Inadequate vascularization of in vitro-engineered tissue constructs after implantation is a major problem in most tissue-engineering applications. In this study we evaluated whether adipose tissue-derived stromal cells (ASCs), similar to bone marrow-derived stromal cells (BMSCs), can support the organization of endothelial cells into prevascular-like structures using an in vitro model. In addition, we investigated the mechanisms leading to the support of endothelial organization by these cells. We cultured human umbilical vein endothelial cells (HUVECs), ASCs, and BMSCs either alone or in combination in fibrin-embedded spheroids for 14 days. We found that BMSCs and ASCs formed cellular networks that expressed alpha smooth muscle actin and, in the case of ASCs, also CD34. Further, BMSCs and ASCs secreted hepatocyte growth factor and tissue inhibitor of metalloproteinase 1 and 2. In addition, ASC-conditioned medium induced HUVEC outgrowth, whereas BMSC-conditioned medium and hepatocyte growth factor-supplemented medium did not. Finally, both BMSCs and ASCs supported HUVEC organization into prevascular-like structures when cocultured. Our results suggest that both BMSCs and ASCs can support the formation of prevascular-like structures in vitro. Further, our findings indicate that cell-cell contacts and reciprocal signaling play an important role in the formation of these prevascular structures.

Vascularization is still one of the most important limitations for the survival of engineered tissues after implantation. In this study, we aim to improve the in vivo vascularization of engineered adipose tissue by preforming vascular structures within in vitro-engineered adipose tissue constructs that can integrate with the host vascular system upon implantation. Different cell culture media were tested and different amounts of human adipose tissue-derived mesenchymal stromal cells (ASC) and human umbilical vein endothelial cells (HUVEC) were combined in spheroid cocultures to obtain optimal conditions for the generation of prevascularized adipose tissue constructs. Immunohistochemistry revealed that prevascular structures were formed in the constructs only when 20% ASC and 80% HUVEC were combined and cultured in a 1:1 mixture of endothelial cell medium and adipogenic medium. Moreover, the ASC in these constructs accumulated lipid and expressed the adipocyte-specific gene fatty acid binding protein-4. Implantation of prevascularized ASC/HUVEC constructs in nude mice resulted in a significantly higher amount of vessels (37 ± 17 vessels/mm 2 ) within the constructs compared to non-prevascularized constructs composed only of ASC (3 ± 4 vessels/mm 2 ). Moreover, a subset of the preformed human vascular structures (3.6 ± 4.2 structures/mm 2 ) anastomosed with the mouse vasculature as indicated by the presence of intravascular red blood cells. Our results indicate that preformed vascular structures within in vitro-engineered adipose tissue constructs can integrate with the host vascular system and improve the vascularization upon implantation.

In vitro adipogenic differentiation of hASCs improves their ability to support endothelial viable cell numbers and suggests that hASCs differentiated for a short period potentially improve angiogenic responses for in vivo implantation.

Adipose regeneration strategies have been hampered by the inability to supply an adequate vascular supply following implantation. Vascularization in vitro, also called prevascularization, is a promising method that could promote the vascularization of engineered adipose tissue constructs upon implantation. In this study we compared the ability of prevascularized-to-non-prevascularized fibrin-based human adipose tissue to promote vascularization. Human adipose tissue-derived stromal cells (ASCs) and different mixtures (1:1, 1:2 and 1:5) of ASCs with human umbilical vein endothelial cells (HUVECs) were cultured in fibrin at two different densities (1.0 × 10(6) and 10 × 10(6) cells/ml) for 7 days. Histological analysis revealed that prevascular structures formed in 1:5 ASC/HUVEC fibrin-based constructs seeded with a total of 10 × 10(6) cells/ml. These constructs and ASC-only constructs were implanted subcutaneously in athymic mice for 7 days and generated lipid-containing grafts. The numbers and densities of blood vessels within the ASC/HUVEC constructs were similar to those of ASC-only constructs. Furthermore, immunostaining studies demonstrated human-derived vasculature within a few of the ASC/HUVEC and ASC-only constructs. A subset of this human-derived vasculature contained erythrocytes, indicating integration with the host vasculature. In conclusion, our study indicated no difference in the rate of vascularization of prevascularized ASC/HUVEC and non-prevascularized ASC-only fibrin-based constructs, suggesting that prevascularization of these fibrin-based constructs does not promote vascularization. Our results further indicated that not only endothelial cells, but also ASCs may contribute to the formation of vascular lumina upon implantation. This finding is interesting, since it demonstrates the possibility of vascularized adipose tissue engineering from a single cell source.