[EVALUATION OF AN OPTIMIZING PROTOCOL FOR FABRICATING A SCAFFOLD DERIVED FROM PORCINE SKELETAL MUSCLE EXTRACELLULAR...

To explore an optimized protocol of decellularization to fabricate an ideal scaffold derived from porcine skeletal muscle acellular matrix.

Serial-step protocol of homogenating-milling-detergent method was used to fabricate decellularized porcine muscle tissue (DPMT) derived from native porcine skeletal muscle tissue from adult pig waist. Histological method was used to assess the effects of decellularization and degreasing. Sirius red staining was used to analyze collagen components. Scanning electron microscopy, BCA assay, and PicoGreen assay were used to evaluate the ultrastructure, total protein content, and DNA content in DPMT. The adipose derived stem cells (ADSCs), NIH3T3 cells, and human umbilical vein endothelial cells (HUVECs) were cultured in extraction liquor of DPMT in different concentrations for 1, 3, and 5 days, then the relative growth rate was calculated with cell counting kit 8 to assess the toxicity in vitro. Live/dead cell staining was used to evaluate the cytocompatibility by seeding HUVECs on the surface of DPMT and co-cultured in vitro for 3 days. For in vivo test, DPMT was subcutaneously implanted at dorsal site of male specific-pathogen free Sprague Dawley rats and harvested after 3, 7, 14, and 28 days. Gross obersvation was done and transverse diameter of remained DPMT in vivo was determined. HE staining and immunohistochemical staining of CD31 were used to assess inflammatory response and new capillary rings formation.

Decellularization of the porcine skeletal muscle tissue by homogenating-milling-detergent serial steps protocol was effective, time-saving, and simple, which could be finished within only 1 day. The decellularizarion and degreasing effect of DPMT was complete. The main component of DPMT was collagen type I and type IV. The DNA content in DPMT was (15.902±1.392) ng/mg dry weight, the total protein content was 68.94% of DPMT dry weight, which was significantly less than those of fresh skeletal muscle tissue[(140.727±10.422) ng/mg and 93.14%] (P<0.05). The microstructure of DPMT was homogeneous and porous. The result of cytocompatibility revealed that the cytotoxicity of DPMT was 0-1 grade, and HUVECs could stably grow on DPMT. In vivo study revealed DPMT could almost maintain its structural integrity at 14 days and it degraded completely at 28 days after implantation. The inflammatory response peaked at 3 days after implantation, and reduced obviously at 7 days. Difference was significant in the number of inflammatory cells between 2 time points (P<0.05). Neovascularization was observed at 7 days after implantation and the number of new vessels increased at 14 days, showing significant difference between at 7 and 14 days (P<0.05).

The homogenating-milling-detergent serial-steps protocol is effective, time-saving, and reproducible. The DPMT reveals to be cell and lipid free, with highly preserved protein component. DPMT has good biocompatibility both in vitro and in vivo and may also have potential in promoting neovascularization.