The Experimental Research Of Perfusion-Decellularized Skeletal Muscle Acellular Matrix Regeneration

Background Skeletal muscle injuries resulting from traumatic accidents, tumor ablation, or degenerative disease result in volumetric muscle loss(VML) with limited treatment options and high morbidity.Although adult skeletal muscle has robust ability to regenerate in response to injury, this regenerative response is dependent upon the severity of muscle insult [1-3]. Generally, if more than 20% of the muscle is lost, the natural repair process will fail and result in an accumulation of scar tissue, dennervation of muscle distal to the defect and a loss of function [1-3]. Clearly, there exists a great need for therapeutic options that can promote the innate ability of skeletal muscle to regenerate and restore function following severe trauma and induce de novo formation of functional muscle. The stem cells were inoculated into Skeletal muscle whole organ acellular matrix(SMWOACM),the tissue engineering lung,liver and heart for living in vivo implantation can be achieved. Skeletal muscle accounted for 40-50% of the body,it is very commom to Skeletal muscle trauma,incuding volumetric muscle loss(VML). Since skeletal muscle is single function of organs,it is easy to obtain tissue engineering muscle by inoculating the stem cells into Skeletal muscle acellular matrix(ECM).Recent studies have shown that homologous tissue of liver and lung biology stents may be more suitable for the organization than a specific organization reconstruction,till now, many people made attempts to get skeletal muscle acellular matrix,nevertheless, there were not been reported for the complete with vascular network of extracellular matrix. Extracellular matrix(ECM)scaffolds derived from organ decellularization are promising, increasingly used biomaterials for tissue engineering. The perfusion-decellularization of skeletal muscle has been poorly assessed and characterized, and the bioactivity and functional capacity of the obtained skeletal muscle ECM(PM-ECM)to remodel, following volumetric muscle loss(VML) injury is unknown. In the present study,PM-ECM was prepared from porcine rectus abdominis(RA). Perfusion-decellularization of porcine RA effectively removed cellular and nuclear material while retaining the intricate three-dimensional microarchitecture and vasculature networks of the native RA while retaining many of the bioactive ECM components and mechanical properties. Why do we choose porcine rectus abdominis as raw materials? Let’s look at the anatomy.(1)structure of rectus abdominis: Rectus abdominis can be used as muscular or musculo-cutaneous flap, pedicled or free flap, indifferent sizes or forms, allowing a great amount of clinical applications, including breast reconstruction after mastectomy, reconstruction of the thoracic wall, reconstruction of the pharynx or oesophagus, reconstruction after tumoral excision in locally advanced soft tissue sarcomas, reconstructive surgery in head and neck area. The rectus is a relatively isolated skeletal muscle surrounded by rectus sheath, with good blood supply. The arterial vascularization of rectus abdominis muscle is realized by the superior epigastric artery(branch of internal mammary artery at the level of the seventh rib) and the inferior epigastric artery(branch of external iliac artery). Inferior epigastric artery has its course between the muscle and the posterior wall of the sheath and at the level of the umbilicus divides into several vessels that anastomose with the superior epigastric artery. There are also anastomoses between the terminal branches of the 4 or 5 lower posterior intercostal arteries and the lumbar arteries and the inferior epigastric artery. porcine rectus abdominis muscle is an very similar to that in human, including the surrounding structure and internal vessel branching patterns.(2)Structure and blood vessels of rectus:the smallest contracting units is the muscle fibers,It is composed of a cylinder, many nuclei, and myotube, Every muscle fiber is surrounded by a thin layer of connective tissue called the endomysium, Approximately 20–80 of these muscle fibers are grouped together in a parallel arrangement called a fiber bundle that is encapsulated by a perimysium, which is thicker than the epimysium enclosing each of the bundled muscle fibers. A distinct muscle is formed by enveloping a large number of muscle fascicles in a thick collagenous external sheath extending from the tendons called theepimysium, major blood vessels of muscle is along the long axis, which give rise to nourish blood vessels at right or oblique angles to the outer membrane, arteriole enter into perimysium vertical muscle fiber, travel perpendicular the muscle fiber axis until giving rise to terminal branches that penetrate the perimysium and immediately branch into numerous capillaries,which in skeletal muscle are approximately 4 μm in diameter, they taper to a larger diameter as they approach post capillary venules. the outflow through a given venule is not derived from its parallel venule,.objective To obtain the PM-ECM support produced by decellularized porcine rectus abdominis(RA) with perfusion; to compare the physicochemical property of PM-ECM with that of SIS material, to finish in vitro repopulating cell detection; to build the model of SD rat abdominal wall, and compare the remediation effect of PM-ECM with that of SIS patch, which was centered with the organic reconstruction, revascularization and biocompatibility in an effort to provide an experimental basis for seeking for the ideal biomaterial.Material and method Part I: the preparation and evaluation of the PM-ECM support perfused with acellular matrix of porcine rectus abdominis(RA). 1. The material-sampling of porcine rectus abdominis was done, the acellular matrix of porcine rectus abdominis was prepared based on the procedure of accellular perfusion. 2. SIS patch was prepared. 3. Determining the content of DNA、SDS、GAG、b FGF and EGFR in PM-ECM, which were to be compared with that of SIS patch, immunohistochemical experimental marking layer connexin, IV collagen, fibronectin, the 3D structure of matrix was observed under electron microscope, and the accellular completeness of PM-ECM was tested. 4. Testing the biomechanics performance of PM-ECM Part II: in vitro bioactivity detection and evaluation of PM-ECM 1. An in vitro cultivation of C2C12 was done to the culture of degradation product ofPM-ECM and SIS, the small chamber method was used to observe the migration of C2C12 with different content of matrix. 2. Comparing the differentiation and proliferation of C2C12 cell in the culture of degradation product of PM-ECM and SIS. 3. The Alamar-blue method was used to observe the metabolism of C2C12 cell at the surface of PM-ECM and SIS. Part III: the effect and evaluation of defect model of repair of rat abdominal wall by PM-ECM and SIS. 1. 36 SD rats were randomly divided into six groups with six rats in each group, an abdominal wall defect model was artificially produced, PM-ECM and SIS was respectively used to repair the defect, and blank control was accordingly set up. 2. A postoperative observation on whether a death had occurred or not and whether a wound rupture or surgical site infection had occurred or not was made. No lax abdominal wall or fistula in the repaired region had been observed. 3. The rats had killed at the 2nd week and at the 8th week to observe whether a seroma had occurred, the sample in the repaired region was cut off for tissue slice, the regeneration of muscle fibers, vessels and neurocytes in repaired region and shrinking of patch was observed.Result The accellular perfusion of skeletal muscle was complete, no cellular structure and composition was observed through the SKWOACM histological and scanning electron microscopic examination, DAPI staining was negative; the DNA content in completely freeze drying PM-ECM was lower than 500ng/mg, by which it can be calculated to determine that 99.5% of DNA in natural skeletal muscle had been removed; all of the residual DNA were short-chain DNA(200-500bp), meeting the accellular golden standard.(2)no residual chemical substance existed, sterilization had no influence on its biological property.(3)the 3D parallel arrangement of extracellular matrix of natural skeletal muscle, periodical ultrastructure of epimysium, perimysium and endomysiumhad been retained, the basilar membrane of skeletal muscle was attached to the internal surface of endomysium. The network having main vessel and blood capillary was retained, a terminal artery and two accompanying terminal veins can be seen; the tissue structure was complete, the internal blood vessel matrix network can endure the 300 mm Hg high pressure liquid perfused through dominating vessel with only few leakages at the distal incisal end.(5) the muscle-tendon joint structure having decellularization was retained, which can directly conduct mechanics.(6)PM-ECM retained many bioactive constituents: it was proved by immunohistochemical staining that PM-ECM contained the basis constituents of skeletal muscle basilar membrane such as IV collagen, laminin, fibronectin.(7)PM-ECM can be made into many ramifications, including slice, particle, fluidization composition, gel; regarding the biomechanics test, the ultimate tensile strength of PM-ECM was similar to that of natural muscle, it had a greater ultimate tensile strength than SIS. 2. The optimum efficiency of myocyte chemotaxis can be achieved with the PM-ECM content at 50ug/ml, the optimum efficiency of myocyte proliferation and differentiation can be achieved with the PM-ECM content at 50ug/ml, it was shown by the in vitro experiment that PM-ECM group was better than SIS group, it was simultaneously proved that, the PM-ECM degradation product can further the in vitro differentiation, proliferation and metabolism of myocyte. 3. In terms of defect model of repair of rat abdominal wall by PM-ECM and SIS, it was shown by observation that, the seroma repaired by biological patch had a high morbidity, the region repaired by PM-ECM was better than the one repaired by SIS patch in terms of neovascularization, myofiber growth, cytopoiesis of nerve fiber, muscle regeneration and shrinking of patch, PM-ECM had a good biosecurity, proving that the in vivo repair of abdominal wall defect of rats by PM-ECM was safe and viable.Experimental conclusion The skeletal muscle vascular perfusion was complete; the 3D parallel arrangement of extracellular matrix of natural skeletal muscle, periodical ultrastructure of epimysium,perimysium and endomysium had been retained; the bioactive factors such as GAG、b FGF、EGFR were retained; 2.the remediation effect on repairing matrix by PM-ECM was better than that of SIS patch, having a good histocompatibility.innovation points 1. the entire-organic decellularization technology for skeletal muscle perfusion was used and the 3D microcirculation structure of extracellular matrix was retained. 2. the skeletal muscular entire-organic decellularization patch repair of abdominal wall defect provided a basis for repair with 3D biological material.