Detoxification of Titanium Implant Surfaces: Evaluation of Surface Morphology and Pre-Osteoblast Cell Compatibility

THE UNIVERSITY OF TEXAS AT DALLASErik Jonsson School of Engineering and Computer Science

MS Thesis DefenseDeepthi Ramesh

Advisor: Dr. Danieli C. RodriguesCommittee Members: Dr. Kelli Palmer, Dr. Taylor Ware

November 28, 2016

DEPARTMENT OF BIOENGINEERINGBiomaterials for Osseointegration and Novel Engineering Lab

OUTLINE

1. Introduction2. Significance3. Background4. Problem 5. Aims

5.1 Aim 15.2 Aim 25.3 Aim 3

6. Conclusion7. Future Work8. References9. Acknowledgments

2

INTRODUCTION

3

INTRODUCTION

Fig. 1 Natural tooth vs. dental implant.

DENTAL IMPLANTS

• Trauma/accident• Cavities• Periodontal diseases

• High success rate (90-95%)

• Faster healing time• Convenience• Aesthetic appeal• Patient comfort & function

Tooth loss due to:

Benefits:

4[1] [2] [3]

INTRODUCTION

DENTAL IMPLANTS

• Bacterial contamination

• Implant design

• Mechanical instability

• Corrosion

• Clinician

Even with high success rates, 5-10% of implants fail.

Fig. 2 Healthy implant vs. failed implant

Failure mechanisms:

5[2] [4] [5]

SIGNIFICANCE

6

More than 500,000 implants placed annually

By age 74, 26% of adults have lost all of their

permanent teeth

70% of adults ages 35-44 have lost at least 1

permanent tooth

SIGNIFICANCE

28%-56% of patients with implants suffer from peri-

implantitis

7[6]

BACKGROUND

8

Graphical Content

BACKGROUND

Material Used:

Surface Design:

Smooth Machined Textured Coated

Fig. 3 Different design of implants.

9[7] [8]

DESIGN & DEVELOPMENT OF DENTAL IMPLANTS

• Titanium (cpTi)• Titanium alloy (Ti-6Al-4V)• Zirconium• Gold• Stainless steel• Cobalt-chromium (CoCr)

• Polymers• Ceramics

Metals Non-metals

10

TITANIUM

Biocompatible

Corrosion resistant

Strong and durable

Osseointegrative (bonds well with bone)

Cost-efficient

Lightweight

Non-ferromagnetic

Ti oxide layer

Fig. 5 Titanium spontaneously forms an oxide layer.

BACKGROUND

Fig. 4 Titanium dental implant.

BACKGROUND

SUCCESS OF DENTAL IMPLANTS

• Biocompatibility

• Nature of implant surface

• Surgical technique

• Implant design

• Patient health

• Oral hygiene

• Osseointegration

Fig. 6 Process of osseointegration.

Fig. 7 Success of dental implant depends greatly on oral hygiene.

11[9] [10]

OSSEOINTEGRATION

• Direct bone anchorage to an implant body which provides mechanical support.

• Integration of implant to supporting bone.

• Mohyi et al. suggested that peri-implantitis is an osseointegration pathology.

Fig. 8 Bone-forming cell adheres onto implant surface.

Fig. 9 Osseointegration is the integration of bone to implant.12[11] [12]

BACKGROUND

BACKGROUND

IMPLANT FAILURES

Late Stage:

Early Stage:

• Implant is placed surgically• Before osseointegration• Bacteria • Poor bone quality & quantity• Surgical trauma• Pre-mature loading

• Abutment & crown placed• After osseointegration• Peri-implantitis • Excessive loading• Inadequate prosthetic construction

1.5 to 21%

1.0 to 28 %

Fig. 10 Different parts of the dental implant.

Fig. 11 Failed implant, loss of bone and inflammation.13[13] [14] [15]

BACKGROUND

PERI-IMPLANTITIS

Causes:

A site specific disease that causes Inflammation & bone loss around implant.

• Formation of bacterial biofilm • Poor oral hygiene• Genetic factors• Residual cement• Smoking• Diabetes

Fig. 12 Process of biofilm formation.

Fig. 13 Peri-implantitis causes bone loss and inflammation.14[16] [17] [18]

Early-colonizing bacteria:

Late-colonizing bacteria:

BACKGROUND

Streptococci & Actinomyces species

Aggregatibacter actinomycetemcomitans (Aa)Porphyromonas gingivalis (Pg)

BACTERIA INVOLVED IN PERI-IMPLANTITIS:

Bacteria mechanisms of action

Creates areas of O₂ depletion

Releases acidic metabolites

Creates acidic environment

Corrosion & hindered osseointegration

Bacteria Biofilm Reduction of pH

Corrosion Implant loss

Fig. 14 Various bacteria associated with biofilm formation.

Fig. 15 Effects of bacterial biofilm.

15[19] [20]

BACKGROUND

TREATMENT FOR PERI-IMPLANTITIS

Mechanical debridement with acidic chemicals

Mechanical Laser CombinationsChemical

Citric acidChlorhexidineDoxycycline

SalineHydrogen peroxide

Er: YAGContinuous CO₂

Implantoplasty Air powder abrasive

Ultrasonic scalerUse metal curettes

Fig. 16 Mechanical debridement. Fig. 17 Laser treatment for peri-implantitis.

16[21] [22] [23] [24] [25] [26] [27]

PROBLEM

17

18

PROBLEM

PREVIOUS STUDIES:

[28] [29] [30] [31]

PROBLEM: Mechanical debridement with acidic chemicals inflicted severe corrosion.

• Cytotoxicity of CA was studied & a significant decrease in cell proliferation.• Damaging effect on surface of implant surfaces.• Chlorhexidine (CHX) - studies revealed CHX inhibits cell proliferation.• Human studies showed that the combined effect of mechanical scrubbing with saline

soaked curettes on peri-implantitis-infected implants resulted stable implants upto 24 months.

.Variation & inconsistencies in literature that study this treatment method.

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Peri-implantitis infected dental implant treated with mechanical debridement with acidic chemicals.

Mechanical abrasion of implant surface

Use of low pH chemicals

Corrosion?

Re-osseointegration?

How does bacterial adhesion change surface?

Bacterial adhesion

PROBLEM

SPECIFIC AIMS

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AIMS

AIM 3: To evaluate bone forming cell activity on cpTi surfaces post-detoxification treatment.

AIM 2: To carry out detoxification/ debridement of cpTi by immersion and rubbing method using chemicals typically employed in the clinical setting.

AIM 1: To contaminate the surface of titanium (cpTi) with a polyclonal culture of peri-implantitis inducing bacteria for biofilm development.

21

AIM 1

22

AIM 1

HYPOTHESIS 1: Immersion of titanium surface in polyclonal bacterial strain culture will lead to biofilm growth on sample surfaces. Bacterial adhesion will create an acidic environment due to production of lactic acid, which will result in surface oxidation.

AIM 1: To contaminate the surface of cpTi disks with a polyclonal culture of peri-implantitis inducing bacteria for biofilm development.

23

cpTi sample preparation

Polyclonal bacterial culture preparation:

S. mutans, S. sanguinis, S. salivarius & Aa

Immersion of cpTi in bacteria

5-day immersion

Retrieve cpTi samples

Surface analysis (SEM & OM)

AIM 1 – METHODOLOGY

24

AIM 1 – RESULTS

After 5 days of immersion a thin white bacterial film was visible on sample surfaces.

Fig 18. Digital image of immersed cpTi samples in BHI medium with S. mutans, S. salivarius, S. sanguinis, Aa bacteria. (A) Day 1 of immersion; (B) Day 5 of immersion.

Fig 19. A contaminated cpTi sample with a visible white biofilm on the surface.

25

AIM 1 – RESULTS

Fig 20. SEM image of uncontaminated sample (A), contaminated with biofilm on surface (B), contaminated and biofilm removed (C).

Fig 21. OM image of uncontaminated sample (A), contaminated with biofilm intact (B), contaminated with biofilm removed (C).

• Clusters of bacteria adhered to the specimen surface, which was seen like a film covering the surface.

• A pit-like feature was observed and is indicated by the yellow arrow in the figure.

• Severe discoloration (yellow and blue) was observed around bacteria clusters on the surface.

• More so after the biofilm was removed as indicated by the yellow arrows.

26

AIM 1 – SUMMARY

HYPOTHESIS 1: Immersion of titanium surface in polyclonal bacterial strain culture will lead to biofilm growth on sample surfaces. Bacterial adhesion will create an acidic environment due to production of lactic acid, which will result in surface oxidation.

Electrochemical attack on Ti oxide layer = oxidation (corrosion)

When oxidized = T³⁺ (purple color) T²⁺ (yellow color)

Low pH

Release of acidic

metabolites

Non-uniform biofilm created

O₂ depletion zones

Discoloration & pitting27[20]

AIM 2

28

AIM 2

HYPOTHESIS 2: Low pH of chemical agents used in detoxification procedures will create an acidic environment, which will cause oxidation, discoloration and pitting of titanium surfaces.

AIM 2: To carry out detoxification/ debridement of cpTi by immersion and rubbing method using chemicals typically employed in the clinical setting.

29

Immersion

Rubbing

cpTi samples Preparation of chemicals:• Citric acid (30%)• Saline (0.9%)• Chlorhexidine ( 0.1%)• Doxycycline (50:50)

Surface analysis (SEM & OM)

AIM 2 – METHODOLOGY

30

AIM 2 - RESULTS

RUBBING METHOD

• Citric acid, inflicted the most significant damage to surface compared to the other chemicals investigated.

• Doxycycline showed mostly minor pitting and no discoloration.

• Chlorhexidine generated discoloration.

• Saline did not change morphology.

Control Citric Acid Saline Doxycycline Chlorhexidine SEM

O

M

Control Citric Acid Saline Doxycycline Chlorhexidine Fig. 22 SEM and OM images of cpTi samples treated by rubbing method.

31

Chemical pHCitric Acid (30%) 1.74

Doxycycline (50:50) 2.74

Chlorhexidine (0.1%) 7.38

Saline (0.9%) 7.44

AIM 2 - RESULTS

IMMERSION METHOD

• Immersion in saline inflicted no negative impact on the surface of titanium.

• Citric acid showed discoloration (indicated by yellow arrows) within cracks present on the surface.

• Doxycycline, resulted in a significant amount of residue left on the sample surface.

• Chlorhexidine created minor discoloration.

Control Citric Acid Saline Doxycycline Chlorhexidine

Control Citric Acid Saline Doxycycline Chlorhexidine

SEM

O

M

Fig. 23 SEM and OM images of cpTi samples treated by immersion method.

32

AIM 2 - SUMMARY

Chemical pH

Citric Acid (30%) 1.74

Doxycycline (50:50) 2.74

Chlorhexidine (0.1%) 7.38

Saline (0.9%) 7.44

HYPOTHESIS 2: Low pH of chemical agents used in detoxification procedures will create an acidic environment, which will cause oxidation, discoloration and pitting of titanium surfaces.

Low pH of chemicals

(citric acid & doxycycline)

Mechanical cyclic force

(rubbing method)

Electrochemical attack on Ti oxide layer = oxidation (corrosion)

When oxidized = T³⁺ (purple color) T²⁺ (yellow color)

Damage to Ti-oxide

layer

Discoloration & pitting

33

AIM 3

34

AIM 3

HYPOTHESIS 3: Acidic chemical agents used in detoxification methods will lead to significant changes in the surface oxide layer decreasing cell proliferation and differentiation of pre-osteoblast cells on specimen surfaces.

AIM 3: To evaluate bone forming cell activity on cpTi surfaces post detoxification treatment.

35

Pre-osteoblast (MC3T3-E1) cell

growth cpTi placed in 24-

welled plateSeeded MC3T3-E1

cells onto each sample (0.05 x 106

cells/well )

Media7-day study

MTT Assay

ALP Assay & Staining

AIM 3 – METHODOLOGY

36

MTT Assay

ALP Assay & Staining

• Colorimetric assay for assessing cell metabolic activity.• For measurement of cell viability and proliferation.

• Colorimetric assay designed to measure alkaline phosphatase.

• High ALP activity = differentiation of pre-osteoblasts to osteoblasts.

AIM 3 – METHODOLOGY

Fig. 24 Progression of bone-forming cells.

37[32] [33]

AIM 3 - RESULTS

MTT ASSAY – Cell viability/proliferation

Fig 25. Cell Viability of pre-osteoblasts on samples treated by rubbing and immersion methods. Control is non treated disks (n=3).

• Overall cell viability for samples that experienced abrasion (rubbing), was lower than on samples that did not experience abrasion (immersion) with the exception of citric acid.

• Significant difference in cell viability between rubbing and immersion methods when treated with doxycycline (p<0.05) & citric acid (p<0.05).

38

Citric Acid (30%) Doxycycline (50:50) Saline (0.9%) Chlorhxidine (0.12%)0

20

40

60

80

100

120

140

160

180 Rubbing Immersion Non treated disks

Cell v

iabi

lity

(%)

*

*

*

*

*

AIM 3 - RESULTS

ALP ASSAY- differentiation• On average, higher ALP activity was seen on cpTi specimens that experienced

mechanical abrasion (rubbing) than with samples that were immersed.

• No statistical difference (p>0.05) was found for ALP activity between rubbing and immersion methods, nor between chemicals used for the two treatment methods.

Fig. 26 ALP activity of pre-osteoblasts on samples treated by rubbing and immersion methods. Control is non treated disks (n=3).39

AIM 3 - RESULTS

ALP STAINING

• All test specimen surfaces were stained to detect ALP enzyme and the differentiated cells were identified by the purple color of the stain.

• Samples treated via rubbing method seemed to have a lot more differentiated cells attached to the surface compared to immersion-treated samples.

Control 1 Doxycycline Chlorhexidine

Rubb

ing

Imm

ersio

n

Saline Citric Acid

Control 2 Doxycycline Chlorhexidine

Saline

Citric Acid

Fig. 27 ALP staining of pre-osteoblasts on treated samples. 40

HYPOTHESIS 3: Acidic chemical agents used as detoxification methods will lead to significant changes in the surface oxide layer decreasing cell proliferation and differentiation of pre-osteoblast cells on specimen surfaces.

AIM 3 - SUMMARY

41

Mechanical abrasion

(Rubbing)Chemicals

Low proliferation

Higher differentiation

No mechanical abrasion

(Immersion)Chemicals

High proliferation

Lower differentiation

Chemicals + mechanical force had a considerable consequence on the proliferation & differentiation of pre-osteoblasts.

CONCLUSION

42

AIM 1: Bacterial Contamination• Bacterial adhesion on titanium surface inflected severe discoloration and

pitting.

AIM 2: Detoxification• Manual rubbing + acidic chemicals exacerbated oxidation = discoloration.• The most damaging treatment found in this study was rubbing & immersion

with citric acid, and the least damaging was immersion with saline.

AIM 3: Cell Compatibility • Combination of manual application of force (rubbing method) and acidic

chemicals resulted in low proliferation rates which indicated cytotoxicity of treated titanium surfaces to pre-osteoblasts.

CONCLUSION

43

Mechanical debridement + irrigation

44

CONCLUSION

Overall observation:

Rubbing + citric acid

= initially low proliferation ( < 70%) = cytotoxicity

= ultimately higher differentiation ( acceptable value)Re-osseointegration?

• If peri-implantitis is not controlled and treated = corrosion of implant surface.

• Coupled with mechanical debridement exacerbates corrosion (discoloration & pitting).

• Saline provided high proliferation & differentiation for both rubbing & immersion

• Based on these results most effective treatment suggestion:

SalineCitric acid

FUTURE WORK

45

The work presented in this research study is a new approach developed to study the synergistic action of bacterial adhesion, detoxification and growth of bone-forming cells.

This experimental setup has the versatility to accommodate different dental implant material as well as different peri-implantitis treatment methods.

ZrO₂ TiZr alloy Ti6Al4V alloy

Include different types of dental implants available in the market.

FUTURE WORK

46

REFERENCES

47

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[1 ] http://www.aaid.com/about/Press_Room/Dental_Implants_FAQ.html.[2] Mellado-Valero, A.; Buitrago-Vera, P.; Sola-Ruiz, M.F; Ferrer-Garcia, J.C. Decontamination of Dental Implant Surface in Peri-Implantitis Treatment: A Literature Review. Med. Oral Patol. Oral Cir. Bucal 2013, 18 (6), e869-e876[3] http://www.lavondental.com/services/dental-implants/[4] Sakka, Salah, Kusai Baroudi, and Mohammad Zakaria Nassani. "Factors associated with early and late failure of dental implants." Journal of investigative and clinical dentistry 3.4 (2012): 258-261.[5] http://www.deardoctor.com/articles/peri-implantitis-can-cause-implant-failure/[6] Bain (2001); www.aaid-implant.org.pdf; Lang et. al (2004); Bain (2001)[7] http://www.intechopen.com/books/biomaterials-science-and-engineering/magnetoelectropolished-titanium-biomaterial[8] http://www.dentalimplantcostguide.com/types-of-dental-implants/[9] http://www.whitehousedental.com.au/what-we-do/dental-implants/[10] http://www.avonortho.com/patient/oral-hygiene[11] http://www.osseointegration.eu/osseointegration/[12] http://pocketdentistry.com/15-dental-implants/[13] http://www.dentaleconomics.com/articles/print/volume-104/issue-10/features/implant-complications-multiple-treatment-modalities-few-financial-options.html[14] http://www.oxygenmedical.no/replacing-missing-teeth-dental-implants/[15] http://www.perioexpertise.com/en/peri-implantitis[16] Mombelli a. Microbiology of dental implant. Adv Dent Res 1993;7:202-6. [17] http://jdr.sagepub.com/content/88/11/982/F1.expansion.html[18] https://commons.wikimedia.org/wiki/File:Periimplantitis_due_to_dental_cement.gif[19] http://www.dentaleconomics.com/articles/print/volume-104/issue-10/features/implant-complications-multiple-treatment-modalities-few-financial-options.html

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[20] Sridhar, S.; Wilson, T. G.; Palmer, K. L.; Valderrama, P.; Mathew, M. T.; Prasad, S.; Jacobs, M.; Gindri, I. M.; Rodrigues, D. C. In Vitro Investigation of the Effect of Oral Bacteria in the Surface Oxidation of Dental Implants. Clin. Implant Dent. Relat. Res. 2015, n/a – n/a.[21] http://periobasics.com/plaque-as-biofilm-and-ecological-plaque-hypothesis.html[22] Subramani, K. & Wismeijer, D. (2012) Decontamination of titanium implant surface and re-osseointegration to treat peri-implantitis: a literature review. International Journal of Oral Maxillofacial Implants 27: 1043–1054.[23] Renvert, S., Polyzois, I. & Maguire, R. (2009) Re-osseointegration on previously contaminated surfaces: a systematic review. Clinical Oral Implants Research 4: 216–227.[24] Tastepe, C.S., Liu, Y., Visscher, C.M. & Wismeijer, D. (2012) Cleaning and modification of intraorally contaminated titanium discs with calcium phosphate powder abrasive treatment. Clinical Oral Implants Research 24: 1238–1246.[25] Kamel, M.S., Khosa, A., Tawse-Smith, A. & Leichter, J. (2013) The use of laser therapy for dental implant surface decontamination: a narrative review of in vitro studies. Lasers in Medical Science 29: 1977– 1985[26] http://clairemccarthy.co/dental-implants-to-probe-or-not-to-probe/[27] http://www.lasermarket.co.uk/laser-suppliers/fotona/fotona-lightwalker-dental-laser/[28] L. F. Guimarães, T. K. Fidalgo, G. C. Menezes, L. G. Primo, and F. Costa e Silva-Filho, “Effects of citric acid on cultured human osteoblastic cells,” Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, vol. 110, no. 5, pp. 665–669, 2010. [29] T.-H. Lee, C.-C. Hu, S.-S. Lee, M.-Y. Chou, and Y.-C. Chang, “Cytotoxicity of chlorhexidine on human osteoblastic cells is related to intracellular glutathione levels,” International Endodontic Journal, vol. 43, no. 5, pp. 430–435, 2010. [30] F. Schwarz, N. Sahm, and J. Becker, “Combined surgical therapy of advanced peri-implantitis lesions with concomitant soft tissue volume augmentation. A case series,” Clinical Oral Implants Research, 2013. [31] F. Schwarz, G. John, S. Mainusch, N. Sahm, and J. Becker, “Combined surgical therapy of peri-implantitis evaluating two methods of surface debridement and decontamination. A two-year clinical follow up report,” Journal of Clinical Periodontology, vol. 39, no. 8, pp. 789–797, 2012. [32] https://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx[33] http://www.promocell.com/fileadmin/knowledgebase/pdf-xls/Osteoblast_Differentiation_and_Mineralization.pdf

REFERENCES

Acknowledgements

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Bone Lab:

Sathya SridharIzabelle de Mello GrindiPavan Preet KaurShant AghyarianJuli SabaDanyal SiddiquiGina QuiramVidya Jayaraman

Lucas RodriguezSutton WheelisJonathan ChariElizabeth BentleyFrederick WangJason ChangThao Hoang

Committee Members:Dr. Danieli C. RodriguesDr. Kelli PalmerDr. Taylor Ware

Collaborators/mentorsDr. Kelli Palmer & LabDr. Heather Hayenga & LabDr. Pilar ValderramaDr. Thomas G. Wilson Jr

51