| BackgroundAs the in-depth research of skin tissue engineering, the culture and transplantation ofepidermal cells and fibroblasts and the development and application of dermal scaffold isrelatively mature. Meanwhile, various artificial skin products have come out in succession,especially various dermal substitutes have been successfully applied in clinical practiceand received good repairing effects. And on this basis researchers tried to repair the woundepidermal and dermal layers at once by constructing active skin substitutes containedepidermal cells and fibroblast with seed cells and dermal scaffold. However, theshortcomings such as its time-consuming process and sophisticated procedure, slowvascularization and low survival rate after the transplantation have limited its clinicalapplication. Therefore, it is urgent to find an approach to quickly construct the skinsubstitutes and accelerate the vascularization in the skin tissue engineering.The construction of tissue engineered skin based mainly on the traditionaltwo-dimensional culture model at present. The routine method was to amplify a sufficientamount of cells firstly and then inoculated cells on the dermal scaffold for two or threeweeks, which could not meet the clinical use in time and easily lead to cell aging anddecrease proliferation activity because it takes a long time. The three-dimensional culturemodel and micro-carrier culture technique provides the effective method for the fast andlarge-scale culture of anchorage-dependent cells, which have been widely applied in thefield of biological pharmacy. In the previous studies, we found that mAM which isprepared from human placental-derived amniotic membrane was suitable as the dermalscaffold because of good histocompatibility, and can rapidly amplify the epidermal stemcells as the micro-carrier. Therefore, this study adopted mAM as the dermal scaffold andprovided an approach to quickly construct active skin substituteswith the three-dimensionalculture technique based on the above researches.Promoting the vascularization process of skin substitutes had been a hot spot in skintissue engineering. At present, researchers mainly adopted the methods of connectingvessels to the dermal scaffold to generate the associated factors or constructed themicro-vascular structure in vitro. However, these methods have some limitations, becausethe neovascularization needed the joint-participation, interaction and regulation of factors,extracellular matrix and associated cells, because the single regulation could not obtain theperfect effect. In addition, there were certain application prospects for constructing endothelial skin substitutes in vitro, but since its complicated operation and high techniquerequired made it hard to popularize in practice.Recent studies have shown that EPC existed in peripheral blood circulation was thekey participant in the formation of new vessels. It not only directly involved in the stemcells angiogenesis of ischemic tissues, but also the formation of the localneovascularization by differentiating into endothelial cells. However, the application ofEPC had been greatly limited because the methods of isolation and culture are not mature.New research has found that SDF-1Α-1a is the chemokine of EPC, which could combinedwith the specific receptor on the surface of EPC, CXCR4(chemokine CXC receptor type4,CXCR4) to make the EPC in the bone marrow enter into the peripheral blood within ashort time, induce the migration of EPC in the peripheral blood to the ischemic hypoxictissues and participate the re-endothelialization of neovascularization and damaged vessels.In this study, we assume to create a new approach to automatically capture the EPC inthe peripheral blood and quickly construct active dermal substitutes by use of thechemotactic effect of SDF-1α, adopting mAM as the dermal scaffold and combining thethree-dimensional RCCS.Materials and Methods1. SDF-1α Plasmid and Lentiviral Packaging1.1Construction of Recombinant PlasmidsUse the PCR method and the cDNA library to obtain the target gene, and then connectthe target gene-SDF-α with the pGC-FU carrier to obtain the recombinant plasmids. Thenthe target plasmid was transfected into293T cells, and the transfected293T samples wouldbe detected by Western Blot.1.2Lentiviral Packaging of SDF-1α-LVThe over-expressed recombinant plasmids and auxiliary original plasmids wereco-transfected into293T cells and obtained the lentivirus concentrated solution. The RealTime-PCR method was used to analyze the Ct value and the expression quantity of sampleset infected by different concentrate of virus and detect the virus titer.2. SDF-1α Lentiviral Transfection in Fibroblast2.1The Evaluation of Transfection EfficiencyThe suitable MOI values was determined by pre-experiment, the human skin fibroblasttransfected by SDF-1α-LV lentivirus, the expressions of green fluorescent after the cells transfection was observed under the fluorescence microscope; the flow cytometry was usedto detect the the transfection efficiency of virus.2.2The Determination of Target Gene and Protein ExpressionThe expression of SDF-1α gene was detected by the Real Time-PCR method and theproteincontent of SDF-1α in the secretion supernatant was determined by ELISA.3. The Experiment on EPC Chemotaxis and MigrationThe migration test and scratch test in the Transwell assay was used to determine thefibroblast of over-expressed SDF-1α and the chemotaxis and migration of supernatant onEPC.4. Construction of the Active Dermal SubstitutesRegarded mAM as the dermal scaffold, and regarded fibroblastof over-expressedSDF-1α as the seed cell, the three-dimensional rotating culture system was applied toconstruct the active dermal substitutes containing the fibroblast. and the adhesionproliferation of fibroblast was observed by the fluorescence microscope and the scanningelectron microscope.5. Transplant the Full-thickness Skin Wound with the Active Dermal SubstitutesTaken the C57BL mouse’s full-thickness skin wound as the model of animaltransplantation, the constructed skin substitutes containing epidermal stem cells weretransplanted into the wounds, detect periodic the number of EPC in the peripheral blood,determin the wound healing rate and carry out the histological detection and the evaluationof vascularization.Results1. The Identification of Lentivirus Transfection in FibroblastThe plasmid sequence of over-expressed SDF-1α was correct and the lentivirus titerwas on average109. When the MOI value reached up to20, the ideal infection efficiencycould be obtained.The infection efficiency of fibroblast was more than90%by the flow cytometry. Atthe fifth days after virus infection, the results of microscopic observation had shown thatthe staining signal of over-expressed SDF-1α group was mainly located in cytoplasm, while the whole cells of GFP group showed green fluorescence. The results of RealTimeRT-PCR had shown that the mRNA expression of over-expressed SDF-1α group was5.3-fold as much as that of the GFP group. The expression quantity of SDF-1α in thesupernatant for transfection cells was173±6pg/10^6by ELISA, but nothing was detectedin the GFP group.2. The Migration and Chemotaxis of EPC by SDF-1αov HDF CellThe migration test in the Transwell assay had shown that EPC had a distinct changeunder the effect of chemotaxis by SDF-1αov HDF. According to the statistic results, thenumbers of migrated cells in the three groups (SDFovgroup, GFP group and non-transfectedgroup) were392±27vs.204±17,181±17,p＜0.01respectively.The scratch test had shown that the supernatant in the SDF-1αov group could enhancethe capability of EPC migration. In24hours, the number of migrated cells in SDF-1αovgroup was (72±8.3), which was much higher than those of GFP group (41±5.7) andnon-transfected group (38±6.7，p＜0.01).3. Construction of Dermal SubstitutesFibroblasts could be adhered to the surface of mAM after planted half an hour. Afterculture for24hours, the shape of fibroblasts displayed well with three-dimensiona growthpatternl. Then the cells would gradually proliferate, which could be densely covered in thesurface of mAM after5-7days. Some particles adhered to each other.Scanning electron microscopy showed that the cells densely adhered to the surface ofmAM. these cells form plump and were spindle shape. Some particles were spherical inshape and curled.4. Transplantation of Dermal Substitutes to WoundThe determination of EPC in the peripheral blood at3rd,5thand7thdays aftertransplantation had shown that the percentage of EPC in the SDF-1α ov group was muchhigher than the other groups. The immunohistochemistry found that the vascularization inthe SDF-1α ov group was much more obvious than others (p＜0.01)and the vasculardensity was significantly higher than the other groups(p＜0.01). The SDF-1α contentdetected by Western Blot at3rd,5thand7thdays had showed that the SDF-1α content ofSDFovHDF-mAM group was significantly higher than other groups. The wound healing velocity of the SDF-1α ov group was significantly faster thanother groups (the healing time of SDF-1α ov group is17.25±0.7d, GFP-HDF group is18.5±1.20d, control group is18.87±1.13d),. After the wound healing, the observationhad shown that the wound of the micro-skin grafting group was smooth and soft with themilder contraction. The histological detection had showed that the particles were filled asthe dermal matrix and the multiple layer of epidermis was very thick.Conclusions1. With the SDF-1α gene over-expression, it has become a new approach to capture EPCand accelerate the vascularization of skin substitutes, which rejected the traditional method(Culture and amplify EPC in vitro, then implant it in the dermal scaffold and transplant atlast.) and provided a new way to promote the vascularization of skin tissue engineering.2. The method was to adopt mAM as the dermal scaffold, and efficiently and quicklyconstruct the skin substitute contained fibroblasts by combining with RCCS, whichimproved the traditional two-dimensional culture model. |
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