The Effects Of Growth Environment Improvement On Hair-induction Of Dermal Papilla Cells And The Mechanism

BackgroundThe hair follicle is composed of epidermal (epithelial) and dermal (mesenchymal) compartments and their interaction play an important role in the morphogenesis and growth of the hair follicle. Complicated cross-talk between these two compartments is also thought to be crucial for successful reconstitution of hair follicles for research or therapeutic purposes. Generally dermal cells are considered as inducers and epithelial cells as responders in the process of hair formation although the signaling in between the two cells type is reciprocal and complicated.The epithelial compartments are comprised of outer root sheath (ORS), inner root sheath (IRS), hair matrix, and hair shaft, all of which arise from the progeny of epithelial stem cells locted in the bulge region of the hair follicle. The keratinocytes in hair matrix are considered as the "hair shaft factory", they are the most rapidly proliferating cell populations found in any mammalian tissue. These cells initiate their terminal differentiation to hair shaft in the precortical hair matrix when they receive a serial of signals released from dermal cells in the bottom. The dermal portion of the hair follicle can be divided into two compartments, the dermal papilla (DP) and dermal sheath (DS). The DP is located at the base of the hair follicle. The DS, or connective tissue sheath, lines the epithelium of the hair follicle from the bulge level downward and is contiguous with the base of the DP through a stalk. The DP contains a large amount of dermal papilla cells (DPCs), which can release a serial of signal or cytokines, and these signal or cytokines can stimulate keratinocytes proliferation and differentiation, and affect the hair growth and hair cycle. Therefore, the DPCs play an important role during the hair follicle development.Due to the importance of hair induction of DPCs, the models which simulate the growth environment to retain or even restore the hair inductive ability of DPCs get more attention. These models are classed into two groups:animal models for hair regeneration and organ culture for hair follicles.The animal model for hair regeneration includes chamber model, flap assay and patch assay. All the models have the same principle:a mixture of dermal and epithelial cells from newborn C57BL/6J mouse is grafted into nude mouse, so the hair regeneration can be observed in vivo, and the in vivo environment which DPCs need is imitated by the animal model. However, all of these models have drawbacks. The chamber model appeared to be the most reproducible, probably due to the method of seeding cell mixtures on a protected granulation tissue bed. Hair shafts were evident as early as 23 days after grafting using this method. Follicle quality and density appeared to be close to normal. While the chamber model provided good-quality results, it used the largest numbers of cells per assay and surgical manipulation was relatively labour intensive. The flap assay yielded similar hair shaft quality and density as the chamber model but the earliest clinical evidence of hair growth was seen at 29 days. This method required less cells than the chamber method but required the use of an intact epidermal sheet and two surgical procedures that were labour intensive. The patch assay had the least acceptable outgrowth of hair. This was because, unlike the other two methods, the cells were placed in an anatomically abnormal location. Hair follicles were disorganized, formed cysts of various sizes, and these cysts often ruptured causing foreign body reactions that ultimately underwent fibrosis, thereby reducing the graft size. However, the advantage of this method was that it required very little labour and yielded rapid histological evidence of hair follicle formation.In vivo models allow follicles to be studied in situ and detailed observations can be made using morphological criteria, immunohistochemistry. However, it is difficult to quantify molecular or biochemical parameters of individual follicles, thus the precise mechanism how individual cytokines participate in hair development is not easy to be investigated. Cell culture involves the isolation and culture of individual cell populations from which many morphological, biochemical, and molecular parameters can be measured. However, cell culture results in the loss of the normal three-dimensional architecture of the organ and as such, it is sometimes difficult to translate observations from cell culture to the whole organ. Organ culture, in which different cell types may be co-cultured goes some way towards addressing some of the disadvantages of cell culture, and organ culture of intact hair follicles can imitate the ecological environment of DPCs and the three-dimensional structure of DP, therefore the hair inductive ability of DPCs can be preserved to the greatest extent. Using this model, we can accurately demonstrate the roles of different drug or cytokines in regulation of hair growth. However, organ culture model still do not fully mimic the in vivo environment, and the hair induction of DPCs is losing continuously during the culture, affecting the growth of hair follicles.In this study,we aim to improve the environment of DPCs in two ways:first, to improve the animal model, which can provide a fine in vivo environment for DPCs, results in preservation of hair induction of DPCs, and promotion of hair regeneration and growth. Second, we try to improve the culture medium for organ culture of hair follicles, making the DPCs within the follicles could restore some of the inductive ability. The results of the study could provide important theoretical and clinical significance for the treatment of hair defect disease.Aim:1. To develop a more simple and effective animal model, which can retain the hair induction of DPCs and stimulate hair regeneration.2. To improve the culture medium for hair follicle organ culture, making the inductive ability of DPCs be restored to some extent, so the anagen phase can be prolonged.Methods:1. Improvement of the animal model for hair regeneration1.1 Preparation of epidermal sheet and dermal cells:Mouse epidermal sheet and dermal cells were isolated from C57BL/6J mice at natal day 0. After sterilization with 75% ethanol, the full thickness of the dorsal skin was removed and incubated in phosphate-buffered saline (PBS) with 0.1% dispase (Invitrogen, Grand Island, NY, USA) at 4℃ overnight. Each skin sample was washed three times with PBS, then the epidermis and dermis were separated with forceps. The epidermis sheet was put in a sterile culture dish containing Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM/10; Invitrogen) temporarily. The dermis was minced and digested in 0.2% collagenase (Sigma, St. Louis, MO, USA) at 37℃ for 1 h. After digestion, an equal volume of DMEM/10 was added, and the cell suspension was filtered sequentially through 100 μm and 40 μm mesh cell strainers. The cell suspension was centrifuged at 200 g for 5 min, then the cell pellet was resuspended in 1 mL DMEM/10 and counted. Finally, the cells were resuspended in 20 μL of DMEM as a slurry.1.2 Procedures for improved flap model (IFM)A lid from a 1.5 mL microcentrifuge tube (Eppendorf, Hamburg, Cologne, Germany) was used, and trimmed into a circle the same size as a 1.5 cm2 silicone plate. Once the dermal cells were prepared, the epidermal sheet was spread onto the smooth side of the plate with its basal side facing up. The slurry of dermal cells was pipetted evenly onto the centre of the basal side of the epidermis using a micropipette. The plate containing the epidermal sheet and dermal cells was placed in an incubator at 37 ℃, and left for 30-60 min to evaporate excess liquid before transplantation. Procedures for grafting:Nude mice were anaesthetized by intraperitoneal injection of 1% pentobarbital sodium, and the graft area of each mouse was cleaned with betadine solution. We created a single incision in the dorsal skin. After subcutaneous dissection, the plate was inserted between the panniculus carnosus of the skin flap and the musculoaponeurotic layer, and the incision was then sutured. Slurries with different numbers of dermal cells were transplanted.1.3 The traditional flap assayThe traditional flap assay was performed as the control group. Briefly, a full-thickness, three sided, rectangular skin flap (8×10 mm) was made in the dorsal skin of recipient mice. The epidermal sheet, attached to a 25×15 mm silicone sheet, was placed onto the wound area with the basement membrane side of the epithelium facing up.1.4 Opening of the flap to allow gross hair observation:When the surface of the skin flap became raised and completely dark in colour, the flap was considered ready for opening. The incision was opened and the flap inverted to expose the graft to the exterior environment, and the wound was closed by suturing the wound edge to the base of the flap.1.5 Mouse cell labelingTo assess the fate of epidermal cells in the grafts, epidermal sheets were obtained from green fluorescent protein (GFP) transgenic mice for some experiments. To track the fate of the dermal cells in the grafts, we labelled the dermal cells with dilinoleyltetramethylindocarbocyanine perchlorate (DiI), a fluorescent cell-tracking dye (Invitrogen) prior to grafting, following the manufacturer’s instructions. The GFP-expressing epidermal sheet was co-transplanted with unlabelled dermal cells, while DiI-labelled dermal cells and unlabelled.1.6 Evaluation of hair reconstitutionHair reconstitution was evaluated by visual and histological observation. For histological observation, the skin on the transplantation site was excised and fixed in 10% formaldehyde at room temperature for 24 h. Tissue was embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin (H&E). The fate of the epidermal and dermal cells in the grafts was assessed by fixing the grafts as before, freezing at -20℃ and sectioning (6-8 μm) for examination under fluorescence microscopy. To evaluate the quality of the regenerated hair shafts, we compared reconstituted hair with normal C57BL/6J hair by scanning electron microscopy.2. Improvement of the culture medium for hair follicle organ culture—The expression of lysozyme in hair follicle and its effect on hair growth in vitro.2.1 The expression change of lysozyme in hair cycleIn order to describe lysozyme expression in mouse vibrissae hair follicles (VFs), immunohistochemistry(IHC) was performed on 4-μm-thick, paraffin-embedded VF tissue sections. The sections were deparaffinized in xylene, and rehydrated by a series of graded ethanol rinses. After antigen retrieval in sodium citrate buffer, endogenous peroxidase activity was blocked and the sections were incubated with rabbit anti-mouse lysozyme primary antibody (SAB 1306215,1:1000; Sigma-Aldrich, St. Louis, MO, USA) overnight at 4℃. Subsequently, goat anti-rabbit antibody conjugated with horseradish peroxidase (sc-3837,1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied as an appropriate secondary antibody for 30 minutes at room temperature. The antibody-antigen complexes were visualized with diaminobenzidine (DAB) and counterstained with hematoxylin. The negative control group was subjected to the same steps as described above, but the primary antibody was replaced by PBS.2.2 Organ culture of VFsAnagen stage follicles were carefully dissected from the upper lip pad of 10 female C57BL/6 mice and maintained individually in 24-well multiwell plates containing 500 ul Williams E medium (Sigma-Aldrich) supplemented with 2 mM 1-glutamine (Gibco; Invitrogen, Carlsbad, CA, USA),10 mg/ml insulin (Sigma-Aldrich),10 ng/ml hydrocortisone (Gibco),1×penicillin and streptomycin solution (Gibco). Forty VFs were equally divided into four groups in each experiment, and incubated with 1-10 μg/ml lysozyme (C-type from hen egg white; Sigma-Aldrich) or culture medium. The experiment was repeated three times.2.3 Length measurement and cycle staging of VFsLength measurements were performed on individual VFs using an inverted microscope (IX71; Olympus Optical Co. Ltd, Tokyo, Japan) and hair shaft elongation was measured from the initial length of the follicles on days 0,1 and 3.2.4 Ki-67 and 5-bromo-2’-deoxyuridine (BrdU) double staining of cultured VFsFor BrdU detection, follicles were incubated with 10 μM BrdU (Roche, Mannheim, Germany) for 12 hours before harvesting. To evaluate the proliferative cells, a Ki-67 and BrdU double staining method was used. Anagen VFs were selected for fixing in 4% paraformaldehyde for 24 hours after 3 days of culture. After paraffin embedding,4 μm sections were deparaffinized and heated in citrate buffer pH 6.0 for 5 min at 100℃ and incubated with a mixture of two primary antibodies in 1% bovine serum albumin in PBST overnight at 4℃, followed by treatment with secondary antibody. The following antibodies were used:rabbit anti-mouse Ki-67 (AB9260,1:1000; Millipore, Bedford, MA, USA) and rat anti-BrdU (ab6326,1:100; Abcam, Cambridge, MA, USA). The secondary antibodies included chicken anti-rabbit TRITC-conjugated antibody (A 15992,1:100; Invitrogen, Grand Island, NY, USA) and chicken anti-rat FITC-conjugated antibody (sc-2991,1:100; Santa Cruz Biotechnology). Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). The sections were photographed under upright fluorescent microscopy (DP71; Olympus).2.5 Culture of DPCsDP were isolated from bulbs of dissected hair follicles of 27 female C57BL/6J mice and transferred onto plastic dishes coated with type 4 collagen for 3 hours, then cultured in DMEM supplemented with 100 U/ml penicillin,100 mg/ml streptomycin and 20% FBS at 37℃ in a humidified atmosphere of 5% CO2. The explants were left for 5 days, and the medium was changed every 3 days. Once cell outgrowth had become subconfluent, cells were harvested with 0.25% trypsin/10 mmol/1 EDTA in Hank’s balanced salt solution, and subcultured with a split ratio of 1:3. DPCs were maintained in DMEM supplemented with 10% FBS for further study.2.6 Western blottingDPCs at passage 2 were incubated with 5-10 μg/ml lysozyme for 24 hours, after which total protein from DPCs was extracted using RIPA lysis buffer (Millipore). Proteins were separated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Amersham, Amersham, Bucks, UK) using a wet transfer system. The blotted membranes were incubated with the respective primary antibodies at 4℃ overnight after they were blocked with 10% normal blocking serum for 2 hours, including rabbit anti-lymphoid enhancer factor 1 (LEF1) (ab85052,1:400; Abeam), rabbit anti-alkaline phosphatase (ALP) (sc-30203,1:1000; Santa Cruz Biotechnology), and rabbit anti-β-actin (ab8227,1:1000; Abeam). The membranes were incubated with secondary antibody (ab6721,1:5000; Abeam) at room temperature for 1 hour. The immune complexes were detected by using a western blotting enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology) and quantified by using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). Assays were repeated three times and relative grayscale densities were calculated.3 Statistical analysisAll data were analyzed by SPSS Statistics version 19 (SPSS Inc, Chicago, IL, USA). Results are expressed as mean ± SEM. For the animal assay, all data were analyzed by unpaired t-test. For organ culture assay, all data were analyzed by one-way ANOVA followed by Bonferroni test if the variance is homogeneous, or by Welch test if the variance is not homogeneous. P< 0.05 was considered to denote statistical significance.Results:1. We have developed a simple and rapid model for hair regeneration by improving the previous flap assay. In this new technique, trauma is reduced and the time needed for hair induction is shortened considerably.2. Lysozyme was mainly expressed in DP and DS regions of VFs, and its production in DP decreased when the VFs entered the catagen phase. In addition, lysozyme has the potential to promote hair growth by enhancing the hair induction of DPCs.Conclusion:1 We improve the growth environment of DPCs in vivo by an improved flap model, therefore the time needed for hair follicle regeneration is shortened obviously.2 We find lysozyme has a poteintail to promote hair follicle growth in vitro.3 The lysozymen can stimulate hair follicle growth by restoring parts of the hair-induction of the cultured DPCs.