Application Of Silk Fibroin And Human Chorionic Mesenchymal Stem Cells(CMCSs) In Skin Tissue Engineering

Due to the lack of sources of autologous skin, tissue engineering is becoming the best effective methods in the treatment of skin damage, which has a broad prospect for clinical application. There are two key points in tissue engineering: one is good source of seed cells the other is bio-degradable scaffolds. Silk fibroin(SF) has many advantages than the other biological materials, such as biocompatibility, biodegradability, good processability, which would be a good choice for skin tissue engineering. Chorionic mesenchymal stem cells(CMSCs) derived from the chorionic villi of human term placenta have drawn considerable interest because of their traits in rich resources, little trauma to the human body, easy to get, and multipotent differentiation potential and good immunomodulatory capacity,leading to its application in tissue engineering as a novel and abundant seeding cells.In conclusion, in this paper CMSCs is adopted as the seed cells, SF nanofibers is selected as the scaffolds for skin tissue engineering, and both the influence of SF nanofiberson biological characteristicsof CMSCs and the potential application of SF nanofibersin repairing skin defects are explored.Part â…  Isolation and culture CMSCs from the human term placenta tissue in vitro and its biological characteristics identificationObjective To isolate and culture CMSCs from term placenta tissue, andtoidentify its biological characteristics.Methods The amnion and chorion tissues were bluntly separated. CMSCs were isolated from the chorion by dissociation using trypsin. Their morphology was observed under inverted microscope. OCT4 SOX2 and NANOG, the specific markers of stem cell, m RNA expression in CMSCs were detected by RT-PCR. Flow Cytometry was used to detect the surface marker of CMSCs. Then under different induction conditions, CMSCs were induced to different mesodermal lineages and specific dye was adopted to determine their differentiation potential.Results(1) CMSCs showed plastic adherence typical fibroblastic morphology under light microscopy and obvious boundary in vitro.F2 RT-PCR results showed that cultured CMSCs expressed stem cell marker genes OCT4 SOX2 and NANOG. F3 The Flow Cytometry analysis showed that CMSCs highly expressed CD90 CD73 CD105 Hbut negative CD34 and HLA-DR.F4 Exposed to osteogenic inductive medium, osteogenic differentiation of CMSCs was identified through extracellular calcium crystals secretion by alizarin red S staining. When CMSCs were cultured in adipogenic inductive medium, intracytoplasmic lipid vacuoles were observed, and confirmed by oil red O staining. After been cultured in chondrogenic inductive medium, chondrogenic differentiation was identified by toluidine blue staining. CMSCs could form the capillary-like structure in matrigel via cultured in angiogenic inductive medium.Conclusion We successfully establish the system to extract and culture human chorionic mesenchymal stem cells in vitro. The results of RT-PCR Flow Cytometry and differentiation induction indicated that the cultured CMSCs which maintained the surfaced markers and the differentiation potential as MSCs could be expanded and used as seed cells for skin tissue engineering.Part â…¡The comparative study between CMSCs and silk fibroin nanofibers and its role in skin tissue engineeringObjective: To evaluate the biocompatibility of SF nanofibers with CMSCs and its role in repairing skin defects.Methods: On the basis of natural silk nanofibers structure, electrospun SF nanofibers with different size were manufactured via adjusting the solution concentration. CMSCs were isolated from human term placenta chorion by trypsin. The cells were cultured and passaged in vitro. Then CMSCs were seeded on silk fibroin nanofibers. The cells were cultured and observed under inverted microscope at different time point. According to the results of CCK8, portray the growth curve and detect the ability of cell proliferation. The behavior of cell state was measured with laser scanning confocal microscopy. Flow Cytometry was used detect the surface marker of CMSCs on SF material.Based on the above results, SF materials with suitable 3D microstructure for cell adhesion and proliferation could be chose and prepared. Then SF scaffolds with optimized structure were transplanted into rat and the wound healing was observed and recorded with H&E staining.Results:(1) We successfully produced SF nanofibers with different nanoscale by changing solution concentration.F2 We firstly produced SF nanofibers with varied 3D structures by combined application of electrospinning parameters freeze-drying technology and ultrasonic wave. After seeding CMSCs on silk fibroin nanofibers, the material could guide the adhesion and growth of cells so as to spread and proliferate effectively. The phenotype of CMSCs was not modified by SF according to the results of Flow Cytometry. Experiments on rat showed that silk fibroin nanofiber had played an efficient role in wound healing, and no obvious inflammation induced by SF materials was observed.Conclusion: We proved the good biocompatibility of silk fibroin nanofibers with CMSCs. Silk fibroin nanofibers can be used as an ideal material in vitro construction of tissue engineering skin and it is feasible to be applied as the covering in skin wound repair as well. This study provided a theoretical basis in the skin tissue engineering application.