| BackgroundLarge-area skin defect is a difficult problem in plastic surgery.Skin expansion,wherein a silicone expander is implanted under the skin and regularly filled with saline to promote cellular proliferation and tissue regeneration,is widely used in reconstructive surgery.This method is used to generate "extra skin tissue",which matches the texture,color,and structure of adjacent healthy skin.However,low expansion efficiency,high retraction,dermal thinning,and local ischemia in expanded skin severely limit its clinical application.Numerus drugs such as theophylline,dimethyl sulfoxide,verapamil,and some growth factors have been used in attempts to circumvent these problems.Nevertheless,these treatments showed limited effects,toxicity,and short half-life in vivo.Transplantation of stem cells to activate wound repair and regeneration is a novel therapeutic strategy that has been received much attention.The process of skin expansion involves repeated microtrauma and repair.Transplantation of bone marrow derived mesenchymal stem cell and adipose-derived stem cell could promote skin regeneration during skin expansion.Hair follicle bulge-derived stem cells(HFSCs),which are present in the bulge region of the hair follicle,are closely associated with skin,and are involved in complex interactions with cells distributed in the epithelia and dermis.HFSCs are involved in skin wound repair,and HFSC transplantation could promote cutaneous wound healing.Further,HFSCs can differentiate into epidermal,sebaceous gland,neuronal,and vascular endothelial cells.Thus,HFSCs,like BMSCs and ADSCs,have multilineage differentiation potential and contribute to wound repair,and are more closely associated with the skin.Further,human HFSCs can be obtained by plucking hair,which is easy to perform and causes minimal harm to the donors.Despite these advantages,the effects of transplanted HFSCs on expanded skin regeneration have not been studied.We hypothesized that HFSC transplantation may promote expanded skin regeneration.In this study,GFP-HFSCs were cultured,locally injected into the expanded skin in a rat scalp expansion model,and subsequently tracked to determine their effects on skin regeneration.These could help us elucidate the mechanism by which HFSC transplantation contributes to tissue regeneration during skin expansion.A large number of animal experiments have been carried out on HFSC.According to previous reports,some studies used HFSC derived from newborn rats,while some studies used HFSC derived from adult rats.However,are the characteristics of HFSC age-related?Which is suitable for cell culture and stem cell therapy?As far as we know,there is no relevant report of such research.We use HFSC from rats in different age to study the influence of age on the characteristics of stem cells,such as migration,self-renewal,proliferation ability and multiple differentiation potential.ObjectiveThe purpose of this research is to explore the role of HFSC transplantation on the regeneration during skin expansion and its mechanism,provide an experimental basis for explaining differences in wound repair at different stages of development,and give a suggestion on how to select the proper stage of donor cells for stem cell therapy.MethodsThis study includes four parts.1)HFSCs from GFP rats were isolated and cultured using an improved microdissection combined with enzymatic digestion method.Defined keratinocyte serum-free medium and a trypsin inhibitor were used to minimize HFSC differentiation.The process of cell growth was shown and recorded by inverted microscope.The cells were identified by the HFSC markers(CK15,CD34,and integrin β1)and Oil Red O staining.2)HFSCs derived from newborn and adult rats were compared to investigate the effects of age on stem cell characteristics,such as morphology,diameter,surface markers,differentiation ability,mortality,growth curve,viability.3)The head expansion model of rats was established with a 1 mL special expander.And a 1 cm×l cm area of scalp under which the tissue expander would be placed was marked.GFP-HFSC transplantation was performed on the 7th day after surgery.Expanders in both groups were routinely inflated(1 mL each time)3 times a week after cell transplantation.The day of cell transplantation was defined as day 0.Tissue specimens were collected on day 7 and day 28.For HFSC group and control group,the detection index includes three aspects:the first is the area,weight,retraction rate,thickness,number of proliferating cells,collagen content,blood flow and new blood vessels in the expanded skin;the second is the cell type differentiated by transplanted GFP-HFSC;and the third is growth factors,including bFGF,EGF,TGF-β and VEGF.5)The growth factors related to fibroblasts in HFSC conditioned medium were detected by ELISA.Then the fibroblasts were treated by stem cell conditioned medium in vitro.The effects of growth factors secreted by HFSCs on the viability,cell migration and collagen of fibroblasts,were detected by CCK-8 test,scratch test and Real-time PCR.Results1)The method of microdissection combined with enzymatic digestion described in this research was successfully used to isolate and culture the bulge HFSCs from the vibrissae of newborn rats.The cells presented the typical morphology of stem cells,such as small cell size and large nuclei;cobblestone,stereoscopic and nest appearance;high colony-forming ability.Mitotic figures were occasionally observed in these cells,revealing an in-vitro proliferative potential.High expression of CD34 and integrin β1 were detected by flow cytometry,and CK15 was detected in the cytoplasm of the cells by immunofluorescence staining.2)Both types of cells exhibited the typical morphology of stem cells,When the cell size was compared using the images,the newborn HFSCs were smaller than adult HFSCs.The cell diameter was further quantified and compared using an automated cell counter.The diameter of the newborn HFSCs were smaller than that of the adult HFSCs at the same passage(pb<0.05).Using a repeated-measures ANOVA,the diameter of the newborn HFSCs did not diff er during P0,P1,P3,and P5(pa>0.05)and an increased tendency of the diameter of adult HFSCs was observed during P0,P1,P3,and P5(pa<0.05).Flow cytometry and cellular immunofluorescence showed no difference in cell surface antigen phenotypes between the two groups(p>0.05).Adipogenic differentiation and oil red O staining displayed that the differentiation potential of newborn HFSC was higher than that of adult HFSC(p<0.05).The mortality rate of the newborn HFSCs were lower than that of the adult HFSCs at P0 and P1(p<0.05).However,the mortality rate did not differ between the newborn and adult HFSCs at P3 and P5(p>0.05).The newborn HFSCs was significantly more than adult HFSCs on the same day(p<0.05).As shown in the CCK-8 results,compared with adult HFSCs,P3 newborn HFSCs exhibited greater viability at all the time points observed(p<0.01).3)Expanded flaps in two groups were compared.Larger area,more weight,and lower retraction rate were shown in HFSC group.H&E staining images showed that the epidermis and dermis were thicker in the HFSC group than those in the control group<p<0.01).Masson staining and Real-time PCR detected more collagens in HFSC group(p<0.01).PCNA positive cells were most located in the hair follicle and basal layer.The number of PCNA positive cells in epidermis and dermis of HFSC group was greater than that of control group(p<0.01).Microcirculatory blood flow detected by full-field laser perfusion imager was significantly higher in HFSC group than in the control group(p<0.01).Immunofluorescence staining images of CD31 positive cells showed significantly more vessels in the HFSC group than those in the control group(p<0.01).The double-immunofluorescence staining was to track the distribution of transplanted GFPHFSCs in the expanded skin.GFP+/CD31+cells,GFP+/CK10+cells,and GFP+/CK14+cells were observed in the HFSC group.However,no GFP+/vimentin+cells were found in the HFSC group.In addition,the protein and mRNA expression levels of EGF,VEGF,bFGF,and TGF-β were higher in HFSC group than those in control group through Western Blot and Real-time PCR.4)The expression of EGF,bFGF and TGF-β were detected by ELISA in HFSC conditioned medium.The scratch test showed that migration ability of fibroblast treated with 50%HFSC-CM was significantly higher than those treated with lower HFSC-CM(0,10 and 25%)or higher HFSC-CM(75%)(p<0.01).The fibroblasts treated with 50%and 75%HFSC-CM had a higher viability compared with those treated with HFSC-CM(0,10 and 25%)using CCK-8 assays.Real-time PCR detected higher mRNA level of Col I and Col Ⅲ in fibroblasts treated with 50%HFSC-CM(p<0.01).Conclusion1)The method of microdissection combined with enzyme digestion was successfully used to isolate and culture HFSCs.2)The characteristics of HFSC are age-related.And HFSCs derived from newborn rats are more suitable for stem cell culture and therapy than those derived from adult rats.3)HFSCs can promote regeneration in epidermis and dermis through directly differentiating into vascular endothelial cells,epidermal cells and outer root sheath cells of hair follicles.4)HFSCs promote the regeneration of expanded skin through the paracrine of growth factors(including EGF,VEGF,bFGF,TGF-β).5)In vitro experiment,HFSCs could promote the migration ability viability,and collagen secretion of fibroblasts through the paracrine of EGF,bFGF and TGF-β. |
