Three-dimensional spherical gelatin bubble-based scaffold improves the myotube formation of H9c2 myoblasts.

Microenvironmental factors including physical and chemical cues can regulate stem cells as well as terminally differentiated cells to modulate their biological function and differentiation. However, one of the physical cues, the substrate's dimensionality, has not been studied extensively. In this study, the flow-focusing method with a microfluidic device was used to generate gelatin bubbles to fabricate highly ordered three-dimensional (3D) scaffolds. Rat H9c2 myoblasts were seeded into the 3D gelatin bubble-based scaffolds and compared to those grown on 2D gelatin-coating substrates to demonstrate the influences of spatial cues on cell behaviors. Relative to cells on the 2D substrates, the H9c2 myoblasts were featured by a good survival and normal mitochondrial activity but slower cell proliferation within the 3D scaffolds. The cortical actin filaments of H9c2 cells were localized close to the cell membrane when cultured on the 2D substrates, while the F-actins distributed uniformly and occupied most of the cell cytoplasm within the 3D scaffolds. H9c2 myoblasts fused as multinuclear myotubes within the 3D scaffolds without any induction but cells cultured on the 2D substrates had a relatively lower fusion index even differentiation medium was provided. Although there was no difference in actin α 1 and myosin heavy chain 1, H9c2 cells had a higher myogenin messenger RNA level in the 3D scaffolds than those of on the 2D substrates. This study reveals that the dimensionality influences differentiation and fusion of myoblasts.

An in situ poly(carboxybetaine) hydrogel for tissue engineering applications.

Hydrogels provide three-dimensional (3D) frames with tissue-like elasticity and high water content for tissue scaffolds. Previously, we reported the design and synthesis protocol of a biodegradable poly(carboxybetaine) poly(CB) hydrogel with a zwitterionic carboxybetaine methacrylate (CBMA) monomer and a disulfide-containing crosslinker via free radical polymerization. We also demonstrated that cells could be successfully encapsulated in the hydrogels without compromising cytoviability. In this study, we evaluated the cytoviability of three commonly used zwitterionic monomers (CBMA, 2-methacryloyloxyethyl phosphorylcholine (MPC) and sulfobetaine methacrylate (SBMA)) and the suitability of being utilized as precursor materials for in situ gel forming implants. These three zwitterionic monomers exhibited lower cell toxicity than other methacrylated monomers. Mixing these monomers with dimethacrylate crosslinkers initiated the gelation process in situ, which was further tested in vivo by injecting the precursor solutions subcutaneously into murine models. Poly(CB) implants retained their original shape up to 3 weeks, while poly(MPC) and poly(SB) hydrogels for shorter periods of time due to lower mechanical strengths. These hydrogels showed minimal inflammation at the injection site. We subsequently showed that the CBMA precursor solution mixed with Arg-Gly-Asp (RGD) and hydroxyapatite (HAp) nanoparticles could be applied in bone tissue engineering. Both in vitro and in vivo studies demonstrated that HAp containing poly(CB) hydrogels greatly enhanced the mineralization process of bone tissue formation. The non-cytotoxic and biodegradable poly(CB) hydrogel conjugated with cell affinity moieties is an excellent material for 3D tissue scaffolds.