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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. Research has shown that a combination of osteogenic and angiogenic cells, such as human umbilical vein-derived endothelial cells and bone marrow mesenchymal stem cells, are critical to stable scaffold microvasculature formation. In order to ultimately translate tissue-engineered microvasculature to clinical use however, technical challenges such as immune matching have created an impetus to achieve microvasculature formation from a single cell source. In this study we evaluated the potential of a single cell type, umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), to undergo both osteogenic and angiogenic differentiation. We then asked whether implantation of UCB-MSCs in a large bone construct would lead to in vivo microvascular network formation in nude mice.
METHODS: In vitro studies were performed by treating UCB-MSCs with either osteogenic or angiogenic conditions. Osteogenic conditions were established by supplementing media with 100nM dexamethasone, 0.05mM ascorbic acid, and 10ng/mL fibroblast growth factor (FGF). Angiogenic conditions consisted of media supplemented with 10ng/mL FGF and 100ng/mL vascular endothelial growth factor (VEGF). Osteogenic differentiation was assessed by evaluating alkaline phosphatase staining after five days. Angiogenic differentiation was assessed using an in vitro Matrigel angiogenesis assay. For in vivo studies, a hybrid scaffold of TCP/PCL (30%TCP:70% PCL; 6x6x6 mm; 65% porosity) with interconnected pores was 3-D printed to serve as an osteoinductive matrix. Groups (N=4) consisted of scaffolds seeded with angiogenic-treated UCB-MSCs, angiogenic-treated and osteogenic-treated UCB-MSCs, and no cells. At 4 weeks, scaffolds were harvested, demineralized, and prepared for histological analysis.
RESULTS: UCB-MSCs treated with osteogenic conditions in vitro demonstrated increased alkaline phosphatase staining compared with non-treated UCB-MSCs (Fig. 1a-b). In addition, UCB-MSCs that underwent in vitro angiogenic treatment demonstrated significant capillary tube formation on Matrigel, a finding not seen with untreated UCB-MSCs (Fig. 1c-d). Histological analysis of implants harvested from nude mice demonstrated the formation of substantial amounts of microvascular beds in the scaffold seeded with angiogenic UCB-MSCs (Fig. 2c) and the scaffolds seeded with angiogenic and osteogenic UCB-MSCs (Fig. 2d), while cell-free scaffolds showed no microvasculature (Fig. 2b).
CONCLUSIONS: The formation of microvasculature represents a crucial element necessary for the ultimate success of any scaffold-guided approach to bone tissue regeneration. The findings in this study demonstrate that UCB-MSCs can be readily differentiated into both osteogenic and angiogenic cell types that are capable of inducing microvascular formation in large bone constructs. This represents an important step in the advancement of clinical strategies for tissue vascularization of large craniofacial defects.