Bone marrow-derived mesenchymal stem cell attenuates skin fibrosis development in mice.

Recent studies showed that mesenchymal stem cell (MSC) transplantation significantly alleviated tissue fibrosis; however, little is known about the efficacy on attenuating cutaneous scar formation. In this study, we established a dermal fibrosis model induced by bleomycin and evaluated the benefit of bone marrow-derived mesenchymal stem cells (BM-MSCs) on skin fibrosis development. Tracing assay of green fluorescent protein (GFP(+) )BM-MSCs showed that the cells disappeared gradually within 24 hours upon administration, which hinted the action of BM-MSCs in vivo was exerted in the initial phase of repair in this model. Therefore, we repeatedly transplanted syngeneic BM-MSCs in the process of skin fibrosis formation. After 3 weeks, it was found that BM-MSC-treated lesional skin demonstrated a unanimous basket-weave organisation of collagen arrangement similar to normal skin, with few inflammatory cells. In addition, lesional skin with BM-MSC treatment exhibited a significant down-regulation of transforming growth factor-ß1 (TGF-ß1), type I collagen and heat-shock protein 47 (HSP47), with higher expression of matrix metalloproteinases (MMPs)-2, -9 and -13. Further experiments showed that α-smooth muscle actin (α-SMA) positive cells, the most reliable marker of myofibroblasts, apparently decreased after BM-MSC transplantation, which revealed that BM-MSCs could attenuate myofibroblast proliferation and differentiation as well as matrix production. Taken together, these findings suggested that BM-MSCs can inhibit the formation process of bleomycin-induced skin fibrosis, alleviate inflammation and favour the remodelling of extracellular matrix.

Future application of hair follicle stem cells: capable in differentiation into sweat gland cells.

BACKGROUND: Sweat glands (SGs) can not regenerate after complete destruction in the severe skin injury, so it is important to find a ideal stem cell source in order to regenerate functional SGs. Hair follicle stem cells (HFSCs) possess the obvious properties of the adult stem cells, which are multipotent and easily accessible. In this research, we attempted to direct the HFSCs suffered from the sweat gland cells (SGCs) special differentiation by a co-operative coculture system in vitro. METHODS: The designed co-culture microenvironment in the transwell was consist of two critial factors: heat shocked SGCs and dermis-like mesenchymal tissue, which appeared independently in the two control groups; after induction, the purified induced SGC-like cells were transplanted into the full-thickness scalded wounds of the nude mice, after 4 weeks, the reconstructed SG-like structures were identified by immunohistochemical and immunofluorescence analysis. RESULTS: A part of HFSCs in experimental group finally expressed SGCs phenotypes, by contrast, the control group 1 which just containing dermis-like mesenchymal tissue failed and the control group 2 consisted of heat shocked SGCs was in a poor efficiency; by immunofluorescence staining and flow cytometry analysis, the expression of HFSCs special biomarkers was down regulated, instead of the positive efficiency of SGCs special antigens increased; besides, the induced SGCs displayed a high expression of ectodysplasin A (EDA) and ectodysplasin A receptor (EDAR) genes and proteins; after cell transplantation, the youngest SG-like structures formed and be positive in SGCs special antigens, which never happened in untreated wounds (P < 0.05). CONCLUSION: The HFSCs are multipotential and capable in differentiating into SGCs which promise a potential stem cells reservoir for future use; our special co-culture microenvironment is promising for HFSCs differentiating; the induced SGCs are functional and could work well in the regeneration of SGs.

Wnt/ß-catenin signaling is critical for dedifferentiation of aged epidermal cells in vivo and in vitro.

Aged epidermal cells have the capacity to dedifferentiate into stem cell-like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/ß-catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell-like cells that were positive for CK19 and ß1 integrin but negative for CK10. In addition, Wnt/ß-catenin pathway was activated during this process. There was increased expression of Wnt-1, Wnt-4, Wnt-7a, ß-catenin, cyclin D1, and c-myc. Secreted frizzled-related protein 1, a Wnt/ß-catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)-3ß inhibitor, of Wnt/ß-catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, ß1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony-forming efficiency, long-term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/ß-catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell-like cells.

Transplantation of human bone marrow-derived mesenchymal stem cells transfected with ectodysplasin for regeneration of sweat glands.

BACKGROUND: Patients with severe full-thickness burn injury suffer from their inability to maintain body temperature through perspiration because the complete destructed sweat glands can not be regenerated. Bone marrow-derived mesenchymal stem cells (BM-MSCs) represent an ideal stem-cell source for cell therapy because of their easy purification and multipotency. In this study, we attempted to induce human BM-MSCs to differentiate into sweat gland cells for sweat gland regeneration through ectodysplasin (EDA) gene transfection. METHODS: The dynamic expression of EDA and EDA receptor (EDAR) were firstly observed in the sweat gland formation during embryological development. After transfection with EDA expression vector, human BM-MSCs were transplanted into the injured areas of burn animal models. The regeneration of sweat glands was identified by perspiration test and immunohistochemical analysis. RESULTS: Endogenous expression of EDA and EDAR correlated with sweat gland development in human fetal skin. After EDA transfection, BM-MSC acquired a sweat-gland-cell phenotype, evidenced by their expression of sweat gland markers by flow cytometry analysis. Immunohistochemical staining revealed a markedly contribution of EDA-transfected BM-MSCs to the regeneration of sweat glands in the scalded paws. Positive rate for perspiration test for the paws treated with EDA-transfected BM-MSCs was significantly higher than those treated with BM-MSCs or EDA expression vector (P < 0.05). CONCLUSIONS: Our results confirmed the important role of EDA in the development of sweat gland. BM-MSCs transfected with EDA significantly improved the sweat-gland regeneration. This study suggests the potential application of EDA-modified MSCs for the repair and regeneration of injured skin and its appendages.

[Effects of estrogen on proliferation and migration of human epidermal stem cells in vitro].

OBJECTIVE: In vivo, the microenvironment of epidermal stem cells (ESCs) is complex, and estrogen might be involved in the micro environment. To investigate the biological effects of estrogen on the proliferation and migration of ESCs in vitro. METHODS: hESCs were isolated from normal human foreskin and cultured. The second generation of hESCs were identified with flow cytometry after being marked with integrin beta1, cytokeratin 19 (CK19), CK14, and CK10 antigens. hESCs of 2 x 10(6) cell density were cultured with ESCs special medium supplemented with 0.1 nmol/L Diethylstilbestrol in group A (estrogen group), with ESCs special medium supplemented with 10 nmol/L Raloxifene hydrochloride in group B (ER blocking agent group), and with ESCs special medium in group C (control group), respectively. The 100 microm "scratch" wounds were created by scraping confluent hESCs plated on Petri dishes with a sterile pipette tip in vitro. The migrating cells from the wound edge were quantified at 24, 48, and 72 hours after incubation. The rates of wound healing were calculated by SigmaScan Pro 5.0 software at 72 hours. The proliferating effect of estrogen on hESCs was determined with MTT method at 24, 48, 72, 96, and 120 hours. RESULTS: Cultured primary hESCs could adhere to the wall showing ovoid in shape and grew into colonies. Flow cytometry showed the positive results for integrin beta1,, CK19, and CK14 (with positive rate of 96.63%, 95.47%, and 94.27%, respectively) and the negative result for CK10 (with positive rate of 1.32%). In group A, the number of cells crossing the wound edge was more than those of group B and group C at 24, 48, and 72 hours. The rates of wound healing were 69.00% +/- 0.05% in group A, 35.00% +/- 0.05% in group B, and 48.00% +/- 0.06% in group C at 72 hours, showing significant differences among groups (P < 0.05). The proliferating speed of hESCs was significantly higher in group A than in groups B and C (P < 0.01), and significantly higher in group C than in group B (P < 0.01) at 24, 48, 72, 96, and 120 hours. CONCLUSION: The estrogen can promote the proliferation and migration of hESCs in vitro. It may be involved in many biological activity of skin.

Calcitonin gene-related peptide regulates the growth of epidermal stem cells in vitro.

Epidermal stem cells are characterized as slow-cycling, multi-potent, self-renewing cells that not only maintain somatic homeostasis, but also participate in tissue regeneration and repair. Various factors can influence the growth of epidermal stem cells. Recently, dysregulation of epidermal stem cells has been reported to be involved in epidermal hyperproliferative diseases and skin tumors. To determine the effect of calcitonin gene-related protein (CGRP), a cutaneous nerve neuropeptide, on the growth of human epidermal stem cells, epidermal stem cells were isolated from human skin and cultured in vitro. Epidermal stem cells grow well and maintain a high proliferative ability in Epilife medium, and express high levels of ß1-integrin. CGRP (10(-8) M) can promote epidermal stem cells to enter the S phase and increase the number of bromodeoxyuridine (BrdU)-labeled cells; the expression of ß-catenin and c-myc genes are deregulated during this process, which can be compromised by CGRP8-37 peptide, an antagonist of CGRP receptor. Experimental evidence suggests that epidermal stem cells can be cultured in vitro for a period of time with preservation of stem cell characteristics. CGRP can stimulate epidermal stem cells to detach from their niche, break quiescence, and undergo division; ß-catenin and c-myc may functionally be involved in the process.

Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells.

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell-like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation-derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c-nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and beta1-integrin emerged at the wound-neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and beta1-integrin+ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10+ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation-derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, beta1-integrin, Oct4 and Nanog in dedifferentiation-derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation-derived cells demonstrated: (1) the numbers of colonies consisting of 5-10 cells and greater than 10 cells were increased 5.9-fold and 6.7-fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 x 10(5) cells versus 1 x 10(12) cells) were all significantly increased and (4) dedifferentiation-derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation-derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.

Regeneration of functional sweat gland-like structures by transplanted differentiated bone marrow mesenchymal stem cells.

Regeneration of sweat glands after deep burns has been an unsolved problem. Owing to lack of perspiration, survivors of an extensive deep burn injury are leading a miserable life in sultry months. It was our contemplation to solve this problem by inducing bone marrow mesenchymal stem cells (MSCs) to acquire the phenotype of sweat gland cells in vitro. Then these cells were transplanted into fresh skin wounds resulting from excision of anhydrotic scars after healing of deep burn injury in five patients. Two to 12 months after the procedure, it was proved that there was recovery of perspiration function in all the MSCs' transplanted areas, as evidenced by positive iodine-starch perspiration test. Histological and biochemical observation confirmed the involvement of MSCs transformed sweat gland cells in the recovery of functional sweat glands, and the components of sweat collected from these areas were similar to that collected from normal skin. This is the first report of successful transplantation of MSCs in regenerating functional sweat glands, which may help solve the problem of depletion of sweat glands in patients surviving extensive deep burns in the future.

Migration of bone marrow-derived mesenchymal stem cells induced by tumor necrosis factor-alpha and its possible role in wound healing.

We aimed to investigate the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the migration ability of mesenchymal stem cells (MSCs) in the context of wound healing. We also explored the role of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) signaling pathways in the migration of MSCs. MSCs were isolated from the bone marrow and cultured. Immunocytochemistry, Western blotting, and reverse transcription-polymerase chain reaction were used to observe the effect of TNF-alpha on the expression of ICAM-1 and VCAM-1 in MSCs. The chemotaxis effect of TNF-alpha on MSCs was investigated by the trans-well system and the inhibition effect of TNF-alpha using its antibody. Western blotting analysis was used to observe the activation of JAK-STAT and mitogen-activated protein kinase signaling pathways, and ERK was inhibited with PD98059 and p38 with SB203580 to observe the effect of TNF-alpha on MSC migration and ICAM-1 expression. The expression of ICAM-1 could be up-regulated by 50 microg/L TNF-alpha (p<0.05), whereas that of VCAM-1 remained unchanged (p>0.05). Also, TNF-alpha showed a chemotaxis effect by enhancing the migration ability of MSCs (p<0.05). TNF-alpha at 50 microg/L increased the expression of phospho-ERK and phospho-p38, and SB203580, but not PD98059, could suppress the chemotaxis effect and up-regulation of ICAM-1 induced by TNF-alpha in MSCs (p<0.05). Thus, TNF-alpha could up-regulate the expression of ICAM-1 in MSCs and enhance the cells' migration ability, and the p38 signaling pathway might be involved in the TNF-alpha-induced migration ability for a role in wound repair and regeneration.

[A new way for isolation and cultivation of sweat gland ductal cells from human split-thickness skin in vitro].

OBJECTIVE: To explore a new method of isolation and culture of eccrine sweat gland ductal cells from human split-thickness skin graft in vitro. METHODS: Human split-thickness skin graft which was presented by volunteer (n = 10) was digested with type II collagenase, and then sweat gland duct were isolated from the split-thickness skin graft, primary cultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2, 95% O2. The cultured eccrine sweat gland ductal cells were identified by analysis CEA, CK8, CK18, CK19 antigens expression with flow cytometry, RT-PCR and Western Blot, and by detecting the electrophysiology with whole cell patch clamp technology. RESULTS: The isolated eccrine sweat gland ductal cells could grow by adhering to the wall, proliferate in vitro after 48 h of adhering to the wall, and confluens after 2 - 4 weeks of adhering to the wall. The FACs analysis showed the expression of CEA was (90.26 +/- 1.12)%, (89.70 +/- 1.43)%, and CK8 was (94.41 +/- 1.84)%, (93.65 +/- 1.63)% in primary cultured sweat gland ductal cells and primary cultured eccrine sweat gland cells, respectively, and there is no significant difference between the two groups (P > 0.05). Immunocytochemistry staining showed CEA, CK8, CK18, CK19 was positive in sweat gland duct cells, RT-PCR revealed that CEA, CK8, CK18 and CK19 gene expression in sweat gland ductal cells, and Western Blot analysis showed the expression of CEA brand, CK8 brand, CK18 brand, and CK19 brand in sweat gland ductal cells, patch clamp indicated that this cells has distinct amiloride sensitive Na(+) channels. CONCLUSIONS: The cultured human eccrine sweat gland duct cells in vitro display the markers and biological characteristics of sweat gland epithelial lineage, and this method of digest the split-thickness skin graft to get the sweat gland duct cells is better than classical dissect sweat gland under dissect microscope.

[Promotive effect of adipose-derived stem cells on the wound model of human epidermal keratinocytes in vitro].

OBJECTIVE: To investigate the migrating effect of adipose derived stem cells (ADSCs) on the wound model of human epidermal keratinocyte (HEKa). METHODS: Rat ADSCs (rADSCs) were isolated and cultured (n = 10), rADSCs were direct co-cultured with HEKa cells in experiment group (experimental group, n = 10). In the control groups, rADSCs were indirect co-cultured with HEKa cells in transwell chamber (indirect group, n = 8), or HEKa was cultured alone (single group, n = 8). Then confluent HEKa cells were scraped to establish a wound model under invert microscope. After scraped 24, 48, and 72 h, cell numbers of which migrated across the edge of the wound was measured, the rate of wound healing was calculated by using SigmaScan Pro 5 software, and the proliferating effect of rADSCs on HEKa were examined by incorporation of [(3)H] thymidine. RESULTS: The cells migrated across the edge of wound after 24 hours in experimental group, indirect group, and single group were (9.2 + or - 0.2), (5.0 + or - 0.3), (4.2 + or - 0.3), and were (58.5 + or - 0.4), (26.5 + or - 0.3), (20.7 + or - 0.5) 48 hours after, and were (125.8 + or - 0.4), (43.0 + or - 0.5), (35.6 + or - 0.5) cells/HP 72 hours after, respectively; the numbers were all significantly higher in experimental group than those in control groups (P < 0.05). The rates of wound healing after scraped 72 hours were 61.0% + or - 3.0%, 35.0% + or - 2.5% and 32.0 + or - 2.1%, the outcome in experimental group was significantly better than in the control groups (P < 0.05). And the thymidine feeding displayed the proliferation of HEKa in the three groups were (1440 + or - 210), (1050 + or - 280) and (1130 + or - 390) cpm/10(5) cell, and there was significant difference between the experimental and the control groups (P < 0.05). CONCLUSIONS: The rADSCs can promote the migration of HEKa by direct contact with it.

Research of PDGF-BB gel on the wound healing of diabetic rats and its pharmacodynamics.

BACKGROUND: One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic patients, a minor skin wound often leads to serious complications. Many experiments had demonstrated that the expression of platelet-derived growth factor (PDGF) and its receptor was decreased in wounds of healing-impaired diabetic mice, indicating that a certain expression level of PDGF is essential for normal repair. MATERIALS AND METHODS: The diabetic rats was induced by a single i.p. injection of streptozotocin and a 1.8 cm diameter full-thickness wound was made on each side of the rat mid-back. Then the rats were randomly divided into five groups, with eight animals in each group as follows: blank control, vehicle control, 3.5 microg PDGF-BB/cm(2) treatment group, 7 microg PDGF-BB/cm(2) treatment group and 14 microg PDGF-BB/cm(2) treatment group for either 7 or 14 consecutive days after wounding. Re-epithelialization area was measured by computerized planimetry, percentage wound closure and percentage wound contraction was calculated, granulation tissue and collagen formation was assessed by Masson trichrome, cell proliferation (proliferating cell nuclear antigen staining) and angiogenesis (Factor VIII related antigen staining) was assessed by immunohistological methods. RESULTS: PDGF-BB treatment improved healing quality, enhanced angiogenesis, cell proliferation and epithelialization, and formed thicker and more highly organized collagen fiber deposition in full-thickness excisional wound of diabetic rats. The effects of topically applied PDGF-BB were dose-dependent. CONCLUSIONS: PDGF-BB is an important future clinical tool, particularly for stimulating soft tissue repair in patients with an impaired capacity for wound healing.

In vivo dedifferentiation of human epidermal cells.

Consistent with our previous study, we herein offer further evidence to demonstrate the dedifferentiation of differentiating epidermal cells into stem cells or stem cells -like in vivo. The epidermal sheets eliminated of basal cells were labeled with 6-diamidino-2-phenylindole (DAPI), and then were transplanted onto the full-thickness skin wounds nude mice. Immunohistochemical examination of the survival sheets showed that some cells were positive for both DAPI and either cytokeratins (CK19, CK14) or beta1 integrin in spinous and granular layers at day 7 after transplantation. Furthermore, there was a significant increase in the percentages of both alpha6briCDdim and alpha6briCD71bri populations in survival epidermal sheet grafts 7 d after transplantation compared with those before xenotransplantation (P<0.05), as determined by flow cytometry. The results collectively indicated that some of the differentiated cells in engrafted epidermal sheets dedifferentiated into stem cells or stem cells-like in vivo, which offer us new evidence and insights into the dedifferentiation.

[Chemotactic effects of burn rat serum on mesenchymal stem cells derived from different sources].

OBJECTIVE: To isolate and culture mesenchymal stem cells ( MSC) from different sources and to investigate the chemotactic effects of burn rat serum on MSC derived from different sources. METHODS: Seventy-two Wistar rats were randomly divided into burn( n = 36, with 30% TBSA full-thickness burns on the back) and sham burn (n = 36, without burns) groups. Bone marrow and peripheral blood of the rats in both groups were collected to isolate and culture MSC. Ratio of MSC, growth speed and cell morphology were observed with inverted microscope. Effects of different serum ( fetal bovine serum, normal rat serum and burn rat serum) on chemotaxis of MSC derived from different sources and their migration ability were subsequently examined with a transwell system. Results MSC were obtained from bone marrow of the rats in both groups. MSC were successfully obtained from bone marrow of all burn rats(100% , P <0.05) , but only from peripheral blood of 7 burn rat(58% ) , and no MSCs were obtained from peripheral blood of 12 rats in sham group( P <0.05). There was small amount of adherent cells 24 hrs after culture, and fusiform shaped adherent cells were sporadically observed in scattered distribution 2-3 days later with inverted microscope. There was no obvious difference in the cell morphology between the 2 groups. In the sham group, the number of MSC migrating to the lower surface of transwell after burn serum treatment [ (94 Il ) cells/ high power field] was significantly greater than that after the treatment with normal rat serum and fetal bovine serum [ (37 +/- 6) , (38 +/- 11) cells/high power field , P <0.01 ] , while no difference in migration ability was found after normal serum treatment compared with that after fetal bovine serum treatment ( P >0. 05). The migration rate of MSCs which were derived from bone marrow in sham group was obviously lower than those derived from bone marrow and peripheral blood from burn rats ( P <0. 05 or 0. 01). Though some difference of the migration ability existed between MSC derived from bone marrow and peripheral blood, there was no statistically significant difference ( P >0. 05). CONCLUSION: MSC can be isolated and cultured from bone marrow and peripheral blood of burn rat, but not from peripheral blood of normal rat. Burn rat serum has a stronger chemotactic effect on MSC. Moreover, the migration ability of MSC derived from burn rat is stronger than that of MSC derived from normal rat.

[Identification and cell phenotype transdifferentiation of adipose-derived stem cells].

OBJECTIVE: To investigate the transdifferentiation of the ADSCs to epidermal cells. METHODS: ADSCs were isolated and cultured from rat adipose tissue by digestion of enzyme. ADSCs was identified by immunocytochemistry and flow cytometry. ADSCs were divided into four groups in order to induce: the condition medium (containing 30% superior of homogenizing rat skin in 10% FBS/DMEM) group, 7 days; 10% FBS/DMEM with EGF (20 ng/ml) group, 7 days; the condition medium for 4 days and then 10% FBS/DEME instead of the condition medium for 3 days group; 10% FBS/DMEM for 7 days group (control group). Cytokeratin 19 and cytokeratin 10 expressions in ADSCs were detected by flow cytometry. RESULTS: (1) The results of immunocytochemistry showed that ADSCs were positive for CD49d and negative for CD106, CD34, CD19, CD10. The results of flow cytometry showed ADSCs were positive for CD49d and CD44. (2) The CK19 expression of ADSCs was 45.32% in the condition medium group, 26.58% in the condition medium with EGF group, 23.37% in te condition medium for 4 days and then 10% FBS/DMEM instead of the condition medium for 3 days gropu and 18.53% in control group, P <0.01. The CK10 expression of ADSCs was 43.56% in the condition medium group, 25.54% in the condition medium with EGF group, 18.20% in the condition medium for 4 days and then 10% FBS/DMEM instead of the condition medium for 3 days group and 2.46% in control group, P < 0.01. CONCLUSIONS: The superior of homogenizing rat skin can induce CK19 and CK10 expressing in ADSCs, and thereby demonstrating ADSCs can differentiate to epidermal cell phenotype in vitro.

Differential expression of matrix metalloproteinases and tissue-derived inhibitors of metalloproteinase in fetal and adult skins.

Matrix metalloproteinases and their tissue-derived inhibitors determine the architecture of the extracellular matrix. In early gestation, the amount and organization of extracellular matrix may be associated with scarless repair of fetal skin wounds. To elucidate the part of the mechanism(s) underlying the phenotypic transition from scarless to scar-forming healing observed during fetal gestation, the ontogeny of matrix metalloproteinase-2, -9, -14 and their tissue inhibitors was characterized in non-wounded fetal human skin with different gestational ages from 13 to 33 weeks and adult skin using reverse transcriptase-polymerase chain reaction, immunohistochemical staining and western blot protocols. We showed that the levels of gene expressions for matrix metalloproteinase-2, -9, -14 and their endogenous inhibitors were significantly more in late gestational and adult skins than that in early gestational skin. Similar results were noted in terms of protein contents of these enzymes and inhibitors in fetal and adult skins. We concluded that the endogenous matrix metalloproteinase-2, -9, -14 and their endogenous inhibitors might be involved in skin development and in maintenance of cutaneous structure and function. Lower protein contents of tissue-derived inhibitor-1, -2 in early gestational skin might provide a predominantly antiscarring signal while higher protein expression of these two inhibitors might be associated with scar-forming healing in late gestational and adult skins.

Recombinant human platelet-derived growth factor enhanced dermal wound healing by a pathway involving ERK and c-fos in diabetic rats.

BACKGROUND: Platelet-derived growth factor (PDGF) has been shown to promote dermal wound healing, however, the molecular mechanisms responsible for are not fully understood. OBJECTIVE: The present study was undertaken to investigate the possible signaling mechanisms by which PDGF improved healing of cutaneous wound in diabetic rats. METHODS: Four full-thickness skin wounds were created on the dorsum of Wistar diabetic rats. Animals were treated with or without recombinant human PDGF (rhPDGF) at 7.0 microg/cm(2) wound or vehicle daily 1 day after wounding. The animals were then killed after various intervals of wounding, and the wounded skin tissues were used for histological evaluation, analysis of the phosphorylation of extracellular signal-regulated kinases (ERK) and the expression of c-fos protein, as well as the labeling indices of proliferative cell nuclear antigen (PCNA). RESULTS: Topical application of rhPDGF significantly accelerated the rate of reepithelialization compared with vehicle-treated or untreated group at 7 days after wounding. At the histological level, the significant increases in the degree of reepithelialization, the thickness of granulation tissue and the density of capillary bud were observed in the wound sites in rhPDGF-treated group at 7 and 14 days after wounding. Moreover, treatment with rhPDGF increased PCNA labeling indices, c-fos protein expression and ERK phosphorylation in the wounded tissues at the indicated time after wounding. CONCLUSION: These results suggest that application of rhPDGF increases cell proliferation, and enhances dermal tissue repair in diabetic skin lesion of rats, which might be partly mediated by ERK activation and c-fos protein expression.

Non-mitogenic acidic fibroblast growth factor reduces intestinal dysfunction induced by ischemia and reperfusion injury in rats.

BACKGROUND: Acidic fibroblast growth factor (aFGF) has potentially therapeutic uses in some diseases, but the mitogenic activity of aFGF has been found to contribute to several human pathologies, so the extensive applications of wild-type aFGF have been limited. The purpose of the present study was to explore the effects and mechanisms of wild-type (aFGF) and non-mitogenic aFGF on gut ischemia-reperfusion injury in rats. METHODS: Rat intestinal ischemia-reperfusion injury (I/R) was produced by clamping the superior mesenteric artery (SMA) for 45 min followed by reperfusion. One hundred and fourteen rats were randomly divided into four groups: sham operation (group C, n = 6), intestinal I/R + 0.1 mL saline (group S, n = 36), intestinal I/R + 4 microg/0.1 mL wild-type aFGF (group W, n = 36) and intestinal I/R + 4 microg/0.1 mL modified aFGF (i.e. non-mitogenic aFGF; group M, n = 36). According to different periods after reperfusion, groups S, W and M were further divided into 0.5-, 1-, 2-, 6-, 12- and 24-h subgroups. The contents of D-lactate and nitrite/nitrate were determined, the changes of intestinal histology were analyzed, the protein expressions of caspase-3, extracellular signal-regulated kinase (ERK)1/2, and p38 were detected by western blot, and apoptotic cells were examined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) assay at 0.5, 1, 2, 6, 12 and 24 h after I/R, respectively. RESULTS: Compared with rats in group S, intestinal histological damage, apoptotic index, d-lactate content and nitrite/nitrate level all decreased significantly in group W and group M rats. However, there was no difference between rats treated with wild-type aFGF and those with non-mitogenic aFGF. The protein expression of caspase-3, ERK1/2, and p38 in saline-treated rats was higher than those in aFGF-treated rats. CONCLUSIONS: Both types of aFGF had protective effects on gut I/R and there was no significant difference between the two aFGF. The protective effects of aFGF may come from the non-mitogenic activity rather than the mitogenic activity of aFGF in tissue repair, indicating a potentially clinical use for the non-mitogenic effects of aFGF in preventing visceral organ injury triggered by I/R injury in the future.

Profiling of genes differentially expressed in a rat of early and later gestational ages with high-density oligonucleotide DNA array.

The early gestational fetus heals dermal wounds rapidly and scarlessly. This phenomenon appears to be intrinsic to fetal skin and is probably modulated by interplay of many genes. We ventured to study differences in gene expression between earlier gestational skin (EGS) and later gestational skin (LGS) with the aid of high-density oligonucleotide DNA array to explore the molecular mechanism underlying scarless healing. Total RNA was isolated from fetal Wistar rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term=21.5 days), and purified to mRNAs. Both the mRNAs from EGS and LGS were reversely transcribed to cDNAs, and were labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes through single primer amplification reaction and Klenow labeling methods. The mixed probes were then hybridized to the oligonucleotide DNA arrays that contained 5,705 DNA fragments representing 5,705 rat genes. After highly stringent washing, the microarray was scanned for fluorescent signals to display the differentially expressed genes between two groups of tissues. Among 5,705 rat genes, there were 53 genes (0.93%) with differentially expressed levels between EGS and LGS; 27 genes, including fibroblast growth factor 8 and follistatin, were up-regulated (0.47%); and 26 genes, containing lymphoid enhancer binding factor-1 and beta-catenin, were down-regulated (0.46%) in fetal skin of scarless period vs. scar-forming period. Analyses of genes related to ion channels, growth factors, extracellular matrix and cellular skeleton, and movement confirmed that our molecular data obtained by oligonucleotide DNA array were consistent with the published biochemical and clinical findings of fetal scarless healing. Stronger expression of fibroblast growth factor 8, follistatin, and weaker expression of lymphoid enhancer binding factor-1 and beta-catenin in EGS vs. LGS were also testified with reverse transcription-polymerase chain reaction and Western blotting methods. Oligonucleotide DNA array was a powerful tool for investigating different gene expression between scarless and scar-forming periods of gestation in the rat fetal skin. Many genes were involved in the phenotypic transition from scarless to scar-forming wound repair during gestation. Further analysis of the obtained genes will help to understand the molecular mechanism of fetal scarless healing.