Objective To observe the effect of radiofrequency ablation technology for the treatment of infected wounds in minipigs. Methods Infected wounds of full-thickness skin defects (about 6.15 cm2/wound) were prepared in 8 6-month-old minipigs (weighing, 30-35 kg) using the method of Davis et al. The 160 wounds were randomly divided into 4 groups (n=40). Infected wounds were debrided with the radiofrequency ablation technology in group A, with the electric knife in group B, and with the scalpel in group C; no treatment was done in group D as a control. The healing rate, healing time, and tissue filling rate were observed; bacterial quantitative examination and histological examination were done at 0, 2, 7, and 14 days after operation. Results All infected wounds were successfully established after 48 hours when Staphylococcus aureus dilution were inoculated. The wounds after radiofrequency ablation technology treatment were fresh and flat with slight bleeding; the healing time of group A was significantly shorter than that of groups B, C, and D (P lt; 0.05), and the healing rate of group A was significantly higher than that of groups B, C, and D at 7 and 14 days after operation (P lt; 0.05). The tissue filling rate of group A was significantly higher than that of groups B, C, and D at 2 days after operation (P lt; 0.05); the tissue filling rates of groups A, B, and C were significantly higher than that of group D at 7 and 14 days after operation (P lt; 0.05). At 0, 2, 7, and 14 days, there were significant differences in the bacterial count per gram tissue among 4 groups (P lt; 0.05), the order from low to high was groups A, B, C, and D. The histological observation showed that the surface of wound was smooth in group A at 0 day, and group A was better than the other groups in wound healing; at 2 days, some exudates were observed in 4 groups, but it was least in group A. There was inflammatory cell infiltration in various degrees in 4 groups at 7 and 14 days; it was lightest in group A with thick epithelium and dense collagen bundles, followed by groups B and C, and it was severe in group D. Conclusion The radiofrequency ablation technology can effectively remove the necrotic tissues of infected wounds, remarkably reduce the number of bacteria, improve the healing rate, and shorten the healing time of wounds.
【Abstract】 Objective To observe the effects of Angelica dahurica extracts on the biological characteristics of human keratinocytes (KC) in vitro and to explore the possible mechanism in promoting wound healing. Methods HaCaT cells of passage 5 from KC were used during the experiment. Different concentrations (5 × 10-2, 5 × 10-3, 5 × 10-4, and 5 × 10-5 g/L) of Angelica dahurica extracts, which was obtained by 95% ethanol from Angelica dahurica raw material, were prepared by DMEM containing 0.25% fetal bovine serum (FBS). After the extracts at different concentrations were respectively used for KC culture for 5 days, the cell proliferation activities were detected by MTT, and DMEM containing 0.25% FBS served as the negative control. According to the cell proliferation activity, the optimal concentration was determined. KC was further treated with Angelica dahurica extracts of the optimal concentration (experimental group) or with DMEM containing 0.25% FBS (control group) for 48 hours. The cell cycle was tested by flow cytometry. Cyclin D1 and Caspase-3 mRNA levels were also detected by real-time fluorescent quantitative PCR technique. Results Angelica dahurica extracts at concentrations of 5 × 10-4, 5 × 10-3,and 5 × 10-2 g/L could significantly enhance KC proliferation, showing significant differences in absorbance (A) values compared with that of control group (P lt; 0.05) with an optimal concentration of 5 × 10-3 g/L. At this concentration, an increased percentage of S and G2/M phase cells and a decreased percentage of G0/G1 phase cells were detected, showing significant differences when compared with control group (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that the cyclin D1 and Caspase-3 mRNA levels of experimental group was significantly down-regulated, showing significant differences when compared with control group (P lt; 0.05). Conclusion Angelica dahurica extracts can promote the proliferation of KC, accelerate the cell cycle of KC by down-regulating mRNA expressions of cyclin D1, and inhibit apoptosis by down-regulating mRNA expressions of Caspase-3. These effects might enhance the process of wound healing by expediting the process of epithelization.
Objective To observe the effects of sal iva on impaired raw surface so as to elucidate the possible mechanism in wound heal ing by comparing with Yunnan baiyao. Methods Six wounds (2.5 cm × 2.5 cm in size) were establ ished at both sides on the back of 6 3-month-old adult Japanese rabbits (weighing 2.0-2.5 kg). According to treatment, 36 wounds were randomly divided into 3 groups: wounds were treated with 0.4 mL normal sal ine (blank control group, n=12), 0.5 g Yunnan baiyao powder (Yunnan baiyao group, n=12), and 0.4 mL sal iva of health adult (sal iva group, n=12) for 15 days, respectively. And the general observation of raw surface, the scar formation time, wound healing rate, and histopathology were used to evaluate the effectiveness of sal iva on wound heal ing. Results The wound healing speeds of sal iva group and Yunnan baiyao group were faster than that of blank control group. The wound healing rates of sal iva group were significantly higher than those of blank control group and Yunnan baiyao group at 5, 8, and 11 days after injury (P lt; 0.05). No obvious hemorrhage or necrosis of raw surfaces was observed in sal iva group, and the raw surfaces generally were covered with epidermis at 15 days after injury. The inflammatory cells and microvessel density in sal iva group were significantly less than those of Yunnan baiyao group and control group (P lt; 0.05). Conclusion Sal iva could obviously improve wound heal ing, which is related to its effects on reducing inflammatory cell infiltration, preventing wound infection, accelerating collagen fibers prol iferation, and promoting vessel reconstruction in the process of wound heal ing.
Objective Extracorporeal shock wave (ESW) can promote angiogenesis and tissue repair. To investigate the influence of ESW therapy on the histological features of diabetic chronic wounds and wound healing. Methods Ninety-six male Sprague Dawley rats with weight (220 ± 20) g were divided into 3 groups (n=32): diabetic control group, ESW treatment group, and normal control group. The diabetic rats were prepared in diabetic control group and ESW treatment group by intraperitoneal injection of Streptozotocin (60 mg/kg). Then a circular full-thickness skin wound of 1.8 cm in diameter was made at the back of diabetic rats to establish the diabetic chronic wound model, and the same wound was made in normal control group. In ESW treatment group, ESW (0.11 mJ/mm2, 1.5 Hz energy, and 500 pulses) was applied to treat the wound at 1 day after wounding; in two control groups, no ESW treatment was given. The wound healing and histological changes were observed by HE and Masson staining at 3, 7, and 14 days after treatment; and the cell proliferation, angiogenesis, and collagen deposition were observed by CD31 and proliferating cell nuclear antigen (PCNA) immunohistochemical staining. Results The wound closure rate in diabetic control group was lower, and the healing time was significantly longer than those in normal control group (P lt; 0.05); at 3, 7, and 14 days after treatment, the inflammatory cell infiltration in wound tissue was obvious, and the relative area density of collagen fibers, wound microvessel density (MVD), and the relative density of PCNA-positive cells were significantly lower than those in normal control group (P lt; 0.05). The wound healing time was significantly shorter and the wound closure rate was significantly higher in ESW treatment group than those in the diabetic control group (P lt; 0.05). At different time points in ESW treatment group, the inflammatory cells signficantly reduced, while the relative area density of collagen fibers, MVD, and relative density of PCNA-positive cells significantly increased when compared with those in diabetic control group (P lt; 0.05). No significant difference in MVD and relative density of PCNA-positive cells was found between ESW treatment group and normal control group (P gt; 0.05). Conclusion Low-energy ESW treatment can inhibit the local inflammatory response, promote cell proliferation, increase angiogenesis and collagen deposition, and enhance granulation tissue formation, and so it can promote chronic wound healing in diabetic rats.
Objective To explore the possible mechanism of nerve growth factor (NGF) mixed insul in on the angiogenesis of burn wounds and the effect on the expressions of Bcl-2 and Bax in diabetic rats. Methods A total of 75 SPF male Wistar rats, weighing 200-220 g, were selected randomly and divided into nomal control (group A, n=15), the rats with diabetic control (group B, n=15), insul in treatment (group C, n=15), NGF treatment (group D, n=15), NGF and insul in treatment (group E, n=15) groups. In groups B, C, D, and E, streptozotocin was given by intraperitoneal injection at dose of 10 mg/kg on the 1st day and 50 mg/kg on the 3rd day to prepare the diabetic rat models. In group A, citric acid buffer at the samedose was given. After 1 month of diabetic models, second degree scald was made on the back of the rats, and then wounds were treated with 3-layer normal sal ine gauze in groups A and B, with 3-layer gauze containing 5 U Novol in 30R and subcutaneous injection of Novol in 30R (4-6 U/kg) everyday in group C, with 3-layer gauze containing 5 mL NGF (25 U/mL) in group D, and with a combination of groups C and D in group E. At 7, 11, 15, and 21 days, the wound heal ing rate was calculated; at 3, 7, 11, 15, and 21 days, the expressions of Bcl-2, Bax, and CD34 were determined and the microvascular density was measured by immunohistochemistry staining. Results All rats survived till experiment was finished. The area of wounds became smaller gradually with time. Group E was better than other groups in the wound heal ing rate (P lt; 0.05), the skin keratosis, the hair growth, and the granulation tissue and collagen fibers growth. With time, the expressions of CD34 and Bcl-2 increased gradually, reached the peak at 15 days and decreased at 21 days; the expression was ber in group E than in other groups (P lt; 0.05). At 3 days, Bax did not express; at 7 days, Bax began to express in new vascular endothel ial cells and the expression increased gradually with time; the expression was weaker in group E than in other groups (P lt; 0.05). Conclusion A combination of NGF and insul in local appl ication can enhance the angiogenesis of the burn wound in diabetic rats and accelerate wound heal ing by increasing the expression of Bcl-2 and decreasing the expression of Bax and restraining apoptosis of the wounds vascular endothel ial cells of diabetic rats.
Objective To observe and explore the effects of adipose-derived stem cells (ADSCs)-hyaluronic acid (HA) composite on heal ing of wound combined with radiation injury. Methods The ADSCs were harvested from the fat tissue in groin of 10 inbred Sprague Dawley (SD) rats and were isolated and cultured by enzyme digestion. The ADSCs-HA composite wasprepared with ADSCs (5 × 106 cells/mL) at passage 6 and HA (10 mg/mL). Thirty inbred SD rats, 15 males and 15 females, were randomly divided into groups A (n=10), B (n=10), and C (n=10). A 2 cm × 2 cm full-thickness skin defect was made on the rat back before 20 Gy 60Co radiation exposure. One week after debridement, wounds were treated by petrolatum gauze in group A as the control group, by HA (0.4 mL) and petrolatum gauze in group B, and by ADSCs-HA composite (0.4 mL) and petrolatum gauze in group C. The microvessel density (MVD) and the distribution of CD90 positive cells were observed at 1st, 2nd, 3rd, and 4th weeks. Results The wound heal ing was slower, and wound did not heal at 4th week and still filled with granulation tissue in group A; the wound heal ing of group B was faster than that of group A, and the wound did not heal completely with depression in the center at 4th week; the wound healed completely with epidermil izated surface and no obvious depression at 4th week in group C. The histological observation showed that MVD was significantly higher in group C than in groups A and B at the 1st, 2nd, and 3rd weeks (P lt; 0.05), and in group B than in group A at the 3rd week (P lt; 0.05); MVD was significantly higher in groups B and C than in group A (P lt; 0.05), but no significant difference was found between groups B and C (P gt; 0.05) at 4th week. No CD90 positive cell was found in groups A and B; CD 90 positive cells were observed in group C and gradually decreased with time. Conclusion ADSCs-HA composite can accelerate heal ing of wound combined with radiation injury by promoting and controll ing wound angiogenesis.
Objective To investigate the effectiveness and mechanism of recombinant human granulocyte-macrophage colony-stimulating factor (rhGMCSF) gel on wound debridement and healing of deep II thickness burn. Methods Between December 2008 and December 2010, 58 patients with deep II thickness burn, accorded with the inclusive criteria, were collected. There were 36 males and 22 females with an average age of 32.4 years (range, 12-67 years). The causes were hot liquid in 38 cases and fire in 20 cases. The time from injury to treatment was 1-3 days (mean, 2.1 days). In this randomized, double-blind, and self-control study, all patients were randomly divided into 2 groups, wounds were treated with rhGMCSF gel (test group) or gel matrix (control group). There was no significant difference in wound area between 2 groups (P gt; 0.05). The time of completed removal eschar and the percentage of removal-area of eschar were recorded at 2, 6, 10, 14, and 18 days during healing process. The time of wound healing was also recorded. Results Compared with control group, the necrotic tissues on the burn wound got soft in test group at 4 days after treatment. At 6 days, they loosened and eventually sloughed off. The wound bed presented in red and white, followed by rapidly growing granulation tissues. Except 18 days after treatment, there were significant differences in the percentage of removal-area of eschar between 2 groups (P lt; 0.05). The time of completed removal eschar in test group [(7.71 ± 2.76) days] was significantly shorter than that in control group [(14.71 ± 3.63) days] (t=13.726, P=0.000). The time of wound healing in test group was (18.41 ± 2.47) days, which was significantly shorter than that in control group [(23.58 ± 3.35) days] (t=15.763, P=0.000). Conclusion Compared with the gel matrix, the rhGMCSF gel may promote wound debridement and healing in deep II thickness burn.
Objective Human acellular amniotic membrane (HAAM) contains collagens, glucoproteins, proteinpolysaccharide,integrin, and lamellar, which can supply rich nutrition to cell prol iferation and differentiation. To explore the possibil ity of HAAM with adi pose-derived stem cells (ADSCs) as a good engineered skin substitute for repairing skin defect. Methods Primary ADSCs were obtained from inguinal fat of 30 healthy 4-month-old SD rats, male or female, weighing 250-300 g, and cultured in vitro and purified. The 3rd passage ADSCs were used to detect CD44, CD49d and CD34 by immunocytochemistry staining. After physical and trypsin preparation, the HAAM was observed by HE staining and scanning electron microscope(SEM) respectively. ADSCs were seeded on epithel ial side of HAAM at the density of 2 × 105/cm2, cocultured, and observed by SEM at different time. MTT test was used to detect viabil ity of cells that seeded on HAAM, the group without HAAM was used as control. Thirty SD rats were made models of full-thickness skin wound and randomly divided into three groups (A, B, and C). Wound was repaired with HAAM/ADSCs composites in group A, with HAAM in group B, and with gauze as control in group C. The rats underwent postoperative assessment of wound heal ing rate and histological observation at the 1st, 2nd, and 4th weeks. Results HE staining showed that the 3rd passage ADSCs was spindle-shaped with an ovoid nucleus which located in the middle of cell; the immunocytochemistry staining showed positive result for CD44 and CD49d and negative result for CD34. There were no residues of cells in the HAAM by HE staining. SEM showed that there were different structures at the two sides of HAAM；one side had compact reticular structure and the other side had fibrous structure. After 3 days of co-culture, ADSCs showed good growth on HAAM; the cells were closely packed onto the HAAM, attached firmly and prol iferated to confluence on the stromal surface of HAAM. MTT test showed that the cells on the HAAM grew well and had b prol iferation vital ity. There was no significant difference between ADSCs cultured in the HAAM and control group (P gt; 0.05). One, 2, 4 weeks after graft, there were significant differences in wound heal ing rate between group A and groups B, C (P lt; 0.05), between group B and group C (P lt; 0.05). HE staining showed that wound healed faster in group A than in groups B, C. Cytokeratin 19 (CK19) immunohistochemical statining showed that there were more CK19 positive cells in group A than in groups B, C. Conclusion The graft of HAAM with ADSCs plays an effective role in promoting the repair of full-thickness skin wound
Objective To investigate the effect of topical external administration of recombinant human epidermal growth factor (rhEGF) when controll ing blood sugar on expression of epidermal growth factor receptor (EGFR) and EGFR mRNA of wound in diabetes mell itus (DM) combined with scald. Methods A total of 136 male Wistar rats weighing (188.57 ± 6.59) g were randamly divided into 4 groups (groups A, B, C, and D, n=34). The rats was made DM model by intraperitoneal injected 60 mg/kg streptozocin in groups A, B, and C; rats were injected buffer alone in group D as control group. After 8 weeks, the rats of 4 groups were placed in 80℃ hot water for 6 seconds for preparation of the back deep II degree scald model. In group A, the blood sugar level was controlled at the level of group D 1 week before scald model; within 24 hours after models preparation, rhEGF was sprayed on wound at 150 U/cm2 . In group B, the rats were given the same treatment as group A except not controll ing blood sugar. In group C, the blood sugar was controlled as group A and wound was suture fixation with 1% silver sulfadiazine cream at 24 hours after the model. In group D, the same treatment as group A was given after injury. The heal ing rate of the wound was detected at 3, 7, 11, 15, and 21 days after injury; the EGFR mRNA expression was determined by mRNA hybridization in situ, and the EGFR protein expression was deterimined by immunohistochemistry and Western blot at 1, 3, 5, 7, 11, 15, and 21 days. Results All the rats survived at the end of experiment. There was no significant difference in the heal ing rate of the wound among the 4 groups at 3 days (P gt; 0.05). The heal ing rate of the wound was significantly higher in groups A and D than in groups B and C (P lt; 0.05) at 7, 11, 15, and 21 days. The expession of EGFR mRNA in 4 groups was observed by hybridization in situ, which mainly distributed in the dermal fibroblasts, capillary endothel ial cells and remnants of skin and wound edge epithel ium of the subsidiary; the expessions reached the peak at 5 days in group A, at 7 days in groups B and C, and at 11 days in group D; and the peak level was significantly higher in groups A and D than in groups B and C (P lt; 0.05). Immunohistochemistry and Western blot showed that the expession of EGFR protein was observed in 4 groups and reached the peak level at 7 days in groups A and B, and at 11 days in groups C and D; showing significant difference between groups B, C and groups A, D (P lt; 0.05). Conclusion External appl ication of rhEGF when controll ing blood sugar can accelerate obviously the wound heal ing in DM combined with scald. After controll ing blood sugar, external appl ication of rhEGF can boost obviously the expressions of EGFR mRNA, EGFR, and the extending process of signal conduction.