The Utilisation of GelMA-based Surface-patterned Scaffolds for Craniofacial Muscle Regeneration Applications

Abstract

In the circumstances of volumetric muscle loss of craniofacial muscles, which can happen due to accidents, for instance, the functionality of muscle tissue could be completely lost. Although muscle flap surgery is the current standard treatment utilised, a novel approach is the use of “tissue engineering” in which scaffolds, cells and additional biomolecules are exploited to aid the muscle regeneration process. In this project, gelatin methacryloyl (GelMA) was synthesised and optimised for muscle regeneration applications. Various parameters involved in the reaction, such as reaction time and methacrylic anhydride (MA) concentration, GelMA concentration, photo-initiator concentration and ultraviolet (UV) exposure time were optimised. The mechanical and biological properties were evaluated and reported for a number of formulations/processing conditions. The data suggested that using 10-15% (w/v) GelMA mixed with 0.1% (w/v) LAP and crosslinked for 2 minutes had the optimum balance between mechanical and biological properties for skeletal muscle regeneration applications. The investigation also thoroughly examined the impact of both the source and the bloom number of gelatin on the properties of the laboratory-synthesised GelMA. Specifically, porcine, bovine, and fish-derived gelatin sources were evaluated, along with porcine gelatin of 175 versus 300 bloom number. Extensive assessment of the mechanical and biological characteristics was carried out using various techniques. The results concluded that bovine-derived GelMA demonstrated superior mechanical properties compared to other groups, while no significant differences were observed in terms of biological properties. All groups showed relatively high metabolic activity and low LDH release which suggests high cell viability and a low cytotoxic response. Additionally, the ability of GelMA hydrogels (derived from porcine, bovine or fish) to promote myoblast differentiation of C2C12 muscle cells and myotube formation was evaluated. Results showed that all groups succeeded in promoting differentiation of myoblasts into myocytes and myotubes, and presented similar myogenesis ability with no significant differences after 21 days of differentiation. A key challenge in skeletal muscle regeneration is enhancing the alignment of the myocytes and myotubes. Therefore, three-dimensional (3D) printed moulds with specific sizes of grooves and ridges (300 µm, 600 µm or 900 µm) were printed using biocompatible commercial resin before GelMA hydrogels and C2C12 cells were cast in the moulds and crosslinked via UV. The influence of these patterns was characterised using DAPI and phalloidin F-actin staining to study the impact of the surface patterns on the alignment of myotubes. The Z-stack images obtained by confocal microscope illustrated that using surface patterns of 300 µm helped in improving the alignment of myotubes in comparison with the other groups. To this end, the investigation of GelMA hydrogels from different species concluded that bovine-GelMA (B-GelMA) presented the greatest mechanical properties in which all groups presented excellent biological properties in terms of promoting cell growth and attachment, as well as cellular differentiation into myocytes and myotubes. The surface pattern of 300 µm grooves and ridges demonstrated the highest cell and myotube alignment compared to larger-sized grooves and ridges. In addition, this myotube alignment, as documented by the RT-qPCR, led to enhanced gene expression of MyoG and MyoD-1.