(M-492) Ultra-Violet Crosslinked Decellularized Heart Matrix Allows for Surface Micropatterning and Cardiomyocyte Maturation In Vitro

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row M - Poster # 492

Presenting Author(s)

VP

Valinteshley Pierre

Graduate Research Assistant
Case Western Reserve University
SHAKER HEIGHTS, Ohio, United States

Primary Investigator(s)

SS

Samuel Senyo

Assistant Professor
Case Western Reserve University, United States

Co-Author(s)

CL

Chao Liu

Graduate Student Researcher
Case Western Reserve University, United States
DW

Douglas Wu

Graduate Student Researcher
Case Western Reserve University
Shaker Heights, Ohio, United States
EE

Elif Ertugral

Graduate Student Researcher
Cleveland State University, United States
Chandra Kothapalli

Professor of Chemical and Biomedical Engineering
Cleveland State University
Cleveland, Ohio, United States

Introduction:: Decellularized heart matrix (DHM) therapy is a tissue engineering strategy to repair the ischemic heart. Myocardial ischemia is a primary risk factor for heart failure due to death of amitotic cardiomyocytes and cellular disorganization which progressively impair cardiac function. Currently there are no existing cures for heart failure. DHM is tissue-derived injectable hydrogel that releases biological cues that stimulate regenerative signaling in cardiac cells. We have shown that DHM regulates cardiomyocyte proliferation and maturation, myofibroblast quiescence, and angiogenesis in a mechanosensitive manner. Despite the limited mechanical properties of natural biomaterials, DHM treatment also stabilizes the injury region by buffering tissue remodeling. Donor cell therapy options are impaired by tissue integration as well as immature gene expression. We hypothesize that design of a DHM-defined bioactive substrate with topographical cues will spatially organize and increase the maturity of seeded synthetic cardiomyocytes for tissue engineering applications. To address this hypothesis, we have developed crosslinkable DHM for softlithography patterning to promote organization and molecular programming of cells. This platform was achieved by chemically modifying DHM with methacryloyl functional groups (DHM-MA) to enable crosslinking via UV. We first characterized DHM-MA degree of methacrylation, crosslinking, and micropatterning integrity. We then evaluated the bioactivity of DHM-MA using a cardiomyoblast cell line. Finally, we evaluated cardiomyoblast organization and maturation on our microgroove DHM-MA of different dimensions. We show modulation of stiffness with chemical formulation and UV time which determine minimum topographical features. The current evidence demonstrates that the biological and mechanical cues from crosslinked DHM-MA promote cardiomyoblast maturation.

Materials and Methods:: Decellularized heart matrix hydrogel was generated using previously established methods [PMID:32590172]. Briefly, segmented porcine left ventricular myocardium was decellularized using 1% SDS, and 1% Triton X-100 in water. Decellularized tissue was freeze dried and then solubilized by pepsin (1mg/ml) digestion in 0.01 M hydrochloric acid buffer for 3 days. Methacrylate anhydride was added to the hydrogel suspension at ~8.5pH under constant agitation for 2 days at 4°C. The suspension was dialyzed and freeze dried. The resultant decellularized heart matrix methacrylate (DHM-MA) was resuspended in PBS at 20 mg/ml. DHM-MA was optimized for fabrication of features of different aspect ratios by varying protein concentration, photoinitiator replenishment (Irgacure 2959 0.5%), pH, buffer, and UV exposure time and intensity. Separately, polydimethylsiloxane molds were fabricated using established methodology [PMID:36300548] to achieve 17 micron tall grooves with widths varying between 10-100 microns for different aspect ratios. DHM-MA was cast on top of the PDMS molds and exposed to UV for 1-30 minutes then sterilized using ethanol. UV-crosslinked DHM-MA was evaluated by a simple bulk indentation assay, AFM, FTIR, TNBS assay, optical profilometry, and protein release assays. To address bioactivity, cardiomyoblasts (H9C2s) undergoing differentiation were exposed to uncrosslinked DHM-MA for 3 days to evaluate retention of DHM maturation effects. Maturation was assayed by immunocytochemistry for cardiac troponin-T. H9C2s were also plated onto crosslinked DHM-MA grooves to evaluate cell attachment and orientation. Cell cycle activity was assessed after 24 hours with BrdU labeling (4 hour incubation).

Results, Conclusions, and Discussions:: We successfully modified DHM with methacryloyl functional groups demonstrated by FTIR (Fig.1A) and TNBS assay. FTIR data shows increase intensity with Amide I and II bands consistent with increased amount of C=O bonds and stretching of amine groups on DHM protein that occurs with methacrylation. Crosslinking required a minimum of 5 minutes to achieve solid substrates that could be handled manually and by visual inspection were semi-translucent (Fig.1B). We confirmed crosslinking by FTIR. We observed that crosslinking resulted in a greater than 60% decrease in the passive protein release rate. We demonstrated that protein release can be tuned by varying crosslinking parameters and protein concentration (n=3). Using atomic force microscopy, we determined the best formulation that generated an appreciable difference in crosslinked DHM-MA stiffness suitable for micropatterning (Fig.1C). Applying the method of softlithography, microgrooves were patterned during DHM-MA crosslinking (Fig.1B). We observed that 50 micron grooves was the minimum limit with our initial protocol. Altering the DHM-MA formulation (degree of methacrylation, crosslink density, intermittent photoinitiator replenishment) allowed for finer topographical features (20 microns). Micropatterns were verified via microscopy and optical profilometry. Micropatterned DHM-MA was further verified by seeding H9C2s on the surface and showing polarized cell orientation and cytoskeletal alignment (Fig.1D; n=2). DHM-MA showed retention of bioactivity on cultured H9C2s with increases in proliferation (n=3, *p< 0.05, One-Way ANOVA) and differentiation (n=3, *p< 0.05, One-Way ANOVA). We are investigating if micropatterned DHM-MA promotes H9C2 maturation. Results so far suggest that DHM-MA could be used as a multipurpose biological and physical regulator of cardiomyocyte phenotype.

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