The Study Of Hair Regeneration By Hair Follicle Cells Implantation

Background:Skin is the largest organ of the human body, and hair follicle is an important appendant organ of the skin. It is cyclicity, and it can regenerate regularity by itself. The cycle exists in all one's life. So, hair follicle is a very important structure to regulating the hair growth. The hair number of a healthy human is about 50-150 thousand. And the growth of hair is effected by age,climate,environment and health status. The cause of baldness includes various kinds of disease,medicine,iatrogenic factor,and so on. Nowadays, the study of hair regeneration not only brings gospel to patients who need hair implantation, but also creates extremely effect to the domain about tissue engineering and cells regeneration. Moreover, it is an excellent model for the study of interaction between epidermis and mesoderm origin cells, regeneration of organ and tissue, chromatogenesis, wound healing and the pathogenesy of skin cancer.The hair follicle is a specific organ in mammals, and it is composed of both dermal and epidermal components. A hair follicle consists of concentric an epithelial sheath, which is hair shaft, inner root sheath, and outer root sheath, surrounded by a dermal sheath that is connected to a dermal papilla (DP) at the base of the follicle. In the middle expansion of the outer root sheath, called the bulge, the epidermal stem cells are pooled. These stem cells interact with the DP at the base of the follicle and give rise to several types of hair follicle cells. During embryogenesis, the dermal condensation that forms the primordium of the DP interacts with the epidermal germ to induce the entire hair follicle. The DP is regarded as essential to hair follicle development and cyclical growth. Either DP cells (DPCs) or cultured dermal condensation cells inserted separately into skin of mice can induce new hair follicles, and either will maintain the phenotype of the donor follicle. Therefore, the transplantation of DPCs in humans is expected to have clinical applications as a cell therapy.Hair undergoes recurring growth cycles throughout life. Two major components, which are follicular epithelium and DP, develop and remodel hair follicles by reciprocal communication. DPCs are considered essential to hair follicle induction. So, in the study of cell surface labels, we knew that CD133,CD34,CD44 and versican concerned with DPCs'inducibility. CD34 and CD133 are all surface markers of human hematopoietic stem cell. CD34 represents the stem cell characteristics, and the hair follicle cells which express CD133 not only have the characteristics of stem cells, but also have the close associateion with the ability to induce hair follicle. CD44 is a kind of cell adhesion molecules, which represents cells adhesion. Study indicate that CD133,CD34,CD44, all were markers of normal tissue juvenile cells. Versican is one of the major extracellular matrix substances which DPCs produce. It was reported that versican-expressing DPCs have hair-inducing ability.DPCs can be harvested by excision of rodent vibrissae and human scalp hair follicles. Because dermal papilla is small, it is difficult to separate hair follicles from adult skin. Nowadays, researchers at home and abroad mainly use microdissection and enzymatic digestion to obtain the DPCs. Although methods continue to improve, but the key steps still have to rely on the separation of microscopic anatomy, which not only requires skill and patience, but also needs work intensity and efficiency low, while increasing the chance of contamination. Therefore, the study of hair follicle regeneration, we need a simple and easy experiment work which can help us to getting considerable hair follicle-inducibility cells for hair tissue engineering and baldness treat with cell therapy. Objective:1. To search a simple and easy experiment method and adequate source to obtain hair follicle cells.2. To confirm the hair follicle cells we getting possess stem cell character and hair follicle-inducibility.3. To attempt to inducing new hair follicle and hair fiber by implanting hair follicle cells in vivo.Methods:1. C57 mouse dermal cells'separation and cultivation. We shuck c57 neonate rats' skins and cut into small fragments,0.5 cm×0.5cm。And the skin fragments were incubated in 0.1% dispase for 16h at 4℃. They were rinsed 3 times by PBS. Then separated the epiderm and derma by microforceps。The derma was cut into trivials, and then was incubated in 0.2% collagenase for 30min at 37℃. And immerse them in a supplemented mixture medium of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum to stop digesting..Then removeled tissue chip and cell aggregate. We collected single cell suspl to centrifugate in 1000 rs/min for 5min, again twice, then removeled supernatant and added DMEM containing 10% fetal. Put 1.5ml cell suspl to inoculate in the 25ml culture flask, added 2ml medium. The culture was incubated at 37℃and 5% CO2 in air, and added the medium after 24h.2. The identification of inducibility of C57 mouse dermal cells.2.1 Cytoactive detection:cell suspension and 0.4% trypanblau mixed at the ratio of 1:1 then dropped in cell countiong plate and observed by microscope. The living cells were uncolored and splendent; the dead cells were colored and cell body expanding.Counting uncolored cell population and total cellular score and the percentage.2.2 Immunohistochemistry detection:The cells were seeded on cover slips, PBS rinsing two times (5 min/times),4% formalin-fixed 30 minutes then the PBS rinse three times (5 min/times).0.1% Triton X-100/0.1% sodium citrate solution, rupture of membrane at room temperature for 10 minutes, PBS washed three times (5 min/ times). Closure of endogenous peroxidase:3%H2O2 were incubated at room temperature for 10 minutes; PBS washed three times (5 min/times). Normal goat serum at room temperature closed to 10 minutes.Anti-CD133/versican (concentration: CD133 1:150; Versican 1:100) incubation:the cells seeded in DAKO antibody diluent diluted antibody solution were incubated overnight in 4℃. Rewarming for 15 minutes.PBS wash three times (5 min/times) DAKO anti-mouse/rabbit incubated at room temperature 40 minutes. PBS washed three times (5 min/times). DAB color, 1-10 minutes at room temperature, under the microscope control. ddH2O washed three times,5 min/times. After hematoxylin, PBS back to blue. Dehydrate, transparent, mounting by neutral gum. The results were to take a photo by otter BK 5000 biological microscope, Nikon D60 camera.2.3 Flow cytometric analysis:The mouse cells which activity were>95% by typranblau stain were seeded in 12-well plates as 106/kong. Cells were collected by 0.25% trypsin. Re-hanging in the solution which were contained 1:50 FITC-CD34 antibody and 1:50 FITC-CD44 antibody or 1:20 FITC-CD133 antibody (in 1:50 mouse IgG/FITC as a control).37℃,30min. Flow cytometry excitation wavelength Ex=488 nm; emission wavelength Em=530 nm. Test positive rate.3. Hair follicle cells implantation. We mixed dermal cells and epidermal cells at the rate of 2:1. Nude mouse 4-6w, female or male. Nude mouse were anesthetized using 0.8% napental. Sterilize the skin of back. The mixed cells, epidermal cells and PBS implanted under the skin of the nude mouse by injecting. Then we observed the hair follicle formation, growth, reconstruction and hair shaft condition. The histologic examination of the skin was performed after the 2weeks,4weeks or 8weeks respectively。Results:1. The dermal part of newborn rat skin was flocc after digestion by dispase, reddish; epiderm part was easily to float, the color was silver. We observed by stereoscopy that the derma liked sponges, in which we can seen a large number of micro-blood vessels, and the epiderma was silver-white, paving stone-like. 2. We could obtain dermal cells of newborn rats by dispase and collagenase digestion, the cell viability was>95%. And about 80% of them could be adherence after inoculated 24h. Initially, they were small round, non-uniform cell size, a larger of nuclear/plasma ratio, then they gradually extent to short shuttle and long spindle-shaped and polygonal after 24h.4 d later, they showed fibroblast-like morphology, nuclear oval-shaped, large. Primary cultured cells were amounted to 70%-80% confluences after 7 days. And with the passage number increased, cell volume is gradually increasing.3. Dermal cells were aggregative. The aggregates formed by the structure after 3d cultivation observed on a smooth surface and shape into a ball. In the microscope, they could be observed in cell aggregates uniformly mixed together, and no obvious substrate material.4. CD133, versican expression in primary dermal cells by immunohistochemistry detection, cytoplasmic membrane coloring, was positive.5. Flow cytometric analysis:The percentage of positive cells CD34 (62.5%); CD44 (41.97%); CD133 (52.1%); IgG (71.9%).6. We could see slightly elevating and pigmentation on the transplant site after implanting 2 weeks Generally observed 2 weeks,and turnout the graft site, we could see the hair fiber by stereomicroscope and which could erupt after 4 weeks, followed by the naked eye it could be gradually observed black hair. The injection site, most of the hair fiber forming under the skin without piercing the skin, the individual hair dries off, but there was new hair shaft grows in 2 weeks.7. Histologic examination:mixed cells were injected after 2 weeks, and transplanted tissue sections stained area. There were a large number of hair follicles was arranged in concentric circles structure, and their centers were keratinized eosinophilic staining material, outside the following order:inner root sheath, outer root sheath, dermal sheath. There was no change in histology in the control group.Conclusion:1. Back skin of newborn C57mice was the tissue of origin, and we can get the dermal cells from it by digestion of dispase and collagenase. Experimental operation is simple, which could reduce the opportunity of pollution, and we could get a large number of isolated cells by one operation. C57 mice are black, and it is easy to observe after implantation in vivo.2. The cells we got were fibroblast-like and adherence easily. We tested that they had stem cell properties, strong ability to induce hair follicles and aggregative growth in vitro.3. We could observe hair follicle regeneration and hair fiber formation by grafting the cells in nude mice.