ABSTRACT
Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED).Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs,and a two-step collagenase digestion was used to treat healthy children's prepuce tissues to prepare fibroblast suspensions.When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2,they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissueengineered skin.A total of 20 heahhy male nude mice aged 3-4 weeks were enrolled into this experiment,and full-thickness skin defects were made on the middle of the back of mice.Then,these mice were randomly divided into 2 groups by using a lottery method,and reconstructed full-thickness tissueengineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively.The whole body and transplantation sites of the nude mice were observed on day 7,14,21 and 28 after transplantation,the wound healing time and rate were compared between the above two groups,and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination.Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen4 (SSEA-4).After 2-week organ culture,the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium,and the histological structure of the epidermis was similar to that of the normal human skin.Compared with the control group,the tissue-engineered skin group showed significantly higher wound healing rates on day 7,14 and 21 after transplantation (57.49％ ± 6.11％ vs.22.93％ ± 4.26％,92.80％ ± 3.10％ vs.54.57％ ± 7.94％,98.83％ ± 0.25％ vs.91.16％ ± 4.79％,respectively;n =10,t =27.36,32.23,11.80,respectively,all P ＜ 0.001),shorter wound healing time [(21.51 ± 1.51) d vs.(28.80 ± 1.14) d,n =10,t =42.23,P ＜ 0.001],with the color of skin grafts closer to that of autologous skin on day 28 after transplantation.Histological examination revealed distinct stratification of the epithelium,obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group,but thin epithelium with some defects,indistinct stratification of the dermis,and inflammatory cell infiltration in the control group.Condusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation,resulting in a more favorable healing of wounds,and is expected to serve as a kind of ideal tissue-engineered skin.
ABSTRACT
Objective To investigate the effects of an ar?turmerone derivative(ATD)on the proliferation and apoptosis of A375 human melanoma cells. Methods Both A375 cells and human skin fibroblasts (HSFs) were cultured with different concentrations(5, 10, 20, 40 and 80μmol/L)of ATD, vincristine and ar?turmerone, separately, for 48 hours in vitro. Subsequently, cell counting kit?8 (CCK?8) was used to evaluate cell proliferation, inverted microscopy to observe cell morphology after acridine orange/ethidium bromide (AO/EB) staining, and a colorimetric method to estimate caspase?3 activity. DNA fragmentation assay and flow cytometry were performed to assess cell apoptosis, and flow cytometry was conducted to analyze cell cycle. Results ATD, vincristine and Ar?turmerone all inhibited the proliferation of A375 cells in a dose?dependent manner(ATD:R2=0.99, F=340.96, P<0.05;vincristine:R2=0.99, F=349.19, P<0.05;ar?turmerone:R2=0.89, F=25.41, P<0.05). The fifty percent inhibitory concentra?tions(IC50s)of ATD, vincristine and ar?turmerone against A375 cells were 15.96 ± 0.02μmol/L, 77.00 ± 0.04μmol/L and 356.95 ± 0.01μmol/L respectively. When the drug concentrations were 5 and 10μmol/L, the proliferation of HSFs was inhibited by 8%± 0.06%and 25%± 0.02%respectively by ATD, by 49%± 0.09%and 34%± 0.07%respectively by ar?turmerone, and by 33%± 0.04%and 29%± 0.08%respectively by vincristine, and the proliferation of A375 cells was inhibited by 26%± 0.06%and 39%± 0.02%respectively by ATD, by 6%± 0.09%and 10%± 0.07%respectively by ar?turmerone, and by 8% ± 0.04% and 17% ± 0.08% respectively by vincristine, with the inhibitory effects of the three drugs being significantly different from that of dimethyl sulfoxide(all P<0.05). ATD showed stronger inhibitory effects on the proliferation of A375 cells, but weaker cytotoxic effects on HSFs compared with ar?turmerone and vincristine(all P<0.05). Meanwhile, ATD, vincristine and ar?turmerone all induced the apoptosis of A375 cells(P<0.05), and caspase?3 activity increased with the increase in drug concentrations(ATD:R2=0.98, F=162.30, P<0.05;vincristine:R2=0.96, F=94.39, P<0.05;ar?turmerone:R2=0.95, F=57.35, P<0.05). The effect of ATD on caspase?3 activity was strongest, followed by that of vincristine and ar?turmerone. As flow cytometry showed, all the three drugs induced cell apoptosis to different degrees, and ATD showed a relatively strong effect on cell apoptosis, especially late apoptosis, compared with the other two drugs. In the ATD group, the number of A375 cells in G1 phase gradually increased, while that in G2 phase and S phase significantly decreased with the increase in drug concentrations. Conclusions ATD exhibited proliferation?inhibiting and apoptosis?inducing effects on A375 cells, and the effects were stronger than those of vincristine and ar?turmerone. It is quite possible that ATD affects cell proliferation and differentiation by activating caspase?3 and arresting cell cycle in the G1 phase.
ABSTRACT
BACKGROUND:We have built the three-dimensional human skin melanoma model with human epidermal keratinocytes and MV3 melanoma cel s co-cultured on the de-epidermized dermis in vitro. OBJECTIVE:To establish a skin model of melanoma by mixed culture of MV3 melanoma cel s and HaCaT cel s on the de-epidermized dermis in vitro. METHODS:MV3 melanoma cel s and HaCaT cel s were mixed with different percentages and inoculated on the surface of de-epidermized dermis fol owed by a liquid culture and air-liquid culture, and then the tissue-engineered skin model was established in vitro. Routine biopsy immunohistochemical observation was performed on the constructed skin melanoma model. RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that MV3 melanoma cel s were distributed on the surface layer of de-epidermized dermis and formed tumor masses, while the HaCaT cel s were mixed growth with tumor cel s and formed a typical epidermoid structure. Some tumor cel s infiltrated into the surface or deep of de-epidermized dermis and showed a tumor foci distribution. The CK10, CK-pan and S-100 proteins were positive for immunohistochemical staining. With the increasing of MV3:HaCaT cel percentage, CK10 and CK-pan gradual y down-moved from the surface, and changed from layer distribution to lumpy distribution, while the staining of S-100 protein was gradual y distributed layer-by-layer, and some area showed tumor-like distribution. The results show that the skin model of melanoma can be in vitro constructed successful y by mixed culture of MV3 melanoma cel s and HaCaT cel s on de-epidermized dermis.
ABSTRACT
Requirement for facility and environment of FDA, regulation of China medical device Quality System, Europe medical device Directive and ISO13485 standard are introduced. The comparison is made to help domestic medical device industry understand the regulation requirement.
Subject(s)
Device Approval , Environmental Monitoring , Industry , Quality ControlABSTRACT
Objective To observe the changes of melanin granules in superficial corneocytes in vitiligo lesions after irradiation with narrow-band ultraviolet B (NB-UVB) by using an adhesive tape stripping technique.Methods Vitiligo lesions were selected from 6 patients and irradiated with NB-UVB every other day for 31 sessions.Superficial corneocytes were obtained by an adhesive tape stripping technique from the vitiligo lesions and perilesional normal skin before every treatment.The morphology,distribution and color of melanin granules were observed after Masson-Fontana silver staining.The percentage of area occupied by melanin granules in superficial corneocytes were calculated by using the Image-Pro Plus 6.0 software.Statistical analysis was conducted by the SPSS11.5 software.Results There were still a few superficial corneocytes containing melanin granules remaining in the vitiligo lesions before treatment.The percentage of area occupied by melanin granules was (5.31 ± 4.12)％ before treatment,(6.24 ± 2.65)％ after 10 treatment sessions,(10.14 ± 5.73)％ after 20 sessions,and (13.05 ± 6.17)％ after 30 sessions,with significant differences between these time points (F =4.334,P ＜ 0.05).Multiple comparisons revealed a significant increase in the percentage of area occupied by melanin granules after 30 treatment sessions compared with those before treatment and after 10 treatment sessions (both P ＜ 0.01 ).The morphology and color of melanin granules in repigmented lesions after treatment differed from those in perilesional normal skin before treatment.Conclusion The adhesive tape stripping technique may serve as a useful tool for the evaluation of repigmentation in vitiligo lesions after phototherapy.
ABSTRACT
Objective To construct tissue-engineered skin containing melanin with mixed culture of human keratinocytes (KCs) and melanocytes (MCs) on de-epidermized dermis (DED) in vitro. Methods Single-cell suspension was obtained by digestion of isolated preputial epidermis with pancreatin. Keratinocyte serum-free medium (K-SFM) and modified M254 culture medium were used to culture KCs and MCs respectively. Third-passage KCs were seeded into cell culture flasks and cultured for 48 hours; then, third-passage MCs were seeded into the same cell culture flasks with the MC/KC ratio being 1: 10 followed by a 5-day coculture. The suspension of third-passage KCs and MCs with the MC/KC ratio of 10:1 were seeded onto the surface of a prepared DED and maintained at the air-liquid interface for 11 days following a 4-day submerged culture.Subsequently, the constructed tissue-engineered skin was examined with HE staining, immunohistochemical staining for keratin and Masson-Fontana staining. Results After coculture in flasks for 5 days, KCs exhibited a typical paving-stone appearance, MCs with projected dendrites were scattered in the extracellular space between KCs. HE staining revealed 3 to 6 layers of cells with the formation of stratum corneum after mixed culture on DED for 15 days. Keratin protein was positive throughout the artificial epidermis, and melanin pigments were located in the basal layer of the epidermis as Masson-Fontana staining showed. Conclusions The co-culture of MCs and KCs can form single-cell layers with the contact between MCs and KCs in flasks, and construct tissue-engineered skin with melanin component on DED in vitro.
ABSTRACT
Objective To establish a double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) for the rapid detection of germ tube-specific antigens of Candida albicans,and to evaluate its specificity and sensitivity.Methods A DAS-ELISA was established with the monoclonal antibody McAb03.2C1-C2 as the primary antibody,and horseradish peroxidase (HRP) labeled McAb03.2C1-C2 as the secondary antibody.The established assay was used to detect germ tube-specific antigens of Candida albicans in sera from 5 patients with systemic Candida albicans infection and from 6 rabbit models at 12,24,48,72hours,on week 1,2 after infection with Candida albicans.Results A good liner relationship was observed between the absorbance value at 495 nm and antigen concentrations when the titer of McAb03.2C1-C2 was 1 ∶ 4000 and the concentration of coated antigen varied from 1.25 to 40 μg/ml.The specificity and sensitivity of the DAS-ELISA were 95％ and 92％ respectively in the detection of germ tube-specific antigens in the rabbit models.The results of detection with DAS-ELISA in serum specimens from the patients were consistent with those with the routine method.Conclusions A DAS-ELISA is primarily established for the rapid detection of germ tube-specific antigens of Candida albicans,and has shown a satisfactory sensitivity and specificity in the animal model experiment.
ABSTRACT
Objective To construct an artificial skin model of melanoma by mixed culture of human keratinocytes and MV3 melanoma cells on de-epidermized dermis (DED) in order to study the effect of keratinocytes on melanoma invasion. Methods Epidermal cell suspension was obtained by a two-step digestion method from the circumcised foreskin of a child, keratinocyte serum-free medium was applied to the culture and passage of keratinocytes. MV3 melanoma cells were cultured and passaged in RPMI 1640 medium. Log-phase keratinocytes and MV3 cells were mixed with a ratio of 3:1 and seeded onto the surface of DED followed by a liquid culture and air-liquid culture for a total of 2 weeks. Thereafter, the artificial tissue model was assessed by HE staining and immunohistochemical staining for S-100 protein, HM64S and keratin. Results HE staining showed that MV3 cells formed band-like tumor masses or foci on the surface of DED, with keratinocytes intermingling among the tumor cells, but no typical epidermis-like structure was observed. Some tumor cells infiltrated into the surface of DED and showed a cluster distribution; some tumor cells invaded the lumen of the DED, and attached to the luminal wall in a ring shape; some tumor cells penetrated through the wall into the surrounding dermal tissue. On the bottom and lateral side of DED, tumor cells were infiltrating dispersedly.The tumor loci stained positive for S-100 protein and keratin, and weakly positive for HMB45. Conclusion Keratinocytes enhance the invasion of MV3 melanoma cells into the skin tissue model of melanoma.
ABSTRACT
BACKGROUND:Study confirmed that the de-epidermized dermis(DED)can be used as dermal substitute and may form epidermal structure after incubating keratinocytes.However,the cell biological activity,tissue structure characteristics and the basement membrane component analysis of dermal substitute have been reported less.OBJECTIVE:To investigate the cell activity and the tissue structure characteristics of DED.METHODS:Skin flap was treated with 56℃ phosphate buffered solution to remove the epidermis,and the dermal cell components were deleted by freezing and thawing with liquid nitrogen to obtain DED.The DED cell activity was detected with tissue culture method,hematoxylin nuclear staining was used to determine the DED cell nuclei,and vimentin immunohistochemistry was applied for fibroblast determinations.The basement membrane and its components were detected using Periodic Acid-Schiff staining and collagen type Ⅳ immunohistochemistry.Van Gieson stain,Weigart stain and those double staining were respectively used to determine DED collagen fibers and elastic fibers.The DED ultrastructure was observed under transmission and scanning electron microscope.RESULTS AND CONCLUSlON:Using tissue culture method,the cultured DED did not exhibit cell growth at 2 weeks.Hematoxylin-eosin staining showed no nuclear in DED,vimentin immunohistochemistry showed no vimentin expressed in DED.Van Gieson staining showed DED collagen fibers were stained as rose red,Weigert staining showed DED elastic fibers were stained as pu rplish black double staining further demonstrated uniform arrangement of collagen fibers and elastic fibers.DED surface and the remaining appendages were strongly positive for Periodic Acid-Schiff staining,and type Ⅳ collagen expression was significant.Transmission and scanning electron microscope results showed that,the DED elastic fibers and collagen overlap arranged with pore intervals,they intercrossed into a network.There is no living cell component in DED,dermal matrix surface and appending organ luminal wall still retain glycogen,type Ⅳ collagen and other basement membrane components,dermal matrix is rich in collagen and elastic fibers.it is a three-dimensional collagen matrix similar to in vivo dermis.
ABSTRACT
Objective To study the in vitro culture condition for melanoblasts from human foreskin tissue. Methods The skin tissue taken from foreskin of children was treated with 0.5% dispase Ⅱ to separate epidermis from dermis, then with trypsin to obtain single cell suspension, which was cultured in modified medium for melanoblasts, i.e., MCDB254 medium supplied with several cell growth factors. Finally, melanoblasts were obtained based on the difference of adhesion speed. The morphology and proliferation of cultured melanoblasts were observed under a light microscope. DOPA staining, immunostaining with anti- S-100 and -tyrosinase related protein 2 (TRP2) antibodies, and transmission electron microscopy were per- formed to identify the cultured melanoblasts. Results The cultured human melanocytes displayed a match-like shape, scattered arrangement, syrmnetric double poles, slim cell body, highly refractive nuclei; meanwhile, the melanoblasts exhibited plentiful cytoplasm, large volume, bipolar or irregular shape and clonal growth. Additionally, the melanocytes were positive for TRP2, S-100 and Dopa staining, while the melanoblasts were positive only for TRP2. Electron microscopy revealed the presence of mature melanin granules (stage Ⅲ-Ⅳ ) in melanocytes but immature melanin granules (stage Ⅰ ) in melanoblasts. Conclu- sion Stable pure culture of melanoblasts has been realized with the reformed medium, which may lay a foundation for the investigation into the mechanism of epidermal pigmentation.
ABSTRACT
Objective To investigate the relationship of fascin-1 protein expression with the metastasis of basal cell carcinoma and squamous cell carcinoma. Methods Skin specimens were obtained from 10 normal human controls, 13 patients with basal cell carcinoma (8 nodular variant and 5 superficial variant) and 24 patients with SCC (11 SCC in situ and 13 invasive SCC). Immunohistochemical staining was performed to analyze the expression of fascin-1 protein. The staining results were quantitatively assessed with computer image analysis system (Image-pro Plus 6.0). Results The optical density of fascin-1 averaged 0.1152±0.04574 in SCC in situ, 0.1257±0.03096 in invasive SCC, and 0.0293±0.00981 in normal controls; the expression of fascin-1 was significantly higher in SCC tissue than in normal control skin (both P<0.05). Increased optical density was also observed for fascin-1 in nodular variant of SCC (0.0808 ±0.05642) and superficial variant of SCC (0.0806±0.04346) compared with the normal controls, whereas no statistical difference was observed between nodular and superficial variant of SCC. Conclusion In BCC and SCC, there is an over expression of fascin-1, which may be linked to the local invasion of carcinoma,
ABSTRACT
Objective To establish an in vitro model for malignant melanoma with a malignant melanoma cell line MV3 on de-epidermized dermis (DED) and to study the invasion mode of melanoma cells. Methods A human de-epidermized dermis was prepared with some elements of basal membrane (BM). Then, the reconstructed BM was identified by periodic acid schiff (PAS) staining and immunochemical staining for collagen Ⅳ. MV3 cells were seeded onto the prepared acellular dermis and maintained at the air-liquid interface for 13-15 days after 3-day submerged culture. Subsequently, the reconstructed malignant melanoma tissue was examined with hematoxylin and eosin (HE) staining and immunohistochemical staining with antibodies to S-100 protein and HMB45. Results No obvious changes were observed by naked eye in DED after the inoculation with MV3 cells. PAS staining and immunochemical staining for collagen Ⅳ confirmed the presence of BM component on the surface of DED and in the cavity of skin appendages in DED. Histological examination and immunochemical staining revealed that on the BM zone, MV3 cells grew into irregularly sized clusters; in the .cavity of skin appendages, they attached onto the BM and aggregated into circular or bandlike shape; and at the lateral side of DED, they invasively and diffusely grew, broke through the BM and intruded into the surrounding tissues of DED. The reconstructed tissue was positive for S-100 protein and weakly positive for HMB45. Conclusions The in vitro model of malignant melanoma could be reconstructed by skin organ culture system. And, the experiment suggests that BM could affect the invasive growth pattern of malignant melanoma cells.
ABSTRACT
Objective To investigate the phenotype,number and distribution of inflammatory cells in early and late stages of spontaneous regression of halo nevi,and to elucidate the immunological mechanisms for spontaneous regression of these nevi.Methods Halo nevi,their surrounding non-lesional skin,and normal control skin were examined by immunohistochemical staining with monoclonal antibodies to CD3,CD4,CD8,CD20,CD1a,CD56 and CD68.Staining results were observed and analyzed by the computer image analysis system,image-pro plus 6.0.Results The number of CD4+,CD8+,CD20+,CD1a+cells,along with the diameter of CD1a+and CD68+ cells was significantly increased in the lesions of early and late stage of spontaneous regression of halo nevi than in non-lesional skin and normal control skin(both P＜0.01).The ratio of CD8+/CD4+ cells in the lesions of late stage of spontaneous regression was also higher than that in the lesions of early stage (2.05∶1 VS 1.82∶1).A massive infiltrate of CD8+ cells was observed in the nests of nevus cells.ConclusionsCD4,CD8,CD20,CD1a,CD56 and CD68 positive cells are all involved in the spontaneous regression of halo nevi,and CD8+ cells may play a predominant role in this process.
ABSTRACT
Objective To construct tissue-engineered skin via in vitro inoculation of epidermal stem cells(ESCS)onto de-epidermized dermis.Methods Skin tissue was obtained from the foreskin of a healthy 6-year-old child.and keratinocytes were isolated by two-step trypsinization method followed by the collection of ESCS via rapid adhesion by collagen Ⅳ.The ESCS were identified by morphological observation and immunohistochemical staining with K19 and integrin β1.To construct tissue-engineered skin,selected ESCS were seeded onto the surface of de-epidermized dermis followed by a one-week culture immersed in the medium and a subsequent 4-week culture at the air-medium interface.The tissue-engqneered skin was evaluated with haematoxylin & eosin(HE)staining as well as keratin immunohistochemistry.Results Micro scopically,cultured ESCs showed a paving stone-like appearance and grew into colonies.Immunohistochemistry revealed that the ESCs were positive for integrin-β1 and keratin 19.After 5 weeks of culture,3-6 layers of epidermal cell were observed on the dermis with the formation of stratum corneum.Keratin protein was observed in the artificial epidermal skin.Conclusion Tissue-engineered skin is successfully constructed with epidermal stem cells and de-epidermized dermis in vitro.
ABSTRACT
Objective To establish a reconstructed human epidermis model in vitro, and to study the process of re-epithelialization. Methods A dermal substrate devoid of epidermis was prepared; a 2 mm skin biopsy explant was transplanted onto the dermal substrates. Visualization of epidermal cell migration was carried out by fluorescence imaging. The proliferation and differentiation of the new epithelial cells were observed using histopathological and immunohistochemical staining (Ki67). EGF was added to the culture medium of the experimental samples but not to that of the controls. Results After 3 days of culture, re-epithelialization was observed on the surface of the dermal substrate. A complete structure resembling regular epidermis was noted in 10 days. As compared to control samples, EGF-treated samples had larger area of re-epithelialization (t= 3.02, P
ABSTRACT
Objective:To reconstruct middle ear structure for open mastoid antrum with external auditorycanal after radical mastoidectomy in one-stage. Method: 71 ears of post-mastoidectomy (discharging 53 ears anddried up 18 ears) were undergone with reconstruction of middle ear. The posterior wall of external auditorycanal, mastoid cavity and chain of ossicles were reconstructed with homologous costal cartilage. Result: 69 ears of71 cases were near normal structure followed up 6 months to 5 years after operations. The result showed hearingimprovement over 15 dB were 55 ears (77.5％) and under 15 dB were 11 cases (15.5％). Five cases (7.0％)were failed. Conclusion: Reconstruction of middle ear with homologous costal cartilage is a ideal surgery toreconstruct hearing structure and avoid infection of middle cavity after radical mastoidectomy.
ABSTRACT
Objective To investigate the effects of staphylococcal enterotoxin A SEA gene on target cells mediated by replicative-deficient recombinant adenovirus vector. Methods Lymphocytes of C57BL/6 mice were infected with various titers of recombinant adenoviruses. Supernatants were collected after 12 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h of incubation and analyzed for proliferation of lymphocytes by MTT assay. IL-2 level in the culture supernatants was measured with ELISA. The killing effect of lymphocytes was also observed by MTT assay. Results Proliferation response and elevated levels of IL-2 were observed in experimental group. The killing effect on B16 cells was stronger in experimental group, which seemed to be dose-dependent with the increase of ratio of lymphocytes/target cells. Conclusions SEA gene can be expressed in lymphocytes of C57BL/6 mice mediated by replicative-deficient recombinant adenovirus vector. The expressing products can activate lymphocytes of C57BL/6 mice, which kill B16 cells in vitro.