(671a) Silk-Extracellular Matrix Hydrogels for Cardiac Tissue Engineering 

Cardiovascular disease is the leading cause of death in the U.S.¹ and rapidly becoming the leading cause of death worldwide.² The development and use of injectable biomaterials for cardiac repair can reduce the invasiveness of the biomaterial application.³ Natural injectable materials, like solubilized cardiac extracellular matrix (cECM), contain appropriate binding sites for cells, yet the mechanics and porosity are difficult to control. In contrast, synthetic materials allow for finer control over mechanics and porosity but do not contain the appropriate diversity of cellular binding sites. Recently, we developed a tunable, elastic, silk hydrogel that is formed via di-tyrosine crosslinking using a horseradish peroxidase (HRP)/hydrogel peroxide (H₂O₂)-based enzymatic crosslinking approach.⁴ Silk fibroin protein does not contain integrin binding sites for cells, but we have previously demonstrated that the addition of cECM to silk sponges enhanced cardiac cell phenotype and function over native silk fibroin alone.⁵ In addition, we have shown that addition of ECM to silk sponges enhances native cell infiltration in a subcutaneous implant study.⁵ Here, we aim to augment these elastic, silk hydrogels with cECM and assess the tunability of this material for both in vivo applications, as well as its utility in our bioreactor systems to modulate cardiac cell function.

To achieve these goals, we formed silk-based hydrogels from silk solution as previously reported.^4,6 Adult porcine hearts were obtained from the local butcher shop and decellularized using sodium dodecyl sulfate as previously described.⁵ Left ventricular cardiac extracellular matrix (cECM) was solubilized in pepsin⁷ and incorporated into silk solution prior to the initiation of the HRP/H₂O₂ reaction. Mechanics of acellular gels and cellularized gels were measured via unconfined compression or tension using dynamic mechanical analysis. To evaluate cellular response to silk-cECM hydrogel, cardiac fibroblasts (CFs, adult Sprague Dawley rats) and neonatal cardiomyocytes (CMs, 1-day old pups, Sprague Dawley) were isolated using procedures approved by the Tufts University IACUC. CFs were activated to a myofibroblast phenotype (typically present in infarct tissue) via treatment with 2.5 ng/ml TGF-beta for 48 hours. For both cell types, viability was assessed by live-dead stain and a DNA assay. Important fibroblast markers, key players in integrin signaling, and proteins important for cardiac maturity and function were assessed by Western blot. Hydrogels were cultured both as unconfined gels in tissue culture plates and within our custom built bioreactor, which is capable of delivering physiologically relevant electromechanical stimulation.^8-10

Addition of cECM did not negatively impact the gelation of the silk-HRP hydrogels and the gelation kinetics can be tuned by altering the concentration of the starting silk solution or the molecular weight of the silk fibroin protein. In unconfined culture, we found that acellular gels demonstrated a time-dependent stiffening in culture, which is useful for the generation of infarct models to study how CF activation and CM viability and function change based on changes to tissue mechanics.

For the culture of activated cardiac fibroblasts, the addition of cECM to elastic silk-HRP gels enhanced and altered gene expression that was modulated by time-dependent stiffening of the hydrogels. Importantly, the addition of cECM augmented protein expression particularly in the proteins important in integrin mediated signaling. In addition, subcutaneous implant studies demonstrate the injectable nature of these formulations and relate the in vitro results to in vivo potential. On-going work is focused on the evaluation of neonatal CMs within the bioreactor system to determine the role of cECM composition, hydrogel mechanical properties, and electromechanical stimulation on CM maturation and function. Future work seek to tune the stiffening process to aid in functional muscle tissue restoration upon injection in vivo.

References:

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