Bone Structure and Formation

Materials Science and Engineering R 58 (2007) 77116
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,*
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
Available online 20 July 2007
Abstract
Bone is a hierarchically structured composite material which, in addition to its obvious biological value, has been well studied by
the materials engineering community because of its unique structure and mechanical properties. This article will review the existing
bone literature, with emphasis on the prevailing theories regarding bone formation and structure, which lay the groundwork for
proposing a new model to explain how intrafibrillar mineralization of collagen can be achieved during bone formation. Intrafibrillar
refers to the fact that growth of the mineral phase is somehow directed by the collagen matrix, which leads to a nanostructured
architecture consisting of uniaxially oriented nanocrystals of hydroxyapatite embedded within and roughly [0 0 1] aligned parallel
to the long collagen fibril axes. Secondary (osteonal) bone, the focus of this review, is a laminated organicinorganic composite
composed primarily of collagen, hydroxyapatite, and water; but minor constituents, such as non-collagenous proteins (NCPs), are
also present and are thought to play an important role in bone formation. To date, there has been no clear understanding of the role of
these NCPs, although it has been generally assumed that the NCPs regulate solution crystal growth via some type of epitaxial
relationship between specific crystallographic faces and specific protein conformers. Indeed, epitaxial relationships have been
calculated; but in practice, it has not been demonstrated that intrafibrillar mineralization can be accomplished via this route. Because
of the difficulty in examining biomineralization processes in vivo, the authors of this article have turned to using in vitro model
systems to investigate the possible physicochemical mechanisms that may be involved in biomineralization.
In the case of bone biomineral, we have now been able to duplicate the most fundamental level of bone structure, the
interpenetrating nanostructured architecture, using relatively simple anionic polypeptides that mimic the polyanionic character of
the NCPs. We propose that the charged polymer acts as a process-directing agent, by which the conventional solution crystallization
is converted into a precursor process. This polymer-induced liquid-precursor (PILP) process generates an amorphous liquid-phase
mineral precursor to hydroxyapatite which facilitates intrafibrillar mineralization of type-I collagen because the fluidic character of
the amorphous precursor phase enables it to be drawn into the nanoscopic gaps and grooves of collagen fibrils by capillary action.
The precursor then solidifies and crystallizes upon loss of hydration waters into the more thermodynamically stable phase,
leaving the collagen fibrils embedded with nanoscopic hydroxyapatite (HA) crystals. Electron diffraction patterns of the highly
mineralized collagen fibrils are nearly identical to those of natural bone, indicating that the HA crystallites are preferentially aligned
with [0 0 1] orientation along the collagen fibril axes. In addition, studies of etched samples of natural bone and our mineralized
collagen suggest that the long accepted deck of cards model of bones nanostructured architecture is not entirely accurate.
* Corresponding author. Tel.: +1 352 846 3336; fax: +1 352 846 3355.
E-mail address: lgowe@mse.ufl.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
78
M.J. Olszta et al. / Materials Science and Engineering R 58 (2007) 77116
Most importantly, this in vitro model demonstrates that a highly specific, epitaxial-type interaction with NCPs is not needed to
stimulate crystal nucleation and regulate crystal orientation, as has long been assumed. Instead, we propose that collagen is the
primary template for crystal organization, but with the important caveat that this templating occurs only for crystals formed from an
infiltrated amorphous precursor. These results suggest that the 25-year-old debate regarding bone formation via an amorphous
precursor phase needs to be revisited.
From a biomedical perspective, in addition to providing possible insight into the role of NCPs in bone formation, this in vitro
system may pave the way toward the ultimate goal of fabricating a synthetic bone substitute that not only has a composition similar
to bone, but has comparable mechanical properties and bioresorptive potential as natural bone. From a materials chemistry
perspective, the non-specificity of the PILP process and capillary infiltration mechanism suggests that non-biological materials
could also be fabricated into nanostructured composites using this biomimetic strategy.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Secondary bone formation; Biomineralization; Hydroxyapatite; Amorphous calcium phosphate; Biomimetic
Contents
1.
2.
3.
4.
5.
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. Intrafibrillar 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 fluorescently tagged poly(aspartate) . . . . . . . . . . .
2.3.2. Turkey tendon preparation and mineralization . . . . . . . . . . . . . .
2.3.3. Confocal microscopy analysis . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Intrafibrillar mineralization of collagen with a CaP PILP process . . . . . . .
3.2. X-ray diffraction (XRD) studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Electron diffraction analysismineral identification . . . . . . . . . . . . . . . .
3.4. Electron diffraction analysiscrystallographic orientation . . . . . . . . . . . .
3.5. SEM analysis of crystal morphology and organization . . . . . . . . . . . . . .
3.6. Confocal studies on mechanism of mineral infiltration . . . . . . . . . . . . . .
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. Clarification of crystal orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Elucidation of mineral morphology. . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Mechanism of alignment of intrafibrillar crystals . . . . . . . . . . . . . . . . . .
4.6. Mechanism of mineral penetration and implications for application . . . . .
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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79
1. Introduction
Bone is a bioceramic composite that has long held the attention of the materials engineer who seeks to duplicate its
enviable mechanical properties, in which both high strength and fracture toughness can be achieved due to the unique
architecture of this organicinorganic composite. This article will not discuss bones mechanical properties, for which
there are many excellent reviews [19] as well as recent contributions [1017]; but instead it will focus on the unique
structure that confers these properties, and to the materials chemistry underlying its formation.
1.1. Primary versus secondary bone
When discussing the structure and formation of bone, one first needs to understand that there are two main stages of
bone formation, referred to as primary and secondary osteogenesis (or synonymously as ossification), and that the
mechanism of bone formation differs substantially in these two stages [18,19]. Epiphysial cartilage, which serves as
the locus for primary bone formation (i.e., endochondral ossification), is a combination of ground substance and very
loose, small (1020 nm in diameter) fibril bundles of collagen [19,20]. There is also a high occurrence of matrix
vesicles, which are believed to deliver either crystals or a high concentration of ions to the mineralization front
[2125]. The mineralization is relatively rapid and unorganized, forming a woven bone microstructure. Although
type-I collagen is present in this ground substance (17% in rat cartilage), it is not organized into lamellae, 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 calcification nodules [27] or calcospherites [28], due to the
spherulitic arrangement of the crystal clusters. Cameron [20] suggested that the collagen fibrils found in cartilage
are too narrow for the mineral to deposit within them, thereby resulting in the observed extrafibrillar mineralization. In
this instance, the collagen does not appear to play an appreciable role in directing the mineralization process, and
therefore this type of bone formation is not the focus of this paper.
In secondary bone formation, the primary woven bone is remodeled into a more optimal structure, such as parallelfibered 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 fibrils; the
collagen fibrils are layered in parallel arrangement within lamellae; the lamellae are arranged concentrically around
blood vessels to form osteons; finally, the osteons are either packed densely into compact bone or comprise a
trabecular network of microporous bone, referred to as spongy or cancellous bone.
The collagen fibrils in secondary bone are secreted by osteoblasts and are larger than those in primary bone, with a
mean diameter of 78 nm [27], and they assemble into a highly organized, close-packed lamellar structure. Close to the
mineralization front, there are also non-collagenous proteins (NCPs), many of which are highly charged from an
abundance of carboxylate groups from aspartic and glutamic acid residues, as well as phosphate from phosphoserine
[3136]. Although in low concentration, these NCPs are also thought to play an important role in the mineralization
process [18,34,3638]. During secondary bone formation, the organization of the crystals is directed by the collagen
fibrils within which they form [37]. This leads to intrafibrillar crystals that are extremely small (only a few unit cells
thick [3942]), which would not normally be thermodynamically stable if it were not for being embedded within the
organic matrix. Crystals may also form on the surface and between collagen fibers, which are referred to as
interfibrillar crystals. According to Martin et al. [43], about 58% of the mineral in canine whole bone is intrafibrillar,
14% interfibrillar, and 28% from within the gaps between the ends of collagen fibrils.
The high degree of mineral loading that is achieved by intrafibrillar mineralization leads to a biocomposite with a
composition of around 65 wt.% mineral phase, 25 wt.% organic, and 10 wt.% water [3,11,18,34,39,43,44]. By
comparison, because bone is such a unique composite, containing a collagenous hydrogel matrix, on a volumetric
basis it consists of about 3343% apatite minerals, 3244% organics, and 1525% water [1]. Even though water is a
minor constituent, its significance should not be overlooked, because it contributes to the overall toughness of the
biocomposite, acting something like a plasticizer [45]. On the other hand, the non-collagenous proteins, which
comprise only 1015% of the organic matrix [18], may be less important in terms of bones mechanical properties, but
apparently play a crucial role in the formation of bone structure.
80
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 significant
variation between bone types of a single organism. It is the nanostructural level of organization that lies at the foundation
of all types of secondary bone (e.g., parallel-fibered, lamellar, fibrolamellar, trabecular, osteonal). Therefore, in order to
truly understand bones exceptional properties, it is necessary to examine its most basic level of organization, the
nanostructured array of hydroxyapatite (HA) crystals embedded within the collagen matrix (level 2 of Fig. 1). This level
81
of structure is created by intrafibrillar mineralization of collagen, and it is the intimate relationship between the selfassembled fibrillar collagen matrix and the uniaxially oriented, nanometer sized, platy HA crystals, that provides bone
with its remarkable mechanical properties and remodeling capabilities. From a materials science perspective, the
interpenetrating nature of the organicinorganic phases makes it difficult to classify bone as one specific type of
composite. For example, the high degree of loading of mineral phase which encases the collagen fibrils suggests that bone
is a fiber-reinforced ceramicmatrix composite. In sharp contrast to this observation is the fact that ultrastructural
examination of deproteinated bone reveals individual 2550-nm-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 fibrils. Before describing the complex
composite structure of bone, the organization of the organic matrix must first be defined. The structure of collagen
alone took many years to resolve, and various permutations of Hodge and Petruskas quarter-stagger model are largely
accepted as providing a reasonable description of the fibrillar organization [4953]. The repetitive nature of the amino
acid sequences of collagen, which consists of (Gly-X-Y-)n, where X and Y are frequently proline and
hydroxyproline residues, allows the protein to assemble into triple helical structures referred to as tropocollagen
molecules [54]. Secondary bonding interactions between tropocollagen units leads to self-organization into fibrillar
structures (see schematic in Fig. 2). Type-I collagen, the primary constituent of secondary bone tissues, assembles its
tropocollagen units in a quarter-staggered array, which leads to hole and overlap zones that can be seen as a periodic
Fig. 2. Schematic by Landis et al. [57] illustrating the mineralization of turkey tendon, based on ex situ observations using high-voltage TEM and
tomographic reconstruction imaging. The cylindrical rods represent tropocollagen units composed of triple helical collagen molecules that assemble
into fibrils in a quarter-staggered fashion, which leaves periodic gaps and grooves within the fibrils. The 67 nm repeat (64 nm when dehydrated)
results from the combination of a 40 nm hole zone and 27 nm overlap zone. The space between assembled tropocollagen units is 0.24 nm. Upon
mineralization, the more electron dense mineral can be seen as striations within the collagen fibrils, and the crystals are oriented such that the
crystallographic c-axes lie parallel to the long axes of the molecules and their (1 0 0) planes are all approximately parallel to each other. Irregularly
shaped, large and small mineral deposits may occupy several hole zones, and preferential growth in the c-axial length follows the long axis of the
collagen (reprinted from [57] (Journal of Structural Biology), with permission from Elsevier).
82
banding pattern when stained for observation by transmission electron microscopy (TEM) (e.g., the banding seen in
levels 13 of Fig. 1).
On the other hand, Hansma and co-workers have recently shown by atomic force microscopy (AFM) of collagen
that there are grooves on the surfaces of the fibrils with a 37 nm height corrugation, which is not consistent with the
1.6 nm overlap mismatch predicted by the Petruska and Hodge model. They suggest, therefore, that the periodic
banding pattern seen in TEM may result from differential deposition of stain in the grooves versus peaks of the fibrils
[55]. This group has additionally shown that the collagen fibrils bend in a tube-like fashion, suggesting that the
composition is not homogeneous laterally across the fibrils, but rather has a relatively hard shell with a softer core [56].
The quarter-stagger arrangement leaves a regular array of 40 nm gaps within each periodic unit, and these are
reportedly [57,58] the locations where crystal nuclei are first observed in systems such as naturally mineralizing turkey
tendon (Fig. 2). Turkey tendon has served as a useful model of bone formation because it mineralizes naturally, but
does not remodel from primary bone. Instead, parallel fibers of collagen mineralize in an intrafibrillar fashion that
appears to be similar to secondary bone formation [57,58]. A variety of modifications to this quarter-staggered model
have also been proposed, such as the alignment of gaps to form grooves, which are proposed to help account for the
fact that the dimensions of the HA crystals extracted from bone are larger than the dimensions of the gap zones where
they are thought to form. The rod-like character of the cylindrical tropocollagen units imparts collagen with liquid
crystalline character [59], which may play a role in vivo in the formation of tissues with complex structural order [60],
such as the gradual splay in collagen (and thus intrafibrillar crystal) orientation across lamellae in osteonal bone (e.g.,
level 4 of Fig. 1 [60]).
1.2.2. Mineral phase
The size of bone crystals reported in the literature varies, with values ranging from length, 3050 nm; width, 15
30 nm; thickness, 210 nm [18,42,48,53,61,62]. Recent AFM studies find that the bone crystals are longer than those
observed by TEM, with widths and lengths ranging from 30 to 200 nm [46]. They suggest that this discrepancy is due
to breakage that occurs during dispersion for TEM sample preparation. While this variability is in part due to sample
preparation, it is likely also a function of the type of mineralized tissue (e.g., animal species, maturation, and location
of the tissue examined). For example, in Fig. 3, even though the individual platelets cannot be discerned with scanning
electron microscopy (SEM), the outward appearance of the bone texture differs for samples from dog (Fig. 3A),
turkey, (Fig. 3B) horse (Fig. 3C), and human bone (Fig. 3D). The inability to resolve the crystallites could also be due
to an extrafibrillar organic matrix (presumably proteoglycans or non-collagenous proteins), which has been observed
by AFM [46]. Regardless of these variations, it is important to realize that the crystals clearly outgrow the dimensions
of the gap zones in the fibrils.
Not only are bone crystallites extremely small, they are often described as poorly crystalline because of the
broad X-ray diffraction peaks (relative to synthetic HA), which is thought to arise from the incorporation of impurities,
such as carbonate, sodium and magnesium ions (46% carbonate; 0.9% Na; 0.5% Mg) [18,63,64], and 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-deficient apatite, with a Ca:P ratio less than 1.67, which is the theoretical value for pure
hydroxyapatite, Ca5(PO4)3(OH) [39,64]. Because bone is a living tissue that is continuously undergoing remodeling
and repair, the small size and/or non-stoichiometry of the crystals presumably bestows the mineral phase with the
solubility needed for resorption of the bone by osteoclasts (bone resorbing cells). For example, bone substitutes made
of synthetic HA, although bioactive (stimulatory for bone formation), are rather slow to resorb due to the low solubility
of HA under physiological conditions [39,66,67].
1.2.3. Intrafibrillar mineralization
In secondary bone formation (and dentin), collagen directs the mineral growth such that platelets of HA grow in the
[0 0 1] direction along the long-axis of the fibril (Fig. 4A), as demonstrated by orientation of the (0 0 2) and (0 0 4)
planes parallel to this axis in selected area electron diffraction (SAED) patterns of fibrils collected from native bone
(Fig. 4B). This example demonstrates the unique structure created by intrafibrillar mineralization, in which the HA
crystallites are embedded within the collagen fibrils. In addition to the uniaxial orientation, platelets are often
illustrated as being coherently aligned in the ab plane, stacked in parallel arrays like a deck of cards, as described by
Traub and co-workers [53]. Although this model of bone structure is generally well accepted and highly cited by
83
Fig. 3. Comparison of bone textures when viewed by scanning electron microscopy (SEM). (A) Reprint from Bagambisa et al. [136] showing the
resting bone surface in a sample of whole bone from dog femur diaphysis. Diagonally from upper left to lower right, the collagen fibers form a
bundle in which mineral is densely invested, sometimes causing bulging and clubbing of the fibers. (reprinted from [136] (Cells and Materials 20
(1)), with permission from the authors). (B) SEM of as-fractured turkey bone. (C) SEM of as-fractured equine bone. (D) SEM of cortical cancellous
chips of human cadaver bone (obtained from Regeneration Technologies Inc.).
researchers within the biomedical community, it is not an entirely accurate representation of the nano structured
architecture of bone because the HA crystals do not have biaxial order (as discussed in Section 3.4).
1.3. Mechanism of bone formation
While the structure of bone is reasonably well defined, its formation remains an enigma. For example, although
collagen can be reconstituted into the native fibrillar structure, in vitro crystallization studies have not been able to
duplicate even bones most fundamental level of nanostructure. There have been several ex vivo studies examining the
early stages of mineralization, both in bone and in naturally mineralizing turkey tendon [27,6875]. Most of these
studies took place in the 1960s and 1970s, when the mechanism of bone formation was hotly debated. One group of
researchers argued that the HA crystals were formed via the traditional solution crystallization process (i.e., nucleation
and growth), while a handful of others pointed to the deposition of an amorphous calcium phosphate (ACP) precursor.
To alleviate this discrepancy in observations, it was also suggested that the amorphous substance observed in early
mineralizing tissues by some researchers is actually paracrystalline mineral (i.e., a loss of long-range crystalline
order as a result of lattice imperfections) [76], which might be undetectable by conventional analytical techniques
[7779]. Various metastable crystalline phases (e.g., octacalcium phosphate (OCP) [80] and brushite [81]) have also
been implicated as transitory precursors to HA in bone and teeth formation. The spectroscopic evidence also shows a
band at 945 cm1 that is attributed to a highly disordered structure, which becomes less prominent as the mineral ages.
It is assumed that intermediate phases such as OCP or TCP could precipitate along the pathway to the most
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Fig. 4. Electron micrographs of equine cortical bone. (A) TEM brightfield image demonstrating intrafibrillar mineralization of type-I collagen fibrils
in natural bone. The native banding pattern of type-I collagen can be observed due to the infiltration of electron dense mineral, and staining is not
needed. The striated appearance results from hydroxyapatite platelets aligned parallel to the long axis of the collagen. Bar = 100 nm. (B) Selected
area electron diffraction (SAED) of a single fibril of crushed equine bone. The arcing of the (0 0 2) and (0 0 4) planes, which are parallel to the long
axis of the collagen fibrils (white arrow) is characteristic of bone. The (1 1 2), (2 1 1) and (3 0 0) planes, indexed using d-spacings and angles relative
to the (0 0 2) plane, form 3 arcs which nearly overlap, combining into what appears to be a ring; however, there is a gap in the ring just behind the
(0 0 2) arc because it is not really a powder ring, but three distinct sets of planes which have very close d-spacings. The appearance of these three
planes simultaneously indicates that there is more than one orientation of the HA platelets in the ab plane. (C) TEM brightfield image of an isolated
collagen fibril showing the characteristic banding pattern of type-I collagen. The SAED pattern (inset) of this fibril demonstrates that the fibril does
not diffract, suggesting that the electron dense phase, which is the only thing providing contrast (the sample was not stained), is amorphous CaP.
Bar = 50 nm.
thermodynamically stable phase of hydroxyapatite, but the central question of this report is whether that pathway
starts with an amorphous phase, and the structural consequences of such a mechanism.
The ACP theory was first proposed by Posner, Termine and their co-workers [70,72,8284], in which they argued
that the two-phase nature of bone mineral may prove to be a valuable aid in understanding the operative mechanisms
of bone metabolism and the molecular basis of bone structure [82]. In their studies, the measurements of crystalline
fraction, as determined by X-ray diffraction peak widths, as well as infrared analysis peak splitting, were made during
different stages of development of chick, rat, and cow bones. In later years, Bonnuci also described an inorganic
substance in bands when observing early stage bone formation by TEM [37], in which a more electron dense material
is seen primarily within the hole zones of collagen, which subsequently becomes needle-like as it transforms into
hydroxyapatite. For the most part, this debate over bone formation was eventually quelled by the landmark paper of
Glimcher et al. [81], entitled Recent studies of bone-mineralis the amorphous calcium-phosphate theory valid?
This group examined bone from the rapid turnover stage of 17-day-old chick embryos, and found intermediate range
in the radial distribution function analysis of XRD data (as compared to 10 A
for a synthetic ACP).
order of up to 25 A
85
Likewise, in support of this argument (the absence of amorphous mineral in calcification), Landis and co-workers
[57,58] examined the early stages of mineralization of turkey tendon, and through tomographic 3D reconstruction of
high-voltage TEM images, provided evidence to suggest that HA crystals nucleate in the hole zones of collagen, in
which a stippled haze of material is notable, a feature which has been observed by others [85]. Continued growth
from these initial mineral deposits occurs primarily in length and width (not in thickness), where the crystal lengths
appear to traverse adjacent collagen hole and overlap zones rather than being restricted to a single hole or overlap
zone. Crystal growth in width across collagen fibrils forms mineral bridges as it apparently follows channels or
grooves, where smaller and larger crystals appear to fuse in coplanar alignment to form larger mineral platelets [57].
These descriptions are highlighted because such observations tie in well with the mechanism we later propose (even
though our mechanism deals with an amorphous precursor). While there is evidence to deny an amorphous phase,
careful examination of these papers indicates a greater degree of ambiguity than is generally recognized. In addition,
more recent results in the field of glass science have improved our understanding of intermediate range order in
amorphous materials and suggest the interpretation of the data in these earlier works on bone mineral may need to be
revised. These issues will be considered in more detail in Section 4, where comparison is made to our new model
system.
Before describing our in vitro model, it is worth pointing out that we also find biological evidence to support the
amorphous precursor mechanism. When examining natural bone samples for comparison to our synthetic materials (to
be described later), we came across some collagen fibrils that contain non-crystalline mineral. For example, the fibril
shown in Fig. 4C displays a marked banding pattern (without staining), but does not exhibit Bragg diffraction, even
with concerted effort (Fig. 4C, inset). It is common practice to refer to a material that yields only a broad diffuse
diffraction ring as amorphous; but given the unique structure of bone mineral, which is at best poorly crystalline
(i.e., exhibits weak long-range periodic correlations) when it reaches full maturity, one must be cautious about
terminology. For example, one cannot rule out the possibility that the non-diffracting material could be
nanocrystalline, such that the diffraction peaks become too broadened to detect; however, it clearly exhibits weaker
long-range periodic correlations than the mineral phase seen in the majority of the fibrils, which show strong Bragg
maxima (such as in Fig. 4A). This particular example was from mature equine bone; therefore this fibril was
presumably from a region of the bone that was recently remineralized during the natural remodeling process of mature
bone.
1.4. Synthetic attempts at mimicking bone formation
While considerable effort has gone into determining the relationship between collagen structure and mineral
orientation, synthetic re-creation of this most fundamental level of bone structure has eluded the materials engineer
seeking to fabricate bone-like composites. It would be desirable to mimic both the composition and structure of bone
for synthetic bone graft substitutes, but attempts at reproducing the intrafibrillar mineralization of collagen scaffolds
have achieved limited success.
Early attempts at mimicking bone formation were performed by introducing reconstituted bovine or porcine type-I
collagen substrates into simulated body fluid (SBF) (which, depending on the recipe, contains NaHCO3, Na2SO4,
MgCl26H2O, NaCl and KCl, but does not contain NCPs). It was believed that the hole zones of the collagen would
serve as nucleation sites, as appeared to be the case for turkey tendon. In support of this, Glimcher et al. [86] indicated
that the hole zones of reconstituted collagen from rabbit bone could nucleate HA, and the resultant structure appeared
similar to the early stages of calcification of embryonic bone. Yet, the reason for this ability to nucleate mineral in a
biological collagen matrix was not clear. It has therefore been assumed that the collagen substrate does not act alone in
directing crystal growth, and that the non-collagenous proteins (NCPs) found in regions of bone growth play an
essential role in calcification due to their ability to bind calcium and their high affinity for collagen [87].
These NCPs, such as osteonectin and various other phosphoproteins (e.g., osteopontin, osteocalcin,
phosphophoryn, and bone sialoprotein), are enriched with acidic amino acids, particularly aspartic acid and
phosphoserine [37,8890]. We note that, although these water soluble proteins that are found in nearly all highly
regulated biomineralization processes are often referred to as the acidic proteins [9194], we will refer to them as
polyanionic proteins [95], to emphasize the charged character of the deprotonated active form of the protein. In
synthetic crystal growth assays, such polyanionic proteins have been shown to both inhibit HA nucleation when in
soluble form, and promote nucleation when attached to a substrate [91,96,97]; thus it has been generally assumed that
86
binding of such proteins to collagen fibrils could be a means of promoting nucleation within the collagen. This
hypothesis prompted the search for epitaxial relationships between NCPs and HA. Indeed, Hoang et al. [98]
demonstrate that the X-ray crystal structure of porcine osteocalcin reveals a negatively charged surface that
coordinates five calcium ions in a spatial orientation that is complementary to calcium ions in a hydroxyapatite crystal
lattice, which they suggest may lead to selective binding characteristics with HA that could play a role in modulating
HA crystal morphology and growth in bone formation. It should be kept in mind, however, that the early mineral phase
of bone is considered to be very poorly ordered, so one must be cautious when extrapolating results based on modeling
structural relationships with a well-ordered HA lattice to the poorly crystalline HA of bone. Calcium binding could
also play an important role in formation of an amorphous precursor, but as will be shown later, a high degree of
selectivity during the ion binding event is probably not required. In any case, it should be noted that, to date,
intrafibrillar mineralization of collagen has not been demonstrated using an epitaxial nucleation approach.
There has been partial success at mimicking some of the nanostructural features of bone by performing the
crystallization in situ during fibrillogenesis, during which some crystals appear to nucleate at the hole zones [99], and
in some cases yield similar electron diffraction patterns indicating uniaxial orientation of HA crystallites [100,101].
While this approach has produced intriguing results, is does not contribute to understanding how bone is formed from a
fundamental point of view because, in bone, fibrillogenesis occurs prior to mineralization. An alternative approach has
been to add anionic polymers or generate anionic functional domains to the collagen, producing mineral composites
with a non-descript mineral morphology similar to bone [102]. Although these studies appear promising, none has
been able to generate an interpenetrating collagenHA composite with a uniaxially oriented apatite phase that fully
infiltrates the organic matrix, thereby achieving the high mineral loading as in bone. However, in a recent paper by
Chen et al. [103], it was shown that demineralized fish bone could then be remineralized to obtain the proper uniaxial
orientation (demonstrated by wide angle X-ray diffraction), particularly when polyglutamate (polyGlu) was added to
the crystallizing solution. They did not examine the mechanism of remineralization (nor determine the role of the
polyGlu), but we suspect that it occurred through a precursor process similar to that described in the present work using
polyaspartate (polyAsp). Our work is experimentally similar to this latter approach of adding anionic polypeptides to
the reaction, not for the purpose of stimulating crystal nucleation on collagen, but rather as a process-directing agent,
in which the solution crystallization is converted into a precursor process.
1.5. Polymer-induced liquid-precursor (PILP) mineralization process
Previous work in our group has shown that the addition of micromolar quantities of anionic polypeptides to a
crystallizing solution can transform the conventional solution crystallization process into a precursor process. This
PILP process was first discovered for calcium carbonate (CaCO3) mineralization, in which it was observed that
droplets of a highly hydrated liquid-phase precursor phase separate as the solution is gradually raised in
supersaturation [104106]. The charged polymer sequesters ions, while inhibiting crystal nucleation, inducing liquid
liquid-phase separation in the crystallizing solution. Droplets of the fluidic amorphous phase accumulate and coalesce,
typically forming mineral films and coatings on a variety of substrates. The precursor phase then solidifies and
crystallizes to a more thermodynamically stable phase as the waters of hydration (and most of the polymeric impurity)
are excluded [107]. Although the term solidification is typically used to indicate solid formation from a melt, we use
it here to describe the conversion of the liquid-phase precursor to an amorphous solid, highlighting the fact that this
process is distinctly different from precipitation from solution.
The important consequence of this PILP process is that the crystals retain the shape delineated by the phase
boundaries of the precursor phase, thus generating a variety of non-equilibrium crystal morphologies. It has long
remained an enigma how biominerals are molded into their elaborate symmetry-breaking crystal morphologies, but
many of the morphological features found in CaCO3 biominerals have now been demonstrated in vitro via this
polymer-induced liquid-precursor (PILP) process, such as the deposition of CaCO3 tablets and thin films [104,108],
molded [109,110] and patterned calcite crystals [111113], and calcite nanofibers [114116]. Therefore, we have
proposed that the PILP process might play a fundamental role in the morphogenesis of calcitic biominerals, in which
the polyanionic macromolecules involved in biomineralization could be considered process-directing agents. Note,
this explanation is distinctly different from a previously held theory, in which the soluble anionic proteins were
considered structure-directing agents that modulate crystal morphology through selective interactions with specific
crystallographic faces [91,92,117,118]. In contrast, with a polymeric process-directing agent, the shaping of the
87
mineral occurs prior to the presence of structural order, and is simply a result of the inhibitory polymers affinity for
ions (and retention of hydration water). The results are striking and, considering the non-specificity of the organic
inorganic interactions, we thought is might be possible to induce mineralization by such a process with calcium
phosphate.
1.6. New hypothesis on the mechanism of bone formation
In the case of vertebrates, which primarily utilize calcium phosphates (CaP) in their hard tissues, we now present
evidence to suggest that the PILP process may also play a fundamental role in the biomineralization of bones and teeth.
Using this PILP process, we have been able to achieve intrafibrillar mineralization of a pre-existing fibrillar type-I
collagen matrix, first with CaCO3 [116,119,120], and now with CaP, allowing us to duplicate the nanostructured
architecture of bone. The evidence presented is based primarily on an in 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 intrafibrillar mineralization with this system deserves attention since other theories seem
to fall short in practice. Section 4 will include a more thorough deliberation on this system as compared to literature
studies on bone formation. For now, our hypothesis on bone formation will be presented before getting to the results so
that the reasoning behind the experimental methodology can be understood.
We propose that hydroxyapatite crystals in mineralized collagenous tissues (i.e., bone and dentin) do not initially
nucleate within the hole zones, but rather a liquid-phase amorphous precursor is drawn into the collagen fibrils via
capillary action, and upon solidification, the precursor crystallizes, leaving the collagen fibrils embedded with
nanoscopic platelets of HA (Fig. 5). This liquid-phase precursor, analogous to the PILP phase we have demonstrated
Fig. 5. Schematic depicting a proposed mechanism of intrafibrillar mineralization of collagen. Each of these pictures represents the hole zone region
of a collagen fibril within the aqueous mineralizing solution containing the polymeric process-directing agent (i.e., polyaspartate). (a) The negatively
charged polymer sequesters ions and at some critical ion concentration generates liquidliquid-phase separation within the crystallizing solution,
forming nanoscopic droplets of a highly hydrated, amorphous calcium phosphate phase. The nanoscopic droplets of this polymer-induced liquidprecursor (PILP) phase adsorb to the collagen fibril, and due to their fluidic character, become pulled up and into the hole zones and interstices of the
collagen fibril by capillary action. (b) The collagen fibril becomes fully imbibed with the amorphous mineral precursor, which then solidifies as the
hydration waters are excluded. (c) The amorphous precursor phase crystallizes, leaving the collagen fibril embedded with nanoscopic crystals of
hydroxyapatite.
88
in vitro, would be induced by polyanionic proteins, which we assume would be one or some combination of the
polyanionic NCPs found associated with bone.
It should be pointed out that this capillary infiltration occurs within an aqueous crystallizing solution, such that the
collagen scaffold is already swollen with imbibed water. In other words, the PILP phase is not soaked up by a dry
collagen sponge, but rather the PILP phase is delineated from the surrounding solution by a distinct phase boundary,
and combination of this interfacial energy with the pore space in the collagen leads to capillary forces, which in this
case, could draw the PILP phase up into the collagen matrix and into the fibrils. This would then provide a means for
infiltrating the collagen with a high loading of mineral ions, which then form a hydrated amorphous solid as some of
the waters of hydration are excluded from the liquid-phase precursor [107]. This solidification process occurs while the
sample is still in the aqueous crystallizing solution. Additional waters of hydration are then driven off as the metastable
amorphous phase transforms into the more thermodynamically stable, anhydrous crystalline state (e.g., calcite in
CaCO3 or HA in CaP). Although this process is described in terms of the steps involved, it is more likely that it occurs
in a continuous fashion with no sharp distinction between the individual stages.
The results described below provide evidence for our proposed mechanism. Scanning electron microscopy,
transmission electron microscopy, X-ray diffraction, and electron diffraction all demonstrate that, by using the PILP
process, we are able to create the first example of a synthetic composite that mimics the nanostructure of bone. We also
provide evidence to suggest that intrafibrillar mineralization is achieved due to the fluidity of an amorphous precursor,
and show that long-held beliefs about the orientation of the crystallites within bone need to be revised. The strong
similarities between our composite and bone suggest that our system may be able to serve as an in vitro model that can
further shed light on the mechanisms involved in bone formation.
2. Methods and materials
2.1. Mineralization of collagen
Calcium phosphate formation by the PILP process was achieved by mixing equal volumes of 9 mM CaCl2 solution
in Tris buffer (pH 7.4) and 4.2 mM K2HPO4 solution in Tris buffer (pH 7.4), to final concentrations of 4.5 mM calcium
and 2.1 mM phosphate. The Trissaline buffer was made by dissolving 8.77 g of NaCl, 0.96 g of Trisbase, 6.61 g of
TrisHCl in 1 l dH2O (adjusted to pH 7.3 using NaOH at 25 8C, which becomes 7.4 at 37 8C). We note that these 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
89
were then removed from the solution, rinsed with ddH2O followed by ethanol, and dried for FE-SEM analysis (with an
amorphous carbon coating).
2.2.2. Transmission electron microscopy (TEM) analysis
Samples were prepared for transmission electron microscopy (TEM) following the protocols performed on bone
and naturally mineralized tendon by Weiner and Traub [41]. This included crushing the samples into a fine-grained
powder in a liquid nitrogen mortar and pestle. A few small drops of ethanol were then placed on the powder, followed
by drawing the slurry into a micropipette. The slurry was transferred to a 3 mm diameter carbon/Formvar coated
copper TEM grid, followed by an optional stain with 1% phosphotungstic acid (PTA) in a PBS buffer on the 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 brightfield
(BF), darkfield (DF) and selected area electron diffraction (SAED) modes. Normal darkfield images were produced by
tilting the beam to the diffracting plane of interest, such that the chosen Bragg beam produces the brightest DF image
possible.
For extraction of the CaP crystals from the composite, the mineralized Cellagen1 sample was sonicated in H2O for
15 min. to remove superficial mineral coatings. Then the sample was gently crushed in liquidN2 into a fine powder,
and 2% NaOCl was added to remove the surrounding collagen. After washing with H2O and ethanol, the nanocrystals
were gently transferred to a 3 mm diameter carbon/Formvar coated copper TEM grid.
2.2.2.1. Selected area electron diffraction (SAED) analysis. To differentiate between HA and OCP, the following
crystallographic formulae [121] were used to calculate the theoretical diffraction angles between planes (h k l) with
respect to the (0 0 2) reference plane, for comparison to the angles measured in SAED patterns What is measured in an
SAED zone pattern is the angles between reciprocal lattice vectors lying in the same planar section of reciprocal space
defined by the zeroth-order Ewald sphere intersection. Therefore, angles between planes in real space can be
correlated with the angles between their normals in reciprocal space. In the case of OCP and HA, the angles provide an
additional measure to distinguish between the two phases, which have very similar d-spacings.
hexagonal system :
cos f
h1 h2 k1 k2 1=2h1 k2 k1 h2 3=4a2 =c2 l1 l2

fh21 k12 h1 k1 3=4a2 =c2 l21 h22 k22 h2 k2 3=4a2 =c2 l22 g
1=2
(1)
where f is the angle between (h1 k1 11) and (h2 k2 l2); unit cell dimensions and angles are given by a, b, c and a, b, g,
respectively.
triclinic system :
cos f
F
Ah1 k1 l1 Ah2 k2 l2
(2)
where
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 gk1 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 final mineral crystalline phase was confirmed
from the collection of XRD d-spacings that correlated well with the Joint Committee on Powder Diffraction Standards
(JCPDS) files for HA. Monochromatized Cu Ka X-ray radiation from a Philips XRD 3720 fixed anode X-ray tube,
90
Table 1
Hydroxyapatite crystal structure parameters, refined by Rietveld analysis
Phase
Crystal
system
Space
group
Lattice
parameters
Atomic coordinates
HA
Hexagonal
P63/m
a = 9.422;
c = 6.88
Atom Ca1: x = 0.333, y = 0.667, z = 0.001; atom Ca2: x = 0.246, y = 0.993, z = 0.25;
atom P: x = 0.4, y = 0.369, z = 0.25; atom O1: x = 0.329, y = 0.484, z = 0.25; atom
O2: x = 0.589, y = 0.466, z = 0.25; atom O3: x = 0.348, y = 0.259, z = 0.073;
ion OH: x = 0, y = 0, z = 0.25
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 refinement [122,123]. The HA crystal model was built using
information from the International Crystal Structure Database (ICSD). The details of the crystallographic model are
listed in Table 1 above. Peak shapes were modeled using the pseudo-Voigt function, and two asymmetry parameters
were refined. In each case, four background parameters, a scale factor, five peak-shape parameters, 2u offset (zero
point correction), sample displacement, cell parameters, and atomic positions were all refined. The occupancies of the
oxygen and hydrogen atoms associated with the hydroxyl (OH) group were refined as a group (i.e., OH occupancy)
using the same reasoning as Knowles et al. [124]. The program was also tested for its accuracy using a three-phase
mixture of known composition of aluminum (20%), alpha-alumina (50%) and monocliniczirconia (30%). Results of
the standard analyses showed 21.4% aluminum, 49.4% Al2O3 and 29.2% ZrO2, confirming the robustness of the
software.
2.3. Confocal microscopy study of polymer penetration depth
2.3.1. Preparation of fluorescently tagged poly(aspartate)
The polymer (poly-(ab)-DL-aspartic acid, sodium salt, Mw = 6000 Da; SigmaAldrich, USA), hereafter referred to
as polyAsp, was fluorescently labeled by dissolving 20 mg of it in 2 ml of 0.1 M sodium carbonate buffer [0.2 M of
Na2CO3 (8 ml) and 0.2 M NaHCO3 (17 ml), Aldrich, USA], then gently adding 200 ml of a fluorescein isothiocyanate
(FITC, SigmaAldrich, USA) solution (5 mg dissolved in 0.5 ml of dimethyl sulphoxide, SigmaAldrich, USA),
which was then sealed from light and incubated at 4 8C in a refrigerator for 24 h. After incubation, the polymer-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 final concentration of polymer was
assumed not to change, although there was likely some loss of the lower molecular weight chains below the 3 kDa
cutoff.
2.3.2. Turkey tendon preparation and mineralization
The common calcanean turkey tendon, which had not yet mineralized, was harvested from young birds (10 weeks
old, obtained from Nicholas Turkey Breeding Farm, Sonoma, CA), and stored in ethanol in a refrigerator one day
before the experiment. The tendon was sliced longitudinally to expose the densely packed, well-aligned collagen
fibers. The tendon sections were laid flat with the freshly cleaved collagen surface exposed to the mineralization
solution (the elastin sheath surrounding the tendon is inhibitory to mineralization). One half of the tendon was used for
the control experiment without phosphate counter-ions, to measure diffusion penetration of polyAsp alone. The other
half was used for mineralization, to test the hypothesis that the polyAsp-containing PILP phase could penetrate further
into the collagen scaffold via capillary forces.
The tendons were mineralized at 37 8C for 4 days with 50 ml of reaction solution whose final concentrations 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 final concentration of 4.5 mM
Ca2+. The FITCPolyAsp solution was added to the mineralization solution or the control experiment at a final
concentration of 100 mg/ml. This value is higher than our previous mineralization protocol, because mineralization of
91
turkey tendon was found to be optimal at a higher polymer concentration, presumably because the collagen is so
densely packed. In addition, this higher polymer concentration could help alleviate any loss of polymer activity from
the hydrophobic fluorophore probe, as well as loss of lower molecular weight chains from the Centricon1 purification
process.
2.3.3. Confocal microscopy analysis
An Olympus Fluoview 500 confocal scanning unit mounted on an IX-81 inverted fluorescence microscope was used
with an argon laser light source (488 nm excitation) and 505525 nm bandpass filter. Scanning depths along the
z-direction were chosen to be 500 mm with a 2.5 mm step size, where the z-direction corresponds to a depth profile into the
cross-section of the tendon. A photomultiplier voltage (PMT) of 400 V was used to optimize the fluorescence intensity,
which is very bright near the surface. Additionally, a larger PMT setting of 500 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 profiles 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. Intrafibrillar mineralization of collagen with a CaP PILP process
In order to mimic the organic matrix of bone, we chose to use a Cellagen1 sponge (ICN Biomedicals), which is a
commercially available hemostatic sponge composed of reconstituted type-I bovine collagen (Fig. 6). This collagen
scaffold was chosen because it contains the 64 nm banding pattern indicating that it has assembled appropriately to
match native type-I collagen. It should be noted that the banding pattern of collagen is seen in TEM only when the
fibrils are stained with an electron dense substance, such as phosphotungstic acid (PTA), as can be seen from the
comparison between Fig. 6A and B. The periodic bands can also be seen in topographic images from field-emission
SEM, as shown in Fig. 6C.
In our control reaction, which did not include the polyaspartate (Polyasp) additive, spherulitic clusters of HA
formed on the surface of the scaffold (Fig. 6D). The 1540 mm diameter clusters were composed of randomly oriented
platelets, which appear to have nucleated heterogeneously in a non-specific fashion, typical of HA grown on a variety
of substrates. This was expected since other mineralization experiments reported in the literature show similar
findings. It was not until the polyAsp agent was added to the solution that an amorphous liquid-phase mineral
precursor was produced, and this had a pronounced effect on mineralization of the collagen. The collagen fibrils took
on a distinctly different appearance in TEM, as seen in the micrograph of Fig. 7A. The added contrast in this unstained
sample is apparently due to the presence of amorphous mineral within the fibril (compare the unstained, nonmineralized fibril of Fig. 6B to the unstained, mineralized fibril of Fig. 7A). The amorphous nature of the mineral
during the early stages of mineralization was determined through both selected area electron diffraction (SAED)
analysis of isolated fibrils (Fig. 7A, inset), and bulk XRD analysis (see Section 3.2).
After a few days, the collagen fibrils took on a more striated appearance, which we believe arises from
crystallization of the precursor within the fibrils (Fig. 7B). In this particular example, PILP droplets can be seen
adsorbed to the fibril. When examined by SEM, the surfaces of the fibrils were generally smooth and featureless
(Fig. 7C). This appearance is typical of mineral formed from the PILP phase, which usually lacks crystal facets. The
featureless appearance, however, makes it less obvious that the sample has mineralized; but the presence of mineral
can be verified using X-ray energy dispersive spectroscopy (EDS), as seen in Fig. 7D. The mineralization was also
evident macroscopically through physical changes seen in the collagen, which became rigid and white as it
mineralized in the solution, and the individual fibers retained the dimensions of the swollen state even when
dehydrated, we surmise because they became permeated with mineral [120]. The fractured fibril in Fig. 7E shows
crystals protruding from the fracture surface which span across the entire diameter of the fibril, demonstrating the full
depth of penetration of the mineral phase. This sample was lightly etched with bleach to remove the surrounding
organic matrix, allowing visualization of the remnant mineral phase. With full deproteination using a more
concentrated bleach solution, the crystals could be extracted and examined by TEM (Fig. 7F), revealing a thin platy
morphology typical of that described for extracted bone crystals.
92
Fig. 6. Electron micrographs of reconstituted type-I collagen from a Cellagen1 sponge, before and after mineralization by the traditional solution
crystallization process. (A) TEM brightfield image of individual collagen fibrils stained with 1% phosphotungstic acid (PTA). The accumulation of
the heavy metal within the hole zones allows the 64 nm banding pattern characteristic of type-I collagen to be observed. Inset: SAED of nonmineralized collagen fibrils shows no diffraction. Bar = 200 nm. (B) TEM brightfield image of an unstained collagen fibril. In the absence of the PTA
electron dense stain, the 64 nm banding pattern is not readily discerned. Bar = 200 nm. (C) High magnification FE-SEM micrograph of collagen
fibrils of the Cellagen1 sponge. Although the Cellagen1 has randomly oriented fibers, in this region the fibers were arranged nearly parallel, which
appears to have allowed for registry in alignment of the bands across neighboring fibrils, as has been seen by others [160]. (D) Scanning electron
micrograph of collagen mineralized without polyAsp (the control) shows only clusters of 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 fibril that appeared to contain amorphous phase discussed above (Fig. 7A), the precursor
mechanism of mineralization was verified on bulk samples with X-ray diffraction. Two sets of XRD time series
measurements were performed (Fig. 8). The first time-series experiment was run with sample sets in parallel, in which
four samples of Cellagen1 were mineralized within the same beaker, but removed at different time points (Fig. 8a).
The second time-series experiment was run in series, with the same sample being examined at consecutive time points
(Fig. 8b). For comparison, XRD patterns for commercial hydroxyapatite (Fig. 8a, 1) and natural bone (Fig. 8a, 5) were
included.
For the parallel time series, the control reaction consisted of the Cellagen1 scaffold mineralized in the absence of
polymer (Fig. 8a, 2). The XRD patterns contained sharp hydroxyapatite peaks similar to the commercial HA standard
(Fig. 8a, 1), as might be expected based on the relatively large (micrometer) dimensions of the crystals seen in Fig. 6D.
When Cellagen1 was mineralized in the presence of polymer additive, broad hydroxyapatite peaks were seen to
emerge after 1 day, which then became sharper by days 2 and 6 (Fig. 8a, 35); but, as is the case for bone (Fig. 8a, 6),
they never developed into the narrow peaks typical of synthetic hydroxyapatite (Fig. 8a, 1). The peak at 338 is 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 solidification of the precursor phase.
93
Fig. 7. Mineralization of Cellagen1 sponge via the PILP process (with 15 mg/ml polyAsp). (A) TEM of a collagen fibril removed during the early
stages of infiltration with calcium phosphate. The appearance of the banding pattern in the unstained fibril suggests that it is entrenched with
amorphous mineral. Bar = 200 nm. The SAED pattern (inset) of this fibril confirms the amorphous nature of the mineral precursor. (B) TEM of
collagen fibrils isolated after the mineralization reaction shows a distinct striated texture that runs parallel to the fibers. Remnant droplets of the PILP
phase can be seen adsorbing to the fibers (arrows). Bar = 100 nm. (C) SEM of PILP mineralized fibrils shows a relatively non-descript mineral
morphology, even after crystallization. (D) Because identifiable crystal features are not seen in surface view, energy dispersive spectroscopy of the
mineralized composites is useful for confirming the presence of mineral, as seen here by the large Ca and P peaks. (E) A fractured fibril reveals the
extent of intrafibrillar mineral penetration, as evidenced by the platy/needle-like HA crystals that protrude from the fracture surface. This 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 fibril, suggesting the precursor had penetrated the entire fibril and subsequently crystallized.
Bar = 100 nm. (F) TEM bright-field image of HA crystals extracted from the mineralized composite using a strong bleach treatment to fully remove
collagen. The platy morphology of the HA can be observed (platelets are laying on a holey carbon film). Some bundles were difficult to separate into
individual crystals and appear much darker. The striations on the thick bundles on the bottom left suggest that the crystals are stacked such that they
are being viewed nearly edge on. The crystals that were isolated (such as those seen at the top) appear to have a platy morphology which resembles
the nanocrystals extracted from bone (see Fig. 1, level 1). Bar = 200 nm.
94
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
c-Axis
(4)
(7)
(4)
(5)
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,
95
any increase in intensity of the diffraction peaks after the sample was placed in the buffer would have to result from
transformation of an already existing phase, as there was no additional source of mineral ions available for further
crystal growth.
At this point, the Cellagen1 product was discontinued and no longer commercially available, so an alternative
collagen scaffold that exhibits a similar fibrillar structure had to be used for further experiments. The calcanean turkey
tendon was chosen because it mineralizes naturally, and therefore provides a useful biological matrix for comparison.
The one concern about using a biological scaffold is that it may have crystals or nuclei already present in the collagen
which could stimulate further crystal growth. But in the control reaction (turkey tendon mineralized without addition
of polymeric process-directing agent), HA clusters formed only on the surface of the turkey tendon (data not shown),
yielding an XRD pattern similar to that shown in Fig. 8a(2) for the synthetic Cellagen1 scaffold. Therefore, it was
concluded that, if any crystal nuclei were present, they did not appreciably affect the mineralization process.
In Fig. 8b(1), the first XRD scan is of the turkey tendon alone. Upon mineralization, the sample was first measured
when mineral was visually evident (at 40 h), but only barely detectable by XRD, with only a broad intensity maximum
centered at about 2u = 328 that has been attributed to amorphous or paracrystalline [76,126] calcium phosphate
(Fig. 8b, 2). Subsequently, the same sample was stored in TRIS buffer (pH 7.4 and 37 8C) for another 24 and 48 h, 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 identification
Phase identification of calcium phosphate compounds, in particular distinguishing OCP and HA, can be difficult
because of their very similar d-spacings. Table 2 lists d-spacings derived from electron diffraction data measured for
our mineralized collagen samples, such as the representative fibril shown in Fig. 9A, as well as from natural bone
(Fig. 4A), for comparison to those of HA and OCP taken from the JCPDS standards. As can be seen, the d-spacings for
our samples and bone could be attributed to either mineral (especially the (0 0 2) and (0 0 4) reflections, which are
identical). We found that selected area electron diffraction (SAED) can be useful for differentiating between the
phases because it provides both the angles and the dimensions of the unit cell, which provides more data for correlation
to the standard unit cells in question. For example, in the example shown in Fig. 9, the platelets are preferentially
oriented in the [0 0 1] direction (as described more fully in the following section). Therefore, one can derive the angles
that each of the planes make with the (0 0 2) plane from the SAED pattern, and compare with the calculated values
based on the hexagonal unit cell of HA, or the triclinic unit cell of OCP (see Eqs. (1) and (2), respectively, in Section
2.2.2).
Table 2
Electron diffraction data from biomimetic and natural bone, measured against standards for hydroxyapatite (HA) and octacalcium phosphate
(OCP), demonstrating a method for distinguishing between HA and OCP, which have very similar d-spacings
Bone
Hydroxyapatite (JCPDS 9-432)
Biomimetic
Natural
)
d-Spacing (A
aa
)
d-Spacing (A
aa
3.44
3.13
2.82
2.81
2.77
2.33
1.99
1.87
1.72
0.0
90.0
65.5
36.3
90.0
90.0
55.9
36.7
0.0
3.44
3.10
2.81
2.77
2.71
2.26
1.92
1.84
1.72
0.0
90.0
65.5
38.0
90.0
90.0
58.3
38.4
0.0
a
b
Octacalcium phosphate (JCPDS 79-0423)
Plane
)
d-Spacing (A
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
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].
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
96
Fig. 9. TEM micrographs of Cellagen1 sponge mineralized by the PILP process (using 15 mg/ml polyAsp), demonstrating the location and
orientation of the HA crystallites. (A) Brightfield TEM image of a single mineralized fibril isolated from the mineralized sponge. The dark streaks
along the long axis of the collagen fibril suggest that platy hydroxyapatite crystals are uniaxially oriented within the collagen. The darker lines are
from those platelets viewed edge on, while other orientations are less easily seen due to the extreme thinness of the platelets. Banding patterns,
although not as pronounced as in the equine bone sample shown in Fig. 1A, can be discerned. Bar = 100 nm. (B) Selected area electron diffraction
(SAED) of the fibril shown in (A) demonstrates that the mineral phase is hydroxyapatite. The (0 0 2) and (0 0 4) planes are oriented parallel to the
long axis of the fibril (arrow), and the arcing, which is the same as in natural bone, indicates that the crystals are tilted with a slight mis-orientation
along their c-axis. Importantly, all fibrils examined had this same pattern, which matches in orientation and intensity the patterns exhibited by
naturally mineralized collagen found in bone and turkey tendon. (CF) Darkfield TEM images of the fibril in (A), illuminated with the (0 0 2),
(1 1 2), (2 1 1), and (3 0 0) diffraction planes, respectively. Platy or needle-like HA crystals can be seen to be uniaxially oriented along the long axis
of the collagen, and bright crystals appear throughout the fibril for each rotational orientation, suggesting that the crystals are not fully aligned in the
ab plane as would be expected for the deck of cards model. Bars = 200 nm.
97
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 intrafibrillar crystal arrangement, we examined individual mineralized fibrils with TEM by
crushing our mineralized collagen sponge samples in liquid nitrogen, employing similar sample preparation protocols
to those described in the literature for bone [129]. As is seen in natural bone, our mineralized fibrils show dark streaks
in brightfield TEM (Fig. 9A); the width of the streaks suggest that these are HA platelets which are being viewed edgeon. When viewed in full screen size (via electronic article), many lighter striations can also be seen throughout the
fibril. Crystals that are not oriented edge-on are more difficult to see since they are extremely thin, but further evidence
of the high mineral loading is demonstrated by application of dark-field TEM imaging (DF-TEM) to the same sample.
Imaging with the (0 0 2) diffracted beam (Fig. 9B) shows that the fibril is well infiltrated with numerous HA crystals
(Fig. 9C). Crystal lengths, as measured by the bright streaks, are 2550 nm, which falls in the range of bone
crystallites, which are reportedly 3050 nm in length [18]. Likewise, DF-TEM images using other diffracted beams
also show bright striations throughout the fibril (Fig. 9CF).
To determine the crystallographic orientation of the intrafibrillar HA crystals, selected area electron diffraction
(SAED) was performed on isolated fibrils. The SAED pattern of the electron dense, 200 nm diameter fibril illustrated
in Fig. 9A indicates that the HA crystals are oriented in the [0 0 1] direction parallel to the long axis of the collagen
fibril (arrow, Fig. 9B). To our surprise, the SAED pattern was indistinguishable from that of bone (compare Fig. 9B to
Fig. 4B and electron diffraction patterns of bone from the literature [130]). Although we anticipated getting crystals
within the collagen fibrils using this capillary-infiltration mechanism, we did not expect them to have the same
orientation as found in bone, given that we did not add any specific nucleating domains or specialized proteins to the
collagen. Every mineralized fibril (n > 20) we imaged using TEM exhibited the same SAED pattern as Fig. 9B, 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 fibrils, the mineralized collagen sample was
deproteinated using 2% sodium hypochlorite (NaOCl) for 15 min [116,120], and the remnant mineral phase was
examined by SEM (Fig. 10). Although it was not possible to be certain that the organic matrix has been completely
removed by this procedure, it served the purpose of exposing the principal mineral component. The intrafibrillar
organization of the crystals was well preserved, as can be seen by the uniaxial alignment of the crystals (Fig. 10A and
B), in which it appears the mineral phase had fully infiltrated the collagen fibrils. The crystals in the image exhibit a
mixed fibrousplaty texture, and apparently had become very elongated as crystal growth traversed along the collagen
fibril. This fibrousplaty texture is strikingly similar to SEM images of real bone presented by Weiner et al. [130], as
well as our example illustrated in Fig. 3D.
To carry the comparison to natural bone further, the bleach treatment was applied to an equine bone sample
(Fig. 10C) as well as turkey bone (Fig. 10D), and the remnant mineral phase examined by SEM. The meandering
crystal texture we had observed in our sample (Fig. 10A and B) can also be seen in the natural samples, although the
fine texture of the biogenic crystals is less pronounced, and distinct crystals are not readily discerned. The most
noticeable difference is the size and organization of the collagen scaffold. The differences between the bleached
samples and the original bone samples (Fig. 3), when imaged with the SEM, are not that obvious given the apparent
continuity of mineral phase; yet physically, the bleached samples easily crumbled and lacked the mechanical integrity
of the original organicinorganic biocomposite.
3.6. Confocal studies on mechanism of mineral infiltration
The evidence that intrafibrillar mineralization has been achieved in our system is strong, but an alternative to our
hypothesized capillary action mechanism could be argued. For example, the polyAsp additive may have diffused into
98
Fig. 10. FE-SEM micrographs of mineralized collagen fibrils 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 fibrils, 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 fibrous structures are clearly the mineral
remnants of the composite after bleach treatment. Nevertheless, EDS spectroscopy was used to confirm the presence of Ca and P peaks, which
dominated the composition of the bleached sample (not shown). Bar = 1 mm. (B) At higher magnification, it is seen that the HA crystals exhibit a
platyfibrous 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 conflicting 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 fibril shape with relatively featureless texture, while the turkey bone seems to have a meandering ultrathin fibrousplaty 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 confined spaces, such as a hydrogel, due to the large decrease in entropy. Many of the unusual
properties of macromolecules are dominated by the entropy component of the Gibbs free energy. On the other hand,
proteins, even though they are biological macromolecules, may behave differently from conventional random coil
polymers, particularly if the protein has a globular conformation and its diffusion and transport properties are more
similar to that of a particle. In the case of the highly anionic, soluble proteins associated with biominerals, less ordered
conformations have been observed [131133]; therefore, they might be expected to behave more like a random coil
polyelectrolyte and be dominated by entropy effects. Clearly, it is hard to predict (and unfortunately difficult to
measure) the state of the NCPs in their natural surroundings during biomineralization, hence the value of an in vitro
model system becomes apparent.
To determine 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 fluorescently 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
99
Fig. 11. Confocal fluorescence 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 fluorescence 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 significant 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 profiles 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
interfibrillar 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 fibrils 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 fluorescence 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 fluorescence markedly more
intense, the depth of penetration had more than doubled. We examined beyond the region of brightest fluorescence
with a PMT detector of higher sensitivity and found that, in fact, some polymer had traversed well beyond 500 mm,
though the fluorescence 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 fluorescence
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
solidification 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 fluid.
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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 first experiments were
successful with respect to demonstrating proof-of-concept that a mineral precursor could be infiltrated into collagen
fibrils [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 intrafibrillar 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 fibrils first absorb the fluidic amorphous precursor
(Fig. 7A), which subsequently crystallizes as the waters of hydration are released during the solidification 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 insufficient 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 fibers, because it is inconceivable that
such peaks could arise solely from the limited supply of ions adsorbed onto the collagen fibrils. 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 fibril which produced a diffraction pattern with only diffuse rings was clearly well infiltrated 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 fiber 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 fibers (and bone) is counterintuitive to the idea that they are infiltrated 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 fibrils, versus imaging the surface of the fibrils by SEM.
Lastly, even after the amorphous mineral has crystallized, the fibers 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 fiber
(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 sufficiently hydrated to be a liquid phase is that it enables the collagen fibrils 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 intrafibrillar
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 fluidity necessary to be transported into the collagen
fibers via capillary action.
The following provides an in depth discussion as to the possible ramifications 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 first 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 field 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 influence 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
fibrils (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 intrafibrillar 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 solidification 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
sufficiently 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. Specifically, 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 infiltrating the
fibrils with the mineral precursor in the first 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., solidification 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 finding a specific 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 identification of an amorphous precursor is the presence of other (crystalline) phases
that make identification of an amorphous phase difficult. 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 infiltrates 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. Clarification 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 fibril (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, first 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 flat platelet lying normal to an electron beam (such as
would occur when the long axis of a collagen fibril 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 intrafibrillar bone and
turkey tendon (as well as in our mineralized fibrils); 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 fibril diameter (in our synthetic sample), indicating that alignment is not parallel
throughout the fibril. 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 fiber (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 fibril, there needs to be a rotation about the
c-axis of the hydroxyapatite crystals from the beam normal, B 1 1 0, by at least 108 for the (2 1 1), (2 1 3), and
(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 fibril, 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 field 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 fibril for each of these planes, at least demonstrating that these additional
diffraction planes are not from crystals in separate regions of the fibril.
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
fibrils 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
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 0).
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 difficult 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 significance of these additional diffraction planes becomes apparent in the recent work by
Rhee et al. [100], who compare the mineralization of chondroitin sulfate (CS) fibers versus collagen fibers in an in vitro
reaction. Although the individual fibers of both scaffolds had orientation of HA crystals in the [0 0 1] direction along
the long axis of the fibers, 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 fibers. 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 significant 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 satisfied 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 specific crystal order within our schematic illustration in Fig. 5 because the
rotational variation is still not defined (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 flat, 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 fibro-platy
texture of the crystals (as seen in our samples) may explain why the bone literature is full of conflicting descriptions of
the morphology of HA crystals in bone, which have been described as needles, rods, filaments 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 identifiable 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 field-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 field 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 deorganified 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 fibers. Even with this apparently higher degree of
deproteination, they also found that remnant fibers 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 fibrillar structure reflecting the collagen template. Periodic bulges along the long
axis of the fibrils, with a spacing of about 70 nm, representing aggregates of crystallites, correspond to the banding pattern of collagen. (b) Higher
magnification of (a), with arrows indicating crystallites periodically bridging the fibrils. 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 deorganified).
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sufficient inter-crystallite bonding to retain the fibril 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 fibrous material. It must be considered to be a collagen fiber-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 fibrils in Cellagen1 leads to a fibrillar 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 fibril 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 extrafibrillar crystals which are larger than
crystals found in normal individuals, while the intrafibrillar 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 fibrils that appear clubbed (reprint provided in Fig. 3A). This is often observed
in our mineralized fibrils as well (Fig. 7C), yet when these same structures are bleached to preferentially remove the
organic phase, the fibro-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 solidification 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
finely 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
solidifies.
Their observations also indicate that a crystal is not confined 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 fibrils 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
fibrils. This alignment appears to occur for reconstituted collagen fibrils 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 intrafibrillar space, and upon solidification, 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 fibrils. In the case of CaP mineralized
108
Fig. 15. An atypical but illustrative example of mineral crystals growing within and emerging from the collagen fiber constraints. This sample was
not treated with bleach, but evidently was so fully mineralized that the crystals protrude beyond the intrafibrillar constraint. (A) At low
magnification, the fibers 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 magnification, the edges of HA crystals can be seen protruding from the surface of the collagen fibrils. 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 fibrils. Bar = 100 nm. (C) In this region, the bulges along the fibril 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 fibrils 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
difficult 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 first place would be the capillary
infiltration mechanism, as discussed below in Section 4.6.
4.5. Mechanism of alignment of intrafibrillar 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
109
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-specific character of the interactions arising from the polymeric process-directing
agent. This is not to say that specific 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 intrafibrillar 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 specific 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 fibrils; 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 fibrils (this is best seen by enlarging the figure to
full screen if an electronic file 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
intrafibrillar crystals cannot be seen on the surface. Interestingly, clubbing can be seen along the fibrils, in which it
appears that the crystal bundles grew in a uniaxial fashion when they were restricted by the fibril compartment, until
they overcame the constraint and emerged from the fibrils (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 fibril, rather than any specific 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 fibrils (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 fibrils 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 confined 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 infiltrate 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 fibrous growth pattern, perhaps with some twisting as they continued
to grow throughout the fibril. 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 fibrils. This
could also result in the rotational misorientations described above.
The diffraction evidence, in combination with the SEM images of mineralized fibrils, 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
110
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 intrafibrillar precursor phase is constrained by the nanoscopic space within
the collagen fibrils, both the dimensions and growth direction of the crystals become limited, leading to nanocrystals
that are stacked in uniaxial arrays within the collagen fibrils.
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 infiltration within the collagen matrix. The collagen scaffold acts like a large
sponge to absorb the fluidic 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 infiltrate 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 infiltrated 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 sufficiently high concentrations to nucleate crystals within the collagen, and there is not sufficient 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 intrafibrillar mineralization.
This capillary mechanism for intrafibrillar 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 solidification 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-fibered), 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
111
that the high degree of intrafibrillar mineralization that occurs in bone formation is achieved by capillary action
applied to a fluidic 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 fibril. The fluidity of
the amorphous precursor phase, which is a function of the polymeric process-directing agent, is thought to play a
crucial role in achieving intrafibrillar mineralization through a capillary infiltration mechanism.
The SAED patterns of individual mineralized fibrils are basically identical to those of bone and naturally
mineralizing turkey tendon. By demonstrating the orientational relationship of platelets along the fibril 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-specific epitaxial nucleation and growth is not necessary to
achieve intrafibrillar crystallization, shedding a new light onto existing theories of calcification 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
specifically 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 first step towards
reaching that goal. Given that this is the first 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
financial 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.
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Materials Science and Engineering R 58 (2007) 77116

Bone structure and formation: A new perspective

Available online 20 July 2007

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

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

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1.2. Nanostructured architecture of secondary bone

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h1 h2 k1 k2 1=2h1 k2 k1 h2 3=4a2 =c2 l1 l2

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

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

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3.2. X-ray diffraction (XRD) studies

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Hydroxyapatite (JCPDS 9-432)

Octacalcium phosphate (JCPDS 79-0423)

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

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

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

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