161. Comparison Of Different Mural Cells On Vascular Networks Of Tissue Engineered Skin Constructs Article Swipe
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· 2023
· Open Access
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· DOI: https://doi.org/10.1097/01.gox.0000938224.47910.4e
· OA: W4377013286
PURPOSE: Skin tissue engineering represents a promising avenue for treating burns, traumatic injuries, skin cancer defects, and diabetic wounds. However, this promise will likely remain limited without proper vascularization. Vascular networks within tissue-engineered skin constructs are necessary for rapid incorporation through vascular anastomosis and inosculation with the host wound bed. Generating these networks that are both stable and operational is dependent upon finding the right culture conditions for the growth of endothelial cells (ECs) with mural cells in different biomaterials. We hypothesize that the mural cells can secrete the growth factors necessary for angiogenesis and provide biomechanics to sustain the network structure. Here, we cultured ECs with different mural cells and scaffolds, mimicking the skin's dermal compartment, and quantified the resulting vascular networks. METHODS: Enhanced green fluorescent protein (EGFP) labeled Human Umbilical Vein Endothelial Cells (HUVECs) were cultured at a density of 9x105 cells/mL in a 6 well plate. Three different types of mural cells, namely, Dental Pulp Stem cells (DPSCs), human dermal Fibroblasts, and human Preadipocytes were mixed with HUVECs at a ratio of 1:2 in a 3D culture. Two types of extracellular matrices (ECMs) were studied: a) collagen mixed with Matrigel and b) collagen mixed with fibrin. These ECMs were mixed in a 1:1 ratio. Thus, in total, 6 different culture conditions were tested in Endothelial Cell Growth Medium (EGM2) in a humidified incubator at 37 °C and an atmosphere with 5% CO2. The media was changed every other day, and ECs network assembling was recorded by EVOS Auto Fl scanning microscope. RESULTS: The preadipocyte co-culture was stable for more than 112 days compared to the DPSCs, and fibroblasts that peaked at 14 and 7 days, respectively. The highest degree of order was observed in the DPSCs culture, where at least three types of networks were present. From the quantitative results of the network, HUVECs-DPSCs have the most extensive networks in terms of the total network length (68573.4 microns) and the number of nodes (390). The presence of lumen (17.9 microns diameter) was also discovered with this culture condition. In addition, we noticed a greater degree of gel contraction with the collagen/fibrin scaffolds than with collagen/Matrigel scaffolds. Concerning cells, the gel contraction between fibroblasts and preadipocytes was not significantly different but higher than those obtained with DPSCs. CONCLUSIONS: These results demonstrate that comprehensive and stable network formation was observed with DPSCs in just two weeks in collagen/fibrin scaffolds. This study lays the groundwork for finding an effective coculture methodology to develop autologous dermal equivalents. Future studies focus on adding epidermal layers to these dermal equivalents to create vascularized skin constructs.
