| Background:Hair follicle morphogenesis is the result of interactions between hair follicle epithelial and dermal cells. Hair follicle stem cells in the epithelial and dermal papilla cells in the dermal compartment are necessary for hair follicle morphogenesis. However, these two compartments is how to interact with each other and which signals and factors involved in it is not clear. Regeneration of fully functional hair follicle is difficult in vitro owing to the requirement of its complex three-dimensional organization. In vitro hair follicle reconstitution models range from monolayer hair follicle cell culture systems to three-dimensional artificial skin models that utilize hair follicle cells. While useful for hair follicle tissue engineering to some extend, there are severe limitations to in vitro work to answer complex questions about signalling of hair follicle development and control of the hair follicle cycling. Hair follicles are supplied with nutrients and oxygen by blood vessels in vivo but they also receive signals, from neighbouring cells (paracrine effects) through signalling factors such as growth factors and cytokines. Loss or reduction in nutrients, oxygen and signalling factors in vitro does not fully mimic in vivo hair follicle development. Therefore, it is very important to establish a suitable in vivo model of hair follicle development to study the hair follicle morphology occur and the cycle molecular mechanisms.Follicular cell implantation is an experimental cell therapy for the treatment of hair loss. The process is based on the demonstration that dermal papilla cells retain the high proliferative capacity and the hair inductive capacity. However, during the process of dermal papilla cells culture, the inductive ability of dermal papilla cells was lost gradually. At present, there are no known cellular markers in dermal papilla cells whose expression correlates with hair induction, and there are no convenient in vitro assays for hair induction. Therefore, it is necessary to construct a convenient, reliable and visual model of hair follicle development to test the hair-inductive potential of follicular cells.Until to now, a number of in vivo mouse models for hair follicle reconstruction were developed. The chamber assay, first introduced by Lichti and Weinberg and updated by Lichti in2008, is one of most established models available for the study of hair follicle reconstitution. Another approach, the hair patch assay, was first introduced by Morris et al. and later refined by Zheng et al. The hair patch assay was able to reconstitute hair follicles by subcutaneous injection of a mixture of epidermal and dermal cells. In the sandwich assay, first reported by Reynolds and Jahoda, dermal papilla cells were inserted between the epidermis and dermis of the donor mice and then transplanted into a skin wound. The flap assay is a recently developed model. In the flap assay, an epidermal sheet was unfolded on a silicon plate, dermal cells were grafted onto the epidermal sheet, then the silicon palte was inserted into a surgically created wound in the skin of nude mice. Each model has its applicability. The chamber model appeared to be the most reproducible and was widely used. Epidermal and dermal cells were transplanted into a chamber in which the two components came into close contact with one other, providing the proper space and environment for hair reconstitution and excluding the intervention of receptor cells. Three weeks after cell transplantation, new hair grew from the grafted site, which provided direct evidence for the evaluation of hair follicle reconstitution. The quality and density of regenerated hair appeared to be close to normal. In experiment, we construct the chamber model of hair follicle development and examine which component, either epithelial or mesenchymal, affects and contributes to hair follicle formation.The chamber model has several deficiencies, however:(1) serious trauma to the animal,(2) frequent infection of skin wounds and subsequent animal death,(3) requires a substantial amount of cells(approximately1×107epidermal cells and1×107dermal cells) and (4) a limit of only one chamber transplanted onto each animal. As a result, the success rate has been affected to some extent.In addition, if cells are difficult to obtain, require special treatment or are transplanted using the chamber assay, the difficulty and cost of the experiment is increased. In our study, we attempted to use mini-chamber transplantation to avoid these deficiencies.Dermal papilla cells are necessary dermal compartment for hair follicle reconstitution. Nowadays, reshearchs at home and abroad mainly use microdissection and enzymatic digestion to obtain the dermal papilla cells from rodent vibrissae and human scalp hair follicle. The work was labor intensity and low efficiency. Therefore, in the study of hair follicle reconstitution, we urgent need for an easy way to obtain a large number dermal papilla cells with the ability of inductive. At natal day1in mice, hair follicle in the back skin was in before the4th stage of morphogenesis, the dermis contains a high proportion of hair-inductive cells that become dermal papillae. The in vitro expansion of natal mice dermal cells provided a logistically more feasible cell source. We co-grafted cultured dermal cells with fresh epidermal cells to determine the efficiency of cultured dermal cells for hair follicle reconstitution. Objective:(1) To construct the chamber model of hair follicle reconstruction.(2) To construct the mini-chamber model of hair follicle reconstruction.(3) To investigate the feasibility of using cultured dermal cells of noenatal mice for hair follicle reconstruction.Methods:Implantation of newborn mice skin cells with chamber method to construct a model of hair follicle developmentMouse epidermal cells and dermal cells were isolated from C57BL/6J mice at natal day0. The full thickness of the dorsal skin was removed, and then incubated in phosphate-buffered saline (PBS) with0.1%dispase at4℃overnight.The skin piece was then split into epidermis and dermis with forceps after being rinsed3times in PBS. Each component was minced. The dermis and epidermis was digested separately in0.2%collagenase at37℃for1h. After digestion, an equal volume of Dulbecco’s Modified Eagle’s Medium supplemented with10%fetal bovine serum (DMEM/10) was added, and the cell suspension was filtered sequentially through100μm mesh cell strainers. The cell suspension was centrifuged at1000rpm for5min, repeared twice and then the cell pellet was resuspended in DMEM. Adjust the concentration of dermal cells and epidermal cells at1×107/ml. dermal cells(1×107)+epidermal cells (1×107) were resuspended with200μl DMEM. Adjust the ratio of epidermal cells and dermal cells, one of the following combinations of cell mixtures were resupended in a total volume of200μl:dermal cells(1×107)+epidermal cells(5×106); dermal cells(1×107)+epidermal cells(1×106);dermal cells (1×107);dermal cells(5×106)+epidermal cells(1×107); dermal cells(1×106) +epidermal cells (1×107);epidermal cells (1×107);Chambers were made by centrifuge tubes. Detailedly, the lids of centrifuge tubes (5ml) were cut,then the central parts of the lids were removed. Affixed a layer of paper tape to the lower end of the lid and a large number of holes were made. Nude mice were anesthetized by intraperitoneal injection of pentobarbital sodium0.4ml/100g (10g/1), then the graft area of sedated mice was decontaminated with Betadine. Pull up the skin on the back with forceps and removed a portion of lifted skin with curved scissors to create hole,chambers were inserted between the skin and the thoracic wall. Finally, suture at the edge of the hole. Cell suspension was transplanted to the chamber, after3days,a small hole was cut at the top of chamber to facilitate the wound dry. One week after cell transplantation, the chambers were removed. Observe hair follicle formation,development and growth of the hair shaft at1,2,3and4weeks after transplantation.4weeks after transplantation, the skin at the transplantation site was excised and fixed in10%formaldehyde, paraffin sections were prepared and the histological sections were stained with hematoxylin and eosin. Plucking all hair at the grafted site and regeneration after hair plucking was observed.Implantation of newborn mice skin cells with mini-chamber method to construct a model of hair follicle developmentFresh mouse dermal cells were isolated from C57BL/6J mice at natal day0. The full thickness of the dorsal skin was removed, and then incubated in phosphate-buffered saline (PBS) with0.1%dispase at4℃overnight.The skin piece was then split into epidermis and dermis with forceps after being rinsed3times in PBS. Epidermis was discarded.Dermal component was minced with forceps and digested in0.2%collagenase at37℃for1h. After digestion, an equal volume of Dulbecco’s Modified Eagle’s Medium supplemented with10%fetal bovine serum (DMEM/10) was added, and the cell suspension was filtered sequentially through100μm mesh cell strainers. The cell suspension was centrifuged at1000rpm for5min, repeared twice and then the cell pellet was resuspended in DMEM.Dermal cells were labeled with Dilinoleyloxacarbocyanine perchlorate (DiI), a fluorescent cell tracking dye prior to grafting according to the manufacturer’s instructions. Epidermal cells were isolated from green fiuorescent protein (GFP) transgenic C57BL/6J mice at natal day0. The process of epidermal cells preparation was similar to the dermal cells.Mini-chambers weare mde by several silicone mini-chambers with a diameter of2.5mm. Mini-chambers were implanted onto the back of athymic nude mice. Briefly, nude mice were anesthetized by intraperitoneal injection of pentobarbital sodium0.4ml/100g (10g/l), then the graft area of sedated mice was decontaminated with Betadine. Pull up the skin on the back with forceps and removed a portion of lifted skin to create hole,mini-chambers were inserted between the skin and the thoracic wall. Finally, purse-string suture at the edge of the hole to prevent the mini-chamber from popping out of the hole. Four mini-chambers were transplanted onto each mouse. One of the following combinations of cell mixtures in a total volume of20ul was transplanted into each mini-chamber through the hole on top:(ⅰ) dermal cells (5×106)+epidermal cells (5×106);(ⅱ) dermal cells (2×106)+epidermal cells (2×106);(ⅲ) dermal cells (1×106)+epidermal cells (1×106);(ⅳ) dermal cells (5×105)+epidermal cells (5×105);(ⅴ) dermal cells(1×105)+epidermal cells (1×105);(ⅵ) dermal cells (5×104)+epidermal cells (5×104). In addition, Dil labeled dermal cells (5×106) and unlabeled epidermal cells (5×106) were transplanted with mini-chamber assay. GFP transgenic epidermal cells (5×106) and unabled dermal cells (5×106)were transplanted with mini-chamber assay. One week after cell transplantation, the mini-chambers were removed. Observe hair follicle formation,development and growth of the hair shaft at1,2,3and4weeks after transplantation.4weeks after transplantation, the skin at the transplantation site was harvested for paraffin section,frozen section and scanning electron microscopy.Cultured dermal cells engrafted with fresh epidermal cellsMouse epidermal cells and dermal cells were isolated from C57BL/6J mice at natal day0. The full thickness of the dorsal skin was removed, and then incubated in phosphate-buffered saline (PBS) with0.1%dispase at4℃overnight.The skin piece was then split into epidermis and dermis with forceps after being rinsed3times in PBS. The dermis was minced and digested in0.2%collagenase at37℃for1h. After digestion, an equal volume of Dulbecco’s Modified Eagle’s Medium supplemented with10%fetal bovine serum (DMEM/10) was added, and the cell suspension was filtered sequentially through100μm mesh cell strainers. The cell suspension was centrifuged at1000rpm for5min, repeared twice and then the cell pellet was resuspended in DMEM. Adjust the concentration of epidermal cells and dermal cells at1×105/ml. lml of cell suspension was inoculated into10cm cell culture dish and6ml medium was added. The culture was incubeted at37℃and5%CO2in air. Change medium after24h and subculture when80%confluency. The epidermis was digested in0.25%trypsin for10min at37℃. The preparation of epidermis cell pellets was similar to that for dermis cell pellets.27receptor nude mice were randomly divided into four groups, four mini-chambers were transplanted onto each mouse. One of the following combinations of cell mixtures (dermal cells:5×106; epidermal cells:5×106) in a total volume of20μl was transplanted into each mini-chamber through the hole on top:fresh epidermal cells and fresh dermal cells (group1); fresh epidermal cells and cultured passage0(PO)dermal cells (cultured for3days, group2); fresh epidermal cells and cultured passage1(PI) dermal cells (group3); fresh epidermal cells and cultured PO dermal cells (cultured overnight, group4). One week after cell transplantation, the mini-chambers were removed.Observe hair follicle formation,development and growth of the hair shaft at1,2,3and4weeks after transplantation.4weeks after transplantation, the skin at the transplantation site was harvested for paraffin section.ResultThe result of transplanting newborn mice skin cells with chamber method1w after cells implantation,the wound was moist without apparent contraction and among that pink and translucent tissue was formed.2w after implantation, the wound healed completely.3w after implantation,black hair grew from the skin was observed.4w after implantation,thick and black hair grew from the skin vertically.Completely developed structure of hair follicle was observed with paraffin section and HE staining.1w after plucking,new hair had regrown.The ratio of cell component was varied, whereas the other component was fixed at1×107cells. When the number of epidermal cells was reduced to1×106cells, the efficiency of hair follicle reconstitution was mostly unchanged. On the other hand, the density of newly formed hair was diminished considerably by reducing the number of dermal cells to5×106cells or lower.Neither epidermal cells nor dermal cells transplanted alone formed hair follicle. Hairs could regrow after plucking and approach their normal length within3weeks.The result of transplanting newborn mice skin cells with mini-chamber method4w after cells implantation, a number of thick and black hairs grow from the skin. The number of transplanted cells was changed, the efficiency of hair follicle reconstitution also changed. when the number of engrafted donor cells including fresh epidermal cells and fresh dermal cells in a ratio of1:1,varied from5×106to2 X106, the efficiency of hair follicle reconstitution was mostly unchanged. There are no hair grow from the skin and only melanogenesis was observed, however, by reducing the number of donor cells to5X104. The skin of hair growth was harvested for paraffin section and HE staining, completely developed structure of hair follicle was observed. Good-quality hair grew from the skin regularly and appeared to be normal was observed under scanning electron microscopic. The grafted sites were fixed and frozen sections were prepared and examined under fluorescence microscopy. All of the dermal papilla in the neonatal follicles observed were Dil positive and all epithelial component in the follicle were GFP positive. The reconstituted hairs could persist for at least6months and could regenerate after plucking.The result of co-grafting cultured dermal cells and fresh epidermal cellsNo regenerated hair growth on the skin was observed in groups2and3; only melanogenesis was observed, and histological examination revealed that no regenerated hair follicles were present in the grafted site. In group4(fresh epidermal cells co-grafted with dermal cells and cultured overnight), hairs of good quality and quantity grew from the grafted sites and were similar to those in group1. Mature hair follicle structures were confirmed histologically.Conclusion(1) Newborn mice skin cells transplanted by chamber method can construct a complete model of hair follicle development,which can be used to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling.(2) Mini-chamber assay is a reliable and practical model, with the advantages of requiring fewer cells, being minimally invasion and resulting in a higher graft take and fewer complications. In particular, fewer cells are required and several mini-chambers can be transplanted onto one animal, reducing experimental cost and difficulty.(3) Fresh dermal cells cultured overnight exclude keratinocyte contamination and retain functional dermal papilla cells for hair follicle reconstitution. |
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