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Octacalcium phosphate (OCP) is resorbable bone regenerative material, but its brittleness makes it difficult to maintain its shape without restraint. We have engineered a scaffold constructed of synthetic OCP and porcine collagen sponge (OCP/Collagen) and investigated whether OCP/Collagen composite could improve bone regeneration. To examine this hypothesis, bone regeneration by the implantation of OCP/Collagen was compared with those by OCP and collagen. Radiographic and histological examination was performed and the percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. OCP/Collagen, OCP, or collagen was implanted into the critical-sized defects in rat crania and fixed at 2, 4, or 8 weeks after implantation. OCP/Collagen improved the handling performance than the granules of OCP, and synergistically enhanced the bone regeneration beyond expectation, which were composed of bone nucleation by OCP and cell infiltration by collagen. Histomorphometrical analysis showed that n-Bone% +/- standard error treated with OCP/Collagen (48.4 +/- 5.14) was significantly higher than those with OCP (27.6 +/- 4.04) or collagen (27.4 +/- 5.69) in week 8. The present study suggests that the combination OCP with collagen elicited the synergistic effect for bone regeneration.

Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N-acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization.

Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering.