Differentiation of rat dermal papilla cells into fibroblast-like cells induced by transforming growth factor ß1.

BACKGROUND: The origin of wound-healing fibroblasts is still debated. Dermal papilla cells (DPCs), which are an important population of stem cells for the regeneration of hair follicles, play a considerable role in cutaneous wound healing. Based on the plasticity of DPCs in wound healing, we hypothesized that DPCs may contribute to the fibroblast population of wound repair. OBJECTIVE: To explore the possibility of differentiation of DPCs into fibroblasts induced by transforming growth factor ß1 (TGF-ß1). METHODS: The fourth passage DPCs were treated with TGF-ß1 (10 ng/mL) for 4 days, and a series of methods was used to observe morphologic changes under an inverted phase contrast microscope, to validate the messenger ribonucleic acid expression change in α-smooth muscle actin (α-SMA) and vimentin by quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR), to analyze the expression of α-SMA and vimentin protein by flow cytometry, and to semiquantitatively measure the expression of fibroblast-specific protein 1 (FSP1) by Western blot. RESULTS: DPCs treated with TGF-ß1 presented fibroblast-like changes in morphology and immunocytochemistry. The effects of TGF-ß1 on α-SMA and vimentin in DPCs were detected on both the transcriptional and the posttranscriptional levels. The results showed that TGF-ß1 significantly downregulated α-SMA expression and enhanced the expression of vimentin at all times tested. Further study revealed that TGF-ß1 could gradually promote the expression of FSP1 in a time-dependent manner. CONCLUSION: DPCs experienced the changes in molecular marker expression in response to TGF-ß1, which may be a key source of fibroblasts in wound healing.

Attenuation of alpha1 collagen production with antisense ribonucleic acid in cultured hypertrophic scar fibroblasts.

BACKGROUND: It has been demonstrated that hypertrophic scar fibroblasts (HSFs) overexpress collagen messenger ribonucleic acid (mRNA) and protein, especially alpha1 collagen. Antisense nucleic acids are effective in inhibiting harmful or uncontrolled gene expression, suggesting that antisense ribonucleic acid (RNA) can effectively downregulate the expression of alpha1 collagen gene and attenuate the scars. AIMS: This study was conducted to observe the effect of recombinant plasmid pREP9-COL1 on alpha1 collagen expression in HSFs and clarify the prospect of antisense RNA on scar treatment. METHODS: The alpha1 collagen gene fragment including the region of 5' UTR to exon (229 bp) was cloned in the eukaryotic expression plasmid pREP9 in the antisense orientation relative to the RSV-LTR promoter to reconstruct the pREP9- COL1 plasmid. Then it was transferred into HSFs through lipofectamine. The expression of alpha1 collagen was examined by immunostaining, reverse-transcriptase polymerase chain reaction, and Western blots. RESULTS: The recombinant plasmid pREP9-COL1 with a correct sequence was constructed successfully; pREP9-COL1 consistently inhibited human alpha1 collagen gene expression at both mRNA and protein levels. CONCLUSIONS: Antisense RNA was effective in downregulating alpha1 collagen expression of HSFs. Therefore, this approach offered a prospect of scar treatment by attenuation of alpha1 collagen production with antisense RNA.

Effect of asiaticoside on hypertrophic scar in the rabbit ear model.

BACKGROUND: Previous observations suggested that asiaticoside had a possible antiscaring effect. However, the precise pathological mechanism still remain unknown. We questioned whether asiaticoside might alleviate the formation of hypertrophic scar by affecting the expression of Transform growth factor beta (TGF-beta)/Smad signaling. AIMS: To investigate the effect of asiaticoside on the expression of TGF-beta/Smad signaling in the rabbit ear model of hypertrophic scar and to clarify the mechanism of asiaticoside on the scar treatment. METHODS: The rabbit model with hypertrophic scar was created and applied topically with a low-dose (0.5%) or high-dose (1%) asiaticoside three times daily for 1, 2 or 3 months and then we examined the changes of macroscopic and histopathologic characteristics of scars, and the expression of TGF-beta(1) and Smad protein was studied by applying reverse transcription-polymerase chain reaction and western blotting. RESULT: Asiaticoside could remarkably alleviate the scar in the rabbit ear model. Western blotting showed that the asiaticoside could decrease TGF-beta(1) expression, and further study revealed that asiaticoside could remarkably enhance the expression of inhibitory Smad7, but it had no effect on the expression of Smad2. CONCLUSION: Asiaticoside suggested a possible antiscaring effect probably by enhancing the expression of inhibitive Smad7.

Influence of skin-derived progenitor cell combining with hyaluronic acid on the wound healing of diabetic rat / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of skin-derived progenitor cell (SKP) combined with hyaluronic acid( HA) on the wound healing in diabetic rats.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>SKP-HA composition can promote wound healing in diabetic rats.</p>

Isolation and differentiation characteristics of dermal multipotent stem cells from humans of different ages cultured in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study isolation, identification and differentiation characteristics of dermal multipotent stem cells from human of different age in vitro culture.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of citrus reticulata blanco extract on fibroblasts from human hypertrophic scar in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of citrus reticulata blanco extract on the proliferation and collagen metabolism of fibroblasts from human hypertrophic scar.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Study on the regular pattern of the distribution of skin epidermal stem cells in the different parts of a healthy human body / 中华烧伤杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Influence of aerosols on the expression of cyclin B1, cyclin C and proliferating cell nuclear antigen in wound tissue healing of burned rat models / 中华外科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>Aerosols can up-regulate the expression of cyclin C and PCNA in skin basal cell nucleus. Therefore the aerosols can accelerate wound tissue healing.</p>

Clinical study and pathological examination on the treatment of deep partial thickness burn wound with negative charge aerosol / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effect of negative charge aerosol (NCA) on the treatment of burn wound.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Negative charge aerosol is safe and effective in promoting wound healing of the patients with partial thickness burns.</p>

Comparison of the effects of rhEGF with rhbFGF on the acceleration of wound healing / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the mechanism and the accelerating effect of rhEGF and rhbFGF on wound healing.</p><p><b>METHODS</b>Twelve New Zealand rabbits with 72 incised wounds on ventral side of 24 ears were randomly divided into two therapeutic groups (rhEGF of 10 ug/cm(2) and rhbFGF of 100 AU/cm(2)) and a control group (1% silver sulfadiazine cream, SD-Ag). The general conditions of the wound healing was observed grossly. Biopsies were harvested at different time points for the pathomorphological examination, the electron microscopic examination, and for assessment of integrin beta1 mRNA expression by in situ hybridization.</p><p><b>RESULTS</b>The expressions of integrin beta 1 mRNA in two therapeutic groups were significantly higher than that of control group. The quality of the wound healing was improved in therapeutic group with its healing time shortened when compared with that in control group (P < 0.05). There was an obvious difference in the number of fibroblasts and capillary gemmules between the therapeutic and control groups (P < 0.05).</p><p><b>CONCLUSION</b>The wound healing and quality could be improved by both rhEGF and rhbFGF, but rhbFGF seemed better to be employed during the early and middle stages of the wound repair for the growth of granulation tissue, while rhEGF should be applied at the late stage of wound repair to accelerate the re-epithelialization of the wound. Combined application of rhEGF with rhbFGF according to time effect could be more beneficial to the wound repair.</p>

A preliminary study on the identification and distribution of epidermal stem cells in different degrees of burn wounds in scalded rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the distribution of epidermal stem cells (ESCs) in different degrees of burn wounds in scalded rats.</p><p><b>METHODS</b>Thirty-two Sprague-Dawley (SD) rats were employed in the study. First degree (I), shallow (shallow II) and deep partial thickness (deep II) and full thickness burn wounds (III) were created on the rat skin. Burn wound samples were harvested at 24 postburn hours (PBHs) from all the wounds and were processed to tissue slices. The tissue slices were stained by immunohistochemistry technique. The expression and distribution of ESCs in different degrees of burn wounds were observed with integrins alpha 2 beta 1 and keratin 10 (K10) as first antibodies.</p><p><b>RESULTS</b>K10 positive cells were found to distribute in the strata spinosum, granulosum and lucidum in the first degree burn wound (I) with large amounts of integrins alpha 2 beta 1 positive cells in the residual basal layer and skin appendages (hair follicles) in shallow partial thickness burn wound (shallow II degree), and there were less integrins alpha 2 beta 1 positive cells in the remaining skin appendages in deep dermis in deep partial thickness burn wound (deep II degree). Finally, integrins alpha 2 beta 1 positive cells were sparsely found in the III degree burn wound.</p><p><b>CONCLUSION</b>The distribution of ESCs in burn wounds was closely related to the depth of burn wound. The residual ESCs might be the origin of burn wound regeneration and reepithelization.</p>

A research of endothelial cell-targeted therapy for cure of hypertrophic scar / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of endothelial cell-targeted therapy to cure post-burn hypertrophic scar.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Clinical significance of the predominant bacterial strains on burn wound during early postburn stage / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the clinical significance of the predominant bacterial colonization on burn wound in our department during recent years, so as to help select optimal antibiotics in burn patients with severe infections.</p><p><b>METHODS</b>This bacterial investigation was carried out in 215 cases of severely burned patients. The bacterial culture and the drug susceptibility test were carried out.</p><p><b>RESULTS</b>(1) One hundred and twenty-two strains of bacteria were cultured, in which 28 strains (23%) were Staphylococcus with negative coagulase, 27 (22%) S. aureus, 17 (14%) Pseudomonas aeruginosa, 11 (9%) Escherichia coli, 10 (8%) Enterobacter, 9 (7%), enterococci, 3 (2.5%) fungi, and 17 (14.5) other bacteria. (2) The resistance of S. aureus to ampicillin, oxacillin and amoxicillin/clavulanic acid was 81%, 38% and 31%, respectively. 11% and 16% of Pseudomonas aeruginosa resistant to Imipenem and Ceftazidime, respectively. (3) The sensitivity of G + cocci to vancomycin and norvancomycin, Chloramphenicol, Teicoplanin, Trimethoprim/Sulfamethoxaz, Rifampin was 100%, 100%, 100%, 94% and 88% respectively, and the Gram-negative bacilli to Meropenem, Imipenem, Amikacin, Cefepime, Cefoperazone/Sulbactam, Ceftazidime were 91%, 90%, 81%, 78%, 71% and 70%, respectively. Furthermore, the sensitivity of Pseudomonas aeruginosa to Cefoperazone/Sulbactam, Ceftazidime, Tobramycin, Meropenem, Amikacin, Ciprofloxacin, Amikacin, Cefepime were between 82% and 91%. MRSA was very sensitive to both vancomycin and norvancomycin.</p><p><b>CONCLUSION</b>The results suggested that Staphylococcus with negative coagulase and S. aureus were the predominant bacteria and Pseudomonas aeruginosa ranked second. The resistance of these bacteria to antibiotics was on the increase. Moreover, colonization of enterococcus and fungi on burn wound increased recently, which were scarce before. This implied the importance of rational and correct use of antibiotics during early postburn stage.</p>

Study of expression of platelet-derived growth factor and its receptor in granulation tissue of burn wound and post-burn hypertrophic scar at excessive stages / 中国康复理论与实践

@#ObjectiveTo investigate the role of platelet-derived growth factor(PDGF) and its receptor in the development of hypertrophic scar. MethodsThe expression of PDGF and its receptor were detected in biopsy specimens of 9 pieces of normal skin, 7 pieces of granulation tissue of burn wound and 34 pieces of hypertrophic scar by immunohistochemical staining using specific polyclonal antibodies.ResultsPDGF and its receptor markedly increased in granulation tissue and hypertrophic scars, reaching the peak in the hypertrophic scars within 6 months and then decreased after the peak, whereas PDGF and its receptor expressed weakly in only a few normal skin specimens, and the differences were significant(P<0.05).ConclusionsThe increasing expression of PDGF and its receptor may be related to the development of hypertrophic scar.