Hydrogel-based microfluidics for vascular tissue engineering

Abstract

Abstract:In this work, we have explored 3-D co-culture of vasculogenic cells within a synthetically modified fibrin hydrogel. Fibrinogen was covalently linked with PEG-NHS in order to improve its degradability resistance and physico-optical properties. We have studied influences of the degree of protein PEGylation and the concentration of enzyme thrombin used for the gel preparation on cellular responses. Scanning electron microscopy analysis of prepared gels revealed that the degree of PEGylation and the concentration of thrombin strongly influenced microstructural characteristics of the protein hydrogel. Human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem cells (hASCs), used as vasculogenic co-culture, could grow in 5:1 PEGylated fibrin gels prepared using 1:0.2 protein to thrombin ratio. This gel formulation supported hASCs and HUVECs spreading and the formation of cell extensions and cell-to-cell contacts. Expression of specific ECM proteins and vasculogenic process inherent cellular enzymatic activity were investigated by immunofluorescent staining, gelatin zymography, western blot and RT-PCR analysis. After evaluation of the optimal gel composition and PEGylation ratio, the hydrogel was utilized for investigation of vascular tube formation within a perfusable microfluidic system. The morphological development of this co-culture within a perfused hydrogel over 12 days led to the formation of interconnected HUVEC-hASC network. The demonstrated PEGylated fibrin microfluidic approach can be used for incorporating other cell types, thus representing a unique experimental platform for basic vascular tissue engineering and drug screening applications.

Digging deeper: structural background of PEGylated fibrin gels in cell migration and lumenogenesis

“…Previously, we have shown that the 5 : 1 PEGylated brin gel possesses a high angiogenic potential compared to the native brin gel. 11,14,15 However, the structural rearrangement caused by brin modication remained unclear. The results presented in this study allowed us to suggest that the inner structure of the PEGylated 5 : 1 brin gel can be presented by exible macroparticles with a smaller space between them.…”

“…10 Natural brin rapidly degrades and is not transparent, so several modications have been suggested to overcome these limitations and, moreover, those have been shown to increase its angiogenic potential. [11][12][13] Among them, PEGylation (modication with functionalized polyethylene glycol (PEG)) is of particular interest.…”

“…they supported the formation of lumens). 11,14,15 However, the structural aspects remain undiscussed despite their evident importance in designing angiogenesis-guiding biomaterials. Earlier, we determined the shape of brinogen in solution using small angle X-ray scattering (SAXS).…”

Time resolved 3D live-cell imaging on implants

“…However, fibrin gels undergo degradation and strong matrix remodeling in vitro , which results in construct shrinkage and weight loss. PEGylation of fibrinogen is one possible method to control the gel stability without impairment of its excellent cytocompatible properties [ 28 ]. In this study PEGylated fibrin was evaluated highly cytocompatible with gingival fibroblasts.…”

“…The PureCol—Matrigel mixture was prepared by mixing the bovine collagen type I solution (PureCol, 5005-100ML, Advanced BioMatrix Inc, USA), Matrigel, and cell culture medium on ice to final concentrations of 0.67 mg/mL and 4.7 mg/mL, respectively. The PEGylated fibrin hydrogel was prepared as previously described [ 28 ]. The human gingival fibroblasts (5000 cells in 150 μL culture medium) were seeded on 50 μL of PEGylated fibrin hydrogels after gelation in 96-well plates and cultured for 24 or 72 hours at 37 °C in a humidified atmosphere with 5% CO 2 .…”