|89th Annual Meeting Abstracts
Thrombin Biomatrix and HUVEC/HSC Enhance Vascularization of a Large PCL-β TCP Bone Construct Implanted in Nude Mice
David Hindin, B.A.1, Michael Stosich, D.M.D., M.S.2, Imad Salhab, M.S.2, Byung-Ho Yang, D.D.S., Ph.D.2, Joseph M. Serletti, M.D.1, Hyun-Duck Nah, D.M.D., Ph.D.1.
1The University of Pennsylvania School of Medicine; Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA, USA, 2Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Purpose - In the scaffold-guided repair of large, voluminous bony defects for craniofacial reconstruction, the creation of vascular networks is critical to the success of subsequent bone formation within the scaffold. Thrombin has been suggested to play a role in the initiation, regulation, and organization of angiogenic molecules. In this study, we assessed the angiogenic effect of thrombin by determining its ability to induce VEGF expression in human umbilical vein-derived endothelial cells (HUVEC) and osteogenic mesenchymal stem cells (MSCs). We then asked whether co-implantation of HUVECs or hematopoietic stem cells (HSCs) with osteogenic MSCs would enhance the in vivo formation of microvascular networks in a large bone construct.
Methods - In vitro studies were performed by exposing HUVECs and MSCs in culture to various concentrations of thrombin (0, 0.5, 1 and 2 U/mL). VEGF gene expression was assessed using RT-qPCR. For in vivo studies, a hybrid scaffold of TCP/PCL (30%TCP:70% PCL; 6x6x6 mm; 65% porosity) with interconnected open pores was 3-D printed to serve as an osteoinductive matrix. Groups (N=4) consisted of scaffold samples seeded with MSCs, MSCs-HUVECs, MSCs-HSCs, and no cells. To promote vasculogenesis, cells were suspended in thrombin and seeded into the microporous constructs, sealed with fibrinogen, and subcutaneously implanted into immunodeficient athymic nude mice. At 8 weeks, mouse blood vessels were perfused with radiopaque microfil. The scaffolds were harvested, demineralized and analyzed for neovasculature by microcomputed tomography (micro-CT).
Results - Thrombin increased VEGF gene expression by several-fold in HUVECs (Fig. 1). This effect was specific for HUVECs and was not observed in MSCs. Implants harvested from nude mice demonstrated the formation of substantial amounts of microvascular beds in the MSC-HUVEC constructs as illustrated by micro-CT and vascular volumetric plotting; MSC, MSC-HSC, and cell-free constructs demonstrated markedly less neovasculature (Fig. 2). Volumetric analysis showed that MSC-HUVEC constructs contained 250% more microvasculature than cell-free constructs, and 180% more microvasculature than MSC or MSC-HSC constructs. Additionally, neovascularization in the cell-free construct was limited to the surface of the construct.
Conclusion - The findings from this study demonstrate that the use of a thrombin biomatrix and the addition of HUVEC cells drastically enhanced microvascular network formation in large bone constructs. Thrombin induced upregulation of VEGF in HUVECs, suggesting an explanation for its angiogenic influences when utilized within the scaffold. The development of neovascularization from noninvasive sources constitutes an important step in the advancement of clinical strategies for tissue vascularization of large craniofacial defects.