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.

Keywords::

• 3D cell culture
• cardiomyocytess
• electrospinning
• Scaffolds

Acknowledgement

We are grateful to the Department of Biotechnology, Government of India, for funding this work. Grant no: DO No. BT/PR11228/BRB/10 677/2008. We thank Dr. Suresh R, Department of Periodontology, Sri Ramachandra University, for his kind gift of collagen. We are also grateful to the faculty and technical experts at the Biomedical Testing Wing at Sri Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, for their help in tensile testing and confocal microscopy.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

Supplementary material available online

Supplementary Videos 1 and 2 Video microscopy of neonatal cardiomyocytes seeded on 2D and 3D cultures.

1. Movie clips of beating cardiomyocytes on 2D (culture flask) culture at different time points. Magnification (20X).

2. Beating cardiomyocytes on 3D collagen nanofibrous scaffold on the 17th day time point, under magnification (20X). Cardiomyocytes were beating continuously well on all time points in the collagen scaffolds.