Creating capillary networks within human engineered tissues: Impact of adipocytes and their secretory products

Abstract

The development of tissue-engineered substitutes of substantial volume is closely associated with the need to ensure rapid vascularization upon grafting. Strategies promoting angiogenesis include the in vitro formation of capillary-like networks within engineered substitutes. We generated both connective and adipose tissues based on a cell sheet technology using human adipose-derived stromal cells. This study evaluates the morphology and extent of the capillary networks that developed upon seeding of human microvascular endothelial cells during tissue production. We posited that adipocyte presence/secretory products could modulate the resulting capillary network when compared to connective substitutes. Analyses including confocal imaging of CD31-labeled capillary-like networks indicated slight differences in their morphological appearance. However, the total volume occupied by the networks as well as the frequency distribution of the structure's volumes were similar between connective and adipose tissues. The average diameter of the capillary structures tended to be 20% higher in reconstructed adipose tissues. Quantification of pro-angiogenic molecules in conditioned media showed greater amounts of leptin (15×), angiopoietin-1 (3.4×) and HGF (1.7×) secreted from adipose than connective tissues at the time of endothelial cell seeding. However, this difference was attenuated during the following coculture period in endothelial cell-containing media, correlating with the minor differences noted between the networks. Taken together, we developed a protocol allowing reconstruction of both connective and adipose tissues featuring well-developed capillary networks in vitro. We performed a detailed characterization of the network architecture within engineered tissues that is relevant for graft assessment before implantation as well as for in vitro screening of angiogenic modulators using three-dimensional models.

Human adipose-derived stromal cells for the production of completely autologous self-assembled tissue-engineered vascular substitutes

“…Fortunately, subcutaneous fat tissue contains a large amount of microvascular EC (MVEC), and in vitro culturing could be avoided [42,43]. Furthermore, skin MVEC have already been isolated and used for tissue engineering [44,45] and would also be an interesting alternative. In a clinical setting, ASC-TEVS could be endothelialized with fat-derived MVEC and DF-TEVS with skin-derived MVEC therefore no additional biopsies would be required.…”

Opportunities and challenges in three-dimensional brown adipogenesis of stem cells

“…Additionally, 3D co-culture with endothelial cells on porous silk protein matrices could maintain the structure and function of ASC-differentiated adipocytes for at least 6 months (Bellas et al, 2013). In this type of co-culture, there is a mutually enhanced effect between the adipocytes and endothelial cells, which mimics in vivo cell-cell interactions and supports improved adipocyte function, endothelial cell proliferation and capillary network formation (Aubin et al, 2015; Yao et al, 2013a). Moreover, an in vivo study demonstrated the long-term stability of volume and weight of the injected HUVEC-adipocytes within collagen/alginate microspheres, indicating that vascularization facilitates the formation, maturity, and maintenance of the adipose tissue (Yao et al, 2013b).…”

New and Improved Tissue Engineering Techniques: Production of Exogenous Material-Free Stroma by the Self-Assembly Technique

“…ASCs extracted from liposuctions can be expanded in culture and used as building blocks for tissue engineering. Both connective and adipose tissues were engineered in vitro using ASCs [31][32][33]. Both allogeneic and xenogeneic ASCs can be transplanted in patients regardless of their immunocompatibility and without the need of immunosuppression therapy, making them an unlimited source for regenerative medicine applications [34].…”