense

fluorescence throughout the cytoplasm and nucleolus of OC

cultured on titanium foils. Comparable fluorescence inten-

sities were not observed in monocytes and control cells cul-

tured on glass. The present study demonstrated that human

osteoclast precursors are able to grow and differentiate to-

ward mature OC on titanium and aluminum. Furthermore, it

established that the mature cells are able to directly corrode

the metal surface and take up corresponding metal ions,

which subsequently may be released and thereby induce the

formation of osteolytic lesions in the periprosthetic bone,

contributing to the loosening of the implant. VC 2010 Wiley

Periodicals, Inc. J Biomed Mater Res Part A: 95A: 1004–1010, 2010.

Key Words: osteoclast, biocorrosion, titanium, aluminum,

osteolysis, Newport Green

INTRODUCTION

Over the past decades, significant developments have beentaking place to provide suitable metals and metal alloys forprosthetic and osteosynthetic implants with optimal me-chanical characteristics and good biocompatibility with min-imal adverse reactions to host tissue.1 Among other metals,pure titanium and titanium alloys, often containing alumi-num and vanadium, are frequently used biomaterials in den-tal, orthopedic, and trauma surgery because of a favorablecombination of strength, good fabrication properties, lowinherent toxicity, and minimal reactivity with biomoleculeswhen compared with other metallic biomaterials.1,2 How-ever, despite the immense progress in providing suitablemetallic biomaterials, their tendency to corrode whenexposed to a physiological environment remains a seriousconcern to the surgical and biomaterial community.2–4 Ele-vated concentrations of metal ions (including titanium) havebeen measured in clinically retrieved capsular and peripros-thetic tissues as well as in distal organs (liver, spleen, andlymph nodes) and body fluids (serum and urine) of totalhip arthroplasty patients.5–7 The biology behind corrosioninside the human body and the distribution of the metalions throughout the organism is not yet fully understood. Ithas been suggested that extracellular body fluids, including

blood serum, have corrosive proprieties and contain metalbinding proteins.3,4 Furthermore, it has long been debatedwhether osteoclasts (OC) are able to corrode orthopedicimplants when entering in contact with the metal surfaces.Using a human in vitro model, we have recently demon-strated that human OC are able to corrode surgical stainlesssteel.8 However, direct evidence of titanium corrosion by OChas not yet been demonstrated. In a water-based environ-ment, titanium and aluminum form thin oxide layers ontheir surface, protecting them from further electrochemicalcorrosion. At physiological pH, the water solubility for alu-minum and titanium ions is in the range of micromolar con-centrations.9 However, in the presence of certain anionsfound in biological fluids, such as phosphates, titaniumforms complexes with lower water solubility, resulting inconcentrations below detectable levels. Thus, we haverecently established a fluorescent method for the detectionof complex, intracellular metal ions, including titanium,using Newport Green DCF diacetate ester.10

OC are highly specialized, often multinucleated cells,which are uniquely capable of bone resorption.11 They dif-ferentiate from marrow-derived mononuclear precursors,which circulate in the CD14þ monocyte fraction of peri-pheral blood, in the presence of macrophage-colony

*These authors contributed equally to this work and should both be considered as second author.†Dieter Cadosch was awarded a Scholarship for International Fees Research (SIRS) by the University of Western Australia. The research was

carried out while the author was a SIRS scholar.

Correspondence to: D. Cadosch; e-mail: dcadosch@gmx.net

1004 VC 2010 WILEY PERIODICALS, INC.

stimulating factor (M-CSF) and the receptor activator of NF-jB ligand (RANK-L), which is expressed by osteoblasts andother bone-related stromal cells.12,13 Mature OC are oftendescribed as multinucleated cells, displaying additional dis-tinct morphological characteristics, such as a ruffled basalborder surrounded by a membrane-stabilizing extensivering of actin filaments, and expressing cathepsin K and tar-tate-resistant acid phosphatase.14–16 However, also mono-nucleated OC can already display the distinct morphological,molecular, and functional properties of mature cells.17,18 Inrecent years, we have established a reliable in vitro modelfor the generation of human OC using human monocytesisolated from peripheral blood and cultured in the presenceof osteoclastic cytokines (M-CSF and RANK-L).8,15,19–21 Thiswell-characterized in vitro model has been used throughoutthis study.

The aims of this study were as follows: first, to investi-gate whether human monocytes are able to grow on alumi-num and titanium and to differentiate into mature OC; andsecond, to test the hypothesis that mature OC are able todirectly corrode pure titanium and aluminum and releasecorresponding metal ions into the peri-implant environment.Aluminum was included as an easy-to-handle and inexpen-sive basic metal foil model; however, titanium was the mainfocus of this study.

MATERIALS AND METHODS

Metal foilsTitanium foils were purchased from EURO Titan, UK, andwere 5 mm in diameter. Commercially available aluminumfoil (Capral Aluminum, NSW, Australia) was wrapped overglass coverslips for easy handling. Before use, the foils werewashed in ethanol, rinsed in distilled water, and steamsterilized.

Isolation of peripheral blood monocytic cellsEthical approval for the use of human blood cells wasgranted by the Ethics Committee of the University of West-ern Australia and the Australian Red Cross Blood Service(ARCBS), Perth, WA, Australia. Human monocytic cells (MC)were obtained from adherent peripheral blood mononuclearcells (PBMCs). Briefly, PBMCs were isolated from buffy coatsof 10 healthy blood donors (ARCBS) through Ficoll-gradientcentrifugation (Amersham Biosciences, Uppsala, Sweden) aspreviously described.21,22 The PBMCs were cultured (37�C,humidified, 5% CO2) in 25 cm2 tissue culture flasks (Sar-stedt) in RPMI-1640 Glutamax medium (RPMI) (Gibco/Invi-trogen, Auckland, NZ), and supplemented with 10% humanserum (ARCBS) and 1% antibiotics (Gibco) (standard me-dium). After 1 h, the nonadherent PBMCs were discarded,and the adherent PBMCs consisting of MC cells werewashed twice with 0.1M phosphate-buffered saline (PBS)pH 7.2 (Gibco). The collected cells were resuspended inRPMI medium and counted manually in a hemocytometer.

Adherent cell cultures on metal foils and coverslipsThe isolated MC were seeded either onto titanium, alumi-num foils (106 cells/foil) or glass coverslips (12-mm diame-

ter) placed into 24-well tissue culture plates (Sarstedt,Nuernbrecht, Germany). After overnight incubation (37�C,humidified, 5% CO2), the cultures were replenished with1500 mL of standard medium. To generate OC, one-half ofthe cell cultures were supplemented with osteoclast differ-entiation cytokines (10 ng/mL recombinant human RANK-Land 10 ng/mL recombinant human M-CSF, R&D, Minneapo-lis, MN) as previously described.15,19 One-half of the cul-tures were left as MC with standard culture medium onlyand no cytokines added. As negative controls, metal foilsand glass coverslips were incubated in standard culturemedium only. The conditioned supernatant was collectedweekly, pooled, frozen at �20�C, and replaced with 1500 mLfresh standard medium. After 21 days, the supernatant wascollected and the cells fixed with 2.5% EM grade glutaralde-hyde (Electron Microscopy Science, Washington, PA) in culturemedium for scanning electron microscopy (SEM). The samplesdesigned for confocal microscopy with Newport Green DCFdiacetate ester staining were fixed with 1% paraformaldehydein PBS before processed further as described later. Six speci-mens were cultured for each condition and all experimentswere carried out in triplicate.

Atomic emission spectrometryThe amount of biocorrosion was quantified by measuringthe concentrations of titanium and aluminum ions releasedinto the corresponding culture supernatant. Ion levels weremeasured using atomic emission spectrometry [InductivelyCoupled Plasma-Optical (Atomic) Emission Spectrometer,Varian VISTA Axial, Mulgrave, Australia; available at Mur-doch University, Perth, WA, Australia]. The detection limitfor both titanium and aluminum was 0.01 mg/L.

Scanning electron microscopyScanning electron microscopy analysis was performed toassess whether MC were able to grow and differentiate ontitanium and aluminum foil. The glutaraldehyde fixed cellsadherent on the foils and glass coverslips were dehydratedthrough graded ethanol, critical point dried (BAL-TEC,Balzers, Liechtenstein) and mounted on SEM stubs (AgarScientific, Stansted, UK) for 4-nm platinum coating beforeexamination by SEM. Documentation and analysis of thespecimens was performed with a high resolution fieldemission SEM [Zeiss 1555 VP-FESEM, 0.1–30 kV, variablepressure (up to 133 Pa, dry only), with BSE, CL, OxfordInstruments EDS and In Lens SE detector capabilities,Oberkochen, Germany; available at the Centre for Microscopy,Characterization and Analysis, University of Western Australia,Perth, WA, Australia].

Fluorescent tartrate-resistant acidic phosphatase andActin StainingTo further characterize the OC cultures, fluorescent tartrate-resistant acidic phosphatase (TRAP) and actin staining wasperformed. The cells, fixed with 1% paraformaldehyde inPBS, were stained for TRAP activity using a fluorescence-based protocol.15 Briefly, the cells were incubated for 15minutes with ELF97 substrate (20 mM, E6569, Molecular

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Probes/Invitrogen, Eugene, OR) in 110 mM acetate buffer(pH 5.2), containing 1.1 mM sodium nitrite, and 7.4 mM tar-trate (all from Sigma-Aldrich). The nuclei were stained withDAPI (40,6-diamidine-20-phenylindole dihydrochloride, 10ng/mL, Roche Diagnostics, Mannheim, Germany). The basalactin ring was stained with AlexaFluor546-labelled phalloi-din (0.2 U/mL in PBS). After mounting (Dako FluorescentMounting Medium, Dako Cytomation, Carpinteria, CA, USA),the samples were analyzed and documented using confocalmicroscopy.

Newport Green DCF diacetate ester stainingTo investigate the ability of OC to corrode titanium and takeup corresponding ions, paraformaldehyde (1% in PBS) fixedcells adherent to the metal foils and glass coverslips wereincubated for 1 h at room temperature with Newport GreenDCF diacetate ester at 1 lM concentration (MolecularProbes/Invitrogen) and washed with PBS.10 Subsequently,the cells were stained with DAPI nuclear stain and mountedin Dako Fluorescent Mounting Medium (DakoCytomation)for confocal microscopy.

Confocal microscopyCell morphology was analyzed using multiphoton confocalfluorescence microscopy, including the UV-laser for detec-tion of DAPI, multiphoton laser for ELF97, phalliodin Alexa-Fluor546 and Newport Green DCF detection and the corre-sponding software for image analysis (Leica TCS SP2 AOBS,Germany).

Calculations and statistical analysisData were analyzed using SPSS for Windows (version 15.0;SPSS Inc., Chicago, IL). Independent sample t-tests wereconducted to determine whether mean ion concentrationswere significantly greater than minimal detection limits. Ap-value of less than 0.05 was considered statisticallysignificant.

RESULTS

This study aimed to investigate whether human OC are ableto directly corrode titanium and aluminum, and release cor-responding metal ions when in contact with metal surfaces.The first step was to evaluate the ability of MC and in vitrogenerated OC to attach, grow on titanium and aluminum.For that purpose, the cells were cultured for 21 days eitheron glass coverslips or on metal foils before processing forSEM and fluorescence microscopy. The experiments demon-strated that human MC and OC attach and grow as well ontitanium and aluminum surfaces, as they grow on glass andculture dishes (Fig. 1). The OC cultured on metal foilsshowed a more vacuolated membrane and were smallerthan control cells cultured on glass (averaged cell diameter21 6 4 lm vs. 28 6 7 lm, p > 0.05). No differences in celldensity were detected between the different cell cultures(p > 0.05). Additionally, OC were stained for TRAP activityand localization of the basal actin ring using confocal mi-croscopy methods (Fig. 2), confirming the osteoclastic prop-

erties of the cells cultured in the presence of osteoclasticcytokines (M-CSF and RANK-L).

Measurement of metal ions in culture supernatant byatomic emission spectrometryThe first finding to provide evidence and quantification ofdirect corrosion of titanium and aluminum by human OCwas realized by measuring the concentration of metal ionsreleased into the corresponding culture supernatant afterevery week of incubation. Increased concentrations of alu-minum were detected in the supernatant of OC cultured onaluminum foils when compared with the levels measured inthe supernatant of MC cultured on aluminum foils and con-trol foils left in culture medium without cells (p < 0.01)

FIGURE 1. Representative SEM images showing human OC growing

on glass (A), titanium foil (B), and aluminum foil (C) after 21 days of

incubation.

1006 CADOSCH ET AL. BIOCORROSION AND UPTAKE OF TITANIUM

(Table I). No differences in ion concentrations were meas-ured between the different collection points (each incuba-tion week) in the supernatant of OC cultures (p > 0.05)(data not shown). Surprisingly, all measurements werebelow detection limits in the culture supernatant of OC andMC cultured on titanium foils as well as in the medium inwhich the foils were left during the same period of time.

Detection of intracellular titanium ions in OCusing confocal microscopyTo demonstrate corrosion of titanium and uptake of corre-sponding ions, the cells cultured on titanium foils werestained using Newport Green DCF diacetate ester and ana-lyzed using confocal microscopy. Images showed intensefluorescence signals in OC cultured on titanium foils [Fig.3(D,E)], when compared with fluorescence intensities in OCand MC cultured on glass [Fig. 3(A–C)]. The microscopyanalysis provided additional relevant information about thesubcellular distribution of the titanium ions in OC culturedon titanium foils, showing intense partially granular andpartially diffuse fluorescence throughout the cytoplasm anddistinct fluorescence of the nuclear heterochromatin. Thissuggests that titanium ions bind to cytoplasmic and nuclearstructures and are therefore not released into the culturesupernatant. The fluorescent Newport Green DCF signal forcells cultured on aluminum was similar to the control levels(data not shown).

DISCUSSION

Despite the remarkable progress in providing suitable me-tallic biomaterials such as titanium and titanium alloys,with minimal bioreactivity and rejection by the body, metalimplants in contact with biological systems remain prone tocorrosion. The aim of this study was to investigate the cor-rosive potency of human OC on titanium and aluminum. Forthat purpose, in vitro generated OC using a well-establishedmodel, and corresponding cultured monocytes were used.Aluminum was used as an inexpensive easy-to-handle metalmodel, whereas titanium was the main focus of thisresearch.

In tissue fluids, corrosion occurs at the metal surfaceas an electrochemical redox reaction, in which oxidation

FIGURE 2. Multiphoton confocal fluorescence microscopy of TRAP (A; green color) and actin (B; red color) stain on OC cultured for 21 days

(Nuclei are stained in blue with DAPI). Arrow 1 indicates the well-documented basal actin ring in a monocucleated osteoclastic cell. Arrow 2 indi-

cates a multinucleated osteoclast in close contact with two other cells, possibly undergoing additional fusion and formation of a cell with more

nuclei. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

TABLE I. Measurement of Aluminium Concentrations

Culture conditionsAluminum

concentrations (mg/L)

Monocytes on glass 0.032 (60.004)Osteoclasts on glass 0.078 (60.024)Monocytes on aluminum 0.162 (60.125)Osteoclasts on aluminum 9.500* (60.384)

Measurement of aluminium concentrations (6SD) in the culture su-

pernatant using atomic emission spectrometry after 21 days incubation.

Osteoclasts cultured on aluminium foils released significantly higher

concentrations when compared with monocyte cultures. Note all meas-

urements of titanium concentrations were below detection limits.

*p < 0.01.

FIGURE 3. Multiphoton confocal fluorescence microscopy image (op-

tical sections through the ‘‘nuclear level’’) showing monocytes cul-

tured for 21 days on glass (A) or titanium foil (B), as well as OC on

glass (C) or on titanium foil (D and E). Note the staining of intracellu-

lar titanium complexes with Newport Green DCF diacetate ester,

detected only in OC cultured on titanium foil, in a granular and dif-

fuse distribution throughout the cytoplasm, as well as in the hetero-

chromatin of the nucleus (D and E; green color). Nuclei are stained in

blue with DAPI. Note the multiple nuclei in the cells on (D) and (E).

[Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

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(electron loss of the metal) is coupled with reduction (elec-tron gain of electrolyte components).23 In terms of how abiological environment can affect corrosion, it is believedthat proteins can influence the electrochemical behavior ofimplant metals/alloys. However, the overall results are notconclusive and the exact effect of proteins on corrosion isstill the topic of much debate.24–28 Beside the electrochemi-cal aspect of corrosion in a physiological environment, ithas been debated whether mature OC themselves are ableto corrode the metal surface.

Using fluorescence microscopy analysis, we have con-firmed that MC isolated from peripheral blood used forthese experiments can differentiate into TRAP positive, mul-tinucleated OC displaying a distinct basal actin ring. Addi-tionally, we have previously demonstrated that the humanOC generated in vitro using the same protocol, are func-tional cells able to resorb dentin and express cathepsin K,as well as a basal acidic compartment.19–21 Using this invitro OC model, our results indicate that osteoclast precur-sors are able to grow and differentiate on titanium and alu-minum, as well as to directly corrode the metal surface asdemonstrated by the release of aluminum ions into the cul-ture supernatant and by the intracellular detection of tita-nium. It is reasonable to assume that a similar process maytake place at the bone–implant interface and represent anadditional mechanism of metal corrosion in vivo that con-tributes to the levels of metal ions (including titanium)measured in the periprosthetic tissues, in distal organs andin the serum from total arthroplasty patients.5–7

Both aluminum and titanium corrode easily in a water-based environment. However, a thin oxide layer is quicklyformed on the surface, protecting the metal from furtherelectrochemical corrosion.9,29 In a physiological context, itmust be considered that functional OC are able to generatean environment more prone to corrosion, by secreting pro-tons into the resorption lacunae.18 The low pH in theresorption compartment with the high concentration of Hþmight destabilize the oxide layer, leading to free metal ionson the surface.9,29 Removal of the metal oxide layer willsubsequently expose new metal to additional corrosionenhanced by the acidic conditions in the osteoclastic resorp-tion pit. Once solubilized in the osteoclastic pit, the metalions are most likely taken up by the OC into the TRAP-containing transcytotic compartment for further process-ing.21,30,31 Usually, resorbed material is eventually releasedby the OC into the extracellular space. This process happensin the case of aluminum, as shown in this study and forother metal ions such as cobalt and chromium as previouslydemonstrated.8 However, certain metal ions may bind to cel-lular structures and form stable complexes that remain inthe cells. This is certainly the case for titanium ions thatbind to cytoplasmic and nuclear structures, as depicted byconfocal fluorescence microscopy. Consequently, titaniumwas not released into the culture supernatant, explainingthe nondetectable titanium levels in our cultures. It is rea-sonable to assume that titanium ions bind to phosphoryl-ated molecules, including phosphorylated intracellular pro-teins.32–35 This hypothesis is further supported in previous

experiments by our group using element-specific electronmicroscopy methods, such as energy-filtered transmissionelectron microscopy, showing the strong affinity of titaniumions for phosphorylated cellular structure. By complexingwith phosphorylated cellular structures, which often corre-spond to active functional states of enzymes or signalingproteins, titanium may interfere with signaling pathways.Additionally, the attachment of titanium to membrane phos-pholipids may change its fluidity and thereby alter cellularfunctions such as migration, protein secretion and the abil-ity to respond to stimuli as shown with other metals suchas aluminum and nickel.36 Titanium–protein complexes maybe eventually released into the extracellular space, and con-ceivably into the systemic circulation in patients, once theOC die and break down. However, additional studies arerequired to show how titanium is translocated from theimplant site into the surrounding tissue and how it accumu-lates in distant organs.

Aseptic loosening of metal joint implants remains a signif-icant problem in orthopedic surgery. More than 10% ofpatients suffer from the condition within 20 years of aprimary hip arthroplasty, with the incidence continuing toincrease.37 Aseptic loosening is suggested to result from anincreased osteolytic activity at the bone-implant interface,which ultimately leads to loss of fixation.38–42 Several studieshave demonstrated the role of titanium wear particles (in thenanometer range) in the initiation and development of asep-tic loosening by inducing an inflammatory response charac-terized by an enhanced production of proinflammatory cyto-kines, such as TNF-a, IL-6, and IL-1a/b, which ultimatelylead to an increased osteolytic activity.38,43–47 In addition topathophysiological mechanisms involving wear particles, wehave previously demonstrated that titanium ions directlyinduce differentiation of osteoclast precursors toward matureOC capable of effective bone resorption.19 Titanium ions arealso supposed to play a role in the recruitment of osteoclastprecursors by inducing the expression of specific cytokines.20

The osteoclastic corrosion described here is likely to contrib-ute to the pathophysiological mechanism mentioned earlierby releasing intracellular titanium ions after cell death.

Summarizing, this study demonstrates that human OCgrow as well on titanium and aluminum as they grow onglass and culture dishes. Human OC are able to directly cor-rode the metal surface and take up corresponding metalions. Aluminum ions are released by OC into the surround-ing extracellular space, whereas titanium ions remain in theOC, as they appear to bind to cellular structures, probablyphosphorylated proteins, phospholipids, and nucleotides.Following cell death, the released titanium ions are likely tocontribute to the pathophysiological mechanisms of asepticloosening by inducing osteoclastic differentiation. Further-more, once titanium–protein complexes are released intothe blood circulation they may act as antigens and triggerinflammatory immune reactions.5,48

CONCLUSIONS

Within the present study, it was confirmed that humanosteoclast precursors were able to grow and differentiate

1008 CADOSCH ET AL. BIOCORROSION AND UPTAKE OF TITANIUM

toward mature OC on aluminum and titanium. The maturecells were able to directly corrode the surface of the metalsand take up metal ions. Similar processes may take place invivo, leading to increased biocorrosion, and release of metalions into the surrounding tissue and into the systemic bloodcirculation.

ACKNOWLEDGMENT

The authors thank T. Knox for her outstanding assistance andeditorial advice.

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48. Martin SF. T lymphocyte-mediated immune responses to chemi-

cal haptens and metal ions: Implications for allergic and autoim-

mune disease. Inter Arch Allergy Immunol 2004;134:186–198.

1010 CADOSCH ET AL. BIOCORROSION AND UPTAKE OF TITANIUM

Chapter 06 Titanium IV ions induced human osteoclast differentiation and enhanced bone resorption in vitro The sum of all aspects of biocorrosion, including the mechanism presented in

Chapter 04 and 05, leads to the release of Ti ions (predominantly Ti in the 4+

oxidation state: Ti(IV)) from surgical implants, with concentrations in the range of 1

μM in tissue and blood. The aim of this study was to investigate the influence of

Ti(IV) ions on differentiation and function of OC and their precursor cells in vitro.

Human monocytes and in vitro generated OC were exposed to 1 μM Ti(IV) ions for

10 days. Thereafter, OC differentiation, activation, and function were evaluated.

Transcription of specific OC genes was measured using quantitative reverse PCR),

which showed increased expression of tartrate-resistant acid phosphatase (TRAP) in

Ti(IV)-treated monocytes from ~20% of the patients. Detection and quantification of

intracellular TRAP activity using phosphatase substrate ELF97 as a fluorescent

substrate revealed a significant increase of TRAP-positive cells in Ti(IV)-treated

monocytes. In addition, the OC phenotype of the TRAP-positive cells was

investigated through functional assays by measuring lacunar resorption of dentine.

Ti(IV)-treated monocytes became functional bone resorbing cells, significantly

increasing their osteo-resorptive activity to similar levels as OC in vitro. These results

suggest that Ti(IV) ions directly (i.e. disconnected from OC differentiation cytokines)

induce differentiation of OC precursors toward mature and functional OC in ~20% of

individuals.

- 31 -

Titanium IV ions induced human osteoclast differentiationand enhanced bone resorption in vitro

Dieter Cadosch,1,2 Erwin Chan,1 Oliver P. Gautschi,1,2 James Meagher,1 Rene Zellweger,2 Luis Filgueira11School of Anatomy and Human Biology, University of Western Australia, Crawley, Australia2Department of Orthopaedic and Trauma Surgery, Royal Perth Hospital, Perth, Australia

Received 10 January 2008; revised 4 May 2008; accepted 3 June 2008Published online 5 August 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32183

Abstract: There is increasing evidence that titanium (Ti)ions are released from orthopedic implants, with concentra-tions in the range of 1 lM in tissue and blood, and mayplay a role in aseptic loosening of orthopedic implants. Thisstudy investigated whether Ti(IV) ions induce differentia-tion of monocytic osteoclast precursors into osteo-resorptivemultinucleated cells and influence the activation and func-tion of in vitro generated osteoclasts. Human monocytesand in vitro generated osteoclasts were exposed to 1 lMTi(IV) ions for 10 days. Thereafter, osteoclast differentiation,activation, and function were evaluated. Transcription ofspecific osteoclastic genes was measured using quantitativereverse transcription polymerase chain reactions, whichshowed increased expression of tartrate-resistant acid phos-phatase (TRAP) in �20% of Ti(IV)-treated monocytes.

Detection and quantification of intracellular TRAP activityusing ELF97 as a fluorescent substrate revealed a significantincrease of TRAP-positive cells in Ti(IV)-treated monocytes.Additionally, as demonstrated on dentin slide cultures,Ti(IV)-treated monocytes became functional bone resorbingcells, significantly increasing their osteo-resorptive activityto similar levels as osteoclasts in vitro. These results suggestthat Ti(IV) ions released by biocorrosion from orthopedicimplants induce differentiation of monocytes towardmature, functional osteoclasts, which may well contributethe pathomechanism of aseptic loosening. � 2008 WileyPeriodicals, Inc. J Biomed Mater Res 91A: 29–36, 2009

Key words: titanium; osteoclast; monocyte; tartrate-resist-ant acid phosphatase; ELF97

INTRODUCTION

Failure of orthopedic joint implants is a significantproblem in orthopedic surgery, leading to pain, lossof function, and ultimately, revision surgery. Failurecan be caused by various factors such as infection,fracture, mechanical failure, or poor surgical tech-nique. However, the most frequent cause of implantfailure is aseptic loosening, which is thought tooccur in over 10% of cases within 20 years of aprimary hip arthroplasty.1 Aseptic loosening issuggested to occur through an increase of osteolyticactivity at the bone–implant interface, leading to lossof fixation and potentially difficult revision sur-gery.2,3 To date, the pathophysiological mechanismof increased osteolysis in the presence of metalimplants is still not fully understood.

Several studies have shown that the net loss ofbone at the tissue–implant interface occurs due toincreased bone resorption by enhanced osteoclasticactivity and/or a decrease in bone formation byosteoblasts. The mechanical wear process in articularcoupling of prostheses is responsible for severeinflammatory reactions and bone resorption, whichhas been recognized as the primary biological mech-anism leading to periprosthetic osteolysis.4 Phagocy-tosis of titanium wear particles by macrophagesinduces their activation, producing mediators thatenhance osteoclast formation.5 However, the poten-tial role of titanium ions released by biocorrosionfrom the implant surface must also be considered.

Titanium as a biomaterial has been extensivelystudied and is frequently used in restorative surgery.Pure titanium (Ti) and several titanium alloys, suchas TiAl6V4, Ti318, and Ti350, are commonly used.After implantation, all metal surfaces are immedi-ately surrounded by a layer of extracellular fluid andproteins. Subsequently, a dense passive oxide film isformed with titanium at the Ti(IV) oxidation state.Ti(IV)O2 provides the benefits of low inherent toxic-ity, low water solubility (in the range of 1 lM con-centration), and a low reactivity with biomolecules

Correspondence to: D. Cadosch; e-mail: dcadosch@anhb.uwa.edu.auContract grant sponsor: National Institutes of Health;

contract grant number: GM072726Contract grant sponsor: AO Foundation; contract grant

number: 05Z34

� 2008 Wiley Periodicals, Inc.

in an aqueous environment.6 However, despite theserelatively inert qualities, titanium has been shown tobe released into surrounding tissues of orthopedicimplants by various mechanisms including wear,corrosion, biological activity, and mechanically accel-erated electrochemical processes such as stress, fret-ting, and fatigue corrosion.7 Indeed, elevated concen-trations of titanium and other metal ions have beenmeasured in clinically retrieved capsular, peripros-thetic tissues, distal organs (liver, spleen, and lymphnodes), and body fluids (serum and urine) in totalhip arthroplasty patients.8,9 Jacobs et al. have meas-ured titanium concentrations up to 11.17 ng/mL inthe serum of patients with titanium alloy prosthesesat 36 months postoperatively.10 Dorr et al. reported ti-tanium levels in the fibrous membrane encapsulatingtitanium-alloy implants up to 22 ng/mL (�0.5 lM).11

Therefore, it can be hypothesized that metal ions maycontribute to aseptic loosening by accelerating osteo-clastic bone resorption and/or inhibiting the functionof osteoblasts. Previous studies have demonstratedthat nontoxic concentrations of metal ions affect thedifferentiation and function of osteoblastic cells invitro.12 Although there have been several studiesinvestigating the effect of metal ions on cell metabo-lism, relatively few have investigated their effects onbone cells. Of those investigating bone cells, mostfocused on osteoblastic cells.12,13 To date, little isknown about the effects of metal ions on humanosteoclast differentiation and function.

Osteoclasts are highly specialized multinucleatedcells, which are uniquely capable of bone resorp-tion.14 They are formed by the fusion of marrow-derived mononuclear precursors which circulate inthe CD14þ monocyte fraction of peripheral blood.15

Osteoclastic differentiation from hematopoietic andcirculating monocytes occurs in the presence of mac-rophage-colony stimulating factor (M-CSF) and thereceptor activator of NF-jB ligand (RANK-L) whichis expressed by osteoblasts and other bone-relatedstromal cells.16 Mature osteoclasts display distinctcharacteristics. First, they have specific morphologi-cal qualities, such as multiple nuclei and a basalruffled border surrounded by a membrane stabiliz-ing the extent ring of actin filaments.17 Second, thereis a specific expression of cathepsin K (CATK),which is found within the lysosomal granules. Last,they express tartrate-resistant acid phosphatase(TRAP), which is regarded as an osteoclastic marker,although there are other cells of the macrophage/dendritic cell lineage that express TRAP under cer-tain conditions.18,19

The aim of this study was to test the hypothesisthat Ti(IV) ions, which represent the majority andmost stable form of titanium ions released by biocor-rosion from titanium implants, induce osteoclastdifferentiation in blood monocytes to become osteo-

resorptive cells. For this purpose, TRAP expressionand activity was assessed.19–21 In addition, the boneresorptive function of the cells was evaluated byusing dentin slide cultures.

MATERIALS AND METHODS

Isolation of peripheral blood monocytic cells

Blood samples of 22 individuals were used for this study.Every sample was examined independently throughout allstudy assays. Human monocytes were obtained from ad-herent peripheral blood mononuclear cells (PBMCs).Briefly, PBMCs were isolated from buffy coats [AustralianRed Cross Blood Service (ARCBS), Perth, WA, Australia]through Ficoll-gradient centrifugation (Amersham Bioscien-ces, Uppsala, Sweden). The PBMCs were cultured (378C,humidified, 5% CO2) in 25-cm2 tissue culture flasks (Sar-stedt, Nuernbrecht, Germany) in RPMI-1640 Glutamaxmedium (RPMI; Gibco/Invitrogen, Auckland, NZ), supple-mented with 10% human serum (HS) (ARCBS) and 1% anti-biotics (10,000 U/mL penicillin G sodium, 10,000 lg/mLstreptomycin sulphate, and 25 lg/mL amphotericin B in0.85% saline; Gibco). After 1 h in culture, the nonadherentPBMCs were discarded and the adherent PBMCs consistingof monocytic cells were washed twice with 0.1M phos-phate-buffered saline (PBS), pH 7.2 (Gibco).

Cell culture conditions

Human osteoclasts were generated in vitro from the ad-herent PBMCs.22 Briefly, the cells (�1.5 to 2 3 106 cells/flask) were cultured in 5 mL of RPMI supplemented with5% HS and 1% antibiotics (standard culture medium) for24 h. Subsequently, the supernatant was removed and thecell cultures rinsed twice with PBS before adding freshmedium. Thereafter, the adherent PBMCs were used onone hand as monocytes. On the other hand, osteoclastswere generated in vitro by culturing the adherent PBMC inthe presence of cytokines (10 ng/mL recombinant humanRANK-L and 10 ng/mL recombinant human M-CSF, R&D,Minneapolis, MN). The monocytes and the in vitro gener-ated osteoclasts were subsequently treated with 100 nM to100 lM TiCl4 (Fluka, Switzerland; atomic spectrometrystandard purity) before processed further as describedlater. Ethics approval for using human blood cells for thisstudy was granted by the Ethics Committee of the Univer-sity of Western Australia and the ARCBS.

Real-time reverse transcription PCR

Cells were collected after 10 days in culture under thedifferent conditions described above. Total RNA wasextracted from cells using Ultraspec1 RNA (Biotecx Labo-ratories, South Loop East, Houston, TX). mRNA wasreverse-transcribed to cDNA using oligo(dT)18 primer andSuperScript III (Invitrogen Life Technologies). cDNA wasamplified by PCR in a thermal cycler (RotorGene 3000,

30 CADOSCH ET AL.

Journal of Biomedical Materials Research Part A

Corbett Research, Mortlake, Australia) using a PlatinumPCR Supermix (Invitrogen) and the following primer pairs:50-CTG GCT GAT GGT GCC ACC CCT G-30 (þ) and 50-CTC TCA GGC TGC AGG CTG AGG-30 (2) for TRAP; 50-CCC GAA GGG AAA CAA GCA-30 (þ) and 50-GCC TGTACC TGT ACA GCA-30 (2) for CATK; and 50-GCC AAGGTC ATC CAT GAC AAC TTT GG-30 (þ) and 50-GCCTGC TTC ACC ACC TTC TTG ATG TC-30 (2) for glyceral-dehyde-3-phosphate dehydrogenase (GAPDH). The ampli-fication conditions were denaturation at 958C for 30 s(10 min for the first cycle), annealing at 538C for 30 s, andextension at 728C for 30 s (5 min for the last cycle) for40 cycles for all osteoclast markers and for GAPDH. Speci-ficity of amplification products was assessed according tothe melting curves and through separation by electropho-resis on 2% agarose gels, stained with ethidium bromide(Sigma-Aldrich). The measurements of gene expression ofuntreated monocytes were used for baseline calculation.The other conditions were calculated according to the rela-tive expression levels, using RotorGene 3000 Software(Corbett Research, Mortlake, Australia).

Detection of TRAP in monocytes and osteoclastswith ELF97 and flow cytometry (FACS)

After 10 days in culture under the different conditionsdescribed earlier, the cells were fixed in culture mediumcontaining 0.5% paraformaldehyde. The phosphatase sub-strate ELF97 (Molecular Probes, Eugene, OR) with a corre-sponding protocol for cells in suspension was used for thedetection of endogenous TRAP activity in monocytes andosteoclasts as previously described by our group.19 Briefly,the fixed cells were washed twice with distilled water andsubsequently incubated with 200 lM ELF97 in 110 mM ac-etate buffer staining solution (pH 5.2, 1.1 mM sodium ni-trate, 7.4 mM tartrate) for 20 min. Additionally, the cellswere characterized for expression of surface markers usingfluorescence-labeled mouse monoclonal antibodies bindingspecifically to human HLA-DR (phycoerythrin fluorescentlabeled; Becton Dickinson Biosciences, San Jose, CA) andCD45 (PerCP fluorescent labeled; Becton Dickinson Bio-sciences) according to the manufacturers’ instructions.Unstained controls and isotype antibodies were used toaccount for autofluoresence and background signal. Thesamples were analyzed in a FACSVantage Cell Sorter(Becton Dickinson Biosciences) equipped with a UV laser.ELF97 was excited at 350 nm and the TRAP related signalcollected with a 530/30 band pass filter (515–545 nm). ThePE and PerCP fluorochromes were excited with the488 nm laser with the signals collected by a 575/26 bp fil-ter and 675/20 bp filter, respectively. Monocytes andosteoclasts were gated according to their forward (FSC)and side scatter (SSC) profile. Approximately 10,000 gatedcells were analyzed. The data were further analyzed andprepared on Flowjo 8.5.3 (Treestar, Ashland, OR).

Bone resorption tests on dentin slides

Functional evidence of osteoclastic function and bone re-sorptive activity was determined by a lacunar resorption

assay system using cell cultures on dentin slides.23 Forthat purpose, whale dentine slides (diameter 15 mm, thick-ness 0.5 mm) were cut from a sperm whale tooth, pur-chased from Kaempf (Osborne Park, Western Australia)(collected prior to 1972, before ban on whale hunting inAustralia). Adherent cells (4 3 105 cells/well) were cul-tured on dentine slides in six-well multiwell plates (Sar-stedt, Nuernbrecht, Germany). The cells were incubated(378C, humidified, 5% CO2) overnight before the additionof cytokines and TiCl4 as described earlier. After 21 daysin culture, dentin slides were washed vigorously in waterand left overnight in 0.25% ammonium hydroxide toremove all cellular material in order to assess the extent ofbone resorption by osteoclasts. Subsequently, the dentinslides were stained with 1% toluidine blue in 1% sodiumborate for 10 min. The stained dentin slides showing theresorption features were documented with a NikonInverted Microscope Eclipse TE 300 (Nikon Instruments,Melville, NY) and a Nikon CCD digital camera. The entiresurface of the dentine was examined for evidence of lacu-nar bone resorption. The images were analyzed usingDXM 1200F-ACT-1 image processing software (Nikon).

Resorption pits were predominantly observed as indi-vidual small pits or, very rarely, as multilocular areas,stained with toluidine blue. As such, a resorption pit wasdefined as a dark-blue excavation of bone surface with aclear rim of unchanged and unstained original surfacebetween neighboring excavations. The extent of boneresorption was determined by calculating the total planarsurface area of discrete resorption pits and by the numberof pits formed. The diameters of 20 pits/slide were meas-ured and their mean used to calculate the surface of thecounted pits. Pit numbers were counted by a single-blinded observer for all experiments.

Statistical analysis

Data were analyzed using SPSS for Windows (version15.0; SPSS, Chicago, IL). A one-way analysis of variance(ANOVA) was used to test mean differences in expressionof TRAP, CATK, and GAPDH, as well as mean differencesin the number of resorption pits and surface area acrossthe various cell cultures. Bonferroni post hoc tests wereused to determine which conditions showed significantdifferences in gene expressions as well as resorption sur-face area and pits. A p-value of less than 0.05 was consid-ered statistically significant.

RESULTS

The aim of this study was to investigate the influ-ence of Ti(IV) ions on differentiation and function ofosteoclasts and their precursor cells. The very firststep was to evaluate the optimal and relevant tita-nium concentration. For this purpose, the monocytesand the in vitro generated osteoclasts were treatedwith different TiCl4 concentrations from 100 nM to100 lM over 10 days. These first experiments

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revealed that a 100 nM concentration had only aminor effect on TRAP expression (1.7 and 2.4 rela-tive increase of gene expression for treated mono-cytes and osteoclasts respectively). A 1 lM concen-tration, which corresponds to the concentrationmeasured in patients with implant loosening, hadthe strongest effect on osteoclastic activity and celldifferentiation (5.0 and 7.1 relative increase of TRAPexpression for treated monocytes and osteoclastsrespectively).10,11 Higher concentrations showed atoxic effect on cell cultures (0.7 and 0.4 relativedecreased expression of GAPDH for 10 and 100 lMconcentrations, respectively). Subsequently, a 1 lMconcentration was chosen as standard condition forall experiments.

Effect of Ti(IV) on expression ofosteoclastic markers

After 10 days incubation, the effects of Ti(IV) onosteoclastic activity and differentiation were assessedby measuring expression of mRNA for TRAP andCATK, osteoclastic markers, in monocytes andin vitro generated osteoclasts from 22 individuals.CATK, which is found in the lysosomal granules,and TRAP are both considered specific osteoclastmarkers.18,19 There were two different patterns ofgene expression among the 22 tested healthy blooddonors. Five donors (22.73%) showed a ‘‘responsive’’pattern characterized by increased expression ofTRAP mRNA and to a lesser extent of CATK mRNAdue to Ti(IV) treatment (p < 0.001). The gene expres-sion was not affected by Ti(IV) in the blood samplesof the other 17 (77.27%) individuals (nonresponsivepattern). Table I shows the combined data of theeffect of Ti(IV) on the gene expression of TRAP andCATK in the different culture conditions all tested‘‘responsive’’ individuals. A one-way ANOVArevealed a statistically significant difference of TRAPand CATK expression between the different cell cul-tures (F(3,56) 5 528.64, p < 0.001 for TRAP and F(3,56)5 280.94, p < 0.001 for CATK). None of the groupswere significantly different in their expression of thehousekeeping gene GAPDH (F(3,56) 5 2.74, p > 0.05).

FACS analysis for TRAP activity in monocytesand osteoclasts

Flow cytometry was used to measure HLA-DRand CD45 expression on the cells and to evaluateTRAP activity. As expected, after 10 days incubation,untreated monocytes expressed high levels of HLA-DR, while all cells developing toward osteoclastsdownregulated HLA-DR expression. All cells main-tained a high level of CD45 expression.22 The effectsof Ti(IV) on the osteoclastic activity and differentia-

tion were also evaluated by measuring the enzy-matic activity of TRAP using ELF97 as a substrateafter 10 days of incubation.19–21 As expected, expres-sion of TRAP activity was not detected in untreatedcultured monocytes. Analysis of in vitro generatedosteoclasts showed TRAP activity at very high levelsfor �43% of the total cells, at lower levels for themajority of cells. The results of TRAP activity, asmeasured by flow cytometry, matched the mRNAexpression analysis, showing the same individualsbeing ‘‘responsive’’ or ‘‘nonresponsive,’’ respectively.In the ‘‘responsive’’ individuals, the Ti(IV)-treatedmonocytes showed an upregulation of TRAP activ-ity, amounting to �67% of cells showing very highTRAP activity. On the other hand, Ti(IV) induced aslight decrease of TRAP activity in the in vitro gener-ated osteoclasts. Figure 1 shows representative FACSresults of up regulated TRAP activity in Ti(IV)-treated cells obtained from a ‘‘responsive’’ donor.

Bone resorptive function

Having revealed that Ti(IV) induces differentiationand maturation of monocytes toward osteoclasts in‘‘responsive’’ individuals, the resorptive function ofthe cells was assessed on dentin slides. For this pur-pose, the cells were cultured on dentin slides for21 days and the extent of lacunar resorption was mea-sured. Dentin resorption was assessed for the numberof resorption pits formed and for the total area ofresorbed dentin surface. Figure 2 shows representa-tive images of resorption pits for the ‘‘responsive’’individuals tested. As expected, untreated monocytesdisplayed only few resorption pits (32 6 5.4 pits/mm2) and a negligible resorbed dentin area (0.35%).In vitro generated osteoclasts revealed an average of822 (626.5) resorption pits/mm2 with a correspond-ing resorption area of 9.08% (p < 0.001). Ti(IV) treat-ment of monocytes resulted in a significantlyincreased number of resorption pits (670 6 21.2 pits/mm2) and resorption area, up to 7.23% of the totaldentine surface when compared with untreatedmonocytes (p < 0.001). Ti(IV) treatment of osteoclasts

TABLE IMeasurements of TRAP, CATK, and GAPDH Expression

TRAP CATK GAPDH

Monocytes 1 6 0.00a 1 6 0.00a 1 6 0.00Osteoclasts 8.13 6 0.19a 3.09 6 0.10a 1.11 6 0.06Monocytes þ Ti(IV) 5.25 6 0.15a 6.25 6 0.16a 1.17 6 0.05Osteoclasts þ Ti(IV) 7.25 6 0.14a 4.83 6 0.19a 0.99 6 0.08

The measurements of TRAP, CATK, and GAPDHexpression by monocytes were used for baseline calcula-tion (5 1). The other conditions were calculated accordingto the relative increase of expression levels (�SE).

ap < 0.05.

32 CADOSCH ET AL.

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showed a slight, although significantly decreasednumber of resorption pits (552 6 15.0 pits/mm2)and total resorption area (6.26%) compared withuntreated osteoclasts (p < 0.001). Quantitative analy-sis of resorbed areas is depicted in Figure 3.

DISCUSSION

Periprosthetic osteolysis is the most relevant factorin aseptic loosening of orthopedic implants. The netbone loss at the metal–tissue interface occurs becauseof one or all of the following events: increasedrecruitment of osteoclast precursors from the bloodcirculation at the bone-implant interface, enhanceddifferentiation and functional activation of osteoclastprecursors into mature multinucleated cells, andfinally longer survival of osteoclasts.24 Several stud-ies have shown the role of wear particles in inducingaseptic loosening of orthopedic implants.4,5,25 Experi-

ments in murine models have shown the inductionof osteoclast differentiation as the cause of boneresorption in the presence of titanium particles.26 Itis well recognized that activated macrophages inperiprosthetic tissue, in response to titanium wearparticles, produce inflammatory cytokines such astumor necrosis factor-a (TNFa), interleukin-6 (IL-6),and interleukin-1a/b (IL-1a/b) that stimulate osteol-ysis.27–29 Although many studies have investigatedthe role of metal wear particles in osteoclastogenesisand aseptic loosening, little is known about the effectof metal ions released by biocorrosion. Our studysuggests that in patients with titanium-containingimplants and periprosthetic osteolysis, Ti(IV) ionsinduce the differentiation of osteoclast precursors,resulting in TRAPþ cells capable of bone resorption.On the other hand, Ti(IV) ions were revealed to haveonly a minor inhibiting effect on the osteoclastic ac-tivity of in vitro generated osteoclasts.

Osteoclasts are derived from hematopoietic pro-genitors of the monocyte-macrophage lineage.30

Figure 1. Expression of TRAP measured by flow cytometric detection of ELF97. The shaded region represents the controlstaining. The continuous line represents the TRAP activity in the stained cells (X-axis 5 fluorescent intensity; Y-axis 5percentage of cells). Note the increased fluorescent intensity for Ti(IV) treatment indicating increased TRAP activity.

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Journal of Biomedical Materials Research Part A

Osteoblasts or bone marrow-derived stromal cellsexpressing RANK-L and M-CSF are involved inosteoclastogenesis through a mechanism involvingcell-to-cell contact with osteoclast precursors.16,31

Our study found a slightly inhibitory effect of Ti(IV)on the osteoclastic capacity of in vitro generatedosteoclasts, which have differentiated under the con-trol of osteoblast-derived factors, such as M-CSF andRANK-L. The effect of the addition of Ti(IV) on theosteoclasts was a decrease of TRAPþ cells and areduction of their resorptive capacity by �30% whencompared with untreated osteoclasts. These observa-tions are in accordance with previously publishedstudies. Nichols et al. showed a decrease in resorp-tion ability and formation of rat bone-marrow–derived osteoclasts when exposed to titanium ions.32

Under the physiological control of osteoblasts, osteo-clasts are responsible for regulated bone resorption,which is an obligatory event not only during bonegrowth but also during bone remodeling and frac-ture healing. These results suggest that Ti(IV) maydisconnect the osteoclast function from the osteoblas-tic control mechanisms while still exhibiting relevantbone resorption.

Primarily, our study indicates that Ti(IV) ionsinduce monocytes to differentiate toward osteoclasts.Under the influence of Ti(IV) the expression of tran-scripts encoding TRAP, and its enzymatic activitywere found to be clearly upregulated in monocytescocultured with Ti(IV) ions. In addition, the osteo-clast phenotype of the TRAPþ cells was investigated

Figure 2. Effects of Ti(IV) on bone resorption. The images show a representative example of resorption pits (dark spots)on dentine slides after 21 days incubation at different culture conditions. (A) Untreated monocytes. (B) Monocytes þTi(IV). (C) Untreated osteoclasts. (D) Osteoclasts þ Ti(IV).

Figure 3. Histogram representing the mean area (percent-age of total area 6 SE) of resorption on dentine slides after21 days in culture under different conditions (a,bp < 0.001).Note the significant increase of dentin resorption bytitanium-treated monocytes.

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through functional assays by measuring lacunarresorption of dentine. The results of the functionaltests were in line with the changes in the formationof TRAPþ cells and gene expression previouslyobserved, revealing an increased osteolytic activityamong the monocytes incubated with Ti(IV). Giventhe advances in the understanding of osteoclastogen-esis within the last few years, it is possible to specu-late on the detailed mechanisms involved in thestimulation of osteoclast differentiation by Ti(IV)ions. Usually, an increased osteoclastic differentiationis primarily due to an increased production ofRANK-L by the osteoblastic cells.30,33–35 However, theprocess described in our study is a direct effect ofTi(IV) ions on the osteoclast precursor cells and dis-connected from the physiological control of osteo-blast-derived factors, such as M-CSF and RANK-L.Several studies have demonstrated the strong affinityof titanium ions for organic phosphates suggestingthat Ti(IV) may bind to phosphorylated proteins, lip-ids, or nucleosides.36,37 It is tempting to speculate thatTi(IV) binds to phosphorylated proteins of yet un-identified signal transduction pathways and/or hasan effect on gene expression by binding to specificnucleotides, which then promotes the differentiationof monocytes toward mature functional osteoclasts.This is likely to occur in periprosthetic tissues, where,because of biocorrosion, titanium ion concentrationsare known to be elevated, as discussed earlier.

Of great interest was the finding that only �20%of the tested healthy blood donors showed aresponse to Ti(IV), leaving the remaining 80% with-out a response to Ti(IV) exposure. This fact was vali-dated by matching results between gene expression,protein synthesis, and functional tests within thetested individuals. It is important to note that clinicalobservations of aseptic loosening report a similarpercentage (10–15%) of patients being affected byperiprosthetic osteolysis after primary arthroplasty.1

Biological diversities between individuals may leadto molecular variations in the signaling pathwaysinvolved in the reaction to Ti(IV) resulting in a tita-nium ‘‘compatibility’’ or ‘‘incompatibility.’’

In summary, we have found that Ti(IV) ionsinduce the differentiation of mononuclear osteoclastprecursors and influence the osteoclastic activity in�20% of individuals. It is reasonable to furtherhypothesize that Ti(IV) ions could also play a role inthe recruitment and survival of monocytes andosteoclasts. Further studies are required to outline acomplete picture of the interaction between metalions and bone cells, and the molecular pathwayswhich are involved in their activation through Ti(IV)ions. The compounding effect of our demonstrationthat Ti(IV) ions are able to increase osteolytic activityin vitro by acting directly on precursors of boneresorbing cells, in addition to the recognized role of

wear particles, indicates that titanium ions are mostlikely to contribute to aseptic loosening. This in turnopens new research perspectives in this field, whichcould offer innovative therapeutic options, such as apossible use of an inhibitor specific to titanium ionsfor the prevention of periprosthetic osteolysis.Finally, studies investigating the effects of specificmetal ions on individual bone metabolism wouldhelp in selecting suitable metals for the orthopedicimplant alloys for each individual patient.

CONCLUSIONS

This study provides strong support for the hy-pothesis that titanium ions induce differentiation ofhuman precursor cells toward active osteoclasts in�20% of individuals. Conversely, titanium ions mayhave only a minor effect on osteoclasts that have dif-ferentiated under the physiological control of osteo-blast-derived factors such as M-CSF and RANK-L.

The authors thank C. Motteram and B. von Katterfeldfor the statistical analysis, as well as G. Cozens and G.Ben-Ary for their excellent technical assistance and advice.The authors gratefully acknowledge K. Heel of Centre forMicroscopy, Characterization and Analysis (CMCA) forher excellent assistance with the cytometric analysis.

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11. Dorr LD, Bloebaum R, Emmanuel J, Medrum R. Histologic,biochemical, and ion analysis of tissue and fluids retrievedduring total hip arthroplastry. Clin Orthop Relat Res 1990;261:82–95.

12. Thompson GJ, Puleo DA. Effects of sublethal metal ion con-centrations on osteogenic cells derived from bone marrowstromal cells. J Appl Biomater 1995:6;249–258.

13. Fanti P, Kindy MS, Mohapatra S, Klein J, Colombo G, Mal-luche HH. Dose-dependent effects of aluminium on osteocal-cin synthesis in osteoblast-like ROS 17/2 cells in culture. AmJ Physiol 1992;263:E1113–E1118.

14. Vaananen HK, Zhao H, Mulari M, Halleen JM. The cell biol-ogy of osteoclast function. J Cell Sci 2000;113:377–381.

15. Fujikawa Y, Quinn JMW, Sabokbar A, McGee JO, AthanasouNA. The human mononuclear osteoclast precursors circulatesin the monocytes fraction. Endocrinology 1996;137:4058–4060.

16. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, BurgessT, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, SullivanJ, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, KaufmanS, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ.Osteoprogeterin ligand is a cytokine that regulates osteoclastdifferentiation and activation. Cell 1998;93:165–176.

17. Akisaka T, Yoshida H, Inoue S, Shimuzu K. Organisation ofcytoskeletal F-actin, G-actin, and gelsolin in the adhesionstructure in cultured osteoclasts. J Bone Miner Res 2001;16:1248–1255.

18. Walsh NC, Cahill M, Carinici P, Kawai J, Okazaki Y, Haya-shizaki Y, Hume DA, Cassady AI. Multiple tissue-specificpromoters control expression of the murine tartrate-resistantacid phosphatase gene. Gene 2003;307:111–123.

19. Filgueira L. Fluorescence-based staining for tartrate-resistantacidic phosphatase (TRAP) in osteoclasts combined withother fluorescent dyes and protocols. J Histochem Cytochem2004;52:411–414.

20. Telford WG, Cox WG, Stiner D, Singer VL, Doty SB. Detec-tion of endogenous alkaline phosphatase activity in intactcells by flow cytometry using the fluorogenic ELF-97 phos-phatase substrate. Cytometry 1999;37:314–319.

21. Benito GE, Sanchez ML, del Pino-Montes J, Calvo JJ, Menen-dez P, Garcıa-Marcos MA, Osdoby P, Orfao A. A new cyto-metric method for the immunophenotypic characterization ofbone-derived human osteoclasts. Cytometry 2002;50:261–266.

22. Meagher J, Zellweger R, Filgueira L. Functional dissociationof the basolateral transcytotic compartment from the apicalphago-lysosomal compartment in human osteoclasts. J Histo-chem Cytochem 2005;53:665–670.

23. Domon T, Yamazaki Y, Fukui A, Ohnishi Y, Takahashi S,Yamomoto T, Wakita M. Three-dimensional distribution of

the clear zone of migrating osteoclasts on dentin slices invitro. Tissue Cell 2002;34:326–336.

24. Greenfield EM, Bi Y, Ragab AA, Goldberg VM, Van De Mot-ter RR. The role of osteoclast differentiation in aseptic loosen-ing. J Orthop Res 2002;20:1–8.

25. Sommer B, Felix R, Sprecher C, Leunig M, Ganz R, HofstetterW. Wear particles and surface topographies are modulatorsof osteoclastogenesis in vitro. J Biomed Mater Res A 2005;72:67–76.

26. Bi Y, Van De Motter RR, Ragab AA, Goldberg VM, AndersonJM, Greenfield EM. Titanium particles stimulate bone resorp-tion by inducing differentiation of murine osteoclasts. J BoneJoint Surg Am 2001;83:501–508.

27. Jiranek WA, Machado M, Jasty M, Jevsevar D, Wolfe HJ,Goldring SR, Goldberg MJ, Harris WH. Production of cyto-kines around loosened cemented acetabular components.Analysis with immunohistochemical techniques and in situhybridization. J Bone Joint Surg Am 1993;75:863–879.

28. Kim KJ, Rubash HE, Wilson SC, D’Antonio JA, McClain EJ.A histologic and biochemical comparison of the interface tis-sues in cementless and cemented hip prostheses. Clin OrthopRelat Res 1993;287:142–152.

29. Stea S, Visentin M, Granchi D, Ciapetti G, Donati ME, Suda-nese A, Zanotti C, Toni A. Cytokines and osteolysis aroundtotal hip prostheses. Cytokine 2000;12:1575–1579.

30. Suda T, Takahashi N, Martin TJ. Modulation of osteoclast dif-ferentiation. Endocr Rev 1992;13:66–80.

31. Udagawa N. Mechanisms involved in bone resorption. Bio-gerontology 2002;3:79–83.

32. Nichols KG, Puleo DA. Effect of metal ions on the formationand function of osteoclastic cells in vitro. J Biomed Mater Res1997;35:265–271.

33. Ragab AA, Nalepka JL, Bi Y, Greenfield EM Cytokines syn-ergistically induce osteoclast differentiation: Support byimmortalized or normal calvarial cells. Am J Physiol CellPhysiol 2002;283:C679–C687.

34. Haynes DR, Crotti TN, Potter AE, Loric M, Atkins GJ, HowieDW, Findlay DM. The osteoclastogenic molecules RANKLand RANK are associated with periprosthetic osteolysis.J Bone Joint Surg Br 2001;83:902–911.

35. Greenfield EM, Bi Y, Miyauchi A. Regulation of osteoclastactivity. Life Sci 1999;65:1087–1102.

36. Ikeguchi Y, Nakamura H. Determination of organic phos-phates by column-switching high performance anion-exchange chromatography using on-line preconcentration ontitania. Anal Sci 1997;13:479–483.

37. Chen CT, Chen YC. Fe3O4/TiO2 core/shell nanoparticles asaffinity probes for the analysis of phosphopeptides usingTiO2 surface-assisted laser desorption/ionization mass spec-trometry. Anal Chem 2005;77:5912–5919.

36 CADOSCH ET AL.

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Chapter 07 Titanium induced production of chemokines CCL17/TARC and CCL22/MDC in human osteoclasts and osteoblasts The most relevant aspect of AL is ultimately the net bone loss at the metal-tissue

interface. Beside enhanced differentiation and functional activation into mature OC

(including the effects of Ti(IV)) and their longer survival, this may occur because of

an increased recruitment of OC precursors to the peri-implant tissues. This study

investigated whether Ti(IV) induces expression of chemokines and cytokines that are

important in osteoclastogenesis in human OC and osteoblasts. Incubation of those

cells with 1 μM Ti(IV) significantly up-regulated expression of CCL17/TARC (thymus

and activation-regulated chemokine), CCL22/MDC (macrophage-derived

chemokine), RANK-L and macrophage colony-stimulating factor (M-CSF) and pro-

inflammatory cytokines as determined by quantitative real-time PCR and ELISA

assays. Additionally, flow cytometry was used to show Ti(IV) related increased

expression of CCR4, the cognate receptor for CCL17 and CCL22 in challenged OC

precursors. These results strongly suggest that Ti(IV) ions play a role in the

recruitment of OC precursors to the bone-implant interface by increasing CCL17 and

CCL22 expression and by up-regulating their cognate receptor. Moreover the

increased expression of RANK-L and M-CSF by osteoblasts together with increased

levels of pro-inflammatory cytokines may enhance OC differentiation and activity.

- 40 -

Titanium induced production of chemokines CCL17/TARCand CCL22/MDC in human osteoclasts and osteoblasts

Dieter Cadosch,1,2 Oliver P. Gautschi,1,2 Erwin Chan,1 Hans-Peter Simmen,3 Luis Filgueira11School of Anatomy and Human Biology, University of Western Australia, Crawley, Western Australia, Australia2Department of Orthopaedic and Trauma Surgery, Royal Perth Hospital, Perth, Western Australia, Australia3Division of Trauma Surgery, University Hospital Zurich, Zurich, Switzerland

Received 23 August 2008; revised 26 October 2008; accepted 13 November 2008Published online 9 February 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32390

Abstract: There is increasing evidence that titanium(Ti(IV)) ions are released from orthopedic implants and playa role in aseptic loosening. This study aimed to investigatewhether titanium induces expression of chemokines andcytokines that are important in osteoclastogenesis in humanosteoclasts and osteoblasts. Incubation of those cells with1 lM Ti(IV) significantly upregulated expression of CCL17/TARC and CCL22/MDC, RANK-L, M-CSF and pro-inflam-matory cytokines as determined by quantitative real-timePCR and ELISA assays. Additionally, flow cytometry wasused to show Ti(IV) related increased expression of CCR4,the cognate receptor for CCL17 and CCL22 in challenged

osteoclast precursors. These results strongly suggest thatTi(IV) ions play a role in the recruitment of osteoclast pre-cursors to the bone-implant interface by increasing CCL17and CCL22 expression and by upregulating their cognate re-ceptor. Moreover the increased expression of RANK-L andM-CSF by osteoblasts together with increased levels of pro-inflammatory cytokines may enhance osteoclast differentia-tion and activity, and subsequently contribute to the patho-mechanism of aseptic loosening. � 2009 Wiley Periodicals,Inc. J Biomed Mater Res 92A: 475–483, 2010

Key words: titanium; osteoclast; osteoblast; CCL17; CCL22

INTRODUCTION

The most frequent cause of joint implant failure inorthopedic surgery is aseptic loosening, which isthought to occur in over 10% of cases within 20years of a primary hip arthroplasty.1 Over the lastdecade, considerable effort has been directed toinvestigate the effects of the metal particles pro-duced by the mechanical wear process occurring inthe articular coupling of prostheses.2–6 The presenceof wear particles is responsible for a severe inflam-matory reaction because of the release of proinflam-matory cytokines by activated macrophages thatfavor differentiation of osteoclast precursors intomature, multinucleated osteoclasts capable of effi-cient bone resorption.5,7 In addition, osteoblastsstimulated with wear particles increase their produc-tion of cytokines, such as receptor activator of NF-jB

ligand (RANK-L) and macrophage colony-stimulat-ing factor (M-CSF) that enhance osteoclast differen-tiation and activation.7 However, to date, the patho-physiological mechanism of increased osteolysis inthe presence of metal implants remains not fullyunderstood. In addition to the wear particles-medi-ated reactions, the potential role of metal ions (suchas titanium) released by biocorrosion from theimplant surface must also be considered in the con-text of aseptic loosening.

Pure titanium and several titanium alloys are com-monly used in trauma and orthopedic surgery. Tita-nium provides the benefits of low inherent toxicity,low water solubility at physiologic pH (in the rangeof 1 lM concentration), and low reactivity with bio-molecules in an aqueous environment.8 Despitethese relatively inert qualities, titanium has beenshown to be released into surrounding tissues ofmetal implants by various mechanisms includingwear, corrosion, and biological activity.9 Indeed, ele-vated concentrations of titanium have been meas-ured in clinically retrieved capsular and peripros-thetic tissues, as well as distal organs (liver, spleen,and lymph nodes) and body fluids (serum andurine) in total hip arthroplasty patients.10,11 In 1990,Dorr et al. reported titanium levels in the fibrous

Correspondence to: D. Cadosch; e-mail: dcadosch@gmx.netContract grant sponsor: National Institute of Health;

contract grant number: 5R01GM072726-02Contract grant sponsor: AO Foundation (Switzerland);

contract grant number: 05Z34

� 2009 Wiley Periodicals, Inc.

membrane encapsulating titanium-alloys implantsup to 22 ng/mL.12 In 1998, Jacobs et al. have meas-ured titanium concentrations up to 11.17 ng/mL inthe serum of patients with titanium-alloy prosthesesat 36 months postoperatively.13

Titanium ions may contribute to implant looseningby affecting the formation of bone surrounding theimplant and/or increasing local osteoclastic boneresorption. With respect to the latter, we have previ-ously demonstrated that titanium ions (in a þ4 oxi-dation state, Ti(IV)), in concentrations similar tothose measured in retrieval studies, induced the dif-ferentiation of osteoclast precursors toward matureand functional osteoclasts.14 Additional past studieshave demonstrated that nontoxic concentrations ofmetal ions affect the differentiation and function ofosteoblastic cells in vitro.6 Despite these studiesrelaying the effect of metal ions on cell differentia-tion and their activation, the effects of metal ionsreleased from orthopedic implants are not com-pletely characterized. Thus, it is reasonable to furtherhypothesize that Ti(IV) could also play a role in theactivation and recruitment of osteoclast precursorsfrom the systemic blood circulation to the bone-implant interface by inducing cytokine and chemo-kine production.

Chemokines are a family of low-molecular weight(6–14 kDa) cytokines, which primarily inducedirected migration of hematopoietic cells throughinteractions with a group of seven transmembrane Gprotein-coupled receptors.15 CCL17/TARC (thymusand activation-regulated chemokine) and CCL22/MDC (macrophage-derived chemokine) are the tworecognized ligands for the chemokine receptor CCR4and are known to be mainly produced by cell line-ages closely related to osteoclasts such as dendriticcells.16 This study investigated whether Ti(IV) ions,potentially released by biocorrosion from titaniumimplants, induce synthesis and release of chemo-kines CCL17 and CCL22 and cytokines, such asRANK-L and M-CSF, by human osteoblasts andin vitro-generated osteoclasts. Furthermore, the effectof Ti(IV) on the expression of the chemokine recep-tor CCR4 in human osteoclast precursors was inves-tigated using flow cytometry.

MATERIALS AND METHODS

Cells

Ethical approval for using human blood cells for thisstudy was granted by the Ethics Committee of the Univer-sity of Western Australia and the Australian Red CrossBlood Service (ARCBS). Blood samples collected from 19healthy individuals were used for this study. Every samplewas examined independently throughout all study assays.

Human monocytes were obtained from adherent periph-eral blood mononuclear cells (PBMCs). Briefly, PBMCswere isolated from buffy coats (ARCBS, Perth, WA, Aus-tralia) through Ficoll-gradient centrifugation (AmershamBiosciences, Uppsala, Sweden). The PBMCs were cultured(378C, humidified, 5% CO2) in 25-cm2 tissue culture flasks(Sarstedt, Nuernbrecht, Germany) in RPMI-1640 Glutamaxmedium (RPMI, Gibco/Invitrogen, Auckland, NZ), supple-mented with 10% human serum (ARCBS) and 1% antibiot-ics (10,000 U/mL penicillin G sodium, 10,000 lg/mL strep-tomycin sulfate, and 25 lg/mL amphotericin B, Gibco)(standard culture medium). After 1 h in culture, the non-adherent PBMCs were discarded, and the adherent PBMCsconsisting of monocytic cells were washed twice with0.1M phosphate buffered saline (PBS) pH 7.2 (Gibco).

The conditionally immortalized human fetal osteoblasticcell line hFOB1.19 (hFOB), obtained from the AmericanType Culture Collection (ATCC; Manassas, VA), was usedas a human osteoblastic cell model. The cells were culturedaccording to ATCC protocols in 25-cm2 tissue cultureflasks (Sarstedt) containing D-MEM/F-12 medium (Gibco)supplemented with 15% (v/v) fetal calf serum (JRH Bio-sciences, Lenexa, KS) and 1% antibiotics (Gibco) at 348C(humidified, 5% CO2). Twenty-four hours prior to in vitroincubations, flasks containing hFOB cells were washedtwice with PBS and reincubated with standard medium.

Cell culture conditions

Human osteoclasts were generated in vitro from the ad-herent PBMCs as previously described.14,17 Briefly, the iso-lated cells were cultured in 5-mL standard culture mediumfor 24 h. Subsequently, the supernatant was removed andthe cell cultures rinsed twice with PBS before adding freshmedium. Thereafter, a set of the adherent PBMCs wereused as monocytes. Osteoclasts were generated in vitro byculturing the alternative set of adherent PBMCs in thepresence of cytokines (10 ng/mL recombinant humanRANK-L and 10 ng/mL recombinant human M-CSF, R&DSystems, Minneapolis, MN).

Monocytes, in vitro-generated osteoclasts (�1.5–2 3 106

cells/flask), and hFOB cells (�1–2 3 105 cells/flask, sub-confluent) were subsequently treated with 1 lM TiCl4(Fluka, Switzerland; atomic spectrometry standard purity).An incubation period of 10 days was chosen to allow themonocytes to differentiate into mature osteoclasts and toinvestigate the longer-term effects of Ti(IV) on cell cul-tures. Our previous experiments revealed that a 1 lM con-centration was the most appropriate condition having thestrongest effect on cellular activity and differentiation.14

Untreated cell cultures (incubated with standard medium)were used as negative control. The cultured cells andsupernatants were collected after 10 days of incubation.The cells were processed immediately as described later,and the supernatant was stored at 2208C until use.

Reverse transcription and real-time PCR

Total RNA was extracted from cells using Ultraspec1

RNA (Biotecx Laboratories, South Loop East, Houston).

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mRNA was reverse-transcribed to cDNA using oligo(dT)18primer and SuperScript III (Invitrogen Life Technologies).cDNA was amplified by PCR in a thermal cycler (Rotor-Gene 3000, Corbett Research, Mortlake, Australia) using aPlatinum PCR Supermix (Invitrogen) and the primer pairslisted in Table I. The amplification conditions were denatu-ration at 958C for 30 s (10 min for the first cycle), anneal-ing at 538C for 30 s, and extension at 728C for 30 s (5 minfor the last cycle) for 40 cycles. Specificity of amplificationproducts was assessed according to the melting curvesand through separation by electrophoresis on 2% agarosegels, stained with ethidium bromide (Sigma-Aldrich). Themeasurements of gene expression by untreated cells wereused for baseline calculation. The other conditions werecalculated according to the relative expression levels, usingRotorGene 3000 Software (Corbett Research, Mortlake,Australia).

ELISA assays

Commercially available ELISA reagents were employedfor the determination of chemokines (CCL17, CCL22) andM-CSF released from monocytes, in vitro-generated osteo-clasts, and hFOB cells into the culture medium (Quanti-kine1, R&D Systems). All procedures were performedfollowing the manufacturer’s instructions. Photometricmeasurements were conducted at an absorbance of 450 nmand recorded using a microplate reader (Multiskan RC,Labsystems OY, Helsinki, Finland). The detection limits ofthe immunoassays were 7 pg/mL for CCL17, 62.5 pg/mLfor CCL22, and 9 pg/mL for M-CSF.

Cytometric bead array analysis

The secretion of IL-1a/b, IL-6, RANK-L, and tumor ne-crosis factor-alpha (TNF-a) into the culture medium wasevaluated using a commercially available Cytometric BeadArray (CBA) kit (BD Biosciences, San Jose, CA). Fluores-

cence intensity was measured with FACSCanto II Flow Cy-tometer (BD Biosciences) and quantified from a calibrationcurve using FCAP Array v1.0 Software (Softflow Technolo-gies, New Brighton, MN). All assays were performedaccording to the manufacturer’s instructions.

Flow cytometry

Flow cytometry (FACS) was used to investigate theinfluence of Ti(IV) on the expression of the chemokinereceptors CCR4, CCR6, and CCR7 in human mononuclearosteoclast precursors. Adherent PBMCs (5 3 105/well)were incubated in the presence of 1 lM Ti(IV) for 2, 4 and6 h. Cell culture and staining were carried out in roundbottom 96-well plates (Sarstedt). Human anti-mouse mono-clonal antibodies specific for chemokine receptors CCR4-PE, CCR6-FITC, and CCR7-APC (R&D Systems) wereused. Unstained controls and isotype antibodies were usedto account for autofluoresence, background signal, andunspecific antibody binding. After staining, the cells werefixed with 0.5% paraformaldehyde. Fluorescent quantifica-tion was performed with FACSCanto II Flow Cytometerusing FACS DIVA Software (BD Biosciences). All eventswere recorded and stopped at 30,000 gated events. Mono-cytes were gated according to their forward and side scat-ter profile. The recorded data were analyzed using Flowjo8.5.3 Software (Treestar, Ashland, OR).

Calculations and statistical analysis

Data were analyzed using SPSS for Windows (version15.0; SPSS, Chicago, IL). A one-way ANOVA was used totest mean differences in expression of genes and secretedcytokines and chemokines in the various cell cultures. Bon-ferroni post hoc tests were used to determine which condi-tions showed significant differences in gene expressions aswell as released proteins. A p-value of less than 0.05 wasconsidered statistically significant.

TABLE IChemokine and Cytokine Specific Primers Sequence

PrimersSequence(50 ? 30)

CCL17 (TARC) Sense CTG CTC TGC TTC TGG GGA CAntisense TGT TTG GCT TTG GGG TCT GC

CCL22 (MDC) Sense GGT CCC TAT GGT GCC AAT GAntisense TTA TCA AAA CAA CGC CAG GC

RANK-L Sense CCT ACG CAC AAG GCG AAG ATG CAntisense CGT AGA CCA CGA TGA TGT CGC C

M-CSF Sense CTG ACC AGC TCA GAG AGAAntisense CTC ATC AAT GTG CAG GA

TNF-a Sense CCA TGA GCA CTG AAA GCA TGAAntisense TCA CAG GGC AAT GAT CCC AAA GTA CTG CCC

IL-1 Sense CCT ACG CAC AAG GCG AAG ATG CAntisense CGT AGA CCA CGA TGA TGT CGC C

IL-6 Sense TTCGGT CCA GTT GCC TTC TCCAntisense GGT TGG GTC AGG GGT GGT TAT T

GAPDH Sense GCC AAG GTC ATC CAT GAC AAC TTT GGAntisense GCC TGC TTC ACC ACC TTC TTG ATG TC

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RESULTS

Effect of titanium (IV) on chemokines andcytokines expression

After 10 days incubation, the influence of Ti(IV)on chemoattractant activity was assessed by meas-uring the mRNA expression of CCL17 and CCL22 inmonocytes, in vitro-generated osteoclasts, and hFOBcells. Additionally, the effect of Ti(IV) on osteoclastdifferentiation and activation was investigated bydetermining the expression of RANK-L, M-CSF,TNF-a, IL-1a/b, and IL-6. Table II shows the com-bined data of gene expression of chemokines andcytokines in the different cell cultures. Interestingly,analysis revealed slightly decreased chemokine lev-els in monocytes exposed to Ti(IV) (p > 0.05),whereas statistically increased expression of CCL17and CCL22 was measured in Ti(IV)-challengedosteoclasts and hFOB cells (p < 0.05). Increasedexpression of TNF-a was revealed in Ti(IV)-treatedosteoclasts when compared with untreated cells (p <0.05). Statistically increased levels of RANK-L andM-CSF were detected in hFOB cells, because of themetal challenge (p < 0.05). None of the culture con-ditions was significantly different neither in theirexpression of the housekeeping gene GAPDH nor inIL-1a/b and IL-6 expression (p > 0.05).

Measurement of chemokines and cytokines inculture media

Chemokines and cytokine concentrations in thesupernatants of monocytes, in vitro-generated osteo-clasts, and hFOB cells treated with or without Ti(IV)were obtained after 10 days of incubation, usingELISA and CBA assays. The measured chemokinesand cytokines were normalized to the individual(untreated cells incubated with standard mediumfrom the same individual) and averaged. Figure 1shows the concentrations of CCL17 and CCL22released within the various culture conditions. The

PCR results correlate with the increased levels ofCCL17 and CCL22 in the culture supernatants ofosteoclasts and hFOB cells incubated with Ti(IV) (p< 0.001). Similarly, increased levels of TNF-a werereleased by osteoclasts challenged with Ti(IV) (p <0.001) (Fig. 2). The production of M-CSF and RANK-L by hFOB cells in response to Ti(IV) was signifi-cantly elevated compared with untreated cells (p <0.001) (Fig. 3). High levels of IL-6 and converselylow levels of IL-1a/b were measured in monocyte,osteoclast, and hFOB cell cultures. However, Ti(IV)exposure did not influence their expression (data notshown).

Chemokine receptor expression in mononuclearosteoclast precursors

Flow cytometry was used to evaluate the effect ofTi(IV) on chemokine receptor CCR4, CCR6, andCCR7 expression in mononuclear osteoclast precur-sors. Untreated cells expressed similar levels ofCCR4, CCR6, and CCR7 (data not shown). Ti(IV)treatment induced no changes in the expression ofCCR6 and CCR7, whereas increasing levels of CCR4over time were exhibited in cells exposed to Ti(IV)ions when compared with untreated mononuclearosteoclast precursors (Fig. 4).

DISCUSSION

Aseptic loosening of orthopedic implants mayoccur because of multiple reasons. The most relevantfactor is ultimately the net bone loss at the metal-tis-sue interface, which occurs because of one or moreof the following events: (1) increased recruitment ofosteoclast precursors from the systemic blood circu-lation, (2) enhanced differentiation and functionalactivation into mature multinucleated cells, and (3)longer survival of osteoclasts.18 In the past decade,several studies using murine and human cell cul-tures models have demonstrated the role of metal

TABLE IIQuantitative Reverse Transcription Polymerase Chain Reaction Measuring the Effect of 1 lM Ti(IV) Ions on

Chemokines and Cytokines Gene Expression

CCL17 CCL22 RANK-L M-CSF TNF-a IL-1 IL-6 GAPDH

Untreated cultures 1 1 1 1 1 1 1 1Monocytes þ Ti(IV) 0.95 6 0.2 0.89 6 0.2 0.88 6 0.3 0.92 6 0.1 1.05 6 0.2 1.06 6 0.1 1.02 6 0.1 1.04 6 0.1Osteoclasts þ Ti(IV) 1.97 6 0.2* 2.23 6 0.3* 1.13 6 0.4 1.09 6 0.1 1.53 6 0.3* 1.06 6 0.1 1.03 6 0.1 1.04 6 0.1hFOB þ Ti(IV) 2.18 6 0.1* 1.65 6 0.3* 1.75 6 0.3* 1.65 6 0.2* 0.94 6 0.1 0.98 6 0.1 0.96 6 0.1 1.03 6 0.1

The measurements of specific mRNA expression by corresponding untreated cells (monocytes, in vitro-generated osteo-clasts, and hFOB cells) were used for baseline calculation (51). The other conditions were calculated according to the rela-tive increase of expression levels (6SE), p < 0.05.

478 CADOSCH ET AL.

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wear particles in inducing a severe inflammatoryreaction through the release of proinflammatorycytokines by activated macrophages and osteoblasts,hence enhancing osteoclastogenesis.2,5,19,20 By usinghuman cell cultures and functional bone resorptionassays, we have previously demonstrated that Ti(IV)ions released by biocorrosion induce differentiationof mononuclear osteoclast precursors into functionalosteoclasts.14 This study further demonstrates theenhanced synthesis and secretion of RANK-L and

M-CSF by hFOB cells and TNF-a by osteoclasts,when exposed to Ti(IV) levels correspondent withthe levels measured in the periprosthetic tissues andserum of patients with total joint replacement.12,13

It is well recognized that activated macrophages,in response to titanium wear particles, produceproinflammatory cytokines, such as TNF-a, IL-6, andIL-1a/b.21–23 TNF-a acts directly on osteoclast pre-cursors, whereas IL-6 and IL-1a/b act indirectly byincreasing the expression of RANK-L and M-CSF by

Figure 1. Histogram showing the mean expression levels (n 5 19, 6SD) of CCL17 (a) and CCL22 (b) in the supernatantof monocytes, in vitro-generated osteoclasts, and hFOB cells after 10 days of incubation with and without 1 lM Ti(IV). Theamount of chemokines release was normalized to the individual (unchallenged cells from the same individual incubatedwith standard medium) and averaged. *p < 0.001.

Figure 2. Mean concentrations (n 5 19, 6SD) of TNF-areleased into the culture supernatant of monocytes andin vitro-generated osteoclasts incubated with and without1 lM Ti(IV). *p < 0.001.

Figure 3. Bar chart showing the mean concentrations (n5 19, 6SD) of M-CSF and RANK-L produced by hFOBcells after 10 days incubation with and without 1 lMTi(IV) ions. *p < 0.001.

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osteoblasts, which in turn, directly affect osteoclastprecursors through a mechanism involving cell-to-cell contact.7 The latter is part of the couplingbetween bone resorption and bone formation, whichis essential not only during bone growth but alsoduring bone remodeling and fracture healing. In thisstudy, IL-1a/b and IL-6 did not appear to play animportant role as their expression was not affectedby Ti(IV) challenge. However, we found that Ti(IV)had a significant effect on the production of TNF-aby in vitro-generated osteoclasts, which have differ-entiated under the control of osteoblast-derived fac-tors, such as M-CSF and RANK-L. As osteoclasts areresponsible for regulated bone resorption, whileunder the physiological control of osteoblasts, theseresults suggest that Ti(IV) may disconnect the osteo-clast function from the osteoblastic control mecha-nisms, shifting the balance toward bone resorptionvia an autocrine/paracrine pathway. Moreover, theresults revealed a significant increased production ofRANK-L and M-CSF by Ti(IV)-treated hFOB. Adirect effect of Ti(IV) ions on the cytokine produc-tion by hFOB is indicated, and it is reasonable toassume that this process may represent an additionalpathway, which may activate focal bone resorptionleading to implant failure.

Given that an increase in osteoclast differentiationacts most likely in concert with increased recruit-ment of osteoclast precursors to tissues containinghigh levels of metal ions, we investigated the effectsof Ti(IV) ions on the chemoattractant ability of bonecells. Most importantly, recent studies demonstratedthat osteoclasts express CCL22 upon activation byRANK-L.24,25 CCL17/TARC and CCL22/MDC arethe two recognized ligands for CCR4.26 This studydemonstrated that Ti(IV) enhanced the synthesis andsecretion of CCL17 and CCL22 in osteoclasts andhFOB cells. Furthermore, an increased expression ofCCR4 in Ti(IV)-challenged osteoclast precursors wasdetected using flow cytometric analysis. Althoughthere have been major advances in understanding

the mechanisms by which the differentiation andactivation of osteoclast precursors is induced, little isknown about the factors which regulate their recruit-ment. In other tissues, chemokines play a major rolein these processes inducing cytoskeletal rearrange-ment, adhesion and directional migration of leuko-cytes, macrophages, and monocytes during homeo-static and inflammatory conditions.27–29 CCL22 hasbeen shown to be expressed by activated macro-phages and mature dendritic cells, whereas CCL17has been shown to be secreted by keratinocytes andendothelial cells.26 In this study, we demonstratedthat mononuclear osteoclast precursors upregulatedCCR4 expression when treated with titanium con-centration similar to those measured in vivo. Theincreased expression of CCL17 and CCL22 ligandsby Ti(IV)-challenged osteoclasts and hFOB cells, asreported here, strongly suggests the pivotal role ofthese chemokines in the recruitment and arrest ofCCR4 expressing osteoclast progenitors to the metal-bone interface. The osteoclast precursors recruited tothe periprosthetic tissues are, as discussed earlier,subsequently exposed to high concentrations of vari-ous cytokines, which synergistically may stimulateosteoclast differentiation and activation.

Although CCR4, also known as skin-homing re-ceptor, is preferentially expressed by Th2 cells,recent publications have demonstrated that CCR4 isalso expressed by Th17 cells.30,31 Circulating CCR4positive T cells may be arrested to the periprosthetictissues through increased concentrations of CCL17and CCL22 by ‘‘Ti(IV)-activated’’ osteoclasts andosteoblasts. This hypothesis is supported by micro-scopic analysis of specimens obtained from revisionsurgery of failed hip replacements, which revealed avaried composition of the pseudomembrane includ-ing up to 26% of lymphocytes.32 Th17 cells haverecently been shown to be a specialized subset ofT cells involved in inflammatory conditions likeautoimmunity, gut inflammation, and rheumatoidarthritis.33 Aseptic loosening has often been closely

Figure 4. Representative flow cytometric detection of the expression of the chemokine receptor CCR4 by mononuclearosteoclast precursors after 2, 4, and 6 h incubation with and without Ti(IV). Note the over time increased fluorescent inten-sity of CCR4 in Ti(IV) challenged cells. (X-axis, fluorescent intensity; Y-axis, percentage of cells).

480 CADOSCH ET AL.

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compared with rheumatoid arthritis in terms of pro-gression, joint/bone destruction, (osteolysis) as wellas involving similar factors such as RANK-L.34 Th17cells link the immune system and bone as they pro-duce both IL-17 and RANK-L in parallel.35 Besidethe osteogenic effect discussed earlier, RANK-L alsoaffects dendritic cells by accelerating autoimmuneand inflammatory conditions through production ofmore proinflammatory cytokines.36,37 Most interest-ingly, it has recently been shown that IL-17 has dualfunctions; causing an inflammatory reaction and anenhanced osteolysis by inducing RANK-L produc-tion in osteoblasts resulting in a loss of nuclear fac-tor-kappa-B/osteprogenin balance.35 In periodontaldiseases, both B and T lymphocytes are the primarysource of RANK-L, which leads to bone loss.38

Beside the direct effects of Ti(IV) ions stimulatingosteoblasts to produce osteoclastogenesis factors(RANK-L and M-CSF) and osteoclasts to expressTNF-a, an increased RANK-L production throughrecruited CCR4 positive T cells may represent otherpossibilities to be considered in the pathomechanismof periimplant osteolysis.

Collectively, these results imply a series of novelpathways through which Ti(IV) ions may contributeto the phenomenon of aseptic loosening in patientswith titanium implants. By incorporating our resultsin the light of current understanding, we postulate ona possible mechanism involving Ti(IV) ions in thepathomechanism of aseptic loosening (Fig. 5). First,Ti(IV) may enhance the expression of CCR4 receptorsamong circulating osteoclast precursors. Second, CCR4positive cells including osteoclast precursors, Th2 andTh17 cells, are subsequently recruited to the bone-implant interface through increased levels of cell sur-face CCL17 and CCL22 ligands. Third, the increasedlevels of RANK-L, M-CSF, and proinflammatory cyto-kines in the periprosthetic tissues through a directeffect of Ti(IV) on osteoblasts and osteoclasts, increaseosteoclast differentiation and activation of local andrecruited cells. Finally, the recruited and activated Tcells (including Th17) release additional proinflamma-tory cytokines (IL-17 and RANK-L), which resultantlyincrease osteoclast differentiation, activation, andinflammation leading to the beginning of a viciouscycle involving amplification of responses.

Figure 5. Postulated pathway involving Ti(IV) ions in the pathomechanism of aseptic loosening. [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com.]

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In summary, this study shows for the first timethat Ti(IV) ions directly enhance expression ofRANK-L and M-CSF in osteoblastic cells. In addi-tion, Ti(IV) directly increases TNF-a expression inosteoclasts. Furthermore, concentrations of Ti(IV),similar to the levels measured in the serum ofpatients with total joint replacement, upregulate theexpression of the chemoattractant cytokines CCL17and CCL22 in osteoclasts and hFOB cells, and theircognate receptor CCR4 in osteoclast precursors. Theseresults strongly suggest the pivotal role of titaniumions in the recruitment of osteoclast precursors andCCR4 positive cells to the periprosthetic tissue and inenhancing osteoclast differentiation and activation.Collectively, these findings indicate the contributionof Ti(IV) through a series of direct and indirect path-ways to the pathomechanism of aseptic loosening inpatients with orthopedic implants. This in turn opensnew research perspectives in this field which couldoffer new therapeutic options, such as a possibleblocking or complexing agent for titanium ions pre-venting periprosthetic osteolysis. Finally, studiesinvestigating the effects of specific metal ions on indi-vidual bone metabolism would assist in selecting suit-able metals for the orthopedic implant alloys for eachindividual patient. The pathomechanism of asepticloosening appears to be very complex, and furtherstudies are required to outline a complete picture ofthe interaction between metal and bone cells andtheir molecular pathways, which are involved in theactivation through metal ions such as Ti(IV).

CONCLUSIONS

This study provides strong support for the hy-pothesis that titanium ions, released by biocorrosionfrom orthopedic implants, play a role in the recruit-ment of osteoclast precursors to the bone-implantinterface by inducing expression and secretion of thechemokines CCL17 and CCL22 and upregulation ofthe CCR4 receptor. Additionally, Ti(IV) directlyincreased the expression of cytokines such asRANK-L, M-CSF, and TNF-a, which in turn enhancedifferentiation and activation of the recruited cells,and therefore contribute to the pathomechanism ofaseptic loosening.

The authors thank T. Knox (PathWest Laboratory Medi-cine WA) for her excellent assistance and G. Cozens (Uni-versity of Western Australia) for the outstanding technicalsupport and advice.

References

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10. Savarino L, Granchi D, Ciapetti G, Cenni E, Nardi Pantoli A,Rotini R, Veronesi CA, Baldini N, Giunti A. Ion release inpatients with metal-on-metal hip bearings in total jointreplacement: A comparison with metal-on-polyethylene bear-ings. J Biomed Mater Res 2002;63:467–474.

11. Urban RM, Jacobs JJ, Tomlinson MJ, Gavrilovic J, Black J,Peoc’h M. Dissemination of wear particles to the liver, spleen,and abdominal lymph nodes of patients with hip or kneereplacement. J Bone Joint Surg Am 2000;82:457–476.

12. Dorr LD, Bloebaum R, Emmanual J, Meldrum R. Histologic,biochemical, and ion analysis of tissue and fluids retrievedduring total hip arthroplasty. Clin Orthop Relat Res1990;261:82–95.

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14. Cadosch D, Chan E, Gautschi OP, Meagher J, Zellweger R,Filgueira L. Titanium IV ions induced human osteoclast dif-ferentiation and enhanced bone resorption in vitro. J BiomedMater Res A 2009;91:29–36.

15. Yoshie O, Imai T, Nomiyama H. Novel lymphocyte-specificCC chemokines and their receptors. J Leukoc Biol 1997;62:634–644.

16. Radstake TR, van der Voort R, ten Brummelhuis M, de WaalMalefijt M, Looman M, Figdor CG, van den Berg WB, BarreraP, Adema GJ. Increased expression of CCL18, CCL19, andCCL17 by dendritic cells from patients with rheumatoid ar-thritis, and regulation by Fc g receptors. Ann Rheum Dis2005;64:359–367.

17. Meagher J, Zellweger R, Filgueira L. Functional dissociationof the basolateral transcytotic compartment from the apicalphago-lysosomal compartment in human osteoclasts. J Histo-chem Cytochem 2005;53:665–670.

18. Greenfield EM, Bi Y, Ragab AA, Goldberg VM, Van De Mot-ter RR. The role of osteoclast differentiation in aseptic loosen-ing. J Orthop Res 2002;20:1–8.

19. Bi Y, Van De Motter RR, Ragab AA, Goldberg VM, Anderson

JM, Greenfield EM. Titanium particles stimulate bone resorp-

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20. Sommer B, Felix R, Sprecher C, Leunig M, Ganz R, Hofstetter

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21. Jiranek WA, Machado M, Jasty M, Jevsevar D, Wolfe HJ,Goldring SR, Goldberg MJ, Harris WH. Production of cyto-kines around loosened cemented acetabular components.Analysis with immunohistochemical techniques and in situhybridization. J Bone Joint Surg Am 1993;75:863–879.

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nese A, Zanotti C, Toni A. Cytokines and osteolysis aroundtotal hip prostheses. Cytokine 2000;12:1575–1579.

24. Lean JM, Murphy C, Fuller K, Chambers TJ. CCL9/MIP-1gamma and its receptor CCR1 are the major chemokineligand/receptor species expressed by osteoclasts. J Cell Bio-chem 2002;87:386–393.

25. Nakamura ES, Koizumi K, Kobayashi M, Saitoh Y, Arita Y,Nakayama T, Sakurai H, Yoshie O, Saiki I. RANKL-inducedCCL22/macrophage-derived chemokine produced fromosteoclasts potentially promotes the bone metastasis of lungcancer expressing its receptor CCR4. Clin Exp Metastasis2006;23:9–18.

26. Ferenczi K, Fuhlbrigge RC, Pinkus J, Pinkus GS, Kupper TS.Increased CCR4 expression in cutaneous T cell lymphoma. JInvest Dermatol 2002;119:1405–1410.

27. Campbell JJ, Butcher EC. Chemokines in tissue-specific andmicroenvironment-specific lymphocyte homing. Curr OpinImmunol 2000;12:336–341.

28. Rossi D, Zlotnik A. The biology of chemokines and theirreceptors. Annu Rev Immunol 2000;18:217–242.

29. Zlotnik A, Yoshie O. Chemokines: A new classificationsystem and their role in immunity. Immunity 2000;12:121–127.

30. Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gat-torno M, Lanzavecchia A, Sallusto F, Napolitani G. Surface phe-notype and antigenic specificity of human interleukin 17-pro-ducing T helper memory cells. Nat Immunol 2007;8:639–646.

31. Baatar D, Olkhanud P, Sumitomo K, Taub D, Gress R, Bira-gyn A. Human peripheral blood T regulatory cells (Tregs),functionally primed CCR4þ Tregs and unprimed CCR4-Tregs, regulate effector T cells using FasL. J Immunol 2007;178:4891–4900.

32. Wooley PH, Nasser S, Fitzgerald RH. The immune responseto implant materials in humans. Clin Orthop Relat Res 1996;326:63–70.

33. Schmidt-Weber CB, Akdis M, Akdis CA. TH17 cells in thebig picture of immunology. J Allergy Clin Immunol 2007;120:247–254.

34. Goldring SR, Schiller AL, Roelke M, Rourke CM, O’Neil DA,Harris WH. The synovial-like membrane at the bone-cementinterface in loose total hip replacements and its proposedrole in bone lysis. J Bone Joint Surg Am 1983;65:575–584.

35. Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y,Kadono Y, Tanaka S, Kodama T, Akira S, Iwakura Y, Cua DJ,Takayanagi H. Th17 functions as an osteoclastogenic helper Tcell subset that links T cell activation and bone destruction.J Exp Med 2006;203:2673–2682.

36. Bachmann MF, Wong BR, Josien R, Steinman RM, OxeniusA, Choi Y. TRANCE, a tumor necrosis factor family member

critical for CD40 ligand-independent T helper cell activation.J Exp Med 1999;189:1025–1031.

37. Fouque-Aubert A, Chapurlat R. Influence of RANKL inhibi-

tion on immune system in the treatment of bone diseases.Joint Bone Spine 2008;75:5–10.

38. Kawai T, Matsuyama T, Hosokawa Y, Makihira S, Seki M,

Karimbux NY, Goncalves RB, Valverde P, Dibart S, Li YP,

Miranda LA, Ernst CW, Izumi Y, Taubman MA. B and T

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Chapter 08 Titanium uptake, induction of RANK-L expression, and enhanced proliferation of human T-lymphocytes

Recently, published evidence has shown that the immune system is closely

connected to the development of bone cells. A variety of T-lymphocyte derived

factors and cytokines, including RANK-L and TNF-α, have been shown to directly

and indirectly promote OC activity and inhibit osteoblast function. The aim of this

study was to investigate Ti(IV) uptake by human T-lymphocytes and its effects on

phenotype and proliferation. Freshly isolated human non-adherent peripheral blood

mononuclear cells, were exposed to Ti(IV). Bioavailability and distribution of Ti(IV) in

T-lymphocytes was determined by EFTEM. The effects of Ti(IV) challenge on non-

activated and phytohemagglutinin (PHA)-activated cells were assessed by flow

cytometric analysis of surface markers, RANK-L production, and proliferation assays.

EFTEM colocalized Ti(IV) with phosphorus in the nucleus, ribosomes, cytoplasmic

membranes, and the surface membrane of T-lymphocytes. Ti(IV) increased

significantly the expression of CD69, CCR4, and RANK-L in a concentration-

dependent manner. Titanium enters T-lymphocytes through a currently unknown

mechanism and binds to phosphorus-rich cell structures. Titanium influences

phenotype and function of T-lymphocytes, resulting in activation of a CD69+ and

CCR4+ T-lymphocyte and secretion of RANK-L.

- 40 -

Titanium Uptake, Induction of RANK-L Expression, and EnhancedProliferation of Human T-Lymphocytes

Dieter Cadosch,1,2,3 Michael Sutanto,1 Erwin Chan,1 Amir Mhawi,1 Oliver P. Gautschi,1,2 Brilliana von Katterfeld,1

Hans-Peter Simmen,3 Luis Filgueira1

1School of Anatomy and Human Biology, University of Western Australia, 35 Stirling Highway, Crawley, Australia 6009, 2Department of Orthopaedicand Trauma Surgery, Royal Perth Hospital, Perth, Australia, 3Division of Trauma Surgery, University Hospital Zurich, Zurich, Switzerland

Received 3 June 2009; accepted 27 August 2009

Published online 6 October 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.21013

ABSTRACT: There is increasing evidence that titanium ions are released from orthopedic implants by biocorrosion. Theaim of this study wasto investigate titanium uptake by human T-lymphocytes and its effects on phenotype and proliferation. Freshly isolated human nonadherentperipheral blood mononuclear cells (NA-PBMC), were exposed to TiCl4 [Ti(IV)]. Bioavailability and distribution of Ti(IV) in T-lymphocyteswas determined by energy-filtered electron microscopy (EFTEM). The effects of Ti(IV) challenge on nonactivated and PHA-activated cellswere assessed by flow cytometric analysis of surface markers, RANK-L production, and proliferation assays. EFTEM colocalized Ti(IV) withphosphorus in the nucleus, ribosomes, cytoplasmic membranes, and the surface membrane of T-lymphocytes. Ti(IV) increased significantlythe expression of CD69, CCR4, and RANK-L in a concentration-dependent manner. Titanium enters T-lymphocytes through a currentlyunknown mechanism and binds to phosphorus-rich cell structures. Titanium influences phenotype and function of T-lymphocytes, resultingin activation of a CD69þ and CCR4þ T-lymphocyte population and secretion of RANK-L. These results strongly suggest the involvement oftitanium ions challenged T-lymphocytes in the complex pathophysiological mechanisms of aseptic loosening of orthopedic implants. � 2009

Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:341–347, 2010

Keywords: titamium; RANK-L; T-lymphocytes; EFTEM; proliferation

For several decades, total joint replacements have beenhailed as a great success in the treatment of arthritis-related joint degeneration, with more than a millionpatients worldwide undergoing primary arthroplastyeach year. Most patients tolerate orthopedic metalimplants well; however, complications resulting fromimmune reactivity to metals have been well docu-mented.1–6 Dermal hypersensitivity to metal is com-mon, with up to 20% of Caucasians being sensitive tonickel.7 Immune reactions to dermal contact andingestion of metals, manifested as skin conditionssuch as eczema, urticaria, erythema, and pruritis, arebelieved to be of a type IV cell-mediated hypersensi-tivity.8 Currently, the role of T-lymphocytes in thesystemic and peri-implant tissue responses, charac-terized by increased osteolysis and implant failure(aseptic loosening) in patients with metallic orthopedicdevices, remains poorly understood, with several con-flicting studies regarding their actual involvement.9

Pure titanium and titanium alloys are commonly usedbiomaterials in trauma and orthopedic surgery (wristand acetabular prosthesis of a total hip arthroplasty).10

Titanium provides the benefits of low water solubility atphysiological pH, low inherent toxicity, and low reac-tivity with biomolecules in an aqueous environment.11

Despite these relatively inert qualities, titanium hasbeen shown to be released in its most stable Ti(IV)oxidation state at micromolar concentrations into thetissues surrounding metal implants by various mecha-nisms including wear, corrosion, and biological activ-ity.12 Once released into the systemic circulation, themetal ions bind to serum proteins and form haptens orhapten-like complexes, which can be transported to and

accumulate in distal organs.13 Indeed, homogenates ofremote organs and clinically retrieved tissues frompatients with titanium alloy total hip arthroplastyhave indicated that significantly increased titaniumconcentrations can occur in the liver, kidney, spleen,and lymph nodes. In patients with titanium implants(used for osteosynthesis and prosthetic), infiltration ofperi-implant tissues with macrophages and T-lympho-cytes, along with minor skin sensitivity has beenshown.14,15 Despite this, little is known about themechanisms of aseptic loosening of titanium implantsand what role T-lymphocytes may play in a titanium-related inflammatory process in joints and bone.

This study investigated whether Ti(IV) ions, whichrepresent the main and most stable form of oxidatedtitanium,11 are taken up by immune cells and whetherthey cause alterations in T-lymphocyte proliferation,phenotype, and expression of the receptor activator ofNF-kB ligand (RANK-L).

MATERIALS AND METHODSIsolation of Nonadherent Peripheral BloodMononuclear CellsEthical approval for the use of human blood cells was granted bythe Ethics Committee of the University of Western Australia(agreement number RA/4/1/1069) and the Australian Red CrossBlood Service (ARCBS), Perth, WA, Australia (agreementnumber 06-03WA-25).

Blood samples were obtained from 18 healthy donors withoutany metallic implants. Every sample was examined independ-ently throughout all study conditions. Human nonadherentperipheral blood mononuclear cells (NA-PBMC) were used as asource for T-lymphocytes. Previous studies have shown that T-lymphocytes make up more than 70% in freshly isolated NA-PBMC, the remaining population being monocytes, dendriticcells and B-lymphocytes.16 Usually, the numbers of T-lympho-cytes increase in culture to more than 95% under specificstimulatory conditions. Briefly, PBMC were isolated from buffy

JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2010 341

Correspondence to: Dieter Cadosch (T: þ61 8 6488 3647; F: þ61 86488 1051; E-mail: dcadosch@gmx.net)

� 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

coats (ARCBS) through Ficoll-gradient centrifugation (Amer-sham Biosciences, Uppsala, Sweden). The PBMC were cultured(378C, humidified, 5% CO2) in 25 cm2 tissue culture flasks(Sarstedt, Nuernbrecht, Germany) in RPMI-1640 Glutamaxmedium (RPMI; Gibco/Invitrogen, Auckland, NZ), supple-mented with 5% human serum (ARCBS) and antibiotics(10,000 units/mL Penicillin G sodium, 10,000 mg/mL Strepto-mycin sulphate and 25 mg/mL Amphotericin B, Gibco) (standardmedium) to remove the adherent monocyte cells. After 1 h inculture, the NA-PBMC were collected and counted manuallyusing a Double Neubauer chamber.

Determination of Cell ViabilityCell viability was determined using Trypan blue exclusionbefore being processed further, as follows. Twenty microlitersof MulticelTM (0.5% trypan blue, Nalgene, TRACE, Australia)was added to 100 mL of NA-PBMC, which had been incubatedfor 24 h in standard medium supplemented with andwithout 100 mM TiCl4 (Sigma/Fluka, Buchs, Switzerland,atomic spectrometry standard purity). Thereafter, the cellswere counted and the percentage of nonstained viable cellscalculated. Using the above described standard culturemedium and the described range of Ti(IV) concentrations,there was no titanium precipitate seen in these experiments.

Energy-Filtered Transmission Electron Microscopy (EFTEM) andElectron Energy Loss Spectrometry (EELS)To investigate Ti(IV) uptake by T-lymphocytes and theircellular distribution, viable NA-PBMC (4� 107/condition)were incubated in standard medium supplemented with100 mM Ti(IV). After 3 days in culture the cells were collected,washed once with 0.1 M phosphate buffered saline pH 7.2(Gibco) and immediately fixed in RPMI medium with 2.5% EMgrade glutaraldehyde (Electron Microscopy Science, Washing-ton, PA, USA). The glutaraldehyde-fixed cells were washed withculture medium and resuspended into 70% ethanol before beingtransferred into hard grade LR White resin (London ResinCompany, London, UK) for overnight incubation at roomtemperature. After 24 h, the medium was replaced by freshresin and the cells reincubated for 1 h at room temperature. Thecells were then embedded in size 2 gelatin capsules andpolymerized at 608C for at least 24 h. Ultrathin sections of30 nm were cut on a Leica Ultracut UCT ultramicrotome (Leica,North Ryde, NSW, Australia), collected on 1000-mesh coppergrids (ProSciTech, Thuringowa, QLD, Australia), and coatedwith a carbon layer (5 nm) to stabilize the sections before theywere analyzed.

The sections were examined using a 300 kV JEOL 3000Ffield-emission TEM (JEOL, Tokyo, Japan) equipped with apostcolumn Gatan Imaging Filter, a TV-rate camera, and aMulti-Scan digital camera (Gatan, Inc., Pleasanton, CA, USA).A 150 mm condenser and a 60 mm objective aperture was usedfor imaging the elements of interest. The selection of theappropriate cellular region was achieved using the TV-rateretractable camera and energy filtered imaging at an energyloss selected to give good structural contrast. For this purpose,the inelastic contrast imaging was done using settings of energylosses between 100 to 150 eV and an energy selecting slit widthof 20 eV. The conventional three windows method was used forthe elemental mapping. For titanium, the two backgroundwindows (pre-edge 1 and pre-edge 2), centered at E¼ 428and E¼ 436 eV, respectively, had been carefully selectedto minimize interference from the overlap with the extendedtail of the nitrogen-specific edge. The titanium-specific window

(post-edge) was centered at E¼ 464 eV (L2,3-titanium ionizationedge). An energy selecting slit width of 8 eV was used. Todetermine the intracellular distribution of titanium relative tothe naturally existing cellular elements, the same cellularregion that was mapped for titanium, was also mapped forcarbon, chlorine, nitrogen, oxygen, phosphorus, and sulphur.All images (256� 256 pixels) were recorded at a nominalmagnification of 10,000�with a CCD camera using the viewingand imaging software ImageJ (National Institutes of Health,Bethesda, MD, USA).

Electron energy loss spectra (EELS) were recorded from thesame cellular region used for elemental mapping. The spectrumand energy dispersion was selected such that only nitrogen,titanium, and oxygen ionization edges were visible in thespectrum.

Proliferation AssaysThe influence of Ti(IV) on cell proliferation was assessed byimmunocolorimetric assay. Briefly, 5� 105 NA-PBMC wereseeded in round bottom 96-well plates (Sarstedt) with 200 mL ofstandard medium per well. TiCl4 was added at a concentrationof 100 mM, and then serially diluted to a final concentrationof 3.125 mM in quadruplets. All proliferation assayswere consequently carried out in duplicate, with and withoutphytohemagglutinin (PHA) (0.1 mg/mL) (L4144, Sigma,St. Louis, MO, USA) activation.

After 24 h, 100 mL of supernatant was removed and freshmedium containing the corresponding concentrations of Ti(IV)ions was added. Samples from the same assay condition werepooled and stored at�808C until processed further for RANK-Lmeasurements as described below. Cell proliferation wasmeasured after 5 days incubation using a cell proliferationenzyme-linked immunosorbent assay (ELISA) 5-bromo-20-deoxyuridine (BrdU) colorimetric kit (Roche, Mannheim,Germany), according to the manufacturers’ instructions. Theplates were read and recorded using a scanning multi-wellspectrophotometer (Multiskan RC, Labsystems OY, Helsinki,Finland) at an absorbance of 405 nm. Data were expressed asthe percentage of absorbance relative to untreated (standardmedium) controls.

Flow Cytometry (FACS)Flow cytometry was used to investigate the influence of Ti(IV)on the expression of specific cellular surface markers. NA-PBMC (5� 105/well) were cultured with and without PHAactivation in the presence of 1 mM or 10 mM Ti(IV) for 24 h. Cellculture and staining were carried out in round-bottom 96-wellplates (Sarstedt). Human specific antimouse monoclonal anti-bodies for lymphocyte specific surface markers were used;CD69-Alexa488 (Clone FN50, Biolegend, Inc., San Diego,CA, USA), CD26-PE (Clone BA5b, Biolegend), CD45-PerCP,CD4-PeCy7, CD25-APC, CD8-PerCP, CD3-APCCy7 (BD Phar-mingen, San Jose, CA, USA), CCR6-FITC, CCR7-APC, andCCR4-PE (RD Systems, Minneapolis, MN, USA). Unstainedcontrols and isotype antibodies were used to account forautofluoresence and background signal. After staining, thecells were fixed with 0.5% paraformaldehyde. Fluorescentquantification was performed with FACSCanto II FlowCytometer using FACS DIVA Software (BD Biosciences). Allevents were recorded and stopped at 30,000 gated events.Lymphocytes were gated according to their forward scatter(FSC) and side scatter (SSC) profile. Subsequently, theT-lymphocytes were selected from the gated lymphocytepopulation by double positive expression of CD3 and CD4,

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and further investigated for their expression of the specificsurface markers mentioned above. The recorded data wereanalyzed using Flowjo 8.5.3 Software (Treestar Inc., Ashland,OR, USA).

Cytometric Bead Array (CBA) AnalysisThe release of RANK-L into the culture medium (collected asdescribed above) was evaluated after 24 h of incubation withTi(IV) using a Single Plex kit (Linco Research, St. Charles,MO, USA) on a Luminex Plate Reader (Luminex, Menlo Park,CA, USA). The assay was performed according to themanufacturers’ instructions. The amount of cytokine releasedwas normalized to the individual (unchallenged cells from thesame individual) and averaged.

Calculations and Statistical AnalysisData were analyzed using SPSS for Windows (version 15.0;SPSS Inc., Chicago, IL, USA). A one-way analysis of variance(ANOVA) was used to test mean differences in proliferationrates across the various cell cultures. Bonferroni post hoc testswere used to determine which conditions showed significantdifferences in gene expression and proliferation rates. Ap-value of <0.05 was considered statistically significant.

RESULTSThe aim of this study was to investigate the influence ofTi(IV) at different micromolar concentrations (between1 and 100 mM) on the function of human T-lymphocytesin vitro. The very first step was to determine the relativetoxicity of Ti(IV) and the upper limits of challenge dose.For that purpose, NA-PBMC were treated with 100 mMTiCl4 for 24 h. These first experiments revealed that atleast 95% of the cells were viable, showing no traces oftrypan blue within the cells. Additionally, no morpho-logical changes were observed at light microscopic level.

As a result, Ti(IV) concentrations up to 100 mM wereconsidered suitable for all experiments.

Uptake and Distribution of Titanium (IV) in T CellsAfter 3 days incubation with 100 mM Ti(IV), thepresence and distribution of Ti(IV) within theT-lymphocytes was investigated using EELS andEFTEM analysis. The treated cells showed an absorp-tion peak around 464 eV in EELS, corresponding to theexistence of titanium atoms (Fig. 1). EFTEM resultsrevealed the presence of titanium in the nucleus,ribosomes, and in the surface and vesicular membranes,constantly colocalized with phosphorus, suggestingcomplexing of titanium with the phosphate group ofnucleotides and phospholipids, and possibly with phos-phorylated proteins (Fig. 2).

Influence of Titanium (IV) on NA-PBMC ProliferationFirst, we were interested in investigating the influenceof titanium on nonactivated T-lymphocytes. After 5 daysincubation, the immunocolorimetric proliferation assayrevealed two distinct spontaneous proliferation pat-terns among the Ti(IV) treated NA-PBMC of the testeddonors (n¼ 18). In a subset of 11 subjects (61%), Ti(IV)exposure at low micromolar concentrations demon-strated a slight yet significantly increased cell prolifer-ation compared with nontreated cells. With increasingtitanium concentrations, there were correspondingand significant decreases in the proliferation rates(‘‘nonresponder’’), even to below the proliferation rateof untreated cells above 25 mM (p<0.05) (Fig. 3a).Conversely, the NA-PBMC of the remaining sevensubjects (39%) demonstrated a continuous, Ti(IV)

Figure 1. EELS histogram. EELS his-togram of an activated T-lymphocyte after24 h incubation with 100 mM Ti(IV). Thehistogram shows an absorption peak at464 eV, which is specific for titanium,providing strong evidence for titaniumavailability.

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concentration-dependent increase in the proliferativeresponse (‘‘responder’’) to the metal challenge (p< 0.05)(Fig. 3b). The measurements were run in triplicate forall blood donors and presented comparable results atall times. Retrospective sample size calculationsindicated that there were sufficient sample numbersfor statistical comparisons between assay and groupvariables.

Second, we wanted to know about the influenceof titanium on the proliferation of PHA-activatedT-lymphocytes (Fig. 3c). These experiments reveal-ed a concentration-dependent, enhanced proliferativeresponse to titanium challenge with a maximum ofaverage activity at a 6.25 mM concentration (p< 0.05).Interestingly, in contrast to the two different prolifera-tion patterns (‘‘nonresponder’’ vs. ‘‘responder’’) noted inthe nonactivated cell cultures, Ti(IV) did not producesignificant variations in the proliferation pattern ofPHA-activated NA-PBMC among the tested blooddonors (p¼0.89). A paired t-test confirmed higher meanproliferation rates of PHA-activated cells challengedwith Ti(IV) concentrations between 6.25 and 50 mM,and at all concentrations when compared to treatedNA-PBMC cells of the ‘‘responder’’ and ‘‘nonresponder’’group, respectively (p<0.05).

Effect of Titanium (IV) on Surface Marker ExpressionNonactivated and PHA-activated cells incubated withTi(IV) were tested for phenotypic changes. Ti(IV) treat-ment did not change the expression of CD45, CD3, andCD4 (data not shown). The expression of the activationmarker CD26 was not changed by Ti(IV) treatment inCD3þCD4þ cells (Fig. 4). However, further analysisrevealed a minor increase of CD25 and a major increaseof CD69 expression in PHA-activated CD3þCD4þT-lymphocytes exposed to Ti(IV). Additionally, expres-sion of chemokine receptors CCR6 and CCR7 was at avery low level in CD3þCD4þ cells, independent ofthe treatment. Interestingly, CCR4 levels increased inCD3þCD4þ T-lymphocytes exposed to Ti(IV), especiallyin PHA-activated cells.

Figure 2. EFTEM images. EFTEM images of different elemental maps at 10,000� magnification of activated T-lymphocytes after 24 hincubation with 100mM Ti(IV): nitrogen (a), phosphorous (b), titanium (c). Image of overlayed titanium (red) map and phosphorus (blue) mapbinding to phosphorus-containing structures (in the white oval) such as DNA in the nucleus, ribosomes in the cytoplasm, and phospholipids ofthe plasma membranes (d).

Figure 3. Proliferation rates. Mean proliferation rates of restingT cells in 61% of individuals (n¼11) were found to be inhibited byTi(IV) at high concentrations (a), while the remainder (n¼7)showed enhanced proliferation (b). Proliferation rates of PHA-activated cells were found to be higher than those of naıve cellcultures, and did not reveal different response patterns within thetested individuals (c). Activated cells exhibited concentration-dependent decreased proliferation with increasing Ti(IV) concen-trations from 6.25 mM (c). The proliferation rates were calculated asthe percentage of the negative control [proliferation rate ofnonactivated NA-PBMC incubated with standard medium contain-ing 0% Ti(IV)]. *p<0.05.

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Measurement of RANK-L in Culture MediaInterestingly, significantly increased levels of RANK-Lwere measured with increasing metal concentrations inthe supernatant of PHA-activated cells, when comparedto metal unchallenged cells (p<0.001 for RANK-L atconcentrations �50 mM) (Fig. 5). There was a Ti(IV)concentration-dependent, but statistically nonsigni-ficant RANK-L production by nonactivated cells at lowlevels (<10 pg/mL).

DISCUSSIONAseptic loosening of metal joint implants remains asignificant problem in orthopedic surgery.10 Over 10% ofpatients suffer from the condition within 20 years of aprimary hip arthroplasty, with the incidence continuingto increase.17 Aseptic loosening is suggested to resultfrom inflammatory and hypersensitivity responses tometals, which lead to increased osteolytic activity at thebone–implant interface and ultimately loss of fixa-

tion.5,6,14,15,18–20 However, little is known about theeffects of Ti(IV) ions on human T-lymphocytes, becausemost published studies have focused on other cells (suchas macrophages, osteoblasts, and osteoclasts) and othermetal ions.21

It is well recognized that titanium degradationproducts including Ti(IV) ions are released by biocorro-sion and accumulate in lymphoid tissues.11,13,14,22,23

However, this is the first study using EFTEM analysis,which is recognized as the gold standard in identifyingtitanium availability in biological specimens,24 to dem-onstrate the ability of human T-lymphocytes to take upTi(IV). EFTEM results indicate a strong Ti(IV) affinityfor phosphorus-containing molecules such as the euchro-matin in the nucleus, ribosomes in the cytoplasm, andphospholipids in the cell membrane. The strong phos-phate binding property for titanium has been discoveredand described previously, and applications for enrich-ment and molecular characterization of phosphorylated

Figure 4. Flow cytometry. Representativeflow cytometric detection of the expression ofactivation markers (CD69, CD26, and CD25)and chemokine receptors (CCR4, CCR6, andCCR7) by CD3þ and CD4þ T lymphocytesexposed to different treatments (PHA þ/�,Ti(IV) þ/�) after 24-h incubation. Note theincreased fluorescent intensity of CD69 andCCR4 in Ti(IV) challenged T lymphocytes.(X-axis¼fluorescent intensity; Y-axis¼percentage of cells).

Figure 5. Expression of RANK-L. Histogramshowing the expression levels of RANK-L in thesupernatant of PHA-activated and nonactivatedNA-PBMC from ‘‘responders’’ (n¼5) after 24 hof incubation with different Ti(IV) concentra-tions (0, 12.5, 50, and 100 mM). The amount ofcytokine release was normalized to the indi-vidual (unchallenged cells from the same indi-vidual) and averaged. Note the significantincrease in RANK-L production in PHA-acti-vated cells treated with 50 and 100 mM Ti(IV).*p<0.05.

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molecules have been abundantly established.25 Ti(IV)may bind to phosphorylated intracellular proteins,which often correspond to active functional states ofenzymes or signaling proteins and interfere with signal-ing pathways. Additionally, it is reasonable to assumethat the attachment of Ti(IV) to membrane phospholi-pids may change its fluidity and thereby alter cellularfunctions such as migration, protein secretion, and theability to respond to stimuli as shown with other metalssuch as aluminum and nickel.26 Despite demonstratingthe availability of titanium in T-lymphocytes, themechanisms by which Ti(IV) ions enter the cells shouldbe further investigated in the future.

As Ti(IV) ions were shown to be nontoxic and taken upby the cells, we investigated the effects of Ti(IV) ions onactivation of T-lymphocytes. Our results indicate thatpreactivated T-lymphocytes are further activated byTi(IV) and increase the expression of CD25 slightly andCD69 significantly. CD69 is involved in cell activationand signaling.27 In patients with Co–Cr hip prosthesis,Granchi et al.28 also noted the expression of CD69 byCD3þ cells. Increase in CD69 by Ti(IV) may enhance ordeviate cell activation. However, the molecular mecha-nisms resulting in increased expression of activationmarkers by Ti(IV) have to be further investigated infuture experiments.

Of great interest was the finding that Ti(IV) inducedtwo distinct proliferation patterns among nonactivatedcells of tested healthy subjects. Approximately 61% ofblood donors (designated as ‘‘nonresponder’’) showed adecrease in spontaneous proliferation rates with increas-ing Ti(IV) concentrations, while the remaining 39%(designated as ‘‘responder’’) revealed a significantlyincreased proliferative response to the metal challenge.Previous studies have indicated either an enhanced ordecreased proliferation of T-lymphocytes exposed totitanium.29,30 However, earlier publications investi-gated a small number of subjects (n¼ 8), and this is thefirst study demonstrating both, a ‘‘responder’’ and a‘‘nonresponder’’ pattern within the same group of testedindividuals. This may indicate an individual dependentimmune reactivity to Ti(IV) among approximately 39% ofindividuals, present before any contact with titanium.Preexisting titanium-specific T-lymphocytes may beresponsible for the metal reactivity in the ‘‘responder’’subjects as suggested by lymphocyte transformationassays.31 However, it is not yet possible to determine aclear relationship between the lymphocyte reactivityassays and the clinical outcome of patients withtitanium implants. Further prospective studies correlat-ing in vitro test results and implant performance arenecessary to determine the clinical screening capacity ofsuch assays and draw more accurate conclusions.

Our study clearly shows that titanium inducessecretion of RANK-L in activated T-lymphocytes.RANK-L plays a crucial role in the activation, matura-tion, and function of osteoclasts—the only bone-resorbing cells.32 Secretion of RANK-L in a boneenvironment will result in bone resorption. Activation

of T-lymphocytes in peri-implant tissues can occur in thecontext of autoimmune diseases such as rheumatoidarthritis and infections.10 Additionally, there is evidencethat titanium itself may become antigenic and induceactivation of specific T-lymphocytes.33 In the presence oftitanium ions that are released from an orthopedicimplant, activated T-lymphocytes may be a major sourceof RANK-L, responsible for enhanced osteoclast activa-tion, and resulting in aseptic loosening.

CONCLUSIONSTitanium (IV) enters T-lymphocytes through a currentlyunknown mechanism and binds to phosphorus-contain-ing molecules. Most likely, this intracellular bindingchanges phenotype and function of T-lymphocytes. Thosechanges may well be involved in the inflammatorypathways and bone resorption in the presence of anorthopedic implant.

ACKNOWLEDGMENTSWe thank C. Motteram for the statistical analysis, as well as T.Knox and G. Cozens for their excellent assistance. We alsogratefully acknowledge the Centre for Microscopy, Charac-terization, and Analysis (CMCA), University of WesternAustralia, for the use of their facilities. This study has beensupported by the National Institutes of Health GrantGM072726 and the AO Foundation Grant 05Z34.

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in patients with metal-to-plastic total hip arthroplasties. ActaOrthop Scand 51:57–62.

2. Kubba R, Taylor JS, Marks KE. 1981. Cutaneous complica-tions of orthopedic implants. A two-year prospective study.Arch Dermatol 117:554–560.

3. Mayor MB, Merritt K, Brown SA. 1980. Metal allergy and thesurgical patient. Am J Surg 139:477–479.

4. Merritt K, Rodrigo JJ. 1996. Immune response to syntheticmaterials. Sensitization of patients receiving orthopaedicimplants. Clin Orthop Relat Res 326:71–79.

5. Thomas P, Bandl WD, Maier S, et al. 2006. Hypersensitivity totitanium osteosynthesis with impaired fracture healing,eczema, and T-cell hyperresponsiveness in vitro: case reportand review of the literature. Contact Dermatitis 55:199–202.

6. Thomas P, Thomsen M. 2008. [Allergy diagnostics in implantintolerance]. Orthopade 37:131–135.

7. Rustemeyer T, von Blomberg BM, van Hoogstraten IM, et al.2004. Analysis of effector and regulatory immune reactivity tonickel. Clin Exp Allergy 34:1458–1466.

8. Hallab NJ, Anderson S, Stafford T, et al. 2005. Lymphocyteresponses in patients with total hip arthroplasty. J OrthopRes 23:384–391.

9. Looney RJ, Schwarz EM, Boyd A, et al. 2006. Periprostehticosteolysis: an immunologist’s update. Curr Opin Rheumatol18:80–87.

10. Radmer S, Andresen R, Sparmann M. 2003. Total wristarthroplasty in patients with rheumatoid arthritis. J HandSurg Am 28:789–794.

11. Tengvall P, Lundstrom I. 1992. Physico-chemical considera-tions of titanium as a biomaterial. Clin Mater 9:115–134.

12. Okazaki Y, Gotoh E, Manabe T, et al. 2004. Comparison ofmetal concentrations in rat tibia tissues with various metallicimplants. Biomaterials 25:5913–5920.

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13. Jacobs JJ, Skipor AK, Patterson LM, et al. 1998. Metal releasein patients who have had a primary total hip arthroplasty. Aprospective, controlled, longitudinal study. J Bone Joint SurgAm 80:1447–1458.

14. Lalor PA, Revell PA, Gray AB, et al. 1991. Sensitivity totitanium. A cause of implant failure? J Bone Joint Surg Br73:25–28.

15. Voggenreiter G, Leiting S, Brauer H, et al. 2003. Immuno-inflammatory tissue reaction to stainless-steel and titaniumplates used for internal fixation of long bones. Biomaterials24:247–254.

16. Vollenweider I, Moser R, Groscurth P. 1994. Development offour donor-specific phenotypes in human long-term lympho-kine-activated killer cell cultures. Cancer Immunol Immun-other 39:305–312.

17. Harris WH. 1994. Osteolysis and particle disease inhip replacement. A review. Acta Orthop Scand 65:113–123.

18. Bauer TW. 2002. Particles and periimplant bone resorption.Clin Orthop Relat Res 405:138–143.

19. Graves SE, Davidson D, Ingerson L, et al. 2004. TheAustralian Orthopaedic Association National Joint Replace-ment Registry. Med J Aust 180:S31–34.

20. Harris WH. 1995. The problem is osteolysis. Clin Orthop RelatRes 311:46–53.

21. Au A, Ha J, Hernandez M, et al. 2006. Nickel and vanadiummetal ions induce apoptosis of T-lymphocyte Jurkat cells.J Biomed Mater Res A 79:512–521.

22. Dorr LD, Bloebaum R, Emmanual J, et al. 1990. Histologic,biochemical, and ion analysis of tissue and fluids retrievedduring total hip arthroplasty. Clin Orthop Relat Res 261:82–95.

23. Urban RM, Jacobs JJ, Tomlinson MJ, et al. 2000. Dissem-ination of wear particles to the liver, spleen, and abdominallymph nodes of patients with hip or knee replacement. J BoneJoint Surg Am 82:457–476.

24. Valadares LF, do Carmo Braganca F, da Silva CA, et al. 2007.Low-energy-loss EFTEM imaging of thick particles andaggregates. J Colloid Interface Sci 309:140–148.

25. Ikeguchi Y, Nakmura H. 2000. Selective enrichment ofphospholipids by titania. Anal Sci 16:541–543.

26. Schuster GS, Caughman GB. 2004. Alterations of cell lipids bymetal salts. J Biomed Mater Res A 70:347–353.

27. Sancho D, Gomez M, Sanchez-Madrid F. 2005. CD69 is animmunoregulatory molecule induced following activation.Trends Immunol 26:136–140.

28. Granchi D, Ciapetti G, Savarino L, et al. 2000. Expression ofthe CD69 activation antigen on lymphocytes of patients withhip prosthesis. Biomaterials 21:2059–2065.

29. Hallab NJ, Caicedo M, Finnegan A, et al. 2008. Th1 typelymphocyte reactivity to metals in patients with total hiparthroplasty. J Orthop Surg 3:6.

30. Kohilas K, Lyons M, Lofthouse R, et al. 1999. Effect ofprosthetic titanium wear debris on mitogen-induced monocyteand lymphoid activation. J Biomed Mater Res 47:95–103.

31. Stejskal VD, Danersund A, Lindvall A, et al. 1999. Metal-specific lymphocytes: biomarkers of sensitivity in man.Neuroendocrinol Lett 20:289–298.

32. Kong YY, Feige U, Sarosi I, et al. Activated T cells regulatebone loss and joint destruction in adjuvant arthritis throughosteoprotegerin ligand. Nature 402:304–309.

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Chapter 09 Discussion Cellular Mechanisms of Corrosion

Besides the electrochemical aspect of corrosion, it has been debated whether mature

OC are able to corrode the metal surface. The results presented in this thesis

(Chapter 04 and 05) demonstrate that OC precursors are able to grow and

differentiate on surgical stainless steel, Al, and Ti, and to directly corrode the metal

surface as demonstrated by resorption pits on the metal surface and the release of

corresponding metal ions into culture media. Both Al and Ti corrode easily in a water

based environment. However, a thin oxide layer is quickly formed on the surface,

protecting the metal from further electrochemical corrosion (11,103). In a

physiological context, it must be considered that functional OC are able to generate

an environment more prone to corrosion, by secreting protons into the resorption

lacunae. The low pH in the resorption compartment with the high concentration of H+

might destabilize the oxide layer, leading to free metal ions on the surface (11,103).

Removal of the metal oxide layer will subsequently expose new metal to additional

corrosion enhanced by the acidic conditions in the osteoclastic resorption pit. Once

solubilized in the osteoclastic pit, the metal ions are most likely taken up by the OC

into the TRAP-containing transcytotic compartment for further processing (104,105).

Usually, resorbed material including metal ions is eventually released by the OC into

the extracellular space. This happens in the case for several metals such as Al, Co

and Cr as shown in Chapter 04 and 05. However, certain metal ions may bind to

cellular structures and form stable complexes that remain in the OC. This is certainly

the case for Ti ions that bind to cytoplasmic and nuclear structures, as depicted by

confocal fluorescence microscopy. However, Ti–protein complexes may be released

into the extracellular space once the OC die and break down. We proposed that this

process may take place at the bone-implant interface, representing an additional

mechanism of metal corrosion in vivo and contributing to the levels of metal ions

measured in the periprosthetic tissues and serum from total arthroplasty patients.

Effects of Metal Ions on Bone Metabolism

Total joint replacement has been a great success with improvements in the surgical

techniques, skeletal fixation, prophylaxis against infections, and fatigue strength of

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the component. However, the formation of periprosthetic osteolytic lesions, impairing

the maintenance of a stable interface between the implant and the bone develops in

~15–20% of all patients within 20 years of a primary hip arthroplasty with reported

failure rates of 13% for the femoral component and 34% for the acetabular

component (57–60). Recently, great effort has been directed toward understanding

the pathophysiological cascade of events initiated by biocorrosion products, resulting

in periprosthetic bone loss and ultimately AL. Normal bone maintenance relies on the

balance between bone formation and bone resorption. The net bone loss at the

metal-tissue interface occurs because of an increased bone resorption or reduced

bone formation. Increased bone resorption can be due to an increase of one or more

of the following events: recruitment of OC precursors from the blood circulation at the

bone-implant interface, their differentiation into mature multinucleated cells, their

functional activation, and finally their survival. Increased recruitment of OC

precursors and their subsequent differentiation into mature OC may be involved in

the pathomechanisms of AL. It is reasonable to assume that some of the OC

precursors recruited to the peri-implant tissues differentiate into mature OC. In line

with this hypothesis, several animal studies have demonstrated that OC precursors in

tissues surrounding subcutaneously implanted wear particle differentiate into mature

OC (61–63). It must be assumed that the increased recruitment of OC precursors is

due to increased concentrations of chemotactic cytokines induced by wear particles

and released metal ions. In fact, more recently, particulate wear debris

(predominantly polyethylene) has been shown to induce chemokine expression in

macrophages, fibroblasts, and osteoblasts, including IL-8, monocyte chemoattractant

protein-1, macrophage inflammatory protein 1, and eotaxin (64–67). The in vitro

experiments detailed in Chapter 07 suggest that Ti(IV) ions enhance the synthesis

and secretion of CCL17/TARC and CCL22/MDC in mature OC. Most importantly,

recent studies have demonstrated that OC also express CCL22/MDC upon activation

by RANK-L (68,69). Taken together, these studies lead to the conclusion that

recruitment of OC precursors is an important mechanism by which wear particles and

possibly metal ions stimulate osteolysis in patients with orthopaedic implants.

Osteoclasts are formed by the fusion of bone marrow-derived mononuclear

precursors, which circulate in the CD14+ monocyte fraction of peripheral blood (70).

Osteoclastic differentiation from hematopoietic and circulating monocytes is a

multistep event that occurs in the presence of M-CSF and RANK-L expressed by

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osteoblasts and other bone-related stromal cells. The process includes precursor

proliferation, commitment to the OC lineage, expression of OC specific genes such

as TRAP, and finally fusion into multinucleated giant cells (71). Several studies have

shown, in both animal and human in vitro models, that periprosthetic osteolysis is

primarily driven by an increased OC differentiation and activity. A similar increased

presence of mature OC has been reported in patients with AL (73). In addition,

synovial fluid of patients with loose implants contains elevated levels of TRAP,

presumably due to secretion of this enzyme by the increased number of OC (74,75).

It is well recognized that phagocytosis of metallic wear particles by macrophages

induces their activation, producing mediators that enhance osteoclastogenesis (26).

The release of proinflammatory cytokines, including TNF-α, IL-6, and IL-1α/β by

activated macrophages has been recognized as one of the primary cellular

mechanisms responsible for increased OC differentiation (76–78). These cytokines

act synergistically enhancing differentiation of OC precursors into mature OC able to

efficiently resorb the peri-implant bone (79,80). TNF-α acts directly on OC

precursors, whereas IL-6 and IL-1α/β act indirectly by increasing the expression of

RANK-L and M-CSF by osteoblasts, which in turn, directly drives osteoclastogenesis

through a mechanism involving cell-to-cell contact (80). The latter is part of the

coupling between bone resorption and formation, which is essential not only during

bone growth but also during bone remodelling and fracture healing. In addition to

activated macrophages exposed to wear particles, the potential role of metal ions

released by biocorrosion from the implant surface must also be considered. The

results presented in Chapter 06 suggest that Ti(IV) ions directly induce the

differentiation of OC precursors toward mature OC in ~20% of individuals. These

results suggest also that Ti(IV) may disconnect the OC function from the osteoblastic

control mechanisms, while still exhibiting relevant bone resorbing capacity.

Taken together these results and the studies discussed above suggest that metal

wear and ions (e.g. Ti(IV)) induce increased differentiation and activation of OC

precursors leading to a large number of mature OC able to effectively resorb the

bone surrounding metal implants, but also potentially enhance biocorrosion of the

implant, resulting in more ion release and the development of a vicious cycle. In

addition to an enhanced recruitment and functional maturation of OC precursors, it is

also reasonable to assume that cytokines released in response to biocorrosion

products (including metal ions) increase OC survival. This hypothesis is supported by

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studies showing that several bone resorptive cytokines increase OC survival (81,82).

For example, IL-1 increases OC survival by activating RANK and thereby inhibiting

apoptosis (83). Greenfield et al. found that conditioned media from marrow cells

incubated with Ti particles slightly increased OC survival (72). The results presented

in Chapter 04 showed an increased expression of IL-1 in the supernatant of OC

cultured on surgical stainless steel and Ti foils. The previous studies along with the

results presented in this thesis suggest that and increased OC survival may further

enhance the complex pathomechanism of increased osteolysis in AL.

Effects of Released Titanium Ions on Osteoimmunology

Dermal hypersensitivity to metal is common, with up to 20% of Caucasians being

sensitive to Ni (97). Immune reactions to dermal contact and ingestion of metals,

manifested as skin conditions such as eczema, urticaria, erythema, and pruritis are

believed to be of a type IV cell mediated hypersensitivity (12). The earliest case of an

allergic manifestation towards an orthopaedic implant was reported by Foussereau et

al., when a patient presented with an eczematous rash over a stainless steel fracture

plate (98). Besides the clinical observations of metal hypersensitivity observed in

some patients with orthopaedic implants, strengthened by the alleviation of

symptoms after the removal of the causative metal implant, evidence for the

involvement of the immune system comes from several histological studies of

retrieved peri-implant tissues (99). These studies have shown the infiltration of

lymphocytes, monocytes, dendritic cells, macrophages, and mast cells into the peri-

implant tissues at various time points after metal implantation. Immunohistochemical

analysis of peri-implant tissues revealed high levels of immune specific markers

correlating with an inflammatory response, including CD3+ T-lymphocytes, CD4+

cells, and cells with abundant MHC class II (HLA-DR) expression (99–101). Another

indicator of immune activity is the finding of increased cytokine levels in the serum of

patients with orthopaedic implants. In that respect, cytokines able to induce bone

resorption such as IL-1β, TNF-α, IL-6, and macrophage-granulocyte colony-

stimulating factor (GM-CSF) have been of particular interest, and elevated serum

cytokine levels have been reported in patients with total hip prosthesis (102). The

results presented in Chapter 08 showed that Ti(IV) induces secretion of RANK-L in

activated T-lymphocytes. RANK-L plays a crucial role in the activation, maturation,

and function of OC. Secretion of RANK-L in a bone environment will result in bone

- 61 -

resorption. Activation of T-lymphocytes in peri-implant tissues can occur in the

context of autoimmune diseases such as rheumatoid arthritis and infections.

Additionally, there is evidence that Ti itself may become antigenic and induce

activation of specific T-lymphocytes (106). In the presence of Ti(IV) ions that are

released from an orthopaedic implant, activated T-lymphocytes may be a major

source of RANK-L, responsible for enhanced OC activation, and contributing to AL.

Summary and Outlook

The constant aging of our population will contribute to an increasing number of

patients with implanted metal devices. The results of this work represent an important

step toward understanding the complex pathophysiology of AL and its prevention. In

addition to the recognized role of wear particles, these results indicate that Ti(IV) ions

are most likely to contribute to AL by enhancing OC differentiation, activation and

recruitment. This work also links the immune system to the bone and to its

involvement in AL. Titanium (IV) enters T-lymphocytes through a currently unknown

mechanism and binds to phosphorus-containing molecules. Most likely, this

intracellular binding changes phenotype and function of T-lymphocytes. Those

changes may well be involved in the inflammatory pathways and bone resorption in

the context of AL. To date, revision surgery remains the only option for the treatment

of failed implants caused by AL. Preoperative studies investigating the effects of

specific metal ions on individual bone metabolism and immune system would help in

selecting suitable alloys for each patient and subsequently reduce the occurrence of

AL. Ongoing studies aim to develop such tests and to investigate whether OC

biocorrosion can be blocked pharmacologically.

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Appendix

Ti22

Titanium Induced Bone Resorption Due to Osteoclast Recruitment and Activation

D. Cadosch1,2, E. Chan1, OP. Gautschi1,2, J. Meagher2, AP. Skirving1, HP. Simmen3 and L. Filgueira21Department of Orthopaedic and Trauma Surgery, Royal Perth Hospital, Perth, Western Australia

2School of Anatomy and Human Biology, University of Western Australia, Australia 3Klinik für Unfallchirurgie, Universitätsspital Zürich, Switzerland

BackgroundThere is increasing evidence that titanium (Ti(IV)) ions are released from orthopedic implants and play a role in aseptic loosening. Our aims were to

investigate, whether Ti(IV) ions induce recruitment and differentiation of monocytes into osteo-

resorptive

multinucleated cells, and influence the activation and function of in vitro

generated osteoclasts.

Materials and MethodsHuman monocytes

and in vitro

generated osteoclasts were exposed to 1µM Ti(IV) ions for ten days.

Thereafter, transcription of specific osteoclast genes, including tartrate-resistant acid phosphatase

(TRAP) and cathepsin

K (CATK), and chemokines

(CCL17/TARC & CCL22/MDC) were measured

using semi-quantitative PCR and ELISA analysis.

The effects of Ti(IV) on osteoclastic activity and differentiation were also evaluated by measuring the enzymatic activity of TRAP using ELF97.

Additionally, functional evidence of osteoclastic activity was determined by a lacunar

resorption assay system using cell cultures on dentin slides.

ResultsAfter Ti(IV) treatment monocytes

from five donors (22.7%) showed an increased expression of TRAP

and CATK.

Detection and quantification of

intracellular TRAP activity revealed a significant

increase of TRAP-positive cells in treated monocytes

(Fig. 1). Ti(IV) treated monocytes

became functional bone resorbing

cells, increasing significantly their

osteo-resorptive

activity (Fig. 2&3). Additionally

, Ti(IV) treated osteoclasts showed significantl

y increased CCL17 and CCL22 expression and

secretion (Fig. 4).

Conclusions This study provides strong support for the hypothesis that Ti(IV) ions, released by bio-corrosion from metal implants, induce differentiation of monocytes towards mature, functional osteoclasts in

approximately 20% of individuals. Additionally, Ti (IV) ions activate the expression and secretion of chemokines in mature osteoclasts. These results suggest enhanced

recruitment of osteoclast precursors from the blood circulation and induced osteoclastic differentiation, which may well contribute to the pathomechanism

of aseptic loosening.

Monocytes+Ti

Osteoclasts+Ti

Monocytes

OsteoclastsFigure 2:

Resorption pits (dark spots) depicting the effects of Ti(IV) on bone resorption on dentine slides after 21 days incubation.

Figure 1:

Expression of TRAP by Flow Cytometric

detection of ELF97. The red line represents TRAP activity in the cells, the shaded region, the control staining. (X-axis=fluorescent intensity; Y-axis=% of cells).

Figure 3:

Mean area (±

SD) of resorption on dentine slides after 21 days in culture. *p<0.001

Figure 4:

Expression levels (±SD) of CCL17 and CCL22 in the culture supernatant after ten days of incubation. *p<0.001.

CCL22CCL17

0

250

500

750

1000

0

2000

4000

6000

8000pg

/mL

pg/m

L

**

+T

itani

um (

IV)

+T

itani

um (

IV)

+T

itani

um (

IV)

+T

itani

um (

IV)

Monocytes Osteoclasts Monocytes Osteoclasts0

2

4

6

8

Reso

rptio

n A

rea

ofTo

tal S

urfa

ce A

rea

(%)

**

+T

itani

um (

IV)

+T

itani

um (

IV)

Monocytes Osteoclasts

*

MonocyteT Cell

Up-regulation CCR4

Peri-Implant Tissues

Endothelium

Circulation

Ti22

⑂

⑂

⑂⑂

Dissemination Biocorrosion

CCL17/22

Recruitment

Osteolysis

Aseptic Loosening

TNF

RANK

RANK-LM-CSF

Osteoclasts

RANK-L

RANK

⑂ ⑂

Osteoblast

⑂⑂ ⑂

Monocyte

TRAP

Monocytes

Osteoclasts

Untreated +Ti(IV)

Postulated pathway involving Ti(IV) ions in the pathomechanism

of aseptic loosening (including data from previous studies, Cadosch et al.

2008).

Titanium IV Uptake, Induction of RANK-L Expressionand Enhanced Proliferation of Human T-Lymphocytes

Dieter Cadosch, MD, PhDc1,2,3, Michael Sutanto1, Erwin Chan, PhD1,Amir Mhawi, PhD1, Hans-Peter Simmen, MD3, Luis Filgueira, MD1

1School of Anatomy and Human Biology, University of Western Australia, Australia 2Department of Surgery, Kantonsspital Winterthur, Winterthur, Switzerland3Division of Trauma Surgery, University Hospital Zurich, Zurich, Switzerland

BackgroundThere is increasing evidence that titanium ions are released from orthopaedic implants by biocorrosion and play a role in aseptic loosening. The aim of this study was to investigate titanium uptake by human T-lymphocytes and its effects on phenotype and proliferation.

ConclusionsTitanium enters T-lymphocytes through a currently unknown mechanism and binds to phosphorus-rich cell structures. Titanium influences phenotype and function of T-lymphocytes, resulting in activation of a CD69+ and CCR4+ T-lymphocyte population and secretion of RANK-L. These results strongly suggest the involvement of titanium ions challenged T-lymphocytes in the complex pathophysiological mechanisms of aseptic loosening of orthopaedic implants.

Materials and MethodsFreshly isolated human non-adherent peripheral blood mononuclear cells (NA-PBMC), were exposed to TiCl4 (Ti(IV)). Bioavailability and distribution of Ti(IV) in

T-lymphocytes was determined by energy-

filtered electron microscopy (EFTEM). The effects of Ti(IV) challenge on non-activated and

phytohemagglutinin-activated (PHA) (0.1 mg/mL) cells were assessed by flow cytometric analysis of surface markers, RANK-L production and proliferation assays (BrdU).

ResultsEFTEM showed Ti(IV) uptake by T-lymphocytes and co-localized Ti(IV) with phosphorus in the

nucleus, ribosomes, cytoplasmic

membranes and the surface membrane of T-lymphocytes (Fig. 1). Ti(IV) induced two distinct proliferation patterns among the resting (non-activated) NA-PBMC of the tested subjects. Approximately 61% showed a decrease in proliferation with increasing Ti(IV) concentrations, while the remaining 39% revealed a significant proliferative response to the metal challenge (Fig 2). Proliferation rates of PHA-activated cells were found to be higher than those of resting cell cultures, and did not reveal different response patterns within the tested individuals (data not shown). Ti(IV) increased significantly the expression of CD69, CCR4 and RANK-L in a concentration dependent manner (Fig. 3 & 4).

Figure 1:

Energy-filtered electron microscopy images of different elemental maps at 10’000 X

magnification of activated T-lymphocytes after 24 hours incubation with 100 μM

Ti(IV): nitrogen (A), phosphorous (B), titanium (C). Image of overlayed

titanium (red) map and phosphorus (blue) map binding to phosphorus-containing structures (yellow arrows) such as DNA in the nucleus, ribosomes in the cytoplasm and phospholipids of the plasma membranes (D). N =

nucleus, C = cytoplasma.

0

0.1

0.2

0.3

0.4

0.5

3.125 6.25 12.5 25 50 100

*

*

*

A: Nitrogen Map B: Phosphorus Map

N N

D: Titanium & Phosphorus MapC: Titanium Map

C

N N

C

CC

Titanium IV Concentrations (μM)

Mea

n p

rolif

erat

ion

rat

es

Ti22

MonocyteT Cell

Up-regulation CCR4

Peri-Implant Tissues

Endothelium

Circulation

Ti22

⑂

⑂

⑂⑂

Dissemination Biocorrosion

CCL17/22

Recruitment

Osteolysis

Aseptic Loosening

TNF

RANK

RANK-LM-CSF

Osteoclasts

RANK-L

RANK

⑂ ⑂

Osteoblast

⑂⑂

⑂

Monocyte

Activation Marker CD69 Chemokine Receptor CCR4

A

B

C

D

A

B

C

D

Figure 3: Representative Flow Cytometric detection of the expression of activation

marker CD69 and chemokine receptor CCR4 by CD3+ and CD4+ T-lymphocytes exposed to different treatments (PHA +/-, Ti(IV) +/-) after 24 hours incubation. Note the increased fluorescent intensity of CD69 and CCR4 in Ti(IV) challenged T lymphocytes (D). (X-axis=fluorescent intensity; Y-axis=percentage of cells). A: untreated T-lymphocytes; B: 0.01M Ti(IV); C: PHA-activated; D: PHA-activated and 0.01M Ti(IV.

Figure 4:

Histogram showing the expression levels of RANK-L (±SD) in the supernatant of non-

activated NA-PBMC after 24 hours of incubation with increasing Ti(IV) concentrations. The amount of cytokine release was normalized to the individual (unchallenged cells from the same individual) and averaged. *p<0.05.

RANK-L

*

pg

/mL

200

180

160

140

120

100

80

60

40

20

*

0 μM 12.5 μM 50 μM 100 μM

Postulated pathway involving Ti(IV) ions in the pathophysiological mechanism of aseptic loosening (including data from previous studies: Cadosch D et al.

2008, 2009 and 2010).

Correspondence: dieter.cadosch@gmx.net

or

dieter.cadosch@usz.ch

Figure 2:

Mean proliferation rates (±SD) of resting (non-activated) T-lymphocytes in 61% of

individuals (n=21) were found to be inhibited by Ti(IV) at high concentrations (data not shown), while the remainder 39% showed enhanced proliferation. The proliferation rates were calculated as the percentage of the negative control (proliferation rate of non-activated NA-PBMC incubated with standard medium containing 0% Ti(IV). *p<0.05.