Engineering a biomimetic periosteum on cortical bone allografts for the reconstruction of critical-sized bone defects in mice

Abstract

Load bearing bone allografts suffer from clinical failure due to low allograft-host tissue integration. Removal of the periosteum, a thin tissue layer on bone with a high regenerative capacity, is responsible for bone allografts' decreased clinical performance. This interdisciplinary project addressed this problem by creating multiple engineered periostea on mice bone allografts. Using a polysaccharide biomaterial, chitosan, tissue engineering scaffolds constructed on these bone allografts were modified with the glycosaminoglycan, heparin, and a chitosan derivative, trimethyl chitosan, to create periostea with different scaffold morphologies yet similar surface chemistries. Cell instructive cues such as growth factors fibroblast growth factor-2 (FGF-2) and transforming growth factor-β1 (TGF-β1) were adsorbed onto the engineered periostea and found to release up to 14 and 7 days in-vitro, respectively. Engineered allografts were found to support adipose-derived stem cell (ASC) adhesion and proliferation. FGF-2 and TGF-β1 delivered from the engineered allografts to ASC supported an osteoprogenitor phenotype in ASC and did not inhibit alkaline phosphatase and receptor activator of nuclear factor-kappaB (RANKL) protein expression. From in vitro results, the nanofiber engineered periosteum was found to be the most cytocompatible scaffold and was selected for subsequent implantation in a pre-clinical mouse critical-sized femoral defect model. We assessed the engineered periosteum's efficacy on modulating allograft healing and incorporation. We confirmed the engineered allografts successfully delivered ASC, FGF-2, and TGF-β1 to the femur defect and found ASC persisted in the femur defect for at least 7 days, similar to other reports in the literature. At week 6, microcomputed tomography results of excised femurs showed no statistical difference in new bone volume formation between experimental groups. However, treatment groups containing ASC showed a trend of at least 24% more bone volume compared to their respective cell-free controls suggesting possible therapeutic effects of ASC. Union ratio and histological analysis both confirmed the nanofiber engineered periosteum did not degrade at 6 weeks and inhibited allograft incorporation. Subsequent relative gene expression experiments showed that ASC maintained an undifferentiated phenotype in response to FGF-2 and TGF-β1 delivered from chitosan nanofibers. Overall, this project developed a novel polysaccharide-based engineered periosteum for delivering growth factors and progenitor cells to a bone defect for regenerative medicine applications.