Objective To supply references to tissue-engineered skin cl inical appl ications with autogenic BMSCs composited collagen membrane to repair swine full-thickness cutaneous deficiency. Methods Twenty mL bone marrow were obtained respectively from 4 swine, autogenic BMSCs were cultured and passed to the 3rd passage. The fresh bovine tendontreated by means of chemically cross-l inked was made 5 cm diameter collagen I (Col I) membrane. The 2 × 107/mL P3 swine autogenic BMSCs labeled DAPI were planted to sterile Col I membrane for 24 hours incubation, then the tissue-engineered skin was constructed. The five full-thickness skin defect of 5 cm diameter was excised to the muscle from forward to backward on the back midl ine two sides of swine. The tissue-engineered skin were implanted in the experimental group, while Col I membrane was implanted in control group. After 3 and 8 weeks of implantation, the two swine wound surface heal ing circumstance was observed and further evaluated with histology analysis and TEM. After 3 weeks of implantation, the experimental group were observed with fluorescence microscopy and staining for glycogen. Results After 3 weeks of implantation, the wound surface of control group were observed nigrescence, scab and putrescence, and after 8 weeks of implantation, also evident putrescence and scar. The wound surface of experiment group was al ive after 3 weeks implantation, appearance was leveled off and flexible without evident scar. The wound surface recovered well after 8 weeks of implantation, wound surface heal ing rate was significantly difference between the two groups (P lt; 0.01). After 3 weeks of implantation, control group were observed acestoma hyperplasia and no epidermal coverage by histology analysis. The experimental group was showed integrity epidermis and dermis structure. The basal layer was crimson and continuously positive with glycogen staining. After 8 weeks of implantation, the experimental group and control group were emerged normal skin structure. After 3 weeks of implantation in control group, a lot of neutrophil ic granulocytes and fibroblasts were noticed, but no epidermal structure was observed under TEM. In the experimental group, a lot of epidermal cells were observed, dermatome connection among epidermal cells and hemidermosome connection between basilar membrane cells and basal membrane were observed in epidermis. In the dermis experimental group, blood capillary endothel ial cells were noticed. Furthermore, considerable collagen fiber deposit was found in the surrounding tissue of fibroblasts. After 3 weeks of implantation, BMSCs labeled with DAPI were located reconstructed epidermal basement membrane and dermis by fluorescence microscopy. Conclusion Tissue-engineered skin which is composited with autogenic BMSCs as seed cells and collagen membrane were potential prospects in appl ication of repairing swine full-thickness cutaneous deficiency.
【Abstract】 Objective To compare the properties of collagen membranes before and after crossl inked and to establ ish the foundation of appl ication of collagen membranes. Methods Fresh bovine tendons were separated and collagen was extracted by washing, smashing and acetic acid dissolving. The collagen protein was determined by ultraviolet spectrophotometer and its characteristics were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), wavelength scanning and amino acids detecting. Collagen membranes were produced by lyophil ization. And then the biocharacteristics of the membranes before and after glutaraldehyde crossl inked were compared. BMSCs separated from volunteer’s bone marrow were seeded on collagen membranes before and after crossl inked by 2×103 in 100 μL medium, seven days after culture, the absorption spectrum of BMSCs was examined, and BMSCs were observed by scanning electron microscope (SEM). Results The contents of collagen protein were 2 mg/mL. The maximum absorption wave length appeared at about 230 nm. SDS-PAGE suggested that molecularweight of main bands was more than 66.2×103, the same as collagen marker from calf skin. There were 21.47% glycine, 12.04% pral ine and 10.18% hydroxyprol ine. No tryptophan was found. Before crossl inked, collagen membranes were in shape of white sponges and with big holes and the range of pH value was from 4.5 to 5.0. SEM showed reticular conformation and pore structure of collagen membranes, but the bore diameter was bigger. Their water-absorbing capacity was 61 times as much as their weight. The mechanical strength was 210 g/cm3. The dissolution time of collagenase was 90 minutes. After crossl inked, collagen membranes became thin, colorless, semi-transparent and compact with better tenacity. Under SEM, compact collagen fiber appeared reticular. There was lower water-absorbing capacity and pH value ranged from 6.5 to 7.0. The mechanical strength was 3 400 g/cm3 and the dissolution time of collagenase became longer. BMSCs could grow better either on before-crossl inked collagen membranes or on after-crossl inked ones. Conclusion As biomaterial scaffolds, after crossl inked collagen membranes were better than before-crossl inked ones.