[Roles of adenosine monophosphate activated protein kinase in skeletal muscle atrophy in rats with severe scald].

Objective: To investigate the expression and phosphorylation level change of adenosine monophosphate activated protein kinase (AMPK) in skeletal muscle of severely scald rats and its roles in skeletal muscle atrophy in severely scalded rats. Methods: The experimental research method was applied. Totally 100 6-week-old male Wistar rats were divided into sham injury group and scald group according to the random number table, with 50 rats in each group. After weighing the body weight, rats in scald group were inflicted with full-thickness scald of 30% total body surface area on the back, and rats in sham injury group were simulated with scald. At 6 h and on 1, 3, 5, and 7 d post injury, 10 rats in each group were taken to measure their body weights and weights of extensor digitorum longus and soleus muscle. At 6 h and on 1, 3, 5, and 7 d post injury, the tibialis anterior muscles were collected, the mRNA expressions of muscle atrophy F-box protein (MAFbx) and muscle-specific RING finger protein 1 (MuRF1) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction; the content of adenosine monophosphate (AMP), adenosine diphosphate, and adenosine triphosphate (ATP) were detected by high performance liquid chromatography, and AMP/ATP ratio and energy charge were calculated; the protein expressions of AMPK-α and phosphorylated AMPK-α (p-AMPK-α) were detected by Western blotting, and the p-AMPK-α/AMPK-α ratio was calculated, with sample number of 4 in each time point of each group. Data were statistically analyzed with analysis of variance for factorial design and least significant difference test. Results: The body weights of rats in 2 groups before injury and at each time point post injury were close (P>0.05). At 6 h post injury, the weight of extensor digitorum longus of rats in scald group was (0.107±0.007) g, which was significantly heavier than (0.086±0.0607) g of sham injury group (P<0.01). On 3 d post injury, the weight of extensor digitorum longus of rats in scald group was (0.083±0.016) g, which was significantly lighter than (0.102±0.005) g of sham injury group (P<0.01). The weight of soleus of rats in 2 groups were close at each time point post injury (P>0.05). Compared with those of sham injury group, the mRNA expression of MAFbx in tibialis anterior muscle of rats in scald group was significantly up-regulated at 6 h post injury (P<0.01), and the mRNA expressions of MuRF1 in tibial anterior muscle of rats in scald group were significantly up-regulated at 6 h and on 1 d post injury (P<0.01). At 6 h and on 7 d post injury, compared with those of false injury group, the AMP/ATP ratios of the tibial anterior muscle of rats in scald group were significantly increased (P<0.05 or P<0.01), and energy charges of the tibial anterior muscle of rats in scald group were significantly decreased (P<0.01). At each time point post injury, the protein expressions of AMPK-α of the tibial anterior muscle of rats in 2 groups were close (P>0.05). The p-AMPK-α/AMPK-α ratios of the tibial anterior muscle of rats in scald group at 6 h and on 7 d post injury were significantly higher than those in sham injury group (P<0.05 or P<0.01). Conclusions: The decrease in energy charge and increase in AMP/ATP ratio of skeletal muscle of rats after severe scald activate AMPK. The activation of AMPK in the early stage of injury is consistent with the up-regulation of MAFbx and MuRF1 expressions and down-regulation of skeletal muscle weight. The above-mentioned changes may be one of the molecular mechanisms of skeletal muscle atrophy in rats with severe scald.

[Clinical effects of pre-expanded anterior perforator flap of transverse cervical artery and pre-expanded thoracic random flap in reconstructing extensive facial and cervical scar in relay].

Objective: To explore the clinical effects of pre-expanded anterior perforator flap of transverse cervical artery in extensive facial and cervical scar reconstruction and contralateral pre-expanded thoracic random flap in relay in donor site repair. Methods: A retrospective cohort study was conducted. From May 2008 to December 2018, 10 patients with extensive facial and cervical scar after burns were treated in the Fourth Medical Center of PLA General Hospital, including 8 males and 2 females, aged 10-55 years. In the first stage of operation, two skin and soft tissue expanders of the same volume (with rated capacity of 250-600 mL) were respectively placed in the right side and left side of the chest according to the size of scar, and then the skin was expanded. The total amount of normal saline injected was 2 to 4 times of the rated capacity of the expander. In the second stage, the defect with area of 12 cm×8 cm-23 cm×15 cm caused by scar resection and release was repaired with unilateral pre-expanded anterior perforator flap of transverse cervical artery with area of 12 cm×9 cm-24 cm×16 cm. The contralateral pre-expanded thoracic random flap with the same area as that of the above-mentioned perforator flap was extended to repair the secondary defect with area of 8 cm×6 cm-17 cm×14 cm formed after transfer of the above-mentioned perforator flap. The exploration of perforating branch of transverse cervical artery, flap transfer and survival, injury repair, and complications were observed. The appearance and related function of donor and recipient sites and satisfaction of patients were followed up. Results: The perforating branches of transverse cervical artery appeared stably in the 10 patients. All the flaps were transferred to the recipient area without tension and survived. Both facial and cervical injuries were repaired successfully with no common complications. During the follow-up of 6 months-8 years, the color and texture of the pre-expanded anterior perforator flap of transverse cervical artery matched with the surrounding tissue, the functions of head raising and neck rotation of patients were significantly improved compared with those before operation, the color and texture of the flap transplanted in the first donor site matched with the original skin, linear scar left at the surgical incision, and 9 patients were satisfied with the restoration of the appearance and function of donor and recipient sites. Conclusions: The color and texture of the pre-expanded anterior perforator flap of transverse cervical artery match well with the face and neck, and the repairable area is large. After the perforator flap is removed, the secondary wound can be repaired with the pre-expanded thoracic random flap at the same time, and the injury of the chest donor site is alleviated. This relay repair method is a good choice for reconstructing extensive facial and cervical scar.

[Imaging judgment of soft tissue and vascular injuries in limbs with high-voltage electric burn and its clinical significance].

The injury mechanism of high-voltage electric burn in limbs is complex and special. The soft tissue and vascular injuries caused by high-voltage electric burn are serious and concealed. It is difficult to judge the severity and extent of injury before surgery, which affects the diagnosis and treatment effects and remains a major problem in burn field. In recent decades, a series of clinical studies have been conducted by scholars at home and abroad, using various imaging methods for the judgment of soft tissue and vascular injuries, which have their own advantages and disadvantages. According to the principle of accuracy, precision, safety, and easy operation, magnetic resonance imaging and magnetic resonance angiography are required at the same time in general for the imaging judgment of soft tissue and vascular injuries in limbs with high-voltage electric burn. The B-mode ultrasonography shall be performed if a precise judgment of vascular injury is needed.

[Effects of insulin therapy on skeletal muscle wasting in severely scalded rats and its related mechanism].

Objective: To explore the effects of insulin therapy on skeletal muscle wasting (SMW) in severely scalded rats and its related mechanism. Methods: Totally 48 male Wistar rats aged 7-8 weeks were divided into simple scald (SS) group and insulin therapy (IT) group according to the random number table, with 24 rats in each group. After weighing the body mass and measuring the blood glycemic level of the tail end with a glucometer, the rats in the two groups were immersed in hot water at 94 ℃ for 12 seconds to make a full-thickness dorsal scald model involving 30% total body surface area. Rats in group IT were subcutaneously injected with 1 U/kg insulin glargine at 8: 00 a day from post injury day (PID) 1 to 7, whilst rats in group SS were given the same amount of normal saline. Rats in the two groups were given 10 mL/kg enteral nutritional emulsion by intragastric infusion at 8: 00 (after insulin administration), 13: 00, and 18: 00 a day respectively from PID 1 to 7. The blood glycemic levels of tail end of rats in the two groups were measured by glucometer before insulin administration on PID 1-4, 6, and 7 and on every morning of PID 8, 9, 11, 12, and 14. The body mass of rats in the two groups on PID 14 without any treatment was weighed. Eight rats from each group were collected respectively on PID 4, 7, and 14 to harvest tibialis anterior muscle (TAM) samples. The mass of TAM on PID 14 was weighed. The ultrastructural changes of TAM myocytes on PID 7 were observed with transmission electron microscope. The apoptotic rates of TAM myocytes on PID 4, 7, and 14 were assessed by the assay of terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphate-biotin nick end labeling, the expressions of cysteine-aspartic protease-3 (caspase-3) of TAM on PID 4, 7, and 14 were detected with immunohistochemistry, and protein expressions of endoplasmic reticulum (ER) stress (ERS) associated proteins glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein-homologous protein (CHOP), and activated caspase-12 of TAM on PID 4, 7, and 14 were detected with Western blotting. Data were processed with completely random design t test, analysis of variance for repeated measurement, analysis of variance for factorial design, t test, and Bonferroni correction. Results: The blood glycemic level and body mass of rats in the two groups before injury were similar (t=0.204, 0.405, P>0.05). There were no statistically significant differences in blood glycemic levels of rats between the two groups on PID 1, 6, 9, 11, 12, and 14 (t=0.229, 3.339, 1.610, 0.178, 0.181, 0.079, P>0.05). Compared with those of group SS, blood glycemic levels of rats in group IT were significantly lower on PID 2, 3, 4, 7, and 8 (t=7.245, 4.165, 4.609, 4.018, 3.995, P<0.05 or P<0.01). On PID 14, the body mass and TAM mass of rats in group IT were (271±19) g and (0.47±0.05) g respectively, both obviously higher than (254±12) g and (0.43±0.04) g of group SS (t=2.159, 2.375, P<0.05). On PID 7, nuclear pyknosis and deformation, chromosome misdistribution, and ER swelling in TAM myocytes of rats in group SS were observed; the apoptotic alterations and ER swelling of TAM myocytes were alleviated in rats of group IT as compared with those of group SS. The apoptotic rates of TAM myocytes of rats in group IT were obviously lower than those of group SS on PID 4, 7, and 14 (t=4.262, 9.153, 9.799, P<0.01). The expressions of caspase-3 in TAM of rats in group IT were obviously lower than those of group SS on PID 7 and 14 (t=10.429, 7.617, P<0.01). Compared with those of group SS, the protein expressions of GRP78 were obviously increased on PID 4 and 14 (t=4.172, 4.437, P<0.05), the protein expressions of activated caspase-12 were obviously decreased on PID 7 and 14 (t=11.049, 11.181, P<0.01), and the protein expressions of CHOP were obviously decreased on PID 4, 7, and 14 (t=13.837, 9.572, 6.930, P<0.01) in TAM of rats in group IT. Conclusions: Insulin therapy may reduce skeletal muscle myocytes apoptosis and SMW by alleviating ERS in rats with severe scald.

[Analysis on treatment of eight extremely severe burn patients in August 2nd Kunshan factory aluminum dust explosion accident].

Objective: To summarize the measures and experience of treatment in mass extremely severe burn patients. Methods: The clinical data and treatment of 8 extremely severe burn patients in August 2 Kunshan factory aluminum dust explosion accident who were admitted in the 100th Hospital of PLA on August 2nd, 2014, were retrospectively analyzed. There were 4 males and 4 females, aging 22-45 (34±7) years, with total burn area of 55%-98% [(89±15)%] total body surface area (TBSA) and full-thickness burn area of 45%-97% [(80±21)%] TBSA. All the 8 patients were accompanied with severe shock, inhalation injury, and blast injury. According to the requirements of former PLA General Logistics Department and Nanjing Military Command, a treatment team was set up including a special medical unit and a special care unit, with Chai Jiake from the First Affiliated Hospital of PLA General Hospital as the team leader, Zheng Qingyi from the 175th Hospital of PLA (the Affiliated Dongnan Hospital of Xiamen University) as the deputy leader, the 100th Hospital of PLA as the treatment base, and burn care, respiratory, nephrology, nursing specialists from the First Affiliated Hospital of PLA General Hospital, and the burn care experts and nursing staff from the 180th Hospital of PLA, 118th Hospital of PLA, 98th Hospital of PLA, and 175th Hospital of PLA, and nurses from the 85th Hospital of PLA, 455th Hospital of PLA, 101th Hospital of PLA, 113th Hospital of PLA as team members. Treatment strategies were adopted as unified coordination by the superior, unified responsibility of team leader, division of labor and cooperation between team members, and multidisciplinary cooperation led by department of burns. With exception of one patient who received deep vein catheterization before admission, the other 7 patients were treated with deep vein catheterization 0.5 to 3.0 hours after admission to correct hypovolemic shock as soon as possible. Eight patients received tracheotomy, and 7 patients were treated with mechanical ventilation by ventilator in protective ventilation strategy with low tide volume and low volume pressure to assist breathing. Fiberoptic bronchoscopy was done one to three times for all the 8 patients to confirm airway injuries and healing status. Escharectomy and Meek dermatoplasty in the extremities of all the 8 patients were performed 3 to 6 days after injury for the first time. Escharectomy, microskin grafting, and covering of large pieces of allogeneic skin on the trunks of 4 patients were performed 11 to 16 days after injury for the second time. The broad-spectrum antibiotics were uniformly used at first time of anti-infective therapy, and then the antibiotics species were adjusted in time. The balance of internal environment was maintained and the visceral functions were protected. One special care unit was on responsibility of only one patient. Psychological intervention was performed on admission. The rehabilitative treatment was started at early stage and in company with the whole treatment. Results: Acute renal injury occurred in 5 patients within 36 hours after injury and their renal function was restored to normal 4 days after injury due to active adjustment of fluid resuscitation program. No pulmonary complications, such as severe pulmonary infection and ventilator-associated pneumonia, occurred in the survived patients. One of the 8 patients died, and the other 7 patients were cured successfully. The wounds were basically healed in 2 patients in 26 or 27 days by 2 or 3 times of operation, and in 5 patients by 4 or 5 times of operation. The basic wound healing time was 26-64 (48±15) days for all the 7 patients. Conclusions: Treatment strategies of unified coordination by the superior, unified responsibility of team leader, division of labor and cooperation between team members, and multidisciplinary cooperation led by department of burns are the bases to successful treatment. Correcting shock as soon as possible is the prerequisite and closing wound as soon as possible is the key to successful treatment. Comprehensive treatment measures, such as maintaining and regulating the function of viscera, improving the body immunity, and preventing and treating the complications, are the important components to successful treatment. It is emphasized that in the treatment of mass extremely severe burn patients, specialist burn treatment should always be in the dominant position, and other related disciplines may play a part in auxiliary function.

[Influences of ulinastatin on acute lung injury and time phase changes of coagulation parameters in rats with burn-blast combined injuries].

Objective: To explore the influences of ulinastatin on acute lung injury and time phase changes of coagulation parameters in rats with severe burn-blast combined injuries. Methods: One hundred and ninety-two Sprague-Dawley rats were divided into pure burn-blast combined injury group, ulinastatin+ burn-blast combined injury group, and sham injury group according to the random number table, with 64 rats in each group. Two groups of rats with combined burn-blast injuries were inflicted with moderate blast injuries with the newly self-made explosive device. Then the rats were inflicted with 25% total body surface area full-thickness scald (hereinafter referred to as burn) on the back by immersing in 94 ℃ hot water for 12 s. Rats in sham injury group were sham injured on the back by immersing in 37 ℃ warm water for 12 s. Immediately after injury, rats in the three groups were intraperitoneally injected with Ringer's lactate solution (40 mL/kg), meanwhile rats in ulinastatin+ burn-blast combined injury group were intraperitoneally injected with ulinastatin (4×10(4)U/kg), once every 12 hours, until post injury hour (PIH) 72. Before injury, at PIH 3, 6, 12, 24, 48, 72, and on post injury day (PID) 7, 8 rats in each group were selected to harvest abdominal aortic blood samples to detect plasma levels of activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, D-dimer, antithrombin Ⅲ (AT-Ⅲ), and α2-antiplasmin (α2-AP). At PIH 24, three rats in each group which were used in detection of coagulation parameters were sacrificed to observe lung injury. At PIH 72, three rats in each group were sacrificed for histopathological observation of lung. Data were processed with analysis of variance of factorial design and least-significant difference test. Results: (1) Compared with those of rats in sham injury group, APTT of rats in pure burn-blast combined injury group significantly prolonged at PIH 72 and on PID 7 (P<0.05 or P<0.01). PT significantly prolonged at PIH 3 and 72 and significantly shortened at PIH 6 (P<0.05 or P<0.01) . Fibrinogen level significantly increased from PIH 12 to PID 7 (P<0.01). AT-Ⅲ level significantly decreased at PIH 6 and 12 (P<0.01), and α2-AP level significantly decreased at PIH 6 and significantly increased from PIH 24 to 72 (P<0.01). Compared with those of rats in pure burn-blast combined injury group, APTT of rats in ulinastatin+ burn-blast combined injury group significantly prolonged at PIH 3 and 6 (P<0.01) while PT significantly shortened at PIH 3, 12, and 72 (P<0.05 or P<0.01). Fibrinogen level significantly decreased at PIH 6 and 12 and significantly increased at PIH 72 (P<0.05 or P<0.01). AT-Ⅲ level significantly increased at PIH 3, 12, 48, and 72 (P<0.05 or P<0.01), and α2-AP level significantly decreased from PIH 12 to 72 (P<0.05 or P<0.01). D-dimer level of rats in sham injury group, pure burn-blast combined injury group, and ulinastatin+ burn-blast combined injury group were respectively (0.084±0.013), (0.115±0.015), (0.158±0.022), (0.099±0.011), (0.099±0.012), (0.089±0.011), (0.124±0.014), and (0.116±0.018) µg/mL, (0.064±0.033), (0.114±0.016), (0.135±0.009), (0.060±0.008), (0.104±0.010), (0.124±0.020), (0.180±0.036), and (0.201±0.032) µg/mL, (0.074±0.013), (0.084±0.035), (0.101±0.050), (0.091±0.046), (0.096±0.034), (0.044±0.019), (0.106±0.049), and (0.118±0.047) µg/mL. Compared with that of rats in sham injury group, D-dimer level significantly decreased at PIH 6 and 12 and significantly increased from PIH 48 to PID 7 (P<0.05 or P<0.01). Compared with that of rats in pure burn-blast combined injury group, D-dimer level of rats in ulinastatin+ burn-blast combined injury group significantly decreased at PIH 3, 48, and 72, and on PID 7 (P<0.05 or P<0.01). (2) At PIH 24, there was a large amount of light red effusion in the thoracic cavity, and both lung lobes were hyperemic and edematous with a small amount of blood clots in the left and middle lobe of rats in pure burn-blast combined injury group. There was a small amount of yellowish effusion in the thoracic cavity of rats in ulinastatin+ burn-blast combined injury group, and the degree of hyperemic and edematous of bilateral lobes was lighter compared with rats in pure burn-blast combined injury group with no clot in the left lobe. No congestion, edema, or bleeding was observed in lungs of rats in sham injury group. (3) At PIH 72, disorganized alveolar structure, collapsed alveolar cavity, edematous and thickening pulmonary interstitium, infiltration of a large amount of inflammatory cells, obvious rupture of alveolar septum, and hyaline thrombus were observed in lungs of rats in pure burn-blast combined injury group. Significantly improved alveolar structure, less collapsed alveolar cavity, improved edematous pulmonary interstitium, less infiltration of inflammatory cells, rupture of alveolar septum, and no thrombus were observed in lungs of rats in ulinastatin+ burn-blast combined injury group. The lung tissue had a well-filled alveolar cavity with no interstitial edema or infiltration of inflammatory cells and no thrombosis in lungs of rats in sham injury group. Conclusions: Ulinastatin has positive therapeutic effects on acute lung injury in rats with severe burn-blast combined injuries through its good regulating effects on coagulation and fibrinolytic disorders caused by burn-blast combined injuries.

[Study on changes of calpain and myocardial damage in rats with burn-blast combined injury].

Objective: To study myocardial damage and rules of calpain change in rats with burn-blast combined injury. Methods: One hundred and twenty-eight male SD rats were randomly divided into control group, burn group, blast group, burn-blast group, with 32 rats in each group. CONTROL GROUP: 37 degrees' warm water for 12 s; Burn group: 94 degrees' boiling water for 12 s; Blast group: 5 g cyclonite explode in 75 cm distance from left chest wall of rat; Burn-blast group: burn group and blast group combined modeling method. At 6, 24, 48, 72 h observation points after injury, abdominal aorta blood samples and myocardial specimen were collected. Left ventricular ejection fraction (EF), left ventricular fractional shortening index (FS) were measured through color Doppler ultrasound instrument; Myocardial tissue was stained with hematoxylin-eosin (HE); serum cardiac troponin I (CTnI) and creatine kinase isoenzyme (CK-MB) were detected; detection of cell apoptosis in myocardial tissue was performed by terminal deoxynucleotidyl transferase-mediated dUTP notch labeling technique (Tunel). Expression levels of calpain mRNA level and protein were detected with Real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western imprinting method analysis; calpain activity was detected by fluorescence spectrophotometry. Results: The injury of burn-blast combined injured rats was obvious, including myocardial interstitial edema, large area of myocardial cell degeneration and disintegration and the number of neutrophil infiltration increased. Cardiac function decreased 24 h after injury in burn group, blast group, burn-blast group; both EF and FS were significant lower than those of control group (all P<0.05). FS at 48, 72 h and EF at 72 h in burn-blast group were significantly lower than those of burn group, blast group at the same time points (all P<0.05); the level of cTnI in burn-blast group rose and was higher than control group at all time points, higher than the burn group, blast group at 48 h (all P<0.05). CK-MB in burn-blast group rats increased after injury, lowered at 24 h and rose again at 48 h. The level was significantly higher than control group and burn group (both P<0.05). Comparing to control group, myocardial apoptosis index in burn group, blast group and burn-blast group were significantly increased (all P<0.05). Those of burn group (25.3±4.0) at 24 h and (28.8±5.3) at 48 h were significantly lowered than burn-blast group (43.3±9.4), (53.3±10.4) at same time points, and burn group (31.9±6.7) at 72 h was significantly higher than blast group (17.3±6.3) (all P<0.05). Compared to control group, Calpain mRNA and protein expression in myocardial tissue were significantly increased in burn-blast group at all time points (all P<0.05). Calpain activity reached the peak at 24 h after injury, then gradually declined, and was significantly higher than control group (all P<0.05). Conclusion: Calpain expression and activity increase in burn-blast combined injured rats which leads to myocardial damage.

[Effect of mitochondrial apoptosis on pulmonary fibrosis in rats with severe scald injury].

OBJECTIVE: To explore the role of mitochondrial apoptosis on pulmonary fibrosis in rats with severe scald injury. METHODS: According to the random digital table, a total number of 32 Wistar rats were divided into 4 groups: sham burn (group A), burn group (group B), 12-week post burn recovery group (group C), and 12-week post burn recovery plus a second burn injury group (group D). In group A and group B, lung tissues were harvested on post burn day 4. After received first burn injury 12 weeks, the group C and group D received separately a second sham burn injury and burn injury. Lung tissues were harvested on post burn day 4 after the second burn injury. All tissues were examined for cells apoptosis by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL). Pulmonary fibrosis was assessed by Masson trichrome staining and Sirius red staining. The protein expression levels of cleaved Caspase-3, Bax and Bcl-2 were assessed by Western blot. RESULTS: Both Masson trichrome staining and Sirius red staining showed obvious pulmonary fibrosis in group C and group D. The apoptosis rates of group B, C and D were significantly higher than that in group A ((15.50±3.30)%, (7.88±3.10)%, (15.88±3.23)% vs (2.10±1.07)%, all P<0.05). Compared to group A, cleaved Caspase-3 levels were significantly higher in group B, C and D ((0.59±0.11), (0.33±0.08), (0.73±0.13) vs (0.16±0.05), all P<0.05). The ratio of Bax/Bcl-2 in group B, C and D also increased significantly ((2.08±0.30), (0.83±0.09), (1.54±0.12) vs (0.64±0.05), all P<0.05). CONCLUSION: Severe burn injury can induce pulmonary fibrosis and mitochondrial apoptosis may play an important role in the development of pulmonary fibrosis.

[Study on culture, identification and differentiation of primary rat skeletal muscle satellite cells].

OBJECTIVE: To establish the isolation, culture and identification methods of primary rat skeletal muscle satellite cells (SMSC) and observe its characterization of differentiation in vitro. METHODS: Skeletal muscle satellite cells were obtained by tissue block culture method in combination with pre-plating techniques, and the purity of these cells was detected by both immunocytochemistry and fluorescence activated cell sorter (FACS) with Pax7 as marker of SMSC. Myogenesis of these cells was induced in differentiation medium and the mRNA expressions of myogenic differentiation gene (MyoD) and Myogenin were determined by Real-time polymerase chain reaction (PCR). RESULTS: Cells crawled out from the edge of tissue blocks after 1 week of culture. After purification by pre-plating techniques, more than 97.6% of the cells expressed Pax7, a unique marker of satellite cells. The mRNA of MyoD and Myogenin showed time-specific expression in the myogenesis induction process in vitro. CONCLUSION: Skeletal muscle satellite cells with high purity and strong differentiation ability can be obtained by means of tissue block culture method.

[Comparative proteomics study on lung tissue in early stage of burn-blast combined injury in rats].

OBJECTIVE: To explore changes in the proteomics of lung tissue in early stage of burn-blast combined injury in rats. METHODS: According to a random digital table, a total number of 18 Sprague Dawley (SD) rats were divided into burn-blast combined injury group (n=9) and blast injury group (n=9). Lung protein samples were collected at 6 h post injury. 2-DE was performed to separate proteins. After silver staining, the protein of differential expression were analyzed by PQ Quest and then identified by matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-TOF-MS). The features of the changes of proteomics of rat lung after burn-blast combined injury were studied by biological spectrometry, protein bank and reference article analysis technique. At same time, pathologic changes of the lung were monitored after injury. RESULTS: After removing death drain during the experiment, each group contained 8 rats and the results were analyzed statistically. Well focused and distinct 2-DE maps with good reproducibility were obtained, means of 736±47 and 782±30 protein spots were detected from the blast injury group and burn-blast combined injury group and the matching rates were 91%. From the two groups, 14 differential protein spots expressions were analyzed by MALDI-TOF-MS, of which 10 proteins were up-regulated and 4 proteins were down-regulated in burn-blast combined injury group. 12 different expression proteins were identified in the lung through 2-DE, mass spectrometry and protein date base, including heat shock 27 protein 1, heat shock 70 protein 1, carbonic anhydrase 2, cytochrome c oxidase, ATP synthase subunit alpha, Ca(2+) -transporting ATPase et al, which took part in stress reaction, metabolism, immune response and cytoskeleton. CONCLUSIONS: Burn-blast combined injury could induce dramatically changes of proteomics in lung tissue at early stage. The mechanism probably involves several proteins associated with oxidative stress, energy metabolism, immune response and cytoskeleton.

Association between PAI-1 polymorphisms and plasma PAI-1 level with sepsis in severely burned patients.

We investigated the association between plasminogen activator inhibitor-1 (PAI-1) polymorphisms and plasma PAI-1 level with sepsis in severely burned patients. A total of 182 patients with burn areas lager than 30% of the body surface area were enrolled in this study. Peripheral blood samples were obtained from 103 patients with sepsis (sepsis group) and 79 patients without sepsis (control group). An allele-specific polymerase chain reaction assay was used to determine PAI-1 polymorphism 4G/5G distribution. Plasma PAI-1 levels were detected using an enzyme-linked immunosorbent assay. The frequency of the 4G/4G genotype and the 4G allele frequency in the sepsis group were 42.7 and 62.1% respectively, which were significantly higher than those in the control group (P < 0.05). Sepsis patients had a significantly higher plasma PAI-1 level than the control group (P < 0.05). Compared with the 5G/5G genotype, PAI-1 concentrations were significantly higher in the 4G/4G genotype (P < 0.05). The study indicates that the 4G/5G promoter polymorphism of PAI-1 gene may be related to the susceptibility to burn sepsis and that the 4G/4G genotype may be an important genetic risk factor of burn sepsis. Additionally, PAI-1 concentrations in the serum are increased in patients with burn sepsis.

Safety of recombinant human granulocyte-macrophage colony-stimulating factor in healing pediatric severe burns.

We explored the safety of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for healing burns in children. Subjects were randomly assigned to two groups: the experimental group received external rhGM-CSF gel, and the control group received rhGM-CSF gel matrix components, applied to the burn surface. Neither group was given any other drugs that promote wound healing. Each day we recorded the pulse, body temperature, and respiration status in the two groups. We detected the blood routine, urine routine, and hepatic and renal function before the patients received drug treatment and after 72 h. The wound scab and healing states in the two groups were recorded every 4 days to evaluate wound healing rate and time taken for complete healing. Adverse reactions and their rate of occurrence were also recorded. The median time of healing was 15 days in the experimental group and 19 days in the control group (log-rank χ(2) = 5.139, P < 0.05). After 10 days, the experimental group healing rate was consistently higher than that of the control group (significantly different using intuitive analysis), suggesting the experimental group method was more effective. There were no obvious adverse reactions. There was no significant difference between the blood routine, urine routine, and liver and kidney function in the two groups before the treatment and after 3 days (P > 0.05). Compared with saline treatment of severe burns, rhGM-CSF can effectively shorten the healing time without significant adverse reactions, and is an effective and safe treatment for burns in children.

Preparation of microencapsulated VEGF gene-modified human umbilical cord mesenchymal stem cells and in vitro culture.

BACKGROUND AND OBJECTIVES: The application of microencapsulated stem cells has been shown to have many advantages in various fields of medical research. However, optimal modes for preparation of microencapsulate stem cells need to be improved, and expression and release of products of microencapsulated gene modified stem cells need to be studied in vitro. AIM OF THE STUDY: To explore the optimal parameters when preparing microencapsulated stem cells, and to investigate the effect of microencapsulation on growth, secretion, and metabolism of genetically modified human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs). MATERIALS AND METHODS: In this study, the parameters of preparation were regulated by observing the microcapsule shape and size. Live/dead cell viability kits and fluorescein isothiocyanate-labeled dextrans (FD) were used to detect the microencapsulated cell viability, and the permeability of microcapsules, respectively. Vascular endothelial growth factor (VEGF) production in the supernatant of microencapsulated and non-microencapsulated VEGF gene-modified hUCMSCs cultures was measured by ELISA. RESULTS: The optimal parameters of preparing microcapsules were regulated as followed: bolus velocity was 6 ml/h, and airflow velocity was 3 L/min. The morphology of microcapsules was a spherical structure with a diameter of 450 ± 30 µm. More than 90% of the cells were viable after 21 days of culture. Low and middle molecular weight FD was able to pass through the microcapsules; however, high molecular weight FD was not. Also, the VEGF concentration in microencapsulated and non-microencapsulated cell culture supernatants exhibited no significant difference at each time point. CONCLUSIONS: Microencapsulated stem cells can be ideally prepared via specifically regulated preparation. Lastly, microencapsulation does not alter growth, secretion, and metabolism of the genetically modified hUCMSCs.

Advances and opportunities for stem cell research in skin tissue engineering.

Skin tissue engineering has made significant progress over recent years, but there are still many factors that hamper its further development; these include the critical choice of seed cells. Many researchers eager to develop new cell-based skin products have focused on the use of stem cells, which have demonstrated many prospects for being put into clinical application. In this paper, we review the recent studies investigating the use of different tissue-derived stem cell as seed cells for skin tissue engineering.

Transplantation of microencapsulated cells expressing VEGF improves angiogenesis in implanted xenogeneic acellular dermis on wound.

BACKGROUND: Cell-based gene therapy using cells that express angiogenic factors is an alternative technique for therapeutic angiogenesis in transplantation of xenogeneic acellular dermis matrix (ADM). However, immune rejection is a substantial obstacle to implantation of genetically engineered allogeneic or xenogeneic cells. OBJECTIVE: To evaluate application of microencapsulated cells that express vascular endothelial growth factor (VEGF) in xenogeneic ADM transplants to improve wound angiogenesis and healing. MATERIALS AND METHODS: NIH3T3 cells were genetically modified to secrete VEGF and enveloped in semipermeable microcapsules. Microencapsulated VEGF-NIH3T3 cells were implanted in defects on the dorsa of guinea pigs with xenogeneic ADM and autologous split-thickness skin grafts. Cell structure and microencapsulation were observed at microscopy, and expression of VEGF was detected using an enzyme-linked immunosorbent assay (ELISA) and immunochemistry. Extent of angiogenesis in the ADM and the survival rate of the composite skin were evaluated after 2 weeks. In addition, expression of human VEGF and CD31 in the implanted acellular dermis was assessed, and microvessel density was calculated. RESULTS: Microencapsulated VEGF-expressing NIH3T3 cells were prepared successfully, and demonstrated proliferation and viability, and expressed VEGF both in vitro and in vivo. Extent of angiogenesis and survival rate of the composite skin containing the microencapsulated VEGF-expressing cells were significantly greater than in controls. Microencapsulated VEGF-expressing NIH3T3 cells augmented early angiogenesis in ADM implanted on wound and improved healing. CONCLUSION: Microencapsulated xenogeneic cell-based gene therapy may be a novel approach to therapeutic angiogenesis in transplantation of xenogeneic ADM skin.

(-)-Epigallocatechin gallate induces apoptosis, via caspase activation, in osteoclasts differentiated from RAW 264.7 cells.

BACKGROUND AND OBJECTIVE: Alveolar bone resorption is a characteristic feature of periodontal diseases and involves removal of both the mineral and the organic constituents of the bone matrix, a process mainly carried out by multinucleated osteoclast cells. (-)-Epigallocatechin gallate, the main constituent of green tea polyphenols, has been reported to induce the apoptotic cell death of osteoclasts and to modulate caspase activation in various tumor cells. In the present study, we investigated the inhibitory effect of (-)-epigallocatechin gallate on osteoclast survival and examined if (-)-epigallocatechin gallate mediates osteoclast apoptosis via caspase activation. MATERIAL AND METHODS: The effect of (-)-epigallocatechin gallate on osteoclast survival was examined by tartrate-resistant acid phosphatase (TRAP) staining in osteoclasts differentiated from RAW 264.7 cells. In addition, we evaluated the apoptosis of osteoclasts by (-)-epigallocatechin gallate using a DNA-fragmentation assay. Involvement of caspase in (-)-epigallocatechin gallate-mediated osteoclast apoptosis was evaluated by treatment with a general caspase inhibitor, Z-VAD-FMK. Moreover, the effect of (-)-epigallocatechin gallate on the activation of caspase-3 was assessed by a colorimetric activity assay and western blotting. RESULTS: (-)-Epigallocatechin gallate significantly inhibited, in a dose-dependent manner, the survival of osteoclasts differentiated from RAW 264.7 cells and induced the apoptosis of osteoclasts. Treatment with (-)-epigallocatechin gallate resulted in DNA fragmentation and induced the activation of caspase-3 in RAW 264.7 cell-derived osteoclasts. Additional treatment with Z-VAD-FMK suppressed these effects of (-)-epigallocatechin gallate. CONCLUSION: From these findings, we could suggest that (-)-epigallocatechin gallate might prevent alveolar bone resorption by inhibiting osteoclast survival through the caspase-mediated apoptosis.

Gender differences in tumor-induced anorectic feeding pattern in Fischer-344 rats.

Gender differences of feeding pattern in normal male and female rats are well recognized. Differences in gender-related feeding patterns have also been established following a variety of experimental manipulations, such as hypothalamic lesions, nicotine infusion, and total parenteral nutrition administration. Anorexia is a common feature during tumor growth. The present study examined whether the feeding indices constituting the feeding patterns differed with the development of cancer anorexia in male and female rats. Sixteen male and 15 female Fischer-344 rats had their food intake (FI) and feeding indices, meal number (MN) and meal size (MZ), continuously measured by a computerized rat eater meter. Viable methylcholanthrene (MCA) sarcoma cells (10(6)) were inoculated subcutaneously in 10 male (M-TB) and 8 female (F-TB) Fischer rats, while the rest were controls and received an equal volume of vehicle. Tumor-bearing (TB) rats became anorectic by Day 18, when the weight of the tumor was approximately 8% of the total body weight (BW). A notable decrease in BW was observed in both M-TB and F-TB. A decrease in FI resulted from different feeding indices between male and female rats. In male rats, lower FI was due to a decrease in both MN and MZ. In female rats, lower FI was solely due to a decrease in MN. The data show that gender differences in feeding patterns, which are an external manifestation of biochemical changes in the brain, occur following development of cancer-related anorexia suggesting that besides other factors, cancer anorexia is also influenced by sex-related hormones.

Effects of pretreatment clinical parameters on bioactive glass implantation in intrabony periodontal defects.

BACKGROUND: The various methods for regeneration of periodontal tissue that have been developed can be classified into guided tissue regeneration and bone implantation. Since the implantation materials have shown both deficiencies and merits, dentists have begun exploring the bioactive glass first used in plastic surgery. This paper examines the effectiveness of this new material on periodontal intrabony defects. METHODS: Clinical effects of bioactive glass implantation in intrabony periodontal defects were evaluated 6 months after surgery in 38 intrabony defects from 38 patients with chronic periodontitis. Twenty-one experimental defects received bioactive glass implantation (test group), while 17 control defects were treated with a flap procedure only (control group). The criteria for comparative observation were preoperative and postoperative probing depth (PD), clinical attachment level (CAL), bone probing depth (BPD), and gingival recession. RESULTS: Reductions in PD were observed in both groups (P<0.01). The reduction in PD was significantly greater in the test group when preoperative PD exceeded 7 mm (P<0.01). Improvements in CAL were also observed in both groups (P<0.01), with the test group showing significantly greater gains (P<0.05). In those cases where preoperative CAL was less than 7 mm, there was no statistically significant difference between the two groups. Reduction in BPD was observed in both groups, with the test group showing significantly greater reduction (P <0.01). There was no significant difference in BPD change, however, when preoperative BPD was < or =7 mm. Significantly greater reduction of BPD in the test group was observed when intrabony defect depth was >4 mm (P <0.05). Significant improvements in PD, CAL, and BPD were noted in the test group when the crestal involvement exceeded 100 degrees. Correlation test between various clinical parameters indicated that greater changes in PD and CAL in the test group were observed when preoperative CAL was large (P<0.001), and greater changes in PD (P<0.05), CAL (P<0.01), and BPD (P<0.05) were noted when preoperative BPD was large. Correlation between crestal involvement and CAL change was noted only in the control group (P<0.01). High correlations were observed between PD changes and CAL changes and between CAL changes and BPD changes in both groups. CONCLUSIONS: Use of a bone substitute in a flap operation resulted in significantly greater improvements in CAL and BPD over flap operation alone and seemed to have positive effects in postoperative PD, CAL, and BPD in those cases with more severe preoperative CAL and BPD.

[Effect of improved topical agents on healing time of deep second-degree burn wound].

OBJECTIVE: With the recognization of the mechanism of wound healing, some topical agents are created and applied in trauma to improve the healing rate of wounds. The main purpose of this study is to investigate the effect of some topical agents on the healing rate of deep second-degree burn wounds. METHODS: One thousand five hundred and sixty-three patients with deep second-degree burn wounds(total burn surface area < or = 10%) were involved in this study from January 1982 to December 1999. According to the application time of different treating measures including supplement of Zn, application of growth factors and collagenase, the patients were divided into 3 groups, wound healing rates were compared. RESULTS: Before 1991, none of special topical agents were used, and the healing time of deep second-degree burn wounds was(23.8 +/- 3.5) days. From 1991 to 1996, with the topical application of SD-Ag-Zn, which can provide Zn for cells taking part in wound healing, the healing time of deep second-degree burn wounds was (20.6 +/- 3.2) days, earlier than no special topical agents (P < 0.05). From 1997 to 1999, growth factors such as basic fibroblast growth factor(bFGF) and epithelial growth factor (EGF) and collagenases were applied in wound treatment combining with SD-Ag-Zn, wound healing time was (16.2 +/- 2.8) days, earlier than no special topical agents (P < 0.01) and simple SD-Ag-Zn application (P < 0.05). CONCLUSION: It indicates that the improvement of topical agents can accelerate wound healing speed.