Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds

The most widely accepted hypothesis to account for maturational changes in the X-ray diffraction characteristics of bone mineral has been the 'amorphous calcium phosphate theory', which postulates that an initial amorphous calcium phosphate solid phase is deposited that gradually converts to poorly crystalline hydroxyapatite. Our studies of bone mineral of different ages by X-ray radial distribution function analysis and 31P n.m.r. have conclusively demonstrated that a solid phase of amorphous calcium phosphate does not exist in bone in any significant amount. 31P n.m.r. studies have detected the presence of acid phosphate groups in a brushite-like configuration. Phosphoproteins containing O-phosphoserine and O-phosphothreonine have been isolated from bone matrix and characterized. Tissue and cell culture have established that they are synthesized in bone, most likely by the osteoblasts. Physiochemical and pathophysiological studies support the thesis that the mineral and organic phases of bone and other vertebrate mineralized tissues are linked by the phosphomonester bonds of O-phosphoserine and O-phosphothreonine, which are constituents of both the structural organic matrix and the inorganic calcium phosphate crystals.     

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.     

Developmental changes in bone mineral structure demonstrated by extended X-ray absorption fine structure (EXAFS) spectroscopy

EXAFS spectra have been recorded above the calcium K edge from bones of mice aged 3 days, 1 week, 1 month, 2 months and 7 months. Spectra indicated that the calcium ion environment in bone mineral changes during development. Results were compared with those obtained from amorphous calcium phosphate and a poorly crystalline hydroxyapatite matured from this amorphous calcium phosphate in the presence of water. Spectra from the older mice closely resembled those of the matured product but those from the younger mice were more like those from the freshly prepared amorphous calcium phosphate.     

Investigation of the mineral phases of bone by solid-state phosphorus-31 magic angle sample spinning nuclear magnetic resonance

Phosphorus-31 magic angle sample spinning NMR spectra have been employed to investigate the structure and composition of the mineral deposits in chicken bone. Three different pulse sequences, Bloch decay, cross-polarization, and dipolar suppression, were employed to obtain spectra from bone specimens of varying age. These were compared to the spectra obtained from a variety of crystalline and noncrystalline synthetic calcium phosphate solids used as reference standards. The results suggest that the most suitable model for the major solid calcium phosphate mineral phase in bone is a hydroxyapatite containing approximately 5-10% CO32- and approximately 5-10% HPO42- groups, the latter in a brushite-like configuration. From the NMR line shapes it was deduced that the fraction of HPO42- groups was highest in the youngest bone and decreased progressively with increasing age of the specimen.     

Solid-state phosphorus-31 nuclear magnetic resonance studies of synthetic solid phases of calcium phosphate: potential models of bone mineral

Phosphorus-31 NMR spectra have been obtained from a variety of synthetic, solid calcium phosphate mineral phases by magic angle sample spinning. The samples include crystalline hydroxyapatite, two type B carbonatoapatites containing 3.2 and 14.5% CO3(2-), respectively, a hydroxyapatite in which approximately 12% of the phosphate groups are present as HPO4(2-), an amorphous calcium phosphate, monetite, brushite, and octacalcium phosphate. Spectra were observed by the standard Bloch decay and cross-polarization techniques, as well as by a dipolar suppression sequence, in order to distinguish between protonated and unprotonated phosphate moieties. The spectra of the synthetic calcium phosphates provide basic information that is essential for interpreting similar spectra obtained from bone and other calcified tissues.     

Bone tissue incorporates in vitro gallium with a local structure similar to gallium-doped brushite

During mineral growth in rat bone-marrow stromal cell cultures, gallium follows calcium pathways. The dominant phase of the cell culture mineral constitutes the poorly crystalline hydroxyapatite (HAP). This model system mimics bone mineralization in vivo. The structural characterization of the Ga environment was performed by X-ray absorption spectroscopy at the Ga K-edge. These data were compared with Ga-doped synthetic compounds (poorly crystalline hydroxyapatite, amorphous calcium phosphate and brushite) and with strontium-treated bone tissue, obtained from the same culture model. It was found that Sr²⁺ substitutes for Ca²⁺ in the HAP crystal lattice. In contrast, the replacement by Ga³⁺ yielded a much more disordered local environment of the probe atom in all investigated cell culture samples. The coordination of Ga ions in the cell culture minerals was similar to that of Ga³⁺, substituted for Ca²⁺, in the Ga-doped synthetic brushite (Ga-DCPD). The Ga atoms in the Ga-DCPD were coordinated by four oxygen atoms (1.90 Å) of the four phosphate groups and two oxygen atoms at 2.02 Å. Interestingly, the local environment of Ga in the cell culture minerals was not dependent on the onset of Ga treatment, the Ga concentration in the medium or the age of the mineral. Thus, it was concluded that Ga ions were incorporated into the precursor phase to the HAP mineral. Substitution for Ca²⁺ with Ga³⁺ distorted locally this brushite-like environment, which prevented the transformation of the initially deposited phase into the poorly crystalline HAP.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations ACP amorphous calcium phosphate - DCPD dicalcium phosphate dihydrate (brushite) - HAP hydroxyapatite - ED-XRF energy dispersive X-ray fluorescence - EXAFS extended X-ray absorption fine structure - Ga-ACP gallium-doped amorphous calcium phosphate - Ga-DCPD gallium-doped brushite - Ga-HAP gallium-doped hydroxyapatite - XANES X-ray absorption near edge structure - XAS X-ray absorption spectroscopy - XRD X-ray diffraction     

The (1)H NMR structure of bovine Pb(2+)-osteocalcin and implications for lead toxicity

Structural information on the effect of Pb(2+) on proteins under physiologically relevant conditions is largely unknown. We have previously shown that low levels of lead increased the amount of osteocalcin bound to hydroxyapatite (BBA 1535:153). This suggested that lead induced a more compact structure in the protein. We have determined the 3D structure of Pb(2+)-osteocalcin (49 amino acids), a bone protein from a target tissue, using (1)H 2D NMR techniques. Lead, at a stoichiometry of only 1:1, induced a similar fold in the protein as that induced by Ca(2+) at a stoichiometry of 3:1. The structure consisted of an unstructured N-terminus and an ordered C-terminal consisting of a hydrophobic core (residues 16-49). The genetic algorithm-molecular dynamics simulation predicted the lead ion was coordinated by the Gla 24 and Gla 21 residues. It is proposed that mineral binding occurs via uncoordinated Gla oxygen ions binding to calcium in hydroxyapatite. A comparison of Pb(2+)- and Ca(2+)-osteocalcin suggests Pb(2+), at a lower stoichiometry, may induce similar conformational changes in proteins and subsequent molecular processes normally controlled by calcium alone. This may contribute to a molecular mechanism of lead toxicity for calcium binding proteins. Lead exposure may alter the amount of mineral bound osteocalcin and contribute to abnormal bone remodeling.     

The presence of γ-carboxyglutamic acid in the proteins associated with ectopic calcification

γ-Carboxyglutamic acid (Gla) is identified in the proteins associated with several types of ectopic calcifications in which hydroxyapatite is the major mineral component. These included the calcified skin and subcutaneous plaques from a patient with dermatomyositis, the calcium containing material extruded from the skin of a patient with scleroderma, and the calcified, atheromatous plaques from aorta. Alkaline hydrolysis of biopsy material from these and from normal tissue revealed the presence of Gla only in the plaque specimens. Since a γ-carboxyglutamic acid-containing protein is normally present in bone and absent in unmineralized tissues, the presence of Gla in soft tissue calcifications is a potentially significant finding, especially in view of its known calcium and phospholipid binding properties.     

Connective tissue mineralization in Abcc6-/- mice, a model for pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by ectopic mineralization. However, the structure of the mineral deposits, their interactions with the connective tissue matrix, and the details of the progressive maturation of the mineral crystals are currently unknown. In this study, we examined the mineralization processes in Abcc6(-/-) mice, a model system for PXE, by energy dispersive X-ray and Fourier transform infrared imaging spectroscopy (FT-IRIS). The results indicated that the principal components of the mineral deposits were calcium and phosphate which co-localized within the histologically demonstrable lesions determined by topographic mapping. The Ca/P ratio increased in samples with progressive mineralization reaching the value comparable to that in endochondral bone. A progressive increase in mineralization was also reflected by increased mineral-to-matrix ratio determined by FT-IRIS. Determination of the mineral phases by FT-IRIS suggested progressive maturation of the mineral deposits from amorphous calcium phosphate to hydroxyapatite. These results provide critical information of the mechanisms of mineralization in PXE, with potential pharmacologic implications.     

Synthesis and in vitro characterization of a tissue-selective fullerene: vectoring C(60)(OH)(16)AMBP to mineralized bone

A tissue-vectored bisphosphonate fullerene, C(60)(OH)(16)AMBP [4,4-bisphosphono-2-(polyhydroxyl-1,2-dihydro-1,2-methanofullerene[60]-61-carboxamido)butyric acid], designed to target bone tissue has been synthesized and evaluated in vitro. An amide bisphosphonate addend, in conjunction with multiple hydroxyl groups, confers a strong affinity for the calcium phosphate mineral hydroxyapatite of bone. Constant composition crystal growth studies indicate that C(60)(OH)(16)AMBP reduces hydroxyapatite mineralization by 50% at a concentration of 1 microM, following a non-Langmuirian mechanism. Parallel studies with C(60)(OH)(30) also indicate an affinity for hydroxyapatite, but at a reduced level (28% crystal growth rate reduction at 1 microM) compared with C(60)(OH)(16)AMBP. This study is the first to demonstrate that a fullerene-based material can be successfully targeted to a selected tissue as a step toward the development of such materials for medical purposes, in general.     

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.     

Microstructural Features of Non-Union of Human Humeral Shaft Fracture

Microstructures of non-unions of human humeral shaft fractures were investigated by using scanning electron microscopy, transmission electron microscopy, and X-ray microdiffraction. The non-union has a trabeculae structural framework similar to woven bone. Among the trabeculae are cavities that are subdivided into small chambers by thin plates of collagen fibrils. Some chambers are filled with variously shaped mineralized particles several micrometers in size. The collagen fibrils in both the trabeculae and the thin plates were only slightly mineralized by hydroxyapatite. Vesicles loaded with noncrystalline calcium phosphate (NCP) were observed in most mineralized particles, and brushite crystals with special morphology were seen to be embedded in some particles in irregular shapes. X-ray microdiffraction results indicated that the mineral phases in the non-unions were mainly NCP in addition to small amounts of hydroxyapatite and brushite. NCP deposition and insufficient mineralization of the collagen fibrils may be two important microstructural features of the non-unions of human humeral shaft fractures different from normally repaired bone callus.     

Structural evidence of a fourth Gla residue in fish osteocalcin: biological implications

Osteocalcin is a small (45 amino acids) secreted protein found to accumulate in bone and dentin of many organisms by interacting with calcium and hydroxyapatite, through the presence of three gamma-carboxylated residues. In this work, we describe the first X-ray crystal structure for a nonmammalian osteocalcin, obtained at 1.4 A resolution, purified from the marine teleost fish Argyrosomus regius. The three-dimensional fit between the A. regius structure and that of the only other known X-ray structure, the porcine osteocalcin, revealed a superposition of the Calpha atoms of their metal chelating residues, Gla and Asp, showing that their spatial distribution is consistent with the interatomic distances of calcium cations in the hydroxyapatite crystals. In both structures, the protein forms a tight globular arrangement of their three alpha-helices while the remaining residues, at N- and C-terminal regions, have essentially no secondary structure characteristics. This study revealed the presence of a fourth gamma-carboxylation at Glu(25), not previously detected in the structure of the porcine osteocalcin or in any other of the sequentially characterized mammalian osteocalcins (human, cow, and rat). A confirmation of the fourth Gla residue in A. regius osteocalcin was achieved via LC-MS analysis. These four doubly charged residues are, together with Asp(24), concentrated in a common surface region located on the same side of the molecule. This further suggests that the known high affinity of osteocalcin for bone mineral may be derived from the clustering of calcium binding sites on this surface of the molecules.     

Osteoporosis in neurodegeneration

Osteoporosis affects bone microarchitecture and reduces bone mass. There are more than 200 million people with osteoporosis worldwide, and the prevalence is slowly increasing. The highest prevalences are found in Scandinavia and USA, also slowly increasing. A parallel increase in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and multiple sclerosis has been noted since the middle of this century. Osteoporosis is more common in patients with each of these neurodegenerative conditions than in the general population. Several metals with neurotoxic properties accumulate in bone and can substitute for calcium in hydroxyapatite, the main mineral component of bone. Especially cadmium, but also lead, aluminum and arsenic affect bone mineral density negatively. Metals with neurotoxic properties have also been found in brain and cerebrospinal fluid from patients with Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and multiple sclerosis, and markers for neurodegeneration such as amyloid beta peptide and amyloid precursor protein have been detected in bone tissue from patients with osteoporosis. A common mechanism contributing to the pathogenesis of both neurodegeneration and osteoporosis can be suspected. The hypothesis that neurodegenerative disorders are associated with osteoporosis is presented and discussed.     

Chick embryo anchored alkaline phosphatase and mineralization process in vitro.

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI-bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X-ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate by GPI-bALP produced hydroxyapatite in a manner similar to that of beta-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6-12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI-bALP in calcium-containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI-bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.     

The reaction between some dyes and synthetic hydroxyapatite I. The uptake of dyes in relation to their structures

Synopsis The uptake of dyes from dilute solutions by synthetic hydroxyapatite and other sparingly soluble calcium compounds has been determined. About 30 dyes, mostly azo-, dis-azo and anthraquinonoid types were used in 95% ethanol or 0.1 M tris buffer. Many had closely related configurations. Chemical groupings possibly responsible for the adsorption of particular dyes by hydroxyapatite have been deduced from an analysis of the results. The uptake of most dyes from alcoholic solutions was, linearly related to the surface area of hydroxyapatite. Calcium carbonate and secondary calcium phosphate took up less stain than hydroxyapatite of similar surface area. With the simpler anthraquinonoid dyes, the uptake was higher from aqueous than alcoholic solutions, but specificity for hydroxyapatite was much less. The increased uptake of dye by powdered bone or dentine when rendered anorganic was proportional to the increased surface area. It was found that several dyes in common use as stains for bone and calcified tissue were only poorly adsorbed by synthetic hydroxyapatite under the particular conditions of these experiments.The experimental data presented could be used as a basis for the development of histochemical reactions for calcified tissue or inclusions. By suitable choice of dyes, solvent and rinsing solution it ought to be possible to differentiate various forms of calcified material.     

On the role of bone damage in calcium homeostasis

Bone serves as the reservoir of some minerals including calcium. If calcium is needed anywhere in the body, it can be removed from the bone matrix by resorption and put back into the blood flow. During bone remodelling the resorbed tissue is replaced by osteoid which gets mineralized very slowly. Then, calcium homeostasis is controlled by bone remodelling, among other processes: the more intense is the remodelling activity, the lower is the mineral content of bone matrix. Bone remodelling is initiated by the presence of microstructural damage. Some experimental evidences show that the fatigue properties of bone are degraded and more microdamage is accumulated due to the external load as the mineral content increases. That damage initiates bone remodelling and the mineral content is so reduced. Therefore, this process prevents the mineral content of bone matrix to reach very high (non-physiological) values. A bone remodelling model has been used to simulate this regulatory process. In this model, damage is an initiation factor for bone remodelling and is estimated through a fatigue algorithm, depending on the macroscopic strain level. Mineral content depends on bone remodelling and mineralization rate. Finally, the bone fatigue properties are defined as dependent on the mineral content, closing the interconnection between damage and mineral content. The remodelling model was applied to a simplified example consisting of a bar under tension with an initially heterogeneous mineral distribution. Considering the fatigue properties as dependent on the mineral content, the mineral distribution tends to be homogeneous with an ash fraction within the physiological range. If such dependance is not considered and fatigue properties are assumed constant, the homogenization is not always achieved and the mineral content may rise up to high non-physiological values. Thus, the interconnection between mineral content and fatigue properties is essential for the maintenance of bone's structural integrity as well as for the calcium homeostasis.     

Biomimetic CoUagen/Hydroxyapatite Composite Scaffolds： Fabrication and Characterizations

Biomimetic collagen/hydroxyapatite scaffolds have been prepared by microwave assisted co-titration of phosphorous acid-containing collagen solution and calcium hydroxide-containing solution. The resultant scaffolds have been characterised with respect to their mechanical properties, composition and microstructures. It was observed that the in situ precipitation process could combine collagen fibril formation and hydroxyapatite （HAp） formation in one process step. Collagen fibrils guided hydroxyapatite precipitation to form bone-mimic collagen/hydroxyapatite composite containing both intrafibrillar and interfibrillar hydroxyapatites. The mineral phase was determined as low crystalline calcium-deficient hydroxyapatite with calcium to phosphorus ratio （Ca/P） of 1.4. The obtained 1% （collagen/HAp = 75/25） scaffold has a porosity of 72% and a mean pore size of 69.4 ~tm. The incorporation of hydroxyapatite into collagen matrix improved the mechanical modulus of the scaffold significantly. This could be attributed to hydroxyapatite crystallites in collagen fibrils which restricted the deformation of the collagen fibril network, and the load transfer of the collagen to the higher modulus mineral component of the composite.     

Dietary calcium,sex hormones,and bone mineral density in men.

OBJECTIVE--To evaluate the factors that determine bone mineral density at axial and appendicular sites in normal men. DESIGN--Measurement of bone mineral density of the radius by single photon absorptiometry and of the lumbar spine and hip by dual photon absorptiometry to assess their relation with various determinants of bone mineral density. Dietary calcium was assessed from a questionnaire validated against a four day dietary record. SETTING--Local community, Sydney, Australia. PATIENTS--48 Men (aged 21-79, median 44) recruited from the local community including 35 male cotwins of twin pairs of differing sex recruited from the Australian National Health and Medical Research Council twin registry for epidemiological studies on determinants of bone mineral density. MAIN OUTCOME MEASURES--Bone mineral density of the axial and appendicular skeleton and its relation to age, anthropometric features, dietary calcium intake, and serum sex hormone concentrations. RESULTS--Dietary calcium intake (g/day) was a significant predictor of bone mineral density of axial bones, explaining 24% and 42% of the variance at the lumbar spine and femoral neck respectively. This effect was independent of weight. In contrast with the axial skeleton, bone mineral density at each forearm site was predicted by weight and an index of free testosterone but not by dietary calcium intake. CONCLUSIONS--Dietary calcium intake has a role in the determination or maintenance, or both, of the axial but not the appendicular skeleton in adult men.     

Collagen Osteoid-Like Model Allows Kinetic Gene Expression Studies of Non-Collagenous Proteins in Relation with Mineral Development to Understand Bone Biomineralization

Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.