Research progress of skin-derived precursor cells / 南方医科大学学报

As a novel population of neural crest-origin precursor cells, skin-derived precursor cells (SKPs) can be isolated from both embryonic and adult dermis. These cells have important values for research and potential clinical application in wound healing, organ regeneration and disease treatment for advantages in the abundance of cell sources, accessibility, potential of multipotent differentiation, and absence of ethical concerns. Here we review the developmental and anatomical origins of SKPs and their potential application in regenerative medicine. SKPs originate from the embryonic neural crest, and their sources may vary in different areas of the body. SKPs are widely found in the dermis, especially in the dermal papilla (DP), which was known as a niche of SKPs. The multipotent SKPs can used for autologous transplantation and are of vital importance in tissue repair.

Experimental research of hair follicle reconstruction with the aid of embryonic mice dermal cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of embryonic dermal signal on the hair-inductive capacity of neonatal mice dermal cells which have been amplified in vitro.</p><p><b>METHODS</b>Embryonic mice dermal cells of embryonic day 14 were added to a chamber on the back of nude mice with neonatal mice dermal cells which had been amplified in vitro for 3 days and freshly isolated neonatal mice epidermal cells. The hair regeneration was compared between the groups with or without embryonic mice dermal cells. Meanwhile, chambers with following cells respectively were constructed as controls: embryonic mice dermal cells + neonatal mice epidermal cells; freshly isolated neonatal mice dermal cells + neonatal mice epidermal cells; amplified neonatal mice dermal cells only; embryonic mice dermal cells only; freshly isolated neonatal mice dermal cells only; neonatal mice epidermal cells only.</p><p><b>RESULTS</b>The number of regenerated hairs with the aid of embryonic mice dermal cells (207 +/- 15. 948) was significantly higher than that (67 +/- 8.963) in the group without embryonic mice dermal cells (n = 3, t = 7.653, P = 0.002).</p><p><b>CONCLUSION</b>Embryonic dermal signal can enhance the hair-inductive capacity of neonatal mice dermal cells which have been amplified in vitro.</p>

Effect of PRP on the proliferation of dermal papilla cells and hair follicle regeneration in mice / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of platelet-rich plasma (PRP) on the proliferation of dermal papilla cells (DPCs) and hair follicle regeneration.</p><p><b>METHODS</b>PRP was prepared using the double-spin method and applied to DPCs. The proliferative effect of activated PRP on DPCs was measured using MTT assay. To understand the influence of activated PRP on the hair-inductive capacity of DPCs, freshly isolated epidermal cells and DPCs of passage 4 were resuspended, mixed with various concentrations of a PRP (0%, 5% or 10%) and were then transferred to a grafting chamber, which was implanted onto the dorsal skin of nude mice. The chambers were removed 1 week after grafting and HF formation was monitored for 4 weeks; the graft site was harvested and processed for histological examination.</p><p><b>RESULTS</b>Activated PRP increased the proliferation benefited the aggregative growth of DPCs. There are significant difference in the yield of hair follicles compared with 10% PRP (344 +/- 27) with 0% PRP (288 +/- 35) in the area of reconstituted skin (P < 0.05). The areas treated with PRP demonstrated an increase in hair follicles density of 19.4%. Ten percent PRP (18 +/- 1) d also can significantly shorten the time of hair formation, compared with 0% PRP (20 +/- 1) d (P < 0.05).</p><p><b>CONCLUSIONS</b>There is a considerable effect of PRP on the time of hair formation and the yield of hair follicles reconstitution.</p>

Inhibition effect of 6-gingerol on hair growth / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of 6-gingerol, the main active component of ginger, on hair shaft elongation in vitro and hair growth in vivo.</p><p><b>METHODS</b>Firstly, Hair follicles were co-cultured with 3 different concentration of 6-gingerol for 5 days and hair elongation in three groups was measured. Secondly, The proliferative effect of 6-gingerol on DPCs was measured using MTT assay. Thirdly, the expression of Bcl-2 and Bax in DPCs were measured using Western blotting. In vivo study, the influence of 6-gingerol on hair growth in C57BL/6 rats was measured through topical application of 6-gingerol on the dorsal skin of each animal.</p><p><b>RESULTS</b>The length of hair shaft in 20 microg/ml 6-Gingerol group (0.50 +/- 0.08 mm) is less than 0 microg/ml (0.66 +/- 0.19) mm and 10 microg/ml (0.64 +/- 0.03) mm 6-Gingerol group (P < 0.05). In cell culture, compared to 0 microg/ml and 5 microg/ml 6-Gingerol, 10 microg/ml 6-Gingerol can significantly inhibited the proliferation of DPCs (P < 0.05). Along with the growth inhibition of DPCs by 6-gingerol, the Bax/Bcl-2 ratio increased obviously. In vivo study, the hair length and density decreased a lot after using 1 mg/ml 6-gingerol.</p><p><b>CONCLUSIONS</b>6-Gingerol can suppress human hair shaft elongation because it has pro-apoptotic effects on DPCs via increasing Bax/Bcl-2 ratio. It might inhibit hair growth by prolonging the telogen stage in vivo.</p>

Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering

OBJECTIVE@#To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase (TERT) and to investigate the expression of TERT in neonatal mouse hypodermal cells.@*METHODS@#The polymerase chain reaction (PCR)-amplified TERT gene was inserted into plasmid pLEGFP-N1. The positive clone was identified by restriction enzyme digestion and sequencing, then was transfected into packaging cells to produce retrovirus particles. Neonatal mouse hypodermal cells were infected with the virus to generate a stable cell line. The TERT mRNA expression level, telomerase activity, and enhanced green fluorescent protein (EGFP) expression level were analyzed.@*RESULTS@#Retroviral vector pLEGFP-N1-TERT was constructed successfully, and a stable cell line of neonatal mouse hypodermal cells expressing EGFP was established. Western blot and immunohistochemical assay showed that the expression level of TERT was significantly elevated in the neonatal mouse hypodermal cells.@*CONCLUSIONS@#A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenous TERT gene in the neonatal mouse hypodermal cells. This protocol has potential applications for skin tissue engineering and cell transplantation therapy.

Implantation of newborn mice skin cells with chamber method to construct a model of hair follicle development / 中华整形外科杂志

<p><b>OBJECTIVE</b>To construct a convenient, reliable and visual model of hair follicle development to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling.</p><p><b>METHODS</b>An open chamber was transplanted into the nude mice dorsal skin, dermal and epidermal cells isolated from newborn C57BL/6 mice skin were mixed at a specific ratio and then injected into the chamber together, 1 week after transplantation, the chamber was removed, and then, hair formation and regeneration after hair plucking was observed.</p><p><b>RESULTS</b>1 week after cells implantation, the wound was moist without apparent contraction and among that pink and translucent tissue was formed. 2 weeks after implantation, the wound healed completely. 3 weeks after implantation, black hair grew from the skin was observed. 4 weeks 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. 1 week after plucking, new hair had regrown. The ratio of cell component was varied, whereas the other component was fixed at 1 x 10(7) cells. When the number of epidermal cells was reduced to 1 x 10(6) cells, 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 to 5 x 10(6) cells or lower. Neither epidermal cells nor dermal cells transplanted alone formed hair follicle.</p><p><b>CONCLUSIONS</b>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.</p>

Preliminary study on inhibition of the hair follicle development by siRNA targeting Wnt10b in the cultured rat embryonic skin / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate whether the suppression of Wnt10b by siRNA could prevent the development of hair follicle in the cultured rat embryonic skin.</p><p><b>METHODS</b>siRNA-Wnt10b was synthesized by chemosynthesis method. The dorsal skin of SD rat at embryos were cultured in DMEM in the presence of different percentage of interfering RNA targeting Wnt10b. Wnt10b/beta-catenin expression was analyzed by real-time PCR everyday and by Western blot on the third day. The cultured embryonic skin underwent paraffin embedding, section, HE staining on the third day,in which the number of de novo hair follicle was calculated and statistically analyzed.</p><p><b>RESULTS</b>Wnt10b gene in the cultured embryonic skin could be knocked down with the siRNA-based method. Beta-catenin mRNA was not greatly influenced by the downregulation of Wnt10b mRNA. The number of de novo hair follicle placode in cultured embryonic skin decreased, along with the downregulation of Wnt10b and beta-catenin proteins expression.</p><p><b>CONCLUSIONS</b>The downregulation of Wnt10b mRNA and protein by siRNA reduces the number of de novo hair follicle placode in the cultured rat embryonic skin. Wnt10b may control cytoplasm beta-catenin concentration at the protein level.</p>

Hair follicle regeneration by injection of follicular cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the mechanisms of hair follicle regeneration by injection of follicular cells isolated from murine skin.</p><p><b>METHODS</b>Epidermis was peeled off from the dermis of 3-5 d C57BL/6J mouse by 0.2% Dispase digestion at 37 degrees C for 2 hours. Dermis was cut into small pieces and digested in 0.2% collagenase at 37 degrees C for 30 minute with low speed stirring to isolate hair follicles from dermis. Hair follicles were collected through filtration, low-speed centrifugation and density gradient centrifugation. Collagenase and trypsin were added to digest hair follicles into dissociated cells which were marked by Dio and injected into the nude mouse skin.</p><p><b>RESULTS</b>2 d after intradermal injection of hair follicle cells, 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 and showed sparsely localized green fluorescence. The contents of the cyst showed bright green fluorescence. 4 d after injection, the skin became slightly thicken with grey appearance, a lots of hair follicles formed with black bulb. 1 weeks 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. 3 weeks after injection, a cyst containing lots of black hairs formed in the injection site. Newly formed hair follicles showed positive for Dio. Sebaceous gland can be seen accompanied with hair follicles. 6 weeks after injection, the cyst contained lots of sheded club hair shafts and hair follicles on the stage of anagen. Cultured follicular cells and injection below 1 x 10(5) failed to regenerate hairs. While the regenerated hair follicle was few when the hair follicle cells were injected subcutaneously.</p><p><b>CONCLUSIONS</b>Follicular cells can aggregate spontaneously and develop synergistically into hair follicles with normal growth cycle after implantation. The regeneration depends on the interactions between follicular cells, as well as on the recipient sites and cell numbers.</p>

A hair prosthesis consisting of allogeneic hair and polypropylene mimicking follicular units: long-term result and histocompatibility in rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a follicular unit-like construct with allogeneic hair, evaluate its histocompatibility and long-term stability after transplantation, and explore the possibility of its clinical application.</p><p><b>METHODS</b>Human hair and medical polypropylene was processed according to the structure of follicular units and prepared into hair prostheses for transplantation. The histocompatibility of polypropylene and human hair in New Zealand rabbits was observed by HE staining and scanning electron microscope, and the loss rate of the hair was recorded to evaluate the long-term result of transplantation.</p><p><b>RESULTS</b>Mild inflammatory cell infiltration around polypropylene and human hair was observed early after the transplantation, accompanied with local epithelial cell proliferation. The prosthesis mimicking the follicular units still showed good histocompatibility one year after the transplantation without degradation of the hair. The loss rate of the hair was averaged (4.1∓4.0)% at one year after the transplantation, and the total appearance of the prosthesis remained satisfactory.</p><p><b>CONCLUSION</b>Allogeneic human hair and polypropylene in the hair prosthesis show good histocompatibility in rabbits. The prosthesis allows good cosmetic effect after transplantation with low rate of hair loss, demonstrating its potential in clinical application.</p>

Effects of different parts of the follicle-unit grafts subjected to controlled injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the shaft elongation and morphological changes of follicle-unit (FUs) grafts subjected to controlled injury in different parts.</p><p><b>METHODS</b>Human FUs were isolated by microdissection under a dissecting microscope. The single hair of anagen FUs were randomly divided into A, B and C groups, and A and B groups were subjected to controlled injury with microsurgery imposed to the dermal papilla and the bulge of FUs, respectively, with C as the control group without any treatment. HE staining was used to detect the histological changes of the cells, and organ culture for 10 days was conducted to observe the morphological changes and elongation of FUs.</p><p><b>RESULTS</b>There were no histological or morphological changes in A, B and C groups. The average elongation of hair shaft was 1.293-/+0.245, 2.116-/+0.423 and 2.235-/+0.379 mm, respectively. There were significant differences between groups A and B (P<0.05) and between groups A and C (P<0.05). No significant difference was found between groups B and C (P>0.05).</p><p><b>CONCLUSION</b>Damage of the dermal papilla should be avoided in hair transplantation surgery.</p>

Follicular cell implantation for induce hair follicle growth in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a convenient and reliable method for inducing hair regeneration by follicular cell implantation for the treatment of alopecia.</p><p><b>METHODS</b>The human hair follicle stem cells were separated and purified by micromanipulation and magnetic cell sorting, and human scalp dermal papilla cells were isolated by enzyme digestion. The two cells were mixed and implanted subcutaneously in nude mice to observe the regeneration of the hair follicles.</p><p><b>RESULTS</b>Formation of intact hair follicle-like structures was observed in the skin sections of the recipient nude mice.</p><p><b>CONCLUSION</b>Follicular cell implantation can induce hair follicle-like structures in nude mice, which provides a means for efficient hair regeneration for treatment of hair loss.</p>

Subcutaneous implantation of embryonic skin cells to construct a model of hair development in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the hair development after subcutaneous implantation of embryonic skin cells in nude mice, and construct a model of hair development.</p><p><b>METHODS</b>Dermal and epidermal cells isolated from embryonic mouse skin were mixed at a given ratio and injected subcutaneously in nude mice, and hair formation after the implantation was observed under a microscope.</p><p><b>RESULTS</b>Formation of the hair follicles and fibers under the skin of the recipient nude mice and emergence of the hair shaft were observed microscopically.</p><p><b>CONCLUSION</b>Embryonic skin cells be used to construct a complete model of hair development after implantation in vivo.</p>

Histocompatibility of a polypropylene matrix for hair follicle transplantation in rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the histocompatibility of a polypropylene matrix implanted subcutaneously for potential hair follicle transplantation in rabbits.</p><p><b>METHODS</b>The polypropylene matrix for harboring the hair follicles was prepared and implanted subcutaneously at the neck of 5 New Zealand white rabbits by means of hair follicle unit transplantation. At 1 week after the transplantation and then on a monthly basis in the following 6 months, full-thickness skin tissues were sampled at the site of grafting to evaluate the histocompatibility of the matrix material using HE staining and scanning electron microscopy.</p><p><b>RESULTS</b>At 1 week after implantation of the matrix material, a small number of inflammatory cells and lymphocytic infiltration were observed around the graft, with mild hyperemia in the proliferative capillaries and mild inflammatory responses. In the following 6 months, the inflammatory cells and lymphocytes around the graft decreased obviously or even disappeared, and such graft rejection responses as tissue lysis and necrosis were not observed. A large quantity of collagen fibers were found to encapsulate the polypropylene material.</p><p><b>CONCLUSION</b>Polypropylene matrix graft has good histocompatibility with the rabbit subcutaneous tissue without producing obvious graft rejection responses, suggesting its feasibility for further experiments of hair follicle transplantation.</p>

Effect of alexandrite laser treatment for hair removal in Tibet mini-pigs / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the histological and ultrastructural changes of the skin and hair follicles following hair removal by alexandrite laser in Tibet mini-pigs.</p><p><b>METHODS</b>Twelve healthy Tibet mini-pigs with dark hair were treated with alexandrite laser for hair removal. The skin specimens were taken immediately and at 1 h and 1, 3, 5, 10, 15, 30, 60 days after the laser treatment for observation under optical and transmission electron microscope.</p><p><b>RESULTS</b>Laser hair removal resulted in extensive coagulation necrosis, carbonization and falling of the subcutaneous hair shafts, and some of the cells in the outer root sheath and hair bulb underwent degenerative and necrotic changes. One hour after laser treatment, the cells in the outer root sheath and bulb exhibited nuclear condensation, fragmentation and or karyolysis characteristic of cell apoptosis. The cell apoptosis reached the peak level on day 3 after the laser exposure, accompanied by endothelial degeneration in the hair papilla vessels, edema and lymphocyte infiltration in the dermal tissues. Tissue reaction and inflammation were relieved on day 5, and the dermal tissue and follicles recovered their normal structures on day 10. At 60 days after the treatment, the hair follicles decreased markedly but the structure of the residue follicles remained normal.</p><p><b>CONCLUSION</b>Alexandrite laser exposure results in selective destruction of the follicles by inducing direct coagulation and cell apoptosis to achieve permanent hair removal. Tibet mini-pigs with black hair can be used as the animal model of clinical laser hair removal.</p>

Effective isolation and culture of the outer root sheath bulge cells, dermal sheath cells and dermal papilla cells derived from human hair follicle / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an effective method for isolating and culturing outer root sheath (ORS) bulge cells, dermal sheath cells (DSCs) and dermal papilla cells (DPCs) derived from human hair follicle.</p><p><b>METHODS</b>Small scalp specimens were incubated in the presence of dispase at 37 degrees celsius; for 2 h, the hair shafts with ORS embedded in the dermal sheath (DS) were extracted under dissecting microscope, and the ORS tissue were inoculated onto Petri dish. The specimens were transected at the interface between the dermis and subcutaneous tissue. The portions of DS and DP (linked with and enclosed by DS) embedded in the adipose tissue were pulled out and incubated with collagenase at 37 degrees celsius; for 6-8 h, and the DP and DSCs were isolated by repeated low-speed centrifugation and cultured respectively on Petri dishes. The cultured ORS bulge cells were identified by immunohistochemistry with K19 antibody and DPCs and DSCs by immunohistochemistry with alpha-actin antibody.</p><p><b>RESULTS</b>Purified ORS bulge cells, DSCs and DPCs could be harvested from the same human hair follicle.</p><p><b>CONCLUSION</b>This new method allows efficient, rapid, and simultaneous isolation and culture of ORS bulge cells, DSCs and DPCs.</p>

Hair follicle cell mixture forms hair follicle-like structures after subcutaneous implantation in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the hair follicle regeneration after subcutaneous implantation of hair follicle cell mixture in nude mice.</p><p><b>METHODS</b>The hair papilla cells, dermal sheath cells, outer root sheath and fibroblasts of human scalp were mixed with the hair follicle epithelial cells and implanted subcutaneously in nude mice to observe the regeneration of the hair follicle.</p><p><b>RESULTS AND CONCLUSION</b>Formation of intact hair follicle-like structures was observed in the skin sections of the recipient nude mice, suggesting the feasibility of this approach for hair follicle regeneration in vivo.</p>

Isolation and in vitro culture of follicular papilla cells from rat vibrissae / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a simple method for isolating and culturing follicular papilla cells from rat vibrissae.</p><p><b>METHODS</b>The intact follicles were obtained and digested in 0.2% collagenase I with agitation on a rotary stirrer at 100 r/min at 37 degrees C; for 3 h. The suspension was centrifuged at 300 r/min and the papilla cells were collected and suspended in DMEM for cell culture. The adhesion efficiency of the dermal papilla cells was evaluated and compared with that of the cells obtained by microdissection.</p><p><b>RESULTS AND CONCLUSION</b>The described procedure allowed efficient and rapid isolation of the dermal papilla cells from rat vibrissae and ensured improved adhesion of the dermal papillae and outgrowth of the cells with reduced labor and risk of contamination. The cells obtained with this procedure were positive for alpha-smooth muscle actin staining.</p>

p53 gene codon 72 polymorphism and susceptibility to keloid / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the relationship between p53 codon 72 polymorphism and susceptibility to keloid in a southern Chinese population.</p><p><b>METHODS</b>The p53 genotypes were determined by polymerase chain reaction-reverse dot blot (PCR-RDB) and DNA direct sequencing in 45 patients with keloid and 60 unrelated healthy controls.</p><p><b>RESULTS</b>The frequency of the p53 Pro allele among keloid patients was significantly higher than that among healthy controls (chi2 = 4.485, P = 0.034). The Pro/Arg and Arg/Arg genotype distribution among keloid patients was not significantly different from that among healthy controls (chi2 = 0.949, 1.346; P = 0.330, 0.246, respectively). However, the Pro/Pro genotype frequency among keloid patients was significantly higher than that among healthy controls (chi2 = 4.375, P = 0.036). The p53 Pro/Pro genotype significantly increased the risk for developing keloid, compared to the combination of Pro/Arg and Arg/Arg genotypes,with the odds ratio (OR) of 2.400 (95%CI: 1.048-5.498).</p><p><b>CONCLUSIONS</b>Determination of the p53 codon 72 genotype may be used as a stratification marker to predicate high-risk individuals for keloid.</p>

Microanatomy study of facial nerve distribution at the temporal region for safe facelifting / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the route and scope of the facial nerve in the temporal region for clinical applications.</p><p><b>METHODS</b>Temporal region dissection was performed on 12 cadavers (24 sides) under light microscope.</p><p><b>RESULTS</b>There are two branches of the facial nerve in the temporal region from the superior margin of the parotid: the temporal branch and the zygomatic branch. Each of them has two to five branches, which run in the deep layer of the superficial temporal fascia. The temporal branch crosses the zygomatic arch to the temporal region, innervating the frontal muscle, the orbicularis oculi muscle, the corrugator supercilii muscle, and the muscle surrounding the ear, etc. The zygomatic branch goes to the lateral canthus, innervating the orbicularis oculi muscle, the upper and lower eyelid and zygomatic muscles. There are communicating branches among the temporal branches, the zygomatic branches and the supraorbital and lacrimal nerves of the ophthalmic nerve.</p><p><b>CONCLUSION</b>The temporal branches and zygomatic branches of the facial nerve run between the deep zone of the superficial temporal fascia and the superficial layer of the profound temporal fascia, where dissection should be avoided during rhytidectomy in order not to damage the facial nerve branches.</p>