Construction Of Tissue-engineered Skin With Human Amniotic Membrane Cells For Repair Of Skin Defect

Great progress has been achieved in the research of natural skin grafts and artificial skin substitutes in recent years.several skin substitutes have been showed to be useful for replacement or reconstruction of the skin,facilitating wound healing in several different clinical settings. Many researcher believe that Stem cell has played an important role in the wound healing of skin, they had used adult stem cells such asBone Mesenchymal stem cells(BMSCs),Adipose mesenchymal stem cell(sADSCs)and Epidermal stem cell(sESCs) to repairing skin defect and achieved better repairing effects.However,The number and their sources are limited. Recent research have found that cells from amnion membrane display stem cells characteristical, In theory they can differentiate into various tissue cells. These characteristics of the amniotic cells have made them a replacement of other stem cells and received extensive attention.Cells from amnion membrane including Human amniotic mesenchymal cells (HAMCs) and Human amniotic epithelial cells (HAECs),Recent reports indicate that HAECs and HAMCs express stem cell markers and have the ability to differentiate toward all three germ layers. cell surface antigens on HAECs include CD29,CD105,CD73,stage-specific embryonic antigens (SSEA) 3 and 4,and tumor rejection antigens 1-60 and 1-81 (TRA 1-60,TRA 1-81). Surface markers thought to be absent on hAECs include SSEA-1, CD34,and CD133. In addition to surface markers, HAECs express molecular markers of pluripotent stem cells, such as octamer-binding protein 4 (OCT-4) and Nanog. The phenotype of the HAMCs was similar to that of BMSCs. CD90, CD105, CD166,CD49e, and Human leucocyte antigen (HLA)ABC positive and CD31,CD34, CD45, CD49d, CD123, and HLA-DR negative. No difference was found among the expression of one of these markers on MSCs from the different sources [1] . Both HAECs and HAMCs inhibited proliferation of activated PBMCs in a dose-dependent manner [2] .These properties, the ease of isolation of the cells, the large number of the amniotic cells and the availability of placenta as a discard tissue, make the amnion a potentially useful and noncontroversial source of cells for transplantation and regenerative medicine. Objective:To investigate the isolation, culture and stem cells characteristics of HAMCs and HAECS , and to investigate the practicability of repairing wounds with tissue-engineered skin combined with HAMSCs and HAECs for its potential application in regenerative medicine.Methods:Term placentas from healthy donor mothers obtained from caesarean sections were minced under sterile conditions. After sequential trypsin and collagenase digestion, cultivated in DMEM, the morphololgy of cells was observed under the microscope.both cells were identified by immunofluorescence, their surface antigen were detected by FCM and their differentiation potential were carryed out under specific conditions. Collagen scaffolds were utilized as the matrix,HAMCs mixed with Collagen to form a dermal layer and then HAECs seeded onto the collagen gel mentioned above to form the skin equivalent, The collagen gel containing the cells was incubated for 3 days.It was then raised onto the liquid-air surface for 2 weeks.Finally we harvested the engineer skin to repair nude mice skin defect .Results: Mesenchymal cells and epidermal cells derived from human amnion membrane, SSEA-4 and OCT-4 positive by immunofluorescence, The phenotype of the cells such as CD29 ,CD 90,CD105 were positive to a certain extent by FCM. They displays a strong proliferation and multipotential capacity .Both type of cells can differentiate into fat cells and osteoblasts.the tissue engineer skin with multiple layer of epidermis,when transplanted onto nude mice ,it receive a satify repair effection .Conclusions: HAMCs and HAECs can isolation, culture and amplification in vitro, and these cells have stem cell properties.construction of tissue engineer skin with these two type cells can promote wound healling.the skin substitute with HAMCs and HAECs has good prospects applications in regenerative medicine and tissue engineering.