Influence and its mechanism of allogeneic dermal papilla cells on the wound healing of full-thickness skin defects in mice
Objective To explore the influence and its mechanism of allogeneic dermal papilla cells(DPCs)on the wound healing of full-thickness skin defects in mice.Methods This study was an experimental study.DPCs were isolated from the whisker follicles of five 6-week-old male C57BL/6J mice by combining microdissection with collagenase digestion and were successfully identified.Eighteen 8-week-old male C57BL/6J mice were divided into phosphate buffer solution(PBS)group and DPC group according to the random number table,with 9 mice in each group,and the full-thickness skin defect wound model was created on the back of all mice.On day 2,4,and 6 after injury,the mice in DPC group were administered 100 μL of cell suspension containing 1×106 DPCs of the 4th passage by subcutaneous injection around the wound,and the mice in PBS group was administered an equal volume of PBS.On day 3,7,10,and 14 after injury,the wound healing and hair growth of mice in two groups were observed,and the residual wound area was measured,and the hair coverage area on the wound of mice in two groups was measured on day 14 after injury.On day 14 after injury,the wound tissue samples of mice in two groups were collected.Hematoxylin-eosin staining was performed to observe the condition of newborn hair follicles and the number was counted,Masson staining was performed to observe the collagen deposition in the dermis and the collagen deposition area was measured,the immunofluorescence method was used to detect the protein expressions of Wnt/β-catenin signaling pathway related molecules β-catenin and lymphoid enhancer binding factor 1(Lef1),and Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction were used to detect the protein and mRNA expressions of β-catenin and Lef1,respectively.The number of samples in each experiment was 3.Results Compared with those in PBS group,the mice in DPC group had accelerated wound re-epithelialization at each time point after injury,and more hair growth on day 10 and 14 after injury.On day 7,10,and 14 after injury,the residual wound areas of mice in DPC group were(13.92±2.90),(3.69±1.78),and(1.09±0.14)mm2,respectively,which were significantly smaller than(26.19±2.06),(10.84±3.59),and(6.75±2.11)mm2 in PBS group,respectively(with tvalues of 5.85,3.09,and 4.63,respectively,P values all＜0.05).On day 14 after injury,the hair coverage area on the wound of mice in DPC group was(62±7)mm2,which was significantly larger than(35±6)mm2 in PBS group(t=2.89,P＜0.05).On day 14 after injury,compared with those in PBS group,the number of newborn hair follicles in the wound tissue of mice in DPC group was significantly increased(t=5.43,P＜0.05),and the dermal collagen deposition area was significantly reduced(t=3.56,P＜0.05).On day 14 after injury,both the immunofluorescence method and the Western blotting detection showed that the protein expressions of β-catenin(with t values of 5.49 and 4.25,respectively,P values all＜0.05)and Lef1(with t values of 7.50 and 11.54,respectively,P values all＜0.05)in the wound tissue of mice in DPC group were significantly higher than those in PBS group;the mRNA expressions of β-catenin and Lef1 in the wound tissue of mice in DPC group were significantly higher than those in PBS group(with t values of 7.68 and 9.67,respectively,P＜0.05).Conclusions DPCs can accelerate the re-epithelialization of full-thickness skin defect wounds in mice by activating Wnt/β-catenin signaling pathway and promote hair follicle regeneration during the process of wound healing.
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