oa Modified Silk with Cell-Adhesive and Non-Thrombogenic Properties as a Tissue Engineering Substrate

Introduction

Replacement of damaged tissue is nowadays an aim of tissue engineering. This technique involves the use of porous or fibrous structures – the so-called scaffolds – that support the colonization with the desired cell type and which are degraded after fulfilling their temporary supporting function. Basic requirements for the prevailing materials used in this field are nontoxicity, low immunogenicity and cell-adhesiveness. Furthermore blood-contacting devices should exhibit low thrombogenicity.

The biopolymer silk, mainly consisiting of the protein silk fibroin, matches some of these criteria but bare silk does not facilitate cellular adhesion and growth and unfortunately the material is prone to platelet attachment.

In our approach, the chemical surface immobilization of a cell adhesive peptide of silk samples reduces thrombocyte adhesion to a large extent while simultaneously promotes specific adhesion and colonization by endothelial cells (ECs).

The specific interaction of the modification was further demonstrated by fibroblast cell culture.

Materials & Methods

The EC specific adhesive peptide Arg-Glu-Asp-Val (REDV) derived from the extra cellular matrix protein fibronectin was used in our experiments. The peptide was chemically immobilized onto silk fabric scaffold by using hexamethylene diisocyanate (HMDI) as an activator for the substrate. Subsequent hydrolysis of pending isocyanate moieties yielded in primary amino functionalities. REDV was subsequently conjugated directly using a short chain amino-reactive crosslinker or via a bifunctional polyethylene glycol (PEG) spacer. Additionally, silk was modified with amino-functional PEG only. Modified as well as untreated specimens were subjected to cell culture using ECs and fibroblasts. In addition samples were challenged with platelet rich plasma in order to evaluate thrombocyte adhesion. Potential changes in material bulk properties and morphology were checked by scanning electron microscopy, gel permeation chromatography and mechanical testing.

Results & Discussion

Coverage of silk fabric with ECs was greatly promoted through REDV-modification by a factor of 17 (directly coupled peptide) and a factor of 20 (PEG-mediated coupling) respectively after 2 weeks growth in comparison to cell colonization of untreated material. Substrate modification also inhibited initial (24 h) fibroblast adhesion. Thrombocyte attachment was strongly reduced 5-fold as a result of PEG-modification, independent of an additionally conjugated peptide. Mechanical (tensile testing) as well as morphological properties (SEM) were not significantly altered by the chemical treatment. The initial activation of silk showed no detectable influence on the composition (GPC).

Conclusion

Taken together, the feasibility of improving the biological performance of silk, an established biomaterial, was shown. We where able to show that the chemical modification left the basic material properties largely unaffected. These findings may contribute to novel tissue engineering approaches that facilitate the endothelialization of cardiovascular implants such as vascular grafts and heart valves.

Figure 1: Covalent immobilization of the REDV peptide – either directly coupled or PEG-mediated – renders silk an excellent substrate for endothelialization. In addition the presence of PEG alone inhibits the attachment of platelets to a large extend.