Investigation of an electrospun, degradable polar hydrophobic ionic polyurethane patch for cardiac tissue regeneration

Chan, Jennifer PY (Institute of Biomaterials and Biomedical Engineering, University of Toronto)
Santerre, J Paul (Institute of Biomaterials and Biomedical Engineering; Faculty of Dentistry, University of Toronto)

Introduction

Coronary artery disease can lead to myocardial infarction, cardiomyocyte death, myocardium damage, and ultimately heart failure, due to the limited regenerative potential of cardiomyocytes (CMs) (1). Cardiac tissue engineering can be used to repair and replace damaged myocardium. Engineered cardiac tissue scaffolds need to be biocompatible, be biodegradable, be functional, and readily accommodate various cell types. They also have to have mechanical properties and thickness representative of native heart tissue in order to promote angiogenesis and tissue regeneration (1-3). However, current cardiac tissue scaffolds have yet to address all these criteria. Previous studies have shown that D-PHI PU (degradable polar hydrophobic ionic polyurethane), a novel immunomodulatory biomaterial developed in our lab, addresses several of the biocompatibility limitations of current biomaterials and could be used as a suitable material to generate a cardiac patch. The hypothesis is that D-PHI PU can be electrospun into a highly oriented scaffold, will ultimately biodegrade, enable the generation of multilayered constructs of oriented tissue that have mechanical properties representative of native heart tissue, be non-toxic to CMs (at least 80% viability), and enable CM growth and maturation as measured by the expression of functional CM markers. The objective of this project is to electrospin D-PHI PU to generate a functional cardiac patch which can support the culture of CMs.

Materials and Methods

D-PHI PU oligomer was synthesized as previously described and combined with monomers (4), integrated with a degradable linear polycarbonate polyurethane (PCNU) (5), and incorporated into an appropriate solvent to generate a polymer solution. The polymer solution was electrospun with in situ UV cross-linking to generate aligned nanofibre scaffolds. The fibre morphology was determined using scanning electron microscopy (SEM); cross-linking efficiency was determined by Fourier transform infrared spectroscopy; water contact angle studies were used for surface analysis; and scaffold stiffness was measured using tensile testing. The in vivo degradation of the D-PHI PU scaffold was determined by performing subcutaneous implants in Wistar rats (Univ. of Toronto animal protocol #20012047), explanting scaffolds after 7, 30, 60, and 90 days, and measuring scaffold fragmentation size over time. In vitro biocompatibility of the D-PHI PU scaffold was performed using human embryonic stem cell derived CMs. Cell viability was determined through live/dead staining, cell adhesion was measured using SEM, and CM phenotype was determined by immunofluorescence staining for cardiac troponin-T (cTnT) and myosin light chain 2 (MLC2) (two key CM functional markers).  The data was analyzed by ANOVA, and a Student’s t-test was used to make group comparisons. For all analysis, significance was assigned for p<0.05.

Results

50:50 D-PHI PU:PCNU scaffolds yielded an average fibre diameter of 410 ± 349 nm (large standard deviation due to the dual process of light curing and fibre formation occurring simultaneously) and an alignment of 0.60 (complete alignment = 1, absolute randomness = 0) (Figure 1A). The cross-linking efficiency of the D-PHI PU/PCNU scaffolds was 93 ± 1 %, which is comparable to the 96 ± 1% obtained for pure D-PHI PU films that were light cured without fibre electrospinning. Water contact angle (a proxy for surface energy changes, where a low angle indicates higher surface energy) of the D-PHI PU/PCNU scaffolds decreased when compared to pure PCNU scaffolds (44 ± 6° vs. 88 ± 7° respectively) indicating greater adhesion and polarity in the blend. This characteristic could help facilitate enhanced cell attachment. The elastic moduli of D-PHI PU/PCNU wet and dry scaffolds were 55 ± 12 MPa and 142 ± 59 MPa, respectively. These moduli were higher than that of human myocardium (0.5 MPa), hence further work is required to generate scaffolds with a lower stiffness. Preliminary histology results showed some cellular infiltration into the scaffold and the formation of some blood vessels around the scaffold after 30 days, but minimal fibre degradation of the scaffold, further analysis is on-going (Figure 2). CMs cultured on Matrigel coated D-PHI PU/PCNU scaffolds showed viability that was comparable to Matrigel coated tissue culture polystyrene (TCPS) (94 ± 2 % vs 93 ± 4 % respectively after 7 days) (Figure 3A). CMs showed good adhesion on D-PHI PU/PCNU scaffolds and aligned in the direction of fibres (Figure 1B). Immunofluorescence staining showed that the majority of CMs continued to express cTnT and MLC2 after 7 days of culture on D-PHI PU/PCNU scaffolds and expression was similar to that on TCPS (Figure 3B).

Discussion and Conclusion

The D-PHI PU/PCNU scaffold is not toxic to CMs and enables CM growth and maintenance of key CM functional markers after 7 days. Degradation studies are still on-going to determine whether the D-PHI PU/PCNU scaffold will degrade on a timescale conducive to cardiac tissue regeneration. Further work is required to decrease the stiffness of the D-PHI PU/PCNU scaffolds and to match the elastic modulus of native myocardium. It is anticipated that a degradable D-PHI PU/PCNU cardiac patch could be used to overcome limitations on biocompatibility, biodegradability, and mechanical properties faced by current engineered cardiac tissue scaffolds used to support cardiac tissue regeneration.


Figure 1: (A) SEM image of 50:50 D-PHI PU:PCNU fibres (B) SEM image of CMs aligned in the direction of fibres on D-PHI PU/PCNU scaffolds after 7 days.

Figure 2: Cross-sectional histology slice of D-PHI PU/PCNU scaffold explanted after being implanted for 30 days subcutaneously and stained with May-Grünwald-Giemsa stain. D-PHI PU/PCNU scaffold stains light pink, cell nuclei stain varying shades of purple, and cell cytoplasm stain blue/pink.

Figure 3: (A) Live/dead staining of CMs on (i) D-PHI PU/PCNU scaffold and (ii) TCPS after 7 days. Calcein AM live stain (green), ethidium homodimer-1 dead stain (red). (B) Immunofluorescence staining of CMs for cTnT (green) and MLC2 (red) on (i) D-PHI PU/PCNU scaffold and (ii) TCPS after 7 days.

Acknowledgements

Kyle Battiston for help with animal work, NSERC CIHR Collaborative grant #381337, and NSERC CREATE TOeP scholarship.

References

(1) Reis LA et al. J Tissue Eng Regen Med. 2016;10(1):11–28. (2) Jawad H et al. J Tissue Eng Regen Med. 2007;1(5):327–42. (3) Parsa H et al. 2016;96:195–202. (4) Sharifpoor S et al. Biomacromolecules. 2009;2729–39. (5) Yang L et al. J Biomed Mater Res - Part A. 2009;91(4):1089–99.

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