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Abstract
Advanced skin regeneration therapies can combine biomaterials, cells, growth factors and advanced biomanufacturing techniques for the fabrication of constructs that will ultimately mimic native skin anatomy. Regardless of the specific tissue-engineering approach for in vitro artificial skin substitute production, to engineer functional skin, the formation of an efficient vascular network is required.
Aiming to develop a strategy to improve constructs microvascularization with fibroblasts support endothelial cells in the formation of self-assembled vascular structures, this study allowed the dissection of human umbilical vein endothelial cells (HUVEC) and neonatal human dermal fibroblasts (NHDFs) behavior in a 3D microenvironment. We addressed for the first time the effect of several culture parameters on cells behavior when embedded on RGD-grafted soft pectin hydrogels. Conditions such as media composition, cell density, cell type to type ratio and polymer concentration were optimized on standard 2D culture conditions. The results obtained allowed us to choose the best conditions to proceed into a 3D experimental setup.
A 3:1 ratio of M199 to DMEM media was selected for HUVEC:NHDFs co-cultures and we also determined that low HUVEC to NHDFs ratios, in 2D environments led to NHDFs spreading in detriment of HUVEC proliferation while higher ratios sustained a controlled environment where HUVECs were able to grow and assemble in spider weblike structures. In a three dimensional context, Cell behavior parameters displayed better outcomes for lower hydrogel formulations (1.5% w/v) and higher cell densities (1.5x107 cells.mL-1).. Fibroblasts formed spheroidss and contracted the matrix, while maintaining the metabolic activity, in a matrix and cell density-dependent way, with 1.5% (w/v) pectin hydrogels embedded with 1x107cells.mL-1 demonstrating microtissues formation.
Based on combination of NHDFs and HUVECs, a cocuture systems were developed in soft pectin hydrogel matrices. Within these, HUVEC survival was increased, and fibroiblast spheroids formation was observed. Although further investigation is needed, we developed a a three-dimensional co-culture system in RGD-grafted soft pectin hydrogel in which fibroblasts support endothelial cells, and established this techniques as a promising strategy for in vitromicrovascularization towards skin regeneration therapies.
