Extracellular muscle regeneration by decellularized bovine muscle and PCL-muscle matrix composite tissue scaffolds

This study has been financially supported by TÜBİTAK under the project named "Extraocular Muscle Regeneration by Decellurized Bovine Extraocular Muscle and PCL-Extraocular Muscle Matrix Composite Tissue Skeleton" (1002 short term R&D support program, project number: 119S915). The aim of this thesis is to develop ideal tissue skeletons suitable for practical use in pathological conditions like severe muscle loss and thyroid ophthalmopathy. Initially, a new hybrid synthetic tissue skeleton composed of poly(caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA) and extracellular matrix powder (ECM) was fabricated and characterized. For this purpose, PLGA membranes were produced by solvent casting method and PCL nanofibers were collected on these membranes by electrospinning (PCL-PLGA). Then, the decellularized ECM was lyophilized, pulverized into powder form, and coated on PCL-PLGA membranes by gelation of ECM (ECM-PCL-PLGA). Membranes were characterized by various analysis methods such as scanning electron microscopy (SEM), energy dispersive X-ray analysis (XRD), Fourier transform infrared spectroscopy (ATR-FTIR) and mechanical strength tests, and the success of production was confirmed. Potency of these skeletal materials to support muscle regeneration was assessed by in vitro and in vivo analyses. In vitro experiments revealed that these materials support attachment and proliferation of mouse myoblast cells (C2C12) according to 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) analysis. In SEM imaging, C2C12 cells were observed to be adhered in an aligned manner similar to muscle tissue along the fibrous network of scaffolds. Real Time Polymerase Chain Reaction (RT-PCR) analyzes were used to examine for differentiation and it was thought that the effect of ECM contents used in the study on differentiation occurred in the first 7 days, and that the cell proliferation mechanism might be more dominant than differentiation in the following days. After ethical approval from Kobay D.H.L. AŞ. (date: 17.01.2019 and number: 336) in vivo experiments were carried out using 30 rabbits and whole thickness defects of 5 mm in length from the insertion were created in vertical recti of both eyes. The grafts produced were sutured to these defects. Muscle tissues were taken from rabbits on the 15th day to follow the early healing response and on the 45th day for the late phase of healing. The muscle tissue forming potentials of the scaffolds was tested by electrophysiology tests and histological staining. No infection was observed in any groups during the surgical procedures, and it was observed that suturing of HK grafts was much easier as compared to others. In the early period, edema and swelling in the graft were evident in some cases of HK grafts, while intense inflammatory reaction, vascular fullness and increased vascularization were observed in the histopathological examination. In the late phase, the upper rectus muscles of the rabbits were separated from the insertion and the contractile responses to the electrical stimulus were evaluated. The best single twitch responses were obtained in the positive control group, while the HK, PCL-PLGA and ECM-PCL-PLGA groups showed similar responses and the response of the PLGA group was found to be low. In the late histopathological examinations, muscle tissue development was similar in the HK, PCL-PLGA and ECM-PCL-PLGA groups, and the corneal distance of the healed muscle fibers was found to be the best in the positive control group, and the furthest distance was detected in the PLGA group. In the late period, the development and differentiation of muscle tissue lagged behind the other groups in the negative control group. The organization of tissue and muscle fibers were observed to be better in HK grafts. In conclusion, decellularized bovine eye muscle and synthetic hybrid tissue scaffolds were found to be biomaterials promoting muscle tissue regeneration. It has been shown that HK, which is a natural material, provides convenience in surgical procedures and can be used as an alternative to autograft by accelerating the development of regular fibers in the late period.