Engineering biomimetic periosteum with β-TCP scaffolds to promote bone formation in calvarial defects of rats

Background: There is a critical need for the management of large bone defects. The purpose of this study was to engineer a biomimetic periosteum and to combine this with a macroporous beta-tricalcium phosphate (beta-TCP) scaffold for bone tissue regeneration. Methods: Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were harvested and cultured in different culture media to form undifferentiated rBMSC sheets (undifferentiated medium (UM)) and osteogenic cell sheets (osteogenic medium (OM)). Simultaneously, rBMSCs were differentiated to induced endothelial-like cells (iECs), and the iECs were further cultured on a UM to form a vascularized cell sheet. At the same time, flow cytometry was used to detect the conversion rates of rBMSCs to iECs. The pre-vascularized cell sheet (iECs/UM) and the osteogenic cell sheet (OM) were stacked together to form a biomimetic periosteum with two distinct layers, which mimicked the fibrous layer and cambium layer of native periosteum. The biomimetic periostea were wrapped onto porous beta-TCP scaffolds (BP/beta-TCP) and implanted in the calvarial bone defects of rats. As controls, autologous periostea with beta-TCP (AP/beta-TCP) and beta-TCP alone were implanted in the calvarial defects of rats, with a no implantation group as another control. At 2, 4, and 8 weeks post-surgery, implants were retrieved and X-ray, microcomputed tomography (micro-CT), histology, and immunohistochemistry staining analyses were performed. Results: Flow cytometry results showed that rBMSCs were partially differentiated into iECs with a 35.1% conversion rate in terms of CD31. There were still 20.97% rBMSCs expressing CD90. Scanning electron microscopy (SEM) results indicated that cells from the wrapped cell sheet on the beta-TCP scaffold apparently migrated into the pores of the beta-TCP scaffold. The histology and immunohistochemistry staining results from in vivo implantation indicated that the BP/beta-TCP and AP/beta-TCP groups promoted the formation of blood vessels and new bone tissues in the bone defects more than the other two control groups. In addition, micro-CT showed that more new bone tissue formed in the BP/beta-TCP and AP/beta-TCP groups than the other groups. Conclusions: Inducing rBMSCs to iECs could be a good strategy to obtain an endothelial cell source for prevascularization. Our findings indicate that the biomimetic periosteum with porous beta-TCP scaffold has a similar ability to promote osteogenesis and angiogenesis in vivo compared to the autologous periosteum. This function could result from the double layers of biomimetic periosteum. The pre-vascularized cell sheet served a mimetic fibrous layer and the osteogenic cell sheet served a cambium layer of native periosteum. The biomimetic periosteum with a porous ceramic scaffold provides a new promising method for bone healing.