Mechanical properties and effect on osteodifferentiation of induced pluripotent stem cells of chitosan/whisker/calcium phosphate cement composite biomaterial

Abstract

To investigate the mechanical properties of the novel compound calcium phosphate cement (CPC) biological material as well as the biological activity and osteogenesis effects of induced pluripotent stem cells (iPS) seeding on scaffold and compare their bone regeneration efficacy in cranial defects in rats. Ac- cording to the different scaffold materials, the experiment was divided into 4 groups: pure CPC scaffold group (group A), CPC∶10% wt chitosan as 2∶1 ratio mixed scaffold group (group B), CPC∶10% wt chitosan∶whisker as 2∶1∶1 ratio mixed scaffold group (group C), and CPC∶10% wt chitosan∶whisker as 2∶1∶2 ratio mixed scaffold group (group D). Mechanical properties (bending strength, work-of-fracture, hardness, and modulus of elasticity) of each scaffold were detected. The scaffolds were cultured with fifth generation iPS-mesenchymal stem cells (MSCs), and the absorbance ( A) values of each group were detected at 1, 3, 7, and 14 days by cell counting kit 8 (CCK-8) method; the alkaline phosphatase (ALP) activity, Live/Dead fluorescence staining and quantitative detection, ALP, Runx2, collagen typeⅠ, osteocalcin (OC), and bone morphogenetic protein 2 (BMP-2) gene expressions by RT-PCR were detected at 1, 7, and 14 days; and the alizarin red staining were detected at 1, 7, 14, and 21 days. Twenty-four 3-month-old male Sprague Dawley rats were used to establish the 8 mm-long skull bone defect model, and were randomly divided into 4 groups ( n=6); 4 kinds of scaffold materials were implanted respectively. After 8 weeks, HE staining was used to observe the repair of bone defects and to detect the percentage of new bone volume and the density of neovascularization. The bending strength, work-of-fracture, hardness, and modulus of elasticity in groups B, C, and D were significantly higher than those in group A, and in groups C, D than in group B, and in group D than in group C ( P<0.05). CCK-8 assay showed that cell activity gradually increased with the increase of culture time, the A values in groups B, C, and D at 3, 7, 14 days were signifiantly higher than those in group A, and in groups C, D than in group B ( P<0.05), but no significant difference was found between groups C and D ( P>0.05). Live/Dead fluorescence staining showed that the proportion of living cells in groups B, C, and D at 7 and 14 days was significantly higher than that in group A ( P<0.05), and in groups C, D at 7 days than in group B ( P<0.05); but no significant difference was found between groups C and D ( P>0.05). RT-PCR showed that the relative expressions of genes in groups B, C, and D at 7 and 14 days were significantly higher than those in group A, and in groups C, D than in group B ( P<0.05); but no significant difference was found between groups C and D ( P>0.05). Alizarin red staining showed that the red calcium deposition on the surface of scaffolds gradually deepened and thickened with the prolongation of culture time; the A values in groups B, C, and D at 14 and 21 days were significantly higher than those in group A ( P<0.05), and in groups C and D than in group B ( P<0.05), but no significant difference was found between groups C and D ( P>0.05). In vivo repair experiments in animals showed that the new bone in each group was mainly filled with the space of scaffold material. Osteoblasts and neovascularization were surrounded by new bone tissue in the matrix, and osteoblasts were arranged on the new bone boundary. The new bone in groups B, C, and D increased significantly when compared with group A, and the new bone in groups C and D was significantly higher than that in group B. The percentage of new bone volume and the density of neovascularization in groups B, C, and D were significantly higher than those in group A, and in groups C and D than in group B ( P<0.05); but no significant difference was found between groups C and D ( P>0.05). The mechanical properties of the new reinforced composite scaffold made from composite chitosan, whisker, and CPC are obviously better than that of pure CPC scaffold material, which can meet the mechanical properties of cortical bone and cancellous bone. iPS-MSCs is attaching and proliferating on the new reinforced composite scaffold material, and the repair effect of bone tissue is good. It can meet the biological and osteogenic activity requirements of the implant materials in the bone defect repair.