Engineering Vascularized Bone Grafts by Integrating a Biomimetic Periosteum and β-TCP Scaffold

[Image: see text] Treatment of large bone defects using synthetic scaffolds remain a challenge mainly due to insufficient vascularization. This study is to engineer a vascularized bone graft by integrating a vascularized biomimetic cell-sheet-engineered periosteum (CSEP) and a biodegradable macroporous beta-tricalcium phosphate (β-TCP) scaffold. We first cultured human mesenchymal stem cells (hMSCs) to form cell sheet and human umbilical vascular endothelial cells (HUVECs) were then seeded on the undifferentiated hMSCs sheet to form vascularized cell sheet for mimicking the fibrous layer of native periosteum. A mineralized hMSCs sheet was cultured to mimic the cambium layer of native periosteum. This mineralized hMSCs sheet was first wrapped onto a cylindrical β-TCP scaffold followed by wrapping the vascularized HUVEC/hMSC sheet, thus generating a biomimetic CSEP on the β-TCP scaffold. A nonperiosteum structural cell sheets-covered β-TCP and plain β-TCP were used as controls. In vitro studies indicate that the undifferentiated hMSCs sheet facilitated HUVECs to form rich capillary-like networks. In vivo studies indicate that the biomimetic CSEP enhanced angiogenesis and functional anastomosis between the in vitro preformed human capillary networks and the mouse host vasculature. MicroCT analysis and osteocalcin staining show that the biomimetic CSEP/β-TCP graft formed more bone matrix compared to the other groups. These results suggest that the CSEP that mimics the cellular components and spatial configuration of periosteum plays a critical role in vascularization and osteogenesis. Our studies suggest that a biomimetic periosteum-covered β-TCP graft is a promising approach for bone regeneration.