Construction Of Tissue-engineered Skin Using Adipose-derived Stem Cells And Acellular Dermal Matrix

Skin defects are main problems in plastics and burns surgery. Usually, autograft of skin or allograft of skin is used to repair skin defects. However, this therapy has many disadvantages, such as insufficient autografts, a severe morbidity of donor site, immune elimination towards allografts, etc. In the past two decades, traumatherapy and tissue regeneration research have got rapid progress as the result of developments of cytobiology and molecularbiology. Scientists have invented many methods for constructing tissue-engineered skin which give us new prospects for wound healing. However, most of these tissue-engineered skin products have their disadvantages, such as poor physical performances, scarce of autologous seed cells, immune exclusive reaction towards xenogenous seed cells, long culturing time, expensive cost, etc. So it is necessary to invent a new method for constructing tissue-engineered skin which will be useful in clinical practice.ObjectiveTo establish a strategy for differentiation of adipose-derived stem cells (ADSCs) into epithelial cells, then to analyse if ADSCs derived epithelial cells have some characteristics of epithelial stem cells.To observe microstructures of human acellular dermal matrix, then to analyse if it is suitable as scaffolds for tissue-engineered skin. To establish a method for construction of tissue-engineered skin using ADSCs derived epithelial like stem cells and acellular dermal matrix, then to observe structures and microstructures of this tissue-engineered skin.To repair experimental animal skin defects using tissue-engineered skin, then to observe therapeutic effectiveness and structures together with microstructures of living tissue-engineered skin.MethodsADSCs isolated from human liposuction tissue were cultured and replicated in vitro. When cultured in the epithelial induction medium, ADSCs might differentiate into epithelial cells. The cell surface markers, cell cycles, cytokeratin 5, cytokeratin 10 and cytokeratin 19 were detected before and after differentiation. Cell growth curve was also drawed before and after differentiation. When cultured in the osteoplastic induction medium, ADSCs derived epithelial cells might further differentiate into osteoblasts. ALP staining and Von Kossa staining were performed on osteoblasts.Microstructures of human acellular dermal matrix were observed. Tissue-engineered skin were constructed using ADSCs derived epithelial like stem cells and acellular dermal matrix. Then structures and microstructures were observed.Nude mice with dorsal skin defects were cured by grafting tissue-engineered skin and therapeutic effectiveness was observed postoperation. PAS staining was performed on the basement membrane of living tissue-engineered skin, and structures of living tissue-engineered skin together with ADSCs derived epithelial like stem cells labeled with DAPI were observed too.Tissue-engineered skin were constructed using swine ADSCs and acellular dermal matrix. Left skin defects on the back of these swines were repaired by grafting tissue-engineered skin. Right skin defects on the back of these swines were repaired by grafting acellular dermal matrix as contrast. Therapeutic effectiveness was observed, and structures together with microstructures of living tissue-engineered skin were observed postoperation.ResultsWhen ADSCs were cultured in the epithelial induction medium, some cells turned round or oval 72 hours later, and most cells turned round or oval resembling a monolayer culture of epithelial cells 7 days later. The cell growth curve was like "S" shape before and after differentiation. Cells replicated reaching the peak point at the seventhsixth day before differentiation, yet cells replicated reaching the peak point at the sixth day after differentiation. There were 87.42% cells in G₀ and G₁ stage before differentiation, yet 96.38% cells in G₀ and G₁ stage after differentiation. After differentiation, few cells was positive in immunofluorescence for CK5, and some cells was positive in immunofluorescence for CK19. RT-PCR revealed that CK19 was positive at mRNA level after differentiation, and CK10 together with CK19 were positive at mRNA level 10 passages after differentiation. The cells were in normal appearance 10 passages after differentiation with normal chromosomal number and normal female karyotype. There was no neoplasm forming at which the 10th passage cells after differentiation were subcutaneous injected into nude mice 2 months before. ALP staining and Von Kossa staining were positive in osteoblasts differentiated from ADSCs derived epithelial cells.Both the basement membrane and the dermis of human acellular dermal matrix had a good many pores. The diameter of pores in the basement membrane was 31.03～82.76um (51.72±15.64um), yet the diameter of pores in the dermis was 115.38～192.31um(143.27±23.95um). Many ADSCs derived epithelial like stem cells could be seen sticking to the basement membrane as we observed the tissue-engineered skin constructed for 3 days using scanning electron microscope. The cells were in round or oval shape and their diameter was 75.62～107.86um (87.11±11.46um), which was less than the diameter of pores in the basement membrane. Structures of the tissue-engineered skin constructed for 2 weeks were similar to those of normal skin except that there was no corneum in the tissue-engineered skin.1 month postoperation, the tissue-engineered skin grafted to the back of nude mice remained living with a thin layer of epithilium. There was a continuous red stained basement membrane fluctuating between the epithelium and the dermis. Two months postoperation, structures of living tissue-engineered skin were similar to those of normal skin, and no epidermoid could be seen in the dermis under eximination using microscope. 2 to 3 layers of cells with strong DAPI staining could be seen at the deep layer of epithelium 1 month postoperation, yet 5 to 6 layers of cells with weak DAPI staining could be seen at the deep layer of epithelium 1 month postoperation. The skin defects on nude mice grafted with acellular dermal matrix remained uncured with a thin layer of crusta covering them, and margins of the skin defects contracted.1 month postoperation, the tissue-engineered skin grafted to the left back of swines remained living with obvious keratinization, but it had no pigmentation. The acellular dermal matrix grafted to the right back of swines converted into guanulation tissue with local crusta, and margins of the skin defects contracted. Structures of living tissue-engineered skin were similar to those of normal skin, and no epidermoid could be seen in the dermis under eximination using microscope. There were epithelial foot and papilla dentis between the epithelium and the dermis. The acellular dermal matrix converted into guanulation tissue with no epithelium covering it under eximination using microscope. Under eximination using transmission electron microscope, there were hemidesmosomes between the basement membrane and the basal cells, keratin filaments in epithelial cells, and desmosomes between the adjacent epithelial cells.conclusionADSCs are a promising source for tissue engineering for their characteristics such as easy acquirability, extensive proliferation, multilineage differentiation, etc.ADSCs can differentiate into epithelial cells in a certain degree, and some cells have some characteristics of epithelial stem cells. So we name these cells as ADSCs derived epithelial like stem cells. When grafted into experimental animals, these cells can replicate well into multilayer shape. In this study, the strategy for differentiation of adipose-derived stem cells into epithelial cells is safe enough.Cells can stick to the basement membrane of acellular dermal matrix well. Both the basement membrane and the dermis of human acellular dermal matrix have a good many pores. The diameter of these pores are similar to the proper diameter of materials which can facilitate epithelial cells and dermal cells regenerating.Because structures of the dermis of acellular dermal matrix make it sure that fibroblasts and endothelial cells grow into acellular dermal matrix, fibroblasts needn't to be grafted into dermis when we construct tissue-engineered skin. This make the construction easier and safer, and also reduce the cost.The tissue-engineered skin we constructed can convert into normal skin tissue when grafted into experimental animals, and without epidermoid forming.In this study, satisfactory effectiveness is achieved when grafting the tissue-engineered skin to repair skin defects on experimental animals. So it may be useful in clinical practice.