The Study Of Constructing The Bio-hydrogel Derived From Decellularized Tissue Containing Mesenchyreal Stem Cells And Exosomes For The Treatment Of Volumetric Muscle Loss

The volumetric craniomaxillofacial skeletal muscle defect, caused by genetic diseases, abnormal growth and development, surgical excision of tumor or trauma, has always influenced the physical functions of stomatognathic system and facial aesthetics enormously. Unfortunately, the existing therapies, including artificial prostheses, free or pedicle myocutaneous flap, inevitably held some deficiencies such as the lack of function, limitation of surgical site or the surgical trauma of donor-site. Therefore, the efficient repair of muscle tissue defect in both oral function and external shape, without any side effects, has become the doctor’s chasing target.In recent years, with rapid development and integration of life sciences and other related disciplines, tissue engineering, as a new interdisciplinary subject, has been regarded to more and more people, and does well in skeletal muscle’s reconstruction field. Muscle tissue engineering is an approach the cells, featured the potential of myogenic formation, are delivered to the tissue defect area by the biocompatible scaffold materials, toregenerate the desired tissue through their owns differentiation potential or inducing effect. Although, so far there are several kinds of cells, biomaterials and methods for the research in this area, a relatively large volume of muscle defect cannot be treated effectively, attributed to the improper microenvironment or the insufficient blood supply.To solve these problems, we constructed a bioactive gelatinous restoration derived from decellularized skeletal muscles, in which the rat umbilical cord mesenchymal stem cells(r UCMSCs) and specific exosomes were wrapped, to repair the volumetric muscle loss.Firstly, we isolated, cultured, identified and purified the r UCMSCs, and prepared the porcine derived decellularized muscle hydrogel(p DMH). We investigated the growth and proliferation of r UCMSCs cultured in p DMH by Alamar Blue and Live/Dead cell staining, and determined the optimum concentration of p DMH accordingly. Then the impacts of p DMH on myogenic differentiation of r UCMSCs were detected by cell morphologic analysis, q RT-PCR and Western Blot. Secondary, r UVECs were cultured and identified, and the influences of the secretions of r UCMSCs cultured in normoxic or hypoxia state on proliferation, transference and angiogenesis of r UVECs by cell counting, wound healing assay, Transwell, tube formation assay and related gene/protein analysis. Based on this, we extracted and identified the exosomes from the conditioned medium of r UCMSCs under hypoxia culture, and explore their influences on biological characteristics of r UVECs in different concentration. The animal grafting experiments were performed to validate the proangiogenic potential of these exosomes. At last, we embed the r UCMSCs and the exosomes into the p DMH to obtain a tissue engineered reparative implant for the volumetric loss of mice’s tibialis anterior, and the short and long-term therapeutic effects were assessed by both histological and functional examination.The experimental results show that the bio-hydrogel derived from porcine decellularized skeletal muscle can supports and promotes the growth, proliferation and myogenic differentiation of r UCMSCs. In addition, the exosomes secreted by r UCMSCs under hypoxia culture could promote the proliferation, transference and angiogenesis of r UVECs in vitro and enhance the vascularization in vivo. And the defect treatment of the decellularized tissue hydrogel restoration, containing the r UCMSCs and the specificexosomes, in mice’s tibialis anterior achieved satisfactory results. All these results indicated that in the present study, we provide a new promising treatment strategy for skeletal muscle defect in oral and maxillofacial region.