Synthetic Octacalcium Phosphate Research Articles

Proteome analysis of rat serum proteins adsorbed onto synthetic octacalcium phosphate crystals

The present study was designed to determine which proteins are selectively adsorbed onto two bone substitute materials, octacalcium phosphate (OCP) and hydroxyapatite (HA) crystals, from rat serum by proteome analysis. Ground crystals of synthetic OCP and commercially available sintered HA, with the same surface area, were incubated in rat serum proteins at 37 °C for 24 h. The proteins from the crystals extracted with guanidine–HCl–EDTA were listed on the basis of the results of liquid chromatography tandem mass spectrometry (LC/MS/MS). A total of 138 proteins were detected from OCP; 103 proteins were detected from HA. Forty-eight proteins were from both crystals. A quantitative analysis of the proteins detected was performed for the extracted two bone formation-related proteins apolipoprotein E (Apo E), a protein known to promote osteoblast differentiation, and complement 3 (C3). HA adsorbed C3 (3.98 ± 0.03 fmol/μg protein) more than OCP (1.81 ± 0.07 fmol/μg protein) did, while OCP adsorbed Apo E (2.42 ± 0.03 fmol/μg protein) more than HA (1.21 ± 0.01 fmol/μg protein) did even after deleting the high-abundance proteins, such as albumin. The results demonstrated that OCP exhibits a similar property but distinct capacity with HA in adsorbing bone formation-related proteins from the serum constituents.

Reconstruction of Critical‐Sized Bone Defect in Dog Skull by Octacalcium Phosphate Combined with Collagen

The present study was designed to investigate whether synthetic octacalcium phosphate (OCP) combined with collagen (OCP/collagen) can repair a critical-sized defect in dog skull. OCP/collagen has been shown to biodegrade and to tend to be replaced by newly formed bone if implanted in rat calvaria defects. An OCP/collagen disk was prepared from pepsin-digested atelocollagen isolated from porcine dermis and synthetic OCP. Two critical-sized defects (20 mm in diameter) were made in a dog skull. Ten disks of OCP/collagen or collagen (control) were implanted in the bone defects and resected with surrounding tissues at 3, 6, or 12 months after the implantation. The specimens were analyzed radiographically, crystallographically, histologically, and histomorphometrically. X-ray diffraction and FTIR analyses showed that OCP tended to convert to a poorly crystallized hydroxyapatite, similar to that of biological apatite, by 3 months. Radiographic and histologic analyses showed that the implantation of OCP/collagen disks initiated new bone formation in the defects at 3 months after implantation. However, there was no promotion of bone formation by control collagen disks even with prolonged implantation up to 12 months. Histomorphometric analysis revealed that the percentage of newly formed bone in the defect implanted with OCP/collagen increased significantly, from 30.91 ± 6.65 at 3 months to 51.22 ± 5.99 at 12 months, although the value tended to reach a plateau at 6 months (44.49 ± 3.34). On the other hand, the percentage of remaining OCP was estimated at approximately 10% at 3 months and remained nearly unchanged thereafter. The results suggest that bone regeneration of a critical-sized bone defect of dog calvaria by OCP/collagen can be enhanced for 3 to 6 months and that OCP/collagen holds potential as a bone substitute material.

Octacalcium phosphate: Osteoconductivity and crystal chemistry

Octacalcium phosphate (OCP), which is structurally similar to hydroxyapatite (HA), is a possible precursor of bone apatite crystals. Although disagreement remains as to whether OCP comprises the initial mineral crystals in the early stage of bone mineralization, the results of recent biomaterial studies using synthetic OCP indicate the potential role of OCP as a bone substitute material, owing to its highly osteoconductive and biodegradable characteristics. OCP tends to convert to HA not only in an in vitro environment, but also as an implant in bone defects. Several lines of evidence from both in vivo and in vitro studies suggest that the conversion process could be involved in the stimulatory capacity of OCP for osteoblastic differentiation and osteoclast formation. However, the osteoconductivity of OCP cannot always be secured if an OCP with distinct crystal characteristics is used, because the stoichiometry and microstructure of OCP crystals greatly affect bone-regenerative properties. Osteoconductivity and stimulatory capabilities may be caused by the chemical characteristics of OCP, which allows the release or exchange of calcium and phosphate ions with the surrounding of this salt, and its tendency to grow towards specific crystal faces, which could be a variable of the synthesis condition. This paper reviews the effect of calcium phosphates on osteoblastic activity and bone regeneration, with a special emphasis on OCP, since OCP seems to be performing better than other calcium phosphates in vivo.

Octacalcium phosphate (OCP) collagen composites enhance bone healing in a dog tooth extraction socket model

The authors have reported that a scaffold constructed of synthetic octacalcium phosphate (OCP) and porcine atelocollagen sponge (OCP/Col) enhanced bone regeneration more than sintered beta-tricalcium phosphate collagen composite or sintered hydroxyapatite collagen composite with a rat calvarial defect model. To aim for clinical application, the present study investigated whether OCP/Col would enhance bone healing in a dog tooth extraction socket model. Six adult, male, beagle dogs were used. The tooth extraction socket model was made by extracting bilateral third maxillary incisors and the subsequent removal of buccal bone. Disks of OCP/Col were implanted into one side of the model and the other side was untreated. The specimens were fixed 1 or 3 months after implantation. In radiographic analysis, the OCP/Col-treated group showed a wider range of radiopacity than the untreated control. Histologically, the OCP/Col-treated group showed more abundant newly formed bone than untreated control, and the implanted OCP was gradually resorbed. In morphometrical analysis, enlargement of the buccal alveolus in the OCP/Col group was significantly greater than in the untreated control. This study showed that implanted OCP/Col would be replaced by newly formed bone and OCP/Col implantation would enhance bone healing in a tooth socket model.

市販β-TCP焼結体と比較した合成リン酸オクタカルシウム（OCP）の骨芽細胞の分化能

市販β-TCP焼結体と比較した合成リン酸オクタカルシウム（OCP）の骨芽細胞の分化能

Morphometrical, Histological and Ultrastructural Analyses of Bone Formation and Resorption Induced by Synthetic Octacalcium Phosphate in Mouse Bone Marrow

Morphometrical, Histological and Ultrastructural Analyses of Bone Formation and Resorption Induced by Synthetic Octacalcium Phosphate in Mouse Bone Marrow

Biological Role of Synthetic Octacalcium Phosphate in Bone Formation and Mineralization

Biological Role of Synthetic Octacalcium Phosphate in Bone Formation and Mineralization

Osteoclast Differentiation Induced by Synthetic Octacalcium Phosphate Through Receptor Activator of NF-κB Ligand Expression in Osteoblasts

Synthetic octacalcium phosphate (OCP) has a potential to enhance new bone formation and exhibits biodegradable characteristics when implanted in experimentally created bone defects. The precise mechanisms of OCP biodegradation remain unclear, though histological observations have revealed that bone-resorbing osteoclasts appear and resorb implanted OCP. To investigate how osteoclasts develop around implanted OCP, we examined osteoclast differentiation using OCP crystals in vitro. Coculturing of mouse bone marrow cells and osteoblasts in OCP-coated cell culture plates induced osteoclast differentiation, whereas that did not occur without coating. Further, addition of bone morphogenetic protein-2 significantly increased the number of osteoclasts in the OCP-coated wells. In the presence of OCP, osteoblasts expressed receptor activator of NF-kappaB ligand (RANKL), an osteoclast differentiation factor. In addition, when half of each culture well was coated with OCP, osteoclasts were formed in both coated and noncoated areas, suggesting that soluble factors mediate osteoclast differentiation induced by OCP. Also, calcium levels in culture medium were significantly decreased in the presence of OCP, while experimental reduction of calcium from 8.0 to 5.0 mg/dL significantly induced RANKL mRNA expression. These results suggest that OCP itself decreases calcium levels around implanted OCP, which induces osteoclast differentiation through RANKL expression by osteoblasts.

Comparative study on bone regeneration by synthetic octacalcium phosphate with various granule sizes

The present study was designed to investigate whether the granule size of synthetic octacalcium phosphate (OCP) and the resultant intergranular spaces between the granules formed by the filling affect its osteoconductive and biodegradable characteristics in a mouse calvaria critical-sized defect up to 10 weeks after implantation. Mercury intrusion porosimetry showed that OCP granules having distinct diameter sizes ranging from 53 to 300 (S-OCP), 300 to 500 (I-OCP) and 500 to 1000 μm (L-OCP) produced distinct intergranular spaces between OCP granules ranging from 28.8 to 176.6 μm. The dissolution rate of OCP, estimated by the phosphate concentration in the culture medium, was the highest in S-OCP, followed by I-OCP and L-OCP, while the specific surface area of OCP decreased. Histological and histomorphometric analyses showed that bone formation around the implanted granules increased significantly with increasing granule size coupled with activating the appearance of TRAP- and cathepsin K-positive osteoclastic cells. The rate of new bone formation formed with L-OCP was two times higher than that formed with S-OCP at 10 weeks after implantation. The results indicated that the osteoconductive and biodegradable properties of OCP can be augmented by increasing the granule size, most probably by thus providing enough spaces between the granules, suggesting that the intergranular spaces formed by the granules may work similarly to pores, as reported in porous ceramic materials. It seems likely that the enhancement of bone formation by OCP is accompanied by simultaneous activation of osteoclastic resorption of OCP.

The Effect of Microstructure of Octacalcium Phosphate on the Bone Regenerative Property

The present study was designed to investigate whether the microstructure of synthetic octacalcium phosphate (OCP) affects its intrinsic bone regenerative properties as a scaffold and its conversion process into hydroxyapatite (HA). Our previous studies indicated that an agregate of OCP crystals, consisting of randomly oriented plate-like crystals, are capable of enhancing both osteoblastic cell differentiation in vitro and bone regeneration. While the transformation of OCP into HA has been considered in relation to the stimulatory capacity of OCP in bone regeneration, little is known about the effect of the microstructure of OCP granules on these capabilities. Two types of OCP granules, with identical diameters (300-500 microm) but composed of crystals with distinct crystal dimensions (4.0 and 26.6 microm length), were prepared (hereafter referred to as fine OCP granules [F-OCP] and coarse OCP granules [C-OCP], respectively). The intergranule distances and the porosity, including the intergranule spaces, were 108.5 microm and 93.7% for F-OCP, and 67.5 microm and 95.7% for C-OCP, as estimated by mercury intrusion. The OCP granules were implanted in mouse critical-sized calvarial defects for up to 14 days. Histological examination demonstrated that osteoblastic cells aligned on the surface of F-OCP at day 7 and formed new bone around the granules up to day 14. On the other hand, cells around C-OCP were sparse at day 7, and resulted in only slight bone formation around the granules at day 14. X-ray diffraction showed that both OCP granules tended to be converted to an apatite structure with similar conversion velocity by the implantation. Adhesion of mouse bone marrow stromal ST-2 cells was markedly inhibited on C-OCP compared to F-OCP in vitro. These results suggested that the microstructure consisting of plate-like crystals of OCP controls cell adhesion on the crystal surfaces and their resultant bone regenerative properties as well as the physicochemical effect associated with the transitory nature of OCP previously reported.

リン酸オクタカルシウム（OCP）の化学量論性がその骨形成能に及ぼす影響について

リン酸オクタカルシウム（OCP）の化学量論性がその骨形成能に及ぼす影響について

Octacalcium phosphate collagen composites enhance bone healing in a dog tooth extraction socket model

Background and Objectives: We have engineered a scaffold constructed of synthetic octacalcium phosphate (OCP) and porcine atelocollagen sponge (OCP/Col), and reported that OCP/Col enhanced bone regeneration more than sintered beta-tricalcium phosphate collagen composite or sintered hydroxyapatite collagen composite with a rat calvarial model. To aim for clinical application, the present study investigated whether OCP/Col would enhance bone healing in a dog tooth extraction socket model.

Synthetic Octacalcium Phosphate Augments Bone Regeneration Correlated with Its Content in Collagen Scaffold

Previous studies have shown that synthetic octacalcium phosphate (OCP) facilitates in vitro osteoblastic cell differentiation in an OCP dose-dependent manner and that a complex of OCP and collagen (OCP/collagen) enhances critical-sized rat calvaria defects more than OCP alone. The present study was designed to investigate whether the bone regenerative properties of OCP/collagen are augmented in an OCP dose-dependent manner, thereby establishing a suitable composition of this composite as a bone substitute material. OCP/collagens with a wide range of mixing ratios from 23:77 to 83:17, including the previously examined composition (77:23), were prepared by blending granules of OCP with atelocollagen and molded into a disk as an implant. A critical-sized defect was made in rat calvaria, and each disk was implanted into the defect for 4 or 12 weeks and then examined radiographically, histologically, and histomorphometrically. Mouse bone marrow-derived stromal ST-2 cells were cultured in dishes pre-coated with OCP/collagen or OCP alone with different OCP contents to determine the capacity of cell attachment and proliferation up to 14 days. Histological and radiographic examinations showed that newly formed bone was observed in relation to OCP granules within the collagen matrix. Histomorphometric analysis confirmed that increasing the amount of OCP in collagen matrices resulted in progressive enhancement of bone regeneration and that the ratio 83:17 generated the maximum repair level of approximately 64% of the defect at 12 weeks. OCP/collagen promoted the proliferation and attachment of ST-2 cells more than OCP alone regardless of OCP content. Fourier transform infrared spectroscopy analysis of the coatings after the incubation indicated that OCP tended to convert to apatite regardless of the presence of collagen. The present study demonstrated that the osteoconductive characteristics of OCP/collagen can be displayed in an OCP dose-dependent manner. The results suggest that collagen promotes the proliferation and attachment of host osteoblastic cells on OCP/collagen composite implants.

Intramembranous bone tissue response to biodegradable octacalcium phosphate implant

Previous studies showed that synthetic octacalcium phosphate (OCP) enhances bone formation coupled with its own osteoclastic biodegradation more than non-biodegradable hydroxyapatite (HA), including sintered HA ceramic, when implanted in animal bone defects. The present study was designed to investigate whether synthetic OCP in granule form has biodegradable characteristics when implanted in the subperiosteal area of mouse calvaria in comparison with non-sintered stoichiometric HA, especially in relatively short periods after implantation. OCP crystals exhibited plate-like morphology, whereas HA crystals had a sphere-like structure. Both crystals had large pore volumes >75% in total, with micropores within the granules. Direct bonding of newly formed bone was discernible in HA until 35 days after implantation by element analysis for calcium and phosphorus. However, histomorphometric analysis demonstrated that bone formation was facilitated on OCP surfaces with greater alkaline phosphatase activity than on HA up to 21 days. The surfaces attacked by tartrate-resistant acid phosphatase positive osteoclast-like cells were significantly greater than those of HA. OCP became encapsulated and replaced with new bone with prolonged implantation periods up to 180 days. The results suggest that the enhanced bone formation in mouse calvaria could be associated with the biodegradable nature of OCP, and that OCP could be used in augmenting intramembranous bone volume.

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.

Bone Regeneration by Synthetic Octacalcium Phosphate and its Role in Biological Mineralization

Octacalcium phosphate (Ca8H2(PO4)6 * 5H2O; OCP) has been advocated to be a precursor of biological apatite crystals in bone and tooth. Recent studies, using physical techniques, showed that OCP is present as a transient phase during biological apatite formation in human dentin, porcine enamel and murine bone. However, there is still a controversy regarding the chemical nature of the first mineral formed in the biominerals. A number of studies have demonstrated that synthetic OCP shows bone regenerative and biodegradable characteristics, rather than other calcium phosphate bone substitute materials, such as hydroxyapatite (Ca10(PO4)6(OH)2; HA) ceramic. It seems likely that synthetic OCP may be an alternative to autogenous bone graft. It is known that OCP contains alternative layers of water molecules and an apatite structure, and that the transition of OCP to HA is likely to be spontaneous and irreversible. The conversion process induces modification of local environment adjacent to OCP surface, including the changes in adsorption of serum proteins and concentration of calcium and inorganic phosphate ions. This article reviews the possible application to bone regeneration by synthetic OCP and the mechanism to enhance bone regeneration in relation to biological mineralization in bone and tooth.

Differences of Bone Regeneration by Various Calcium Phosphate/Collagen Composites

Our previous studies showed that synthetic octacalcium phosphate (OCP) enhances bone regeneration more than hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). Also, a synthetic bone substitute constructed of synthetic octacalcium phosphate (OCP) and porcine atelocollagen sponge (OCP/Col) showed stable bone regeneration. The present study was designed to investigate the difference of bone regeneration by OCP/Col and other calcium phosphate-collagen composites. OCP/Col, β-TCP$-collagen% composite (β-TCP/Col), or HA$-collagen% composite (HA/Col) sponge was prepared from pepsin-digested atelocollagen isolated from the porcine dermis and OCP, β-TCP, or HA granules, respectively. A standardized critical-sized defect was made in the rat calvarium, and various calcium phosphate-collagen composites were implanted into the defect. The rats were fixed at four weeks after implantation and radiographic and histological examinations were performed by undecalcified cross sections of implants. Radiographic examination showed that uniform radiopaque masses were observed in the created defects treated with OCP/Col, whereas granulous and foggy radiopacity was observed in β-TCP/Col and HA/Col. Histological examination showed that newly formed bone was observed in the reticulum of OCP/Col and around the implanted OCP. The regenerated bone by β-TCP/Col or HA/Col seemed to be less than that by OCP/Col and would not to be nucleated by the granules of β-TCP or HA. The present study indicated that bone regeneration by OCP/Col was different from those of β-TCP/Col and HA/Col. Application of OCP/Col would be expected for clinical use in the future.

Effect of Octacalcium Phosphate Ionic Dissolution Products on Osteoblastic Cell Differentiation

Our previous studies suggested that synthetic octacalcium phosphate (OCP) enhances bone regeneration more than hydroxyapatite (HA). However, the molecular mechanisms to induce osteogenic phenotype in osteoblast by OCP have not been identified. OCP tended to convert into an apatite structure in vivo and in vitro, and its process was accompanied by calcium consumption from the surrounding solution and the release of phosphate ions into the solution at a physiological condition. The present study was designed to investigate whether the dissolution of ionic products of OCP affects on proliferation and differentiation of mouse bone marrow stromal ST-2 cells in vitro. The number of cells treated with OCP-conditioned medium was slightly decreased in comparison to that of control at day 7. On the other hand, the level of alkaline phosphatase activity increased in OCP-conditioned medium. These results demonstrated that OCP is capable of inducing osteoblastic cell differentiation in ST-2 cells.

Bone Regenerative Property of Octacalcium Phosphate in Mouse Critical Sized Calvarial Defects

Bone regeneration by calcium phosphates has been known to be intricately dependent on material properties or implanted milieu of host animals, such as site and species. Critical sized calvarial defects of mouse were recently used as the model for investigating bone regeneration ability and the mechanisms. The purpose of the present study is to investigate whether the critical sized mouse calvarial defects can be utilized to examine bone regeneration with synthetic octacalcium phosphate (OCP). OCP , prepared by wet synthesis methods, was sieved 0.3 ~ 0.5 mm in diameter and used for the animal experiment. At 14 days after surgery, histological examination showed that implantation of OCP grafted defects significantly enhanced bone formation compared with the control defect. OCP tended to convert to hydroxyapatite with time. The tartrate-resistant acid phosphatase (TRAP) positive osteoclastic cells were observed around the OCP particles. The results suggest that the mouse critical sized calvarial bone defects are useful model to investigate the bone formation by the OCP implantation.