Introduction: Octacalcium phosphate (OCP) has been shown to exhibit higher osteoconductivity than other calcium phosphate materials experimentally[1] and recently been applied to filling human maxillary bone defect with the combination by collagen[2]. The osteoconductivity of OCP is controlled by the advancement of hydrolysis of OCP to hydroxyapatite (HA)[3]-[5]. OCP/gelatin composite obtained from the co-precipitation of OCP with gelatin molecules raises the osteoconductivity of this material in rat calvaira defect[6]. The co-presence of gelatin molecules affects the hydrolysis rate of OCP and the crystal morphology in the water[7]. Thus, OCP-apatite conversion may be of critical importance to display the osteoconductivity of this material[3],[5]. In the present study, the structural characteristics of OCP and its hydrolyzates were investigated in the presence of gelatin using physical techniques, such as Raman spectroscopy. The bioactivity of OCP/gelatin composite including co-precipitated OCP with gelatin was also studied.
Materials and Methods: OCP was synthesized by a wet method previously established[1] in the absence or the presence of gelatin up to 7.0%. The precipitates recovered were further hydrolyzed at 65 oC up to 48 hours. X-ray diffraction (XRD) analysis, Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy and scanning electron microscopy (SEM) were carried out to characterize the crystals obtained. The crystallite size in OCP (100) plane was determined from the OCP primary peak in XRD using the Scherrer equation. The collagen orientation as an index of the quality of regenerated bone in critical-sized rat calvaria defect was examined by the picrosirius red staining of the decalcified tissue sections at 8 weeks after the implantation of OCP/gelatin sponge disks (9 mmφ and 1mmh) containing the co-precipitated OCP crystals with gelatin. All procedures were approved by the Institutional Animal Research Committee.
Results: The structure of OCP was preserved even in the preparation with higher concentration of gelatin by XRD and FTIR. Raman bands derived from ν1PO4 stretch around 970 cm-1 of OCP were recognized in these preparations. The crystallite size and the aspect ratio of OCP crystals were regulated with gelatin. The hydrolysis induced an intensity change of ν1HPO4 stretch around 1010 cm-1 to 1100 to cm-1 in Raman, the coalescence of the splitting two ν3 P–O bands around 1030 cm-1 and 1110 cm-1 in FTIR, and marked reduction of the (100) peak in XRD. The quantitative analysis by the picrosirius red staining of the regenerated bone in rat calvaria defect indicated that the collagen orientation was recognized by the inclusion of the co-precipitated OCP.
Discussion and Conclusion: The previous study reported that the distinct morphology of OCP, obtained in the controlled reaction in the absence of gelatin molecules, markedly affects osteoblastic attachment in vitro and bone regeneration in mouse calvaria defect[8]. The present results show that the crystal growth of OCP is influenced by the presence of the gelatin. The results suggest that the osteoconductivity of OCP/gelatin containing the co-precipitated OCP could be stemmed from the OCP characteristics modified by the interaction between the precipitating OCP crystal surfaces and the gelatin molecules in the solution.
This study was supported in part by Grants-in-aid (23106010, 23390450 and 25670829) from the Ministry of Education, Science, Sports and Culture of Japan (MEXT).
References:
[1] Suzuki O. et al., Tohoku J Exp Med 164: 37-50, 1991
[2] Kawai T. et al., Tissue Eng Part A 20: 1336-1341, 2014
[3] Suzuki O. et al., Biomaterials 27: 2671-2681, 2006
[4] Kobayashi K et al., ACS Appl Mater Interfaces 6: 22602-22611, 2014
[5] Miyatake N et al. Biomaterials 30:1005-14, 2009
[6] Handa T et al. Acta Biomater 8:1190-1200, 2012
[7] Ezoe Y et al. J Nanopart Res 17:127, 2015
[8] Honda Y et al. Tissue Eng Part A 15:1965-1973, 2009