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MSR-343; No of Pages 40
Materials Science and Engineering R xxx (2007) xxxxxx www.elsevier.com/locate/mser
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,*
Department of Materials Science and Engineering, University of Florida, Gainesville, FL 32611, USA Center for Biomaterials, Department of Oral Rehabilitation, Biomaterials and Skeletal Development, University of Connecticut Health Center, Farmington, CT 06030, USA c 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, USA e Department of Materials Science and Engineering, University of North Texas, Denton, TX 76203, USA
b a
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 intrabrillar mineralization of collagen can be achieved during bone formation. Intrabrillar 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 bril 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 specic crystallographic faces and specic protein conformers. Indeed, epitaxial relationships have been calculated; but in practice, it has not been demonstrated that intrabrillar mineralization can be accomplished via this route. Because of the difculty 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 intrabrillar mineralization of type-I collagen because the uidic character of the amorphous precursor phase enables it to be drawn into the nanoscopic gaps and grooves of collagen brils by capillary action. The precursor then solidies and crystallizes upon loss of hydration waters into the more thermodynamically stable phase, leaving the collagen brils embedded with nanoscopic hydroxyapatite (HA) crystals. Electron diffraction patterns of the highly mineralized collagen brils are nearly identical to those of natural bone, indicating that the HA crystallites are preferentially aligned with [0 0 1] orientation along the collagen bril 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. Most importantly, this in vitro model demonstrates that a highly specic, 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
* Corresponding author. Tel.: +1 352 846 3336; fax: +1 352 846 3355. E-mail address: lgowe@mse.u.edu (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
Please cite this article in press as: M.J. Olszta et al., Bone structure and formation: A new perspective, (2007), doi:10.1016/j.mser.2007.05.001
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M.J. Olszta et al. / Materials Science and Engineering R xxx (2007) xxxxxx
template for crystal organization, but with the important caveat that this templating occurs only for crystals formed from an inltrated 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-specicity of the PILP process and capillary inltration 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Primary versus secondary bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Nanostructured architecture of secondary bone. . . . . . . . . . . . . . . . . . . . 1.2.1. Collagen matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2. Mineral phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.3. Intrabrillar mineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Mechanism of bone formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Synthetic attempts at mimicking bone formation . . . . . . . . . . . . . . . . . . 1.5. Polymer-induced liquid-precursor (PILP) mineralization process . . . . . . . 1.6. New hypothesis on the mechanism of bone formation. . . . . . . . . . . . . . . Methods and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mineralization of collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Scanning electron microscopy (SEM) analysis . . . . . . . . . . . . . . 2.2.2. Transmission electron microscopy (TEM) analysis . . . . . . . . . . . 2.2.3. X-ray diffraction (XRD) analysis . . . . . . . . . . . . . . . . . . . . . . . 2.3. Confocal microscopy study of polymer penetration depth . . . . . . . . . . . . 2.3.1. Preparation of uorescently tagged poly(aspartate) . . . . . . . . . . . 2.3.2. Turkey tendon preparation and mineralization . . . . . . . . . . . . . . 2.3.3. Confocal microscopy analysis . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Intrabrillar mineralization of collagen with a CaP PILP process . . . . . . . 3.2. X-ray diffraction (XRD) studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Electron diffraction analysismineral identication . . . . . . . . . . . . . . . . 3.4. Electron diffraction analysiscrystallographic orientation . . . . . . . . . . . . 3.5. SEM analysis of crystal morphology and organization . . . . . . . . . . . . . . 3.6. Confocal studies on mechanism of mineral inltration . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Summary of mineralization results using the PILP in vitro model system . 4.2. Amorphous to crystalline transformation: comparison to existing literature 4.3. Clarication of crystal orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Elucidation of mineral morphology. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Mechanism of alignment of intrabrillar crystals . . . . . . . . . . . . . . . . . . 4.6. Mechanism of mineral penetration and implications for application . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000
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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 rst needs to understand that there are two main stages of bone formation, referred to as primary and secondary osteogenesis (or synonymously as ossication), 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 ossication), is a combination of ground substance and very loose, small (1020 nm in diameter) bril 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, and crystals do not form in close association with the collagen. Instead, clusters of hydroxyapatite form within the proteoglycan matrix [26], which are referred to as calcication nodules [27] or calcospherites [28], due to the spherulitic arrangement of the crystal clusters. Cameron [20] suggested that the collagen brils found in cartilage are too narrow for the mineral to deposit within them, thereby resulting in the observed extrabrillar 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 parallelbered 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 by Weiner 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 brils; the collagen brils are layered in parallel arrangement within lamellae; the lamellae are arranged concentrically around blood vessels to form osteons; nally, 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 brils 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 brils within which they form [37]. This leads to intrabrillar 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 bers, which are referred to as interbrillar crystals. According to Martin et al. [43], about 58% of the mineral in canine whole bone is intrabrillar, 14% interbrillar, and 28% from within the gaps between the ends of collagen brils. The high degree of mineral loading that is achieved by intrabrillar 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 signicance 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.
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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).
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 signicant 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-bered, lamellar, brolamellar, 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 collagen matrix (level 2 of Fig. 1). This level
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of structure is created by intrabrillar mineralization of collagen, and it is the intimate relationship between the selfassembled brillar 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 difcult to classify bone as one specic type of composite. For example, the high degree of loading of mineral phase which encases the collagen brils suggests that bone is a ber-reinforced ceramicmatrix composite. In sharp contrast to this observation is the fact that ultrastructural examination of deproteinated bone reveals individual 2550-nm-wide HA crystals [5,4648], implying that bone may 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 brils. Before describing the complex composite structure of bone, the organization of the organic matrix must rst be dened. 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 brillar 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 brillar 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 brils in a quarter-staggered fashion, which leaves periodic gaps and grooves within the brils. 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 brils, 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).
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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 brils 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 brils [55]. This group has additionally shown that the collagen brils bend in a tube-like fashion, suggesting that the composition is not homogeneous laterally across the brils, 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 rst 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 bers of collagen mineralize in an intrabrillar fashion that appears to be similar to secondary bone formation [57,58]. A variety of modications 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 intrabrillar 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 nd 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 extrabrillar 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 brils. 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 nonstoichiometry 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-decient 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. Intrabrillar 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 bril (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 brils collected from native bone (Fig. 4B). This example demonstrates the unique structure created by intrabrillar mineralization, in which the HA crystallites are embedded within the collagen brils. 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
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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 bers form a bundle in which mineral is densely invested, sometimes causing bulging and clubbing of the bers. (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.).
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 dened, its formation remains an enigma. For example, although collagen can be reconstituted into the native brillar 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
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Fig. 4. Electron micrographs of equine cortical bone. (A) TEM brighteld image demonstrating intrabrillar mineralization of type-I collagen brils in natural bone. The native banding pattern of type-I collagen can be observed due to the inltration 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 bril 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 brils (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 brighteld image of an isolated collagen bril showing the characteristic banding pattern of type-I collagen. The SAED pattern (inset) of this bril demonstrates that the bril 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.
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 rst 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 in the radial distribution function analysis of XRD data (as compared to 10 A for a synthetic ACP). order of up to 25 A
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Likewise, in support of this argument (the absence of amorphous mineral in calcication), 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 brils 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 eld 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 nd 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 brils that contain non-crystalline mineral. For example, the bril 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 brils, which show strong Bragg maxima (such as in Fig. 4A). This particular example was from mature equine bone; therefore this bril 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 intrabrillar 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 uid (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 calcication 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 calcication due to their ability to bind calcium and their high afnity 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
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binding of such proteins to collagen brils 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 ve 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, intrabrillar 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 brillogenesis, 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, brillogenesis 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 inltrates 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 sh 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 rst 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 uidic amorphous phase accumulate and coalesce, typically forming mineral lms and coatings on a variety of substrates. The precursor phase then solidies 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 solidication 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 lms [104,108], molded [109,110] and patterned calcite crystals [111113], and calcite nanobers [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 specic crystallographic faces [91,92,117,118]. In contrast, with a polymeric process-directing agent, the shaping of the
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mineral occurs prior to the presence of structural order, and is simply a result of the inhibitory polymers afnity for ions (and retention of hydration water). The results are striking and, considering the non-specicity 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 intrabrillar mineralization of a pre-existing brillar type-I collagen matrix, rst 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 vitro model 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 intrabrillar 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 brils via capillary action, and upon solidication, the precursor crystallizes, leaving the collagen brils 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 proposed mechanism of intrabrillar mineralization of collagen. Each of these pictures represents the hole zone region of a collagen bril 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 liquidprecursor (PILP) phase adsorb to the collagen bril, and due to their uidic character, become pulled up and into the hole zones and interstices of the collagen bril by capillary action. (b) The collagen bril becomes fully imbibed with the amorphous mineral precursor, which then solidies as the hydration waters are excluded. (c) The amorphous precursor phase crystallizes, leaving the collagen bril embedded with nanoscopic crystals of hydroxyapatite.
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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 inltration 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 brils. This would then provide a means for inltrating the collagen with a high loading of mineral ions, which then form a hydrated amorphous solid as some of the waters of hydration are excluded from the liquid-phase precursor [107]. This solidication 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 rst example of a synthetic composite that mimics the nanostructure of bone. We also provide evidence to suggest that intrabrillar mineralization is achieved due to the uidity 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 mM K2HPO4 solution in Tris buffer (pH 7.4), to nal 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 ion concentrations 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 the crystallization to simulate physiological conditions. A 1 mm 1 mm piece of Cellagen1 sponge (approximately 1 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
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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 ne-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 nonmineralized 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 JEOL TEM at 200 kV in brighteld (BF), darkeld (DF) and selected area electron diffraction (SAED) modes. Normal darkeld 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 supercial mineral coatings. Then the sample was gently crushed in liquidN2 into a ne 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 dened 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. h1 h2 k1 k2 1=2h1 k2 k1 h2 3=4a2 =c2 l1 l2
2 2 2 2 2 2 2 2 2 fh2 1 k1 h1 k 1 3=4a =c l1 h2 k2 h2 k 2 3=4a =c l2 g 1=2
hexagonal system :
cos f
(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. cos f F Ah1 k1 l1 Ah2 k2 l2
triclinic system : where
(2)
F h1 h2 b2 c2 sin2 a k1 k2 a2 c2 sin2 b l1 l2 a2 b2 sin2 g abc2 cos a cos b cos g k1 h2 h1 k2 ab2 c cos g cos a cos bh1 l2 l1 h2 a2 bc cos b cos g cos ak1 l2 l1 k2 and Ah k l fh2 b2 c2 sin2 a k2 a2 c2 sin2 b l2 a2 b2 sin2 g 2hkabc2 cos a cos b cos g 2hlab2 c cos g cos a cos b 2kla2 bccos b cos g cos ag 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 nal mineral crystalline phase was conrmed from the collection of XRD d-spacings that correlated well with the Joint Committee on Powder Diffraction Standards (JCPDS) les for HA. Monochromatized Cu Ka X-ray radiation from a Philips XRD 3720 xed anode X-ray tube,
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Table 1 Hydroxyapatite crystal structure parameters, rened by Rietveld analysis Phase HA Crystal system Hexagonal Space group P63/m Lattice parameters a = 9.422; c = 6.88 Atomic coordinates 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
operated at 40 kV and 20 mA was used, together with a diffractometer scan step size of 2u = 0.018, and dwell time of 2 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 renement [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 rened. In each case, four background parameters, a scale factor, ve peak-shape parameters, 2u offset (zero point correction), sample displacement, cell parameters, and atomic positions were all rened. The occupancies of the oxygen and hydrogen atoms associated with the hydroxyl (OH) group were rened 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, conrming the robustness of the software. 2.3. Confocal microscopy study of polymer penetration depth 2.3.1. Preparation of uorescently tagged poly(aspartate) The polymer (poly-(ab)-DL-aspartic acid, sodium salt, Mw = 6000 Da; SigmaAldrich, USA), hereafter referred to as polyAsp, was uorescently 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 uorescein 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-dye solution 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 nal 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 bers. The tendon sections were laid at 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 nal concentrations were 4.5 mM of Ca2+ and 2.1 mM of PO43. The stock solutions were prepared as 9 mM CaCl22H2O and 4.2 mM K2HPO4 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 nal concentration of 4.5 mM Ca2+. The FITCPolyAsp solution was added to the mineralization solution or the control experiment at a nal concentration of 100 mg/ml. This value is higher than our previous mineralization protocol, because mineralization of
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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 uorophore probe, as well as loss of lower molecular weight chains from the Centricon1 purication process. 2.3.3. Confocal microscopy analysis An Olympus Fluoview 500 confocal scanning unit mounted on an IX-81 inverted uorescence microscope was used with an argon laser light source (488 nm excitation) and 505525 nm bandpass lter. Scanning depths along the z-direction were chosen to be 500 mm with a 2.5 mm step size, where the z-direction corresponds to a depth prole into the cross-section of the tendon. A photomultiplier voltage (PMT) of 400 V was used to optimize the uorescence intensity, which is very bright near the surface. Additionally, a larger PMT setting of 500 V was 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 mineralized samples, examining an overall depth of 650 mm; while a 120 mm z-offset was used for the control sample, to an overall depth of 590 mm. (Control samples had much lower intensity proles in the cross section and hence the z-offset was lower in order to include the faint edge of the highest intensity.) Samples did not photobleach during the short analysis time. 3. Results 3.1. Intrabrillar 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 brils 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 eld-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 oriented platelets, which appear to have nucleated heterogeneously in a non-specic fashion, typical of HA grown on a variety of substrates. This was expected since other mineralization experiments reported in the literature show similar ndings. 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 brils 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 bril (compare the unstained, nonmineralized bril of Fig. 6B to the unstained, mineralized bril 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 brils (Fig. 7A, inset), and bulk XRD analysis (see Section 3.2). After a few days, the collagen brils took on a more striated appearance, which we believe arises from crystallization of the precursor within the brils (Fig. 7B). In this particular example, PILP droplets can be seen adsorbed to the bril. When examined by SEM, the surfaces of the brils 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 veried 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 bers retained the dimensions of the swollen state even when dehydrated, we surmise because they became permeated with mineral [120]. The fractured bril in Fig. 7E shows crystals protruding from the fracture surface which span across the entire diameter of the bril, 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.
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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 brighteld image of individual collagen brils 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 nonmineralized collagen brils shows no diffraction. Bar = 200 nm. (B) TEM brighteld image of an unstained collagen bril. In the absence of the PTA electron dense stain, the 64 nm banding pattern is not readily discerned. Bar = 200 nm. (C) High magnication FE-SEM micrograph of collagen brils of the Cellagen1 sponge. Although the Cellagen1 has randomly oriented bers, in this region the bers were arranged nearly parallel, which appears to have allowed for registry in alignment of the bands across neighboring brils, as has been seen by others [160]. (D) Scanning electron micrograph of collagen mineralized without polyAsp (the control) shows only clusters of HA which nucleated heterogeneously on the surface of the Cellagen1 sponge. Bar = 50 mm.
3.2. X-ray diffraction (XRD) studies In addition to the isolated bril that appeared to contain amorphous phase discussed above (Fig. 7A), the precursor mechanism of mineralization was veried on bulk samples with X-ray diffraction. Two sets of XRD time series measurements were performed (Fig. 8). The rst 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 too broad 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 solidication of the precursor phase.
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Fig. 7. Mineralization of Cellagen1 sponge via the PILP process (with 15 mg/ml polyAsp). (A) TEM of a collagen bril removed during the early stages of inltration with calcium phosphate. The appearance of the banding pattern in the unstained bril suggests that it is entrenched with amorphous mineral. Bar = 200 nm. The SAED pattern (inset) of this bril conrms the amorphous nature of the mineral precursor. (B) TEM of collagen brils isolated after the mineralization reaction shows a distinct striated texture that runs parallel to the bers. Remnant droplets of the PILP phase can be seen adsorbing to the bers (arrows). Bar = 100 nm. (C) SEM of PILP mineralized brils shows a relatively non-descript mineral morphology, even after crystallization. (D) Because identiable crystal features are not seen in surface view, energy dispersive spectroscopy of the mineralized composites is useful for conrming the presence of mineral, as seen here by the large Ca and P peaks. (E) A fractured bril reveals the extent of intrabrillar mineral penetration, as evidenced by the platy/needle-like HA crystals that protrude from the fracture surface. This sample was 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 bril, suggesting the precursor had penetrated the entire bril and subsequently crystallized. Bar = 100 nm. (F) TEM bright-eld 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 lm). Some bundles were difcult 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.
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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 Equine bone Commercial HA 0 mg/ml Pasp, day 6 15 mg/ml Pasp, day 6 a-Axis 9.4521 9.4324 9.4603 9.4550 (4) (7) (4) (5)
a
c-Axis 6.8792 6.8894 6.8598 6.8759 (6) (5) (7) (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 the mineral phase was 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 was four times longer.
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 XRD measurements 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,
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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 brillar 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 was concluded that, if any crystal nuclei were present, they did not appreciably affect the mineralization process. In Fig. 8b(1), the rst XRD scan is of the turkey tendon alone. Upon mineralization, the sample was rst 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, and the 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 at 2u = 25.98 and 53.18, respectively. The fact that the same sample was examined clearly indicates that there was a gradual transformation taking place from a poorly ordered precursor into a distinctly crystalline phase with apatitic structure. 3.3. Electron diffraction analysismineral identication Phase identication of calcium phosphate compounds, in particular distinguishing OCP and HA, can be difcult 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 bril 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) reections, 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 Biomimetic ) d-Spacing (A 3.44 3.13 2.82 2.81 2.77 2.33 1.99 1.87 1.72
a b
Hydroxyapatite (JCPDS 9-432) Natural aa 0.0 90.0 65.5 36.3 90.0 90.0 55.9 36.7 0.0 ) d-Spacing (A 3.44 3.10 2.81 2.77 2.71 2.26 1.92 1.84 1.72 aa 0.0 90.0 65.5 38.0 90.0 90.0 58.3 38.4 0.0 0.02 2.10 2.11 1.12 3.00 3.10 2.22 2.13 0.04 3.44 3.08 2.80 2.78 2.72 2.26 1.94 1.84 1.72 0.0 90.0 66.5 36.9 90.0 90.0 56.4 37.5 0.0 Plane ) d-Spacing (A a
b
Octacalcium phosphate (JCPDS 79-0423) Plane ) d-Spacing (A ab
0.02 3.12 7.10 3.22 7.00 6.20 8.22 6.42 0.04
3.43 3.05 2.83 2.77 2.69 2.26 1.95 1.84 1.72
0.0 22.1 87.9 28.7 90.0 86.6 51.9 50.9 0.0
Denotes angle of plane measured with respect to (0 0 2) plane oriented along the collagen c-axis. Denotes angle calculated for the hexagonal HA and triclinic OCP systems [121].
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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) Brighteld TEM image of a single mineralized bril isolated from the mineralized sponge. The dark streaks along the long axis of the collagen bril 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 bril 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 bril (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 brils 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) Darkeld TEM images of the bril 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 bril 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.
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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 HA versus 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 intrabrillar crystal arrangement, we examined individual mineralized brils 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 brils show dark streaks in brighteld TEM (Fig. 9A); the width of the streaks suggest that these are HA platelets which are being viewed edgeon. When viewed in full screen size (via electronic article), many lighter striations can also be seen throughout the bril. Crystals that are not oriented edge-on are more difcult to see since they are extremely thin, but further evidence of the high mineral loading is demonstrated by application of dark-eld TEM imaging (DF-TEM) to the same sample. Imaging with the (0 0 2) diffracted beam (Fig. 9B) shows that the bril is well inltrated 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 bril (Fig. 9CF). To determine the crystallographic orientation of the intrabrillar HA crystals, selected area electron diffraction (SAED) was performed on isolated brils. The SAED pattern of the electron dense, 200 nm diameter bril 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 bril (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 brils using this capillary-inltration mechanism, we did not expect them to have the same orientation as found in bone, given that we did not add any specic nucleating domains or specialized proteins to the collagen. Every mineralized bril (n > 20) we imaged using TEM exhibited the same SAED pattern as Fig. 9B, with the 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 brils, 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 intrabrillar 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 inltrated the collagen brils. The crystals in the image exhibit a mixed brousplaty texture, and apparently had become very elongated as crystal growth traversed along the collagen bril. This brousplaty 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 ne 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 inltration The evidence that intrabrillar 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
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Fig. 10. FE-SEM micrographs of mineralized collagen brils treated with 2% NaOCl solution to remove surrounding collagen and examine the morphology of the remaining mineral phase. (A) Bleach treatment on the PILP mineralized Cellagen1 sponge reveals a high degree of mineral penetration throughout the brils, with HA crystals that are highly elongated and appear to be a nearly continuous phase. Collagen has a distinctly different texture when viewed by SEM (it is smooth, and readily succumbs to beam damage), thus these brous structures are clearly the mineral remnants of the composite after bleach treatment. Nevertheless, EDS spectroscopy was used to conrm the presence of Ca and P peaks, which dominated the composition of the bleached sample (not shown). Bar = 1 mm. (B) At higher magnication, it is seen that the HA crystals exhibit a platybrous texture, with a meandering growth pattern that may explain the rotational misorientation of the crystals seen in diffraction. One can imagine that crystals such as these could be perceived as either needles or platelets when viewed by TEM, depending on the location and orientation of the crystals. This could perhaps explain why the literature is so conicting regarding the morphology of bone crystals. Bar = 100 nm. (C) Natural cortical bone samples from equine femur and (d) turkey femur after bleach treatment. The remnant mineral in the equine bone is coherent and maintains the bril shape with relatively featureless texture, while the turkey bone seems to have a meandering ultrathin brousplaty texture. Bar = 1 mm.
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 conned 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 difcult 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 whether the polymer can diffuse into the collagen scaffold independently or whether the capillary action is required (at least in our in vitro system), we conducted preliminary experiments on the diffusion characteristics of uorescently tagged polyAsp when it was applied to a collagen substrate in solution versus when it was present in the PILP phase (Fig. 11). Using confocal microscopy, we examined the depths of penetration of the
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Fig. 11. Confocal uorescence microscopy showing depth of penetration of FITC-labeled polyAsp into dense collagen scaffolds (sliced turkey tendon). Micrographs on the left were imaged with 400 PMT, while micrographs on the right were imaged at 500 PMT and offset to a greater depth to capture the uorescence below the line of excessive intensity near the surface. (A) Control samples (without phosphate counterion) consistently yield penetration depths of around 100 mm. (B) Tendon mineralized using the PILP process show very high intensities near the surface, and then continued penetration at larger depths, but with signicant loss of intensity. A z-offset of 120 mm was used for the control sample (overall depth of 590 mm) and 180 mm was used for the mineralized sample (overall depth of 650 mm). Note: all the control samples had much lower intensity proles and hence the offset was lower to include the faint edge of the highest intensity. All depth scale bars = 500 mm.
polymer into dense collagen scaffolds, to determine how far the polyAsp alone could diffuse into a collagen scaffold, as opposed to polyAsp that was pulled into the collagen scaffold by capillary forces that act on the phase boundaries of the PILP phase. The control sample was maintained under identical solution conditions, excluding the phosphate counterion to avoid PILP phase formation. For this study, a very dense collagen scaffold was needed in order to avoid diffusion of polymer through the large interbrillar pore spaces found in most synthetic collagen scaffolds. Therefore, we chose to mineralize turkey tendon obtained from young birds prior to its natural mineralization. The tendon provides a very uniform and dense collagen scaffold due to the parallel alignment of the brils that is more typical of the packing density of lamellar collagen in bone. Four tendon samples were tested, with a representative example shown in Fig. 11. Only the interior of the turkey tendon mineralizes (the surrounding sheath is inhibitory), so the tendon was split down the middle and opened up, such that two equivalent samples could be placed face up in the solution, ensuring uniformity of the collagen scaffold in the parallel penetration experiments. The confocal images in Fig. 11 represent a vertical optical slice into the tendon, with the vertical scale bar marking the depth of penetration of the FITC-labeled polyAsp. As seen in Fig. 11A, the collagenous tendon that was treated with FITCPolyAsp alone, with no PILP phase, exhibits a uorescence signal to a depth of $100 mm, while the same tendon exposed to PILP phase shows a signal at a much greater depth. Not only was the uorescence markedly more intense, the depth of penetration had more than doubled. We examined beyond the region of brightest uorescence with a PMT detector of higher sensitivity and found that, in fact, some polymer had traversed well beyond 500 mm, though the uorescence intensity there was substantially reduced. This deep penetration was never seen in the control samples using the same detector PMT. While this data provides evidence for the role of capillary forces in our in vitro model, further work is needed to quantify the forces involved and the rates of diffusion. At present, it is unclear why there is a sharp intensity falloff delineating regions of strong and weak uorescence intensity in many (but not all) samples. The observation may be related to the fact that transport of the polymer becomes hindered as the PILP phase begins to solidify. In addition, much of the polyAsp gets excluded during solidication of PILP phase (found in CaCO3 PILP studies [110]), which could leave an excess concentration of polymer remaining toward the outer surface of the collagen, while the mineral ions of the phase continue to be transported inward, so long as the phase remains a uid.
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4. Discussion Our biomimetics laboratory has a keen interest in the molecular mechanisms involved in biomineralization, both within invertebrates and vertebrates, and has taken the approach of using in vitro model systems to examine the types of interactions that can occur between organic and inorganic species in complex media. More recently, we have turned our attention toward trying to understand the mechanisms involved in bone formation. Our rst experiments were successful with respect to demonstrating proof-of-concept that a mineral precursor could be inltrated into collagen brils [115,116,119,120], however, the reactions were based on CaCO3 because, at that time, we only knew how to generate a PILP phase for CaCO3. Given that CaCO3 is not the mineral system of bone, we have now pursued mineralization of collagen with a calcium phosphate (CaP) PILP phase to provide a more applicable in vitro model system for studying potential mechanisms involved in bone formation. 4.1. Summary of mineralization results using the PILP in vitro model system As has been illustrated repeatedly in the literature [66,134,135], type-I collagen can serve as a heterogeneous substrate for nucleating large hydroxyapatite crystal clusters when placed in a solution supersaturated with respect to calcium phosphate (Fig. 6D). These clusters are no different from HA that nucleates on any compatible substrate. This form of mineralization is not what occurs in the body, and thus serves as a negative control reaction and is one indication that intrabrillar mineralization must involve more than merely growing the crystals directly from solution. When the Cellagen1 scaffold was mineralized in the presence of the anionic polyAsp additive (which we consider a simple mimic of Asp-rich NCPs), we propose that the collagen brils rst absorb the uidic amorphous precursor (Fig. 7A), which subsequently crystallizes as the waters of hydration are released during the solidication and crystallization stage of the process (Fig. 7B). The X-ray diffraction studies show initially broad peaks that sharpen with time (Fig. 8a). The data alone, however, are insufcient to conclude that the mineral is amorphous, because the initial broad peaks (Fig. 8a, 4) could arise from a collection of ordered crystals of nanometer dimensions, and subsequent growth of the small ordered crystals to larger ordered crystals could account for the narrowing of the XRD peaks as the mineral phase matures, as has been suggested for bone [18]. Therefore, XRD patterns were acquired sequentially, with interim removal of early stage mineralized collagen from solution to ensure that crystals did not grow from addition of ions entering from the crystallizing solution (Fig. 8b). In other words, if crystal growth were to occur, it would have to derive from mineral phase already present within the collagen bers, because it is inconceivable that such peaks could arise solely from the limited supply of ions adsorbed onto the collagen brils. The peak narrowing in this XRD series suggest that crystals are indeed forming, which we interpret to mean they are growing from an already present amorphous phase. Evidence for an amorphous phase is also provided by TEM analysis (Fig. 7A inset), because the isolated collagen bril which produced a diffraction pattern with only diffuse rings was clearly well inltrated with mineral, since this sample had not been stained. In other words, the strong contrast must have arisen from a more electron-dense mineral phase, which preferentially stains the hole zones and reinforces the banding pattern. Interestingly, when these samples are observed in the SEM, the surface of each ber is relatively smooth and featureless (i.e., non-faceted), much like that seen in the resting surface of natural bone (Fig. 3A) [136]. The smooth appearance of the mineralized bers (and bone) is counterintuitive to the idea that they are inltrated with platelets of HA, but this is a result of the limited resolution of SEM. When the fully mineralized substrate has been fully deproteinated, platy hydroxyapatite crystals are observed by TEM, highlighting the difference between viewing nanocrystals that are within the brils, versus imaging the surface of the brils by SEM. Lastly, even after the amorphous mineral has crystallized, the bers retain the smooth outward appearance, yet appear striated when imaged in transmission by TEM. When they are fractured and bleached to remove collagen, needlelike crystals can be seen protruding from the fracture surface, which lie parallel to the long axis of the ber (Fig. 7E). It is well-known that the [0 0 1] direction of hydroxyapatite is the fast growth direction during crystallization [18,64]. Assuming therefore that the high aspect dimension of the crystals corresponds to the [0 0 1] direction of hydroxyapatite, the visual appearances of the structure corresponds well with the electron diffraction analysis (Fig. 9), which indicates that the crystal orientation is the same as that observed in bone. Altogether, the model system provides a picture of how a simple polyeletrolyte can transform the conventional solution crystallization process (i.e., nucleation and growth of large hydroxyapatite crystals on the surface of collagen) into a precursor process (crystal growth from organization of ions derived from an amorphous precursor), and the
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dramatic differences that can result. For this reason, we designate the role of the polymer as a process-directing agent (as opposed to a structure-directing agent, which implies a structural relationship between a crystallographic face and the additive). In the case of the PILP process, the additional consequence of forming an amorphous precursor that is sufciently hydrated to be a liquid phase is that it enables the collagen brils to become impregnated with the mineral precursor, which we propose occurs via capillary action (Fig. 5). At this point, it is important to note that calcium phosphate commonly forms an amorphous gel-like precursor at moderate supersaturations [39,64]; but the PILP phase apparently differs, because the negative control reaction (which utilizes concentrations of calcium phosphate that normally form the gel phase) does not lead to intrabrillar mineralization. Therefore, we suggest that the primary role of the polymer may be to provide a means for stabilizing a more highly hydrated amorphous phase that imparts it with the uidity necessary to be transported into the collagen bers via capillary action. The following provides an in depth discussion as to the possible ramications of these data to bone formation. 4.2. Amorphous to crystalline transformation: comparison to existing literature Recent advances in the area of glass science have resulted in an improved understanding of short- and mediumrange order in glasses. It has been recognized that amorphous glasses may have different types of medium-range order. For example, molecular simulations of amorphization of silica have shown that the resulting amorphous glass can have different types of medium-range order that is similar to one of the different polymorphs of crystalline silica [137,138]. Differences in medium-range order can be observed through the rst strong diffraction peak (FSDP) which occurs at values of the scattering vector less than the peak corresponding to nearest-neighbor correlations [139]. Importantly, the presence of this Bragg-like peak at low values of the scattering vector suggest medium-range periodic correlations, even in these amorphous materials. Peak-width calculations suggest that these correlations can extend for up to 3 nm, and the radial distribution functions for these glasses show peaks at these distances [140]. Direct comparison of the RDFs obtained from simulated silica glass and various crystal polymorphs shows that the local structures are identical at distances up to 0.75 nm, while they diverge at larger distances [141]. There are several implications of these results for the eld of biominerals. First, previous studies have shown that there can also be variation in the local order of amorphous biominerals. The presence of a transient amorphous CaCO3 precursor phase has been detected in many species (sponge spicules [142,143], sea urchin larval spicules [144,145] and adult regenerating spines [146], mollusk larvae [147,148] and adult nacre [149]). As shown by Hasse et al. [148] and Weiner et al. [143,150], there can be a great deal of variability in the structure of biogenic amorphous phases, and in some cases, there appears to be differences in short-range order of the amorphous phase (as determined by EXAFS and vibrational spectroscopy) that appear to inuence whether the precursor transforms into calcite or aragonite. Another implication concerns the presence of an amorphous precursor in bone formation. While past studies have used the correlations present in the radial distribution function of newly formed bone to argue for the lack of an amorphous precursor, the results found for silica glass suggest the presence of these correlations, even up to 3 nm, does not necessarily rule out the presence of an amorphous phase [137,138]. Thus, the question of whether or not there is an amorphous precursor during bone formation needs to be re-visited in light of recent advances in understanding of the structure of glasses. Evidence provided in Section 3 and discussed further below suggests that it is appropriate to consider the precursor phase as amorphous, at the same time recognizing that medium-range order is likely to be present. In our system, the presence of an amorphous CaP precursor was determined by electron diffraction of isolated brils (Fig. 7A), as well as by XRD of bulk scaffolds (Fig. 8). The time series experiments (Fig. 8a, 35) demonstrate that the XRD patterns gradually transform from broad diffuse peaks to sharper, more crystalline peaks. It should be noted that the pattern of our mineralized collagen (Fig. 8a, 5) appears similar to the XRD pattern of natural bone (Fig. 8a, 6), and, even in the later stage, both have much broader peaks than collagen mineralized without the polymeric process-directing agent (Fig. 8a, 2). The broad peaks are usually attributed to the extremely small dimensions of the HA crystallites, which is presumably due to the limited space available to intrabrillar mineral. Broad diffraction peaks could also arise from weak long-range periodic correlations, or from crystal strain (which is seen in PILP-formed crystals due to the presence of polymeric impurity and shrinkage during the solidication process [106]).
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As previously mentioned, there have been observations of amorphous calcium phosphate deposits, described as inorganic substance in bands (ISBs), detected in the hole zones in early mineralized bone [27], as well as in our sample of mature equine bone (Fig. 4C). In contrast, Glimcher et al. argued that the presence of peaks at distances of up to 2.5 nm in the radial distribution function of newly forming bone of chick embryos indicated that an amorphous phase was not present [81]. These results have often been interpreted as indicating that an amorphous phase plays no role in the mineralization process. However, the recent results on silica glass described above indicate that amorphous phases can show correlations even at these distances, and thus the presence of these peaks in the RDF from bone does not eliminate the possibility that an amorphous phase is present. Glimcher et al. recognized the possibility of a transient amorphous precursor, as indicated by the following excerpt from page 116 of their paper: It is, of course, possible that an amorphous phase exists as a transient intermediate in the formation of bone mineral, with lifetime so short that it disappears or transforms to a more stable phase before any X-ray diffraction evidence for its presence can be found, even in the rapidly frozen and anhydrously processed samples used in our studies. . . However, such a transient phase must differ from the (amorphous calcium phosphate) phase originally proposed. . ., which was envisioned as one phase of a two-phase ACP and (HAP) system, with the ACP being sufciently stable and long-lived that it could readily be detected as the major constituent of bone mineral in young animals. It is apparent from this excerpt that the amorphous phase they were considering was very different from the amorphous precursor we observe in our in vitro system. Specically, they were looking for a stable and long-lived amorphous phase that co-exists with crystalline hydroxyapatite in a two-phase system, and discount the possibility of such an amorphous phase being present. However, they do accept the possibility of a transient amorphous precursor to the crystalline phase, which is the mechanism we are proposing. Crane et al., who recently provided Raman spectroscopic evidence of a transient OCP (and possibly ACP) precursor in bone indicate that unless mineralizing tissue is examined spectroscopically almost immediately after it is harvested, the observed mineral will be a carbonated apatite [128]. This question as to the mineralization pathway in bone formation still remains open, because even if the amorphous phase is short lived, our results suggest that it may play a critical role in inltrating the brils with the mineral precursor in the rst place. The inability to detect transient precursors can often arise from ex vivo evaluation, highlighting the usefulness of in vitro models for examining mechanistic issues that cannot be readily examined in vivo within the complex biological environment. In our proposed mechanism, the PILP process starts off as an amorphous liquid phase. We suspect that local ordering might arise fairly quickly upon interaction with the collagen, given that the crystallographic orientation is so strongly modulated. Therefore, intermediate-range order as measured in the chick embryonic bone [81] would not be surprising. It is also important to note that the amorphous phase itself is dynamic. The material changes composition steadily throughout the whole process as the polymeric process-directing agent and hydration water are excluded during the transformational stage of the process (i.e., solidication and crystallization) [107]. In our mechanism, it seems appropriate to consider the PILP phase as amorphous in the initial stages; further studies are needed to determine when and how long-range periodic correlations develop during the amorphous to crystalline transformation. It is clearly important to be cautious when employing an in vitro system to build an understanding of more complex biological phenomena. For example, in judging whether this in vitro PILP system can be considered a possible model for bone formation, one needs to compare our diffraction data to the data available for the early stages of natural bone formation, particularly considering that the landmark study mentioned above gave the perception that the early stages of bone in chick embryo did not consist of amorphous mineral. Therefore, it is instructive to take a closer look at their XRD data (which is provided as a reprint in Fig. 12 [81]), and compare to our results (Fig. 8). Looking beyond the earliest stage of our system, the patterns are strikingly similar in terms of both peak widths and their evolution. Both systems start from a very poorly ordered mineral phase, and then evolve into what is often described as the poorly ordered hydroxyapatite of bone. It is clear that bone formation does occur via transformation of some type of very poorly ordered precursor, as illustrated by the X-ray diffraction data of Fig. 12. It seems that the amorphous precursor theory was abandoned in part because of the lack of understanding at the time of medium-range order in amorphous materials, and perhaps because of a misperception of the mechanism described by Glimcher et al. [81]. Much of the bone community has since turned their efforts towards nding a specic nucleating protein for hydroxyapatite, but in our opinion, this mechanism would not be any more consistent with the XRD data from both bone and our composite.
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Fig. 12. From the prior work of Glimcher and co-workers [81], XRD patterns of early stage bone formation in chick tibial mid-diaphyses. (a) 17-day chick embryo, periosteal bone scrapings; (b) 5-week post-hatch chick, 2.12.2 g/cm3 density fraction; (c) 2-year old chick, 2.22.3 g/cm3 density fraction; (d) synthetic HA (reprinted from [81], (Journal of Crystal Growth 53 (1)), with permission from Elsevier).
These data clearly show that the initial broad peaks narrow over time, indicating that the mineral develops through some type of phase transformation process. While the XRD results may be consistent with either a solution crystallization mechanism or an amorphous precursor mechanism, the amorphous precursor mechanism is more consistent with the totality of the data, at least for our in vitro system. Another issue to consider in identication of an amorphous precursor is the presence of other (crystalline) phases that make identication of an amorphous phase difcult. For example, in the chick bone, as each concentric lamella is laid down consecutively during secondary bone formation, only a very small fraction of the bulk sample would still be amorphous as the prior layers will have already begun to take on order. In other words, the intimate association between the collagen and the mineral precursor will likely cause local ordering to occur on a fairly rapid time scale (this might even be enhanced by the vacuum drying process that was used to remove water prior to the Glimcher XRD measurements). Indeed, in our in vitro experiment, the amorphous phase that inltrates the collagen crystallizes more quickly then the amorphous phase that ends up depositing on the bottom of the petri dish. In conclusion, one should keep in mind that the absence of evidence is not the same as evidence of absence. One should not expect to be able to detect the presence of an amorphous phase if partially ordered mineral was also present, since both XRD and RDF analysis are averaging techniques. This is only possible in our in vitro system because the evolution of the mineral is synchronized such that mineral is produced uniformly across the entire collagen scaffold at one time. 4.3. Clarication of crystal orientation As we carefully analyzed the electron diffraction data of these samples (Fig. 9), some concern arose over the accuracy of the long-held views of the orientation of crystals within bone. As in bone, the arcing of the (0 0 2) and (0 0 4) diffraction maxima suggests that the HA crystals are not perfectly oriented uniaxially along the c-axis of the
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collagen bril (the angle subtended by the arcs indicates a misorientation of up to 158 from the c-axis). This misorientation has been described as tilting of the crystals, rst reported by Weiner and Traub in their studies on the structure of bone [129], and later corroborated by Landis and co-workers on samples of naturally mineralizing turkey tendon through three-dimensional tomographic imaging with TEM [57]. The d-spacings of the three diffracting planes are extremely close (see Table 1), so a combination of the three arcs generates a pattern of what appears to be an almost continuous ring. It is important to realize, however, that this is not really one continuous ring, but corresponds to the nearly overlapping arcs corresponding to diffraction by the (1 1 2), (2 1 1) and (3 0 0) planes. As mentioned in the introduction, the existing deck of cards model of bone nanostructure suggests that the HA platelets are not only uniaxially oriented, but they are often illustrated as being coherently aligned within the ab plane such that all platelets appear to be parallel [57,58,100,129,151,152]. Upon closer analysis of our electron diffraction data, which are identical to that from natural bone and turkey tendon, we conclude that these intensely diffracting spots/ arcs from the (2 1 1) and (3 0 0) planes (as well as the weaker (2 1 0) and (3 1 0) spots) are not consistent with the biaxial orientation that is commonly illustrated in the literature [101,129,153155]. Both synthetically and in bone, HA forms platy crystals that express the (1 0 0) faces. Therefore, a at platelet lying normal to an electron beam (such as would occur when the long axis of a collagen bril is oriented transverse to the electron beam, and the deck of cards coplanar crystals are aligned with their (1 0 0) faces normal to the beam), should only diffract from the (0 0 2), (1 1 2), and (1 1 0) planes, with the maxima at angles of 08, 368, and 908, respectively (i.e., the (h h l) type planes), from the (0 0 2) spot. Due to the shape factor of these nanometer sized platelets, slight misorientations of the crystals could cause additional planes to diffract, thereby appearing in the diffraction pattern. Yet, if this were the case, the d-spacing of these planes should change depending on the misorientation. This is not the case here, as the d-spacings for each plane match those listed in Table 1. These planes are readily observed in the diffraction patterns of intrabrillar bone and turkey tendon (as well as in our mineralized brils); but in addition, spots/arcs from the (2 1 1), (2 1 0), (3 0 0) and (3 1 0) also appear, diffracting at angles of 658, 908, 908 and 908 from the (0 0 2) when the beam is normal to the (1 0 0) face. In particular, the (2 1 1), (1 1 2) and (3 0 0) maxima are very intense (Figs. 4B and 9B) and are providing additional information that should not be ignored, since the presence of these diffracting planes indicates that the crystals are not stacked in parallel planar arrays. While parallel stacks of crystals may occur locally, the selected area diffraction covers the entire bril diameter (in our synthetic sample), indicating that alignment is not parallel throughout the bril. With such a high degree of rotational disorder, the crystals should probably not be represented as having biaxial order, which would not really be expected for the uniaxial symmetry of a cylindrical ber (Table 2). The hexagonal symmetry is not readily apparent in the platelet morphology of HA, so in Fig. 13, the hexagonal unit cell is superimposed on a hydroxyapatite platelet to demonstrate the crystallographic orientation of the platelets in bone. From the (0 0 1) projection on the top of the unit cell (Fig. 13), it can be shown that, in order for these extra planes to be illuminated in the diffraction pattern from a single mineralized bril, there needs to be a rotation about the 0, by at least 108 for the (2 1 1), (2 1 3), and c-axis of the hydroxyapatite crystals from the beam normal, B 1 1 (2 1 0); 158 for the (3 1 0); 308 for the (3 0 0). Notethese rotational angles about the c-axis are not the same as the 158 azimuthal tilts from the c-axis, the latter of which has been acknowledged and clearly described in the literature [129]. One might question whether this misorientation could be a side-effect from drying of the samples, but we note that in the analysis of naturally mineralizing tissues, of which researchers took great care to preserve the structural morphology, identical diffraction patterns to our synthetic samples were obtained [129,156]. It should also be noted that when our samples are tilted in the electron beam (up to 368), such that the crystals are rotated about the c-axis of the bril, the diffraction patterns obtained at various positions along this rotation path all consistently contained the appearance of the (0 0 2), (1 1 2), (2 1 1), and (3 0 0) planes at each rotation, again indicating that the platelets are not arranged in a deck of cards, but instead have some rotational disorder about the c-axis. Although we cannot determine from TEM analysis if there is any pattern to the rotation (such as helical twist from a collagen substructure), dark eld TEM images based on the (1 1 2), (2 1 1) and (3 0 0) diffracting planes (Fig. 9DF, respectively) illustrate that the platelets appear throughout the bril for each of these planes, at least demonstrating that these additional diffraction planes are not from crystals in separate regions of the bril. This interpretation is consistent with the visual images of the crystals seen in Section 3.5. Likewise, the visualization of mineral crystals provided by the Landis et al. [57] study using tomographic imaging of mineralizing turkey tendon show similar crystal organization. They state that while not shown by the artwork. . . the orientation or alignment of a particular crystal may be shifted up to about 208 along any of its three coordinate planes from a true
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Fig. 13. Schematic illustration demonstrating the orientation of the relevant planes expressed in the SAED patterns of HA crystals within collagen brils when the beam is directed perpendicular to the large (1 0 0) faces of the platy crystals (beam is parallel to the (1 1 0) faces expressed at the edge of the platelets). This schematic demonstrates that if the crystals were organized according to the deck of cards model, that the (1 1 2), (2 1 1), (3 1 0) and (3 0 0) planes could not mutually diffract. Bottom: three-dimensional model with the HA hexagonal unit cell projected onto an oriented HA platelet, with the platelets oriented such that the (0 0 2) and (1 1 2) planes mutually diffract. In order for diffraction to simultaneously occur for all of these planes combined, some platelets would have to be rotated about the c-axis in order for the additional planes to be parallel to the beam. Top: projections of the (0 0 1) basal plane onto the top of HA platelets illustrate the intersection of the (0 0 2) plane with each plane that simultaneously diffracts (highlighted in bold). The dotted line is the beam direction normal to the (1 0 0) face of the hydroxyapatite crystal (i.e., the 0). crystals are all oriented in the same direction, and the (1 1 2) plane has been chosen as a reference plane which diffracts at a beam direction of 1 1 The angle between the line of intersecting planes and the beam direction is the number of degrees of rotation that would be required for that plane to diffract. Note: This schematic only shows one quadrant of planes, but the three symmetrically equivalent families of planes at other rotations in the hexagonal unit cell could also contribute to these diffraction planes.
parallel direction with respect to the collagen molecule long axis. In their experiment, the disorder was visually observed but not correlated with diffraction patterns, and not represented in their schematic (presumably because it would be difcult to do so). We feel that the perception of perfectly ordered, biaxially aligned crystals tends to persist because of such visual representations. As a case in point, the signicance of these additional diffraction planes becomes apparent in the recent work by Rhee et al. [100], who compare the mineralization of chondroitin sulfate (CS) bers versus collagen bers in an in vitro reaction. Although the individual bers of both scaffolds had orientation of HA crystals in the [0 0 1] direction along the long axis of the bers, the CS pattern is totally lacking diffraction from the (3 0 0) plane [100]. They simply describe this pattern as being more single-crystalline in nature, and suggest that these differences arise from a less ordered arrangement of the collagen bers. Certainly, the very broad 608 arc for the (0 0 2) plane does indicate poor order of the collagen in their system (which is not the case in bone), but the other signicant difference, which is not discussed, is the variable degree of rotational disorder between CS versus the collagen, as indicated by the missing (3 0 0) plane. As is often done when attempting to show bone-like orientation of HA in mineralized scaffolds, most researchers are satised with the uniaxial orientation of the HA mineral in the [0 0 1] direction. Yet, what is often overlooked, and what brings this analysis into focus, is that these additional planes in the SAED diffraction patterns are providing important information regarding the ultrastructure of the interpenetrating phases of the composite. In general, the perfection that is represented in most of the biomineralization literature is misleading because many of the features may be better explained through processes that do not lead to perfect ordering, as is discussed below. We have deliberately avoided showing any specic crystal order within our schematic illustration in Fig. 5 because the rotational variation is still not dened (such as whether there is a gradual helical shift or if it is random). Instead, we prefer to demonstrate the morphology and organization of the crystals in bone using SEM analysis of deproteinated samples (see below), because this is a more accurate representation, and because it can contribute to understanding of how this nanostructured architecture is formed. 4.4. Elucidation of mineral morphology As can be seen in the deproteinated samples shown in Fig. 10, the mineral phase appears to be a coherent and nearly continuous structure, unlike the traditional view of at, isolated platelets of HA stacked like a deck of cards.
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Bagambisa et al. [136] have made similar observations in their study of the ultrastructural organization of bone, in which they indicate that the mineral aggregates coalesce to a dense and continuous mineral mass. There is no apparent suggestion of individual crystal entities in mature bone domains. In Fig. 10C and D, crystal facets can hardly be detected in the natural bone samples, demonstrating the striking difference between visualizing bone HA crystals via TEM versus SEM. The edges of the extracted platelets seen in TEM are probably not distinct facets in the crystallographic sense, but rather the rough and irregular edges may just be fracture surfaces. Likewise, the bro-platy texture of the crystals (as seen in our samples) may explain why the bone literature is full of conicting descriptions of the morphology of HA crystals in bone, which have been described as needles, rods, laments and plates. In large part, that has probably been a result of imaging very thin platelets edge-on in the TEM, which gives rise to the appearance of thin needles; but given the not easily identiable morphology of the crystals seen here, it is clear that the non-descript morphology of the mineral phase could confound this problem. In the natural bone samples, individual crystallites could not be discerned in the deproteinated samples. In contrast, Rosen et al. [47] examined the ultrastructure of anorganic (deproteinated) bovine bone using eld-emission low voltage SEM (FE-LVSEM), and were able to observe individual crystallites once the collagen was removed (Fig. 14a). The advantage of FE-LVSEM is the depth of eld provided by SEM relative to TEM. As in our deproteination study, the nanostructural arrangement of the crystals when viewed by SEM versus TEM differ in outward appearance. In their TEM analysis, the orientation of the crystallites is more readily elucidated in the deorganied bone relative to the whole bone sample (compare Fig. 14c and d, reproduced from same article), and it appears that the rotational arrangements of crystallites (e.g., being on edge or en face to the section, which can be distinguished by the dark striations vs. the lighter gray platelets, respectively), varied throughout, with some regions containing stacks of crystals, but the rotational variation did not appear to alternate in any regular fashion. The deproteination conditions employed in their study (information not provided) likely differed from ours, and led to a more porous appearance due the separation of individual crystallites within the remnant bers. Even with this apparently higher degree of deproteination, they also found that remnant bers were continuous and self supporting, demonstrating that there is
Fig. 14. The ultrastructure of anorganic bovine bone (reproduced from Fig. 1 of an article by Rosen et al. [47]). (a) Field-emission low voltage SEM image showing the natural bone mineral crystallites comprising a brillar structure reecting the collagen template. Periodic bulges along the long axis of the brils, with a spacing of about 70 nm, representing aggregates of crystallites, correspond to the banding pattern of collagen. (b) Higher magnication of (a), with arrows indicating crystallites periodically bridging the brils. From Fig. 4 of same article, TEM diffraction contrast images of the anorganic trabecular bone (c) and (d) unstained whole bone specimen (i.e., not deorganied).
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sufcient inter-crystallite bonding to retain the bril structure after dissolution of the protein template, suggesting that the mineral phase, like the collagen matrix, is continuous. They came to a similar conclusion that bone is not merely a mineral crystallite-reinforced brous material. It must be considered to be a collagen ber-reinforced mineral matrix composite or an interpenetrating composite network (of collagen and the mineral phase). It is noted that the mineralized collagen of bone (Fig. 4) has a more pronounced banding pattern than that seen on our samples (Fig. 7B and 9B). One possible explanation for the difference is that the synthetic reconstitution of collagen brils in Cellagen1 leads to a brillar organization that does not exactly duplicate that of natural collagen, leading to slight differences in the way in which the mineral is distributed throughout the bril compared to natural bone. Even so, the reconstituted collagen seems to work well towards providing a nanoporous compartment for mineral, even though one might expect slightly different morphologies of the molded crystals, as seen in Fig. 10. This may be true in nature as well, where different bones, or different species, may yield slightly different crystal morphologies based on the variability between biological compartments (e.g., Fig. 10C versus D). This issue may be relevant to some disease states, such as osteogenesis imperfecta (brittle bone disease), which arises from defects in the type-I collagen molecules [157]. This apparently leads to excess extrabrillar crystals which are larger than crystals found in normal individuals, while the intrabrillar crystals are smaller than those in age-matched normal bone. An additional morphological consideration pertains to the intra- versus inter-crystalline morphology of the mineral phase. One gets an entirely different impression of the mineral when the surface topography is viewed. For example, Bagambisa et al. [136] have shown the resting surface of a dog canine femur to be very rough, describing it as a smooth but somewhat globular surface with brils that appear clubbed (reprint provided in Fig. 3A). This is often observed in our mineralized brils as well (Fig. 7C), yet when these same structures are bleached to preferentially remove the organic phase, the bro-platy HA crystals are observed (Fig. 10A and B), illustrating that while the surface morphology appears non-descript, the crystals on the interior are more plate-like, and especially when extracted and examined by TEM (Fig. 7F). The morphology in our system is consistent with the description provided by Landis et al. [57] of crystals in mineralizing turkey tendon when viewed by 3D tomographic reconstructions (see Section 1.3). The descriptions in this paper are based on the assumption that the crystals are nucleating de novo, rather than from an amorphous phase. But their images are not inconsistent with how the collagen might appear if the crystals were forming via solidication of a highly hydrated amorphous precursor. For example, the material surrounding the dark crystal outlines is described as a stippled haze, which is attributed to viewing the particles face-on where they contribute only very slightly to electron scattering, such that a somewhat dark (electron dense) haze is observed. They suggest that this likely accounts for the nely stippled, periodic density observed in many micrographs of collagen mineralization. This less pronounced non-uniform electron density could also be accounted for by the presence of an amorphous precursor. Their descriptions also indicate that individual crystals have irregular edges and are of a highly variable length and width, and viewing of stereoscopic pairs shows that twisted threads of mineral appear to bridge the crystal planes, where smaller and larger crystals appear to fuse in coplanar alignment to form larger mineral platelets. Such a fusion process could arise from a precursor phase, in which the PILP phase often exhibits cementatious properties as it solidies. Their observations also indicate that a crystal is not conned by the length of either the collagen hole or overlap zone, which is consistent with the interconnected crystal array seen in our etching studies (Fig. 10). They suggest that a process of interconnection and fusion may produce larger and generally coplanar units from smaller particles. Considerable attention has been given to this issue of how the crystal dimensions in bone do not match the dimensions of the hole zones in collagen. This dilemma has been addressed by suggesting that the collagen brils order themselves such that the hole zone regions are in register across adjacent molecules to form extensive parallel channels or grooves (as represented in Fig. 2), which could provide space for crystal development in width across brils. This alignment appears to occur for reconstituted collagen brils as well, if they can be organized in close packed parallel arrays (as seen in Fig. 6C). However, this alignment is probably not necessary for answering this question, because if a mineral precursor is being pulled into the collagen by capillary forces, it is likely being pulled all throughout the intrabrillar space, and upon solidication, leaves mineral in its path, in and around all the collagen molecules, regardless of the dimensions of the hole zones. On the other hand, the hole zones will likely contain more mineral due to more space. This is seen in our prior work with CaCO3 mineralized collagen [120], where a pronounced banding of increased mineral density could be seen along the brils. In the case of CaP mineralized
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Fig. 15. An atypical but illustrative example of mineral crystals growing within and emerging from the collagen ber constraints. This sample was not treated with bleach, but evidently was so fully mineralized that the crystals protrude beyond the intrabrillar constraint. (A) At low magnication, the bers are fairly aligned in this region and appear linear except for periodic bulges in some regions that disrupt the linearity. Bar = 2 mm. (B) At higher magnication, the edges of HA crystals can be seen protruding from the surface of the collagen brils. This sample was not treated with bleach, but evidently was so fully mineralized that the crystals protrude beyond the scaffolds constraint. The crystal edges appear to be oriented roughly parallel to the brils. Bar = 100 nm. (C) In this region, the bulges along the bril appear to arise from the outgrowth of crystal clusters. The clusters resemble those of the normal growth pattern of spherulitic hydroxyapatite. Bar = 1 mm. (D) It appears that the collagenous constraints have fully broken down in the upper region to the right, yielding a random mesh of HA polycrystals on the surface. Some of this nonconstrained meshwork can also be seen in regions of (C). Bar = 1 mm.
collagen, clubbing along the brils is also seen, and it is particularly pronounced in Fig. 15 which had undergone excessive mineralization. Interestingly, in the Landis et al. study [57], strand-like features were detected (in addition to the collagen), which remained after the mineral component was removed from the volume renderings of reconstruction. It was suggested that these may be non-collagenous proteins that are involved in regulating crystal formation. They state that, it is difcult to understand how other molecules in addition to collagen can be accommodated in the available volume. Perhaps the organic material might be occluded from the crystal surfaces. . . These organic envelopes could be easily understood as arising from exclusion of the polymeric-process-directing agent as the PILP phase crystallizes [110], and the mechanism for bringing those NCP components into the collagen in the rst place would be the capillary inltration mechanism, as discussed below in Section 4.6. 4.5. Mechanism of alignment of intrabrillar crystals If one is convinced that this in vitro system is a valid model of bone formation, then it may be able to shed some light on the issue of how crystallographic orientation is regulated during bone formation. Importantly, we have not added any protein that would be considered to provide an epitaxial relationship with HA [98,158], therefore, the uniaxial crystal orientation appears to be derived only through interaction with the surrounding collagen matrix (and/or
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polyAsp), but only when the collagen interacts with an amorphous precursor phase. Although one might argue that polyAsp could serve as a nucleating template, we deliberately used the achiral (a, b, D, L) form of polyAsp in these studies, which should not possess a regular secondary structure; likewise, polyacrylate can be used in place of polyAspartate, highlighting the non-specic character of the interactions arising from the polymeric process-directing agent. This is not to say that specic interactions could not occur in bone formation, but in this model system, which was devoid of NCPs, the collagen appears to be the primary template. At this point, we cannot say with certainty whether the collagen is directing the crystallographic orientation through structure-based chemical interactions, or if it is simply a result of constrained growth in the naturally favored [0 0 1] direction of hydroxyapatite. In either case, we demonstrate that intrabrillar mineralization with uniaxial crystal orientation can be achieved through a relatively simple process, and without epitaxial nucleation of NCPs. In our view, it seems unlikely that Mother Nature would have adopted a more complex system when not necessary. This is not to say that the variety of NCPs in bone do not serve complex functions; but rather, a variety of interactions may have evolved for other more specic functions (such as kinetic controls) than have been examined here. It is interesting to consider that collagen serves as the primary template of crystal orientation, because this has never been considered to be the case given that collagen alone does not orient crystals grown via the traditional nucleation and growth process. It is possible that an epitaxial mechanism occurs when the crystals are nucleated from the amorphous phase, but the literature does not point to any possible epitaxial relationships between collagen and HA; therefore we are presently inclined to believe that simple surface energetics (e.g., charge density) may dominate the crystal nucleation event, that along with the constrained environment, leads to moderately well aligned crystals (although not perfectly aligned, as is the impression given by much of the biomineralization literature). If we believe that collagen alone is responsible for templating the crystal orientation, then the question becomes how might this occur? To answer this question, we turn to one unusual sample (Fig. 15). This sample was not presented in Section 3 because it is not representative of the typical morphology of the mineralized brils; nevertheless, it does show some interesting features that may provide insight into the crystal growth process. As can be seen in Fig. 15A, the edges of some platelets can be seen protruding from the surface of the brils (this is best seen by enlarging the gure to full screen if an electronic le is available). The edges of the crystals appear to be uniaxially oriented parallel to the collagen (Fig. 15B), thus the crystal organization is likely representative of the more typical samples in which intrabrillar crystals cannot be seen on the surface. Interestingly, clubbing can be seen along the brils, in which it appears that the crystal bundles grew in a uniaxial fashion when they were restricted by the bril compartment, until they overcame the constraint and emerged from the brils (Fig. 15C). Where the crystal bundles emerge, they appear to splay outwards, resembling the more typical spherulitic clusters of HA. In other words, it appears that the uniaxial orientation of the crystals may simply result from a uniaxial constraint of the common spherulitic growth pattern of HA within the collagen bril, rather than any specic epitaxial type interaction. In this example, where the constraint apparently broke down, the crystals were able to spread laterally, resulting in the more typical clusters of HA (Fig. 15C). In some regions of this sample, there was a meshwork of mineral platelets on the surface where it appears that there was too much mineral to be constrained within the collagen brils (Fig. 15D), perhaps caused by the high supersaturation that was used. A breakdown in constraint is not likely to occur in bone because the supersaturation is lower in the physiological environment, and the collagen brils are tightly packed into parallel arrays. This mechanism of constrained crystal growth would not be expected to lead to the deck of cards arrangement of crystals as described for bone, and in fact, it does not. However, this type of crystal arrangement is consistent with the electron diffraction patterns of our samples, as well as bone, thus demonstrating that the former description of bone nanostructure may be inadequate, as described in the Section 4.3. This conned growth mechanism would account for the rotational variation indicated by the SAED patterns, because one should not really expect biaxial-ordered coplanar arrays of crystals when the symmetry is based on bundles of cylinders (tropocollagen rods), because the precursor phase could presumably inltrate around all sides of these cylinders. Both azimuthal and rotational misorientations can be seen in the deproteinated samples shown in Fig. 10, and the images seems to give the impression that crystals are traversing along the collagen following a brous growth pattern, perhaps with some twisting as they continued to grow throughout the bril. Landis et al. [57] observed an apparent curving of neighboring mineral platelets in their 3D TEM reconstructions, which they attributed to crystals located toward the surface of the cylindrical brils. This could also result in the rotational misorientations described above. The diffraction evidence, in combination with the SEM images of mineralized brils, lead us to propose that the crystals may be simply be nucleating and growing from the precursor phase in a fashion typical of HA grown on many
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substrates, but due to the constraints of the collagen matrix, develop into roughly parallel uniaxial arrays. In other words, there is a large body of literature which illustrates that HA commonly grows in the [0 0 1] direction, which is either due to a preferential nucleation from its basal plane, or alternatively, the [0 0 1] growth direction predominates because the faster growth direction can squeeze out other crystallographic orientations when spatially constrained. In either case, the point here is that when an intrabrillar precursor phase is constrained by the nanoscopic space within the collagen brils, both the dimensions and growth direction of the crystals become limited, leading to nanocrystals that are stacked in uniaxial arrays within the collagen brils. 4.6. Mechanism of mineral penetration and implications for application From the confocal study, it appears that capillary action is responsible for the deeper penetration of the polymer, which carries along with it the sequestered ionic constituents and hydration waters that make up the PILP phase, thus leading to the high degree of mineral inltration within the collagen matrix. The collagen scaffold acts like a large sponge to absorb the uidic amorphous mineral phase, and since capillary forces are strong and can act over large distances, much greater penetration depths can be achieved than by simple diffusion of the polymer. It is worth noting that the reaction solution in the presence of the polymer remains fairly clear, unlike the control reaction, which forms a cloudy solution from precipitation of an amorphous calcium phosphate gel (which is typically seen at these relatively high ion concentrations). The polymer-stabilized nanoscopic droplets of PILP phase are too small to scatter light, and apparently inltrate the collagen before being visually observed. For example, light scattering studies on the PILP process with CaCO3 [159] found that the PILP droplets start off with nanoscopic dimensions, and if given time, will slowly grow in size due to aggregation and coalescence (until they become too solid-like to fully coalesce). With the collagen scaffold, the droplets are apparently rapidly soaked up, such that a mineral coating is not apparent on the surface until after the interior has been fully inltrated with the mineral precursor (although evidence of some remnant droplets can be seen in Fig. 7B). It should be pointed out that the collagen is in an aqueous solution and already in the swollen state; therefore it is the phase boundaries of the PILP phase that are necessary to create the interface which is acted upon by capillary forces. This is evident from the control reaction, which shows that ions from the traditional solution crystallization medium do not permeate the hydrated collagen at sufciently high concentrations to nucleate crystals within the collagen, and there is not sufcient diffusion of the anionic polymer into the collagen to help stimulate nucleation of these solution born ions. The point to be made here is that the polymer is not simply a chelator of ionsit induces the formation of a new phase, and this appears to be critical for achieving intrabrillar mineralization. This capillary mechanism for intrabrillar mineralization has important implications in terms of utilizing this biomimetic process for the fabrication of bone substitute materials. In principle, it seems that the limiting factor for mineralizing bulk large-scale collagen scaffolds may arise from the solidication rate of the precursor phase, rather than diffusive transport. Therefore, optimization of the polymeric process-directing agent and other reaction conditions are being actively pursued for the fabrication of biomimetic bone graft substitutes which have the potential to be both load-bearing and bioresorbable. As described in the Introduction, it is the metastability of the mineral phase of bone which allows it to be readily resorbed by osteoclasts. Likewise, it is the interpenetrating nanostructured architecture that lies at the foundation of bones unique mechanical properties. Once the materials chemist is able to fabricate a collagen scaffold that is more representative of bone (parallel-bered), the goal of making a synthetic bone graft substitute which can replace the current gold standard may be reachable. Presently, the gold standard for bone substitutes is to retrieve autograft tissue from another location on the patients body (typically from the Iliac crest), but the donor site morbidity and limited supply makes this a less than ideal approach. Therefore, allograft (cadaver) tissue has become a popular alternative, but it is also limited by short supply, as well as the inherent concerns regarding disease transmission [67]. Therefore, regardless of whether this in vitro system mimics the process of bone formation, it clearly mimics the nanostructure of bone, and thus may provide valuable attributes that enable the fabrication of biomimetic bone graft substitutes. 5. Conclusions The nanostructured architecture of secondary bone has been duplicated through the use of a polymer-induced liquid-precursor (PILP) process. Based on these results from a relatively simple in vitro model system, we hypothesize
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that the high degree of intrabrillar mineralization that occurs in bone formation is achieved by capillary action applied to a uidic amorphous precursor phase that is induced by the highly anionic NCPs of the bone matrix. By using carboxylate-rich biomimetic polypeptides, corollaries to the NCPs in natural bone and dentin, we have demonstrated that an amorphous mineral precursor phase can be used to create a highly loaded mineral/organic composite with 25 100 nm long platy HA crystals oriented in the [0 0 1] direction along the long axis of the collagen bril. The uidity of the amorphous precursor phase, which is a function of the polymeric process-directing agent, is thought to play a crucial role in achieving intrabrillar mineralization through a capillary inltration mechanism. The SAED patterns of individual mineralized brils are basically identical to those of bone and naturally mineralizing turkey tendon. By demonstrating the orientational relationship of platelets along the bril with respect to not only to the d-spacings and (0 0 2) and (0 0 4) planes, but to the d-spacings and angles of all of the diffracting planes (e.g., (0 0 2), (0 0 4), (1 1 2), (2 1 1), (2 1 0), (3 1 0), and (3 0 0)), we have demonstrated a method which can be used to easily distinguish between OCP and HA. Careful analysis of the diffraction patterns with consideration of all the diffracting planes suggest that the platelets in bone are not as well organized as is typically perceived, in which there is considerable rotational and tilting disorder, consistent with the structures visually observed through etching studies. The ability to form uniaxially oriented arrays of hydroxyapatite nanocrystals, matching the nanostructured architecture of natural bone, demonstrates that site-specic epitaxial nucleation and growth is not necessary to achieve intrabrillar crystallization, shedding a new light onto existing theories of calcication of mammalian collagenous structures. We propose that because the amorphous mineral phase is shaped by the collagen prior to crystallization, in conjunction with the fact that HA commonly crystallizes as platelets of preferred orientation, that the collagen primarily acts as a highly organized container to constrain the growth of the HA along its fast growing [0 0 1] axis. Thus while uniaxial orientation appears to be directed by the collagen, perhaps it is not as precisely nor specically controlled as formerly perceived. Certainly the hierarchical levels of bone structure and formation involve a complex assortment of proteins and cellular control which we have not addressed, but an understanding of the underlying materials chemistry that may be occurring at this nanostructural level is an important rst step towards reaching that goal. Given that this is the rst time that this most fundamental level of bone structure has been duplicated in the lab, we feel it is appropriate to revisit the old debate regarding bone formation via an amorphous precursor, because we have now demonstrated how easily this could be achieved, and that it is not necessary to invoke the epitaxial nucleation mechanism that has dominated the literature for many years. By synthetically recreating the most fundamental level of structure of bone in a petri dish, we feel that this biomimetic approach might one day allow for the fabrication of bioceramic composites that not only have the composition and organization of bone, but also match its unique bioresorbable and mechanical properties. Acknowledgements This work was primarily supported by the National Science Foundation under Grant No. ECS-9986333, in the cross-cutting program Nanoscale Exploratory Research: Biosystems at the Nanoscale, followed by the Nanoscale Interdisciplinary Research Team from NSF grant No. BES-0404000. We would also like to acknowledge partial nancial support from a UF Pittman Graduate Student Fellowship, as well thank the Major Analytical Instrumentation Center (MAIC) at UF for maintaining and providing exceptional analytical equipment and guidance. Special thanks to Colin D. Medley, graduate student in the Department of Chemistry at UF, for assistance with the confocal microscopy analysis. References
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Materials Science and Engineering R xxx (2007) xxxxxx www.elsevier.com/locate/mser

Bone structure and formation: A new perspective

triclinic system : where

c-Axis 6.8792 6.8894 6.8598 6.8759 (6) (5) (7) (6)

Octacalcium phosphate (JCPDS 79-0423) Plane ) d-Spacing (A ab

0.02 3.12 7.10 3.22 7.00 6.20 8.22 6.42 0.04

3.43 3.05 2.83 2.77 2.69 2.26 1.95 1.84 1.72

0.0 22.1 87.9 28.7 90.0 86.6 51.9 50.9 0.0

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