Experimental Study On Platelet-Rich Plasma Loaded Human Adipose Derived Stem Cells Promoting Pressure Ulcer Healing In Mice

Objective:To investigate the effect of platelet-rich plasma loaded human adipose-derived stem cells on pressure ulcer wound healing in mice and to explore potential therapeutic mechanisms to provide an experimental basis for pressure ulcer stem cell therapy.Methods:1.Harvest subcutaneous adipose tissue from clinical thinning flap operation.HADSCs were isolated from human adipose tissue by mechanical separation and type I collagenase digestion,and incubated at 37℃in 5%CO2 saturated humidity incubator with L-DMEM Culture medium containing 10%FBS and 1%double antibody(penicillin and streptomycin).After cell subculture to the third generation,adipose-derived stem cells were transfected with a green fluorescent protein-carrying Lentivirus(virus titer of 1×108TU/ml).Multiplicity of infection(MOI)were set to 1,10,25,50,75,100.The best infection complex was took for cell transfection.2.The effects of three differentiation method were identified by alizarin red,alisin blue and oil red staining respectively.The expression of cell surface markers CD90,CD105,CD73,CD34,CD11b,CD19,CD45 and HLADR were detected by flow cytometr.3.70ml of peripheral blood was collected from volunteers,which was centrifuged three times to prepare platelet-rich plasma.The platelet was count and compared with that of normal peripheral blood.4.The model of pressure ulcer was established by taking the wild-type C57BL/6J female mice(6-8 weeks).Briefly,After abdominal anesthesia,shave dorsal hair.A template was used to mark the location of the magnetic plates to assure a consistent placement on each animal.The skin was gently pulled up and placed between 2 round ceramic magnetic plates that had 12 mm diameter and were 5.0 mm thick,with an average weight of 4.8 g.After 12hours of repression of skin ischemia,the magnet was removed for reperfusion with 12hours.After two cycles histological examination was performed on the first day and the fifth day respectively,On the first day,normal skin structure was destroyed,tissue edema and a small amount of inflammatory cells infiltrated;on the 5th day,a large number of inflammatory cells infiltrated,degeneration and necrosis of the tissue cells,and the establishment of a pressure sore model was confirmed.According to this method,wild type C57BL/6J female mice(6-8 weeks)were established pressure ulcer model,the all models divided into 4 groups according to the random table method,15 rats in each group,which were divided into platelet-rich plasma-derived adipose-derived stem cell treatment group(hADSCs+PRP),adipose-derived stem cell treatment group(hADSCs),Platelet-rich plasma group(PRP)and phosphate buffered saline(NC).On the first day after the pressure ulcer model was established,the ASC+PRP group resuspended the 3rd generation of adipose derived stem cells(1 x 10~6)with 100μl of PRP and the 3rd group of adipose derived stem cells(1 x 10~6)to the edge of the wound and the basal part,PRP group and NC group were treated with 100μl PRP or PBS,respectively.5.After transplant hADSCs to the pressure ulcer wound,the wounds healing of each group were observed.Photographs were taken on days 1,5,9,13,17 and 21.Calculating wound size with Image J software.Wound healing rate was calculate with the formula,that is:(original wound size-remaining wound size)/original wound size×100%,Image J software calculates the wound size.Mice were sacrificed on day 5,11 and 21.Hematoxylin and eosin(HE)staining was used to observe the skin structure of the wounds.Masson staining was used to observe the deposition and arrangement of collagen in each wound.The relative expression of collagen was randomly selected from 5 fields under the microscope x 400 times,and the area of red was calculated by Image J software.CD31immunohistochemical staining for detect the number of neovascularization,and the number was determined by Image J software after selecting 5 fields of vision.S100immunohistochemical staining was used to observe the skin nerve regeneration of the wounds.Five fields were selected and the percentage of positive expression was calculated by Image J software.At day 11,wound tissue was cut and making frozen slice to see if the cells survived.6.Statistical analysisThe experimental results were expressed as mean±standard deviation(x±S),and SPSS 17.0 statistical software was used for statistical analysis.After the normality test of measurement data,One-Way ANOVA was used for comparison between groups.LSD was used for multiple comparisons of variance in groups and Dunnett’s T3 test was used for variances;At the same time,the two groups were compared using independent sample t test;P value<0.05 was considered statistically significant.Results:1.Adipose-derived stem cells were successfully isolated from human subcutaneous adipose tissue.Primary cells took a long time to grow adherently and there are a lot of impurities cells.48 hours later,most of the cells adhered to bottle bottom and grew completely adherently after 72 hours and impurities cells significantly reduced.Cells showed spindle-shaped,polygonal-shaped growth,Cross-linked between the visible;the teo generation cells adherent growth faster,we can see cells completely adherent after 24hours,which were spindle-shaped growth.The lentiviral transfection assay showed that when the MOI was 25,the cell infection rate was as high as about 90%,and all cells were transfected with the virus concentration.After 72 hours of transfection,cells were infected by inverted microscopy Rate of more than 90%,cells grow well.2.Human adipose-derived stem cells(hADSCs)differentiated into osteogenic,chondrogenic and adipogenic cells after induced by three lines.Flow cytometry showed that CD90,CD105 and CD73 were positive,while CD34,CD11b,CD19,CD45 and HLADR were negative.3.Platelet-rich plasma was successfully separated and the platelet count in platelet rich plasma was 641.2±74.4×10~9/L and normal whole blood platelet was 208.4±68.9×10~9/L(t=5.781,P=0.029).4.pressure ulcer model established successfully,skin irritation and gradually see the inflammatory exudate around the wound exudation.HE staining of the first day after modeling can be observed normal tissue structure of the skin damage,the epidermis and subcutaneous fat layer disappeared,a small amount of inflammatory cell infiltration can be seen in the dermis,HE staining of 5 days shows a large number of degeneration and necrosis of the dermis,infiltration of inflammatory cells.Compared with hADSCs group,PRP group and NC group,the wounds of hADSCs+PRP group healing were the fastest,and the wound surface was almost completely healed after 9 days,while some blood crusts were still seen in hADSCs group,and PRP group and NC group were still covered with crusts.Compared with the other groups,the wounds of hADSCs+PRP group were completely covered by the hair at 21 days.Most of the wounds grew black with most of the hair growing in black.The wounds in the hADSCs group and the wounds were not seen.There were no obvious exposed wounds,the NC group and the PRP group Still visible large skin exposed,no hair growth.The wound completely healed time:hADSCs+PRP group(8.20±0.75)days,hADSCs group(13.20±1.17)days,PRP group(14.40±1.02)days,NC group(15.60±1.63)days.The wound size of day 5 in each group were as follows:hADSCs+PRP group(0.42±0.02),hADSCs group(0.57±0.04),PRP group(0.69±0.03)and NC group(0.86±0.03).Day 9 as follows:hADSCs+PRP group(0.04±0.01),ASCs group(0.12±0.01),PRP group(0.40±0.02)and NC group(0.47±0.02).Day 13 as follows:hADSCs+PRP and hADSCs groups healed,PRP group(0.12±0.02),NC group(0.22±0.03).The wound size of hADSCs+PRP group compared with other groups on day 5,9,13,statistical significance(P<0.01).The histological results showed that inflammatory cells infiltrated on the 5th day,but the infiltration of inflammatory cells in hADSCs+PRP group was the lightest,and collagen deposition was obvious.There were less collagen fibers in PRP group and NC group,there were statistical differences in each group(P<0.05).On the 11th day,the wounds of the hADSCs+PRP group were significantly reduced,collagen was deposited in a large amount,and the arrangement was more orderly than that of other groups.The gap between the collagen was broadened.Some hair follicles were regenerated.The necrotic callus was observed in hADSCs and PRP groups.Compared with PRP group and NC group,the collagen distribution in PRP group and NC group remained disorderly.A large number of infiltration of inflammatory cells were observed in NC group,there was significant difference between the groups(P<0.01).On day 21,hADSCs+PRP group had the smallest number of abnormal wounds.Most of the wounds had recovered normal skin structure.A large number of hair follicles and other skin appendages were formed.The epidermis and dermis were densely packed,the epidermis thinned,the dermis was thick and collagen was arranged in order.There was no obvious accessory structure in the skin of the PRP group and the NC group,however,the arrangement of the collagen fibers in the NC group was more disorderly and lower than PRP group.The results of immunohistochemical staining of CD31 showed that the neovascularization of hADSCs+PRP group are more than that in hADSCs group,PRP group and NC group at different points in time(P<0.05).On day 11,the number of new blood vessels in each group increased than that in the fifth day.The number of new blood vessels in hADSCs+PRP group was significantly different from other groups(P<0.05).The number of all groups reduce,but the hADSCs+PRP group has the highest number of blood vessels on day 21(P<0.01),and there was no significant difference between PRP group and NC group.The nerve regeneration of pressure ulcer wound was inferred by detecting the expression of S100,a characteristic marker of Schwann cells.S100 immunohistochemistry results showed that on the 11th day,S100-labeled Schwann cells were scattered in the skin layers of hADSCs+PRP group and hADSCs group,but hADSCs+PRP group still had statistically significant differences compared with other groups(P<0.01).At day 21,S100 immunohistochemistry showed that hADSCs+PRP group and hADSCs had the most positive expression area and covered the whole layer of skin,the former was especially,The positive expression was found in the PRP group only in the dermis,and the positive expression area was very few in the control group,and there was a statistical difference between the groups(P<0.01).The results of wound cell tracking showed that ASC was still planted on the wound surface after 11 days,there were more remaining hADSCs in hADSCs+PRP group.Conclusion:Platelet rich plasma loaded human adipose derived stem cells transplantation can reduce inflammatory infiltration,increase collagen deposition,promote angiogenesis and nerve regeneration,and Increase the survival time of hADSCs,thereby accelerating the healing of pressure ulcers and improving the quality of healing in mice.