Immunogenicity Of Seeding Cells In Tissue Engineered Skin And Transplanting Tissue Engineered Skin To SCID Mice

Tissue engineered skin is one of the most efficacious methods in treatment of incurable ulcers and a large area burn. Seeding cells isolated from skin biopsy and natural or synthetic materials are used to construct tissue engineered skin. Autogenic cells can avoid immunological rejection, but their disadvantages are additional trauma sustained at the patient's donor site and they limit the commercialization of tissue engineered skin. Little is known about the immunogenicity of tissue engineered skins which are constructed by allogenic cells and extracellular matrix. Keratinocytes (KC) and fibroblasts (FB) are the most important seeding cells to construct tissue engineered skins. These two cells all express MHC-Ⅰantigens but do not express MHC-Ⅱantigens and costimulatory molecules in steady state, so they can not present antigens to T cells. Some experiments showed that in the influence of interferon-γ, KC and FB expressed HLA-DR, which is one of most important structures of professional antigen presenting cells (APC) in presenting antigens. In addition, some researchers considered that KC could be induced to express B7 molecules and transmit costimulatory signal to T cells.Whether allogenic cells are rejected by host immune system, depends on allogenic cells are recognized by professional APC or not. Langerhans cells (LC) are the professional APC in skin, respiratory tract and digestive tract. LC intake antigens and migrate to local lymphatic tissues, where they become mature to activate T cells .The concrete mechanism how LC recognize antigens is not totally clear. With the development of LC culture in vitro, this problem could be solved.Why did the research of tissue engineered skin transplantation immunology develope so slowly? Because it was limited by ethics when tissue engineered skin was transplanted to hunan body directly. So there need a suitable experimental animal which can mimic human immune environment. Severe combined immunodeficiency(SCID) mice, which have no functional T cells and B cells, can be transplanted in human immunocells and tissues to reconstruct human immune system. Some experiments showed that human lymphocytes transplanted in SCID mice could reject allotransplants. The human skin transplanted in SCID mice sustained normal human skin structure for a long term. These characteristic could make SCID mice to be a powerful tool to study tissue engineered skin transplantation immunology.KC or FB were mixed with allogenic lymphocytes, and proliferation of allogenic lymphocytes was detected. The surface molecules of KC were detected by flow cytometry. The phenotype of LC was influenced by allogenic KC and FB. The SCID mice which have been transplanted on human tissue engineered skin, were implanted in allogenic splenic lymphocytes. At different time, the tissue was cut and detected the evidence of immunologic rejection.Methods:(1)KC and FB were cultured and passaged in vitro. Different passages KC or FB were mixed with allogenic lymphocytes and proliferation of lymphocytes was detect by BrdU-ELISA kit. LC in KC were marked with CD1a and were detected by flow cytometry. CD80 and CD86 expressed on KC were detected by flow cytometry in different culture system.(2) Human peripheral blood monocytes were induced to differentiate dendritic cells(LC1, LC2) by cytokines. LC1 and LC2 were detected CD1a, HLA-DR, CD80, CD86 and CD83 by flow cytometry and identified by electron microscope. LC1 or LC2 cocultured with purified KC and FB were detected to determine how the phenotype of LC1 or LC2 changed by flow cytometry.(3)The growth condition of SCID mice was observed when SCID mice were implanted in human splenic lymphocytes. The classification of human lymphocyes in SCID mice was detected by flow cytometry. The human IgG in SCID mice was detected by ELISA kit.(4)Tissue engineered skin, human foreskin and acellular dermic scaffold were transplanted to SCID mice and detected their growth by histology and immunohistochemistry. After two weeks the SCID mice were implanted in allogenic human splenic lymphocytes and detected the evidence of immunological rejection by histology and immunohistochemistry. Results:(1)Primary KC to passage 4 KC and primary FB could stimulate allogenic lymphocytes to proliferate significantly. The LC mixed in KC decressed gradually and no LC could be detected by flow cytometry after passage 5 KC. CD80 and CD86 could not be detected on passage 5 KC cultured in serum free medium and medium containing serum or PHA.(2) Human peripheral blood monocytes were induced to differentiate LC by GM-CSF, IL-4 and TGF-β1. The cells expressed CD1a at high level , expressed HLA-DR,CD80,CD86 at low level, and did no express CD83. Birbeck granules could be observed in the cells by electron microscope. After coculturing with purified KC and FB, LC did not change the expression of CD80, CD86 and CD83, and could not stimulate autogenic lymphocytes to proliferate.(3)The SCID mice implanted in 3×107 human splenic lymphocytes, did not show visible graft versus host reaction. Human T cells, B cells and NK cells could be detected in SCID mice blood after two weeks. The percentages of T cells and B cells achieved peak at the 8th week, which were 18.2±0.4% and 7.5±0.3%. The human IgG were 182.51±4.96 ug/ml at the 10 th week.(4)The tissue engineered skin and human foreskin transplanted to SCID mice could sustain normal structure of human skin. The acellular scaffold embedded in hypodermis of SCID mice could sustain its structure for a long term. After implanted in allogenic lymphocytes , the tissue engineered skin and acellular scaffold did not showed visible immunological rejection, but human foreskin showed visible immunological rejection.Conclusion:In this study, after mixing with allogenic lymphocytes, the purified tissue engineered skin seeding cells could not stimulate allogenic lymphocytes to proliferate significantly.The expression of CD80, CD86 and CD83 could not be upregulated when immature LC were co-cultured with purified seeding cells. Human splenic lymphocytes implanted in SCID mice could proliferate and secrete human IgG. After implanting allogenic lymphocytes, the tissue engineered skin did not show visible immunological rejection. So the immunogenicity of tissue engineered skin was very weak, and could not be rejected by host immune system.