Human versus porcine tissue sourcing for an injectable myocardial matrix hydrogel

Abstract

Heart failure (HF) after myocardial infarction (MI) is a leading cause of death in the western world with a critical need for new therapies. A previously developed injectable hydrogel derived from porcine myocardial matrix (PMM) has had successful results in both small and large animal MI models. In this study, we sought to evaluate the impact of tissue source on this biomaterial, specifically comparing porcine and human myocardium sources. We first developed an analogous hydrogel derived from human myocardial matrix (HMM). The biochemical and physical properties of the PMM and HMM hydrogels were then characterized, including residual dsDNA, protein content, sulfated glycosaminoglycan (sGAG) content, complex viscosity, storage and loss moduli, and nano-scale topography. Biochemical activity was investigated with in vitro studies for the proliferation of vascular cells and differentiation of human cardiomyocyte progenitor cells (hCMPCs). Next, in vivo gelation and material spread were confirmed for both PMM and HMM after intramyocardial injection. After extensive comparison, the matrices were found to be similar, yet did show some differences. Because of the rarity of collecting healthy human hearts, the increased difficulty in processing the human tissue, shifts in ECM composition due to aging, and significant patient-to-patient variability, these studies suggest that the HMM is not a viable option as a scalable product for the clinic; however, the HMM has potential as a tool for in vitro cell culture.

Figure 1

Histological analysis of human heart decellularization. Human myocardial tissue fresh (A–C), unsuccessfully decellularized with previously developed porcine myocardial matrix (PMM) protocol (D–F), and fully decellularized with the optimized protocol for human myocardial tissue (G–I). Shown are Hematoxylin and Eosin staining (A, D, G), Hoechst staining for dsDNA (B, E, H), and Oil Red O staining for lipids (C, F, I). The top row shows clear distinguishable nuclei and significant lipid staining in the fresh human myocardial tissue. The middle row indicates incomplete decellularization with positive Hematoxylin in purple, Hoechst, and Oil Red O staining in red. The bottom row shows the optimized human protocol with successful removal of dsDNA and lipid content. Scale bars apply to all images and are 100μm.

Figure 2

Quantification of DNA in the myocardial matrix materials. Plotted is the quantified dsDNA isolated from PMM or HMM per mg of dry ECM. DNA content is very low in both materials and there is no significant difference in the dsDNA content between PMM and HMM.

Figure 3

Quantification of sulfate glycosaminoglycan (sGAG) content from PMM and HMM as assessed with a DMMB assay. The PMM contains significantly more sGAG than in HMM. *p<0.0001.

Figure 4

PAGE showing molecular weight bands created by the standard ladder (A), rat tail collagen as a control (B), PMM (C), and HMM (D). While PMM and HMM have similar bands and are predominantly collagen, there are some minor differences.

Figure 5

Characterization of the mechanical properties using a parallel plate rheometer. Complex viscosity of the liquid form was assessed at 25°C over a range of shear rates (A). Both materials are plotted on a log-log scale and are shear thinning in the liquid form. Storage modulus (G') and loss modulus (G”) were measured after incubation for 24 hours at 37°C and example curves over a range of frequencies are plotted (B).

Figure 6

Scanning electron microscopy images of both PMM (A) and HMM (B) gels after incubation at 37°C for 24 hours. The materials produced a complex mesh of nano-scale fibers that were formed via self-assembly. Scale bars for the large images are 4μm and the smaller inset images are 400nm.

Figure 7

Proliferation of rat aortic smooth muscle cells (RASMCs) and human coronary artery endothelial cells (HCAECs) on cell culture coatings of PMM or HMM. Cells were cultured in growth medium for 3 or 5 days and relative proliferation was quantified with a PicoGreen assay. *p<0.05.

Figure 8

RT-PCR for early cardiac markers for human cardiomyocyte progenitor cells (hCMPCs) after culture for four days. Cells were cultured on 2D coatings made from gelatin, PMM, or HMM in proliferation media. *p<0.05.

Figure 9

hCMPCs after 4 days of culture on PMM (A, C) or HMM (B, D) coatings. Nuclei were stained with Hoechst and shown in blue. An early cardiac marker, Nkx2.5, was stained in green (A, B) and cardiac troponin T was also labeled in green (C,D). The scale bar is 100 μm for all images.

Figure 10

In vivo gelation of PMM (A) and HMM (B) in the endocardium of the left ventricle of a Sprague Dawley rat. The injection bolus is indicated with (*) and is surrounded by healthy myocardium. Tissue sections are stained with Hematoxylin and Eosin (H&E). Image scale bars are 100μm.