【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.
Objective Epidermal stem cells (ESCs) can actively partici pate in wound heal ing and enhance reepithel ial ization. To establ ish ideal diabetes mell itus (DM) rat models and to investigate the expression of keratin 19 (K19),β1-integrin, β-catenin, and prol iferating cell nuclear antigen (PCNA) in ESCs of DM rat model, then to study the potential mechanism of difficult recovering wounds of diabetic skin. Methods Twenty male SD rats (weighing 250-300 g) were dividedinto DM group and normal control group randomly (n=10). The DM rat model was made by intraperitoneal injected 65 mg/kg streptozocin (STZ), the normal control group was not treated. At 4 weeks after injection, pancreatic tissue was harvested for HE staining in two groups. The ESCs isolated from full-thickness skins of the back of two group rats were culutured and identified. The 2nd passage of ESCs were obtained for immunocytochemical staining of K19, β1-integrin, β-catenin, and PCNA. Meanwhile, the cell cycle were measured by flow cytometry. The cell colony formation rates were detected after 1 week. Results The achievement ratio of DM rat model was 90% with good stabil ity. HE staining showed that the number of islet cells significantly decreased with degeneration and necrosis in DM group; the structure of islet cell was clear without degeneration and necrosis in normal control group. The integral absorbance values of positive expression for K19, β1-integrin, β-catenin, and PCNA in ESCs of DM group (82.63 ± 14.77, 21.59 ± 4.71, 6.49 ± 6.58, and 90.77 ± 12.44, respectively) were significantly lower than those in the normal control group (151.24 ± 42.83, 54.48 ± 17.43, 116.39 ± 9.26, and 110.62 ± 20.67, respectively) (P lt; 0.01). The clone forming efficiency of ESCs in DM group (6.43% ± 1.01% ) was significantly lower than that in the normal control group (11.37% ± 1.62%) (P lt; 0.01). Flow cytometry indicated that 88.89% of cultured ESCs in the DM group were in resting state/ pre-DNA-synthetic gap (G0/G1), and the apoptosis rate was 3.98%; 91.50% in the normal control group and the apoptosis rate was 0. Conclusion The DM rat model can be effectively induced by intraperitoneal injected 65 mg/ kg STZ. The decreased amount and the low prol iferation and differentiation capacity of ESCs may be one of the important mechanisms of difficult recovering wounds of DM rats.
【Abstract】 Objective To search for a feasibil ity of repairing full-thickness cutaneous deficiency with tissueengineered skin substitute composited by human epidermal stem cells and fibroblasts in fibrin frame. Methods Epidermal stem cells and fibroblasts were harvested from human epidermis and dermis by trypsin digestion. Cells were cultured and subcultured in non-serum medium. Epidermal stem cells (5×104/mL) and dermal fibroblasts (1×104/mL) in 0.5 mL medium were coagulated in 0.5 mL fibrin frame to construct tissue engineered skin substitute. The tissue engineered skin substitute was grafted onto full-thickness cutaneous deficiency of nude mice. Forty-five male mice, 4-5 week old, weighted 20 g on average, were randomly divided into 5 groups. Oil yarn (group C), fibrin frame membrane without cell inoculation (group F), composite skin substitute with epidermal stem cells (group S) and composite skin substitute with fibroblasts (group Fb) were used as controls, while tissue engineered skin substitute (group T) was experimental group. The wounds were observed 1, 3, 6, 8 weeks after surgery. Samples were harvested 3, 6, 8 weeks after surgery, and were examined by means of histology、immunohistochemistryand scanning electron microscopy (SEM). Results Four weeks after cell culture, there were some round cells in the culture capsule of epidemic cells, and some fusiform cells in the culture capsule of fibroblast. Six days after cells were cultured in the BrdU culture medium, there were some BrdU positive cells appeared. There were some CK19 positive cells and Nestin positive cells appeared in the chaff of group T before transplanting. The new formed skin of group T grew faster and had less scar than other groups. Six weeks after surgery, the average thickness of new formed skin was (0.460 ± 0.049) mm in group C, (0.480 ± 0.055) mm in group F, (0.540 ± 0.043) mm in group S, (0.510 ± 0.032) mm in group Fb, (0.660 ± 0.047) mm in group T. The thickness of new formed skin in group T was thicker than other groups (P lt; 0.05). By histology and SEM observation, 3, 6, 8weeks after surgery, the new formed cuticular layer, fibroblast and blood vessels in the group T were more than those in theother groups. The al ignment of blood vessels and collagen fibers in group T were much regular than those in the other groups. Three weeks after surgery, the new formed skin of group T had a continuous color zone of positive collagen Ⅳ staining, while no continuous color zone was found in the other groups. Six weeks after surgery, CK14 positive cells appeared in the new formed skin of group T, while no positive cell was found in the other groups. Conclusion Tissue engineered skin substitute which is composited with epidermal stem cells and fibroblasts in fibrin frame has potential prospects in appl ication of repairing fullthickness cutaneous deficiency with advantage of faster wound heal ing.
Objective To provide theoretical evidence for clinical application of the epidermal stem cells after an investigation on changes of the epidermal stem cells during the survival process after the fullthickness skin autograft. Methods On the backs of 42 Wistar rats, orthotopic transplantation models (1.5 cm×1.5 cm) of the fullthickness skin autograft were made. According to the time of the specimen taking, at 1, 3, 5, 7, 14, 21 and 30 days after operation, the rats were randomly divided in 7 groups (Groups 1-7). Specimens taken in each group before operation were used as controls. At each time point, the gross observation was made on the transplanted skin flaps, from which the skin tissues were harvested at each time point before and after operation. The routine pathological and the immunohistochemical examinations were performed on the specimens, which were stained by HE and were observed for immunohistochemical changes and the changes in the cells positive for integrinβ-1 and p63. Results All the fullthickness skin autografts survived 3 days after operation except the skin autograft in 1 rat in both Group 5 and Group 6, which was infected around the transplanted skin flap. In Groups 1-4, cell edema, inflammatory cell infiltration, and increased fibrocytes were observed. In Groups 5-7, the maturity degree of the epithelial cells became higher and higher, and the fibrocyte proportion was lowered. In each group the cell positivity rate for integrin β1 was lower than the cell positivity rate for p63. The positive cells were arranged in disorder, distributed into the layers of the epidermis and gradually concentrated in the basal layer of the epidermis and the bulge of the folliculus pili. The positive cells were also found in the other layers of the epidermis.The positive cells were gradually decreased in number, and reached the lowest level in Group 2. There was a significant difference in the above variables in Groups 1,2,3,5,6 and 7 between before and after operations (P＜0.05). Conclusion During the survival process of the fullthickness skin autograft, the proportion of theepidermal stem cells is gradually decreased at first; Then, the proportion isgradually increased, even beyond the normal level; finally, the proportion is decreased again. The distribution of the epidermal stem cells appear in disorder, almost distributed in the layers of the epidermis; finally, the almost normal distribution can be found.
Objective To observe the epidermal width, the amount variation and distribution character of epidermal stem cells(ESCs) and the wound healing rate at different periods of diabetes mellitus(DM) rats after trauma, thento study the correlation of them. Methods Forty-eight Wistarrats were divided into DM group and normal control group randomly(n=24).TheDM rats were induced by streptozocin (STZ) and then made chronic healing wound by special perforex.At the 3rd, 4th, 7th,14th and 21st days after trauma, the healing rate was calculated, the wound edge and granulation tissue were obtained for haematoxylin-eosin (HE) staining and immunohistochemical staining of keratin 19(K19) and β1 integrin. Then the epidermal width, the area and the gradation value of positive unit(PU) were measured. Results At the 3rd, 7th,14th and 21st days after trauma, the wound healing rates of normal rats were 24.48%±3.37%, 50.46%±1.26%, 92.82%±2.12% and 99.41%±0.66% respectively, while those of DM rats were 2.43%±1.02%, 40.59%±1.65%, 80.77%±3.57% and 85.40%±0.94% respectively, showing significant differences (Plt;0.01). Before trauma, there was no significant difference in the epidermal width between normal rats and DM rats (Pgt;0.05). However, at the 3rd, 7th, 14th and 21st days after trauma, the epidermal widths of normal rats were 26.43±3.21, 33.29±3.52, 31.53±3.35 and 26.01±3.19 μm respectively, while those of DM rats were23.58±2.33, 31.02±3.38, 33.72±5.49 and 21.80±4.02 μm respectively,the epidermal widths in DM rats were significantly lower than those in normal rats(Plt;0.01). The average PU value of K19 in normal rats were 91.68%, 93.14%, 72.27% and 70.31% respectively, while those in DM rats were 40.29%, 40.79%, 29.94% and 10.37% respectively. The average PU value of β1 integrin in normal rats were 49.6%, 91.16%, 77.13% and 57.17% respectively, while those inDM rats were 38.94%, 24.16%, 61.36% and 38.83%. The results indicated that the average PU values of K19 and β1 integrin in DM rats were significantly lower than those in normal rats(Plt;0.05). Conclusion The amountand activity decrease of ESCs may be one of the important mechanisms of difficult recovering wounds of DM rats.
Objective To investigate the culture method forepidermal stem cells in vitro. Methods The epidermis was separated from the dermis, and shaken for 10 min in 0.05% trypsin at 37℃ to dissociate into single cells. Epidermal stem cells were selected by rapid attachment to collagen Ⅳ for 10-15 min and cultured on collagen Ⅳ or 3T3 feeder layer. All the cells were grown in DMEM without calcium, supplemented with 10% chelexed fetalbovine serum, 10 μg/L epidermal growth factor, 0.05 mmol/L CaCl2 and 0.8 mg/L hydrocortisone. Cultures were observed for colony formation under a phase constrast microscope. The phenotypes of epidermal stem cells were detected by flow cytometry and immunocytochemistry staining. Results The cells selectedby rapid adherence to collagen Ⅳ formed large colonies at 7～8 days, expressedK19 antigen. The percentages of cells at the G0 and G1 phases of the cell cycle and the percentage of α6briCD71dim cells in the experimental groups were higher than those in the control group. It indiciated that there was a significant difference between the experimental groups and the control groups(P＜0.05). ConclusionThe humanepidermal stem cells can be selected by rapid attachment to collagen Ⅳ, and they can be expanded in culture if the appropriate conditions are maintained.
ObjectiveTo observe the possibility and mechanism of microRNA (miRNA)-203 inducing the human epidermal stem cells to differentiate into sweat gland cells. MethodsFive normal human foreskin tissues were harvested to prepare a single cell suspension by 0.25% trypsin-EDTA digestion method, then the human epidermal stem cells were isolated and cultured by type IV collagen differential adherent method. The cell morphology was observed by inverted phase contrast microscope. The monoclonal antibodies of integrin β1 (ITGB1), cytokeratin19 (CK19), CK1, CK10, CK18, and carcinoembryonic antigen (CEA) were used for identification by immunocytochemical staining. Double stranded mimics of has-miR-203 were transfected into the human epidermal stem cells with Lipofectamine 2000 (experimental group) and the human epidermal stem cells transfected with nonsense miRNA mimics served as control group. The monoclonal antibodies of ITGB1, CK19, CK1, CK10, CK18, and CEA were used for identifying the cells after transfection by immunocytochemical staining; the mRNA relative expressions of miRNA-203, P63, ITGB1, CK19, CK1, CK10, CK18, and CEA were detected by real-time fluorescence quantitative RT-PCR before transfected and at 72 hours after transfected. The protein relative expressions of P63, ITGB1, CK19, CK1, CK10, CK18, and CEA were detected by Western blot. The mRNA expression of miRNA-203 and the mRNA and protein expressions of P63 were analyzed respectively with Pearson correlation. ResultsThe CK19 and ITGB1 were positively expressed before transfection, but CK1, CK10, CK18, and CEA were expressed positively after transfection. The mRNA relative expression of miRNA-203 after transfection in experimental group was significantly higher than that before transfection (P<0.05). The mRNA and protein relative expressions of CK1, CK10, CK18, and CEA after transfection in experimental group were significantly higher than those before transfection and control group (P<0.05), while the mRNA and protein relative expressions of P63, CK19, and ITGB1 were significantly lower than those before transfection and control group (P<0.05). These indicators showed no significant difference between the control group and before transfection (P>0.05). The expression level of miRNA-203 was negatively correlated with the mRNA and protein relative expressions of P63 before and after transfection, the correlation coefficients before transfection were -0.91 (t=3.862, P=0.042) and -0.96 (t=5.971, P=0.009) respectively; the correlation coefficients after transfection were -0.92 (t=4.283, P=0.031) and -0.95 (t=5.842, P=0.011) respectively. ConclusionmiRNA-203 can induce epidermal stem cells to differentiate into sweat gland cells by targeting inhibition of P63 probably.
ObjectiveTo explore the phenotypic changes of epidermal stem cells (ESCs) differentiating into sweat glands cells (SGCs) in vitro and its mechanisms. MethodsESCs and SGCs were isolated and cultured in vitro, which were identified using immunofluorescence staining. ESCs at passage 2 were divided into 4 groups: ESCs and SGCs co-cultured by Transwell plates in group A, ESCs cultured by simply adding sweat supernatant in group B, ESCs and SGCs co-cultured on Transwell plate adding epidermal growth factor (EGF) (60 ng/mL) in group C, and ESCs and SGCs co-cultured on transwell plate adding PD98059 (10 mmol/L) in group D. The inverted microscope was used for observing the morphology of ESCs, flow cytometry for detecting ESCs positive phenotype, and Western blot for exploring mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway. ResultsThe morphology observation and immunofluorescence staining suggested that cultured cells were ESCs and SGCs. The inverted phase contrast microscope observation showed that cells had similar morphological changes, with flat polygonal shape at 9 days in groups A, C, and D; cells had slow morphological change in group B, and had similar change to that of other groups at 12 days. Significant decreasing of β1-integrin expression and increasing of carcino-embryonic antigen (CEA) expression of ESCs were observed in group A when compared with group B, which was inhibited by EGF (group C) and enhanced by PD98059 (group D), and there were significant differences among groups A, C, and D (P<0.05). High level of ERK expression was displayed in 4 groups, but it was significantly lower in group B than the other 3 groups (P<0.05). The expression of phosphorylation ERK was the highest in group A and was the lowest in group C, showing significant difference among 4 groups (P<0.05). ConclusionESCs can be induced to differentiate into SGCs with the phenotypic changes under the condition of co-cultured by Transwell plates. The MAPK/ERK pathway plays a key role in the differentiation of ESCs into SGCs.