Proliferation of Myoblast Skeletal Cells on Three-Dimensional Supermacroporous Cryogels

Abstract

Cardiac and skeletal muscle tissue engineering provides a smart approach to overcome problems associated with organ transplantation and cardiac tissue and also lays a platform for superior alternative approaches in muscle regeneration. The aim of the study was to demonstrate cryogel scaffold potential in the field of skeletal muscle and cardiac tissue engineering. Poly-hydroxyethyl methacrylate (pHEMA)-gelatin cryogel scaffold was synthesized using cryogelation technique and such a designed material is being reported first time. Rheology study of the pHEMA-gelatin (HG) suggested that the cryogel scaffolds were stable at different temperatures and phase angle remained constant in both dry and wet state. HG cryogel was able to bear increased stress without leading to deformation. Monitoring the hydration of HG scaffold showed shift from a stiff to a more pliable material and upon continuing hydration, shear modulus remained constant with no further change observed. However, the change in phase angle <0.24º indicates a gradual increase in stiffness of the material over time. Scaffold synthesised using such polymer combinations gave cells a native environment for proliferation and surface stiffness have shown to help in differentiation of the cells. Myoskeletal cell lines were cultured on these scaffolds to check the biocompatibility and cell proliferation. Alamar blue assay performed over a period of 3 weeks analysed the metabolic activity of cells which showed more than 60% increase in the total cellular activity. DNA content of cells was found to be directly related to number of cells present at a given time point and this was found to have increased by more than 50% in 3 weeks. Since in 3-D scaffold the surface area is more in comparison to 2-D, hence better cell proliferation is observed. Hoechst and DAPI staining showed tubular structure and alignment of the cells during formation of the tubules shows promising cellular response to the cryogel matrix. The mechanical strength, stiffness and elastic measurements of the scaffold indicated potential application of these materials for skeletal and cardiac tissue engineering.

Chitosan–amylopectin/hydroxyapatite and chitosan–chondroitin sulphate/hydroxyapatite composite scaffolds for bone tissue engineering

“…Hoechst staining was performed according to the method of Singh et al (2010) [34]. Hoechst 33342 stain has been used to observe the growth and proliferation of live cells on the developed scaffolds.…”

Heat-shock-induced cellular responses to temperature elevations occurring during orthopaedic cutting

“…DNA content was determined following recovery periods of 7 and 14 days, using a Hoechst DNA assay [25]. Briefly, cells were subjected to cell lysis by three cycles of freeze -thawing in molecular grade water.…”

Chondrogenic differentiation of canine myoblasts induced by cartilage-derived morphogenetic protein-2 and transforming growth factor-β1 in vitro

“…Myoblasts have been considered a candidate cell source for tissue engineering and reparative medicine by virtue of their potential to differentiate into chondrocytes, fibroblasts, osteoblasts and other tissues of mesenchymal origin (19,20). Compared to the above-mentioned cell sources, myoblasts represent a more promising source for cartilage engineering, as they are relatively abundant and easily accessible with minimal donor site morbidity (21).…”

“…For chondrogenic differentiation, 3.0x10 5 myoblasts in a 15-ml conical polypropylene tube were centrifuged at 500 g for 5 min at 20˚C (15) and cultured at 37˚C with 5% CO 2 in 0.5 ml DMED-high glucose (Sigma) medium containing 1% FBS, insulin (6.25 µg/ml; Sigma) and ascorbate 2-phosphate (50 µg/ml; Sigma). Human CDMP-2 at different concentrations (10,20,50 and 100 ng/ml; PeproTech EC), TGF-β1 (10,20,30 and 50 ng/ml; Sigma) and different concentrations of CDMP-2 (10, 20, 50 and 100 ng/ml) together with TGF-β1 (20 ng/ml) were added to the cultured pellets. The medium was changed every 3 days for 21 days.…”