Effects of allogeneic mouse adipose-derived mesenchymal stem cell-microporous sheep acellular dermal matrix on healing of wound with full-thickness skin defect in mouse and the related mechanism

Objective:To explore the effects of allogeneic mouse adipose-derived mesenchymal stem cell(ADSC)-microporous sheep acellular dermal matrix(ADM)on wound healing of full-thickness skin defect in mice and the related mechanism.Methods:One Kunming mouse was sacrificed by cervical dislocation to collect adipose tissue from the inguinal region.Mouse ADSCs were isolated from the adipose tissue and cultured in vitro.Cells in the third passage were identified by cell adipogenic and osteogenic differentiation.The expressions of CD34,CD73,CD90,and CD105 were analyzed by flow cytometer.After one sheep was sacrificed with the skin of its back cut off,microporous sheep ADM was prepared by using acellular processing and freeze-thaw method.A round and full-thickness skin defect wound,with a diameter of 12 mm,was made on the back of each of 36 Kunming mice.The wounds were covered by microporous sheep ADM.The mice were divided into ADSC group and control group with 18 mice in each group according to the random number table method after surgery.A volume of 0.2 ml of DMEM/F12 culture medium containing 1×10^(6)ADSCs was injected between microporous sheep ADM and the wound of each mouse in ADSC group,while 0.2 ml of DMEM/F12 culture medium was injected between microporous sheep ADM and the wound of each mouse in control group.At post-surgery day(PSD)12 and 17,the wound healing rate in each group was calculated respectively;wound vascularization in 2 groups of mice was observed under the reverse irradiation of back light;and the granulation tissue in the wound in ADSC group was observed by means of hematoxylin-eosin staining.At PSD 7,the thickness of the granulation tissue in the wound was measured in each group of mice.At PSD 12 and 17,the immunohistochemical method was used to detect the expression of VEGF in each group of mice.The number of samples was 6 in each group at each time point in the above experiments.The data obtained were processed with t-test and factorial design ANOVA.Results:(1)After 7 days of adipogenic induction,red lipid droplets were observed in the cytoplasm with oil red O staining.After 21 days of osteogenic induction,black calcium deposition was observed in the medium stained with silver nitrate.The expression levels of CD73,CD90,CD 105 and CD34 in cells were 97.82%,99.32%,97.35%and 5.88%respectively.The cells were identified as ADSCs.(2)The wound healing rates of ADSC group at PSD 12 and 17[(78±6)%,(98±3)%]were significantly higher than those of control group at PSD 12 and 17[(60±9)%,(90±4)%,t=4.26,4.46,p<.01].(3)At PSD 7,no vessels obviously grew into the center of the wound in both groups of mice,while the granulation tissue already covered the wound in ADSC group.At PSD 12,the wound in ADSC group was more well-perfused than control group.At PSD 17,it was observed that large vessels were crossing through the whole wound in ADSC group,while large vessels were observed without crossing through the whole wound in control group.(4)In ADSC group,at PSD 7,the wound was covered with thin granulation tissue,and the granulation tissue was obviously thickened at PSD 12.At PSD 17,the granulation tissue was covered by epidermis.At PSD 7,the thickness of the granulation tissue in the wound in ADSC group[(0.62±0.05)mm]was significantly greater than that in control group[(0.31±0.04)mm,t=12.27,p<.01].(5)At PSD 12 and 17,the expression levels of VEGF in the wound in ADSC group[(80.7±2.2),(102.8±2.6)/mm^(2)]were significantly than those in control group[(59.5±2.4),(81.5±2.6)/mm^(2),t=15.95,14.14,p<.01].Conclusions:Allogeneic mouse ADSC-microporous sheep ADM can promote angiogenesis and the growth of granulation tissue in the wound with full-thickness skin defect in mice,thus accelerating wound healing.The mechanism is probably related with the increase in the expression of VEGF.