Objective:To study the effect of seeding cells of tissue-engineered skins,keratinocytes and fibroblasts,on the phenotypes of langerhans cells (LC).Methods:Peripheral blood mononuclear cells were induced by cytokines,granulocyte macrophage-colony stimulating factor (GM-CSF),interleukin 4 (IL-4),transforming growth factor β1 (TGF-β1) to generate LCs,which were subsequently cocultured with allogenic keratinocytes or fibroblasts.Then the phenotypes of langerhans cells was detected by flow cytometer (FCM),and the degree of proliferation of autogeneic lymphocytes was assessed after stimulation with LCs.Results:Cultured LCs had low level expression of HLA-DR,CD80,CD86 and no expression of CD83.Cocultured with allogenic keratinocytes or fibroblasts,phenotypes of LCs did not change significantly.Simultaneously,LCs could not stimulate autogeneic lymphocytes proliferation.Conclusion:These data indicate that LCs cocultured with seeding cells of tissue-engineered skins remain immature state.It is suggested that seeding cells of tissue-engineered skins have low immunogenicity and are unable to induce significant immunological rejection of host after transplantation.

Objective To investigate the influence of Wnt-3a signaling on aggregative growth of dermal papilla cells.Methods Recombinant adenovirus pAdEasy-1/Wnt-3a was constructed at first,and used to transfect cultured dermal papilla cells(DPCs).Then,the growth pattem of DPCs was observed by inverse microscopy;laser confocal microscopy and Western blot were utilized to detect the expressions of Wnt-3a,β-catenin and versican in low-passage DPCs,high-passage DPCs and transfected high-passage DPCs.Results The over-expression of Wnt-3a protein could effectively induce the aggregative growth of DPCs.Laser confocal microscopy showed that the fluorescence intensity of β-catenin and versican increased from 34.16±9.62 and 18.81±9.59 in high-passage DPCs to 87.35±17.28 and 92.18±18.39 in transfected high-passage DPCs(t=11.98,17.88,both P＜0.0 1),respectively.Also,Western blot proved a significant increase of expression intensity of β-catenin(15.27±2.17 vs 60.09±7.24,t=10.10,P＜0.01)and versican(19.32±2.46 vs 58.46±2.78,t=20.63,P＜0.01)in transfected high-passage DPCs compared with untransfected high-passage DPCS.ConclusionsWnt-3a signaling plays an important role in modulating aggregative growth of DPCs,which is mainly through the classical pathway with β-catenin as the key protein.As an important downstream gene of Wnt signaling,vesican is an indispensable part for the modulation of aggregative growth of DPCs.

Objective To evaluate antibacterial and antifungal effect of the composite chitosan artificial skin in vitro. Methods The standard strains were used in the experiment, including Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853) and Candida albicans (ATCC10231). Twelve agar plates were prepared for each standard strain, which were equally divided into the trial and the control groups. In the trial groups, 50 ?L composite chitosan dermal substitute was added to each prepared agar plate, two samples for each plate. In the control groups, composite collagen-gelatin dermal substitute was used. After the plates were incubated at 35 ℃ for 18 ~ 24 h, the antibacterial or antifungal rings of every sample were measured. Results The composite chitosan dermal substitute showed the antibacterial effect on Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC25922), and Pseudomonas aeruginosa (ATCC27853) (P

Objective To rapidly isolate and culture sweat gland epithelial cells in vitro and to observe the characteristics of the cells. Methods The secretory coils of sweat glands were dissected and picked out under an anatomical microscope, then digested by collagenase. The harvested epithelial cells of sweat gland were observed for their growth characteristics and identified by immunohistochemistry. Results The cultured epithelial cells grew very well. About 3 weeks later, a distinctive cobblestone appearance was observed in the culture. The antibody-staining showed the cells were positive for epithelial membrane antigen and cytokeratin, but negative for actin, which confirmed that the cells were sweat gland epithelial cells. Conclusion A method is established for rapid isolation and culure of sweat gland epithelial cells in vitro.

Objective To investigate the clinical outcomes of surgical excision combined with recom binant interferon aipha-2b in the treatment of acral malignant melanoma (MM). Methods Fifteen patients with acral MM admitted to the department since 2004 were recruited into this study. The tumors varied from 1.8 mm to 3.9 mm in invasion depth. Thin tumors with an invasion depth of 1.8 - 2.0 mm were excised with a margin of lcm beyond the tumors, and those with an invasion depth of 2.0 - 3.9 mm were excised with a margin of 2 cm beyond the tumors. After excision, 4 cases of minor excision were sutured directly, 10 cases of large excision were repaired with adjacent skin grafts and flaps, 1 patient with the involvement of the first metatarsophalangeal joint underwent toe amputation followed by the repair of planta wound surface with the remaining skin on the dorsa of toes. Patients received intramuscular recombinant interferon alpha-2b for 3 months (3 million units daily for the first 3 days and 6 million units for the remaining days) following operation. Results There were 6 cases of MM in situ and 9 cases of invasive MM in this study. All the skin grafts and flaps survived. Within the 3-year follow up, relapse was observed only in 1 patient with invasive MM. Recovery was achieved in the functions of feet in all patients. Conclusion The excision of tumors with a margin determined by tumor thickness plus intramuscular interferon alpha-2h may improve the survival of patients with cutaneous MM in planta pedis with avoidance of amputation.

Objective To investigate the effect of hepatocyte growth factor (HGF) on migration and apoptosis of,as well as phosphorylated-Akt (p-Akt) expression in cultured human eccrine sweat gland epithehal cells (hESGc).Methods The first generation of hESGc were cultured in keratinocyte serum free medium (KSFM) and treated with various concentrations (2,20,40μg/L) of HGF for different durations.Then,cell scratch test was performed to detect cell migration,a double staining flow cytometry assay using annexin VFITC/propidium iodide to detect cell apoptosis.and Western blot to measure the expression of p-Akt.Results HGF of 2μg/L had no effect on the migration of hESGc,while that of 20 μg/L and 40μg/L could promote the migration of hESGc by 33.2% and 228.2%.respectively.The average number of cells migrating into the scrach zone was significantly lower in untreated cell group than that in 20 and 40μg/L HGF-treated cell group (17.3±5.5 vs 23.0±6.3 and 56.7±7.9,t=2.653, 15.858,P<0.05,0.01, respectively).The apoptosis rate was 14.76% in untreated cells,14.16%,13.5% and 8.87% in cells treated with HGF of 2,20 and 40μ/L, respectively;there was a significant difference between untreated cells and 40μg/L HGF-treated cells (t=7.852,P<0.01).HGF could activate the phosphorylation of Akt protein and increase the expression of p-Akt.Conclusion HGF could promote the migration of,inhibit the apoptosis of,and stimulate the p-Akt expression in.hESGc.

Objective To study histologic characteristics of bilayer skin substitute reconstructed by cells from human hair follicle and whether the skin appendage can be induced based on the bilayer skin substitute.Methods After composite chitosan bilayer skin substitute was reconstructed with dermal papilla cells and outer root sheath cells or dermal sheath cells and outer root sheath cells,its histologic characteristics was investigated in vitro and after transplanted onto SD albino rats.Results Composite chitosan bilayer skin substitute reconstructed by cells from hair follicle had closely arranged epithelium cells and outstanding cornification;Epithelial cords linked with epidermis could be seen in dermis.However,there was no certain hair follicle-like structure formation either in vitro or in vivo.Conclusion Hair follicle cells are good source for skin substitute reconstruction,but it can not induce skin appendage formation through skin substitute by now.

OBJECTIVETo investigate the practicability of repair of full skin loss of rabbits with composite chitosan artificial skin.

METHODSDermal substitute was prepared aseptically by mixing fibroblasts with composite dermal matrix gel. Keratinocytes were then seeded on the substitute and submersion - cultured thereafter for 1 week in keratinocyte culture medium. The composite was further cultured for 1 approximately 2 weeks on the surface of the culture liquid to form artificial skin. The composite chitosan artificial skin was then grafted onto the full skin loss wound of rabbits. Histological changes were undertaken periodically by tissue sampling from the grafted wound. The systemic reaction of rabbits to the artificial skin was observed.

RESULTSAll the grafted wounds healed very well without any suppuration, bleeding or infection under the grafted skin. No obvious immune rejection was seen. The artificial skin could cover the wounds for a long time with good elasticity and easy to be manipulation.

CONCLUSIONThe composite chitosan artificial skin could be an optimal biological dressing with good histocompatibility and easy to be manipulation.

Animals ; Burns ; surgery ; Cells, Cultured ; Chitin ; analogs & derivatives ; metabolism ; Chitosan ; Dermatologic Surgical Procedures ; Humans ; Keratinocytes ; cytology ; metabolism ; Male ; Rabbits ; Skin ; injuries ; Skin Transplantation ; methods ; Skin, Artificial ; Transplantation, Heterologous ; Wound Healing