Octacalcium Phosphate Coatings Research Articles

Mechanical octacalcium phosphate coatings on the plasma electrolytic oxidized pure titanium for bio-implant use

In this study, OCP powder was used for mechanical coating on the plasma electrolytic oxidized (PEO) implant surface to improve bioactivity. PEO treatment was performed on the surface of the implant using a solution mixed with calcium acetate and calcium glycerophosphate, and creates porosity through a pulsed DC power supply. For compositional analysis, energy dispersive X-ray spectrometer analysis was employed to calculate OCP content and X-ray diffractometer analysis was used to confirm the crystal structure. Also, wettability, surface roughness, hardness, elastic modulus, and corrosion resistance testing were performed. Significantly, the reduction in OCP particle size decreased, and pore filling with OCP smaller particles increased as mechanical coating time increased from 0 to 12 h. Surface and cross-sectional morphology showed the presence of OCP on the substrate. Also, surface roughness, contact angles, hardness, and elastic modulus were reduced as mechanical coating time increased. Corrosion resistance was found to be the highest for mechanical OCP coating for 6 h surface treatment on the PEO-treated CW-Ti.

Surface Properties of Octacalcium Phosphate Nanocrystals Are Crucial for Their Bioactivities.

The fundamental structure–biofunction relationship of calcium phosphates (CaPs) remains unclear despite their clinical successes as important biomaterials. Herein, a series of CaP coatings with gradual change of topography and crystallinity is constructed by electrochemical deposition, and the roles of the two basic physicochemical properties are scrutinized for further understanding the mechanism behind the superior bioactivities of octacalcium phosphate (OCP). We observe a distinct modulation on cell proliferation on the prepared CaP coatings for different cells. The magnitude of the modulation seems to depend on the cellular size, and the effect is attributed mainly to the microstructure of the coatings. On the other hand, the crystallinity manifests its significance for the osteogenic property of the OCP coatings. Further transmission electron microscopy analysis and density functional theory calculations reveal a surface rich in HPO42– for the high-crystalline OCP nanocrystals. The results highlight that the nanocrystal surface properties of the OCP coatings, including the periodic structure and the HPO42– composition, may play significant roles surpassing the ion release effect in determining its osteogenic property, probably via surface spatial and/or chemical recognitions. The present findings shed light on the fundamental understanding of the structure–biofunction relationship for CaP biomaterials.

Effect of pH of coating solution on the adhesion strength and corrosion resistance of hydroxyapatite and octacalcium phosphate coated WE43 alloy.

Hydroxyapatite (HAp) and octacalcium phosphate (OCP) layers were formed on Mg- 4mass% Y- 3mass% rare earth (WE43) alloy by a chemical solution deposition method at various pH values of pH 5.5, 6.2, 7.5, and 8.6. Adhesion strength of HAp and OCP layers was evaluated before and after immersing in a medium for 14 days by a pull-off test. The corrosion resistance of these coatings was measured by polarization tests performed in a simulated body fluid (SBF). XRD analysis demonstrated that HAp coating layers were formed at pH 7.5 and 8.6, while OCP coating layers were formed at pH 5.5 and 6.2. Adhesion test results showed that the as-coated pH7.5-HAp layer had the highest adhesion strength of 8.6 MPa, which was attributed to the very dense structure of the coating layer. The as-coated pH8.6-HAp layer showed the adhesion strength of 6.5 MPa. The adhesion strength of the as-coated pH5.5- and pH6.2-OCP layers was 3.9 and 7.1 MPa, respectively, that was governed by the thick and fragile property of the layers. After immersing in the medium for 14 days, the adhesion strength of pH7.5- and pH8.6-specimens decreased to 5.8 and 5.6 MPa, respectively. The pitting corrosion and formation of Mg(OH)2 under the HAp layers were responsible for the decrease of adhesion strength. The polarization tests in SBF at 37 °C showed that the corrosion current density decreased with the HAp and OCP coatings, indicating the improvement of the corrosion resistance of WE43 alloy. The HAp coatings improved the corrosion resistance more efficiently than the OCP coatings.

Facile preparation of nanostructured octacalcium phosphate coatings on micro-arc oxidized magnesium with different functionalities for bone repair application

Magnesium (Mg) has been considered as the next-generation orthopedic implant material due to its biodegradability, but high degradation rate and severe cytotoxicity hinder its clinical application. Preparing micro-arc oxidation (MAO)/octacalcium phosphate (OCP) composite coating is considered as an ideal approach to solve the problems. In this work, nanostructured OCP coatings with variable structures were prepared facilely on MAO coated Mg by chemical deposition (CD) in a simple trace PO43− ion contained Ca(NO3)2 solution. The results show that nanostructured OCP grows and covers the MAO coating completely after CD. The morphology and thickness of OCP coating can be regulated by varying deposition time. The thinnest OCP coating with a fine structure is observed after 0.5 h of deposition, which shows the best cytocompatibility. Extending deposition time roughens the surface structure and thickens the coatings. The thickest OCP coating with a coarse structure can be obtained after 2 h of deposition, which shows the best corrosion resistance and mineralization. These results clearly indicate the functionality of the OCP coating can be easily tailored only by varying deposition time to meet different clinical requirements.

Strontium substituted octacalcium phosphate coatings by electrochemical deposition and their dose-dependent bioactivities

Octacalcium phosphate coatings substituted with different amounts of strontium were synthesized by electrochemical deposition, and variations in coating morphology, crystal structure, composition, solubility, and bioactivities were investigated. The physicochemical properties and bioactivities of the coatings were observed to be modulated by Sr2+ dopant in a dose-dependent manner. While enhanced cell proliferation was detected for substitution at 5% level and higher, unique elevated alkaline phosphatase activity was observed for the 1% Sr2+-substituted coating, indicating a superior osteoinductivity than observed with the other coatings. The electrolyte recipes also demonstrated a high affinity for Sr2+ substitution.

Effect of pH of Coating Solution on Adhesion Strength of Hydroxyapatite and Octacalcium Phosphate Coatings on AZ31 Magnesium Alloy

Hydroxyapatite (HAp) and octacalcium phosphate (OCP) coatings were formed on a Mg-3Al-Zn (AZ31) alloy with a chemical solution deposition method using a Ca-EDTA solution at various pH levels. The adhesion strength of the coatings was examined using the pull-off method. The microstructures of HAp and OCP coatings were measured X-ray diffraction (XRD). The morphology and composition of the surface and cross section of the samples before and after the adhesion test were characterized using scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS), and a 3D profilometer. The results showed that plate-like OCP crystals grew from a continuous OCP layer on the surface of the AZ31 substrate in the case of a pH 6.3 coating solution. At pH values of 7.5 and 8.6, the HAp coating showed a two-layer structure with a HAp rod-like outer layer and a HAp continuous inner layer. Regardless of the pH of the coating solutions, a very thin Mg (OH)2 intermediate layer was formed between OCP or HAp coating and substrate. The highest adhesion strength of the coatings was 6.7±0.5 MPa, achieved at a coating solution pH value of 7.5. A part of Mg (OH)2 layer remained on the substrate, indicating that the delamination occurred in the Mg (OH)2 intermediate layer. The primary particles in the inner layer formed at pH 7.5 was smaller than those at pH 8.6. This result indicates that the initial corrosion of substrate AZ31 at pH 7.5 was more rapidly than that at pH 8.6, presumably leading to the formation of mixed layer of Mg (OH)2 and calcium phosphate. Further investigation is necessary to understand the better adhesion strength at pH 7.5 than that at pH 8.6. This good adhesion could be due to the flawless and rod-like uniform crystal, which had the densest and finest structure on the surface.

Enhanced biocompatibility and improved osteogenesis of coralline hydroxyapatite modified by bone morphogenetic protein 2 incorporated into a biomimetic coating

Objectives(1) To determine whether the biocompatibility of coralline hydroxyapatite (CHA) granules could be improved by using an octacalcium phosphate (OCP) coating layer, and/or functionalized with bone morphogenetic protein 2 (BMP-2), and (2) to investigate if BMP-2 incorporated into this coating is able to enhance its osteoinductive efficiency, in comparison to its surface-adsorbed delivery mode. MethodsCHA granules (0.25 g per sample) bearing a coating-incorporated depot of BMP-2 (20 μg/sample) together with the controls (CHA bearing an adsorbed depot of BMP-2; CHA granules with an OCP coating without BMP-2; pure CHA granules) were implanted subcutaneously in rats (n = 6 animals per group). Five weeks later, the implants were retrieved for histomorphometric analysis to quantify the volume of newly generated bone, bone marrow, fibrous tissue and foreign body giant cells (FBGCs). The osteoinductive efficiency of BMP-2 and the rates of CHA degradation were also determined. ResultsThe group with an OCP coating-incorporated depot of BMP-2 showed the highest volume and quality or bone, and the highest osteoinductive efficacy. OCP coating was able to reduce inflammatory responses (improve biocompatibility), and also simple adsorption of BMP-2 to CHA achieved this. ConclusionsThe biocompatibility of CHA granules (reduction of inflammation) was significantly improved by coating with a layer of OCP. Pure surface adsorption of BMP-2 to CHA also reduced inflammation. Incorporation of BMP-2 into the OCP coatings was associated with the highest volume and quality of bone, and the highest biocompatibility degree of the CHA granules. Clinical significanceHigher osteoinductivity and improved biocompatibility of CHA can be obtained when a layer of BMP-2 functionalized OCP is deposited on the surfaces of CHA granules.

Micromorphological effect of calcium phosphate coating on compatibility of magnesium alloy with osteoblast

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were developed to control the degradation speed and to improve the biocompatibility of biodegradable magnesium alloys. Osteoblast MG-63 was cultured directly on OCP- and HAp-coated Mg-3Al-1Zn (wt%, AZ31) alloy (OCP- and HAp-AZ31) to evaluate cell compatibility. Cell proliferation was remarkably improved with OCP and HAp coatings which reduced the corrosion and prevented the H2O2 generation on Mg alloy substrate. OCP-AZ31 showed sparse distribution of living cell colonies and dead cells. HAp-AZ31 showed dense and homogeneous distribution of living cells, with dead cells localized over and around corrosion pits, some of which were formed underneath the coating. These results demonstrated that cells were dead due to changes in the local environment, and it is necessary to evaluate the local biocompatibility of magnesium alloys. Cell density on HAp-AZ31 was higher than that on OCP-AZ31 although there was not a significant difference in the amount of Mg ions released in medium between OCP- and HAp-AZ31. The outer layer of OCP and HAp coatings consisted of plate-like crystal with a thickness of around 0.1 μm and rod-like crystals with a diameter of around 0.1 μm, respectively, which grew from a continuous inner layer. Osteoblasts formed focal contacts on the tips of plate-like OCP and rod-like HAp crystals, with heights of 2–5 μm. The spacing between OCP tips of 0.8–1.1 μm was wider than that between HAp tips of 0.2–0.3 μm. These results demonstrated that cell proliferation depended on the micromorphology of the coatings which governed spacing of focal contacts. Consequently, HAp coating is suitable for improving cell compatibility and bone-forming ability of the Mg alloy.

Corrosion Behavior of Calcium Phosphate-Coated Biomedical Magnesium Alloy Under in Vitro and In Vivo Environments

Magnesium alloys attract attention as a biodegradable/bioabsorbable medical device because of the biodegradability and good mechanical integrity. Corrosion rate of the magnesium alloys should be controlled depending on the implantation part of body as well as the biocompatibility should be improved. Hydroxyapatite (HAp) and octacalcium phosphate (OCP) coatings were developed to control the corrosion rate and to improve the hard tissue compatibility. Corrosion behavior of HAp- and OCP-coated Mg-3mass% Al-1mass% Zn (AZ31) was examined in cell culture medium (in vitro) or in subcutaneous tissue of mouse (in vivo). Effect of medium-replacement and addition of gelatin in medium on the corrosion behavior was also examined. Chemically polished AZ31 was used as a reference. Concentration of Mg ions in medium was quantified. Apparent corrosion initiation of substrate AZ31 was delayed for ca. 1 week with HAp and OCP coatings in medium while Mg ion release from as-polished AZ31 initiated from the beginning. Filiform corrosion took place, but visible growth of filiform corrosion stopped within several days. Afterwards, Mg ion release continued for 1 year with almost a constant corrosion rate. The corrosion rate of AZ31 alloy in medium was reduced by more than 50% with HAp and OCP coatings. HAp coating showed superior corrosion protection effect than OCP coating. After 1 year of immersion, cross section of the immersed specimens showed that uniform corrosion took place underneath the coatings. The wall of filiform corrosion pits was covered with thick corrosion products layer. These results indicate that corrosion continued for long time under the coating and corrosion products layer in medium. The periodical medium replacement during immersion test did not significantly change the initial corrosion rate for 4 weeks. However, the medium-replacement rather reduced the corrosion rate of HAp-coated AZ31 comparing to the result without medium-replacement. Deposition of calcium phosphate from medium was enhanced with the medium-replacement, leading to the improvement of corrosion protection effect of HAp coating. As-polished AZ31 caused the formation of gas cavity in subcutaneous tissue of mouse due to H2 gas generated in corrosion reaction. Such a gas cavity formation was not observed around the implanted HAp- and OCP-coated specimens, indicating that corrosion of substrate AZ31 was also reduced by HAp and OCP coating in subcutaneous tissue. After 16 weeks of implantation, the specimens were retrieved from the mouse and surface and cross section was observed using SEM. Macro- and micro-pits were observed on the surface and underneath the coatings. Unlike in medium, corrosion pits appeared to grow in depth direction rather than in lateral direction in subcutaneous tissue. Because body fluid circulation in the subcutaneous tissue is limited, to simulate the soft tissue environment, gelatin was added in medium and HAp-coated AZ31 was immersed for 4 weeks. Then, the similar corrosion morphology with macro- and micro-pits was obtained in the gelatin-added medium. These results indicate that corrosion of HAp- and OCP-coated AZ31 is localized in diffusion limited environment. The developed HAp and OCP coatings reduced the corrosion rate of Mg alloy in medium and in soft tissue. The corrosion morphology, especially shape of local corrosion, depended on the corrosion environment. The decrease of mass transfer in the environment caused the localization of corrosion of Mg alloy.

A facile construction of gradient micro-patterned OCP coatings on medical titanium for high throughput evaluation of biocompatibility.

Base on a superhydrophobic-superhydrophilic micro-patterned template, a facile construction of gradient micro-patterned octacalcium phosphate (OCP) coatings on titanium has been firstly developed for high-throughput evaluation of biocompatibility. The gradient OCP coatings with tunable crystal morphologies involving scattered-flower-like, scattered-flower-ribbon-like, short-ribbon-like and long-ribbon-like were fabricated in different micro-units on the same surface. The significant difference of mineralization behavior of the gradient OCP coatings in the micro-patterns was observed visually and efficiently. In vitro cultures of MC3T3-E1 cells showed that the number and morphology of cells selectively adhered on the micro-units of gradient structure of OCP coatings were distinctly different, indicating that the cells are sensitive to the different structures of OCP coatings on medical titanium. The gradient micro-patterned construction is potentially a powerful method for not only high-throughput screening of the biocompatibility of various biomaterials, but also efficient development of advanced biomaterials by controlling cell immobilization and inducing cell response.

Antiresorption implant coatings based on calcium alendronate and octacalcium phosphate deposited by matrix assisted pulsed laser evaporation

The integration of an implant material with bone tissue depends on the chemistry and physics of the implant surface. In this study we applied matrix assisted pulsed laser evaporation (MAPLE) in order to synthesize calcium alendronate monohydrate (a bisphosphonate obtained by calcium sequestration from octacalcium phosphate by alendronate) and calcium alendronate monohydrate/octacalcium phosphate composite thin films on titanium substrates. Octacalcium phosphate coatings were prepared as reference material. The powders, which were synthesized in aqueous medium, were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The transfer was conducted with a KrF* excimer laser source (λ=248nm, τFWHM≤25ns) in mild conditions of temperature and pressure. XRD, FTIR and SEM analyses confirmed that the coatings contain the same crystalline phases as the as-prepared powder samples. Osteoblast derived from stem cells and osteoclast derived from monocytes of osteoporotic subjects were co-cultured on the coatings up to 14 days. Osteoclast displayed significantly reduced proliferation and differentiation in the presence of calcium alendronate monohydrate, pointing to a clear role of the coatings containing this bisphosphonate on inhibiting excessive bone resorption. At variance, osteoblast production of alkaline phosphatase and type I pro-collagen were promoted by the presence of bisphosphonate, which also decreased the production of interleukin 6. The positive influence towards osteoblast differentiation was even more enhanced in the composite coatings, thanks to the presence of octacalcium phosphate.

Self-healing property of hydroxyapatite and octacalcium phosphate coatings on pure magnesium and magnesium alloy

Hydroxyapatite (HAp) and octacalcium phosphate (OCP) coatings were formed on AZ31 alloy and pure Mg, and the surfaces were scratched to expose the metal surface. The specimens were immersed in a cell-culture medium or 0.9% NaCl. Release of Mg ions was not accelerated by scratching. In both solutions, a corrosion product layer containing Mg and P was formed in the scratches; i.e., the coatings self-healed. The corrosion morphology of HAp-coated AZ31 was examined by periodic medium replacement or using a gelatine-added medium. The former enhanced calcium phosphate precipitation; the latter increased corrosion localization.

In vitro and in vivo biocompatibility and corrosion behaviour of a bioabsorbable magnesium alloy coated with octacalcium phosphate and hydroxyapatite

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were formed on Mg–3mass% Al–1mass% Zn (AZ31) magnesium alloy by a single-step chemical solution deposition method. Chemically polished AZ31 (Cpol-AZ31) and HAp- and OCP-coated AZ31 (HAp- and OCP-AZ31) were immersed in a medium for 52weeks or implanted in transgenic mice for 16weeks to examine the long-term corrosion behaviour and in situ inflammation behaviour. In the medium, Mg-ion release was restricted for the initial several days and the corrosion rate thereafter was suppressed by approximately one-half with the HAp and OCP coatings. HAp-AZ31 showed a ∼20% lower corrosion rate than OCP-AZ31. Tissues of the transgenic mouse emit fluorescence in proportion to the degree of inflammation in situ. The luminescence intensity level was too low to be a problem regardless of the coatings. A thinner fibrous tissue layer was formed around OCP- and HAp-AZ31 than around Cpol-AZ31, indicating that the HAp and OCP coatings suppressed corrosion and foreign-body reaction in vivo. Visible pits were formed in filiform and round shapes in vitro and in vivo, respectively. Corrosion was observed underneath the coatings, and almost uniform corrosion took place in vitro, while local corrosion was predominant in vivo. These differences in corrosion morphology are attributed to the adhesion of tissues and the lower diffusivity on the surface in vivo than that in vitro. Dissolution behaviour of OCP crystals in vivo was different from that in vitro. It was demonstrated that the HAp and OCP coatings developed have great potential for a biocompatible and corrosion protection coating.

Hydroxyapatite coatings electrodeposited at near-physiological conditions

Calcium phosphate (CaP) ceramics are used in orthopedics and dentistry due to their excellent biocompatibility and osseointegration. Plasma spraying has been the most common process for application of hydroxyapatite (HAp) coatings. Although electrodeposition (ED) has many potential advantages, very little has been published on ED of HAp at near-physiological conditions. Here we report the ED of uniform HAp–OCP (octacalcium phosphate) coatings on Ti–6Al–4V substrate at 37°C, pH=7.4, following surface pretreatment. Thermodynamic calculations were first used to predict which CaP phases may precipitate from solution under different experimental conditions. Microscopic observation revealed that the coating formed in this study is similar to that formed by chemical deposition, and better mimics the biological apatites in terms of structure and phase content than coatings currently used in orthopedics.

Chemical and crystallographic characterizations of hydroxyapatite- and octacalcium phosphate-coatings on magnesium synthesized by chemical solution deposition using XPS and XRD

Hydroxyapatite (HAp) and octacalcium phosphate (OCP) coatings deposited on a pure magnesium (Mg) substrate from an aqueous solution containing C10H12N2O8Na2Ca (Ca-EDTA) and KH2PO4 were precisely analyzed using X-ray photoelectron spectroscopy (XPS) and high-resolution X-ray diffractometry (XRD) to clarify their chemical and crystallographic characteristics. The calcium phosphate coatings comprised single-phase HAp or OCP containing small amounts of sodium (Na). The intermediate layer formed underneath the coatings consisted of Mg(OH)2 mixed with calcium phosphate. The atomic ratios of calcium to phosphorus ([Ca]/[P]) in the HAp and OCP coatings were notably smaller than those of stoichiometric compounds. The expansion along the a-axis compared to that of stoichiometric HAp was observed in XRD results. Also, we did not detect Na compounds in the coating. Therefore, it is presumed that Na existed as a substitute of Ca in the OCP and HAp structures.

Rapid biomimetic deposition of octacalcium phosphate coatings on zirconia ceramics (Y-TZP) for dental implant applications

Octacalcium phosphate (OCP) coatings on zirconia oral implants have a great potential to improve the osseointegration of already existing ceramic implants, owing to high osteoconductive characteristics of OCP and its possibility of use as a drug delivery system. Such OCP coatings can be prepared with a simple two-step biomimetic procedure investigated in our study. In the first step, zirconia discs were immersed into the solution with a pH 7.4 and after 1h of soaking a thin nanostructured calcium phosphate (Ca-P) layer was precipitated on the ceramic substrate via three stages: (i) precipitation of an amorphous Ca-P; (ii) precipitation of the OCP; and (iii) the transformation of the OCP to apatite. This Ca-P layer later served as a template for the rapid deposition of a thicker OCP coating in the second step of the synthesis where the substrate was immersed into the solution with pH 7.0. The main benefits of the method are a relatively quick synthesis, simplicity and a good reproducibility. Moreover, the coatings show good tensile adhesion strength according to the tape tests (ASTM D-3359). In addition, mild physiological conditions of the synthesis may allow incorporation of biologically active molecules in the coating.

Comparative study of hydroxyapatite and octacalcium phosphate coatings deposited on metallic implants by PLD method

The aim of the presented study was an analysis of two apatite coatings: hydroxyapatite (HA) and octacalcium phosphate (OCP) as coatings materials for metallic implants. Both layers were deposited by means of the PLD method. As a target material, synthetic, powdered and pressed hydroxyapatite was used. HA was deposited on 316L steel substrate in two temperature ranges for obtaining different coatings: 150±30°C and 430±30°C for OCP and HA, respectively. As an intermediate layer, the nanocrystalline diamond layer (NCD) was deposited. Examined calcium phosphate layers were tested for adhesion of osteoblast cell culture (MG-63). Analytical methods (AFM, FTIR) showed the usefulness of the PLD method for deposition of the apatite layers on metallic implants. Both examined layers showed biocompatibility with human osteoblast cells and presented favorable conditions for their proliferation.

Application of New Statistical Approach to Study Drug Release from OCP Coating on Titanium Sheets

In this study, commercially pure titanium (Ti-cp) discs were used as substrates. Octacalcium phosphate (OCP) layers were deposited by immersion of Ti-cp discs up to 7 days into Solution for Bioactivity Evaluation (SBE) and characterized by SEM-EDS and XRD. The OCP coatings were doped with cephazolin or cefalexin by individual immersion in 300 ppm of each solution for 24 h at room temperature. The non-existence of mathematical models to explain drug release from these matrixes made the choosing of correct model, a complex process. Five non-linear mathematical methods were employed in order to identify the possible drug release mechanism using Akaike and Bayesian Information Criterion (AIC and BIC respectively) and its derivatives. The best model was Peppas & Sahlin that consider two stages in release: pure diffusion in first stage, and drug dissolution and migration through the porous matrix at second stage.

Dose-Dependent Osteogenic Effect of Octacalcium Phosphate on Mouse Bone Marrow Stromal Cells

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.

Dose-Dependent Osteogenic Effect of Octacalcium Phosphate on Mouse Bone Marrow Stromal Cells

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.