Addition of a collagen mimetic peptide to octacalcium phosphate/gelatin composites enhances bone formation by accelerating calcification

Octacalcium phosphate/gelatin (OCP/Gel) composites exhibit osteogenicity primarily due to OCP bioactivity; however, further improvement of their capacity is necessary for various bone defects. This study was conducted to investigate whether the addition of a collagen-mimetic peptide (CMP; (Gly-Pro-Hyp)₁₀), which forms a triple helix at physiological temperature, further enhances the osteogenic potential of OCP/Gel. Following the synthesis of OCP, spongy Gel and 44 wt% OCP-containing Gel were prepared from 3 w/v% gelatin solution with or without 0.1 w/v% CMP: 3%Gel, 3%Gel/CMP, OCP/3%Gel, and OCP/3%Gel/CMP. The materials were characterized physicochemically and evaluated in vitro and in vivo. Raman spectroscopy confirmed that the CMP-triple helix was maintained in OCP/Gel sponges. 3D hybrid spheroid culture of mesenchymal stromal cells (MSCs) with the materials showed that OCP/3%Gel/CMP and OCP/3%Gel increased alkaline phosphatase activities by day 7 compared to OCP alone. CMP inclusion in OCP/3%Gel altered gene expression for cytoskeletal organization and immunomodulation in MSCs, as assessed by transcriptomic and PCR analyses of extracted mRNA. Histomorphometry and transmission electron microscopy using non-decalcified sections revealed that the calcified matrix formation area was maximized in OCP/3%Gel/CMP (40.0%), followed by OCP/3%Gel (32.7%) and OCP (21.9%), and that osteoblasts were closely associated with crystal formation within the calcified deposits nucleated in the 3%Gel/CMP matrix during OCP hydrolysis. OCP/3%Gel/CMP enhanced both osteoblasts and osteoclasts accumulation and bone formation more than OCP/3%Gel at 4 weeks after implantation in the rat transcortical femoral defects. The results suggest that CMP-triple helix inclusion may induce Gel substrate calcification, thereby enhancing osteogenesis by the OCP/Gel composite. STATEMENT OF SIGNIFICANCE: A composite of octacalcium phosphate and gelatin (OCP/Gel) was the focus of this study highlighting the effects of a collagen mimetic peptide (CMP, (Gly-Pro-Hyp)10) addition. Gelatin adopts a random-coil structure at physiological temperatures but forms a triple-helix-like structure during cooling gelation of OCP/Gel preparation. Although this structure may be preserved through dehydrothermal cross-linking, it remains less robust than that of native collagen. Using a CMP maintaining a triple-helix structure at physiological temperatures, how this CMP addition affects the gelatin biocompatibility and enhances the osteogenecity of OCP/Gel was studied. Unexpectedly, adding this specific CMP to OCP/Gel promoted not only gelatin matrix calcification but also provided a nucleation site for osteoblastic mineralization in Gel, thereby accelerating overall bone formation by OCP/Gel.