EGFL7-overexpressing epidermal stem cells promotes fibroblast proliferation and migration via mediating cell adhesion and strengthening cytoskeleton.

Epidermal growth factor (EGF)-like family members mediate a wide range of biological activities including cell proliferation and migration. Increasing evidence indicated that EGF plays an important role in the process of wound healing by stimulating fibroblast motility. The aim of this study was to see whether EGF-like domain 7 (EGFL7)-overexpressing epidermal stem cells (EGFL7-ESCs) would promote fibroblast proliferation and migration. We found that mRNA and protein levels of EGFL7 expression were significantly increased in EGFL7-ESCs. The protein expression of EGFL7 was significantly elevated in conditioned media (CM) of EGFL7-ESCs compared to ESCs CM or vector-ESCs CM. The cell count and cell viability of EGFL7-ESCs CM-treated fibroblasts were also significantly increased compared to control. In addition, EGFL7-ESCs CM-treated fibroblasts showed elevated migration compared with control. Moreover, the expressions of ß1-integrin, ß-tubulin, ß-actin, and Vimentin were increased, while that of E-cadherin was decreased in EGFL7-ESCs CM-treated fibroblasts. These results indicate that EGFL7-ESCs contribute towards promoting fibroblast migration through enhancing cell adhesion, strengthening cytoskeleton, and reducing intercellular aggregation. These findings suggest that the stimulating effect of EGFL7-ESCs on fibroblast proliferation and migration may provide a useful strategy for wound healing.

Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway.

Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro Importantly, Jag1 overexpression improves diabetic wound healing in vivo These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound.

Effects of skin-derived precursors on wound healing of denervated skin in a nude mouse model.

Denervated skin could result in impaired healing of wounds, such as decubitus ulcers and diabetic foot ulcers. Other studies indicated that cutaneous fiber density is reduced after inner nerve transection and that neuropeptide level depletes after denervation, leading to reduced cell proliferation around the wound and thus wound healing problems. Recent studies have revealed that skin-derived precursors (SKPs), which form a neural crest-related stem cell population in the dermis of skin, participate in cutaneous nerve regeneration. We hypothesized that injecting SKPs into denervated wound promotes healing. A bilateral denervation wound model was established followed by SKP transplantation. The wound healing rate was determined at 7, 14, and 21 d after injury. Cell proliferation activity during wound healing was analyzed by proliferating cell nuclear antigen immunohistochemistry (IHC). Nerve fiber density was measured by S-100 IHC. The contents of nerve growth factor, substance P, and calcitonin gene-related peptide were examined by enzyme-linked immunosorbent assay. The rate of epithelization in the SKP-treated group was faster than that in the control group. Wound cell proliferation and nerve fiber density were obviously higher in the SKP-treated group than in the control group. In addition, the content of neuropeptides was higher in the SKP-treated group than in the control group during wound healing. In conclusion, SKPs can promote denervated wound healing through cell proliferation and nerve fiber regeneration, and can facilitate the release of neuropeptides.

Directed differentiation of skin-derived precursors into fibroblast-like cells.

Skin-derived precursors (SKPs), which are located at skin's dermis, display multi-lineage potential and can produce both neural and mesodermal progeny in vitro. SKPs are considered to take part in dermal reconstruction and may be an important source of fibroblast during wound repairing. To explore the possibility of differentiation of SKPs into fibroblasts, the 3(rd) passage SKPs were treated with 0, 20, 40, 100, or 500 ng/ml human recombinant connective tissue growth factor (CTGF) for 48 h or treated with 100 ng/ml CTGF for 0, 24, 48, 72, or 96 h. Subsequently, a series of methods were to be used to observe cells immunocytochemistry changes under fluorescence microscope, to validate the mRNA expression change of collagen I, collagen III, fibroblast-specific protein 1 (FSP-1) and alpha smooth muscle actin (α-SMA) by quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR), to analyze the expression of collagen I and collagen III protein by Enzyme-linked immunosorbent assay (ELISA), to semiquantitatively measure the expression of FSP-1 and α-SMA by western-blot. After differentiation, cells showed that positively staining for collagen I, collagen III, α-SMA, and FSP-1, which are markers for fibroblasts, but negative expression for neural precursors. The effects of CTGF on collagen I, collagen III, FSP-1 and α-SMA in SKPs were detected both on the transcriptional and posttranscriptional levels. These findings indicate that SKPs can be induced to differentiate into fibroblast-like cells with CTGF treatment that may be a key source of fibroblast in wound healing.

An improved method for the isolation and culture of rat epidermal stem cells.

The management of burns and injuries using novel treatment strategies involving epidermal stem cells (ESC) requires a better understanding of the biology of these cells, in particular, their isolation and the maintenance of their unique characteristics in culture. The purpose of this study was to describe an improved method for isolating putative ESC from fetal rat skin and to maintain them long term in culture. Single ESC suspensions were obtained from fetal rat skin by enzyme digestion containing 0.5% neutral protease. The target cells were harvested by rapid adherence on type IV collagen plates and were cultured in complex DMEM. After primary isolation, cells were continuously cultured in K-serum free medium. After reaching 70-80% confluence, the cells were digested with 0.25% trypsin at 37°C for 5-10 minutes, and passaged at a ratio of 1:2. The cultured ESC showed good growth, resulting in cell viability of over 98%. Four days later, clones containing 100-200 cells were detected, showing cobblestone-like characteristics. The rapidly adherent cells were positive for keratin 15, 19 and P63. Eighty three percent of cells expressed ß1 integrin. The growth-curve showed that the rapidly adherent cells were in the exponential growth phase. The protocol described in this paper provides a simplified and effective method to isolate and maintain long-term culture of epidermal stem cells from fetal rat skin. This method should be valuable for isolating and studying ESC from various transgenic rat lines that are currently available.

Effects of human bone marrow mesenchymal stem cells on burn injury healing in a mouse model.

OBJECTIVE: To investigate the feasibility and safety of human bone marrow mesenchymal stem cells (BM-MSCs) transplantation on the improvement of burn wound healing. METHOD: Human BM-MSCs were injected into the skin of the mouse models, and the new blood vessels growth, the engraftment of BM-MSCs and the speed of healing were observed. Moreover the body weight and activity were tested after BM-MSCs transplantation. RESULTS: We found that wound surface healing was significantly accelerated when BM-MSCs were applied to the wound surface in mice. Moreover, both the number and density of new blood vessels were increased in the BM-MSC-treated group. The engraftment of BM-MSCs was also investigated using GFP-labeled cells and no GFP-positive cells were observed in tissues other than the location of BM-MSC injection. We also found that both body weight and activity were quickly restored in BM-MSC-treated mice, and no tumor growth was found. CONCLUSION: The present results suggest that BM-MSC transplantation can effectively improve wound healing in a mouse model of burn injuries. Use of BM-MSCs might therefore facilitate development and improvement of burn injury treatments in future.

BMP-7 attenuates TGF-ß1-induced fibroblast-like differentiation of rat dermal papilla cells.

Dermal papilla cells (DPCs) show phenotypic plasticity during wound healing. The multipotency of DPCs is well recognized, but the signaling pathways that regulate the differentiation of these cells into fibroblasts are poorly understood. A preliminary experiment showed that transforming growth factor beta1 (TGF-ß1) can induce DPCs to differentiate into fibroblast-like cells, which suggests that DPCs may be a source of wound-healing fibroblasts. Bone morphogenetic protein-7 (BMP-7), a member of the TGF-ß superfamily, can prevent and reverse fibrosis by counteracting the TGF-ß1-mediated profibrotic effect. To determine whether BMP-7 attenuates the TGF-ß1-induced differentiation of DPCs into fibroblasts, we established an in vitro system for DPC differentiation and recorded the gene expression patterns that distinguished DPCs from fibroblasts. The proportion of fibroblast-like cells was significantly enhanced in DPCs treated with TGF-ß1, as evidenced by immunocytochemistry, flow cytometry, quantitative real-time reverse transcriptase polymerase chain reaction, and Western blot analysis. BMP-7 and TGF-ß1 administration substantially decreased fibroblast-like differentiation, indicating inhibition of TGF-ß1-induced differentiation. The antagonistic BMP-7- and TGF-ß1-activated signaling pathways can be used to promote wound healing or suppress hypertrophic scarring.

Fibroblast growth factor-binding protein facilitates the growth and migration of skin-derived precursors.

BACKGROUND: Fibroblast growth factors (FGFs) are important regulators of cell proliferation, migration, and differentiation during wound healing. FGF-binding protein (FGF-BP) plays a critical role in activating FGFs by releasing them from the extracellular matrix. Although previous studies have demonstrated a pivotal role for FGF-BP in wound healing and angiogenesis, little is known about the biologic effects of FGF-BP on skin stem cells that contribute to wound healing. OBJECTIVE: To investigate the effects of FGF-BP on the growth and migration of skin-derived precursors (SKPs). METHODS: FGF-BP was titrated to determine the optimal concentration that maximally stimulated cell proliferation. Cellular phenotype and telomerase activity were compared in the presence and absence of FGF-BP. The effect of FGF-BP on cell migration was observed by intravenously transplanting SKPs to adult mice. RESULTS: Cell proliferation was maximally stimulated by FGF-BP at a concentration of 10 ng/mL without changing the intrinsic characteristics of SKPs. Low levels of telomerase activity were detected, and FGF-BP decreased the rate at which telomerase activity was downregulated. In vivo, FGF-BP remarkably enhanced the migration of SKPs to skin lesion sites. CONCLUSION: FGF-BP exerts a positive effect on the growth and migration of SKPs, suggesting a potential role for SKPs in wound healing.

Expression of Smad protein by normal skin fibroblasts and hypertrophic scar fibroblasts in response to transforming growth factor beta1.

BACKGROUND: Smad proteins are important intracellular mediators of transforming growth factor (TGF)-beta signaling. Little has been known about the specific relationship between TGF-beta and TGF-beta/Smad signaling in hypertrophic scars. OBJECTIVE: The objective was to investigate the expression of Smads and the specific relationship between TGF-beta and TGF-beta/Smad signaling in hypertrophic scars. METHODS: In this study, we initially determined the endogenous protein levels of Smad2 and Smad7 in hypertrophic scar fibroblast (HSFs) and normal skin fibroblast (NSFs). Second, we stimulated HSFs and NSFs with recombinant human TGF-beta1 for 24 hours to determine whether the TGF-beta1 could potentiate its effect by further stimulating the production of Smad by reverse transcription-polymerase chain reaction and Western blot analysis. RESULTS: When compared with NSFs, the endogenous expression of Smad2 in HSFs was up-regulated and TGF-beta1 could further stimulate the production of Smad2. Although the levels of Smad7 were similar between HSFs and NSFs, TGF-beta1 up-regulated the expression of Smad7 for NSFs only, with no discernible effect on HSFs. These changes were paralleled by a significant increase in cytoplasm-to-nuclear translocation of Smad2. CONCLUSION: These data substantiated the model of an autocrine positive loop in hypertrophic scars pathogenesis. The authors have indicated no significant interest with commercial supporters.

Basic fibroblast growth factor (bFGF) alleviates the scar of the rabbit ear model in wound healing.

Studies suggest a possible antiscarring effect of basic fibroblast growth factor (bFGF) during wound healing. However, little is known about the precise pathological mechanisms of bFGF. In particular, there is only limited information available about the mechanism of exogenous administration of bFGF to scar formation. To investigate the effect of bFGF on the hypertrophic scar in the rabbit ear model and to clarify the mechanisms of bFGF on treatment for scar in wound healing, the rabbit ear model of wound healing was created and treated topically with bFGF once daily for 3 months; then we examined the changes of macroscopic and histopathological characteristics of scars and the expression of collagen and collagenase-1 (matrix metalloproteinase-1). The results of macroscopic and histologic characteristics revealed a significant difference between scars treated with bFGF and control scars. The expression of collagen in the scars treated with bFGF was decreased, as compared with the scars treated with saline. Further study revealed that bFGF could remarkably enhance expression of matrix metalloproteinase-1. bFGF could improve the quality of wound healing and remarkably alleviate the scar in the rabbit ear model in wound healing, which suggests that bFGF exerted a net negative effecton scar formation in wound healing. The evidence should contribute to a better understanding of the biological activities of bFGF during hypertrophic scar formation.

Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells.

OBJECTIVE: To investigate the influence of hair follicle dermal papilla cells (DPCs) on biological features of composite skin. METHODS: In the test group, xenogeneic acellular dermal matrix was employed as the frame, DPCs were seeded on the subcutaneous side, and epithelial stem cells onto the dermal papilla side of the dermal frame so as to construct a composite skin. In the control group, there was no DPC in the frame. The two kinds of composite skin were employed to cover skin defects on the back of the nude mice. Wound healing was observed 4 weeks after grafting and area was analyzed and contraction rate was calculated. The tissue samples in the grafted area were harvested for HE staining and the state of the composite skin was observed. The stress-strain curve of the sampled skin was measured, so as to calculate the maximal breaking power of the sample. The data were collected and statistically analyzed. RESULTS: HE staining indicated that the epithelial depth was increased (more than 10 layers of cells) in test group, with only 6-7 layers in control group. The skin contraction rate in test group on the 4th week after skin grafting (3.94+/-0.013)% was much lower than that in control group (29.07+/-0.018)% (P<0.05). It was indicated by biomechanical test that the stress-strain curve of the composite skin in the test group was closer to that of normal nude mice skin in comparison to that in control group. The maximal breaking force of the composite skin in test group was (1.835+/-0.035)N (Newton), while that in control group was (1.075+/-0.065)N (P<0.01). CONCLUSION: Reconstruction of epidermis in composite skin was promoted by dermal DPCs seeded in the dermal matrix frame. As a result, there was less skin contraction in the composite skin with DPCs, so that the biological characteristics of the skin were improved.

[Influence of skin-derived progenitor cell combining with hyaluronic acid on the wound healing of diabetic rat].

OBJECTIVE: To study the effect of skin-derived progenitor cell (SKP) combined with hyaluronic acid( HA) on the wound healing in diabetic rats. METHODS: SKP of Spraque-Dawley (SD) neonate rats were isolated and cultured and mixed with HA. The differentiation characteristics of SKP in the culture were observed. Sixty SD rats were injected intraperitoneally with 65 mg/kg streptozotocin( STZ) to induce diabetes. Two symmetrical full-thickness cutaneous wounds( 1.0 cm in diameter) were made on the back of each SD rat and randomly divided into A (n = 20, with treatment of 100 mircol SKP-HA) , B (n = 20, with treatment of 100 mirol HA) , and C ( n = 20, with treatment of DMEM/F12 culture medium) groups. Tissue samples from wound in each group were harvested on 1, 2, 3, 4 weeks after the treatment. Wound healing rate, changes in histomorphology, the content of hydroxyproline ( HYP) , and immigration of labelled SKP were determined and analyzed. RESULTS: SKP grew well when cultured with HA. The characteristics of SKP to differentiate into lipocyte, neuron, and neurogliocyte remained in the culture. Compared with that in C group, epithelization in the wounds of A and B groups appeared earlier. The wound healing rate in A group [ (72.1 +/- 2. 8)% ] and B group [ (53.7 +/- 2. 9)% ] were obviously higher at 2 post-treatment weeks(PTW) than that in group C [(42. 5 +/- 1.5)% ( P <0.05) , and that in A group was obviously higher compared with B and C groups at 3 PTW ( P < 0. 05 or 0. 01). The wound healing rates in A and B groups were (100. 00 +/- 0.00) % at 4 PIW, which were obviously higher than that of group C( P <0.01) . There was no obvious difference in the HYP content among the 3 groups at 1 PIW ( P > 0. 05) , but it was obviously higher in A and B groups than that in C group at 2,3,4 PTW( P <0.01) , and that in A group was significantly higher than that in B group at 3 and 4 PTW( P <0. 01). SKP survived well on the wound, and migrated towards the dermis as time elapses. CONCLUSION: SKP-HA composition can promote wound healing in diabetic rats.

[Isolation and differentiation characteristics of dermal multipotent stem cells from humans of different ages cultured in vitro].

OBJECTIVE: To study isolation, identification and differentiation characteristics of dermal multipotent stem cells from human of different age in vitro culture. METHODS: Skin samples( 1 cm x 1 cm) were harvested from fetus, infant, adult and elderly. The original clones were screened in stem cell medium. The diameter and number of clones were recorded. Analysis of each clone and determination of the expression of various related proteins were carried out. RESULTS: The number of suspended clones from normal skins of fetus, infant, adult and the elderly were (20. 1 +/-2. 5) x 102 , (15. 8 +/-5. 7) x 102, (10. 8 +/-1.3) x 10(2), (6.2 +/- 1.4) x 10(2), respectively ( P <0.01), while the diameter of the clones from them were (83 +/-12) microm, (55 +/- 10) microm, (46 +/- 12) Lm, (42 +/-8) microm, respectively ( P <0.05). Cloned cells from fetus, infant, adult and elderly could differentiate into neuron cell , neuroglia cell, smooth muscle cell, and adipocyte. The clones from fetus were inclined to differentiate into neuron cells, but those from infant were inclined to differentiate into neuroglia cells, and those from adult and elderly were inclined to differentiate into adipocytes. After 1 month of culture, the clone forming rate of the cells from fetus, infant, adult and elderly were 41. 1% , 25.5% ,17.7% ,15.2% , respectively. The individual clone cells also showed ability of multidirectional differentiation. Nestin, fibronectin, c-Myc, STAT3 and hTERT protein were expressed in all clones. CONCLUSION: Multipotent stem cells with multi-direction differentiation and proliferation can be efficiently isolated from dermis of human of different age in stem cell culture medium. The number, proliferation and differentiation of dermal multipotent stem cells can be affected by age.

A study of using tissue-engineered skin reconstructed by candidate epidermal stem cells to cover the nude mice with full-thickness skin defect.

OBJECTIVE: To study and explore the feasibility of using candidate epidermal stem cells with reconstruct tissue-engineered skin for a skin defect. METHODS: After the candidate epidermal stem cells were selected directly by rapid adhesion to type IV collagen within 10min from keratinocytes isolated from foreskin epidermis, the TES was constructed by seeding large-scale cultured candidate epidermal stem cells onto a fibroblast-containing dermal substrate, then grafted onto athymic immunodeficient mice with full-thickness skin defects. All specimens were harvested after 1 week, 2 weeks and 4 weeks of transplantation to evaluate by gross, histological, transmission electron microscopic and immunohistochemical techniques its potential to reconstitute a full-thickness skin defect. RESULTS: The transplanted skin developed a well-differentiated epidermis composed of stratum basale, prickle cell layer, granular layer and stratum corneum and clearly defined dermis with the morphological features of intact skin. The continuous and integral basement membrane zone (BMZ) was established; hemidesmosomes, basal lamina and anchoring fibrils were detected. In the dermis, the collagen of dermal substitute degraded gradually and fibroblasts were aligned in order; lymphocytes, organelle debris, differentiated microvasculature and hyperactive collagen fibrillogenesis were observed. The immunohistochemistry suggested that the keratinocytes of the TES were originated from the human candidate epidermal stem cells and not from the mice. The constructed TES was similar to the uninjured skin in morphological features, which suggested the constructed TES by combining cultured candidate epidermal stem cells with the dermal substrate could satisfy the need for the restoration of skin defects.

[Effects of citrus reticulata blanco extract on fibroblasts from human hypertrophic scar in vitro].

OBJECTIVE: To investigate the effects of citrus reticulata blanco extract on the proliferation and collagen metabolism of fibroblasts from human hypertrophic scar. METHODS: Human hypertrophic scar fibroblasts from two burn patients obtained from plastic surgery were cultured in vitro and divided into experimental group (n = 12, with basic culture medium and 2.5, 5.0, 10.0,25.0 mg/L citrus reticulata blanco extract, respectively, 3 bottles for each concentration of citrus reticulata blanco extract ), control group 1 (n = 3, with basic culture medium) , and control group 2 ( n = 3, with basic culture medium and 5% ethyl alcohol). The cell proliferation in each group was observed with MTT method, then the inhibition rate was calculated. Apoptosis and its index ( AI) in each group were determined after TUNEL staining . The changes in the content of ICTP and PINP in each group were observed by radioimmunity. RESULTS: The inhibition rate in the experimental group with the citrus reticulata blanco extract in concentration of 2. 5, 5.0, 10.0, 25. 0 microg/ ml were (7. 100+/-0.038)% , (8. 100+/- 0. 048)% , (10. 900+/-0. 055)%, (15.900+/-0. 097) %, respectively, which were significantly higher than those in other two groups ( P <0.05 ). The Al (69. 7% , 71.7%, 86.4% , 95.2% ), ICTP [(17.2+/-0.6), (18.3+/-0.6), (19.8+/-0.5), (23.2+/-0.6) microg/L] and PINP [ (101.7+/-1.4) , (107. 8+/-1. 1) , (111.6+/-1.2) , (124. 6+/-1.3) microg/L] in experimental group with the citrus reticulata blanco extract in concentration of 2.5, 5.0, 10.0 , 25.0 mg/L were also obviously higher than other two control groups( P <0.05) ,but these indices in control 1 group were similar to those in control 2 group( P >0. 05). CONCLUSION: The citrus reticulata blanco extract might be beneficial for the management of hypertrophic scar through inhibition of the proliferation of fibroblasts in hypertrophic scar, by promoting apoptosis and collagen degradation.

[Study on the regular pattern of the distribution of skin epidermal stem cells in the different parts of a healthy human body].

OBJECTIVE: To investigate the regular pattern of the distribution of skin epidermal stem cells (ESCs) in the different parts of a healthy human body, and to evaluate the feasibility of the identification of ESCs by P63 and CD29 with single and double labeling. METHODS: Full-thickness skin samples from 21 parts (including scalp, dorsum of foot, sole of foot, pubic region, and scrotum) of 5 healthy persons were harvested for the study. Immunohistochemistry method with biotin-streptavidin-horseradish peroxidase (SP) was employed with P63 and CD29 as the first antibody to carry out single and double labeling. The staining results were subjected to image analysis. The distribution of the ESCs in the skin from the above parts was observed and expressed as positive unit (PU) value. RESULTS: It was found by P63 single labeling and P63 and CD29 double labeling that the PU value in the dorsum of foot was the lowest while that in the scalp was the highest among all the parts of a healthy body. It was also found by CD29 single labeling that the PU value in the dorsum of foot was the lowest [(11.9 +/- 1.5)%] while highest in the scalp [(29.1 +/- 5.0)%]. The PU value in the hairy region of a human body was evidently higher than that in the non-hairy region (P < 0.01), when examined by P63 and CD29 single and double labeling. But there was no difference in the PU values between the trunk and limbs by means of P63 and CD29 single and double labeling (P > 0.05). CONCLUSION: There are more ESCs in the skin from the scalp, mons pubis and scrotum than other parts of the body. Single P63 or CD29 labeling exhibits higher sensitivity but lower specificity in the identification of ESCs. While the double labeling method exhibits higher specificity but lower sensitivity. Above all, it seems that the double labeling may be a simple and effective method for the identification of ESCs.

[Protective effects of urinastatin on organ function in patients with severe burn].

OBJECTIVE: To investigate the protective effects of urinastatin on organ function in severe burn. METHODS: Seventy-two cases with comparative severity in burn injury were randomly divided into urinastatin treatment group (n=36) and control group (n=36). Patients in control group received routine therapy, while those in treatment group received intravenous dripping of urinastatin twice a day for 5 to 7 days. The dosage of urinastatin was 300 kU in severe burns and 200 kU in moderate burns. Both polyvinylpyrrolidone iodine (PVP-I) and Jiedu Shaoshang cream were used for wound dressing. The clinical findings were assessed and variables indicating functions of internal organs, including liver (alanine aminotransferase), kidney (blood urea nitrogen, serum creatinine), heart (aspartate aminotransferase, lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, creatine kinase, isoenzymes of creatinine kinase), and coagulation (prothrombin time, international normalized ratio, activated partial thromboplastin time, fibrinogen), as well as blood routine test, troponin, myoglobin, arterial blood analysis, bacteria culture were measured. RESULTS: In treatment group, vital signs and general condition were satisfactory at shock phase and peri-operative stages in all the patients. Edema of burn wound subsided rapidly. The 28-day mortality rate was 0. There was significant difference between the two groups (all P<0.05). CONCLUSION: Urinastatin has protective effects on multiple organs in severe burn injury.

[Influence of aerosols on the expression of cyclin B1, cyclin C and proliferating cell nuclear antigen in wound tissue healing of burned rat models].

OBJECTIVE: To investigate the influence of aerosols on the expression of cyclin B(1), cyclin C and proliferating cell nuclear antigen (PCNA) in wound tissue healing of burned rat models. METHODS: Sprague Dawley (SD) rats were inflicted as the deep partial thickness burn models. Rats were randomly divided into experimental group and control group. The experimental group were treated with aerosols. Samples were collected in 1 approximately 10 postburn days. Immunohistochemistry and image analysis methods were conducted to examine the expression of cyclin B(1), cyclin C and PCNA in both experimental and control groups. RESULTS: The expression of cyclin C in experimental group was detected in nucleus of skin basal cell on the second postburn day, increased evidently at the fifth days and sustained at high expression level up to the tenth days after treatment. The expression of cyclin C in experimental group was significantly higher than control group (P < 0.05). The expression of PCNA was first observed in skin basal cell nucleus and hair follicle cell nucleus in both experimental and control group on the third postburn day. The expression of PCNA increased evidently at the fifth days in experimental after treatment and that increased evidently at the seventh days in control group, which showed there were lots of active proliferation cell. And the difference of the expression of PCNA between experimental and control group was significant (P < 0.01). The expression of cyclin B(1) was detected in nucleus and cytoplasm of skin basal cell in both groups on the third postburn day, and no difference between the experimental and control group (P > 0.05). CONCLUSIONS: Aerosols can up-regulate the expression of cyclin C and PCNA in skin basal cell nucleus. Therefore the aerosols can accelerate wound tissue healing.

[Clinical study and pathological examination on the treatment of deep partial thickness burn wound with negative charge aerosol].

OBJECTIVE: To investigate the effect of negative charge aerosol (NCA) on the treatment of burn wound. METHODS: Patients with superficial or deep partial thickness burn only were enrolled in the study, and they were randomly divided into trial group (T, including 180 cases of superficial thickness burn and 100 cases of deep partial thickness burn), control group (C, including 30 cases with superficial thickness burn and 30 with deep partial thickness burn), and self control group (SC, including 10 cases with superficial thickness burn and 10 with deep partial thickness burn). The patients in T and SC groups were treated with NCA for 1.5 hours, 1-2 times a day, from 6 postburn hour (PBH) to 2 postburn day (PBD), while those in C group received conventional treatment. For those in SC group, some of the wounds were covered with sterile schissel, while other wounds without schissel covering. The general changes in the wounds during NCA treatment were observed, and bacterial culture before and after NCA treatment was performed. The healing time was recorded and the blood biochemical parameters were determined. Rat model with deep partial thickness scald was established, and the rats were also divided into T and C groups, and received treatment as in human. Tissue samples were harvested from the wounds of rats in the 2 groups before and 1, 2, 3 weeks after treatment for pathological examination. RESULTS: There was no infection and little exudation in the patients in T group. No bacteria were found in the wound before and after NCA treatment. The healing time of the wounds of patients with superficial and deep partial thickness burn in T group was 6.3 +/- 1.6 d and 15.1 +/- 3.1 d, respectively, which was obviously shorter than those in C group (11.3 +/- 1.4 d and 21.2 +/- 1.4 d, P < 0.01). In SC group, the healing time of those with sterile schissel coverage was also significantly shorter than those without covering (P < 0.01). There was no obvious change in the liver and kidney functions and blood biochemical parameters among the patients. Pathological examination showed that the skin structure was almost recovered in the rats in T group 3 weeks after treatment, while those in C group was not. CONCLUSION: Negative charge aerosol is safe and effective in promoting wound healing of the patients with partial thickness burns.

[A research of endothelial cell-targeted therapy for cure of hypertrophic scar].

OBJECTIVE: To investigate the feasibility of endothelial cell-targeted therapy to cure post-burn hypertrophic scar. METHODS: A hypertrophic scar animal model was made. Intralesional injecting of VEGF monoclonal antibody was performed for three weeks. The changes of scar in volume and morphology were observed. RESULTS: 1. The volume of scar decreased. 2. The number of the capillary, the amount of collagen I and collagen III decreased. 3. Transmission electron microscope examinations demonstrated many dead or apoptotic fibroblasts and endothelial cells. Fibrocytes were seen relatively common. CONCLUSION: VEGF induces the growth and development of hypertrophic scar in that it induces excessive and uncontrollable angiogenesis, which favors excessive collagen synthesis. Endothelial cell-targeted therapy may be a promising method to cure post-burn hypertrophic scar.