Beads of collagen-nanohydroxyapatite composites prepared by a biomimetic process and the effects of their surface texture on cellular behavior in MG63 osteoblast-like cells

The aim of this work was to develop a novel method for preparing a three-dimensional bone-like matrix comprising nanohydroxyapatite crystals and fibrous collagen and to apply it for bone tissue engineering. Hydroxyapatite and collagen are the major components of natural hard bone. Therefore, they have been used extensively in orthopedic surgery as bone-filling materials. According to the principle of complex coacervation, three-dimensional collagen beads can be formed by extruding collagen solution into chondroitin sulfate A (CSA) solution. Subsequently, the collagen beads thus formed are soaked in simulated body-fluid solution to biomimic the formation process of natural bone matrix via the fabrication of collagen-nanohydroxyapatite beads. We also investigate the effect of the collagen-nanohydroxyapatite matrix on the proliferation and differentiation of MG63 cells. The presence of crystalline hydroxyapatite structure on the surface of fibrous collagen was confirmed by X-ray diffraction. MG63 cells cultured on the collagen-nanohydroxyapatite beads proliferate at the normal rate. Moreover, alkaline phosphatase (ALP) activity and the expression levels of three osteogenic genes, namely, type I collagen osteopontin and osteocalcin, in MG63 cells were significantly higher when the cells were cultured on collagen-nanohydroxyapatite beads than when they were cultured on collagen alone. The results of this study reveal that, in the presence of nanohydroxyapatite, the three-dimensional cell beads not only provide a substrate for cell growth but could also enhance the osteoblast-like cell differentiation of MG63 cells.     

The structural and biological properties of hydroxyapatite-modified titanate nanowire scaffolds

Hydroxyapatite-modified titanate nanowire scaffolds as alternative materials for tissue engineering have been developed via a titanate nanowire matrix assisted electrochemical deposition method. The macroporous titanate nanowire matrix on Ti metal was fabricated by a hydrothermal method, and then followed by an electrochemical synthesis of hydroxyapatite nanoparticles on titanate nanowire. The incorporation of titanate nanowire matrix with high oriented hydroxyapatite nanoparticles generates hierarchical scaffolds with highly osteogenic, structural integrity and excellent mechanical performance. As-prepared porous three dimensional interconnected hydroxyapatite-modified titanate nanowire scaffolds, mimicking the nature's extracellular matrix, could provide a suitable microenvironment for tissue cell ingrowth and differentiation. The ceramic titanate nanowire core with HA nanoparticle sheath structure displays superhydrophilicity, which facilitates the cell attachment and proliferation, and induces the in vitro tissue-engineered bone. Human osteoblast-like MG63 cells were cultured on the hydroxyapatite-modified titanate nanowire scaffolds, and the results showed that the scaffolds highly promote the bioactivity, osteoconductivity and osteoblast differentiation.     

Calcium phosphate-chitosan composite scaffolds for bone tissue engineering

Macroporous calcium phosphate-chitosan composite scaffolds were fabricated and evaluated for use in bone tissue engineering. Human osteoblast-like MG63 cells were cultured on the composite scaffolds, and their response to the materials was studied. Cell morphology, total protein content, and expression of classic markers for osteoblast differentiation were characterized. MG63 cells on the hydroxyapatite scaffolds nesting chitosan sponges (HC1) showed significantly higher alkaline phosphatase (ALP) level and osteocalcin (OC) production during the 11-day culture period, compared with the control culture on tissue culture plates. Cells on the chitosan scaffolds incorporated with hydroxyapatite powders (HC2) exhibited lower ALP activity during the 11-day culture period and OC secretion during the first 7 days, in comparison with that on HC1. The addition of calcium phosphate glass as in HC3 scaffolds increased the ALP and OC levels of MG63 cells. Our study indicated that the hydroxyapatite-matrix composite scaffolds might enhance the phenotype expression of MG63 cells, in comparison with chitosan-matrix scaffolds. Soluble calcium phosphate glasses should be added to the scaffolds to prevent chitosan from fast degradation that may affect the differentiation of osteoblast cells.     

Mineralisation of chitosan scaffolds with nano-apatite formation by double diffusion technique

The study of inorganic crystal assembly in organic matrices has given rise to increasing interest in various fields of materials science to the natural process of biomineralisation. To mimic the formation of hydroxyapatite as natural bone, a double diffusion technique is utilised in this study to nucleate the hydroxyapatite crystals onto three-dimensional porous polymeric scaffolds. The porous polymer scaffolds were produced from chitosan by a thermally induced lyophilisation technique, which yields highly porous, well-controlled anisotropic open pore architecture. The nucleation of hydroxyapatite crystals was initiated at ambient conditions on the surface of the polymer scaffold, which was in contact with a calcium solution chamber, due to diffusion of phosphate ions through the scaffold. The morphology of the mineralised scaffold as analysed by scanning electron microscopy shows that apatite crystals were not only formed on the surface of the scaffold, but also in the pore channels and attached to the pore walls. The X-ray diffraction and Fourier transformed infrared analyses confirmed the phase purity of the formed apatite crystals. The transmission electron microscopy analysis reveals the microstructure of the entangled nano-apatite in the chitosan polymeric matrix. The in-vitro cytocompatibility tests with osteoblast-like cells (Saos-2) demonstrated that the biomineralised scaffold is a suitable substrate for cell attachment and migration in bone tissue engineering.     

Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes

Response of different types of cells on materials is important for the applications of tissue engineering and regenerative medicine. It is recognized that the behavior of the cell adhesion, proliferation, and differentiation on materials depends largely on surface characteristics such as wettability, chemistry, charge, rigidity, and roughness. In this study, we examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surfaces with different micropore sizes (0.2-8.0 microm in diameter). Cell adhesion and proliferation to the PC membrane surfaces were determined by cell counting and MTT assay. The effect of surface micropore on the MG63 cells was evaluated by cell morphology, protein content, and alkaline phosphatase (ALP) specific activity. It seems that the cell adhesion and proliferation were progressively inhibited as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. Increasing micropore size of the PC membrane results in improved protein synthesis and ALP specific activity in isolated cells. There was a statistically significant difference (P<0.05) between different micropore sizes. The MG63 cells also maintained their phenotype under conditions that support a round cell shape. RT-PCR analysis further confirmed the osteogenic phenotype of the MG63 cells onto the PC membranes with different micropore sizes. In results, as micropore size is getting larger, cell number is reduced and cell differentiation and matrix production is increased. This study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells.     

Genetic effect of anatase on osteoblast-like cells

Titanium is the gold standard among materials used for prosthetic devices, because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. Three allotropic forms of titanium dioxide exist, that is brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biological effects. Here we are testing the effect of AC on osteoblast-like cells (MG63) by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to AC. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cell lines (MG-63) cultured on AC, several genes whose expression was significantly up- or downregulated. They cover a broad range of functional activities: signaling transduction, immunity, cell cycle regulation, lysosomes composition and vesicular transport, cell adhesion, cytoskeleton and extracellular matrix components, proliferation, and apoptosis. The data reported constitute, to our knowledge, the first genetic portrait of AC effects. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.     

Surface and biological evaluation of hydroxyapatite-based coatings on titanium deposited by different techniques.

Hydroxyapatite coatings have been deposited on titanium cp by plasma spray, sol-gel, and sputtering techniques for dental implant applications. The latter two techniques are of current interest, as they allow coatings of micrometer dimensions to be deposited. Coating morphology, composition, and structure have been investigated by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). All coatings were homogeneous and exhibited a rough morphology suitable for implant applications. The sputtered (after annealing), plasma spray, and sol-gel coatings all showed diffraction peaks corresponding to hydroxyapatite. The surface contaminants were observed to be different for the different coating types. The sputtered coatings were found to have a composition most similar to hydroxyapatite; the sol-gel deposits also showed a high concentration of hydroxyl ions. A discrepancy in the Ca/P ratio was observed for the plasma spray coatings, and a small concentration of carbonate ions was found in the sputter-deposited coatings. The in vitro cell-culture studies using MG63 osteoblast-like cells demonstrated the ability of cells to proliferate on the materials tested. The sol-gel coating promotes higher cell growth, greater alkaline phosphatase activity, and greater osteocalcin production compared to the sputtered and plasma-sprayed coatings.     

Generation of mesoscopic patterns of viable Escherichia coli by ambient laser transfer.

We have generated mesoscopic patterns of viable Escherichia coli on Si(1 1 1), glass, and nutrient agar plates by using a novel laser-based transfer process termed matrix assisted pulsed laser evaporation direct write (MAPLE DW). We observe no alterations to the E. coli induced by the laser-material interaction or the shear forces during the transfer. Transferred E. coli patterns were observed by optical and electron microscopes, and cell viability was shown through green fluorescent protein (GFP) expression and cell culturing experiments. The transfer mechanism for our approach appears remarkably gentle and suggests that active biomaterials such as proteins, DNA and antibodies could be serially deposited adjacent to viable cells. Furthermore, this technique is a direct write technology and therefore does not involve the use of masks, etching, or other lithographic tools.     

Flow cytometry analysis of effects of glass on response of osteosarcoma cells to plasma-sprayed hydroxyapatite/CaO-P(2)O(5) coatings.

Multilayered coatings composed of mixtures of hydroxyapatite (HA) and P(2)O(5)-based bioactive glasses offer potential clinical benefits in orthopedic and dental surgery. In this study double-layer plasma-sprayed coatings were prepared and the biological response evaluated in tissue culture using two human osteosarcoma cell lines, MG63 and HOS TE85 (HOS). The cells were cultured on the materials and the effects on cell growth were determined using a spectrophometric assay of a mitochondrial enzyme that is active in viable cells. While none of the materials influenced the growth of the MG63 cells, the HOS cells appeared to undergo less proliferation on all the HA materials. Flow cytometry analysis was carried out using rabbit antibodies against osteonectin, osteopontin, bone sialoprotein, fibronectin, and collagen type I to measure the effects of the materials on key cellular functions. The results showed that the materials downregulated the expression of these extracellular matrix antigens by MG63 cells whereas they had less effect on the HOS cells compared to the same cells incubated on a plastic surface. Notably, with both cell lines the composite with the higher percentage of glass restored the production of connective tissue proteins to levels that were more similar to those present in the control cells.     

The influence of titania/hydroxyapatite composite coatings on in vitro osteoblasts behaviour

The biocompatibility of titania/hydroxyapatite (TiO2 /HA) composite coatings, at different ratio obtained by sol-gel process, were investigated studying the behaviour of human MG63 osteoblast-like cells. The biocompatibility was evaluated by means of cytotoxicity and cytocompatibility tests. Cytotoxicity tests, i.e., neutral red (NR), MTT and kenacid blue (KB) assays, were performed to assess the influence of the material extracts on lysosomes, mitochondria and cell proliferation, respectively. Cell proliferation, some preliminary indications of cell morphology, alkaline phosphatase activity, collagen and osteocalcin production of MG63 cells, cultured directly onto TiO2/HA substrates, were evaluated. The results showed that these materials have no toxic effects. Cell growth and morphology were similar on all the materials tested: on the contrary, alkaline-phosphatase-specific activity and collagen production of osteoblasts cultured on TiO2/HA coatings were significantly higher than uncoated titanium and polystyrene of culture plate and were influenced by chemical composition of the coatings. In particular, TiO2/HA coating at 1:1 ratio (w/w) seems to stimulate more than others the expression of some differentiation markers of osteoblastic phenotype. TiO2/HA coatings resulted to be bioactive owing to the presence of hydroxyl groups detected on their surface that promote the calcium and phosphate precipitation and improve the interactions with osteoblastic cells.     

The impact of critical point drying with liquid carbon dioxide on collagen-hydroxyapatite composite scaffolds

Collagen-hydroxyapatite composites for bone tissue engineering are usually made by freezing an aqueous dispersion of these components and then freeze-drying. This method creates a foamed matrix which may not be optimum for growing cell colonies larger than a few hundred micrometres due to the limited diffusion of nutrients and oxygen, and the limited removal of waste metabolites. Incorporating a network of microchannels in the interior of the scaffold which may permit the flow of nutrient-rich media has been proposed as a method to overcome these diffusion constraints. A novel three-dimensional printing and critical point drying technique previously used to make collagen scaffolds has been modified to create collagen-hydroxyapatite scaffolds. This study investigates the properties of collagen and collagen-hydroxyapatite scaffolds and whether subjecting collagen and hydroxyapatite to critical point drying with liquid carbon dioxide results in any changes to the individual components. Specifically, the hydroxyapatite component was characterized before and after processing using wavelength-dispersive X-ray spectroscopy, X-ray diffraction and infrared spectroscopy. Critical point drying did not induce elemental, crystallographic or molecular changes in the hydroxyapatite. The quaternary structure of collagen was characterized using transmission electron microscopy and the quarter-staggered array characteristic of native collagen remained after processing. Microstructural characterization of the composites using scanning electron microscopy showed the hydroxyapatite particles were mechanically interlocked in the collagen matrix. The in vitro biological response of MG63 osteogenic cells to the composite scaffolds were characterized using the Alamar Bluetrade mark, PicoGreentrade mark, alkaline phosphate and Live/Deadtrade mark assays, and revealed that the critical point dried scaffolds were non-cytotoxic.     

脱细胞天然骨基质与诱导性成骨细胞的体外相容性

背景：前期工作表明TritonX-100处理的脱细胞骨基质已满足组织学和免疫学方面的修复要求。如果细胞能在材料表面很好地生长,将利于进一步进行体内动物实验。 目的：采用细胞培养法在体外评估脱细胞骨基质与诱导后成骨细胞的生物相容性。 方法：第3代骨髓基质干细胞经成骨诱导分化培养液诱导分化为成骨细胞,接种于TritonX-100处理的脱细胞骨基质及羟基磷灰石表面,检测成骨细胞的碱性磷酸酶表达并用扫描电镜观察材料表面的细胞生长情况。 结果与结论：碱性磷酸酶活性分析均表明,TritonX-100处理的脱细胞骨基质在培养48 h之后比羟基磷灰石更利于诱导成骨细胞生长;扫描电镜下可见,成骨细胞在脱细胞骨基质表面呈现立体生长方式,细胞呈球形,并且聚集成簇。体外实验结果显示成骨细胞与脱细胞天然骨基质有较好的生物相容性。     

Direct and indirect effects of P2O5 glass reinforced-hydroxyapatite composites on the growth and function of osteoblast-like cells

Human osteoblast-like cells were plated on hydroxyapatite and P2O5-glass reinforced hydroxyapatite composite discs. They were also cultured in the presence of media obtained by incubating the discs in the absence of cells. The effects of these direct and indirect interactions were examined by measuring cell proliferation and the expression of certain key extracellular matrix antigens. One composite was found to initially delay cell growth, while the extract of a different composite appeared to down-regulate DNA synthesis. Flow cytometry analysis showed that growth directly on the discs had little effect on collagen type I, but reduced fibronectin and osteocalcin levels. The extracts of the materials generally had less effect, although one extract obtained from the glass-reinforced hydroxyapatite significantly down-regulated fibronectin. These in vitro studies thus suggest that there were only few differences overall in the growth of the cells directly on the glass-reinforced compared with the hydroxyapatite discs and also only relatively small effects of the extracts on the cells. However, the flow cytometry results suggest that both the materials and the extracts may have a potentially important influence on connective tissue production, and that these effects are both material- and antigen-specific.     

Alendronate-hydroxyapatite nanocomposites and their interaction with osteoclasts and osteoblast-like cells

The direct synthesis of hydroxyapatite in the presence of bisphosphonates is quite difficult due to the great affinity for calcium of these compounds, which are widely used in the treatment of pathologies related to bone loss. We recently developed a new method which allowed to synthesize alendronate-hydroxyapatite composite nanocrystals with a bisphosphonate content up to about 7 wt%. Herein we report the results of an in vitro study aimed to investigate the effects of alendronate incorporation into hydroxyapatite on bone cells response. Osteoblast-like MG63 cells and human osteoclasts were cultured on nanocrystals at different alendronate content (3.9, 6.2, 7.1 wt%). MG63 cells cultured on the composite nanocrystals display normal morphology, good proliferation and increased values of the differentiation parameters. In particular, when cultured on composites at relatively high alendronate contents, osteoblasts display increased values of alkaline phosphatase activity (ALP), collagen type I, and osteocalcin production, as well as significant decrease of matrix metalloproteinases (MMP-1 and MMP-13) production, with respect both to the control and to pure hydroxyapatite nanocrystals. It follows that the presence of alendronate enhances osteoblast activation and extracellular matrix mineralization processes, without any abnormal collagen degradation. The osteoclast number on the composite nanocrystals decrease indicating that the bisphosphonate exerts its inhibitory effect on osteoclast proliferation even when incorporated into hydroxyapatite.     

Hierarchical mesoporous silica nanofibers as multifunctional scaffolds for bone tissue regeneration

Mesoporous materials with pore sizes between 2 and 50?nm have elicited widespread interest in catalysis, separation, adsorption, sensors, and drug delivery applications due to its highly ordered pore size along with high hydrothermal stability and easily modifiable surface functionalities. Fabricating these mesoporous materials as continuous fibers offers exciting vistas for biomedical applications especially in tissue engineering. The aim of the present study was to fabricate, characterize, and evaluate the cellular and gene expression of mesoporous silica with a long ordered fibrous morphology to support regeneration of bone tissue. Tetraethyl orthosilicate, polyvinyl pyrrolidone, and the tri-block copolymer P-123 were subjected to electrospinning to fabricate continuous ordered mesoporous silica nanofibers by optimizing solution and operation parameters. Mesoporous silica fibers with an average diameter of 470?nm and mesopores of dimension 5.97?nm were obtained. The combination of micropores, mesopores, macropores, and the nanofibrous morphology imparted excellent bioactivity to the mesoporous silica fibrous scaffolds as demonstrated by the proliferation of human osteoblast-like cells (MG63) and by the maintenance of its phenotype. The upregulation of collagen I, alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein signifies the maturation of MG63 cells on the silica scaffold. Hence, these novel scaffolds are promising new biomaterials for orthopaedic applications.     

Bioglass-derived glass-ceramic scaffolds: study of cell proliferation and scaffold degradation in vitro

Cell support function as well as cell proliferation on highly porous Bioglass(R)-derived glass-ceramic scaffolds (designed for bone tissue engineering) have been assessed in vitro using osteoblast-like cells (MG 63) cultured for up to 6 days. The biodegradation and mechanical stability of the scaffolds in the cell-culture medium have also been investigated. It was found that the scaffolds had excellent cell supporting ability, with cells effectively infiltrating into and surviving at the center of the scaffolds. A quantitative study using the AlamarBlue assay revealed that the proliferation of cells on the glass-ceramic materials was comparable to that on the noncrystallized Bioglass. While the crystalline phase in the glass-ceramic scaffolds transformed into a biodegradable amorphous calcium phosphate phase during cell culture, the mechanical strength of the scaffolds was maintained when compared with that of scaffolds incubated in simulated body fluid or immersed in cell-free culture medium. It is believed that the attached cells and collagen secreted by cells could fill the micropores and microcracks on the surface of the foam struts, thus contributing to the mechanical stability of the degrading scaffolds. In summary, the developed glass-ceramic scaffolds possess the most essential features of a scaffold for bone tissue engineering: they are capable to support and foster relevant cells, able to provide temporary mechanical function, and biodegradable.     

Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue engineering

High porosity of scaffold is always accompanied by poor mechanical property; the aim of this study was to enhance the strength and modulus of the highly porous scaffold of nanohydroxyapatite/polyamide66 (n-HA/PA66) by coating chitosan (CS) and to investigate the effect of CS content on the scaffold physical properties and cytological properties. The results show that CS coating can reinforce the scaffold effectively. The compress modulus and strength of the CS coated n-HA/PA66 scaffolds are improved to 32.71 and 2.38 MPa, respectively, being about six times and five times of those of the uncoated scaffolds. Meanwhile, the scaffolds still exhibit a highly interconnected porous structure and the porosity is approximate about 78%, slightly lower than the value (84%) of uncoated scaffold. The cytological properties of scaffolds were also studied in vitro by cocultured with osteoblast-like MG63 cells. The cytological experiments demonstrate that the reinforced scaffolds display favorable cytocompatibility and have no significant difference with the uncoated n-HA/PA66 scaffolds. The CS reinforced n-HA/PA66 scaffolds can meet the basic mechanical requirement of bone tissue engineering scaffold, presenting a potential for biomedical application in bone reconstruction and repair.     

Behavior of human osteoblast-like cells in contact with electrodeposited calcium phosphate coatings

Calcium-deficient hydroxyapatite (Ca-def-HAP) thin films were elaborated on Ti6Al4V substrates by electrodeposition. The coatings exhibit two different morphologies and crystallinities. Human osteoblast-like cells (MG-63) were cultured on the surfaces of these materials; the cell content and viability were evaluated up to 28 days. The scanning electron microscopy and biological investigations showed cells with a normal morphology, good proliferation, and viability from 7 to 21 days. But after 28 days, the number of live cells decreases in both cases; however, this decrease is less important in the case of calcium phosphate (CaP) coating surface when compared with the control (cell culture plastic). The cells cultured on Ca-def-HAP coating exhibit more cellular extensions and extracellular matrix. RT-PCR for type I collagen, alkaline phosphatase, and osteocalcin studies were also carried out, and was found that the CaP enhances gene expression of ALP and OC and thus the differentiation of osteoblast-like cells. Moreover, this study shows that the difference in the morphology of CaP coatings has no effect on the biocompatibility.     

Nanocrystalline diamond: In vitro biocompatibility assessment by MG63 and human bone marrow cells cultures

Nanocrystalline diamond (NCD) has a great potential for prosthetic implants coating. Nevertheless, its biocompatibility still has to be better understood. To do so, we employed several materials characterization techniques (SEM, AFM, micro-Raman spectroscopy) and cell culture assays using MG63 osteoblast-like and human bone marrow cells. Biochemical routines (MTT assays, Lowry's method, ALP activity) supported by SEM and confocal microscopy characterization were carried out. We used silicon nitride (Si3N4) substrates for NCD coatings based on a previous demonstration of the superior adhesion and tribological performance of these NCD coated ceramics. Results demonstrate an improved human osteoblast proliferation and the stimulation of differentiated markers, like ALP activity and matrix mineralization, compared with standard polystyrene tissue culture plates. The nanometric featuring of NCD, associated to its chemical affinity are key points for bone regeneration purposes.     

A microarray approach to the identification of polyurethanes for the isolation of human skeletal progenitor cells and augmentation of skeletal cell growth

The present study has examined the efficacy of a polymer microarray platform to screen a library of polyurethanes for applications such as human skeletal progenitor cell isolation and surface modification of tissue engineering scaffolds to enhance skeletal cell growth and differentiation. Analysis of polyurethane microarrays incubated with adult human bone marrow-derived STRO-1+ skeletal progenitor cells identified 31 polyurethanes (from the entire library of 120 polyurethanes) capable of binding to the STRO-1+ cells. Four polyurethanes (out of the 31 identified in the previous screen) were able to selectively immobilise cells of the STRO-1+ fraction from the heterogeneous human bone marrow mononuclear cell population. These four polyurethanes were highly selective for the STRO-1+ fraction of human bone marrow as they failed to bind STRO-1+ immature osteoblast-like MG63 cells, the STRO-1+ fraction of human fetal skeletal cells and differentiated osteoblast-like SaOs cells. Culture of human bone marrow-derived STRO-1+ cells on fibres of Polyglycolic acid (PGA) fleece surface modified by polyurethane adsorption, in osteogenic conditions, enhanced the expression of early osteogenic genes. Similarly, surface modification of PGA fleece fibres by polyurethane adsorption increased the responsiveness of MG63 cells, cultured on this scaffold, to 1,25 dihydroxy Vitamin D3, as demonstrated by enhanced Osteocalcin expression.