Experimental Study On The Preparation And Evaluation Of Extracellular Matrix Scaffold From Human Adipose Tissue And Its Degradation In Soft Tissue Defect Model In Rat

Objective: 1.To prepare extracellular matrix scaffold of human adipose tissue and evaluates the effectiveness of decellularization, component, mechanical properties; 2.To investigate the biocompatibility and adipogenic differentiation of human adipose-derived stem cells(hADSCs) on extracellular matrix scaffold of human adipose tissue; 3.To establish a model of soft tissue defect in rat and to explore the biological degradation of extracellular matrix scaffold from human adipose tissue in the soft tissue defect model.Methods: 1.Adipose tissue donated voluntarily by surgical patients was used as experimental material, the material was cut into pieces about 25 g, and after repeated freeze-thaw, enzymatic digestion, organic solvent extraction, vacuum freeze-drying, adult adipose tissue extracellular tissue matrix was prepared. Observations about the traits of extracellular matrix scaffold were made; the HE staining, Masson staining and DAPI fluorescence staining were used to test the effectiveness of the decellularization; immunohistochemistry was used to detect the reservations of extracellular matrix(IVcollagen, laminin), scanning electron microscopy was employed to observe the ultrastructure of extracellular matrix scaffold, and universal mechanical testing machine was used to measure the mechanical properties of the scaffolds; 2.Enzyme digestion method was used to extract human adipose-derived stem cells, cultured the cells and observed the cell morphology, dected the immunoprofile of the cells, inductived hADSCs to adipocytes and osteoblasts then oil red O staining and alizarin red staining. The 3rd passage hADSCs were co-cultured with extracellular matrix scaffold. CCK8 was introduced to assay cell proliferation activity, and scanning electron microscopy was used to observe cellular adhesion.The cells on the scaffold were induced to adipocytes and observed by freezing section and oil red O staining after 14 days, RT-PCR detected the expression of LPL and PPARγ mRNA; 3.18 female Wistar rats of clean grade level were needed in the study,removed a part of sacrospinal muscle on the left back in rats to established soft tissue defect model, observed wound healing of muscle defect in 4, 8 and 12 weeks. Established the soft tissue defects in another 18 rats, the scaffolds were implanted into the soft tissue defects. Another scaffolds were implanted into the subcutaneous tissue on the right back.Results: 1.The extracellular matrix scaffold of adipose tissue was porous sponges with a well-defined 3-D architecture. There was no obvious cell structure in HE and Masson staining, and matrix proteins were closely arranged. DAPI fluorescence staining showed no obvious positive cell nucleus. The cells in adipose tissue were completely removed. Immunohistochemical staining showed that type IV collagen and laminin staining were positive results. Scanning electron microscope showed that the scaffold was porous structure, the pores were interconnected, and the pore size was about 50-60 um. Universal mechanical testing machine showed that the maximum load value of the scaffold was lower than that of adipose tissue; 2.The collagenase digestion method can be used to isolate human adipose-derived stem cells from adipose tissue. These cells were able to grow adhering to the wall. The primary generation was short spindle shaped and the 2rd passage hADSCs were long spindle shape. Flow cytometry showed that CD44, CD90 were positive, CD34, CD45 were negative. After inductived hADSCs to adipocytes and osteoblasts, oil red O staining and alizarin red staining were positive. After the 3rd passage hADSCs were co-cultured with extracellular matrix scaffold,CCK-8 detection showed that the A value increased with time, the cells grew well, and the matrix scaffold was non-toxic. Scanning electron microscope showed that a large number of cells adhered to the scaffold. The cells on the scaffold were induced to adipocytes and observed by freezing section and oil red O staining, a large number of red dye droplets were observed in the scaffold. RT-PCR displayed LPL, PPARγ mRNA significantly high expression; 3.After tissue defect model was established, the soft tissue defect was persistent existence in 4, 8 and 12 weeks. It failed to complete repair. At 4, 8 and 12 weeks after implantation, the scaffolds were harvested, surface of scaffolds implanted in soft tissue defect and subcutaneous tissue formed capsule, and these scaffolds were evaluated by means of gross and comparation of wet weight. Obviously, the degradation of scaffold in defect group was significantly faster than that in subcutaneous group, the inflammatory infiltration of HE staining in defect group was significantly higher than that in subcutaneous group, the scaffold in two groups were not completely degraded.Conclusions: 1.The extracellular matrix scaffold of human adipose tissue prepared by decellularization technology had three-dimensional space structure. The cells in it were completely removed, kept main ingredients of extracellular matrix and mechanical properties did not decline too much; 2.hADSCs proliferation, adhesion and differentiation are good in scaffold. The extracellular matrix scaffold has the superior biological properties; 3.Soft tissue defect could be established. The extracellular matrix scaffold has good biodegradability.