Directed Reconstruction Of Hair Follicles

BackgroundAt present, autograft of hair follicles is the most important treatment for hair loss, especially for male pattern baldness. But there are some problems for this treatment, such as formation of scar in donor site, scantiness of donor site, low density of transplantations, low survival rate of transplantations, so the tissue engineered hair follicles provide a promising method for repairing hair loss. A hair follicle is composed of epithelial cells including matrix, out root sheath inner root sheath and dermal cells including dermal papilla and deral sheath. Dermal papilla cells are a kind of fibroblast cells, that can aggregate during growing in mediums containing FCS. According to the different phages of hair cycle, the shape of the dermal papilla change periodically. It plays an important role in the formation of hair follicle and hair cycle, so establish the culture model of dermal papilla cells in vitro is very important to study the characteristics of dermal papilla cells and induction of hair follicles. The epithelial cells is the base for producing hair fibers, so it is absolutely necessary for reconstructing hair follicles. But the method of isolating these cells is just fit to be used in a lab, it can not meet the needs required by tissue engineering.Objectives(1) To study the method for isolating hair follicle cells from skin of mouse.(2) To study the effect of epidermal cells, hair follicle cells and transplanting sites on hair follicle reconstruction results.(3) To study the effect of biodegradable material on hair follicle reconstruction results.(4) To study factors decide the direction of reconstructed hair follicles.Methods(1) Isolation of hair follicle cells from skin of mouseBack skin was removed from 3-5day-old C57 BL/6 mice, rinsed 3 times in phosphate buffered saline (PBS), and laid flat in 0.2% dispase solution at 37℃for 2 hours. The epidermis was then peeled away from the dermis and conduced paraffin sectioning for H&E staining. The dermis rinsed three times in PBS, cut into small pieces with scissors, took one piece for paraffin sectioning for H&E staining. Pieces of dermis were digested in 0.2% collagenase I at 37℃with agitation at 100 rpm for 30m. Then, completed medium was added and filtered through a 250μm mesh. Collected filtrate and centrifugated at 150g for 5m, sediment was resuspended in culture medium to centrifugation at 20g for 3m, repeated 2 times. Sediment resuspended in culture medium with equal volume 9% Ficoll to make hair follicle suspension. Add 5 ml 9% Ficoll into a 15 ml conical tube, then add 10 ml hair follicle suspension(containing about hair follicles isolated from 5-10 mice) and centrifugat ed at 40g for 5m. Sediment was resuspended in culture medium and centrifugated at 40g for 5m, repeated 3 times. Sediment was collected and digested in 0.2% collagenaseⅠat 37℃for 1h, then added PBS and centrifugated at 450g for 5m. Next,0.125% trypsin was added into the sediment and digested at 37℃for 10m, vibrated intermittently. Added completed medium into the digest and filtered through 25 um mesh, the filtrate was centrifugated at 450g for 5m. Washed the resulting sediment by resuspending in complete medium followed by centrifugation at 450g for 5m, repeated 2 times. Cell viability was tested by Typan Blue Staining method. Expression of CD34 and CD117 was tested by Flow Cytometry.Plate fresh isolated hair follicle cells on 35mm culture dish at 1×105个/ml and incubated at 37℃in 5% CO2 for 1 hour, remove medium containing slowly attaching cells, such as keratinocytes, added completed medium for cell culture and passaged as routine procedure. Alkaline phosphatase(ALP) andα-Smooth Muscle Actin(α-SMA) staining was performed according to the manufacture's instruction.(2)Injection of hair follicle cellsBack skin was removed from 3-5day-old C57 BL/6 mice, rinsed 3 times in phosphate buffered saline (PBS), and laid flat in 0.2% dispase solution at 37℃for 2 hours. The epidermis was then peeled away from the dermis and digested in 0.125% trypsin at room temperature for 10m, pipetting intermittently to dissociated epidermal cells. Added complete medium and filtered through 25um mesh. Washed the resulting sediment by resuspending in completed medium followed by centrifugation at 450g for 5m, repeated 2 times.Epidermal cells and hair follicle cells were labeled with Dil and DiO respectively according the manufacture's induction.0.1 ml of epidermal cells(1×107/ml), hair follicle cells(1×107/ml), epidermal cells(1×107/ml)+hair follicle cells(1×107/ml), hair follicle cells(1×106/ml), hair follicle cells(1×105/ml), hair follicle cells(1×104/ml),1 passaged hair follicle cells(1×107/ml),2 passaged hair follicle cells(1×107/ml),3 passaged hair follicle cells(1×107/ml) and epidermal cells(1×107/ml) +foot pat fibroblast cells(1×107/ml) was injected into the dermis of 5 sites on the back skin of one nude mouse respectively. The injection site was harvested for paraffin section and frozen section after 3 weeks. The injection site of hair follicle cells(1×107/ml) was harvested after 2d,4d, 1w,3w and 6w.5 sites were marked on the back skin of 1 nude mouse, a puncture was made by 20 ml syringe. A pipette tip was inserted through the puncture and 0.1ml of hair follicle cells(1 X 107/ml) was injected under the skin. The injection site was harvested for paraffin section and frozen section after 3 weeks.(3) Hair follicle cells transplanted with biodegradable materialCollagenⅠsponge was cut into pieces of 1×1cm.0.1ml of hair follicle cells(1×107/ml) was dropped on each piece and transplanted under the skin of nude mouse. Transplanting site was harvested after 4w for paraffin section and frozen section.PLGA micro-tube was cut into pieces of 1.5cm and inserted under the skin of nude mouse.0.1ml of hair follicle cells(1×107/ml) was injected into the micro-tube. Transplanting site was harvested after 4w for paraffin section and frozen section.CollagenⅠsponge, collagenⅠsponge cocultured with hair follicle cells for 1w and PLGA micro-tube were detected by scanning electronic microscope.(4)Chamber transplantation of hair follicle cellsOpen chamber transplantation of hair follicle cells the chamber was made by a 5ml tube lid removed the central part. A rounded full-thickness skin was cut off from the back skin of nude muse and plugged the chamber with rim under skin. The chamber was exposed to air and covered by tap with holes.0.1ml of hair follicle cells(1×107/ml) was dropped on the chamber which was removed after lw. Transplanting site was harvested after 4w for paraffin section and frozen section.Closed chamber transplantation of hair follicle cells The chamber was made by a 5ml tube lid removed the central part and transplanted under the skin through a cut on the back of the nude mouse.0.1ml of hair follicle cells(1×107/ml) was dropped on the chamber which was removed after 1w. Transplanting site was harvested after 4w for paraffin section and frozen section.Results(1) Isolation of hair follicle cells from skin of mouse Most hair follicles of the back skin of 3d C57BL/6j mice were fully developed. Hair shafts emerged on the skin surface of 5d C57BL/6j mice. Because of the Dispase's selective digestion for basement membrane zone, epidermis can easily peeled off from the dermis. Dermis contained lots of hair follicles with integrated structure without epidermis retaining. These hair follicles can collected by filtration, differential centrifugation and density gradient centrifugation. By digestion with collagense and trpsin, hair follicles were dissociated into single cells with viability higher than 85%.Flow cytometry showed 2.5% cells were positive for CD34 and 8.3% cells were positive for CD117. Primary culture showed self aggregated property which was gradually disappeared with passaged culture. Cells after 3 passage showed no self aggregated property. Aggregated cells were positive for ALP andα-SMA. No ALP expression was found in cells after 4 passage which were still positive forα-SMA.(2) Injection of hair follicle cellsA cyst containing grey tissue formed in the injection site 3w after intradermal injection of epidermal cells. H&E stained paraffin section showed no hair follicles, frozen section showed a little red fluorescence. No change was found in the injection site 3w after intradermal injection of foot pat fibroblast cells. H&E stained paraffin section showed no hair follicles, frozen section showed bright green fluorescence. A cyst containing grey tissue formed in the injection site 3w after intradermal injection of epidermal cells plus foot pat fibroblast cells. H&E stained paraffin section showed no hair follicles, frozen section showed bright green fluorescence with a little red fluorescence.2d after intradermal injection of hair follicle cells, H&E stained paraffin section showed a cyst was formed containing lots of round and elliptical cells and homogeneous eosin stained cell-free tissues. The cyst wall was composed of many spindle shaped fibroblast cells. Cell clusters scattered through the inner wall with central keratinization, the extracellular matrix increased. The contents of the cyst showed the bright green fluorescence, the cyst wall showed sparsely localized green fluorescence.4d after injection, the skin slightly evaluated with grey appearance, a lots of hair follicle formed with black bulb,1w after injection, the injection site became black and evaluated with a lots of black hair follicles and hyperproliferation of capillary blood. Newly formed hair follicles showed bright green fluorescence.3w after injection, the cyst wall became thin and dense lined with epidermis-liked structure. Newly formed hair follicles localized centrally with bulb pointing to the outer wall. Sebaceous gland structure can be seen accompanied with hair follicles. 6w after injection, the cyst contained lots of sheded club hair shafts and hair follicles on the stage of anagen.The paraffin section results of injection of epidermal cells plus hair follicle cells were similar to that of epidermal cells alone. Frozen section showed the red fluorescence had very limited distribution on the background of green fluorescence. The dermal papillae showed strong green fluorescence. Passaged 1,2,3 hair follicle cells intradermal injection showed no hair follicles formed.Hypodermal injection of hair follicle cells formed only several grey cyst under the skin with a few hairs localized randomly.(3) Hair follicle cells transplanted with biodegradable materialScanning electronic microscopy showed the inner diameter of the PLGA micro-tube was 1.5mm. The thickness of the tube wall was lmm containing lots of interconnected holes with diameter varied from 10um to 50um. The diameter of interconnected holes varied from 100um to 500um. Few cells attached to the collagen sponge after 1w cocultured with hair follicle cells.4w after transplantation of collagen sponge cocultured with hair follicle cells, a few hair follicles formed with hair shafts showed green fluorescence.4w after transplantation of PLGA micro-tube containing hair follicle cells, the material became degenerate with capillary blood growing on the surface. Paraffin section showed no hair follicle formed.(4)Chamber transplantation of hair follicle cellsThe wound became contracted 1w after transplantation of opened chamber alone. 3w after removing the chamber, the wound contracted into a small scar with diameter of 2mm. The wound showed no contraction lw after transplantation of opened chamber with hair follicles. Black sediments formed in the wound. Hairy skin formed 4w after transplantation. Paraffin H&E stained section showed the structure of newly formed hairy skin was integrated with fully developed epidermis, dermis, hair follicles and sebaceous gland. Frozen section showed bright green fluorescence only localized in epidermis and hair follicles.Newly formed hair follicles formed and randomly localized on the surface of fascia with hyperproliferation of capillary blood under the skin 4w after transplantation of closed chamber with hair follicles. The whole tissue of newly formed with or without hair follicles showed bright green fluorescence.Conclusions(1) Dispase can selectively digest basement membrane to isolate epidermis from dermis without epidermal component attaching to the dermis. Hair follicle cells were mainly composed of dermal papillae cells and contained 2.5% hair follicle stem cells which was positive for CD34 and 8.3% melanocytes which was positive for CD117.(2) These hair follicle cells can reconstructed fully developed normal hair follicle by injection into the nude mouse skin. Hair follicle reconstruction depended on close interaction between cells. (3) Collagen sponge and PLGA micro-tube did not support directed reconstruction of hair follicles.(4) Hair follicle developmental space provide possibility of directed reconstruction, ail-liquid interface determines the direction of newly formed hair follicles. Open chamber transplantation with hair follicle cells can reconstructed normal hairy skin with normal distribution of hair follicles, so it is suitable for clinical application.