Electrospun type 1 collagen matrices preserving native ultrastructure using benign binary solvent for cardiac tissue engineering

Abstract

Electrospinning is a well-established technique that uses a high electric field to fabricate ultrafine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers, due to its widespread occurrence in nature and its biocompatibility. Electrospinning of collagen alone has been reported, with fluoroalcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE), but the resultant collagen lost its characteristic ultrastructural integrity of D-periodicity 67 nm banding, confirmed by transmission electron microscopy (TEM), and the fluoroalcohols used were toxic to the environment. In this study, we describe the use of glacial acetic acid and DMSO to dissolve collagen and generate electrospun nanofibers of collagen type 1, which is non-toxic and economical. TEM analysis revealed the characteristic feature of native collagen triple helical repeats, showing 67 nm D-periodicity banding pattern and confirming that the ultrastructural integrity of the collagen was maintained. Analysis by scanning electron microscopy (SEM) showed fiber diameters in the range of 200-1100 nm. Biocompatibility of the three-dimensional (3D) scaffolds was established by MTT assays using rat skeletal myoblasts (L6 cell line) and confocal microscopic analysis of immunofluorescent-stained sections of collagen scaffolds for muscle-specific markers such as desmin and actin. Primary neonatal rat ventricular cardiomyocytes (NRVCM) seeded onto the collagen scaffolds were able to maintain their contractile function for a period of 17 days and also expressed higher levels of desmin when compared with 2D cultures. We report for the first time that collagen type 1 can be electrospun without blending with copolymers using the novel benign solvent combination, and the method can be potentially explored for applications in tissue engineering.

Review of 3D cell culture with analysis in microfluidic systems

“…90 Benign aqueous based solvents have been explored in limited applications and has been shown to minimize effects on protein structure. 91,92 Early incorporation of ber scaffolds into microuidic devices was done with PDMS-based substrates. One of the earliest was reported in 2008 by Lee et al, who fabricated a channel in a bottom layer of PDMS and then used a top layer of PDMS to sandwich acrylic acid modied polyurethane bers into the device (Fig.…”

Recent concepts in biodegradable polymers for tissue engineering paradigms: a critical review

“…In a study by Elamparithi, a novel collagen type I scaffold developed by electro spinning in the absence of copolymers showed higher levels of desmin. This scaffold was seeded with primary neonatal rat ventricular cardio-myocytes (NRVCM) and exhibited sustained cardiac contractile function over duration of 17 days 201 . The prospect of limiting myocardial damage and facilitating repair and regeneration was addressed in this study 202 .…”

3D and 4D Bioprinting of the Myocardium: Current Approaches, Challenges, and Future Prospects

“…Some methods used cell mixtures from both types as well. Rat myoblast cells (L6 cell line) have been shown to differentiate into functioning cardiac tissue in a variety of experiments [ 25 , 49 ]. In Jakab et al's [ 27 ] study, embryonic myocardium was harvested from leghorn chickens and cardiomyocytes were bioprinted on a biopaper hydrogel.…”