| Hair loss not only affects the appearance of people’s appearance, but also exerts a negative effect on people’s self-confidence. Therefore, the causes and treatment of hair loss have become urgent problem to resolve.Many hormones and neural factors affect hair growth:androgens, estrogens, thyroid hormones, glucocorticoids, retinoids, prolactin, and growth hormone. Hair follicle is a sensitive target organ to androgens. However, testosterone and its active metabolite, dihydrotestosterone, act through androgen receptors in the dermal papilla and have the most dramatic effects. Skin cells contain both isoenzymes of5a-reductase, the enzyme that catalyzes the conversion of testosterone to the more potent and active dihydrotestosterone. It is found in hair follicles and the prostate gland. Individual hair follicles in different regions of the body respond differently to androgens. Androgen-dependent areas, such as the beard, respond to androgens by secreting mitogens and increasing the hair growth. However, in hair follicles of the balding skin, androgen stimulation causes the opposite effect by leading to the synthesis of inhibitory factors, thus causing what is referred to as androgenetic alopecia. In genetically predisposed males and females, androgenetic alopecia is caused by progressive shortening of the anagen stage and an increase in the number of hair follicles in telogen. In addition, the hairs are gradually miniaturized in the presence of androgens, whereas large pigmented terminal hairs are replaced by thin depigmented hairs (vellus-like hairs). However, even after baldness ensues, the miniaturized follicles are still present and cycling, which in theory would make baldness a reversible process. Thus, the goals in treatment of androgenetic alopecia would be prolonging anagen, stimulating telogen follicles to enter anagen, reversing miniaturization, and possibly generating new follicles.The treatment of hair loss includes medication and surgery. Now, only2two medicines which is accepted by medical field get clinical verification to be effective to treat hair loss. The oral medicine is finasteride and drug for exterior use is minoxidil. Its action principle is to suppress male hormones and the initial purpose is to treat prostatic hyperplasia. Compared with hair transplantation, all sorts of medicines to treat hair loss has obvious disadvantage. First, the drugs are only effective to slight hair loss but not severe hair loss. Second, it is easy to produce drug resistance and drug withdrawal symptoms aggravate or other side effects to the body.The major surgical therapy for hair restoration is autologous hair transplantation, which moves individual hair follicles from a part of the body called the donor site to bald or balding part of the body known as the recipient site. It is primarily used to treat male pattern baldness. In this condition, grafts containing hair follicles that are genetically resistant to balding are transplanted to bald scalp. Hair transplantation differs from skin grafting in that grafts contain almost all of the epidermis and dermis surrounding the hair follicle, and many tiny grafts are transplanted rather than a single strip of skin. Orentreich observed that transplanted follicles did not undergo progressive miniaturization and were therefore not androgen-sensitive like other follicles in the bald region. This phenomenon was termed donor dominance, indicating the donor follicle phenotype of androgen insensitivity is maintained after transplantation and the mechanism of androgenetic alopecia is therefore determined by the follicle type rather than by the scalp region in which the follicle resides. Because donor follicles do not undergo miniaturization, hair transplantation is a permanent treatment for hair loss that lasts until the onset of senescent alopecia late in life. The early hair transplant procedures employed large, round grafts that were not esthetically satisfactory for most patients. Today, the state-of-the-art technique is follicular unit transplantation, first described by Limmer, which uses follicular unit grafts of one to four hairs that are dissected with a stereomicroscope from a long strip of donor scalp. When performed by a skilled hair transplant surgeon, these transplants yield excellent results that are virtually undetectable. The treatment is therefore limited by the amount of donor hair available, which is usually not enough to completely cover the bald region of most patients. Also, despite the considerable skill of the surgeon, the maximum hair density achievable is never the prebalding, youthful density. Other drawbacks include the donor site scar, which can be unacceptably wide in some patients and is of special concern for patients who wear their hair short. In addition, although the modern transplant procedure is noninvasive, relatively painless, and associated with a quick recovery, many people are unwilling to undergo what they see as major surgery.Oliver’s research shows that the hairs fall off and no longer continue to grow after the dermal papilla is complete resection. It shows that dermal papilla is a must to maintain the function of hair follicle. Oliver, then transplant the dermal papilla to the follicle of which the bulb has been cut and new hair grows after3weeks. The hair bulb which was cut without transplanting hair follicle dermal papilla was unable to grow new hair. Later, further study found that dermal papilla cells can induce hair when combine with not only the hair follicle epithelial cells, but other types of epithelial cells, such as:cutin cells, renal capsule, etc.Hair follicle development in the embryo arises through a complex interaction between the embryonic ectoderm and its underlying mesoderm and is mediated by soluble factors secreted by both cell types. The first morphologically recognizable step is the appearance of epidermal placodes, which are regularly spaced thickenings in the embryonic epithelium. Clusters or condensates of dermal cells then form beneath the placodes. Peglike downgrowths of epidermis begin to form from the placodes, and they push the dermal condensates down as they grow into the embryonic dermis. Further maturation ensues as the developing follicles grow deeper into the dermis. The epidermal component begins to differentiate into the various follicular epithelial substructures such as the outer root sheath, inner root sheath, and matrix. In the meantime, the dermal condensate forms the dermal papilla as it becomes more compact, and it becomes almost completely enveloped by the developing follicular epithelium. A portion of the condensate forms the dermal sheath. An increasingly detailed picture of the molecular mechanisms that regulate hair follicle formation has revealed a variety of intercellular signaling pathways that are active during follicle morphogenesis, many of which are the same pathways that are used repeatedly throughout mammalian development. Based on the mechanism of hair development, the scientists’ further experimental results show that, by using the hair induced cells and the model which constructs an environment similar to the body enable the interaction between epithelial cells and mesenchymal cells, the hair reconstitution can be achieved.The technique of hair reconstruction which uses the induced hair to replace the autologous hair tissue is expected to become cellular therapy in the treatment of alopecia in the future. Hair reconstitution assays are useful for assessing the hair inductive properties of isolated cell populations. Early reconstitution assays consisted of transposing dermal tissue or cells beneath or in close contact to the upper half of an amputated hair follicle in vivo. The inserted dermal tissue regenerates a new DP and helps to restore the truncated epithelial structure with new hair shaft growth. This type of model restores a pre-existing injured hair follicle rather than regenerating a hair follicle de novo. It is also laborious to dissect and amputate hair follicles and insert dermal population. Only large follicles can be used in this model so there are many limitations in its application. In the subsequent studies, dermal tissues or cells are implanted into the dermis of the skin in order to induce the formation of hair follicles from the overlying epidermis. The origin of the dermal tissue determines the type of new hair follicles. Questions still exist in some studies whether these implanted dermal tissues or cells induce de novo hair follicles, or transform pre-existing hair follicles into another type. Hair formation by implantation of DP cells into glabrous skin favors de novo hair neogenesis. Recently, several hair regeneration models are established using dissociated cell aggregates or single cells from hair follicles as the epidermal and dermal components. This type of model gives us more flexibility to select and manipulate epidermal and dermal populations. The animal models for hair reconstruction include:chamber method, flap method, and method. The chamber assay, first introduced by Lichti and Weinberg in1993and updated in2008, is one of most established methods available for the study of hair follicle reconstitution. The flap assay is a recently developed refinement that combines features of both the sandwich and the ear wound assays (dermal cells were subcutaneously transplanted through a small incision in the ear). In the flap assay, dermal cells were grafted onto an epidermal sheet that was inserted into a surgically created wound in the skin of nude mice. 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. But the disadvantage of this method is that, when be induced subcutaneously, the hair does not have the normal polarity. Besides, no hair can grow out the skin surface. So this model needs to be further improved.In this experiment, based on the original hair reconstitution model, We made improvement in order to make hair reconstruction in " minimally invasive " condition, finally can meet the normal physiological structure and appearance of the hair organization.Objective1. set up the method to isolate and identify the hair inducing cell of neonatal C57mice2. hair reconstitution by the endo-chamber methode3. hair reconstitution by Patch assay followed by FUT (hair follicle unit transplantation) to achieve a proper hair direction and densityMethod1. Isolation, culture and identification of hair follicle induced cellsneonatal C57BL/6mice within1day are put into the75%alcohol for30seconds to sterilize. We shuck their skins and washed in PBS (containing1%penicillin and streptomycin) for3times cleaning. The skins are put into a new culture dish, then added0.1%dispase enzyme for digestion at37℃for1.5hours. Thus the epidermis and dermis can be isolated and cut into pieces, followed by0.2%collagenase digestion for30min at37℃, oscillation every10minutes. Then add the DMEM medium (containing10%fetal bovine serum) and use the200mesh to filtration, removing the tissue debris and cell aggregates. The filtrate was collected in centrifuge tubes, which is centrifuged at1200round per minute for5minutes. Then we got the dermal and epidermal cell sedimentations respectively. The dermal and epidermal cell sedimentations are resuspended and mixed with the ratio of2:1. The concentration of the mixed cell suspension is adjusted at1×106/μL and prepared with150μL as a unit to be used in the following experiment.2. Study on endo-chamber assay for hair reconstructionThe lid of a1.5ml eppendorf tube is cut and hollowed centrally. Then it is sterilized by high temperature and pressure.The nude mouse was abdominal cavity anesthetized by10%sodium pentobarbital. After successful anesthesia, it was disinfected at the dorsal site. A transverse incision about lcm long near the end was made and a subcutaneous lacuna was created by blunt separation to insert the chamber.7days later the skin was cut open to remove the chamber and the wound was left untreated for natural healing. The skin healing and hair growth were daily general observation and then tissue biopsy of the hair follicle structure.3. Combining the injection method and FUT (hair follicle unit transplantation) technique for the redistribution of hair follicleThe nude mouse was abdominal cavity anesthetized by10%sodium pentobarbital. After successful anesthesia, it was disinfected at the dorsal site. The prepared hair inducing cell suspension was injected subcutaneously at the dorsal site.2weeks later, the induced hairs formed and be taken out slightly. The hairs were devided into the hair units and planted at the skin surface at dorsal of nude mouse. The whole process was imitating the course of the hair transplantation surgery. Periodic characteristics of hair growth were observed followed by the tissue biopsy.Result:1. Cells of dermal papilla and dermal sheath is a special mesenchymal cells and with some specific marker. Although the function of many protein marker is unclear, but these markers have been widely used for the identification of dermal papilla and dermal sheath cells, as well as the identification of induction ability of hair. Versican and alkaline phosphatase (AKP) specifically expressed in dermal papilla and associated with hair induced ability. Dermal mesenchymal cells after separating and digestion was cultured for24hours, the expression of Versican and AKP was positive, indicating that still has the ability to induce hair.2.Built-in cell model can be successfully carried out hair reconstruction, compared with the traditional method of the chamber, has the advantages of small wound, fast recovery, low infection rate and the influence suffer from the environmental factors is small, is a more optimized hair induced model In addition, there is the hair reconstruction and the formation of new skin tissue in the wound at the same time, we speculate that this is due to inducted cell firstly formed the skin tissue with hair follicles and other subsidiary organs, Then the hair began to grow.3. By using FUT technology, we achieve the hair redistribution in nude mice body using reconstruction of Hair tissue, and attain the expected growth of hair density and direction. The survival hair after transplantation has the normal physiological structure and Periodic characteristics of hair growth, is no different from normal tissue. Therefore, combining the injection method and FUT technique, is expected to become a new method in the clinical treatment of hair loss in the future.Conclusion:1. Cells of dermal papilla and dermal sheath is a special mesenchymal cells and with some specific marker. Although the function of many protein marker is unclear, but these markers have been widely used for the identification of dermal papilla and dermal sheath cells, as well as the identification of induction ability of hair. Versican and alkaline phosphatase (AKP) specifically expressed in dermal papilla and associated with hair induced ability. Dermal mesenchymal cells after separating and digestion was cultured for24hours, the expression of Versican and AKP was positive, indicating that still has the ability to induce hair.2.Built-in cell model can be successfully carried out hair reconstruction, compared with the traditional method of the chamber, has the advantages of small wound, fast recovery, low infection rate and the influence suffer from the environmental factors is small, is a more optimized hair induced model. In addition, there is the hair reconstruction and the formation of new skin tissue in the wound at the same time, we speculate that this is due to inducted cell firstly formed the skin tissue with hair follicles and other subsidiary organs, Then the hair began to grow.3. By using FUT technology, we achieve the hair redistribution in nude mice body using reconstruction of Hair tissue, and attain the expected growth of hair density and direction. The survival hair after transplantation has the normal physiological structure and Periodic characteristics of hair growth, is no different from normal tissue. Therefore, combining the injection method and FUT technique, is expected to become a new method in the clinical treatment of hair loss in the future. |
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