| ObjectiveEpidermal cells combined with automicroskin were transplanted to repairthe full-thickness defects wound of rats, to research wound healing quality, whichwould provide the experimental foundation for clinical study.Methods1. Isolation, cultivation and marking of rat epidermal cells(1) Epidermal cells from dispase, trypsin-treated adult rat dorsal skin weregrown in serum-free medium. The cells were identified by immunohistochemistry.They were randomly divided into experimental group and control groupsubcultured by trypsinization with0.25g/L trypsin or2.5g/L trypsin respectively.The survival rate was assessed by trypan blue staining and the absorbance valueof adherent cells was analyzed by MTT assay. The subcultured cell morphologywas observed with inverted microscope. Cellular cycle was detected by flowcytometry and the senescent cell rate was calculated by SA-β-gal staining.(2) Epidermal cells were transfected with lentiviral vector carrying RFP atdifferent multiplicity of infection (MOI=0,5,20,50,100). The expression of RFPin keratinocytes was observed under fluorescence microscopy. The transfectionefficiency was detected by flow cytometry to evaluate the most proper MOI. Theeffect of RFP lentivirus on cell proliferation was analyzed by MTT assay.2. Establishment of wound model and wound repair(1) Establishment of wound model: The rats were anesthetized, shaved,disinfected. Sixteen adult SD rats were randomly divided into model group andcontrol group. A piece of full-thickness skin of3.0cm×3.3cm was removed fromthe dorsum of each rat, thus forming the wound with full-thickness skin defect.One steel wire frame was sutured with the wound margin skin in model group, butnot in control group. All the wounds covered with allograft. The woundcontraction rates were observed at7，14and21days post-operation, respectively.(2) Wound repair: Fifty adult SD rats were randomly divided into5groups(n=10) to model the wound with full-thickness skin defect, microskin with expansion ratio of1:10, microskin with expansion ratio of1:20combined withepidermal cell suspensions, microskin with expansion ratio of1:20, epidermal cellsuspensions, transplant noting as control group. All the wounds covered withallograft from twenty-five adult Wistar rats. The allogeneic skin graft was madeinto part-thickness. Wound healing rate was measured. Observed large sheet ofallogeneic skin conditions and shedding time. Biopsies were collected for HEstaining, laminin and type IV collagen immunohistochemistry examination on14,21days after operation. In microskin with expansion ratio of1:20combined withepidermal cell suspensions, epidermal cell transfected with lentiviral vectorcarrying RFP. The fluorescence in wound was detected by whole body imagingtechnique of small animals.Results1. Isolation, cultivation and marking of rat epidermal cells(1) The cells were positive for CK14and CK19. The survival rate ofexperimental group [(94.6±1.7)%] was significantly higher than that of controlgroup [(66.2±2.6)%, t=15.815], P<0.05]. The absorbance value of adherent cellsin experimental group (0.205±0.015,0.225±0.014,0.265±0.021) was significantlyhigher than that of control group (0.176±0.015,0.196±0.011,0.221±0.019, tvalues respectively2.947,3.517,3.476, P values all below0.05). The5thpassaged cells in experimental group with pebble-like appearance while cells incontrol group got large, proliferated slow, and gradually covered the bottom of theflask into a sheet. The cellular percent of S+G2/M phase in experimental group[(30.6±2.3)%] was significantly higher than that of control group [(17.0±5.6)%,t=3.890, P<0.05]. The senescent cells rate in experimental group [(10.9±2.0)%]was significantly lower than that of control group [(75.9±4.1)%, t=24.624,P<0.05].(2) Fluorescence began to appear after transfection24h, fluorescenceenhanced after transfection48h, the higher level was observed after transfection72h. The RFP positive expression was1.68%,17.15%,47.53%,69.90%at72hafter infection with RFP lentivirus with MOI=5,20,50,100. At72h afterinfection, the absorbance value in MOI=0,5,20,50,100were respectively1.14±0.143,1.05±0.073,1.02±0.090,1.03±0.141,0.912±0.102, with MOI=0the non-transfection group and MOI=5the absorbance value was significantly higherthan that of MOI=100group (F=3.022, P<0.05).2. Establishment of wound model and wound repair(1) Establishment of wound model: The wound contraction ratio decreasedsignificantly at7,14and21days post-operation in model group group(1.3±0.5)%,(1.9±0.9)%,(2.6±1.3)%than those of control group (8.6±1.2)%,(37.4±2.6)%,(65.0±3.7)%(t=15.911,36.581,44.847, P<0.01).(2) Wound repair:①General observation: Allogeneic skins were red5days after operation. Itwas dry and began to fall14days after operation. Allogeneic skins were off after21days in four experiment groups. The time of allogeneic skins completely off incontrol group was late. When the allogeneic skins were completely off, there werepatches of epithelial in the wound in microskin with expansion ratio of1:10,microskin with expansion ratio of1:20combined with epidermal cell suspensions,microskin with expansion ratio of1:20, epidermal cell suspensions groups. Thewound healing rate was(84.3±11.9)%in microskin with expansion ratio of1:10group,(74.2±8.0)%in microskin with expansion ratio of1:20combined withepidermal cell suspensions group,(59.2±10.8)%in microskin with expansion ratioof1:20group,(53.8±11.5)%in epidermal cell suspensions group,(22.7±5.5)%in control group. There were significant difference in these groups (F=34.446,P>0.05). The wound healing rate in microskin with expansion ratio of1:20groupwas lower than that of microskin with expansion ratio of1:10and microskin withexpansion ratio of1:20combined with epidermal cell suspensions groups(P<0.05). Wound in epidermal cell suspensions group was also healing but theepithelial thin and easily liquefied. The majority of the wound in control groupwas granulation.②Histological observation: HE staining showed that the new epithelialmade the allogeneic skin leave wound14d after grafting in microskin withexpansion ratio of1:10, microskin with expansion ratio of1:20combined withepidermal cell suspensions, microskin with expansion ratio of1:20, epidermal cellsuspensions groups. The allogeneic skin off, a tratified epidermis had formed21dafter grafting in microskin with expansion ratio of1:10, microskin with expansionratio of1:20combined with epidermal cell suspensions, microskin with expansion ratio of1:20groups. In epidermal cell suspensions group, epidermis containsmultiple vacuoles. There was no epithelial under the allogeneic skin in controlgroup after14d. After21d, allogeneic skin off, the wound showing granulationtissue. The positive staining pattern for laminin and type IV collagen were seen inepidermis-dermal conjunction in microskin with expansion ratio of1:10,microskin with expansion ratio of1:20combined with epidermal cell suspensions,microskin with expansion ratio of1:20groups. There was no expression inepidermal cell suspensions and control groups, some positive staining only indermis neovascularization.③Observed in vivo imaging of small animals, wound fluorescence imagingin fluorescently labeled cell transplantation group.Conclusions1. Rat epidermal cells can be obtained by using a modified enzyme digestionmethod in better vitality and proliferation.2. The lentiviral vector containing RFP gene was infected the rat epidermiscells efficiently. MOI value is50, as may be appropriate for epidermis cellstransfected.3. Using steel wire frame could effectively prevent full thickness skindefective wound contracture.4. When the expansion ratio of microskin increased, in combination withepidermal cells can increase the rate of wound healing, reducing the exposedwounds. It improves the efficiency of autologous skin for the treatment ofextensive burns to bring new ideas. |
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