Hybrid hydrogel templates for cardiac tissue engineering

Abstract

Cardio-mimetic hydrogel-based biomaterials are inevitable in bioengineering cardiac tissue. The research focused to engineer and evaluate biologically favorable and cardiac compatible hydrogel scaffolds using the natural polysaccharides such as alginate, starch and carboxy methyl cellulose reinforced with synthetic polymers PVA and PEG for cardiac tissue engineering (CTE). Two panel of hydrogel scaffolds viz ACPV and ASPG were synthesized. ACPV hybrid hydrogel system was prepared by interpenetrating alginate and cellulose with the synthetic polymer PVA and the subsets ACPV1 and ACPV2 were prepared by varying the composition of the co-polymers. Similarly, PEG was used in ASPG system instead of PVA and the two subsets ASPG1 and ASPG2 were prepared by altering the constituent ratio of co-polymers. Divalent cation, Ca2+ was used as the crosslinking agent in both the preparations. ATR-IR analysis, water profiling, surface morphometry studies, contact angle and tensile strength measurements and biostability studies evaluated the physicochemical and mechanical properties of the hydrogels. The findings showed that all the hydrogels exhibited appreciable properties with respect to the presence of surface functional groups, optimum pore architecture and water holding capacity. The hydrogels were amphiphilic and biodegradable with appreciable mechanical properties. Cell material interaction was determined by direct contact assay, MTT assay and by evaluating the extent of cell adsorption and penetration onto hydrogel surfaces, using H9c2 cells. Direct contact assay demonstrated that the cells did not show any deviation from the normal morphology suggesting the biocompatibility of the hydrogels. Cytotoxicity studies of H9c2 cells cultured with hydrogel extract revealed viability of >80% in all subsets. Both ACPV and ASPG hydrogels displayed superior adhesion and spreading of H9c2 cells. Both ACPV and ASPG hydrogel systems promoted plasma protein adsorption on their interstices where the major fraction of adsorbed protein was contributed by albumin. The hydrogel surfaces were functionalized for antioxidant and antimicrobial activity by loading them with ascorbic acid and antibiotics viz, amikacin and vancomycin respectively. The antibiotic loaded ACPV and ASPG hydrogel subsets displayed significantly increased zone of inhibition against Gram-positive and Gram-negative bacteria. ACPV and ASPG hydrogels loaded with ascorbic acid displayed appreciable antioxidant response in vitro cell free system as evident from DPPH and nitric oxide scavenging assay. Interestingly, the hydrogels showed inherent antioxidant potential as they demonstrated significantly decreased level of oxidative stress in RAW267.4 cells upon direct contact with ACPV and ASPG hydrogels. Immunocompatibility of ACPV and ASPG hydrogels were evaluated by studying the expression of inflammatory mediators such as IL6, NF-Kβ, IL-10, TGF-β, TNF-α and IKβ using RAW 264.7 cells. The activation of RAW cells upon contact with the hydrogels were minimal suggesting the immunocompatibility. Immunofluorescence revealed that ACPV hydrogels exhibited immunomodulatory effects when compared with ASPG system based on the expression status of IL6, NF-Kβ, IL-10, TGF-β, TNF-α and IKβ. The genes and regenerative pathways involving these genes were assessed by NetworkAnalyst program. The study concluded that the selected genes were intimately associated with 370 genes and 120 pathways related to inflammation and/or immunomodulation revealing their potential network.