Tunable methacrylated decellularized heart matrix: a versatile scaffold for cardiac tissue engineering

Abstract

Therapeutic tissue regeneration remains a significant unmet need in heart failure and cardiovascular disease treatment, which are among the leading causes of death globally. Decellularized heart matrix (DHM) offer promising advantages for tissue engineering, including low immunogenicity and seamless integration into biological processes, facilitating biocompatibility. However, DHM is challenged by weak mechanical properties that limit its utility to biomedical applications like tissue engineering. To address this limitation, we functionalized DHM with methacryloyl functional groups (DHMMA) that support UV-induced crosslinking to enhance mechanical properties. By modulating the degree of methacryloyl substitution, a broad range of stiffness was achieved while maintaining cell viability on crosslinked DHMMA. Additionally, we show that increasing UV exposure time and pH increases DHMMA stiffness. Furthermore, topographical features transferred on DHMMA via soft lithography facilitated physical orientation of cells in culture. We demonstrate DHMMA as a scaffold with tunable stiffness and matrix-degradation properties suitable for cell survival and microfabrication for cardiac tissue engineering applications.

Keywords: biomaterials; decellularized heart matrix; matrix protein release; methacrylation; ultra-violet crosslinking.

FIGURE 1

Schematic illustration of DHMMA fabrication process: (A) Decellularization, solubilization, and (B) Methacrylation.

FIGURE 2

Chemical characterization of DHMMA. (A) Solutions of DHM/DHMMA from TNBS reaction to determine (B) degree of substitution. (C) FTIR spectra comparing DHM and DHMMA formulations. Arrow indicating the peak between 1720–1740 cm^-1 increasing for all DHMMA formulations. Bar graph data represented as mean ± standard deviation, n = 3. Significance level: **p < 0.01, and ***p < 0.001. Scale bar = 10 mm.

FIGURE 3

Physical characterization of crosslinked DHMMA. (A) Crosslinked DHMMA after 10 min of UV exposure (scale bar = 1 mm). (B) Representative SEM images at ×500 (left) and ×2000 (right) magnification (scale bar = 50 µm). (C) Histogram representation of changes in DHMMA cross-section with methacrylation reaction conditions. (D) Young’s Modulus of DHMMA from AFM analysis (mean ± standard error mean). (E) DHMMA compressed by 0.6 mm to determine the maximum stress. (F) Swelling ratio after immersion in DI water for 24 h at 37°C. All data represented as mean ± standard deviation, n = 3 gels. Significance level: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Passive and enzymatically induced matrix protein release. (A) Protein release from crosslinked DHMMA immersed in DI water at 37°C for 30 days with sampling every 3 days (B) DHMMA exposed to digestion buffer for 24 h at 37°C with sample aliquots taken at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. Data represented as mean ± standard deviation, n = 3 gels. Statistics: Two-way ANOVA.

FIGURE 5

Physical characterization of DHMMA crosslinked for 30 s, 60 s, and 300 s. (A) Crosslinked $\text{DHMM}{\mathrm{A}}_{\text{pH}}^{\text{CB}}$ compressed by 0.6 mm to determine (B) stiffness. (C) $\text{DHMM}{\mathrm{A}}_{\text{pH}}^{\text{CB}}$ exposed to digestion buffer for 24 h at 37°C with sample aliquots taken at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. All data represented as mean ± standard deviation, n = 3 gels. Significance level: *p < 0.05.

FIGURE 6

Varying DHMMA suspension pH prior to UV crosslinking. (A) Crosslinked hydrogels after adjusting $\text{DHMM}{\mathrm{A}}_{\text{pH}}^{\text{CB}}$ suspension pH from acidic to basic. (B) Compression of $\text{DHMM}{\mathrm{A}}_{\text{pH}}^{\text{CB}}$ after pH adjustment to determine (C) maximum stress (pH 5 was not solid enough to undergo compression). (D) $\text{DHMM}{\mathrm{A}}_{\text{pH}}^{\text{CB}}$ after pH adjustment exposed to digestion buffer for 24 h at 37°C with sample aliquots taken at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. All data represented as mean ± standard deviation, n = 3. Significance level: *p < 0.05. Scale bar = 1 mm.

FIGURE 7

Live staining of H9C2 cells on crosslinked DHMMA. (A,B) H9C2 cells adhere and spread on crosslinked DHMMA and remain viable 72 h post-seeding. (C) MTT assay of H9C2 cells on crosslinked DHMMA after 72 h post-seeding. All data represented as mean ± standard deviation, n = 3. Significance level: ns (p > 0.05) for all conditions. Scale bar = 100 µm. TCP = tissue culture plastic.

FIGURE 8

Crosslinked DHMMA for soft-lithography applications. (A) Fabrication process of micropatterned $\text{DHMM}{\mathrm{A}}^{\text{PBS}}$ using soft-lithography. (B) Micropatterned $\text{DHMM}{\mathrm{A}}^{\text{PBS}}$ . (C) Optical profilometry analysis of 100 μm and 50 µm groove patterns. (D) H9C2 cells aligning on micropatterned $\text{DHMM}{\mathrm{A}}^{\text{PBS}}$ . Scale bar = 100 µm. TCP = tissue culture plastic.