A Rat Model Of Skin Wound Repair By Hair Follicle Upper Segment Autograft And Its Mechanism

Background Skin autograft is the most effective method to treat skin wounds like burn and extensive avulsion.The current clinical trend is micro-skin autograft,so as to maximize the amplification of epidermal stem cells to repair the wound.However,lack of source of autograft skin is still a problem seriously obstructing the treatment of extensive skin avulsion,which makes this one of the urgent bottleneck problems that regenerative medicine needs to solve.In recent years,with the development of the study on skin appendages,researchers have found that hair follicles are the largest pool of stem cells in the skin,and suggested that the stem cells of follicle bulge located in the upper segment of the hair follicle have a great clinical application prospect in wound repair.It has been observed in clinical practice that scalp skin flaps containing hair follicles have the effect of promoting wound repair.It was first noticed in burn department that skin chronic wounds grafted with flaps containing hair follicles healed faster than those grafted with flaps without hair follicles.A clinical trial compared the effect of micro-skin graft,with and without hair follicles,on wounds of equal area,and found that the wounds with hair follicles repaired faster than the control group.On week 18,the repair area ratio was about 2.27:1.Also,studies have shown that when skin or hair follicle is damaged,hair follicle stem cells can also migrate to the damaged site and repair tissue defects through proliferation and differentiation.In conclusion,we proposed the following hypothesis: transplanting the upper segments of the hair follicle containing stem cells to the skin wounds can promote wound healing.Objective1.To establish a rat model of back skin wound and observe the effect on its repair of hair follicle upper segment autograft,with hair shaft removed.2.To explore the expression patterns of proteins SDF-1 secreted by dermal papilla cells of human scalp in different stages of hair follicle cycle.To observe the effect of exogenous SDF-1 on the number and morphology of local skin hair follicles when applied by intradermal injection on the back of rats.Methods1.A wedgy skin stripe was cut from the tentacle pad on one side of the rat following the direction hair follicles grew.Tentacle follicles were isolated from the stripe to prepare hair shaft-free upper segments.2.Rat models of back skin wound were established.3.Experimental grouping and wound treatment: rats were randomly divided into three groups.In the experimental group,8-9 upper segments of the tentacle follicles were attached to the wound on each rat’s own back;in the control group,the back skin cut from each rat’s wound was trimmed into skin flaps about 0.3cm×0.3cm,and 8-9 skins were affixed to the autologous back skin wounds;the blank group didn’t undergo any transplantation.4.The wounds were bandaged and immobilized;the rats were fitted with anti-bite collars to prevent them from gnawing on their own wounds and kept in separated cages to prevent them from biting the wounds of each other.5.On the 3rd,7th,14 th and 21 st days after the surgery,the wounds were sampled,prepared into paraffin sections to observe their repair.Morphological changes were observed by HE staining,and expression of epidermal stem cell marker CK15 was observed by fluorescent immunohistochemistry.6.Human scalps were prepared into sections and the hair growth cycle stage of the follicles in the sections were identified.The expression of the key secreted proteins of the dermal papilla cells in human scalp hair follicles was observed through fluorescence immunoassay.7.Establishment of rat model of hair follicle formation induced by exogenous SDF-1.(1)The skin of the middle posterior segment of the backs of SD rats were prepared with an electric shaving knife.After hair removal with hair removal cream,a 1.5cm×1.5cm square area was drawn with markers on each back.(2)Experimental grouping and wound treatment: rats were randomly divided into three groups.The SDF-1 injection group was injected intradermally with 300μl of exogenous SDF-1(concentration of 500ng/ml)continuously for 10 days;the PBS injection group was injected intradermally with 300μl of PBS continuously for 10 days;the blank group didn’t undergo any treatment.(3)Post-treatment and observation: the rats were kept in separated cages and the changes of the hair on their backs were observed every day.On the 12 th day of the experiment,the rats were executed and equal area of the experimental zones skin tissue were sampled from each group and prepared into paraffin sections.After HE staining,the morphology,size and structure of hair follicles were observed under the microscope and the number of hair follicles were counted to perform statistical analysis.Results1.The rat tentacles were easy to be extracted with clear morphological structure and discernible to the naked eye.2.After the upper segments of the tentacle follicles were autografted to the wound on the rats’ backs,no visible skin island formation was observed.On day 15,microscopically,new epidermal layers form on the upper surface of the transplanted hair follicle upper segments were observed,CK15 was positively expressed,and the migration trails of hair follicle stem cells were seen.3.In the human scalp hair follicle,the expression of SDF-1 was mainly expressed in the dermal papilla at the stage of catagen.4.SDF-1 and PBS were respectively applied by intradermal injection into the back skin of SD rats after hair removal for 10 days.SDF-1 injection group,PBS injection group and blank group all grew hair.The thickening of back skin of the experimental group was more obvious than that of the control group and the blank group.The number of hair follicles and hair follicle units in the SDF-1 injection group was significantly higher than that in the PBS injection group and the blank group,with statistically significant differences.However,there was no statistically significant difference between the PBS group and the blank group.Conclusions1.Autograft of the rat tentacle follicle upper segments to the back skin wound can produce a new epidermal layer,and the new epidermis is probably the result of migration and differentiation of hair follicle stem cells.2.SDF-1 can induce the formation of hair follicles on the backs of rats.