Adhesion, proliferation and viability of human umbilical vein endothelial cells cultured on the surface of biodegradable non-woven matrices modified with RGD peptides

Abstract

Tissue engineering is a promising area for the production of small-diameter vascular grafts. In recent years, a number of strategies have been developed to make the polymer surfaces of vascular prostheses capable to selectively adhesion of endothelial cells. The arginine–glycine–aspartic acid (RGD) sequence (a cell adhesion site that is present on many extracellular matrix proteins) is the promising target for modification. The efficiency of attachment of endothelial cells can be influenced both by the structure of RGD peptide and the extent of linker group.Aim: to determine the optimal method for modification of non-woven matrices of polyhydroxybutyrate/ valerate and polycaprolactone (PHBV/PCL) by RGD-peptides leading to the increasing of adhesion, viability and proliferation of endothelial cells.Materials and methods. Electrospinning was used to produce 4 mm diameter tubular polymer matrices from PHBV/PCL. Modification of surface of polymer scaffolds was performed using 4,7,10-trioxa-1,13-tridekandiamin, hexamethylenediamine, glutaraldehyde, ninhydrin, ascorbic acid, a cyclic peptide c [RGDFK], RGDK, AhRGD. The quality of modification was assessed by ninhydrin test and determination of arginine-containing peptide. The structure of the surface of matrices before and after modification was studied by scanning electron microscopy. Adhesion, viability and proliferation of Human umbilical vein (HUVEC) endothelial cells cultured for 7 days on the surface of matrices in the presence of RGD and without one were examined using fluorescence and laser scanning microscopy after the cells were pre-stained with fluorescent nuclear dyes (ethidium bromide and Hoechst 33342), and also by special kits for proliferation assessment (Click-iTTM Plus EdU Alexa FluorTM 488 Imaging Kit).Results. RGD peptides bound to the matrix surface via a long linker (4,7,10-trioxa-1,13-tridecanediamine) were characterized by the increased bioavailability and activity. High level of cell adhesion, viability and proliferation were noted on the surface of RGDK and c[RGDFK] modified matrices, whereas their paired analogues with a short linker (hexamethylenediamine) showed low results of cellular viability even against satisfactory cell adhesion.Discussion. Non-woven matrices based on PHBV/PCL and modified using 4,7,10-trioxa-1,13-tridecanediamine showed better results in case of adhesion of HUVEC and subsequent preservation of cell viability and proliferation. RGD-containing peptides of RGDK and c [RGDFK] were more tropic to endothelial cell receptors.

Biodegradable Patches for Arterial Reconstruction Modified with RGD Peptides: Results of an Experimental Study

“…Previous studies also reported that these combinations improved adhesion, viability, and proliferation of both endothelial colony-forming cells and human umbilical vein endothelial cells on the surface of electrospun PHBV/PCL scaffolds. 25 , 26 Thus, we tested the performance of TTDDA/RGDK-, TTDDA/AhRGD-, and TTDDA/c[RGDFK]-modified patches upon the implantation into the rat abdominal aorta. Unmodified PHBV/PCL patches and commercially available KemPeriplas-Neo patches were used as controls.…”

“…Modification of biodegradable patches with RGD-containing peptides was performed as previously described. 25 Briefly, aminemodified patches were sequentially rinsed with a mixture of isopropanol/ddH 2 O (1:1), ddH 2 O, 0.1% Triton X-100, and ddH 2 O. Samples were incubated in 2% aqueous glutaraldehyde (Sigma, USA) at 24 °C for 3 h followed by a quick rinse with ddH 2 O. Patches were incubated in a solution of RGDK, AhRGD, or c[RGDFK] (0.2 mg/mL) in 50 mM carbonate buffer (pH = 8.5) containing 2.5 mM sodium cyanoborohydride at 24 °C for 4 h. Finally, samples were sequentially washed with 0.1% Triton X-100 and ddH 2 O.…”

“…After being fixed with formalin, the patches were embedded in paraffin followed by staining with hematoxylin and eosin and Alizarin Red S as described previously. 25 Slides were visualized using the Axio Imager A1 light microscope (Carl Zeiss).…”

“…Snap-frozen tissue blocks were sectioned and stained for CD31 (ab119339, Abcam), CD34 (ab185732, Abcam), collagen type IV (ab6586, Abcam), and vWF (ab8822, Abcam) as described previously. 25 Slides were visualized using the LSM 700 confocal laser scanning microscope (Carl Zeiss) as described previously. 25 5.13.…”

“…25 Slides were visualized using the LSM 700 confocal laser scanning microscope (Carl Zeiss) as described previously. 25 5.13. Statistical Analysis.…”