The material design of octacalcium phosphate bone substitute: increased dissolution and osteogenecity.

Octacalcium phosphate (OCP) has been advocated as a precursor of bone apatite crystals. Recent studies have shown that synthetic OCP exhibits highly osteoconductive properties as a bone substitute material that stems from its ability to activate bone tissue-related cells, such as osteoblasts, osteocytes, and osteoclasts. Accumulated experimental evidence supports the proposition that the OCP-apatite phase conversion under physiological conditions increases the stimulatory capacity of OCP. The conversion of OCP progresses by hydrolysis toward Ca-deficient hydroxyapatite with Ca2+ ion incorporation and inorganic phosphate ion release with concomitant increases in the solid Ca/P molar ratio, specific surface area, and serum protein adsorption affinity. The ionic dissolution rate during the hydrolysis reaction was controlled by introducing a high-density edge dislocation within the OCP lattice by preparing it through co-precipitation with gelatin. The enhanced dissolution intensifies the material biodegradation rate and degree of osteogenecity of OCP. Controlling the biodegradation rate relative to the dissolution acceleration may be vital for controlling the osteogenecity of OCP materials. This study investigates the effects of the ionic dissolution of OCP, focusing on the structural defects in OCP, as the enhanced metastability of the OCP phase modulates biodegradability followed by new bone formation. STATEMENT OF SIGNIFICANCE: Octacalcium phosphate (OCP) is recognized as a highly osteoconductive material that is biodegradable by osteoclastic resorption, followed by new bone formation by osteoblasts. However, if the degradation rate of OCP is increased by maintaining the original osteoconductivity or acquiring a bioactivity better than its current properties, then early replacement with new bone can be expected. Although cell introduction or growth factor addition by scaffold materials is the standard method for tissue engineering, material activity can be augmented by introducing dislocations into the lattice of the OCP. This review article summarizes the effects of introducing structural defects on activating OCP, which was obtained by co-precipitation with gelatin, as a bone substitute material and the mechanism of improved bone replacement performance.

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.

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.

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).

Comparative analysis of bovine serum albumin adsorption onto octacalcium phosphate crystals prepared using different methods.

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.

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.

Comparative study on the resorbability and dissolution behavior of octacalcium phosphate, ß-tricalcium phosphate, and hydroxyapatite under physiological conditions.

The dissolution behaviors of octacalcium phosphate (OCP), ß-tricalcium phosphate (ß-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 ß-TCP and HA remained unchanged after the implantation. The DS of the SBF solution remained slightly supersaturated with respect to OCP and ß-TCP, but slightly undersaturated in the Tris-HCl buffer. These findings suggest that previously reported OCP and ß-TCP biodegradation could be induced through cell-mediated osteoclastic resorption rather than a simple dissolution process.