Tuning composition and architecture of biomimetic scaffolds for enhanced matrix synthesis by murine cardiomyocytes

Abstract

A major onset of heart failure is myocardial infarction, which causes the myocardium to lose cardiomyocytes and transform into a scar tissue. Since mammalian infarcted cardiac tissue has a limited ability to regenerate, alternative strategies including implantation of tissue-engineered scaffolds at the site of damaged myocardium have been explored. The goal is to enable in situ cardiac reconstruction at the injured myocardium site, replace the lost cardiomyocytes, deliver the required biomolecules, and remodel the extracellular matrix (ECM). ECM synthesis and deposition by cardiomyocytes within such scaffolds remains categorically unexplored. Here, we investigated the survival, ECM synthesis and deposition, and matrix metalloproteinases (MMPs) release by cardiomyocytes within three-dimensional (3D) substrates. Rat cardiomyocytes were cultured for three weeks within two structurally different substrates: 3D collagen hydrogels or polycaprolactone (PCL) nanofibrous scaffolds. The concentration and composition of the hydrogels was varied, while PCL nanofibers were surface-modified with various ECM proteins. Results showed that myocyte attachment and survival was higher within collagen hydrogels, while myocyte alignment and beating was noted only within PCL scaffolds. Total protein synthesis by myocytes within PCL scaffolds was significantly higher compared to that within collagen hydrogels, although more protein was deposited as matrix within hydrogels. Significant ECM synthesis and matrix deposition, TIMP-1, and MMP release were noted within modified collagen hydrogels and PCL nanofiber scaffolds. These results were qualitatively confirmed by imaging techniques. Results attest to the prominent role of scaffold composition and architecture in influencing cardiomyocyte phenotype, matrix synthesis and cytokines release, with significant applications in cardiac tissue remodeling strategies.

Cardiomyogenic differentiation of human bone marrow‐derived mesenchymal stem cell spheroids within electrospun collagen nanofiber mats

“…The PLGA/fibrin composite scaffolds also led to higher expressions of cardiac specific markers, such as troponins, actinin, tropomyosin, and desmin when compared to that in pure PLGA scaffold. We previously noted significant improvement in cell survival, alignment, and matrix synthesis, by rat cardiomyocytes within ECM‐coated, aligned PCL nanofibers, compared to uncoated counterparts . Specifically, the beating frequency and the average number of beating cells were significantly higher in collagen (type I) coated PCL nanofibers, compared to the uncoated PCL nanofibers .…”

“…Here, higher percentage of exclusive expression of MSC stemness (CD90), early (GATA4) or late (cTnT, cTnI) cardiac phenotypes would indicate the stemness or homogeneity of the differentiated cells during differentiation, while their co‐expressions would indicate cell heterogeneity. LIVE/DEAD® viability/cytotoxicity kit (Molecular Probes) was used to determine the cellular viability and toxicity at various culture conditions and at each time points (days 1, 12, and 28), using protocols described earlier …”

“…LIVE/ DEAD ® viability/cytotoxicity kit (Molecular Probes) was used to determine the cellular viability and toxicity at various culture conditions and at each time points (days 1, 12, and 28), using protocols described earlier. 24 Cell alignment analysis Cell alignment was assessed using an in-built angle measurement function of ImageJ, where the angles were measured with respect to a reference axis from calcein AM-stained live images of the cells at days 12 and 28 of culture within collagen nanofiber mats. The reference axis was selected based on the direction in which maximum numbers of cells aligned in each field of view.…”

Sensitivity of neural stem cell survival, differentiation and neurite outgrowth within 3D hydrogels to environmental heavy metals

“…A LIVE/DEAD® viability/cytotoxicity kit (Life Technologies) was used to quantify the number of surviving cells within each culture condition (n = 6 wells/concentration/metal/time point) as we explained earlier (Gishto et al, 2014). Experimental details are available in supplementary methods.…”

Materials for cardiac tissue engineering

“…In this context, different natural and synthetic biocompatible and biodegradable materials (Figure 1) have appeared in the experimental set. -Natural materials such as fibrin [75], chitosan [76], alginate [10] or collagen mixtures [77] [110]. (I) Immunofluorescence against βIII tubulin (green), cTnI (white), and elastin (red) labelled nerve fibres (arrows), M, myocardium and P, patch, respectively [111].…”