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This study aimed to investigate how the chemical elements in relation to octacalcium phosphate (OCP) hydrolysis affect the osteoblastic differentiation in the presence of serum fetuin. The adsorption of fetuin onto OCP was examined in buffers having different degrees of supersaturation (DS) with respect to OCP and hydroxyapatite (HA) at pH 7.4 and 37 °C. The osteoblastic differentiation of mesenchymal stem cells (MSCs) was evaluated in cultures with OCP and 0 to 0.8 mg/mL of fetuin. The amount of fetuin adsorbed increased with increasing DS in the buffer. In the MSC culture, the coexistence of OCP and 0.2-0.4 mg/mL of fetuin close to serum level increased alkaline phosphatase activity; however, the activity was suppressed by 0.2-0.8 mg/mL of fetuin. Transmission electron microscopy revealed de novo crystal formation on OCP in supersaturated buffer and culture media with respect to OCP and HA at lower fetuin concentrations. Infrared spectroscopy and DS estimation indicate that the hydrolysis of OCP with de novo apatite formation was promoted in the culture media at 0.2-0.4 mg/mL of fetuin. These results suggest that OCP may promote osteoblastic differentiation if the suitable conditions are attained regarding the chemical elements and fetuin adsorption around OCP.

This study was designed to investigate how the strength of the interaction between octacalcium phosphate (OCP) and modified chondroitin-A sulfate (CS-A), a glycosaminoglycan, regulates the adsorption-desorption of cytokines and subsequently affects the osteoblastic differentiation of mesenchymal stem cells (MSCs) in vitro. The utilization of cytokines produced by cells, such as macrophages, stimulated by the hydrolysis of OCP, is expected to enhance the bone regeneration capacity of the OCP. CS-Na was used to modify CS-A on the OCP immobilized with the amino group through electrostatic interactions. On the other hand, N -hydroxysuccinimide (NHS)-esterified CS-A was used to form the covalent bond between CS-A and the amino group on the surface of OCP. X-ray diffraction and Raman spectroscopy indicated that the CS-A-modified OCP maintained its structure, regardless of the modification process. The remaining ratio of the modified CS-A in the buffer suggests that increasing the immobilized density of the amino group and the modification using NHS ester may enhance interaction strength between OCP and CS-A. The adsorption amount and retention rate of recombinant human bone morphological protein-2 (rhBMP-2, an endogenous cytokine model) increased onto CS-A-modified OCP under physiological conditions when the interaction strength between OCP and the protein was stronger. The higher interaction strength between the OCP and CS-A could be associated with the enhanced adsorption affinity for the lower-molecular-weight basic protein. The alkaline phosphatase activity of MSCs increased depending on the remaining rate of rhBMP-2 adsorption on the CS-A-modified-OCP. Fourier transform infrared spectroscopy and chemical analysis indicated that the hydrolysis of the OCP progressed regardless of CS-A modification after MSC incubation. The present study suggests that stronger interactions between the OCP and glycosaminoglycans could contribute to the capture and retention of endogenous cytokines on the surface to further promote osteoblastic differentiation under the chemical environment induced by the hydrolysis of the OCP.

Previous research has found that octacalcium phosphate (OCP) increases macrophage accumulation and alters the initial inflammatory response. However, the role of the immune response induced by OCP in osteogenesis remains unknown. This study investigated the behavior of macrophages and bone regeneration capacity during the early inflammatory stage of OCP-mediated osteogenesis. To assess the change in macrophage polarization and osteogenic capacity, we used a standardized rat defect model filled with OCP or calcium-deficient hydroxyapatite (CDHA)-a material obtained through the hydrolysis of the original OCP. OCP or CDHA granules were incubated with RAW264 cells for 5 days to investigate the effect of physicochemical characteristics on macrophage cytokine/chemokine expression in vitro. Our in vivo results show that due to the OCP implantation, macrophages in the rat tibial defect area tend to polarize to the M2 phenotype (anti-inflammatory) and inhibit the formation of the M1 phenotype (pro-inflammatory). In comparison to CDHA, OCP exhibited superior bone regeneration potential due to its rapid promotion of cortical bone healing and stimulation of macrophage-related growth factors. Furthermore, our in vitro results have shown that OCP regulates the expression of macrophage chemokines over time. Compared to incubation with CDHA, incubation with OCP caused changes in the ionic microenvironment. These findings suggest that the OCP-mediated macrophage polarization and secretion profile not only regulate immune function but also positively affect osteogenesis., (© 2022 Wiley Periodicals LLC.)

Calcium phosphate (CaP) materials influence macrophage polarization during bone healing. However, the effect of the crystal phase of CaP materials on the immune response of bone remains unclear. In this study, the effect of the crystal phases of CaP materials on the regulation of macrophage polarization was investigated. Human THP-1 cells and mouse RAW 264 cells were cultured with octacalcium phosphate (OCP) and its hydrolyzed form Ca-deficient hydroxyapatite to assess the expression of pro-inflammatory M1 and anti-inflammatory M2 macrophage-related genes. OCP inhibited the excessive inflammatory response and switched macrophages to the anti-inflammatory M2 phenotype, which promoted the expression of the interleukin 10 ( IL10 ) gene. In contrast, HL stimulated an excessive inflammatory response by promoting the expression of pro-inflammatory M1 macrophage-related genes. To observe changes in the microenvironment induced by OCP and HL, inorganic phosphate (Pi) and calcium ion (Ca 2+ ) concentrations and pH value in the medium were measured. The expression of the pro-inflammatory M1 macrophage-related genes (tumor necrosis factor alpha ( TNFα ) and interlukin 1beta ( IL1β )) was closely related to the increase in ion concentration caused by the increase in the CaP dose. Together, these results suggest that the microenvironment caused by the crystal phase of CaP materials may be involved in the immune-regulation capacity of CaP materials.

The microstructure of biomaterials influences the cellular and biological responses in the bone. Octacalcium phosphate (OCP) exhibits higher biodegradability and osteoconductivity than hydroxyapatite (HA) during the conversion process from OCP to HA. However, the effect of the microstructure of OCP crystals on long tubular bones has not been clarified. In this study, two types of OCPs with different microstructures, fine-OCP (F-OCP) and coarse-OCP (C-OCP), were implanted in rat tibia for 4 weeks. F-OCP promoted cortical bone regeneration compared with C-OCP. The osteoclasts appearance was significantly higher in the C-OCP group than in the control group (defect only) at 1-week post-implantation. To investigate whether the solubility equilibrium depends on the different particle sizes of OCPs, Nano-OCP, which consisted of nanometer-sized OCPs, was prepared. The degree of supersaturation (DS) tended to decrease modestly in the order of C-OCP, F-OCP, and Nano-OCP with respect to HA and OCP in Tris-HCl buffer. F-OCP showed a higher phosphate ion concentration and lower calcium ion concentration after immersion in the buffer than C-OCP. The crystal structures of both OCPs tended to be converted to HA by rat abdominal implantation. These results suggest that differences in the microstructure of OCPs may affect osteoclastogenesis and result in osteoconductivity of this material in long tubular bone by altering dissolution behavior.

This study hypothesized that distant octacalcium phosphate (OCP) scaffolds may enhance osteocyte differentiation in newly formed bone matrices. The results obtained were compared with those of Ca-deficient hydroxyapatite (OCP hydrolyzate, referred to as HL hereafter). Granular OCP and HL, 300-500 µm in diameter, were implanted in critical-sized rat calvarial defects for eight weeks and subjected to histology, immunohistochemistry, histomorphometry, and transmission electron microscopy (TEM). Early osteocyte differentiation from an osteoblastic cell line (IDG-SW3) was examined using materials without contacting the surfaces for 10 days. The material properties and the medium composition were analyzed through selected area electron diffraction (SAED) using TEM observation and curve fitting of Fourier transform infrared (FT-IR) spectroscopy. The number of positive cells of an osteocyte earlier differentiation marker podoplanin (PDPN) in bone matrices, along the direction of bone formation, was significantly higher in OCP than that in HL. The ultrastructure around the OCP surfaces observed by TEM showed the infiltration of some cells, including osteocytes adjacent to the OCP surface layers. The OCP structure remained unchanged by SAED analysis. Nanoparticle deposition and hydrolysis on OCP surfaces were detected by TEM and FT-IR, respectively, during early osteocyte differentiation in vitro. The medium saturation degree varied in accord with ionic dissolution, resulting in possible hydroxyapatite formation on OCP but not on HL. These results suggested that OCP stimulates early osteocyte differentiation in the bone matrix from a distance through its metastable chemical properties. STATEMENT OF SIGNIFICANCE: This study demonstrated that octacalcium phosphate (OCP) implanted in critical-sized rat calvaria bone defects is capable of enhancing the early differentiation of osteocytes embedded in newly formed bone matrices, even when the surface OCP is separated from the osteocytes. This prominent bioactive property of OCP was demonstrated by comparing the in vivo and in vitro performances with a control material, Ca-deficient hydroxyapatite (OCP hydrolyzate). The findings were elucidated by histomorphometry, which analyzed the differentiation of osteocytes along the parallel direction of new bone growth by osteoblasts. Therefore, OCP should stimulate osteocyte differentiation through ionic dissolution even in vivo owing to its metastable chemical properties, as previously reported in an in vitro study (Acta Biomater 69:362, 2018)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Effect of the simultaneous hydrolysis of octacalcium phosphate (OCP) and poly (lactic-co-glycolic acid) (PLGA) was investigated on its osteoconductivity. PLGA soaked in phosphate buffered saline with 0%, 20%, and 40% OCP at 37°C for eight weeks indicated that when the OCP dose was increased, 1) the weight loss of PLGA increased, 2) the glass transition temperature of the PLGAs decreased, 3) the saturation degree in the saline moved to nearly saturated condition with respect to hydroxyapatite (HA) but was undersaturated with respect to OCP, and 4) OCP tended to convert to HA by X-ray diffraction and Fourier transform infrared spectroscopy. OCP/PLGA composites of 20% and 40% with more than 92% porosity were produced by combining OCP granules with 1,4-dioxane-solubilizing PLGA followed by lyophilization and then subjected to four- and eight-week in vivo implantation tests in 3 mm diameter rat femora defects. Microfocus X-ray computed tomography, histochemical and histomorphometric analyses showed that while bone formation was very limited with PLGA implantation, the extent of repair tended to increase with increasing OCP content in the PLGA, coupled with PLGA degradation, and bridge the defects with trabecular bone. Tartrate-resistant acid phosphatase-positive osteoclast-like cells were accumulated four weeks after implantation, while osteocalcin-positive osteoblastic cells appeared later at eight weeks, especially in 40% OCP/PLGA. These results suggest that OCP hydrolysis, with phosphate ion release, enhances PLGA hydrolysis, probably through the acid catalysis function of the protons supplied during the hydrolysis of OCP, thereby inducing PLGA biodegradation and new bone formation in the femoral defects. STATEMENT OF SIGNIFICANCE: Octacalcium phosphate (OCP) enhances osteoblasts and osteocytes differentiations during its hydrolysis accompanying inorganic ions exchange in this material. The present study found that the advancement of OCP hydrolysis under physiological conditions had an effect on poly (lactic-co-glycolic acid) (PLGA) degradation through its chemical environmental change around OCP, which was ascertained by the decreases in weight loss and glass transition temperature of PLGA with increasing the dose of OCP co-present. Rat femur-penetrated standardized severe defects were found to repair through bridging the cortical region defect margin. PLGA degradation could be enhanced through an acid catalyst function by protons derived from inorganic phosphate (Pi) ions through OCP hydrolysis under bone forming condition, resulting in showing a prominent bone regenerative capacity in OCP/PLGA composite materials., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

This study compared bovine serum albumin (BSA) adsorption onto octacalcium phosphate (OCP) materials prepared from two wet preparations in the absence (w-OCP) and presence (c-OCP) of gelatin. Raman spectroscopy was used to analyze the BSA adsorption onto OCPs in a 150 mM Tris-HCl buffer containing 0.5 mM calcium and inorganic phosphate (Pi) ions at pH 7.4 and at 37°C. The degree of supersaturation of the supernatants after the adsorption was determined by measuring the ion composition. The results showed that BSA adsorption onto w-OCP was higher than that for c-OCP. The calcium ion concentration of the supernatant decreased for both w-OCP and c-OCP, whereas the Pi ion concentration increased, approaching OCP equilibria at different saturation levels. BSA adsorbed even onto c-OCP, which included a small amount of gelatin during c-OCP preparation. These results indicate that the biodegradability of w-OCP and c-OCP may be modulated through interactions with serum proteins.

Previous studies have reported that octacalcium phosphate (OCP) enhances osteoblast differentiation and osteoclast formation during the hydrolysis process to hydroxyapatite (HA). However, the crystal phases that affect the crosstalk between osteoclasts and osteoblasts are unknown, which should determine the bone substitute material's property of OCP. The present study was designed to investigate whether the chemical composition and crystal structure of calcium phosphates affect osteoclast formation and the osteoclast-osteoblast crosstalk. Biodegradable β-tricalcium phosphate (β-TCP) was used as the control material. Osteoclasts were cultured on HA/OCP or HA/TCP disks and their cellular responses were assessed. Both OCP and β-TCP had a similar ability to create multinucleated osteoclasts. However, OCP promoted the expression of complement component 3a (C3a), a positive coupling factor, in osteoclasts, whereas β-TCP enhanced that of EphrinB2 (EfnB2) and collagen triple helix repeat containing 1 (Cthrc1). During osteoclast culture, phosphate ions were released from the crystals, and OCP-HA conversion was advanced in HA/OCP mixtures and OCP. X-ray diffraction analysis revealed no remarkable changes in the crystal structures of HA/TCP mixtures and β-TCP before and after osteoclast culture. These results indicate that the distinct chemical environment induced by the calcium phosphate phases affects the crosstalk between osteoclasts and osteoblasts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1001-1013, 2019., (© 2019 Wiley Periodicals, Inc.)

Effect of octacalcium phosphate/gelatin composite (OCP/Gel) on angiogenesis was studied by its implantation in rat calvaria critical-sized defect in relation to bone regeneration for 2 and 4 weeks. The implantation of OCP/Gel disks was analyzed by radiomorphometry using a radiopaque material perfusion (Microfil®) method and histomorphometry by hematoxylin and eosin-staining before and after the decalcification. Effect of the OCP dose in the range up to 4 mg per well on the capillary-like tube formation by human umbilical vein endothelial cells (HUVECs) was also examined in a transwell cell culture. The results showed that the blood vessels formation by OCP/Gel group was significantly higher at 2 weeks than other groups but decreased at 4 weeks during the advancement of new bone formation. The capillary-like tube formation was highest in an OCP dose of 1 mg per well while other OCP doses above or below 1 mg did not show such a stimulatory effect. The results established both in vivo and in vitro confirmed that OCP has a positive effect on angiogenesis during bone regeneration in a suitable dose ranges, suggesting that the angiogenesis stimulated by OCP could be involved in the OCP/Gel-enhanced bone regeneration. STATEMENT OF SIGNIFICANCE: We have reported that octacalcium phosphate (OCP) materials display stimulatory capacities on the bone tissue-related cells. However, the effect of OCP on the angiogenesis and its relation to the OCP-enhanced bone regeneration is unknown. This study confirmed the capacity of OCP on angiogenesis before increasing the new bone mass after the implantation of a composite of OCP and gelatin (OCP/Gel). The blood vessels formation took place associated with the area beginning of the new bone formation, which finally decreased together with development of bone formation. Because OCP was ascertained stimulating the capillary-like tube formation in HUVEC culture with a certain OCP dose, the present study is the first report showing the capacity of OCP on angiogenesis during the OCP/Gel-enhanced bone regeneration., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Three-dimensional (3-D) cell culture can better mimic physiological conditions in which cells interact with adjacent cells and the extracellular matrix than monolayer culture. We have developed a 3-D cell culture device, the Oxy chip, which can be used to generate and supply oxygen to cell spheroids to prevent hypoxia. Here, we used the Oxy chip to generate hybrid spheroids comprising calcium phosphate (CaP) particles (hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) or octacalcium phosphate (OCP)) and mesenchymal stem cells (MSCs, C3H10T1/2 cells or D1 cells) that can be used to analyze cell differentiation mechanisms. We showed that the 3-D cell-cell and cell-material interactions and oxygenation offered by the Oxy chip promoted osteoblastic differentiation of MSCs. We also used histomorphometric analysis of hematoxylin and eosin staining, quality analyses by μCT and collagen orientation observation with picrosirius red staining in bone regeneration following implantation of three CaPs in a critical-sized defect in mouse calvaria. The in vivo bone formation capacity of the three tested CaP materials was OCP ≥ β-TCP > HA: the newly formed bone by OCP had a structure relatively close to that of the calvaria intact bone. When MSCs were 3-D cultured with the CaP materials using the Oxy chip, the in vitro osteogenic capacity of these materials was highly similar to trends observed in vivo. The in vitro alkaline phosphatase activity of D1 cells had the highest correlation with in vivo bone volume (R = 0.900). Chemical and FTIR spectroscopic analyses confirmed that differentiation of D1 cells could be associated with amorphous calcium phosphate (ACP) precipitation concomitant with OCP hydrolysis. Taken together, hybrid spheroid cultures using the Oxy chip can be used to screen and predict bone forming potential of bone substitute materials. STATEMENT OF SIGNIFICANCE: An oxygen permeable-culture chip (Oxy chip) can be used to induce formation of cell spheroids by mesenchymal stem cells (MSCs). Use of the Oxy chip avoids hypoxia in the spheroid core and enhances MSC osteoblastic differentiation relative to conventional spheroid culture methods. The present study showed that the Oxy chip mimics the in vivo environment associated with bone formation and can be used to generate hybrid spheroids consisting of calcium phosphates and MSCs that are useful for analyzing cell differentiation mechanisms. Bone formation analysis following implantation of calcium phosphate materials in mouse calvaria defects showed positive correlation with the in vitro results. We propose that hybrid spheroids cultured on the Oxy chip can be used to screen and predict the bone forming potential of bone substitute materials., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The present study was designed to characterize co-precipitated fluoridated apatitic materials from octacalcium phosphate (OCP) precursor and to investigate their surface reactions with polyols including glycerol in the presence of fluoride ions. Laboratory-synthesized fluoridated apatite crystals (LS-FA) were obtained in a solution containing fluoride (F) from 25 to 500 ppm. LS-FAs and commercially available fluoroapatite (FA) and hydroxyapatite (HA) were characterized by physical techniques, such as X-ray diffraction. LS-FA obtained in the presence of 100 ppmF (100 ppm-LS-FA) had an apatitic structure, but its solubility was close to HA in a culture medium (α-MEM) despite the fact it contains over 3 wt % of F. 100 ppm-LS-FA, FA, and HA were then subjected to the human serum albumin (HSA) adsorption test at pH 7.4 (in a 150 mM Tris-HCl buffer) and the dissolution and re-mineralization experiments in the presence of xylitol, D-sorbitol, or glycerol, and F under acidic and neutral conditions. Adsorption affinity of HSA was estimated as highest for FA and lowest for LS-FA. LS-FA, FA, and HA were immersed in a lactic acid solution with the polyols and/or F ion-containing solution up to 200 ppm to analyze the dissolution behavior. LS-FA had the highest dissolution tendency in the conditions examined. Glycerol enhanced the dissolution of phosphate from apatite crystals in particular from LS-FA. The results suggest that the apatite crystals, obtained through the hydrolysis of OCP in the presence of F, provide a more reactive surface than FA or HA under physiological environments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2235-2244, 2018., (© 2017 Wiley Periodicals, Inc.)

The activity and the distribution of osteogenic cells around octacalcium phosphate (OCP) granules combined with gelatin matrix (OCP/Gel) onlaid on rat calvaria were analyzed histomorphometrically and immunohistochemically during vertical bone augmentation under mechanical or nonmechanical environment until 8 weeks. OCP/Gel disk was placed in subperiosteal pocket on the calvaria with or without polytetrafluoroethylene (PTFE) support. The latter is a nonmechanical stress model to alleviate the mechanical stress from the subcutaneous tissues. Onlay grafts of gelatin (Gel) sponge disk and OCP granules were also carried out for the comparison purpose. When bone augmentation was evaluated in first area from bone surface (area until 150 μm high from bone surface) and second area above the newly formed bone (area until 150 µm high from the first area), bone formation was enhanced most in first area followed by second area of OCP/Gel with PTFE. The appearance of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells was suppressed more in the newly formed tissue with PTFE than those without PTFE with an emphasis of the presence of gelatin. Although Runx2 positive-cells were accumulated more in both OCP/Gel with and without PTFE, osteocalcin-positive cells were abundant in OCP/Gel with PTFE than that without PTFE, suggesting that nonmechanical stress condition is more suitable for osteoblast differentiation. The appearance of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-positive cells was restrained in OCP/Gel and Gel with PTFE while osteoprotegerin-positive cells were most accumulated in OCP/Gel without PTFE. The results suggest that OCP/Gel composite under the mechanical stress-alleviated condition can enhance bone augmentation through balanced osteoblastic and osteoclastic cellular activities. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1322-1333, 2018., (© 2018 Wiley Periodicals, Inc.)

Octacalcium phosphate (OCP) has been shown to act as a nucleus for initial bone deposition and enhancing the early stages of osteoblastic differentiation. However, the effect on differentiation at the late stage into osteocytes has not been elucidated. The present study was designed to investigate whether OCP can promote the differentiation lineage from osteoblasts to late osteocytes using a clonal cell line IDG-SW3 compared to commercially available sintered β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) in a transwell cell culture. Special attention was paid to detect the progress of OCP hydrolysis associated with ionic dissolution products from this material. OCP induced the appearance of an alkaline phosphatase (ALP) peak in the IDG-SW3 cells compared to β-TCP and HA and increased SOST/sclerostin and FGF23 gene expression after 35 days of incubation. Analyses by X-ray diffraction, curve fitting of Fourier transform infrared spectra, and acid phosphate inclusion of the materials showed that OCP tended to hydrolyze to an apatitic structure during the incubation. Since the hydrolysis enhanced inorganic phosphate ion (Pi) release from OCP in the media, IDG-SW3 cells were further incubated in the conditioned media with an increased concentration of Pi in the presence or absence of phosphonoformic acid (PFA), which is an inhibitor of Pi transport within the cells. An increase in Pi concentration up to 1.5 mM raised ALP activity, while its positive effect was eliminated in the presence of 0.1 to 0.5 mM PFA. Calcium ions did not show such an effect. These results indicate the stimulatory capacity of OCP on osteoblastic differentiation toward osteocytes., Statement of Significance: Octacalcium phosphate (OCP) has been shown to have a superior osteoconductivity due to its capacity to enhance initial stage of osteoblast differentiation. However, the effect of OCP on the late osteoblastic differentiation into osteocyte is unknown. This study showed the capacity associated with the structural change of OCP. The data show that OCP released inorganic phosphate (Pi) ions while the hydrolysis advanced if soaked in the media, determined by chemical and physical analyses, and enhanced osteocytes differentiation of IDG-SW3 cells more than hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). Conditioned elevated Pi-containing media in the absence of OCP enhanced the osteocyte differentiation in the range of the concentration induced by OCP, the effect of which was cancelled by the inhibitor of Pi-transporters., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The present study was designed to investigate whether composite of coprecipitating octacalcium phosphate and gelatin (C-OCP/Gel) has an effect in repairing critical-sized defect of rat calvaria with oriented regenerative bone if implanted. The materials were prepared through two steps to disperse homogenous and well-elongated OCP toward long axis of the crystals in gelatin (Gel) matrix with the distinct concentration 17-44 wt %: OCP precipitates recovered from the coprecipitated with Gel molecules in aqueous solution (referred to as C-OCP hereafter) were mixed again in fresh aqueous Gel solution with various mixing ratio to form C-OCP/Gel for implantation. C-OCP/Gel disks with 9 mm diameter and 1 mm thickness after the dehydrothermal treatment was implanted in 9 mm diameter rat calvaria critical-sized defect. The histology, the histomorphometry in the regenerated bone and the quantitative analysis of the orientation of collagen with picrosirius red staining were carried out. It was found that C-OCP/Gel is capable of not only inducing sufficiently regenerative bone over 80% of the defect coupled with practically complete material biodegradation but also forming oriented bone significantly in relation to the amount of C-OCP in Gel matrix until 12 weeks after the implantation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1029-1039, 2017., (© 2016 Wiley Periodicals, Inc.)

Recently it was reported that the implantation of octacalcium phosphate (OCP) and collagen composite (OCP-collagen) was effective at promoting bone healing in small bone defects after cystectomy in humans. In addition, OCP-collagen promoted bone regeneration in a critical-sized bone defect of a rodent or canine model. In this study, OCP-collagen was implanted into a human mandibular bone defect with a longer axis of approximately 40 mm, which was diagnosed as a residual cyst with apical periodontitis. The amount of OCP-collagen implanted was about five times greater than the amounts implanted in previous clinical cases. Postoperative wound healing was satisfactory and no infection or allergic reactions occurred. The OCP-collagen-treated lesion was gradually filled with radio-opaque figures, and the alveolar region occupied the whole of the bone defect 12 months after implantation. This study suggests that OCP-collagen could be a useful bone substitute material for repairing large bone defects in humans that might not heal spontaneously. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

The present study was designed to investigate the local microenvironment of octacalcium phosphate in a granule form upon biomolecules adsorption utilizing an indicator-immobilized-gel-sheet for measuring pH. We previously showed that octacalcium phosphate enhances bone regeneration during its progressive hydrolysis into hydroxyapatite if implanted in bone defects. The gel-sheet was made from a photocrosslinkable prepolymer solution, which can easily immobilize a pH indicator (bromothymol blue; BTB) in the hydrogel. The indicator-immobilized-gel-sheet was mounted on a biochip which was made of polydimethylsiloxane (PDMS) with a flow channel. The pH value was calculated by detecting the color changes in the gel-sheet and displayed as the pH distribution. After pre-adsorption of bovine albumin, β-lactoglobuline or cytochrome C onto octacalcium phosphate granules, the granules with the gel-sheet were further incubated in Tris-HCl buffer solution in the absence or presence of fluoride, known as an accelerator of octacalcium phosphate hydrolysis. pH values of the gel-sheet surrounding octacalcium phosphate granules showed a decrease from pH 7.4 to 6.6 in relation to the proteins adsorbed. Overall, the proposed pH-sensitive gel can be used to detect the pH around octacalcium phosphate granules with a high spatial resolution.

Objective: Composite of synthetic octacalcium phosphate and collagen (OCP/Col) enhances bone regeneration if implanted into human, canine, or rodent bone defects. This study was designed to investigate the influence on eruption of the permanent successor tooth and alveolar height of OCP/Col implantation into extraction cavities of deciduous teeth., Design: Disks of OCP/Col prepared from synthetic granules of octacalcium phosphate (OCP) and porcine atelocollagen, and commercially available sintered porous β-tricalcium phosphate (β-TCP) was implanted into the prepared defect of eleven male beagle dogs by extraction of two deciduous premolars. Untreated group had nothing implanted into the prepared defect, and physiological group (Physiol group) was observed without any treatment. Periodical macroscopic and radiographic examinations were performed until the period equivalent to the physiological eruption of permanent successor teeth (P3 and P4)., Results: Although no unerupted permanent successor teeth were observed among the OCP/Col, Untreated, and Physiol groups, two permanent successor teeth were unerupted in the β-TCP group. No significant difference of the alveolar heights or the eruptive position in P3 and P4 was observed between OCP/Col and Physiol groups., Conclusion: OCP/Col implantation in the alveolar region that included the unerupted permanent successor teeth would cause no disturbance of permanent tooth eruption and preserve the alveolar ridge., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The purpose of this study was to compare the biodegradation rate and quality of regenerated bone among four materials. A short time period of 8 weeks was chosen to examine early bone healing. The rod-shaped implants of commercially available two β-tricalcium phosphate (β-TCP) ceramics with porosity 60% and 71-80%, respectively, laboratory prepared octacalcium phosphate/gelatin composite (OCP/Gel), which has been proven to have a highly osteoconductive and biodegradable property in rat calvarial defect, and gelatin sponge (Gelatin) were implanted in rabbit tibia defect of 6 mm diameter and 7 mm depth for 2, 4 and 8 weeks. Analyses by μCT, histomorphometry and push-in test were carried out to evaluate the extent of the tissue regeneration and the material biodegradation in the long bone. OCP/Gel and Gelatin were completely resorbed but only OCP/Gel induced cortical bone bridge until 8 weeks that has strength compatible to that of the natural bone. β-TCP (71%-80%) and β-TCP (60%) were not completely resorbed and never induced the amount of new bone formation beyond that by OCP/Gel. The results indicate that the new bone having enough strength could be regenerated if the material shows not only higher biodegradation rate but also higher osteoconductivity. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2833-2842, 2016., (© 2016 Wiley Periodicals, Inc.)

The dissolution behaviors of octacalcium phosphate (OCP), β-tricalcium phosphate (β-TCP), and hydroxyapatite (HA) were compared by implanting the materials in rat subcutaneous pouches for 8 weeks using a filter chamber or immersing them in simulated body fluid (SBF) or Tris-HCl buffer for 2 weeks at pH 7.4 and 37(o)C. X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and chemical analysis were conducted on these materials. Degree of supersaturation (DS) in the two solutions immersed with each calcium phosphate material was calculated from their chemical compositions. The results showed that OCP partially converted to apatitic crystals, while β-TCP and HA remained unchanged after the implantation. The DS of the SBF solution remained slightly supersaturated with respect to OCP and β-TCP, but slightly undersaturated in the Tris-HCl buffer. These findings suggest that previously reported OCP and β-TCP biodegradation could be induced through cell-mediated osteoclastic resorption rather than a simple dissolution process.

The present study investigated whether alginate (Alg) hydrogel microbeads have a role in maintaining mouse bone marrow stromal ST-2 cells and release the cells after being stimulated by synthetic octacalcium phosphate (OCP), which is a mineral crystal capable of stimulating osteoblastic differentiation during a conversion process to hydroxyapatite (HA). The ST-2 cell suspension in the alginate solution, which contained various concentrations of OCP granules with diameters less than 53 μm, was extruded drop-wise into a stirred gelation solution containing BaCl2 using an encapsulator with nitrogen gas stream. The Alg-microbeads (Alg/OCP · ST-2 microbeads) that were generated, which had a diameter of approximately 400 μm, were incubated for up to 14 d and then assessed for osteoblastic differentiation. Alg-microbeads with cells were also incubated to identify the possible conversion from OCP to HA. Osteoblast differentiation markers in ST-2 cells, alkaline phosphatase (ALP) and collagen type I, were up-regulated in the presence of higher amounts of OCP. X-ray diffraction analysis and Fourier transform infrared spectroscopy confirmed that the OCP tended to convert to HA over time, suggesting that the OCP in Alg-microbeads interacts three-dimensionally with ST-2 cells and stimulates its osteoblastic differentiation. The release of ST-2 cells from the microbeads was also estimated. ST-2 cells were identified outside of the microbeads, although the cell number tended to decrease with increasing OCP. These results suggest that Alg/OCP microbeads could be used as a vehicle to activate osteoblastic cells and deliver them to sites where bone regeneration is needed.

Synthetic octacalcium phosphate (OCP) has been suggested to be a useful biomaterial for the regeneration of hard tissues, including bone. However, it remains unknown whether OCP induces dentine formation by dental pulp. We investigated biomineralization of dental pulp exposed to synthetic OCP in vitro and in vivo. When dental pulp was exposed directly to OCP, rapid formation of reparative dentine (RD) was induced and expression of dentine sialoprotein synthesis was observed in dental pulp adjacent to newly synthesized RD. OCP inhibited the proliferation of rat pulp cells and also promoted their odontoblastic differentiation in vitro, as alkaline phosphatase activity, mineralization of pulp cells and the expression level of dentine sialophosphoprotein were enhanced. Direct contact between OCP and pulp cells is required for OCP to exhibit its effects in vitro. The expression level of Runx2, a transcription factor whose downregulation is closely related to odontoblast differentiation, was downregulated in pulp cells cultured with OCP. Structural changes of OCP during culture were determined by Fourier transform infrared spectroscopy. OCP tended to be converted to carbonate hydroxyapatite after incubation with or without pulp cells, which may be analogous to biological apatite crystals. Taken together, our data suggest that synthetic OCP supports RD formation by dental pulp and downregulation of Runx2 may be involved in that stimulatory activity. Furthermore, OCP-apatite conversion is involved in this stimulatory capacity of OCP., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: To improve the success rate of rotator cuff repair, we investigated whether octacalcium phosphate (OCP) with gelatin (Gel) vehicle had a positive effect on tendon-to-bone healing., Methods: We assessed the histologic characteristics of the tendon-to-bone healing using the rabbit rotator cuff repair model. We divided the shoulders into 3 groups: control (without OCP/Gel composite), OCP/Gel composite (OCP+group), and Gel alone without OCP (Gel group) to evaluate the effectiveness of gelatin., Results: Both the number of newly formed tendon fibers and the Sharpey fibers at the repair site increased in the OCP+group compared with those in the other 2 groups on hematoxylin-eosin staining (P < .05). On immunohistochemical evaluation, both the bone and the fibers in the OCP+group demonstrated that type I collagen was picked up, whereas the newly formed tendon fibers and Sharpey fibers revealed type III collagen., Conclusion: Treatment with OCP made collagen fibers and the Sharpey fibers, constituted by type I and type III collagens, increase at the tendon-to-bone insertion. It might be beneficial for the healing of rotator cuff tendon to bone., (Copyright © 2015 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

The present study was designed to investigate the extent of osteoconductive property of a mechanical mixture of octacalcium phosphate (OCP) and amorphous calcium phosphate (ACP). OCP was mixed with ACP in granules that had a diameter of 300 and 500 μm, respectively, and at 25, 50, or 75 wt %. The physicochemical characteristics and the osteoconductive properties of the mixtures were compared with OCP alone or ACP alone through implantation into rat critical-sized calvaria defects for up to 12 weeks and simulated body fluid (SBF) immersion for 2 weeks. The mixtures of OCP and ACP, in particular the OCP 25 wt % and ACP 75 wt % (O25A75), had higher radiopacity compared to ACP and OCP alone. O25A75 induced greater enhancement of bone regeneration than ACP alone at 8 weeks and that than OCP alone at 12 weeks. X-ray diffraction and Fourier transform infrared (FTIR) analyses of the retrieved mixtures showed that ACP, OCP, and O25A75 tended to convert to hydroxyapatite (HA) after the implantation, while the structure of OCP remains without complete conversion after SBF immersion. Analyses by FTIR curve fitting of the solids and the degree of supersaturation of the SBF supported the observation that the existence of ACP enhances the kinetics of the conversion. Scanning electron microscopy found that the surface of O25A75 had distinct characteristics with OCP and ACP after SBF immersion. The results suggest that the extent of the osteoconduction of OCP could be controlled by the copresence of ACP most probably through the prevailing dissolution-precipitation of the surface of ACP crystals to form HA.

Objective: Synthetic octacalcium phosphate and porcine atelocollagen composites significantly enhanced bone regeneration more than β-tricalcium phosphate collagen composite and hydroxyapatite collagen composite in a rat cranial defect model. However, the long-term stability and quality of octacalcium phosphate collagen (OCP/Col) composites-derived regenerated bone, when implanted in a canine alveolar cleft model, have yet to be elucidated. The present study investigated the longterm stability and quality of bone regenerated by OCP/Col., Design: Disks of OCP/Col or collagen were implanted in a canine alveolar-cleft model (n = 6). Then, bone regeneration in the implanted areas was investigated macroscopically, radiographically, and histologically at 10 months after implantation. In addition, three-dimensional quantitative images of regenerated bone were analyzed by microcomputed tomography., Results: Macroscopically, the OCP/Col treated alveolus was clearly augmented, and radio-opacity in the OCP/Col implanted area was comparable to that of the original alveolus bone. On histological analysis, the area was mostly filled with newly formed bone, and a few granules of implanted OCP/Col were enclosed in it. In the microcomputed tomography analysis, the regenerated bone volume in the OCP/Col group was larger than that in the collagen group. OCP/Col-derived bone consisted of outer cortical and inner cancellous structure with dense trabeculae and seemed like the original bone structure., Conclusions: OCP/Co composites could be a useful bone regenerative material to substitute for autogenous bone because their implantation could elicit high bone regeneration and active structural reconstitution.

We have previously demonstrated that octacalcium phosphate (OCP) collagen composite (OCP/collagen) promotes bone regeneration in a critical-sized bone defect of a rodent or canine model. This study was designed to investigate the bone regeneration of OCP/collagen in human bone defect as a first clinical trial. Two patients who had a radicular cyst or apical periodontitis consented to participate in our clinical study, and OCP/collagen was implanted into the defects after operation. Radiographic examination showed effective bone healing in each bone defect at 3 or 6 months. Likewise, computed tomography value significantly increased after implantation. Postoperative wound healing was uneventful, and neither infection nor allergic reaction against OCP/collagen was observed for the entire period. This study demonstrated that OCP/collagen would be safely used and enhanced bone regeneration in human bone defects. To reinforce the efficacy of OCP/collagen as a bone substitute material, it should be compared with other suitable comparators in the future.

The present study was designed to investigate whether three sodium hyaluronic acid (HyA) medical products, Artz(®), Suvenyl(®) and a chemically modified derivative of sodium HyA Synvisc(®), can be used as suitable vehicles for an osteoconductive octacalcium phosphate (OCP). OCP granules (300-500 μm diameter) were mixed with these sodium HyAs with molecular weights of 90 × 10(4) (Artz(®)), 190 × 10(4) (Suvenyl(®)) and 600 × 10(4) (Synvisc(®)) (referred to as HyA90, HyA190 and HyA600, respectively). OCP-HyA composites were injected using a syringe into a polytetrafluoroethylene ring, placed on the subperiosteal region of mouse calvaria for 3 and 6 weeks, and then bone formation was assessed by histomorphometry. The capacity of the HyAs for osteoclast formation from RAW264 cells with RANKL was examined by TRAP staining in vitro. Bone formation was enhanced by the OCP composites with HyA90 and HyA600, compared to OCP alone, through enhanced osteoclastic resorption of OCP. HyA90 and HyA600 facilitated in vitro osteoclast formation. The results suggest that the osteoconductive property of OCP was accelerated by the HyAs-associated osteoclastic resorption of OCP, and therefore that HyA/OCP composites are attractive bone substitutes which are injectable and bioactive materials., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The present study investigated the role of mitogen-activated protein kinase (MAPK) signaling in osteoblastic differentiation of stromal ST-2 cells induced by synthetic octacalcium phosphate (OCP) incubation. Since our previous studies revealed that OCP consumes calcium ions in media during conversion to hydroxyapatite, the effect of the ions on ST-2 cell differentiation with or without OCP crystals was analyzed. The effect of presence or absence of MAPK inhibitors was also analyzed. OCP increased alkaline phosphatase (ALP) activity and the mRNA expression of differentiation markers via the p38 signaling pathway. The PD98059 MAPK inhibitor increased ALP activity and differentiation marker genes in cells cultured in OCP-coated wells. Reduction of calcium ions in the medium by EGTA increased the ALP activity without OCP in the presence of phosphate ion concentrations up to 7.5 mM. OCP may enhance osteoblastic differentiation through the p38 signaling pathway via the reduction of calcium ions induced by its physicochemical property.

Implantation of octacalcium phosphate (OCP), a hydroxyapatite precursor, has been reported to induce chondrogenesis in vivo. In this study, we examined the effects of OCP on the chondrogenic differentiation of mouse chondroblastic ATDC5 cells in vitro. Contrary to our expectation, chondrogenic differentiation of ATDC5 cells evaluated by the mRNA expression of Col2a1, Acan and Col10a1 was suppressed by OCP. Among Sox9, Sox5 and Sox6, essential transcription factors for chondrogenesis, the expression of Sox6 mRNA was markedly lowered by OCP. Whereas ATDC5 cells dissolved OCP to liberate calcium and inorganic phosphorus, increased calcium or phosphate in the medium had little effect on the differentiation of these cells. Direct contact of ATDC5 cells with OCP was required to suppress the expression of Col2a1 and Sox6 mRNAs, whereas the introduction of Sox6 short interfering RNA lowered the expression of Col2a1 mRNA. On the other hand, the forced expression of Sox6 protein partially but significantly, restored the expression of Col2a1 mRNA suppressed by OCP. These results indicate that OCP suppresses the chondrogenic differentiation of ATDC5 cells, at least in part, at the Sox6 transcription level.

The present paper reviews biomaterial studies of synthetic octacalcium phosphate (OCP) as a scaffold of osteoblastic cells. OCP crystals have been suggested to be one of precursor phases in hydroxyapatite (HA) crystal formation in bone and tooth. The recent intensive biomaterials and tissue engineering studies using synthetic OCP disclosed the potential function of OCP as a bioactive material as well as synthetic HA materials due to its highly osteoconductive and biodegradable properties. In vitro studies showed that OCP crystals exhibit a positive effect on osteoblastic cell differentiation. In vivo studies confirmed that the materials of OCP in a granule forms and OCP-based composite materials with natural polymers, such as gelatin and collagen, enhance bone regeneration if implanted in various model bone defects with critical-sized diameters, defined as a defect which does not heal spontaneously throughout the lifetime of the animals. One of particular characteristics of OCP, found as a mechanism to enhance bone regeneration in vivo, is a process of progressive conversion from OCP to HA at physiological conditions. The OCP-HA conversion is accompanied by progressive physicochemical changes of the material properties, which affects the tissue reaction around the crystals where osteoblastic cells are encountered. Mesenchymal stem cells (MSCs) seeded in an OCP-based material enhanced bone regeneration in the rat critical-sized calvaria defect more than that by the material alone. The overall results reveal that OCP crystals have an effect on osteoblastic cell differentiation including the differentiation of MSCs in vivo. The evidence collected experimentally in the laboratory was presented.

Objective: The present study evaluated the efficacy of bone regeneration between synthetic octacalcium phosphate (OCP) granules combined with porcine atelocollagen (OCP/Col) and β-tricalcium phosphate (β-TCP)., Study Design: A disk of OCP/Col (20 mm diameter, 2.5 mm thick) or commercially available sintered porous β-TCP was implanted into a critical-sized calvarial defect (20 mm diameter) of adult male canines (n = 10). The newly formed bone in the defect was analyzed radiographically, crystallographically, histologically, and histomorphometrically at 6 months after implantation., Results: Histomorphometry showed that there was significantly more newly formed bone in OCP/Col-treated defects than for β-TCP (P < .05). X-Ray diffraction patterns of implanted OCP/Col were similar to those of original bone and different from those of implanted β-TCP., Conclusions: These results suggest that OCP/Col implantation in canine critical-sized defect enhanced bone regeneration more than β-TCP, which is the most commonly used synthetic bone substitutes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The present study was designed to investigate the extent to which calcium phosphate bone substitute materials, including osteoconductive octacalcium phosphate (OCP), display cytotoxic and inflammatory responses based on their dissolution in vitro. Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics, which are clinically used, as well as dicalcium phosphate dihydrate (DCPD) and synthesized OCP were compared. The materials were well characterized by chemical analysis, x-ray diffraction and Fourier transform infrared spectroscopy. Calcium and phosphate ion concentrations and the pH of culture media after immersion of the materials were determined. The colony forming rate of Chinese hamster lung fibroblasts was estimated with extraction of the materials. Proliferation of bone marrow stromal ST-2 cells and inflammatory cytokine TNF-α production by THP-1 cells grown on the material-coated plates were examined. The materials had characteristics that corresponded to those reported. DCPD was shown to dissolve the most in the culture media, with a marked increase in phosphate ion concentration and a reduction in pH. ST-2 cells proliferated well on the materials, with the exception of DCPD, which markedly inhibited cellular growth. The colony forming capacity was the lowest on DCPD, while that of the other calcium phosphates was not altered. In contrast, TNF-α was not detected even in cells grown on DCPD, suggesting that calcium phosphate materials are essentially non-inflammatory, while the solubility of the materials can affect osteoblastic and fibroblastic cellular attachment. These results indicate that OCP is biocompatible, which is similar to the materials used clinically, such as HA. Therefore, OCP could be clinically used as a biocompatible bone substitute material.

The present study was designed to determine whether the osteoconductivity of octacalcium phosphate-collagen (OCP/Col) composite can be improved by controlling the granule size of OCP. The granules of synthetic OCP, with diameters in the range of 53 to 300, 300 to 500, and 500 to 1000 μm, were used as an inorganic source of composite materials mixed with atelo-Col. After vacuum dehydrothemal treatment, OCP/Col disks were implanted into critical-sized calvaria defects in Wistar rats for 4, 8, and 12 weeks and examined radiographically, histologically, histomorphometrically, and histochemically. The materials were characterized according to mercury intrusion porosimetry and scanning electron microscopy. X-ray diffraction was performed before and after implantation. The dissolution of OCP crystals in a Col matrix was determined by immersing OCP/Col disks in a culture medium. OCP/Col had a constant pore size (~30 μm) regardless of OCP granule size. OCP in the Col matrix tended to convert to hydroxyapatite (HA) during the implantation. OCP/Col with the smallest granules of OCP enhances both bone regeneration and biodegradation the most through tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cellular resorption of OCP granules. The smallest OCP granules in the Col matrix showed the highest dissolution and had the greatest potential to form HA. The results indicated that the size of the included OCP granules can controll the osteoconductivity of OCP/Col. The overall results suggest that the physicochemical property of OCP crystals is a factor that determines the bone regenerative capacity of OCP/Col in critical-sized calvaria large bone defects in rats.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although the efficacy of the in vivo osteogenic capabilities of synthetic octacalcium phosphate (OCP) crystal implantation can be explained through its stimulatory capacity for the differentiation of the host osteoblastic cell lineage, direct evidence that OCP supports bone regeneration by osteogenic cells in vivo has not been shown. Mesenchymal stem cells (MSCs) isolated from 4-week-old male Wistar rat long bones were pre-incubated in osteogenic or maintenance medium in the presence or absence of basic fibroblast growth factor (bFGF). OCP/Collagen (OCP/Col) or collagen disks were seeded with MSCs that had been pre-incubated in osteogenic medium containing bFGF, which exhibited the highest differentiation induction, and then incubated for an additional day. The disks were implanted in critical-sized calvaria defects of 12-week-old male Wistar rats and the specimens were analysed radiographically, histologically, histomorphometrically, and by micro-computed tomography (CT) imaging at 4 and 8 weeks after the implantation. The OCP/Col·MSCs group rapidly induced more bone regeneration, even within 4 weeks, compared to the OCP/Col group without MSCs. The bone mineral density of the OCP/Col·MSCs group was also greater than the OCP/Col group. The Col·MSCs group did not exhibit prominent osteogenicity. These results indicate that OCP crystals in a collagen matrix efficiently promote exogenously introduced osteogenic cells to initiate bone regeneration if the cells are pre-treated in a suitable differentiation condition.

Purpose: 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., Materials and Methods: 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., Results: 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., Conclusion: 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., (© 2009 Wiley Periodicals, Inc.)

Osteoclasts are involved in bone resorption, and its activation is considered one of the causes of osteoporosis. The pit assay is the principal method for evaluating osteoclast function by measuring hydroxyapatite resorption in vitro. However, the pit assay requires time and trained techniques, including the pit image analysis, and there is no other easy method for evaluating bone resorption. In this study, we developed a novel approach to quantify the bone resorption activity using a calcium phosphate (CaP) coating labeled with fluorescent polyanion. Fluoresceinamine-labeled chondroitin polysulfate or Hoechst 33258-labeled deoxyribonucleic acid was used for CaP labeling. When macrophage cell line RAW264 was cultured on the labeled CaP under the stimulation with the receptor activator of the NF-κB ligand (RANKL), RAW264 cells differentiated into osteoclastic cells and the fluorescence intensity of the culture supernatant and pit area increased in a time- and dose-dependent manner. Furthermore, drugs for osteoporosis treatment, such as pamidronate and β-estradiol, inhibited fluorescein release by the cells stimulated with RANKL. A positive correlation between the fluorescence intensity and pit area was observed (r=0.917). These results indicated that this new method using fluorescent polyanion-labeled CaP is a standardized useful assay system for the evaluation of bone resorption activity., (2010 Elsevier Inc. All rights reserved.)

The present study was designed to investigate whether mechanical testing in conjunction with microcomputed tomography (microCT) analysis can be used to evaluate the quality of regenerated bone enhanced by the implantation of a composite composed of granular octacalcium phosphate and collagen (OCP/Col) matrix. Previous studies confirmed that the granules of OCP alone or OCP in Col matrix tend to mature into bone-like hydroxyapatite and enhance bone regeneration coupled with its own biodegradation, if implanted in various bony sites. OCP/Col was implanted in rat calvaria critical-sized bone defect for 4 to 12 weeks for microindentation, mechanical testing, microCT imaging, and histological examinations. The microindentation testing of the regenerated bone revealed a progressive increase of the Vickers hardness showing the highest value in 12 weeks. The Vickers hardness was in good agreement with both the parameters of microCT and the mechanical property; the quality of regenerated bone increased progressively with the implantation periods. The regenerated bone exhibited a mature bone-like matrix structure with osteocytes histologically. The quality of the regenerated bone was compatible to that of normal control calvaria bone regarding the mechanical properties. The results indicate that microindentation testing can be used to evaluate the quality of regenerated bone in the initial regeneration and the development of bone enhanced by OCP granules within Col matrix. It appears that the implantation of OCP/Col could be a model study to determine the quality of the regenerated bone.

The present study was designed to investigate whether the formation of octacalcium phosphate (OCP) is accelerated on titanium (Ti) surface by an electron cyclotron resonance (ECR) plasma oxidation at various pressures and temperatures. X-ray diffraction (XRD) of Ti-oxidized substrates showed that the rutile TiO(2) phase on its surfaces appeared at 300 degrees C and was crystallized when the oxidation temperature increased up to 600 degrees C. The thickness of TiO(2) film on the substrates increased progressively as the temperature increased. The oxidized Ti surfaces were soaked in calcium and phosphate solutions supersaturated with respect to both hydroxyapatite (HA) and OCP but slightly supersaturated with dicalcium phosphate dihydrate (DCPD). OCP crystals with a blade-like morphology were deposited as the primary crystalline phase on Ti substrates, while DCPD was included as a minor constituent. The amount of OCP deposition was maximized under 0.015 Pa in 300 degrees C. On the other hand, the oxidation temperature did not show a significant effect on the deposit in the range examined. The phase conversion from OCP to HA, determined by XRD, was demonstrated to occur even at 1 day and to advance until 7 days by immersing the Ti substrate with the deposit in simulated body fluid at 37 degrees C. The present results suggest that ECR plasma oxidation could be used to improve a Ti surface regarding its bioactivity due to the enhancement of osteoconductive OCP deposition., ((c) 2010 Wiley Periodicals, Inc.)

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 microm (L-OCP) produced distinct intergranular spaces between OCP granules ranging from 28.8 to 176.6 microm. 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., (Copyright 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The present study was designed to investigate whether the amount of octacalcium phosphate (OCP) affects the characteristics of alginate (Alg)/OCP scaffolds regarding the pore formation and its distribution, and the thermodynamic stability from OCP to hydroxyapatite (HA) in an in vitro physiological environment. Alg/OCP composites with weight ratios of 100/0, 75/25, 50/50, and 25/75 were prepared through mixing the ground synthesized OCP crystals with an Alg solution and applying lyophilization. Analysis of X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and mercury intrusion porosimetry verified that the crystalline phase of OCP and the porosities were retained regardless of the OCP amount. On the other hand, the elastic modulus, determined by mechanical testing, and, interestingly, the pore size increased with increasing the OCP amount. The immersion of the composites in a simulated body fluid up to 14 days revealed that OCP in Alg matrices tends to convert to HA with enhancing the calcium consumption depending on the OCP amount. The results indicated that the inclusion of OCP crystals in the Alg matrix by the mixing process controls the character of the pore distribution in Alg/OCP composites while maintaining the transitory nature of OCP.

Previous studies have suggested that the biodegradability of octacalcium phosphate-collagen (OCP/Col) composite by osteoclasts is accelerated in association with mechanical stress suffered by the host tissue around the implant. The present study was designed to investigate whether alleviation of mechanical stress restores the bone regenerative properties of OCP/Col, as previously shown in nonload-bearing sites. OCP/Col discs supported with a polytetrafluoroethylene (PTFE) ring, which has a higher modulus than OCP/Col, were implanted in a rat subperiosteal pocket for up to 12 weeks. The structural features of the implant and biological responses were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, histomorphometry, histochemistry, and tissue mRNA expression around the implants. The effect of compression was analyzed using mouse stromal ST-2 cells by deforming the cell-seeded OCP/Col discs in vitro with or without a PTFE ring. The results clearly indicated the restoration of bone formation by the alleviation of mechanical stress and the upregulation of osteoblast-related genes, such as osterix on the other hand, the implantation of OCP/Col on calvaria or in an in vitro test without PTFE support resulted in the upregulation of osteoclast-related genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, in the tissues or receptor activator of the nuclear factor-kappaB ligand (RANKL) in ST-2 cells. The results confirmed that calvaria augmentation is enhanced by implanting OCP/Col if suitable conditions regarding mechanical stress are provided.

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.

The present study was designed to investigate whether octacalcium phosphate (OCP)-precipitated alginate (Alg) promotes osteoblastic cell proliferation and bone regeneration in comparison with Alg itself. Alg, known to lack mammal cell attachment capability, was used as the matrix to test the distribution effect of OCP in a three-dimensional environment. A series of Alg/OCP scaffolds with different pore sizes was prepared by centrifuging Alg gels precipitated by OCP crystals. The scaffolds had a bimodal distribution of pores (ultrafine pores: approximately 100 nm; relatively large pores: from 6.0 to 51.7 microm) and over 86% porosity. The osteoconductive capability of Alg/OCP was determined by examining mouse bone marrow stromal ST-2 cell proliferation after 3 days in vitro and bone regeneration in mouse calvaria critical-sized defect after 21 days. The analyses showed that ST-2 cell proliferation and bone regeneration increased with an increase in the pore size and reached the highest level in the 51.7 microm pore scaffold. The results suggest that OCP-precipitated Alg provides a better scaffold for osteoblasts to attach and proliferate in a three-dimensional environment and promotes bone regeneration, indicating that OCP is a candidate material to modify the surface of non-cell-interactive polymeric scaffolds, such as Alg, into an osteogenic condition.

Metastatic bone disease is often associated with bone pain, pathologic fractures, and nerve compression syndromes. Effective therapies to inhibit the progression of bone metastases would have important clinical benefits. Therefore, we developed a novel calcium phosphate-binding liposome for a bone-targeting drug delivery system. We synthesized a novel amphipathic molecule bearing a bisphosphonate (BP) head group to recognize and bind to hydroxyapatite (HA). We demonstrated that the liposomes having BP moieties show high affinity for HA. Doxorubicin-loaded liposomes adsorbed on the surface of HA significantly reduce the number of viable human osteosarcoma MG63 cells. This shows that the liposomes can be excellent carriers for anticancer drugs because they specifically target bone tissue. This calcium phosphate-binding liposome system could be used with many drugs for bone-related diseases such as osteoporosis, rheumatoid arthritis, and multiple myeloma.

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.

The present study was designed to investigate whether the stoichiometry of octacalcium phosphate OCP affects its osteoconductive and immune response characteristics in rat bone marrow. Those characteristics of synthetic, well-grown OCP but with a non-stoichiometric composition were compared with those of a slightly hydrolyzed OCP (low crystalline OCP: LC-OCP), the fully hydrolyzed apatitic product of OCP or biodegradable beta-tricalcium phosphate (beta-TCP) ceramic, by their implantation in rat tibia for 56 days. The physicochemical aspect of implants and biological responses were analyzed by X-ray diffraction, histomorphometry, immunohistochemistry and expression of mRNA around the implants. The remarkable findings were that: (1) the highest bone formation rate was obtained for beta-TCP whereas the lowest for LC-OCP at Day 14; (2) the rates were reversed and reached the highest for LC-OCP until Day 56; (3) the early expression of ostoeoclast markers TRAP and cathepsin-K was suppressed with LC-OCP; (4) the expression of inflammatory markers IL-beta1 and TNF-alpha was suppressed with LC-OCP. The results confirmed that the partially hydrolyzed OCP with Ca/P molar ratio 1.37 (LC-OCP) enhances bone formation most, suppressing early osteoclast activity and reducing inflammation.

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.

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.