Control over the In vitro spreading, cell fate and proliferation of myoblast Cells in multicomponent self-assembled peptide hydrogels

摘要

Introduction Cell replacement therapy (CRT) has considerable potential for tissue repair, particularly for the regeneration of skeletal muscle in dystrophy or following trauma; This technology is rich in potential for improving the quality of life of a patient. When therapeutic cells transplanted into the damaged tissue manage to survive, they integrate within the host tissue and provide symptomatic relief. However, one of the major limitations has been the restricted availability of appropriately specified cells suitable for transplantation and unacceptably high rates of cell death following transplantation. The engineering of an environment structurally similar to the native extracellular matrix (ECM) is necessary in order to closely match the chemical and physical conditions within the extracellular niche. Recently, nanostructured hydrogels formed by Fluorenylmethyloxycarbonyl self-assembling pepticles (Fmoc-SAPs) have shown great potential for such biological applications due to their inherent biocompatibility, propensity to form higher order structures and rich chemical functionality. Importantly, it is possible to incorporate biologically relevant macromolecules to tune the morphological and mechanical properties of the target materials. Our research aims to creative a natural ECM mimic underpinned by self-assembled Fmoc-peptide scaffolds and through the incorporation of other, larger components that can be used for the growth and proliferation of myoblast cells. To investigate the combination of multiple components in Fmoc-SAP systems, we considered three important extracellular matrix (ECM) protein-derived pepticles; the fibronectin-derived arginine-glycine-aspartate (RGD), and two macromolecules to study in the work. Fucoidan is a sulphated, fucose-rich polysaccharide and it has been shown that fucoidan suppressed the expression of the myogenic regulatory factors and the myocyte enhancer factors as well as the modification of morphology during differentiation of myoblast cells; Versican is involved in developing muscle for cell adhesion, migration, and myogenesis. MATERIALS AND METHODS Solid phase peptide synthesis was used to generate the SAPs in a crystalline powder. We used and four groups of peptide hydrogel to determine the effect of the inclusion of macromolecules. (1) Fmoc-FRGDF alone; (2) co-assembly of Fmoc-FRGDFffucoidan; (3) co-assembly of Fmoc-FRGDF/versican conditioned media and (4) co-assembly of Fmoc-FRGDF/empty vector (pcDNA3.1) conditioned media used as a control of group. We then used a suite of mechanical, spectroscopic and microscopic techniques to characterise both systems to determine the outcome of the assembly process, and thier suitability for biological applications. C2C12 myoblast cells were used to determine the biological properties of the hydrogels. RESULTS Our results indicate that all approaches form stable hydrogels. Characterisation analyses determine that the underlying structure of the hydrogels is maintained and stable nanofibrous networks are formed for both homogeneous and heterogenous systems. Small-angle X-ray scattering (SAXS) was then used to detail important structural differences between the fibrils in the respective systems. Staining showed that from day 1 to day 3, cells had an area over 10,000 μm2 in the three systems except the Fmoc-FRGDF/fucoidan hydrogel, where smaller, still viable cells were observed. Moreover, Fmoc-FRGDF/Fucoidan group has the most single nuclear cells, while Fmoc-FRGDF/versican group have more multinuclear cells indicating a potential transition to a multinuclear myotubular morphology. CONCLUSIONS we will show that by incorporation of macromolecules into Fmoc-SAPs, hydrogels were formed with the potential to control myoblast cell fate. This work highlights the promising application of Fmoc-SAP hydrogels to produce a supply of cells for the treatment of muscle diseases such as Duchenne muscular dystrophy.