| Background:The development of skin tissue engineering brings great hope to those who were suffered from skin demage,which may caused by burning,chemical damage and so on.Mesenchymal stem cells(MSCs)is a hotspot in seed-cells research.Those MSCs which were separated from human adipose tissue are called human adipose-derived stem cells(hASCs).hASCs are easy to obtain and it can also proliferate fastly and differentiate cross-germlayer and so on,which make it a very good kind of tissue engineering seed-cells.Studies have shown that hASCs can effectively reduce the inflammatory response,promote the deposition of granulation tissue,the re-epithelialization,the vascular reestablishment and the wound contraction.hASCs are able to differentiate into epidermis cells,vascular endothelial cells and thus promote wound healing.Previous studies have shown that stem-cells transplantes alone leads to a low survival rate and short living time of transplanted cells,which is considered to be a major factor in limiting stem-cell therapy.The development of biomedical tissue engineering brings us improved scaffold material and it provides a new strategy for stem-cell regeneration therapy.Extracellular matrix Matrigel is a soluble basement membrane matrix,it is a gelatinous protein mixture which secreted by the sarcoma cells of Engelbreth-Holm-Swarm(EHS)mice,the main components include:laminin,collagen IV,nestin,heparin sulfate glycoprotein and many growth factors which can be expressed spontaneously in EHS sarcoma such as transforming growth factor-?(TGF-?),fibroblast growth factor,tissue fibrogenologen activating factor and so on.The structure of Matrigel is similar to those complex extracellular environment found in different tissues,which make Matrigel possess injectability and excellent histocompatibility,so for many years Matrigel has been used as the substrate of the cultured cells by cell biologists to study cell growth,differentiation and functional morphology.Therefore,we speculate that Matrigel is a good kind of injectable scaffold material which can provide a three-dimensional support so that can protect stem-cells from the trauma microenvironment.Objective:In this study,hASCs were inoculated into Matrigel,and the hASCs-Matrigel composite was transplanted into the dorsal full-thickness defective skin of the nude mice by local injection.By analyzing the results we can discuss the related mechanism,and finally to provide a new idea and experimental basis for clinical treatment of skin trauma,especially refractory skin trauma.Methods:Part 1:The adipose tissue derived from human adipose aspiration was selected as the source of hASCs.hASCs were extracted from human adipose tissue by enzyme digestion then cultured in HG-DMEM medium to the third generation.The morphology of primary cells was observed by inverted microscope.Cell phenotype was identified by flow cytometer(FCIM).The ability of osteoblast differentiation and adipogenic differentiation of stem-cells was confirmed by adding different inducers.Part 2:Human adipose-derived stem cells were inoculated into Matrigel,and the nude mice were used to establish an animal model with dorsal full-thickness detective skin,then we randomly divided these nude mice into three groups:Group hASCs+Matrigel,Group hASCs and Control Group(PBS).Through the healing curve,which was determined by general observation,we can assess the healing ability of each group.Through the whole-body fluorescent imaging system we can observe the survival of the stem-cells in nude mice.The specimens were taken at 1d,7d and 14d after operation and then detected with HE staining and immunohistochemistry.the expression of vascular endothelial growth factor(VEGF),epidermal growth factor(EGF),b-fibroblast growth factor(b-FGF)and platelet derived growth factor-bb(PDGF-bb)were detected by qPCR,so that we can observe the survival and transformation of hASCs after injected into nude mice.Results:Part 1:After cultured in vitro for one week,most of the cells were polygonal,spindle-shaped or irregularly triangular,and grew into spiral or bundle.The proliferation rate of hASCs became faster after passage and up to 90%fusion in a week,and its immunophenotype expressed a high degree of consistency with umbilical cord blood stem cells.Continued culture showed that hASCs possessed good osteogenic and adipogenic differentiation ability.Osteogenic induction can reach more than 90%differentiation in 20 days;adipogenic induction can reach more than 90%differentiation in 10 days.Part 21.Through the treated wound area we made a healing curve and gather statistics,then we found that Group hASCs+Matrigel has the shortest healing time(significant statistical differences with Group hASCs and Control Group),Group hASCs's healing time is followed(significant statistical differences with Control Group),while Control Group has the longest healing time.2.The whole-body fluorescent imaging results showed that the cells' fluorescent signal of Group hASCs+Matrigel were stronger than those in Group hASCs,and the hASCs in Group hASCs+Matrigel were gradually converged to the wound area over time,while those in Group hASCs were more dispersed.3.Each group gathered specimens in day 1,7 and 14 after operation and then take HE staining.The results showed that the wound in Group hASCs+Matrigel had been completely healed on the 14th day after operation,the epidermal healing was good and was arranged as stratified epithelium,a small amount of inflammatory cell infiltration was visible.In Group hASCs,there were still some wounds that failed to be healed,the granulation tissue grew well and the restored part of the epidermis was good repaired,the epidermal was arranged as stratified epithelium,more infiltration of inflammatory cells infiltration is visible than Group hASCs+Matrigel in granulation tissue and wound surface.The Control Group had a considerable part of wound that not be healed,and the quality of the healed part epidermis is worse than Group hASCs+Matrigel and Group hASCs,a pile of inflammatory cell infiltration was observed,and we found scar tissue around the unhealed area.4.Each group gathered specimens in day 1,7 and 14 after operation and then take CD31 staining.The results showed that the wound in Group hASCs+Matrigel had been completely healed on the 14th day after operation,the epidermal healing was good and was arranged as stratified epithelium,new blood vessels were visible in the superficial damage layer and the deep vascular grew vertically to the surface of the wound,the density of new blood vessels was large,a small amount of inflammatory cell infiltration was visible.In Group hASCs,there were still some wounds that failed to be healed,the granulation tissue grew well,the density of new blood vessels was not as large as Group hASCs+Matrigel but still rich,the restored part of the epidermis was good repaired,the epidermal was arranged as stratified epithelium,more infiltration of inflammatory cells infiltration is visible than Group hASCs+Matrigel in granulation tissue and wound surface.The Control Group had a considerable part of wound that not be healed,a pile of inflammatory cell infiltration was observed,and has less new blood vessels than Group hASCs+Matrigel and Group hASCs.The qPCR results showed that in day 7 and 14,the gene expression of VEGF,EGF,bFGF and FDGF-bb from Group hASCs+Matrigel and Group hASCs were increased,while Control Group had no significant changes.The increasement from Group hASCs+Matrigel was the most significant(statistical differences with Group hASCs and Control Group,while Group hASCs also had statistical differences with Control Group).Conclusion:1.We obtained human adipose tissue from human adipose aspiration,and then used the classical enzyme digestion to isolate hASCs.After cell culture and passage and cell identification we obtained the third generation of hASCs,which was the seed cells we need in the experiment.2.hASCs-Matrigel composite can increase the survival rate of cells in the traumatic skin and prolong the survival time of cells as well,thus the effect of hASCs on healing wound is enhanced.3.The local injection of hASCs to treat full-thickness defective skin of nude mice can locally induce microvascular regeneration,and the three-dimensional support environment provided by Matrigel can enhance the paracrine effect of hASCs,lead to a stronger expression of angiogenesis factor,and make a increasement number of capillaries in wound area.4.After being transplanted into defective skin of nude mice,hASCs can differentiate into many different cells such as epidermal cells,fibroblasts and endothelial cells. |
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