Effect of Octacalcium Phosphate Crystals on the Osteogenic Differentiation of Tendon Stem/Progenitor Cells In Vitro.

Synthetic octacalcium phosphate (OCP) activates bone tissue-related cells, such as osteoblasts, osteoclasts, and vascular endothelial cells. However, the effect of OCP on tendon-related cell activation remains unknown. This study examined the response of rat tendon stem/progenitor cells (TSPCs) to OCP and related calcium phosphate crystals in vitro. TSPCs were cultured with OCP and Ca-deficient hydroxyapatite (CDHA) obtained from the original OCP hydrolysis to assess the activity of alkaline phosphatase (ALP) and the expression of osteogenesis-related genes. Compared with CDHA, the effect of OCP on promoting the osteogenic differentiation of TSPCs was apparent: the ALP activity and mRNA expression of RUNX2, Col1a1, OCN, and OPN were higher in OCP than in CDHA. To estimate the changes in the chemical environment caused by OCP and CDHA, we measured the calcium ion (Ca2+) and inorganic phosphate (Pi) ion concentrations and pH values of the TSPCs medium. The results suggest that the difference in the osteogenic differentiation of the TSPCs is related to the ionic environment induced by OCP and CDHA, which could be related to the progress of OCP hydrolysis into CDHA. These results support the previous in vivo observation that OCP has the healing function of rabbit rotator cuff tendon in vivo.

Octacalcium Phosphate/Gelatin Composite (OCP/Gel) Enhances Bone Repair in a Critical-sized Transcortical Femoral Defect Rat Model.

BACKGROUND: Bone grafting is widely used to treat large bone defects. A porous composite of a bioactive octacalcium phosphate material with gelatin sponge (OCP/Gel) has been shown to biodegrade promptly and be replaced with new bone both in animal models of a membranous bone defect and a long bone defect. However, it is unclear whether OCP/Gel can regenerate bone in more severe bone defects, such as a critical-size transcortical defect. QUESTIONS/PURPOSES: Using an in vivo rat femur model of a standardized, transcortical, critical-size bone defect, we asked: Compared with a Gel control, does OCP/Gel result in more newly formed bone as determined by (1) micro-CT evaluation, (2) histologic and histomorphometric measures, and (3) osteocalcin staining and tartrate-resistant acid phosphatase staining? METHODS: Thirty-four 12-week-old male Sprague-Dawley rats (weight 356 ± 25.6 g) were used. Gel and OCP/Gel composites were prepared in our laboratory. Porous cylinders 3 mm in diameter and 4 mm in height were manufactured from both materials. The OCP/Gel and Gel cylinders were implanted into a 3-mm-diameter transcortical critical-size bone defect model in the left rat femur. The OCP/Gel and Gel were randomly assigned, and the cylinders were implanted. The biological responses of the defect regions were evaluated radiologically and histologically. At 4 and 8 weeks after implantation, CT evaluation, histological examination of decalcified samples, and immunostaining were quantitatively performed to evaluate new bone formation and remaining bone graft substitutes and activity of osteoblasts and osteoclast-like cells (n = 24). Qualitative histological evaluation was performed on undecalcified samples at 3 weeks postimplantation (n = 10). CT and decalcified tissue analysis was not performed blinded, but an analysis of undecalcified specimens was performed under blinded conditions. RESULTS: Radiologic analysis revealed that the OCP/Gel group showed radiopaque regions around the OCP granules and at the edge of the defect margin 4 weeks after implantation, suggesting that new bone formation occurred in two ways. In contrast, the rat femurs in the Gel group had a limited radiopaque zone at the edge of the defect region. The amount of new bone volume analyzed by micro-CT was higher in the OCP/Gel group than in the Gel group at 4 and 8 weeks after implantation (​​4 weeks after implantation: OCP/Gel versus Gel: 6.1 ± 1.6 mm 3 versus 3.4 ± 0.7 mm 3 , mean difference 2.7 [95% confidence interval (CI) 0.9 to 4.5]; p = 0.002; intraclass correlation coefficient [ICC] 0.72 [95% CI 0.29 to 0.91]; 8 weeks after implantation: OCP/Gel versus Gel: 3.9 ± 0.7 mm 3 versus 1.4 ± 1.1 mm 3 , mean difference 2.5 [95% CI 0.8 to 4.3]; p = 0.004; ICC 0.81 [95% CI 0.47 to 0.94]). Histologic evaluation also showed there was a higher percentage of new bone formation in the OCP/Gel group at 4 and 8 weeks after implantation (​​4 weeks after implantation: OCP/Gel versus Gel: 31.2% ± 5.3% versus 13.6% ± 4.0%, mean difference 17.6% [95% CI 14.2% to 29.2%]; p < 0.001; ICC 0.83 [95% CI 0.53 to 0.95]; 8 weeks after implantation: OCP/Gel versus Gel: 28.3% ± 6.2% versus 9.5% ± 1.9%, mean difference 18.8% [95% CI 11.3% to 26.3%]; p < 0.001; ICC 0.90 [95% CI 0.69 to 0.97]). Bridging of the defect area started earlier in the OCP/Gel group than in the Gel group at 4 weeks after implantation. Osteocalcin immunostaining showed that the number of mature osteoblasts was higher in the OCP/Gel group than in the Gel group at 4 weeks (OCP/Gel versus Gel: 42.1 ± 6.5/mm 2 versus 17.4 ± 5.4/mm 2 , mean difference 24.7 [95% CI 16.2 to 33.2]; p < 0.001; ICC 0.99 [95% CI 0.97 to 0.99]). At 4 weeks, the number of osteoclast-like cells was higher in the OCP/Gel composite group than in the Gel group (OCP/Gel versus Gel: 3.2 ± 0.6/mm 2 versus 0.9 ± 0.4/mm 2 , mean difference 2.3 [95% CI 1.3 to 3.5]; p < 0.001; ICC 0.79 [95% CI 0.35 to 0.94]). CONCLUSION: OCP/Gel composites induced early bone remodeling and cortical bone repair in less time than did the Gel control in a rat critical-size, transcortical femoral defect, suggesting that OCP/Gel could be used as a bone replacement material to treat severe bone defects. CLINICAL RELEVANCE: In a transcortical bone defect model of critical size in the rat femur, the OCP/Gel composite demonstrated successful bone regeneration. Several future studies are needed to evaluate the clinical application of this interesting bone graft substitute, including bone formation capacity in refractory fracture and spinal fusion models and the comparison of bone strength after repair with OCP/Gel composite to that of autologous bone.

Angio-osteogenic capacity of octacalcium phosphate co-precipitated with copper gluconate in rat calvaria critical-sized defect.

The objective of this study is to investigate the effects of octacalcium phosphate (OCP)-induced bone regeneration on angiogenesis regulated by the inclusion of copper ions in OCP in vitro and in vivo. Calcium (Ca)-deficient Cu-OCPs, containing 0.01 wt% Cu (low-Cu-OCP) and 0.12 wt% Cu (high-Cu-OCP), were synthesized with co7pper gluconate salt. The lattice parameters of Cu-OCPs tended to decrease slightly with Cu inclusion, as estimated by Rietveld analysis. Cu ions were released in OCP when the materials were incubated in the medium for human umbilical vein endothelial cells (HUVECs). The solubility of Cu-OCPs, estimated by the degree of supersaturation, was slightly higher than that of the original OCP. Cu-OCP tended to hydrolyze to an apatite structure while maintaining the crystal plate-like morphology when incubated with mesenchymal stem D1 cells in osteogenic media for 14 days. The specimens were characterized by selected area electron diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. Low-Cu-OCP significantly enhanced the HUVEC capillary cross-linking density. D1 cell differentiation was inhibited with the inclusion of Cu, even at low concentrations. The composite of low-Cu-OCP with a gelatin sponge (low-Cu-OCP/Gel) significantly enhanced angiogenesis coupled with bone regeneration when implanted in a rat calvarial critical-sized defect for 4 weeks, compared with the corresponding amount of Cu-containing Gel (Cu/Gel) or OCP/Gel materials through angiography and tissue histomorphometry. These results support the proposition that angiogenesis stimulated by low-Cu-OCP is closely related with enhanced bone regeneration.

Mutual chemical effect of autograft and octacalcium phosphate implantation on enhancing intramembranous bone regeneration.

This study examined the effect of a mixture of octacalcium phosphate (OCP) and autologous bone on bone regeneration in rat calvaria critical-sized defect (CSD). Mechanically mixed OCP and autologous bone granules (OCP+Auto), approximately 500 to 1000 µm in diameter, and each individual material were implanted in rat CSD for 8 weeks, and subjected to X-ray micro-computed tomography (micro-CT), histology, tartrate-resistant acid phosphatase (TRAP) staining, and histomorphometry for bone regeneration. Osteoblastic differentiation from mesenchymal stem cells (D1 cells) was examined in the presence of non-contacting materials by alkaline phosphatase (ALP) activity for 21 days. The material properties and medium composition before and after the incubation were determined by selected area electron diffraction (SAED) under transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), and chemical analysis. The results showed that while bone formation coupled with TRAP-positive osteoclastic resorption and cellular ALP activity were the highest in the Auto group, a positive effect per OCP weight or per autologous bone weight on ALP activity was found. Although the OCP structure was maintained even after the incubation (SAED), micro-deposits were grown on OCP surfaces (TEM). Fibrous tissue was also exposed on the autologous bone surfaces (SEM). Through FT-IR absorption, it was determined that bone mineral-like characteristics of the phosphate group increased in the OCP + Auto group. These findings were interpreted as a structural change from OCP to the apatitic phase, a conclusion supported by the medium degree of saturation changes. The results demonstrate the mutual chemical effect of mixing OCP with autologous bone as an active bone substitute material.

Macrophage Polarization Related to Crystal Phases of Calcium Phosphate Biomaterials.

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 (Ca2+) 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 (IL1ß)) 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.

Bone Tissue Response to Different Grown Crystal Batches of Octacalcium Phosphate in Rat Long Bone Intramedullary Canal Area.

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.

Biophysical and biochemical nature of amorphous protein oligomers determines the strength of immune response and the generation of T-cell memory.

Here, we used domain 3 of dengue virus serotype 3 envelope protein (D3ED3), a natively folded globular low-immunogenicity protein, to ask whether the biophysical nature of amorphous oligomers can affect immunogenicity. We prepared nearly identical 30 ~ 50 nm-sized amorphous oligomers in five distinct ways and looked at any correlation between their biophysical properties and immunogenicity. One oligomer type was produced using our SCP tag (solubility controlling peptide) made of 5 isoleucines (C5I). The others were prepared by miss-shuffling the SS bonds (Ms), heating (Ht), stirring (St) and freeze-thaw (FT). Dynamic light scattering showed that all five formulations contained oligomers of approximately identical sizes with hydrodynamic radii (Rh) between 30 and 55 nm. Circular dichroism (cd) indicated that the secondary structure content of oligomers formed by stirring and freeze-thaw was essentially identical to that of the native monomeric D3ED3. The secondary structure content of the Ms showed moderate changes, whereas the C5I and heat-induced (Ht) oligomers exhibited a significant change. The Ms contained D3ED3 with intermolecular SS bonds as assessed by nonreducing size exclusion chromatography (SEC). Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti-D3ED3 IgG titre. Ht, St and FT were only mildly immunogenic, similar to the monomeric D3ED3. Cell surface CD marker analysis by flow cytometry confirmed that immunization with Ms generated a strong central and effector T-cell memory. Our observations indeed suggest that controlled oligomerization can provide a new, adjuvant-free method for increasing a protein's immunogenicity, yielding a potentially powerful platform for protein-based (subunit) vaccines.

Osteogenic capacity of octacalcium phosphate involving macrophage polarization.

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.

Differentiation of committed osteoblast progenitors by octacalcium phosphate compared to calcium-deficient hydroxyapatite in Lepr-cre/Tomato mouse tibia.

This study aimed to investigate the accumulation and differentiation of mesenchymal stem cells (MSCs) around octacalcium phosphate (OCP) compared with those around calcium-deficient hydroxyapatite (CDHA), a material obtained through hydrolysis of the original OCP. Leptin receptor (Lepr)-expressing bone marrow-derived MSCs around the OCP and CDHA were pursued utilizing genetically modified Lepr-cre/Tomato mice. OCP and CDHA granules were implanted into the tibia defect of the mice for 10 weeks and subjected to histomorphometric and immunohistochemical analyses. The structural properties of OCP and CDHA after inoculation into mouse subcutaneous tissue (until 4 weeks) or culture mediums (14 days) were analyzed using physicochemical techniques. In vitro osteoblastic differentiation of primary MSCs was examined with the materials for 14 days. While Lepr-cre/Tomato positive cells (red) accumulated around both OCP and CDHA, Lepr and osteocalcin double-positive osteoblastic cells (yellow) were significantly more abundant around OCP than around CDHA in the early implantation period. OCP enhanced the osteoblastic differentiation of MSCs more than CDHA in vitro. Physicochemical and structual analyses provided evidence that OCP tended to convert to the apatitic phase in the tested physiological environments. The higher osteoconductivity of OCP originated from a capacity-enhancing osteoblastic differentiation of committed osteoblast progenitors in bone marrow accompanied by OCP hydrolysis. STATEMENT OF SIGNIFICANCE: MSCs play a key role in bone regeneration through osteoblastic differentiation. Calcium phosphates have been widely applied as bone substitute materials, and OCP has a better ability to promote osteoblast differentiation of MSCs than that of HA in vitro. However, it is not clear how MSCs accumulate in the bone marrow and differentiate into osteoblasts during bone regeneration in vivo. In this study, we focused on the leptin receptor, a marker of bone marrow-derived MSCs. Using genetically modified mice labeled with the red fluorescent protein Tomato, we observed the accumulation of MSCs around calcium phosphates implanted in tibia bone defects and their differentiation into osteoblasts.

Proteomic identification of serum proteins to induce osteoconductivity of hydroxyapatite.

We performed proteomic analysis of rat serum proteins adsorbed on hydroxyapatite (HAp) and α-alumina (α-Al2O3) in order to identify proteins that specifically adsorb onto HAp and control cellular responses. Proteins with either or both molecular weight of 22-32 kDa and computed isoelectric point of 5.0-5.5 were preferentially adsorbed on HAp. In total, 182 proteins were adsorbed on both HAp and α-Al2O3, of which 14 were highly enriched on HAp, whereas 68 were adsorbed only on HAp. Therefore, 82 (14+68) proteins were further evaluated by bioinformatics and literature-based analyses. We predicted that hepatocyte growth factor and angiopoietin-like protein 3 (ANGPTL3) are candidate proteins responsible for the osteoconductivity of HAp. Although ANGPTL3 promoted the attachment and spreading of MC3T3-E1 cells, it did not promote their proliferation and differentiation. Our results suggest that specific adsorption of ANGPTL3 on HAp induced osteoconductivity by enhancing the attachment and spreading of osteoblasts.

Involvement of distant octacalcium phosphate scaffolds in enhancing early differentiation of osteocytes during bone regeneration.

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).

Impact of simultaneous hydrolysis of OCP and PLGA on bone induction of a PLGA-OCP composite scaffold in a rat femoral defect.

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.

Octacalcium phosphate bone substitute materials: Comparison between properties of biomaterials and other calcium phosphate materials.

Octacalcium phosphate (OCP) is a material that can be converted to hydroxyapatite (HA) under physiological environments and is considered a mineral precursor to bone apatite crystals. The structure of OCP consists of apatite layers stacked alternately with hydrated layers, and closely resembles the structure of HA. The performance of OCP as a bone substitute differs from that of HA materials in terms of their osteoconductivity and biodegradability. OCP manifests a cellular phagocytic response through osteoclast-like cells similar to that exhibited by the biodegradable material ß-tricalcium phosphate (ß-TCP). The use of OCP for human cranial bone defects involves using its granule or composite form with one of the natural polymers, viz., the reconstituted collagen. This review article discusses the differences and similarities in these calcium phosphate (Ca-P)-based materials from the viewpoint of the structure and their material chemistry, and attempts to elucidate why Ca-P materials, particularly OCP, display unique osteoconductive property.

Chemical Stability-Sensitive Osteoconductive Performance of Octacalcium Phosphate Bone Substitute in an Ovariectomized Rat Tibia Defect.

The purpose of this study was to investigate whether the chemical condition of osteoporotic serum affects the chemical stability of octacalcium phosphate (OCP) and its osteoconductive property. The in vitro chemical dissolution in osteoporotic ovariectomized (OVX)-simulated conditions was analyzed. OCP and its composite form with gelatin (OCP/Gel), containing specific amounts of OCP (either 17% or 44% by weight), were used as experimental materials. The degrees of supersaturation (DS) of the OVX-simulated buffer solutions, containing distinct inorganic phosphate (Pi) ion concentrations, after immersing OCP or OCP/Gel, were determined. The rod-shaped OCP/Gel was then implanted into the OVX and Sham rat tibia defects, exhibiting a similar shape and size, and assessed at 4, 8, and 12 weeks. Increasing Pi concentration in OVX-simulated buffer solution increased the DS, with respect to OCP, upon the introduction of OCP and 44% OCP/Gel, but decreased the DS to a slightly saturated condition with 17% OCP/Gel, indicating that increasing the OCP in the Gel matrix tends to inhibit the hydrolysis of OCP into hydroxyapatite (HA). Histomorphometric analyses of bone formation and the appearance of osteoblasts and osteoclast-like cells, together with OCP resorption, confirmed that while 44% OCP/Gel showed higher bone formation than 17% OCP/Gel at intramedullary bone defect sites in Sham rat tibia, both OCP/Gels tended to enhance cortical bone formation in the OVX group, concomitant with the higher resorption of OCP within 17% OCP/Gel. The appearance of osteoclast-like cells in the OVX group increased as the OCP dose decreased from 44% to 17% in the Gel matrix, with an approximately 4 times higher bone formation rate, 8-12 weeks after the implantation. Additional in vitro assays showed that bone marrow mesenchymal stem cells isolated from OVX and wild-type (WT) rats treated with OCP had similar proliferation and differentiation rates, up to 21 days. These results show that OCP can enhance cortical bone repair even in osteoporotic bone if suitable thermodynamic metastable dissolution conditions are provided in relation to the mass of OCP.

Effect of calcium phosphate phases affecting the crosstalk between osteoblasts and osteoclasts in vitro.

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 ß-tricalcium phosphate (ß-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 ß-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 ß-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 ß-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.

Angiogenesis involvement by octacalcium phosphate-gelatin composite-driven bone regeneration in rat calvaria critical-sized defect.

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.

Culture of hybrid spheroids composed of calcium phosphate materials and mesenchymal stem cells on an oxygen-permeable culture device to predict in vivo bone forming capability.

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), ß-tricalcium phosphate (ß-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 ≥ ß-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.

Osteogenic cellular activity around onlaid octacalcium phosphate-gelatin composite onto rat calvaria.

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.

Capacity of octacalcium phosphate to promote osteoblastic differentiation toward osteocytes in vitro.

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 ß-tricalcium phosphate (ß-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 ß-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 ß-tricalcium phosphate (ß-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.

Surface reactivity of octacalcium phosphate-derived fluoride-containing apatite in the presence of polyols and fluoride.

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.