Bone Structure and Formation

Bone structure and formation: A new perspective

Matthew J. Olszta a,1, Xingguo Cheng a,b, Sang Soo Jee a, Rajendra Kumar a,c,Yi-Yeoun Kim a,d, Michael J. Kaufman e, Elliot P. Douglas a, Laurie B. Gower a,*

a Department of Materials Science and Engineering, University of Florida, Gainesville, FL 32611, USAb Center for Biomaterials, Department of Oral Rehabilitation, Biomaterials and Skeletal Development,

University of Connecticut Health Center, Farmington, CT 06030, USAc Department of Environmental Engineering (BioEngineering Initiative), Montana Tech of the University of Montana, Butte, MT 59701, USA

d Discovery Research, Specialty Minerals, Inc., Bethlehem, PA 18017, USAe Department of Materials Science and Engineering, University of North Texas, Denton, TX 76203, USA

Available online 20 July 2007

Abstract

Bone is a hierarchically structured composite material which, in addition to its obvious biological value, has been well studied by

the materials engineering community because of its unique structure and mechanical properties. This article will review the existing

bone literature, with emphasis on the prevailing theories regarding bone formation and structure, which lay the groundwork for

proposing a new model to explain how intrafibrillar mineralization of collagen can be achieved during bone formation. Intrafibrillar

refers to the fact that growth of the mineral phase is somehow directed by the collagen matrix, which leads to a nanostructured

architecture consisting of uniaxially oriented nanocrystals of hydroxyapatite embedded within and roughly [0 0 1] aligned parallel

to the long collagen fibril axes. Secondary (osteonal) bone, the focus of this review, is a laminated organicinorganic composite

composed primarily of collagen, hydroxyapatite, and water; but minor constituents, such as non-collagenous proteins (NCPs), are

also present and are thought to play an important role in bone formation. To date, there has been no clear understanding of the role of

these NCPs, although it has been generally assumed that the NCPs regulate solution crystal growth via some type of epitaxial

relationship between specific crystallographic faces and specific protein conformers. Indeed, epitaxial relationships have been

calculated; but in practice, it has not been demonstrated that intrafibrillar mineralization can be accomplished via this route. Because

of the difficulty in examining biomineralization processes in vivo, the authors of this article have turned to using in vitro model

systems to investigate the possible physicochemical mechanisms that may be involved in biomineralization.

In the case of bone biomineral, we have now been able to duplicate the most fundamental level of bone structure, the

interpenetrating nanostructured architecture, using relatively simple anionic polypeptides that mimic the polyanionic character of

the NCPs. We propose that the charged polymer acts as a process-directing agent, by which the conventional solution crystallization

is converted into a precursor process. This polymer-induced liquid-precursor (PILP) process generates an amorphous liquid-phase

mineral precursor to hydroxyapatite which facilitates intrafibrillar mineralization of type-I collagen because the fluidic character of

the amorphous precursor phase enables it to be drawn into the nanoscopic gaps and grooves of collagen fibrils by capillary action.

The precursor then solidifies and crystallizes upon loss of hydration waters into the more thermodynamically stable phase,

leaving the collagen fibrils embedded with nanoscopic hydroxyapatite (HA) crystals. Electron diffraction patterns of the highly

mineralized collagen fibrils are nearly identical to those of natural bone, indicating that the HA crystallites are preferentially aligned

with [0 0 1] orientation along the collagen fibril axes. In addition, studies of etched samples of natural bone and our mineralized

collagen suggest that the long accepted deck of cards model of bones nanostructured architecture is not entirely accurate.

www.elsevier.com/locate/mser

Materials Science and Engineering R 58 (2007) 77116

* Corresponding author. Tel.: +1 352 846 3336; fax: +1 352 846 3355.

E-mail address: [email protected] (L.B. Gower).1 Present address: Department of Materials Science and Engineering, Penn State University, State College, PA 16802, USA.

0927-796X/$ see front matter # 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.mser.2007.05.001

Most importantly, this in vitro model demonstrates that a highly specific, epitaxial-type interaction with NCPs is not needed to

stimulate crystal nucleation and regulate crystal orientation, as has long been assumed. Instead, we propose that collagen is the

primary template for crystal organization, but with the important caveat that this templating occurs only for crystals formed from an

infiltrated amorphous precursor. These results suggest that the 25-year-old debate regarding bone formation via an amorphous

precursor phase needs to be revisited.

From a biomedical perspective, in addition to providing possible insight into the role of NCPs in bone formation, this in vitro

system may pave the way toward the ultimate goal of fabricating a synthetic bone substitute that not only has a composition similar

to bone, but has comparable mechanical properties and bioresorptive potential as natural bone. From a materials chemistry

perspective, the non-specificity of the PILP process and capillary infiltration mechanism suggests that non-biological materials

could also be fabricated into nanostructured composites using this biomimetic strategy.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Secondary bone formation; Biomineralization; Hydroxyapatite; Amorphous calcium phosphate; Biomimetic

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

1.1. Primary versus secondary bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

1.2. Nanostructured architecture of secondary bone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

1.2.1. Collagen matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

1.2.2. Mineral phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

1.2.3. Intrafibrillar mineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

1.3. Mechanism of bone formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

1.4. Synthetic attempts at mimicking bone formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

1.5. Polymer-induced liquid-precursor (PILP) mineralization process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

1.6. New hypothesis on the mechanism of bone formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

2. Methods and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

2.1. Mineralization of collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

2.2. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

2.2.1. Scanning electron microscopy (SEM) analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

2.2.2. Transmission electron microscopy (TEM) analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

2.2.3. X-ray diffraction (XRD) analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

2.3. Confocal microscopy study of polymer penetration depth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

2.3.1. Preparation of fluorescently tagged poly(aspartate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

2.3.2. Turkey tendon preparation and mineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

2.3.3. Confocal microscopy analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

3.1. Intrafibrillar mineralization of collagen with a CaP PILP process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

3.2. X-ray diffraction (XRD) studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

3.3. Electron diffraction analysismineral identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

3.4. Electron diffraction analysiscrystallographic orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

3.5. SEM analysis of crystal morphology and organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

3.6. Confocal studies on mechanism of mineral infiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

4.1. Summary of mineralization results using the PILP in vitro model system . . . . . . . . . . . . . . . . . . . . . . . . 100

4.2. Amorphous to crystalline transformation: comparison to existing literature . . . . . . . . . . . . . . . . . . . . . . . 101

4.3. Clarification of crystal orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

4.4. Elucidation of mineral morphology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

4.5. Mechanism of alignment of intrafibrillar crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

4.6. Mechanism of mineral penetration and implications for application . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

M.J. Olszta et al. / Materials Science and Engineering R 58 (2007) 7711678

1. Introduction

Bone is a bioceramic composite that has long held the attention of the materials engineer who seeks to duplicate its

enviable mechanical properties, in which both high strength and fracture toughness can be achieved due to the unique

architecture of this organicinorganic composite. This article will not discuss bones mechanical properties, for which

there are many excellent reviews [19] as well as recent contributions [1017]; but instead it will focus on the unique

structure that confers these properties, and to the materials chemistry underlying its formation.

1.1. Primary versus secondary bone

When discussing the structure and formation of bone, one first needs to understand that there are two main stages of

bone formation, referred to as primary and secondary osteogenesis (or synonymously as ossification), and that the

mechanism of bone formation differs substantially in these two stages [18,19]. Epiphysial cartilage, which serves as

the locus for primary bone formation (i.e., endochondral ossification), is a combination of ground substance and very

loose, small (1020 nm in diameter) fibril bundles of collagen [19,20]. There is also a high occurrence of matrix

vesicles, which are believed to deliver either crystals or a high concentration of ions to the mineralization front

[2125]. The mineralization is relatively rapid and unorganized, forming a woven bone microstructure. Although

type-I collagen is present in this ground substance (17% in rat cartilage), it is not organized into lamellae, andcrystals do not form in close association with the collagen. Instead, clusters of hydroxyapatite form within the

proteoglycan matrix [26], which are referred to as calcification nodules [27] or calcospherites [28], due to the

spherulitic arrangement of the crystal clusters. Cameron [20] suggested that the collagen fibrils found in cartilage

are too narrow for the mineral to deposit within them, thereby resulting in the observed extrafibrillar mineralization. In

this instance, the collagen does not appear to play an appreciable role in directing the mineralization process, and

therefore this type of bone formation is not the focus of this paper.

In secondary bone formation, the primary woven bone is remodeled into a more optimal structure, such as parallel-

fibered or lamellar bone, which in the case of humans is organized into concentric lamellae that make up the osteons of

the Haversian canal system [29]. When discussing the extraordinary structure of bone, people are usually referring to

secondary bone, which is often described in terms of its hierarchical levels of structure [30]. An excellent review was

provided byWeiner et al. [5], who broke down the structure of bone into seven levels of hierarchy (Fig. 1), starting with

nanoscopic platelets of hydroxyapatite (HA) that are oriented and aligned within self-assembled collagen fibrils; the

collagen fibrils are layered in parallel arrangement within lamellae; the lamellae are arranged concentrically around

blood vessels to form osteons; finally, the osteons are either packed densely into compact bone or comprise a

trabecular network of microporous bone, referred to as spongy or cancellous bone.

The collagen fibrils in secondary bone are secreted by osteoblasts and are larger than those in primary bone, with a

mean diameter of 78 nm [27], and they assemble into a highly organized, close-packed lamellar structure. Close to the

mineralization front, there are also non-collagenous proteins (NCPs), many of which are highly charged from an

abundance of carboxylate groups from aspartic and glutamic acid residues, as well as phosphate from phosphoserine

[3136]. Although in low concentration, these NCPs are also thought to play an important role in the mineralization

process [18,34,3638]. During secondary bone formation, the organization of the crystals is directed by the collagen

fibrils within which they form [37]. This leads to intrafibrillar crystals that are extremely small (only a few unit cells

thick [3942]), which would not normally be thermodynamically stable if it were not for being embedded within the

organic matrix. Crystals may also form on the surface and between collagen fibers, which are referred to as

interfibrillar crystals. According to Martin et al. [43], about 58% of the mineral in canine whole bone is intrafibrillar,

14% interfibrillar, and 28% from within the gaps between the ends of collagen fibrils.

The high degree of mineral loading that is achieved by intrafibrillar mineralization leads to a biocomposite with a

composition of around 65 wt.% mineral phase, 25 wt.% organic, and 10 wt.% water [3,11,18,34,39,43,44]. By

comparison, because bone is such a unique composite, containing a collagenous hydrogel matrix, on a volumetric

basis it consists of about 3343% apatite minerals, 3244% organics, and 1525% water [1]. Even though water is a

minor constituent, its significance should not be overlooked, because it contributes to the overall toughness of the

biocomposite, acting something like a plasticizer [45]. On the other hand, the non-collagenous proteins, which

comprise only 1015% of the organic matrix [18], may be less important in terms of bones mechanical properties, but

apparently play a crucial role in the formation of bone structure.

M.J. Olszta et al. / Materials Science and Engineering R 58 (2007) 77116 79

1.2. Nanostructured architecture of secondary bone

It should be kept in mind that the higher levels of bone structure are species dependent, and there is even significant

variation between bone types of a single organism. It is the nanostructural level of organization that lies at the foundation

of all types of secondary bone (e.g., parallel-fibered, lamellar, fibrolamellar, trabecular, osteonal). Therefore, in order to

truly understand bones exceptional properties, it is necessary to examine its most basic level of organization, the

nanostructured array of hydroxyapatite (HA) crystals embedded within the collagenmatrix (level 2 of Fig. 1). This level


Fig. 1. The hierarchical levels of structure found in secondary osteonal bone, as demonstrated by Weiner and Wagner [5] (reprinted from [5], with

permission from Annual Reviews, www.annualreviews.org).

of structure is created by intrafibrillar mineralization of collagen, and it is the intimate relationship between the self-

assembled fibrillar collagen matrix and the uniaxially oriented, nanometer sized, platy HA crystals, that provides bone

with its remarkable mechanical properties and remodeling capabilities. From a materials science perspective, the

interpenetrating nature of the organicinorganic phases makes it difficult to classify bone as one specific type of

composite. For example, the high degree of loading ofmineral phasewhich encases the collagenfibrils suggests that bone

is a fiber-reinforced ceramicmatrix composite. In sharp contrast to this observation is the fact that ultrastructural

examination of deproteinated bone reveals individual 2550-nm-wideHAcrystals [5,4648], implying that bonemay be

better described as a nanoparticle-reinforced polymermatrix composite. It is this nanostructured architecture that is the

essence of bone, both in terms of mechanical properties and bioresorbability.

1.2.1. Collagen matrix

Only through tedious diffraction analysis and innovative microscopy techniques have researchers been able to

determine the intimate relationship between the HA platelets and collagen fibrils. Before describing the complex

composite structure of bone, the organization of the organic matrix must first be defined. The structure of collagen

alone took many years to resolve, and various permutations of Hodge and Petruskas quarter-stagger model are largely

accepted as providing a reasonable description of the fibrillar organization [4953]. The repetitive nature of the amino

acid sequences of collagen, which consists of (Gly-X-Y-)n, where X and Y are frequently proline and

hydroxyproline residues, allows the protein to assemble into triple helical structures referred to as tropocollagen

molecules [54]. Secondary bonding interactions between tropocollagen units leads to self-organization into fibrillar

structures (see schematic in Fig. 2). Type-I collagen, the primary constituent of secondary bone tissues, assembles its

tropocollagen units in a quarter-staggered array, which leads to hole and overlap zones that can be seen as a periodic


Fig. 2. Schematic by Landis et al. [57] illustrating the mineralization of turkey tendon, based on ex situ observations using high-voltage TEM and

tomographic reconstruction imaging. The cylindrical rods represent tropocollagen units composed of triple helical collagen molecules that assemble

into fibrils in a quarter-staggered fashion, which leaves periodic gaps and grooves within the fibrils. The 67 nm repeat (64 nm when dehydrated)

results from the combination of a 40 nm hole zone and 27 nm overlap zone. The space between assembled tropocollagen units is 0.24 nm. Upon

mineralization, the more electron dense mineral can be seen as striations within the collagen fibrils, and the crystals are oriented such that the

crystallographic c-axes lie parallel to the long axes of the molecules and their (1 0 0) planes are all approximately parallel to each other. Irregularly

shaped, large and small mineral deposits may occupy several hole zones, and preferential growth in the c-axial length follows the long axis of the

collagen (reprinted from [57] (Journal of Structural Biology), with permission from Elsevier).

banding pattern when stained for observation by transmission electron microscopy (TEM) (e.g., the banding seen in

levels 13 of Fig. 1).

On the other hand, Hansma and co-workers have recently shown by atomic force microscopy (AFM) of collagen

that there are grooves on the surfaces of the fibrils with a 37 nm height corrugation, which is not consistent with the

1.6 nm overlap mismatch predicted by the Petruska and Hodge model. They suggest, therefore, that the periodic

banding pattern seen in TEM may result from differential deposition of stain in the grooves versus peaks of the fibrils

[55]. This group has additionally shown that the collagen fibrils bend in a tube-like fashion, suggesting that the

composition is not homogeneous laterally across the fibrils, but rather has a relatively hard shell with a softer core [56].

The quarter-stagger arrangement leaves a regular array of 40 nm gaps within each periodic unit, and these are

reportedly [57,58] the locations where crystal nuclei are first observed in systems such as naturally mineralizing turkey

tendon (Fig. 2). Turkey tendon has served as a useful model of bone formation because it mineralizes naturally, but

does not remodel from primary bone. Instead, parallel fibers of collagen mineralize in an intrafibrillar fashion that

appears to be similar to secondary bone formation [57,58]. A variety of modifications to this quarter-staggered model

have also been proposed, such as the alignment of gaps to form grooves, which are proposed to help account for the

fact that the dimensions of the HA crystals extracted from bone are larger than the dimensions of the gap zones where

they are thought to form. The rod-like character of the cylindrical tropocollagen units imparts collagen with liquid

crystalline character [59], which may play a role in vivo in the formation of tissues with complex structural order [60],

such as the gradual splay in collagen (and thus intrafibrillar crystal) orientation across lamellae in osteonal bone (e.g.,

level 4 of Fig. 1 [60]).

1.2.2. Mineral phase

The size of bone crystals reported in the literature varies, with values ranging from length, 3050 nm; width, 15

30 nm; thickness, 210 nm [18,42,48,53,61,62]. Recent AFM studies find that the bone crystals are longer than those

observed by TEM, with widths and lengths ranging from 30 to 200 nm [46]. They suggest that this discrepancy is due

to breakage that occurs during dispersion for TEM sample preparation. While this variability is in part due to sample

preparation, it is likely also a function of the type of mineralized tissue (e.g., animal species, maturation, and location

of the tissue examined). For example, in Fig. 3, even though the individual platelets cannot be discerned with scanning

electron microscopy (SEM), the outward appearance of the bone texture differs for samples from dog (Fig. 3A),

turkey, (Fig. 3B) horse (Fig. 3C), and human bone (Fig. 3D). The inability to resolve the crystallites could also be due

to an extrafibrillar organic matrix (presumably proteoglycans or non-collagenous proteins), which has been observed

by AFM [46]. Regardless of these variations, it is important to realize that the crystals clearly outgrow the dimensions

of the gap zones in the fibrils.

Not only are bone crystallites extremely small, they are often described as poorly crystalline because of the

broad X-ray diffraction peaks (relative to synthetic HA), which is thought to arise from the incorporation of impurities,

such as carbonate, sodium and magnesium ions (46% carbonate; 0.9% Na; 0.5% Mg) [18,63,64], and non-

stoichiometry of the biogenic mineral. The carbonated form of apatite has the mineral name of Dahllite, which is

sometimes used in the bone literature [5,18,65], but more commonly biological apatite is referred to hydroxyapatite.

Bone mineral is a calcium-deficient apatite, with a Ca:P ratio less than 1.67, which is the theoretical value for pure

hydroxyapatite, Ca5(PO4)3(OH) [39,64]. Because bone is a living tissue that is continuously undergoing remodeling

and repair, the small size and/or non-stoichiometry of the crystals presumably bestows the mineral phase with the

solubility needed for resorption of the bone by osteoclasts (bone resorbing cells). For example, bone substitutes made

of synthetic HA, although bioactive (stimulatory for bone formation), are rather slow to resorb due to the low solubility

of HA under physiological conditions [39,66,67].

1.2.3. Intrafibrillar mineralization

In secondary bone formation (and dentin), collagen directs the mineral growth such that platelets of HA grow in the

[0 0 1] direction along the long-axis of the fibril (Fig. 4A), as demonstrated by orientation of the (0 0 2) and (0 0 4)

planes parallel to this axis in selected area electron diffraction (SAED) patterns of fibrils collected from native bone

(Fig. 4B). This example demonstrates the unique structure created by intrafibrillar mineralization, in which the HA

crystallites are embedded within the collagen fibrils. In addition to the uniaxial orientation, platelets are often

illustrated as being coherently aligned in the ab plane, stacked in parallel arrays like a deck of cards, as described by

Traub and co-workers [53]. Although this model of bone structure is generally well accepted and highly cited by


researchers within the biomedical community, it is not an entirely accurate representation of the nano structured

architecture of bone because the HA crystals do not have biaxial order (as discussed in Section 3.4).

1.3. Mechanism of bone formation

While the structure of bone is reasonably well defined, its formation remains an enigma. For example, although

collagen can be reconstituted into the native fibrillar structure, in vitro crystallization studies have not been able to

duplicate even bones most fundamental level of nanostructure. There have been several ex vivo studies examining the

early stages of mineralization, both in bone and in naturally mineralizing turkey tendon [27,6875]. Most of these

studies took place in the 1960s and 1970s, when the mechanism of bone formation was hotly debated. One group of

researchers argued that the HA crystals were formed via the traditional solution crystallization process (i.e., nucleation

and growth), while a handful of others pointed to the deposition of an amorphous calcium phosphate (ACP) precursor.

To alleviate this discrepancy in observations, it was also suggested that the amorphous substance observed in early

mineralizing tissues by some researchers is actually paracrystalline mineral (i.e., a loss of long-range crystalline

order as a result of lattice imperfections) [76], which might be undetectable by conventional analytical techniques

[7779]. Various metastable crystalline phases (e.g., octacalcium phosphate (OCP) [80] and brushite [81]) have also

been implicated as transitory precursors to HA in bone and teeth formation. The spectroscopic evidence also shows a

band at 945 cm1 that is attributed to a highly disordered structure, which becomes less prominent as the mineral ages.It is assumed that intermediate phases such as OCP or TCP could precipitate along the pathway to the most


Fig. 3. Comparison of bone textures when viewed by scanning electron microscopy (SEM). (A) Reprint from Bagambisa et al. [136] showing the

resting bone surface in a sample of whole bone from dog femur diaphysis. Diagonally from upper left to lower right, the collagen fibers form a

bundle in which mineral is densely invested, sometimes causing bulging and clubbing of the fibers. (reprinted from [136] (Cells and Materials 20

(1)), with permission from the authors). (B) SEM of as-fractured turkey bone. (C) SEM of as-fractured equine bone. (D) SEM of cortical cancellous

chips of human cadaver bone (obtained from Regeneration Technologies Inc.).

thermodynamically stable phase of hydroxyapatite, but the central question of this report is whether that pathway

starts with an amorphous phase, and the structural consequences of such a mechanism.

The ACP theory was first proposed by Posner, Termine and their co-workers [70,72,8284], in which they argued

that the two-phase nature of bone mineral may prove to be a valuable aid in understanding the operative mechanisms

of bone metabolism and the molecular basis of bone structure [82]. In their studies, the measurements of crystalline

fraction, as determined by X-ray diffraction peak widths, as well as infrared analysis peak splitting, were made during

different stages of development of chick, rat, and cow bones. In later years, Bonnuci also described an inorganic

substance in bands when observing early stage bone formation by TEM [37], in which a more electron dense material

is seen primarily within the hole zones of collagen, which subsequently becomes needle-like as it transforms into

hydroxyapatite. For the most part, this debate over bone formation was eventually quelled by the landmark paper of

Glimcher et al. [81], entitled Recent studies of bone-mineralis the amorphous calcium-phosphate theory valid?

This group examined bone from the rapid turnover stage of 17-day-old chick embryos, and found intermediate range

order of up to 25 A in the radial distribution function analysis of XRD data (as compared to 10 A for a synthetic ACP).


Fig. 4. Electronmicrographs of equine cortical bone. (A) TEM brightfield image demonstrating intrafibrillar mineralization of type-I collagen fibrils

in natural bone. The native banding pattern of type-I collagen can be observed due to the infiltration of electron dense mineral, and staining is not

needed. The striated appearance results from hydroxyapatite platelets aligned parallel to the long axis of the collagen. Bar = 100 nm. (B) Selected

area electron diffraction (SAED) of a single fibril of crushed equine bone. The arcing of the (0 0 2) and (0 0 4) planes, which are parallel to the long

axis of the collagen fibrils (white arrow) is characteristic of bone. The (1 1 2), (2 1 1) and (3 0 0) planes, indexed using d-spacings and angles relative

to the (0 0 2) plane, form 3 arcs which nearly overlap, combining into what appears to be a ring; however, there is a gap in the ring just behind the

(0 0 2) arc because it is not really a powder ring, but three distinct sets of planes which have very close d-spacings. The appearance of these three

planes simultaneously indicates that there is more than one orientation of the HA platelets in the ab plane. (C) TEM brightfield image of an isolated

collagen fibril showing the characteristic banding pattern of type-I collagen. The SAED pattern (inset) of this fibril demonstrates that the fibril does

not diffract, suggesting that the electron dense phase, which is the only thing providing contrast (the sample was not stained), is amorphous CaP.

Bar = 50 nm.

Likewise, in support of this argument (the absence of amorphous mineral in calcification), Landis and co-workers

[57,58] examined the early stages of mineralization of turkey tendon, and through tomographic 3D reconstruction of

high-voltage TEM images, provided evidence to suggest that HA crystals nucleate in the hole zones of collagen, in

which a stippled haze of material is notable, a feature which has been observed by others [85]. Continued growth

from these initial mineral deposits occurs primarily in length and width (not in thickness), where the crystal lengths

appear to traverse adjacent collagen hole and overlap zones rather than being restricted to a single hole or overlap

zone. Crystal growth in width across collagen fibrils forms mineral bridges as it apparently follows channels or

grooves, where smaller and larger crystals appear to fuse in coplanar alignment to form larger mineral platelets [57].

These descriptions are highlighted because such observations tie in well with the mechanism we later propose (even

though our mechanism deals with an amorphous precursor). While there is evidence to deny an amorphous phase,

careful examination of these papers indicates a greater degree of ambiguity than is generally recognized. In addition,

more recent results in the field of glass science have improved our understanding of intermediate range order in

amorphous materials and suggest the interpretation of the data in these earlier works on bone mineral may need to be

revised. These issues will be considered in more detail in Section 4, where comparison is made to our new model

system.

Before describing our in vitro model, it is worth pointing out that we also find biological evidence to support the

amorphous precursor mechanism.When examining natural bone samples for comparison to our synthetic materials (to

be described later), we came across some collagen fibrils that contain non-crystalline mineral. For example, the fibril

shown in Fig. 4C displays a marked banding pattern (without staining), but does not exhibit Bragg diffraction, even

with concerted effort (Fig. 4C, inset). It is common practice to refer to a material that yields only a broad diffuse

diffraction ring as amorphous; but given the unique structure of bone mineral, which is at best poorly crystalline

(i.e., exhibits weak long-range periodic correlations) when it reaches full maturity, one must be cautious about

terminology. For example, one cannot rule out the possibility that the non-diffracting material could be

nanocrystalline, such that the diffraction peaks become too broadened to detect; however, it clearly exhibits weaker

long-range periodic correlations than the mineral phase seen in the majority of the fibrils, which show strong Bragg

maxima (such as in Fig. 4A). This particular example was from mature equine bone; therefore this fibril was

presumably from a region of the bone that was recently remineralized during the natural remodeling process of mature

bone.

1.4. Synthetic attempts at mimicking bone formation

While considerable effort has gone into determining the relationship between collagen structure and mineral

orientation, synthetic re-creation of this most fundamental level of bone structure has eluded the materials engineer

seeking to fabricate bone-like composites. It would be desirable to mimic both the composition and structure of bone

for synthetic bone graft substitutes, but attempts at reproducing the intrafibrillar mineralization of collagen scaffolds

have achieved limited success.

Early attempts at mimicking bone formation were performed by introducing reconstituted bovine or porcine type-I

collagen substrates into simulated body fluid (SBF) (which, depending on the recipe, contains NaHCO3, Na2SO4,

MgCl26H2O, NaCl and KCl, but does not contain NCPs). It was believed that the hole zones of the collagen would

serve as nucleation sites, as appeared to be the case for turkey tendon. In support of this, Glimcher et al. [86] indicated

that the hole zones of reconstituted collagen from rabbit bone could nucleate HA, and the resultant structure appeared

similar to the early stages of calcification of embryonic bone. Yet, the reason for this ability to nucleate mineral in a

biological collagen matrix was not clear. It has therefore been assumed that the collagen substrate does not act alone in

directing crystal growth, and that the non-collagenous proteins (NCPs) found in regions of bone growth play an

essential role in calcification due to their ability to bind calcium and their high affinity for collagen [87].

These NCPs, such as osteonectin and various other phosphoproteins (e.g., osteopontin, osteocalcin,

phosphophoryn, and bone sialoprotein), are enriched with acidic amino acids, particularly aspartic acid and

phosphoserine [37,8890]. We note that, although these water soluble proteins that are found in nearly all highly

regulated biomineralization processes are often referred to as the acidic proteins [9194], we will refer to them as

polyanionic proteins [95], to emphasize the charged character of the deprotonated active form of the protein. In

synthetic crystal growth assays, such polyanionic proteins have been shown to both inhibit HA nucleation when in

soluble form, and promote nucleation when attached to a substrate [91,96,97]; thus it has been generally assumed that


binding of such proteins to collagen fibrils could be a means of promoting nucleation within the collagen. This

hypothesis prompted the search for epitaxial relationships between NCPs and HA. Indeed, Hoang et al. [98]

demonstrate that the X-ray crystal structure of porcine osteocalcin reveals a negatively charged surface that

coordinates five calcium ions in a spatial orientation that is complementary to calcium ions in a hydroxyapatite crystal

lattice, which they suggest may lead to selective binding characteristics with HA that could play a role in modulating

HA crystal morphology and growth in bone formation. It should be kept in mind, however, that the early mineral phase

of bone is considered to be very poorly ordered, so one must be cautious when extrapolating results based on modeling

structural relationships with a well-ordered HA lattice to the poorly crystalline HA of bone. Calcium binding could

also play an important role in formation of an amorphous precursor, but as will be shown later, a high degree of

selectivity during the ion binding event is probably not required. In any case, it should be noted that, to date,

intrafibrillar mineralization of collagen has not been demonstrated using an epitaxial nucleation approach.

There has been partial success at mimicking some of the nanostructural features of bone by performing the

crystallization in situ during fibrillogenesis, during which some crystals appear to nucleate at the hole zones [99], and

in some cases yield similar electron diffraction patterns indicating uniaxial orientation of HA crystallites [100,101].

While this approach has produced intriguing results, is does not contribute to understanding how bone is formed from a

fundamental point of view because, in bone, fibrillogenesis occurs prior to mineralization. An alternative approach has

been to add anionic polymers or generate anionic functional domains to the collagen, producing mineral composites

with a non-descript mineral morphology similar to bone [102]. Although these studies appear promising, none has

been able to generate an interpenetrating collagenHA composite with a uniaxially oriented apatite phase that fully

infiltrates the organic matrix, thereby achieving the high mineral loading as in bone. However, in a recent paper by

Chen et al. [103], it was shown that demineralized fish bone could then be remineralized to obtain the proper uniaxial

orientation (demonstrated by wide angle X-ray diffraction), particularly when polyglutamate (polyGlu) was added to

the crystallizing solution. They did not examine the mechanism of remineralization (nor determine the role of the

polyGlu), but we suspect that it occurred through a precursor process similar to that described in the present work using

polyaspartate (polyAsp). Our work is experimentally similar to this latter approach of adding anionic polypeptides to

the reaction, not for the purpose of stimulating crystal nucleation on collagen, but rather as a process-directing agent,

in which the solution crystallization is converted into a precursor process.

1.5. Polymer-induced liquid-precursor (PILP) mineralization process

Previous work in our group has shown that the addition of micromolar quantities of anionic polypeptides to a

crystallizing solution can transform the conventional solution crystallization process into a precursor process. This

PILP process was first discovered for calcium carbonate (CaCO3) mineralization, in which it was observed that

droplets of a highly hydrated liquid-phase precursor phase separate as the solution is gradually raised in

supersaturation [104106]. The charged polymer sequesters ions, while inhibiting crystal nucleation, inducing liquid

liquid-phase separation in the crystallizing solution. Droplets of the fluidic amorphous phase accumulate and coalesce,

typically forming mineral films and coatings on a variety of substrates. The precursor phase then solidifies and

crystallizes to a more thermodynamically stable phase as the waters of hydration (and most of the polymeric impurity)

are excluded [107]. Although the term solidification is typically used to indicate solid formation from a melt, we use

it here to describe the conversion of the liquid-phase precursor to an amorphous solid, highlighting the fact that this

process is distinctly different from precipitation from solution.

The important consequence of this PILP process is that the crystals retain the shape delineated by the phase

boundaries of the precursor phase, thus generating a variety of non-equilibrium crystal morphologies. It has long

remained an enigma how biominerals are molded into their elaborate symmetry-breaking crystal morphologies, but

many of the morphological features found in CaCO3 biominerals have now been demonstrated in vitro via this

polymer-induced liquid-precursor (PILP) process, such as the deposition of CaCO3 tablets and thin films [104,108],

molded [109,110] and patterned calcite crystals [111113], and calcite nanofibers [114116]. Therefore, we have

proposed that the PILP process might play a fundamental role in the morphogenesis of calcitic biominerals, in which

the polyanionic macromolecules involved in biomineralization could be considered process-directing agents. Note,

this explanation is distinctly different from a previously held theory, in which the soluble anionic proteins were

considered structure-directing agents that modulate crystal morphology through selective interactions with specific

crystallographic faces [91,92,117,118]. In contrast, with a polymeric process-directing agent, the shaping of the


mineral occurs prior to the presence of structural order, and is simply a result of the inhibitory polymers affinity for

ions (and retention of hydration water). The results are striking and, considering the non-specificity of the organic

inorganic interactions, we thought is might be possible to induce mineralization by such a process with calcium

phosphate.

1.6. New hypothesis on the mechanism of bone formation

In the case of vertebrates, which primarily utilize calcium phosphates (CaP) in their hard tissues, we now present

evidence to suggest that the PILP process may also play a fundamental role in the biomineralization of bones and teeth.

Using this PILP process, we have been able to achieve intrafibrillar mineralization of a pre-existing fibrillar type-I

collagen matrix, first with CaCO3 [116,119,120], and now with CaP, allowing us to duplicate the nanostructured

architecture of bone. The evidence presented is based primarily on an in vitromodel system; therefore, caution must be

exercised when trying to extrapolate the results to the complex physiological environment. Nevertheless, the fact that

we have been able to achieve intrafibrillar mineralization with this system deserves attention since other theories seem

to fall short in practice. Section 4 will include a more thorough deliberation on this system as compared to literature

studies on bone formation. For now, our hypothesis on bone formation will be presented before getting to the results so

that the reasoning behind the experimental methodology can be understood.

We propose that hydroxyapatite crystals in mineralized collagenous tissues (i.e., bone and dentin) do not initially

nucleate within the hole zones, but rather a liquid-phase amorphous precursor is drawn into the collagen fibrils via

capillary action, and upon solidification, the precursor crystallizes, leaving the collagen fibrils embedded with

nanoscopic platelets of HA (Fig. 5). This liquid-phase precursor, analogous to the PILP phase we have demonstrated


Fig. 5. Schematic depicting a proposedmechanism of intrafibrillar mineralization of collagen. Each of these pictures represents the hole zone region

of a collagen fibril within the aqueous mineralizing solution containing the polymeric process-directing agent (i.e., polyaspartate). (a) The negatively

charged polymer sequesters ions and at some critical ion concentration generates liquidliquid-phase separation within the crystallizing solution,

forming nanoscopic droplets of a highly hydrated, amorphous calcium phosphate phase. The nanoscopic droplets of this polymer-induced liquid-

precursor (PILP) phase adsorb to the collagen fibril, and due to their fluidic character, become pulled up and into the hole zones and interstices of the

collagen fibril by capillary action. (b) The collagen fibril becomes fully imbibed with the amorphous mineral precursor, which then solidifies as the

hydration waters are excluded. (c) The amorphous precursor phase crystallizes, leaving the collagen fibril embedded with nanoscopic crystals of

hydroxyapatite.

in vitro, would be induced by polyanionic proteins, which we assume would be one or some combination of the

polyanionic NCPs found associated with bone.

It should be pointed out that this capillary infiltration occurs within an aqueous crystallizing solution, such that the

collagen scaffold is already swollen with imbibed water. In other words, the PILP phase is not soaked up by a dry

collagen sponge, but rather the PILP phase is delineated from the surrounding solution by a distinct phase boundary,

and combination of this interfacial energy with the pore space in the collagen leads to capillary forces, which in this

case, could draw the PILP phase up into the collagen matrix and into the fibrils. This would then provide a means for

infiltrating the collagen with a high loading of mineral ions, which then form a hydrated amorphous solid as some of

thewaters of hydration are excluded from the liquid-phase precursor [107]. This solidification process occurs while the

sample is still in the aqueous crystallizing solution. Additional waters of hydration are then driven off as the metastable

amorphous phase transforms into the more thermodynamically stable, anhydrous crystalline state (e.g., calcite in

CaCO3 or HA in CaP). Although this process is described in terms of the steps involved, it is more likely that it occurs

in a continuous fashion with no sharp distinction between the individual stages.

The results described below provide evidence for our proposed mechanism. Scanning electron microscopy,

transmission electron microscopy, X-ray diffraction, and electron diffraction all demonstrate that, by using the PILP

process, we are able to create the first example of a synthetic composite that mimics the nanostructure of bone.We also

provide evidence to suggest that intrafibrillar mineralization is achieved due to the fluidity of an amorphous precursor,

and show that long-held beliefs about the orientation of the crystallites within bone need to be revised. The strong

similarities between our composite and bone suggest that our system may be able to serve as an in vitro model that can

further shed light on the mechanisms involved in bone formation.

2. Methods and materials

2.1. Mineralization of collagen

Calcium phosphate formation by the PILP process was achieved by mixing equal volumes of 9 mM CaCl2 solution

in Tris buffer (pH 7.4) and 4.2 mMK2HPO4 solution in Tris buffer (pH 7.4), to final concentrations of 4.5 mM calcium

and 2.1 mM phosphate. The Trissaline buffer was made by dissolving 8.77 g of NaCl, 0.96 g of Trisbase, 6.61 g of

TrisHCl in 1 l dH2O (adjusted to pH 7.3 using NaOH at 25 8C, which becomes 7.4 at 37 8C). We note that these ionconcentrations are higher than physiological values, but given that the polymeric process-directing agent we use has

not been optimized as one would assume has occurred biologically, we consider this system acceptable for the

intended purpose of demonstrating proof-of-concept. Micromolar aliquots of polymer (polyaspartic acid, sodium salt,

Mw = 6200 Da) were added to each solution to achieve various concentrations of the process-directing agent (0, 15 and

75 mg/ml). Each solution was adjusted to pH 7.4 using 0.1 N NaOH or HCl, and then incubated at 37 8C during thecrystallization to simulate physiological conditions. A 1 mm 1 mm piece of Cellagen1 sponge (approximately1 mm thick) was placed in the crystallizing solution and, after mineralization, was removed and rinsed with DI-H2O

and ethanol to remove extraneous salts. The mineralization process was usually allowed to proceed for 4 days, except

for an experiment where the phase of the mineral was examined by XRD at different time points (1, 2, and 6 days). For

this experiment, side-by-side samples were run in parallel in the same crystallizing dish, with removal of separate

samples for diffraction studies at each time period. After drying in air at room temperature, the samples were prepared

for further analysis. Another set of diffraction experiments was performed in series (with the same sample examined at

consecutive time points), using turkey tendon as the collagen scaffold, as described below for the confocal microscopy

analysis in Section 2.3.2.

2.2. Characterization

2.2.1. Scanning electron microscopy (SEM) analysis

Samples were prepared for scanning electron microscopy (SEM) by mounting the dried samples on an aluminum

stub covered in double-sided copper tape, then sputter coated with either Au/Pd or amorphous carbon. The samples

were analyzed using either a 6400 JEOL SEM or a 6330 JEOL FEG-SEM at 15-20 kV.

For the bleach etching studies, the mineralized Cellagen1 samples were placed in 2% NaOCl solution for 15 min.

The natural bone samples were soaked in bleach for 20 h, owing to the high packing density of the material. Samples


were then removed from the solution, rinsed with ddH2O followed by ethanol, and dried for FE-SEM analysis (with an

amorphous carbon coating).

2.2.2. Transmission electron microscopy (TEM) analysis

Samples were prepared for transmission electron microscopy (TEM) following the protocols performed on bone

and naturally mineralized tendon by Weiner and Traub [41]. This included crushing the samples into a fine-grained

powder in a liquid nitrogen mortar and pestle. A few small drops of ethanol were then placed on the powder, followed

by drawing the slurry into a micropipette. The slurry was transferred to a 3 mm diameter carbon/Formvar coated

copper TEM grid, followed by an optional stain with 1% phosphotungstic acid (PTA) in a PBS buffer on the non-

mineralized collagen sample (mineralized samples were not stained). Before analysis, all samples were sputter coated

with a thin layer of amorphous carbon. The samples were analyzed using a 200CX JEOLTEM at 200 kV in brightfield

(BF), darkfield (DF) and selected area electron diffraction (SAED) modes. Normal darkfield images were produced by

tilting the beam to the diffracting plane of interest, such that the chosen Bragg beam produces the brightest DF image

possible.

For extraction of the CaP crystals from the composite, the mineralized Cellagen1 sample was sonicated in H2O for

15 min. to remove superficial mineral coatings. Then the sample was gently crushed in liquidN2 into a fine powder,

and 2% NaOCl was added to remove the surrounding collagen. After washing with H2O and ethanol, the nanocrystals

were gently transferred to a 3 mm diameter carbon/Formvar coated copper TEM grid.

2.2.2.1. Selected area electron diffraction (SAED) analysis. To differentiate between HA and OCP, the following

crystallographic formulae [121] were used to calculate the theoretical diffraction angles between planes (h k l) with

respect to the (0 0 2) reference plane, for comparison to the angles measured in SAED patterns What is measured in an

SAED zone pattern is the angles between reciprocal lattice vectors lying in the same planar section of reciprocal space

defined by the zeroth-order Ewald sphere intersection. Therefore, angles between planes in real space can be

correlated with the angles between their normals in reciprocal space. In the case of OCP and HA, the angles provide an

additional measure to distinguish between the two phases, which have very similar d-spacings.

hexagonal system : cosf h1h2 k1k2 1=2h1k2 k1h2 3=4a2=c2l1l2

fh21 k21 h1k1 3=4a2=c2l21h22 k22 h2k2 3=4a2=c2l22g1=2(1)

where f is the angle between (h1 k1 11) and (h2 k2 l2); unit cell dimensions and angles are given by a, b, c and a, b, g,respectively.

triclinic system : cosf FAh1k1l1Ah2k2l2

(2)

where

F h1h2b2c2 sin2 a k1k2a2c2 sin2 b l1l2a2b2 sin2 g abc2cosa cos b cos gk1h2 h1k2 ab2c cos g cosa cos bh1l2 l1h2 a2bc cos b cos g cosak1l2 l1k2

and

Ahk l fh2b2c2 sin2 a k2a2c2 sin2 b l2a2b2 sin2 g 2hkabc2cosa cos b cos g 2hlab2c cos g cosa cos b 2kla2bccos b cos g cosag

2.2.3. X-ray diffraction (XRD) analysis

X-ray diffraction analysis was used to determine the crystal structures of samples mineralized in the absence and

presence of polymeric additives, along with those of comparative reference samples of synthetic hydroxyapatite

(SigmaAldrich) and equine cortical femur bone used as standards. The final mineral crystalline phase was confirmed

from the collection of XRD d-spacings that correlated well with the Joint Committee on Powder Diffraction Standards

(JCPDS) files for HA. Monochromatized Cu Ka X-ray radiation from a Philips XRD 3720 fixed anode X-ray tube,


operated at 40 kVand 20 mAwas used, together with a diffractometer scan step size of 2u = 0.018, and dwell time of2 s/step, over a 2u range of 10608.

2.2.3.1. Rietveld analysis. The raw data from the XRD scans were inputted into the MAUD Rietveld analysis

software program developed for crystallographic refinement [122,123]. The HA crystal model was built using

information from the International Crystal Structure Database (ICSD). The details of the crystallographic model are

listed in Table 1 above. Peak shapes were modeled using the pseudo-Voigt function, and two asymmetry parameters

were refined. In each case, four background parameters, a scale factor, five peak-shape parameters, 2u offset (zero

point correction), sample displacement, cell parameters, and atomic positions were all refined. The occupancies of the

oxygen and hydrogen atoms associated with the hydroxyl (OH) group were refined as a group (i.e., OH occupancy)using the same reasoning as Knowles et al. [124]. The program was also tested for its accuracy using a three-phase

mixture of known composition of aluminum (20%), alpha-alumina (50%) and monocliniczirconia (30%). Results of

the standard analyses showed 21.4% aluminum, 49.4% Al2O3 and 29.2% ZrO2, confirming the robustness of the

software.

2.3. Confocal microscopy study of polymer penetration depth

2.3.1. Preparation of fluorescently tagged poly(aspartate)

The polymer (poly-(ab)-DL-aspartic acid, sodium salt, Mw = 6000 Da; SigmaAldrich, USA), hereafter referred toas polyAsp, was fluorescently labeled by dissolving 20 mg of it in 2 ml of 0.1 M sodium carbonate buffer [0.2 M of

Na2CO3 (8 ml) and 0.2 M NaHCO3 (17 ml), Aldrich, USA], then gently adding 200 ml of a fluorescein isothiocyanate(FITC, SigmaAldrich, USA) solution (5 mg dissolved in 0.5 ml of dimethyl sulphoxide, SigmaAldrich, USA),

which was then sealed from light and incubated at 4 8C in a refrigerator for 24 h. After incubation, the polymer-dyesolution was centrifuged in a Centricon1 spin column with molecular weight cutoff of 3 kDa to remove the un-reacted

dye. As there is only minimal loss of polymer during the centrifugation, the final concentration of polymer was

assumed not to change, although there was likely some loss of the lower molecular weight chains below the 3 kDa

cutoff.

2.3.2. Turkey tendon preparation and mineralization

The common calcanean turkey tendon, which had not yet mineralized, was harvested from young birds (10 weeks

old, obtained from Nicholas Turkey Breeding Farm, Sonoma, CA), and stored in ethanol in a refrigerator one day

before the experiment. The tendon was sliced longitudinally to expose the densely packed, well-aligned collagen

fibers. The tendon sections were laid flat with the freshly cleaved collagen surface exposed to the mineralization

solution (the elastin sheath surrounding the tendon is inhibitory to mineralization). One half of the tendon was used for

the control experiment without phosphate counter-ions, to measure diffusion penetration of polyAsp alone. The other

half was used for mineralization, to test the hypothesis that the polyAsp-containing PILP phase could penetrate further

into the collagen scaffold via capillary forces.

The tendons were mineralized at 37 8C for 4 days with 50 ml of reaction solution whose final concentrations were4.5 mM of Ca2+ and 2.1 mM of PO4

3. The stock solutions were prepared as 9 mMCaCl22H2O and 4.2 mMK2HPO4

in 0.5 M Tris-buffer (SigmaAldrich, USA), with the solution pH adjusted to 7.4. For the control experiment, the

calcium-containing solution was mixed with TRIS-buffer (without phosphate ions) to a final concentration of 4.5 mM

Ca2+. The FITCPolyAsp solution was added to the mineralization solution or the control experiment at a final

concentration of 100 mg/ml. This value is higher than our previous mineralization protocol, because mineralization of


Table 1

Hydroxyapatite crystal structure parameters, refined by Rietveld analysis

Phase Crystal

system

Space

group

Lattice

parameters

Atomic coordinates

HA Hexagonal P63/m a = 9.422;

c = 6.88

Atom Ca1: x = 0.333, y = 0.667, z = 0.001; atom Ca2: x = 0.246, y = 0.993, z = 0.25;

atom P: x = 0.4, y = 0.369, z = 0.25; atom O1: x = 0.329, y = 0.484, z = 0.25; atom

O2: x = 0.589, y = 0.466, z = 0.25; atom O3: x = 0.348, y = 0.259, z = 0.073;

ion OH: x = 0, y = 0, z = 0.25

turkey tendon was found to be optimal at a higher polymer concentration, presumably because the collagen is so

densely packed. In addition, this higher polymer concentration could help alleviate any loss of polymer activity from

the hydrophobic fluorophore probe, as well as loss of lower molecular weight chains from the Centricon1 purification

process.

2.3.3. Confocal microscopy analysis

An Olympus Fluoview 500 confocal scanning unit mounted on an IX-81 inverted fluorescence microscope was used

with an argon laser light source (488 nm excitation) and 505525 nm bandpass filter. Scanning depths along the

z-directionwere chosen to be 500 mmwith a 2.5 mmstep size,where the z-direction corresponds to a depth profile into thecross-section of the tendon. A photomultiplier voltage (PMT) of 400 V was used to optimize the fluorescence intensity,

which is very bright near the surface. Additionally, a larger PMT setting of 500 Vwas used to examine the greater depths

of penetration where the intensity declined. In this case, a 180 mm offset in the z-direction was used for the mineralizedsamples, examining an overall depth of 650 mm; while a 120 mm z-offset was used for the control sample, to an overalldepth of 590 mm. (Control samples hadmuch lower intensity profiles in the cross section andhence the z-offsetwas lowerin order to include the faint edge of the highest intensity.) Samples did not photobleach during the short analysis time.

3. Results

3.1. Intrafibrillar mineralization of collagen with a CaP PILP process

In order to mimic the organic matrix of bone, we chose to use a Cellagen1 sponge (ICN Biomedicals), which is a

commercially available hemostatic sponge composed of reconstituted type-I bovine collagen (Fig. 6). This collagen

scaffold was chosen because it contains the 64 nm banding pattern indicating that it has assembled appropriately to

match native type-I collagen. It should be noted that the banding pattern of collagen is seen in TEM only when the

fibrils are stained with an electron dense substance, such as phosphotungstic acid (PTA), as can be seen from the

comparison between Fig. 6A and B. The periodic bands can also be seen in topographic images from field-emission

SEM, as shown in Fig. 6C.

In our control reaction, which did not include the polyaspartate (Polyasp) additive, spherulitic clusters of HA

formed on the surface of the scaffold (Fig. 6D). The 1540 mm diameter clusters were composed of randomly orientedplatelets, which appear to have nucleated heterogeneously in a non-specific fashion, typical of HA grown on a variety

of substrates. This was expected since other mineralization experiments reported in the literature show similar

findings. It was not until the polyAsp agent was added to the solution that an amorphous liquid-phase mineral

precursor was produced, and this had a pronounced effect on mineralization of the collagen. The collagen fibrils took

on a distinctly different appearance in TEM, as seen in the micrograph of Fig. 7A. The added contrast in this unstained

sample is apparently due to the presence of amorphous mineral within the fibril (compare the unstained, non-

mineralized fibril of Fig. 6B to the unstained, mineralized fibril of Fig. 7A). The amorphous nature of the mineral

during the early stages of mineralization was determined through both selected area electron diffraction (SAED)

analysis of isolated fibrils (Fig. 7A, inset), and bulk XRD analysis (see Section 3.2).

After a few days, the collagen fibrils took on a more striated appearance, which we believe arises from

crystallization of the precursor within the fibrils (Fig. 7B). In this particular example, PILP droplets can be seen

adsorbed to the fibril. When examined by SEM, the surfaces of the fibrils were generally smooth and featureless

(Fig. 7C). This appearance is typical of mineral formed from the PILP phase, which usually lacks crystal facets. The

featureless appearance, however, makes it less obvious that the sample has mineralized; but the presence of mineral

can be verified using X-ray energy dispersive spectroscopy (EDS), as seen in Fig. 7D. The mineralization was also

evident macroscopically through physical changes seen in the collagen, which became rigid and white as it

mineralized in the solution, and the individual fibers retained the dimensions of the swollen state even when

dehydrated, we surmise because they became permeated with mineral [120]. The fractured fibril in Fig. 7E shows

crystals protruding from the fracture surface which span across the entire diameter of the fibril, demonstrating the full

depth of penetration of the mineral phase. This sample was lightly etched with bleach to remove the surrounding

organic matrix, allowing visualization of the remnant mineral phase. With full deproteination using a more

concentrated bleach solution, the crystals could be extracted and examined by TEM (Fig. 7F), revealing a thin platy

morphology typical of that described for extracted bone crystals.


3.2. X-ray diffraction (XRD) studies

In addition to the isolated fibril that appeared to contain amorphous phase discussed above (Fig. 7A), the precursor

mechanism of mineralization was verified on bulk samples with X-ray diffraction. Two sets of XRD time series

measurements were performed (Fig. 8). The first time-series experiment was run with sample sets in parallel, in which

four samples of Cellagen1 were mineralized within the same beaker, but removed at different time points (Fig. 8a).

The second time-series experiment was run in series, with the same sample being examined at consecutive time points

(Fig. 8b). For comparison, XRD patterns for commercial hydroxyapatite (Fig. 8a, 1) and natural bone (Fig. 8a, 5) were

included.

For the parallel time series, the control reaction consisted of the Cellagen1 scaffold mineralized in the absence of

polymer (Fig. 8a, 2). The XRD patterns contained sharp hydroxyapatite peaks similar to the commercial HA standard

(Fig. 8a, 1), as might be expected based on the relatively large (micrometer) dimensions of the crystals seen in Fig. 6D.

When Cellagen1 was mineralized in the presence of polymer additive, broad hydroxyapatite peaks were seen to

emerge after 1 day, which then became sharper by days 2 and 6 (Fig. 8a, 35); but, as is the case for bone (Fig. 8a, 6),

they never developed into the narrow peaks typical of synthetic hydroxyapatite (Fig. 8a, 1). The peak at 338 is toobroad to resolve the separate (2 1 1), (1 1 2) and (3 0 0) peaks, but an overall peak shift is seen relative to synthetic HA,

as is also found for bone. This peak shift for bone has been attributed to carbonate substituents [125]. In our system, the

crystallizing solution is not purged of carbon dioxide, so this could be a source of carbonate impurities which likely

become entrapped during solidification of the precursor phase.


Fig. 6. Electron micrographs of reconstituted type-I collagen from a Cellagen1 sponge, before and after mineralization by the traditional solution

crystallization process. (A) TEM brightfield image of individual collagen fibrils stained with 1% phosphotungstic acid (PTA). The accumulation of

the heavy metal within the hole zones allows the 64 nm banding pattern characteristic of type-I collagen to be observed. Inset: SAED of non-

mineralized collagen fibrils shows no diffraction. Bar = 200 nm. (B) TEM brightfield image of an unstained collagen fibril. In the absence of the PTA

electron dense stain, the 64 nm banding pattern is not readily discerned. Bar = 200 nm. (C) High magnification FE-SEM micrograph of collagen

fibrils of the Cellagen1 sponge. Although the Cellagen1 has randomly oriented fibers, in this region the fibers were arranged nearly parallel, which

appears to have allowed for registry in alignment of the bands across neighboring fibrils, as has been seen by others [160]. (D) Scanning electron

micrograph of collagen mineralized without polyAsp (the control) shows only clusters of HAwhich nucleated heterogeneously on the surface of the

Cellagen1 sponge. Bar = 50 mm.


Fig. 7. Mineralization of Cellagen1 sponge via the PILP process (with 15 mg/ml polyAsp). (A) TEM of a collagen fibril removed during the early

stages of infiltration with calcium phosphate. The appearance of the banding pattern in the unstained fibril suggests that it is entrenched with

amorphous mineral. Bar = 200 nm. The SAED pattern (inset) of this fibril confirms the amorphous nature of the mineral precursor. (B) TEM of

collagen fibrils isolated after the mineralization reaction shows a distinct striated texture that runs parallel to the fibers. Remnant droplets of the PILP

phase can be seen adsorbing to the fibers (arrows). Bar = 100 nm. (C) SEM of PILP mineralized fibrils shows a relatively non-descript mineral

morphology, even after crystallization. (D) Because identifiable crystal features are not seen in surface view, energy dispersive spectroscopy of the

mineralized composites is useful for confirming the presence of mineral, as seen here by the large Ca and P peaks. (E) A fractured fibril reveals the

extent of intrafibrillar mineral penetration, as evidenced by the platy/needle-like HA crystals that protrude from the fracture surface. This samplewas

treated with bleach to remove surrounding collagen to reveal the mineral phase (fracture had occurred prior to the bleach treatment), and crystals can

be seen in the middle and all across the diameter of the fibril, suggesting the precursor had penetrated the entire fibril and subsequently crystallized.

Bar = 100 nm. (F) TEM bright-field image of HA crystals extracted from the mineralized composite using a strong bleach treatment to fully remove

collagen. The platy morphology of the HA can be observed (platelets are laying on a holey carbon film). Some bundles were difficult to separate into

individual crystals and appear much darker. The striations on the thick bundles on the bottom left suggest that the crystals are stacked such that they

are being viewed nearly edge on. The crystals that were isolated (such as those seen at the top) appear to have a platy morphology which resembles

the nanocrystals extracted from bone (see Fig. 1, level 1). Bar = 200 nm.

One could argue that the broad peaks grow in intensity with time due to the conventional nucleation and growth of

nanocrystallites from solution; to eliminate this possibility, a series of XRDmeasurements was performed on the same

sample at increasing times (Fig. 8b). In these experiments, a single sample was mineralized just to the point at which

mineral could be observed. It was then removed from the crystallizing solution and placed in buffer.With this protocol,


Fig. 8. X-ray diffraction (XRD) studies run in series (a) vs. parallel (b) of the mineralization process. (a) XRD of calcium phosphates crystallized in

the absence and presence of polyaspartic acid. The bottom XRD pattern (1) is of a commercial hydroxyapatite (HA) standard showing the typical

diffraction planes present in a randomly oriented powder pattern of HA. Pattern (2) is of mineralized collagen in the absence of polyaspartic acid,

which serves as a control sample that only produces HA clusters on the surface of the Cellagen1 sponge (Fig. 6D). Therefore, the same planes are

expressed as the standard HA due to the high degree of crystallinity and random orientation of the crystallites in the clusters. XRD patterns (35)

show stages of the PILP mineralization process, in which collagen samples were removed from the mineralizing solution at 1, 2 and 6 days

respectively. The sharp peak for (5) is from adventitious OCP crystals that nucleated and grew on the collagen surface. Pattern (6), which is of equine

bone, is shown for comparison of peak widths. It is readily apparent that the 15 mg/ml Pasp sample and the equine bone have comparable values.

Lattice parameters/sample a-Axis c-Axis

Equine bone 9.4521 (4)a 6.8792 (6)

Commercial HA 9.4324 (7) 6.8894 (5)

0 mg/ml Pasp, day 6 9.4603 (4) 6.8598 (7)

15 mg/ml Pasp, day 6 9.4550 (5) 6.8759 (6)

a Error in the last decimal in parenthesis.

(b) To determine if amorphous mineral is present in the collagen prior to crystallization, the mineralization experiment was repeated on collagen in

turkey tendon. Pattern (1) is for the turkey tendon alone, prior to mineralization. When mineralized, samples were removed from the solution at 40 h

(pattern 2), at the visual onset of mineralization, and incubated in Tris buffer only (without crystallization ions) for an additional 48 h. Pattern 3 is for

24 h and pattern 4 is for 48 h of immersion in TRIS buffer only. As can be seen, the lack of mineral peaks in the 40 h sample (pattern 2) indicated that

themineral phasewas initially amorphous or at most paracrystalline. The appearance of peaks with time (24 and 48 h immersion in TRISpatterns 3

and 4, respectively) shows that amorphous mineral phase gradually transformed into poorly crystalline HA. The scattering hump at the lower angles

(238) is from the collagen and/or glass substrate, which is more pronounced than the (a) series since the counting time for these experiments wasfour times longer.

any increase in intensity of the diffraction peaks after the sample was placed in the buffer would have to result from

transformation of an already existing phase, as there was no additional source of mineral ions available for further

crystal growth.

At this point, the Cellagen1 product was discontinued and no longer commercially available, so an alternative

collagen scaffold that exhibits a similar fibrillar structure had to be used for further experiments. The calcanean turkey

tendon was chosen because it mineralizes naturally, and therefore provides a useful biological matrix for comparison.

The one concern about using a biological scaffold is that it may have crystals or nuclei already present in the collagen

which could stimulate further crystal growth. But in the control reaction (turkey tendon mineralized without addition

of polymeric process-directing agent), HA clusters formed only on the surface of the turkey tendon (data not shown),

yielding an XRD pattern similar to that shown in Fig. 8a(2) for the synthetic Cellagen1 scaffold. Therefore, it wasconcluded that, if any crystal nuclei were present, they did not appreciably affect the mineralization process.

In Fig. 8b(1), the first XRD scan is of the turkey tendon alone. Upon mineralization, the sample was first measured

when mineral was visually evident (at 40 h), but only barely detectable by XRD, with only a broad intensity maximum

centered at about 2u = 328 that has been attributed to amorphous or paracrystalline [76,126] calcium phosphate(Fig. 8b, 2). Subsequently, the same sample was stored in TRIS buffer (pH 7.4 and 37 8C) for another 24 and 48 h, andthe XRD scan repeated. At this time, crystalline peaks began to emerge (Fig. 8b, 3 and 4), with overlapping peaks from

the (2 1 1), (1 1 2) and (3 0 0) planes appearing between 2u = 32348, as well as clear (0 0 2) and (0 0 4) maxima at2u = 25.98 and 53.18, respectively. The fact that the same sample was examined clearly indicates that there was agradual transformation taking place from a poorly ordered precursor into a distinctly crystalline phase with apatitic

structure.

3.3. Electron diffraction analysismineral identification

Phase identification of calcium phosphate compounds, in particular distinguishing OCP and HA, can be difficult

because of their very similar d-spacings. Table 2 lists d-spacings derived from electron diffraction data measured for

our mineralized collagen samples, such as the representative fibril shown in Fig. 9A, as well as from natural bone

(Fig. 4A), for comparison to those of HA and OCP taken from the JCPDS standards. As can be seen, the d-spacings for

our samples and bone could be attributed to either mineral (especially the (0 0 2) and (0 0 4) reflections, which are

identical). We found that selected area electron diffraction (SAED) can be useful for differentiating between the

phases because it provides both the angles and the dimensions of the unit cell, which provides more data for correlation

to the standard unit cells in question. For example, in the example shown in Fig. 9, the platelets are preferentially

oriented in the [0 0 1] direction (as described more fully in the following section). Therefore, one can derive the angles

that each of the planes make with the (0 0 2) plane from the SAED pattern, and compare with the calculated values

based on the hexagonal unit cell of HA, or the triclinic unit cell of OCP (see Eqs. (1) and (2), respectively, in Section

2.2.2).


Table 2

Electron diffraction data from biomimetic and natural bone, measured against standards for hydroxyapatite (HA) and octacalcium phosphate

(OCP), demonstrating a method for distinguishing between HA and OCP, which have very similar d-spacings

Bone Hydroxyapatite (JCPDS 9-432) Octacalcium phosphate (JCPDS 79-0423)

Biomimetic Natural Plane d-Spacing (A) ab Plane d-Spacing (A) ab

d-Spacing (A) aa d-Spacing (A) aa

3.44 0.0 3.44 0.0 0.02 3.44 0.0 0.02 3.43 0.0

3.13 90.0 3.10 90.0 2.10 3.08 90.0 3.12 3.05 22.1

2.82 65.5 2.81 65.5 2.11 2.80 66.5 7.10 2.83 87.9

2.81 36.3 2.77 38.0 1.12 2.78 36.9 3.22 2.77 28.7

2.77 90.0 2.71 90.0 3.00 2.72 90.0 7.00 2.69 90.0

2.33 90.0 2.26 90.0 3.10 2.26 90.0 6.20 2.26 86.6

1.99 55.9 1.92 58.3 2.22 1.94 56.4 8.22 1.95 51.9

1.87 36.7 1.84 38.4 2.13 1.84 37.5 6.42 1.84 50.9

1.72 0.0 1.72 0.0 0.04 1.72 0.0 0.04 1.72 0.0

a Denotes angle of plane measured with respect to (0 0 2) plane oriented along the collagen c-axis.b Denotes angle calculated for the hexagonal HA and triclinic OCP systems [121].


Fig. 9. TEM micrographs of Cellagen1 sponge mineralized by the PILP process (using 15 mg/ml polyAsp), demonstrating the location and

orientation of the HA crystallites. (A) Brightfield TEM image of a single mineralized fibril isolated from the mineralized sponge. The dark streaks

along the long axis of the collagen fibril suggest that platy hydroxyapatite crystals are uniaxially oriented within the collagen. The darker lines are

from those platelets viewed edge on, while other orientations are less easily seen due to the extreme thinness of the platelets. Banding patterns,

although not as pronounced as in the equine bone sample shown in Fig. 1A, can be discerned. Bar = 100 nm. (B) Selected area electron diffraction

(SAED) of the fibril shown in (A) demonstrates that the mineral phase is hydroxyapatite. The (0 0 2) and (0 0 4) planes are oriented parallel to the

long axis of the fibril (arrow), and the arcing, which is the same as in natural bone, indicates that the crystals are tilted with a slight mis-orientation

along their c-axis. Importantly, all fibrils examined had this same pattern, which matches in orientation and intensity the patterns exhibited by

naturally mineralized collagen found in bone and turkey tendon. (CF) Darkfield TEM images of the fibril in (A), illuminated with the (0 0 2),

(1 1 2), (2 1 1), and (3 0 0) diffraction planes, respectively. Platy or needle-like HA crystals can be seen to be uniaxially oriented along the long axis

of the collagen, and bright crystals appear throughout the fibril for each rotational orientation, suggesting that the crystals are not fully aligned in the

ab plane as would be expected for the deck of cards model. Bars = 200 nm.

From a comparison of both the angles and spacings shown in Table 2, there is no longer any question as to the

identity of the mineral phase, which is clearly HA, and not OCP. The issue of HAversus OCP is particularly relevant to

discussions on bones and teeth because some researchers have suggested that OCP serves as a precursor to HA in the

mineralized tissues of vertebrates [127,128].

3.4. Electron diffraction analysiscrystallographic orientation

To determine the intrafibrillar crystal arrangement, we examined individual mineralized fibrils with TEM by

crushing our mineralized collagen sponge samples in liquid nitrogen, employing similar sample preparation protocols

to those described in the literature for bone [129]. As is seen in natural bone, our mineralized fibrils show dark streaks

in brightfield TEM (Fig. 9A); the width of the streaks suggest that these are HA platelets which are being viewed edge-

on. When viewed in full screen size (via electronic article), many lighter striations can also be seen throughout the

fibril. Crystals that are not oriented edge-on are more difficult to see since they are extremely thin, but further evidence

of the high mineral loading is demonstrated by application of dark-field TEM imaging (DF-TEM) to the same sample.

Imaging with the (0 0 2) diffracted beam (Fig. 9B) shows that the fibril is well infiltrated with numerous HA crystals

(Fig. 9C). Crystal lengths, as measured by the bright streaks, are 2550 nm, which falls in the range of bone

crystallites, which are reportedly 3050 nm in length [18]. Likewise, DF-TEM images using other diffracted beams

also show bright striations throughout the fibril (Fig. 9CF).

To determine the crystallographic orientation of the intrafibrillar HA crystals, selected area electron diffraction

(SAED) was performed on isolated fibrils. The SAED pattern of the electron dense, 200 nm diameter fibril illustrated

in Fig. 9A indicates that the HA crystals are oriented in the [0 0 1] direction parallel to the long axis of the collagen

fibril (arrow, Fig. 9B). To our surprise, the SAED pattern was indistinguishable from that of bone (compare Fig. 9B to

Fig. 4B and electron diffraction patterns of bone from the literature [130]). Although we anticipated getting crystals

within the collagen fibrils using this capillary-infiltration mechanism, we did not expect them to have the same

orientation as found in bone, given that we did not add any specific nucleating domains or specialized proteins to the

collagen. Every mineralized fibril (n > 20) we imaged using TEM exhibited the same SAED pattern as Fig. 9B, withthe crystallographic orientation of the HA platelets consistently matching that observed in natural bone (Fig. 4B).

3.5. SEM analysis of crystal morphology and organization

To examine the morphology of the mineral phase within the fibrils, the mineralized collagen sample was

deproteinated using 2% sodium hypochlorite (NaOCl) for 15 min [116,120], and the remnant mineral phase was

examined by SEM (Fig. 10). Although it was not possible to be certain that the organic matrix has been completely

removed by this procedure, it served the purpose of exposing the principal mineral component. The intrafibrillar

organization of the crystals was well preserved, as can be seen by the uniaxial alignment of the crystals (Fig. 10A and

B), in which it appears the mineral phase had fully infiltrated the collagen fibrils. The crystals in the image exhibit a

mixed fibrousplaty texture, and apparently had become very elongated as crystal growth traversed along the collagen

fibril. This fibrousplaty texture is strikingly similar to SEM images of real bone presented by Weiner et al. [130], as

well as our example illustrated in Fig. 3D.

To carry the comparison to natural bone further, the bleach treatment was applied to an equine bone sample

(Fig. 10C) as well as turkey bone (Fig. 10D), and the remnant mineral phase examined by SEM. The meandering

crystal texture we had observed in our sample (Fig. 10A and B) can also be seen in the natural samples, although the

fine texture of the biogenic crystals is less pronounced, and distinct crystals are not readily discerned. The most

noticeable difference is the size and organization of the collagen scaffold. The differences between the bleached

samples and the original bone samples (Fig. 3), when imaged with the SEM, are not that obvious given the apparent

continuity of mineral phase; yet physically, the bleached samples easily crumbled and lacked the mechanical integrity

of the original organicinorganic biocomposite.

3.6. Confocal studies on mechanism of mineral infiltration

The evidence that intrafibrillar mineralization has been achieved in our system is strong, but an alternative to our

hypothesized capillary action mechanism could be argued. For example, the polyAsp additive may have diffused into


the interstices of the collagen to stimulate crystal nucleation, analogous to the concept of NCPs serving as epitaxial

templates to stimulate crystal nucleation in bone. We consider the possibility unlikely though, because polymers do

not readily diffuse into confined spaces, such as a hydrogel, due to the large decrease in entropy. Many of the unusual

properties of macromolecules are dominated by the entropy component of the Gibbs free energy. On the other hand,

proteins, even though they are biological macromolecules, may behave differently from conventional random coil

polymers, particularly if the protein has a globular conformation and its diffusion and transport properties are more

similar to that of a particle. In the case of the highly anionic, soluble proteins associated with biominerals, less ordered

conformations have been observed [131133]; therefore, they might be expected to behave more like a random coil

polyelectrolyte and be dominated by entropy effects. Clearly, it is hard to predict (and unfortunately difficult to

measure) the state of the NCPs in their natural surroundings during biomineralization, hence the value of an in vitro

model system becomes apparent.

To determine whethe

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