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2021 Vol. 37, No. 12

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Expert Forum
New bioactive materials for promoting wound repair and skin regeneration
Lyu Guozhong, Zhao Peng
2021, 37(12): 1105-1109. doi: 10.3760/cma.j.cn501120-20211029-00373
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Abstract:
The modulation of microenvironment is a key technology towards promoting wound repair and skin regeneration. In recent years, a series of new bioactive materials that modulate the microenvironment and cell behaviors have been developed, demonstrating highly efficient capability of inducing wound repair and skin appendage regeneration. This article summarizes the research development of related new bioactive materials and their mechanisms of action.
Original Articles·Treatment of Special Wounds
Clinical effects of partially de-epithelized local flaps in repairing tubercular chest wall defects
Li Pengcheng, Jia Chiyu, Li Dongjie, Chen Liping, Zheng Mengli, Shen Chuan'an
2021, 37(12): 1110-1115. doi: 10.3760/cma.j.cn501120-20210929-00337
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Abstract:
  Objective  To explore the clinical effects of partially de-epithelized local flaps in repairing tubercular chest wall defects.  Methods  A retrospective observational study was conducted. From April 2010 to February 2021, twelve patients who met the inclusion criteria were admitted to the Department of Burns and Plastic Surgery of the Eighth Medical Center of PLA General Hospital, including 9 males and 3 females with age of (42±18) years. The sizes of tubercular chest wall defects of patients were ranged from 4 cm×3 cm×2 cm to 16 cm×8 cm×5 cm, which were all repaired with partial de-epithelized local flaps. The widths of flaps were equal to the widths of the defects, and the lengths of flaps were 2 cm longer than those of the defects. In one patient, the local flap was too large to close the donor site directly by suturing, so an autologous back free medium thickness skin graft was used for repair. In other patients, the collection areas of local flaps were small, and the donor areas of flaps were directly closed. The duration of operation, intraoperative bleeding, and postoperative drainage volume and indwelling time of drainage tube were observed and recorded. In two weeks after operation, the survival, color, and texture of flaps, the presence of subcutaneous hydrops and skin ulcer, and donor site healing including wound disruption, local infection, hematoma were observed. Chest X-ray, CT scan, or nuclear magnetic resonance imaging was performed in one month after operation to check whether new local hydrops and bone destruction occurred in the chest wall defects and the concomitant tuberculose focus of patients. All patients were followed up for more than 6 months to record whether the surgical incisions of the chest wall defects of the patients were complicated by hypertrophic scar, redness, swelling, and sinus.  Results  In surgery, the patient had (104±18) min of operation duration, (119±53) mL of intraoperative bleeding, (134±49) mL of cumulative drainage of drainage tube, and (5.3±1.7) days of drainage tube indwelling time. In two weeks after operation, all the grafted local flaps survived, and the color and texture of flaps were similar to the surrounding normal skin. One patient had fluid leakage from the incision of chest wall defect area with the incision partially dehisced, which healed well after a phase Ⅱ operation; no wound infection, subcutaneous hydrops, or wound rupture occurred in other patients. The incisions of donor sites in all the patients healed well and no wound disruption, local infection, or hematoma occurred. One month after operation, no new bone destruction was observed in the operative region by chest imaging examination. Patients were followed up for 6 to 96 months, with one patient having wound swelling, ulceration, and sinus in the operative area of the chest wall defect in 12 months after surgery, which healed after phase Ⅱ operation; the incisions of chest wall defect wounds in other patients healed well and had no scar, redness and swelling, or sinus.  Conclusions  Partially de-epithelized local flap could be used in repairing tubercular chest wall defect wounds, with the advantages of flexible flap design, minimal donor site injury, and good postoperative wound healing.
Clinical effects of flaps with cervical cutaneous branch of transverse cervical artery in repairing neck radiation ulcers
Li Gang, Zhang Zhi, Li Yeyang, Wang Jinlun
2021, 37(12): 1116-1121. doi: 10.3760/cma.j.cn501120-20200807-00371
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Abstract:
    Objective   To explore the clinical effects of flaps with cervical cutaneous branch of transverse cervical artery in repairing neck radiation ulcer.    Methods   The retrospective observational research was conducted. From January 2016 to December 2019, 8 cases with neck radiation ulcer were admitted to Guangzhou Red Cross Hospital of Jinan University and repaired with flaps based on cervical cutaneous branch of transverse cervical artery. There were 6 males and 2 females, aged 52-75 years. The ulcers occurred 14.5 years after radiotherapy on average, with ulcer areas of 5.0 cm×3.0 cm-7.0 cm×6.0 cm on admission. The wound areas were ranged from 6.0 cm×5.0 cm to 13.0 cm×6.5 cm after ulcers and fibrotic tissue around were resected. Seven cases underwent resection of flaps and wound repair operation on the first stage, and the other 1 case underwent pre-expansion of flap donor area on the first stage and resection of flap and wound repair operation on the second stage, with flap sizes of 8.0 cm×7.0 cm-15.0 cm×8.5 cm. The wounds in the donor areas of flaps in 7 patients were sutured directly, and the wound in the donor area of flap in the other 1 patient was repaired with thin split-thickness skin graft from thigh after being sutured partially. The preoperative ulcer tissue was collected for pathological examination, and the postoperative survival of the flaps and healing of the flap donor areas were observed. The flaps, the recurrence of the neck ulcers and neck function, and the scar hyperplasia in the donor areas of flaps were observed during follow-up.    Results   Preoperative pathological examination of ulcer tissue showed that full-thickness necrosis occurred in ulcer skin, obvious fibrotic tissue hyperplasia, collagenization, and small-scale calcification in the base and surrounding tissue of the ulcers, and a large amount of chronic inflammatory cells and a small amount of acute inflammatory cells infiltration were observed in intercellular substance, which excluded the recurrence of tumor. All the flaps in 8 cases survived, the wounds were repaired effectively, and the postoperative donor areas of flaps healed well. During postoperative follow-up of 6-24 months, the flaps had good appearances without recurrence of ulcer, the movement function of neck was significantly improved, and no obvious scar hyperplasia was observed in the donor areas of flaps.    Conclusions   Radiation ulcer in the neck is a serious long-term complication of neck after radiotherapy, which is difficult to heal with conservative treatment. The flap with cervical cutaneous branch of transverse cervical artery is close to the neck, with rich blood supply, constant anatomy, and is easy to cut. Neck radiation ulcers treated with the flaps showed good wound healing and improved functions, with no obvious scar hyperplasia.
Original Articles
Prospective study on the analysis of intestinal microflora changes and prediction on metabolic function in severe burn patients at early stage by 16S ribosomal RNA high-throughput sequencing
Guo Zaiwen, Song Mingming, Zhang Jie, Liu Lu, Yang Yunxi, Shao Yiming, Li Linbin, Sun Bingwei
2021, 37(12): 1122-1129. doi: 10.3760/cma.j.cn501120-20200916-00414
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Abstract:
  Objective  To analyze the changes of intestinal microflora and to predict the metabolic function of intestinal microflora in severe burn patients at early stage by 16S ribosomal RNA (rRNA) high-throughput sequencing.  Methods  In this prospective observational study, 48 patients with severe burns who met the inclusion criteria were admitted to Department of Burns and Plastic Surgery of Affiliated Hospital of Jiangsu University from January 2018 to December 2019 were included in burn group, and 40 healthy volunteers who met the inclusion criteria and underwent physical examination at the Physical Examination Center of Affiliated Hospital of Jiangsu University in the same period were included in healthy group. Fecal samples were collected from patients in burn group in about 1 week after admission and from volunteers in healthy group on the day of physical examination. The 16S rRNA V4 gene sequencing was performed in the feces of patients in burn group and volunteers in healthy group to analyze the relative abundance of various bacteria. The operational classification unit (OTU) was divided by Mothur software to analyze the dominant bacteria. The OTU number, Chao1 index, Ace index, and Shannon index of fecal microflora were analyzed by QIIME1.9.0 software. The principal component analysis for relative abundance of fecal microflora was performed by Canoco Software 5.0. The metabolic function of fecal microflora was predicted by Kyoto Encyclopedia of Genes and Genomes. Data were statistically analyzed with independent sample t test, and Mann-Whitney U test, and Bonferroni correction.  Results  The relative abundance of Bacteroides, Enterococcus, Acinetobacter, Macrococcus, and Staphylococcus in feces of patients in burn group was significantly higher than that of volunteers in healthy group (Z=-5.20, -2.37, -5.17, -4.41, -6.03, P<0.05 or P<0.01), and the relative abundance of unclassified-Helicobacillae, Prevotella, Cecobacteria, unclassified-Rumencocci, Pseudobutyrivibrio, Brautia, and unclassified-Digiestive Streptococcaceae (Z=-8.03, -3.21, -7.63, -5.88, -8.05, -8.05, -6.77, P<0.01) and other 12 species of bacteria in the feces of volunteers in healthy group was significantly higher than that of patients in burn group. The diversity of fecal microflora of volunteers in healthy group was better than that of patients in burn group, the main dominant microflora of volunteers in healthy group were Bacteroides, unclassified-Helicobacillae, Prevotella, unclassified- Enterobacteriaceae, Brautia, Parabacteroides, Escherichia coli, etc., and the main dominant microflora of patients in burn group were Bacteroides, Prevotella, unclassified-Enterobacteriaceae, and Parabacteroides. The OTU number, Ace index, Chao1 index, and Shannon index of fecal microflora of patients in burn group were 149±47, 199±45, 190±45, 2.0±0.9, which were significantly lower than 266±57, 323±51, 318±51, 3.8±0.5 of volunteers in healthy group (t=10.325, 11.972, 12.224, 11.662, P<0.01). The relative abundance of fecal microflora of patients in burn group and volunteers in healthy group was clearly divided into two groups by principal component 1, and the contribution rate of principal component 1 was 32.50%, P<0.01. The fecal microflora of volunteers in healthy group were more concentrated on principal component 2, the fecal microflora of patients in burn group were dispersed in principal component 2, and the contribution rate of principal component 2 was 13.44%, P>0.05. The metabolic levels of alanine-aspartate-glutamate, arginine- proline, cysteine-methionine, glycine-serine-threonine, phenylalanine, tryptophan, and tyrosine in amino acid, tricarboxylic acid cycle, glucose and mannose, galactolipin, glycolysis/gluconiogenesis, starch and sucrose in carbohydrate of fecal microflora of patients in burn group were significantly lower than those of volunteers in healthy group (Z=-4.75, -4.54, -4.75, -4.62, -3.71, -3.28, -4.19, -3.82, -4.72, -4.35, -4.75, -4.71, P<0.01). The levels of lipoic acid metabolism and coenzyme Q synthesis of fecal microflora of patients in burn group were significantly higher than those of volunteers in healthy group (Z=-6.07, -4.51, P<0.01). The metabolic level of arachidonic acid of fecal microflora of patients in burn group was similar to that of volunteers in healthy group (P>0.05).  Conclusions  There are significant differences in intestinal microflora between severe burn patients at the early stage and healthy people, and the species and diversity of microflora are decreased, and the nutrient metabolism level is decreased in burn patients by 16S rRNA high-throughput sequencing.
A prospective randomized controlled study of the effectiveness of artificial dermis combined with split-thickness skin for repairing wounds with bone and tendon exposure in hands and feet
Di Haiping, Mu Xinling, Shi Jijing, Xue Jidong, Liu Lei, Guo Haina, Xing Peipeng, Xia Chengde
2021, 37(12): 1130-1136. doi: 10.3760/cma.j.cn501120-20210325-00103
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Abstract:
    Objective   To explore the clinical effects of artificial dermis combined with split-thickness skin for repairing wounds with bone and tendon exposure in hands and feet.    Methods   A prospective randomized controlled study was conducted. From October 2018 to February 2020, 82 patients with bone and tendon exposed wounds in hands and feet admitted to the Department of Burns of Zhengzhou First People′s Hospital who met the inclusion criteria were selected. All the patients were divided into flap group (41 cases, including 27 males and 14 females) and artificial dermis+split-thickness skin group (41 cases, including 29 males and 12 females) according to the random number table, with age of (37±7) years. After complete debridement of wounds of patients in the two groups, the wounds of patients in flap group were transplanted with anterolateral femoral free flaps; the wounds of patients in artificial dermis+split-thickness skin group were grafted with artificial dermis with continuous negative pressure suction applied, and then grafted with split-thickness skin from autologous lateral thigh once the vascularization of artificial dermis was completed. One week after autologous skin graft/flap grafting, the survival of wound graft was observed and the graft survival rate was calculated. The complete wound healing time, number of operation, length of hospital stay, hospitalization cost, and the occurrence of surgery-related complications during hospitalization after autologous skin graft/flap grafting were recorded, and the incidence of complications was calculated. Six months after autologous skin graft/flap grafting, the scar hyperplasia of recipient area was evaluated by Vancouver Scar Scale (VSS), while the recovery of hand and foot function was evaluated by Total Action Mobility (TAM) System Rating method and American Orthopaedic Foot and Ankle Society Ankle and Hindfoot Function Scale (AOFAS-AHS), respectively. Data were statistically analyzed with chi-square test, Fisher's exact probability test, and independent sample t test.    Results   One week after autologous skin graft/flap grafting, the survival rates of wound grafts were similar in the two groups (P>0.05). The complete wound healing time and length of hospital stay were (29±5) and (35±5) d for patients in artificial dermis+split-thickness skin group, respectively, which were significantly longer than (22±4) and (28±5) d in flap group (t=6.96, 6.22, P<0.01). Compared with those in flap group, the number of operations was fewer (t=7.39, P<0.01), the incidence of surgery-related complications during hospitalization after autologous skin graft/flap grafting was lower (P<0.01), but there was no significant change in hospitalization cost of patients in artificial dermis+split-thickness skin group (P>0.05). Six months after autologous skin graft/flap grafting, the VSS scores of recipient area of patients in the two groups were similar (t=0.32, P>0.05); the TAM score of hand function and AOFAS-AHS score of foot function of patients in artificial dermis+split-thickness skin group were 40±6 and 62±12, respectively, which were significantly higher than 34±6 and 53±11 of flap group (t=4.66, 3.41, P<0.01).    Conclusions   The combined application of artificial dermis and split-thickness skin results in fewer number of operation compared with using flaps in the repair of wounds with bone and tendon exposure in hands and feet, reducing the incidence of surgery-related complications and improving the postoperative hand and foot joint function of patients, without significant scar hyperplasia, although it may also prolong the wound healing time and length of hospital stay accordingly.
Effects of regional citrate anticoagulation in continuous veno-venous hemofiltration of severe burn patients
Wang Zhiyong, Feng Shihai, Fan Baoli, Ma Wei, Jia Xiangcheng, Geng Hui
2021, 37(12): 1137-1142. doi: 10.3760/cma.j.cn501120-20200816-00381
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Abstract:
  Objective  To investigate the effects of regional citrate anticoagulation in continuous veno-venous hemofiltration (CVVH) of severe burn patients.  Methods  A retrospective non-randomized controlled study was conducted. From January 2017 to August 2020, sixty-eight severe burn patients who met the inclusion criteria were treated with CVVH in Affiliated Hospital of Nankai University. According to the different methods of blood anticoagulation in CVVH treatment, patients were divided into citrate group (n=40) and heparin group (n=28). In the citrate group, 32 males and 8 females were (40±18) years old with total burn area of (62±14)% total body surface area (TBSA); in the heparin group, 22 males and 6 females were (38±16) years old with total burn area of (57±20)%TBSA. Creatinine level, C-reactive protein (CRP) value, and urea nitrogen level in serum of patients were recorded at 0 (immediately), 48, and 96 h after CVVH treatment in 2 groups, urea clearance index was calculated based on urea nitrogen level at 0, 48, and 96 h after CVVH treatment in 2 groups, platelet count (PLT), prothrombin time (PT), and activated partial thromboplastin time (APTT) in total coagulation of patients were recorded. The frequency of forced hemofiltration termination caused by adverse reactions such as severe hypocalcemia, aggravated wound bleeding, and new bleeding on non-wound surface of patients was recorded within 96 h of CVVH treatment. The duration of daily CVVH use from the beginning to the end was recorded. Data were statistically analyzed with chi-square test, analysis of variance for repeated measurement, independent samples t test, and Bonferroni correction.  Results  There were no significant differences in urea nitrogen level, creatinine level, and CRP value in serum of patients between 2 groups at 0 h after treatment (P>0.05). At 48 and 96 h after treatment, urea nitrogen level, creatinine level, and CRP value in serum of patients in citrate group were significantly lower than those in heparin group (t=3.366, -2.315, 2.942, -2.657, 2.011, -2.441, P<0.05), and urea clearance index of patients in citrate group was significantly higher than that in heparin group (t=1.017, 2.233, P<0.05). There were no statistically significant differences in PLT, PT, and APTT of patients between 2 groups at 0 h after treatment (P>0.05). At 48 and 96 h, PLT of patients in citrate group was significantly higher than that in heparin group (t=-3.417, -4.143, P<0.05 or P<0.01), PT of patients in citrate group was significantly shorter than that in heparin group (t=2.760, -3.655, P<0.01), APTT of patients in citrate group was significantly shorter than that in heparin group (t=3.719, 5.146, P<0.05 or P<0.01). Within 96 h of treatment, there was 1 case of hypocalcemia and 1 case of aggravated wound bleeding resulting in forced hemofiltration termination in citrate group, but there was no new bleeding on non-wound surface; in heparin group, there was no hypocalcemia, but 7 cases of aggravated wound bleeding and 2 cases of new bleeding on non-wound surface (both at the tracheotomy site) resulting in forced hemofiltration termination. The use time of blood purification filter of patients in citrate group was (11.7±4.8) h, obviously longer than (6.6±2.5) h in heparin group (t=3.310, P<0.01).  Conclusions  The use of regional citrate anticoagulation in CVVH treatment of severe burn patients has the advantages including little effect on coagulation function and high safety, can effectively prolong the use time of filter and improve the therapeutic effect, but this conclusion still needs to be further verified in clinical application.
Clinical effects of transplantation of turbocharged bipedicle deep inferior epigastric perforator flap in breast reconstruction
Song Dajiang, Li Zan, Zhang Yixin, Zhou Bo, Lyu Chunliu, Tang Yuanyuan, Yi Liang, Luo Zhenhua, Wang Zhiyuan, Hua Zhanqiang, Feng Guang
2021, 37(12): 1143-1148. doi: 10.3760/cma.j.cn501120-20200824-00390
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Abstract:
    Objective   To explore the clinical effects of transplantation of turbocharged bipedicle deep inferior epigastric perforator (DIEP) flap in breast reconstruction.    Methods   A retrospective observational study was used. From December 2008 to December 2016, 24 patients who met the inclusion criteria were treated in the Department of Plastic Surgery of Hunan Cancer Hospital, all patients were female, aged 28-51 (36.5±1.6) years. All cases received turbocharged bipedicle DIEP flap for two-staged breast reconstruction. According to the patterns of turbocharged vessels anastomosis, the turbocharged bipedicle DIEP flaps with length of (27.5±0.3) cm and width of (12.8±1.4) cm, were divided into three types: distal end of pedicle anastomosis type, main branch of pedicle anastomosis type, and muscular branch of pedicle anastomosis type. After complete hemostasis in the donor region, the anterior sheath was repaired with intermittent suture, and umbilical reconstruction was completed. Two negative pressure drainage tubes were indwelled, and subcutaneous tissue and skin were sutured layer by layer. The specific ways of vascular anastomosis of the flap pedicle with the internal thoracic vessels of recipient site included anastomosing the proximal end of one artery and one vein, anastomosing the proximal and distal end of one artery and one vein, and anastomosing the proximal end of one artery and two veins. Postoperatively, the survival and blood supply of flaps were observed. The patients were followed up to observe the reconstructed breast shape satisfaction, donor site complications, abdominal wall function, and scar hyperplasia.    Results   All turbocharged bipedicle DIEP flaps for two-staged breast reconstruction survived well, with good blood supply. During follow-up for 14 to 56 (20±6) months, the shape of reconstructed breasts was satisfied. Only linear scar was left in the donor sites of abdomen with no complications, and the function of abdominal wall was not affected.    Conclusions   For patients with clear indications, transplantation of free turbocharged bipedicle DIEP flap is a safe, reliable, and satisfactory choice for breast reconstruction with autologous tissue.
Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat
Lyu Shangjun, Fan Ronghui, Wu Dan, Peng Xi
2021, 37(12): 1149-1157. doi: 10.3760/cma.j.cn501120-20210601-00208
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Abstract:
  Objective  To investigate the effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat (hereinafter referred to as burn serum).  Methods  The experimental research method was applied. Ten gender equally distributed Wistar rats aged 7-8 months were taken to prepare normal rat serum (hereinafter referred to as normal serum), another twenty gender equally distributed Wistar rats aged 7-8 months were taken to prepare burn serum after full- thickness burn injury of 30% total body surface area, and primary cardiomyocytes were isolated and cultured from 180 Wistar rats aged 1-3 days by either gender and used in the following experiments. The cells were divided into normal serum group and burn serum group according to the random number table (the same grouping method below) and cultured with the corresponding serum. At post culture hour (PCH) 1, 3, 6, 9, and 12, trypanosoma blue test was used to detect the cell survival rate. The cells were divided into burn serum alone group, burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group, which were treated with burn serum alone or burn serum added with the corresponding final molarity of glutamine and cultured for the time screened in the experiment before, and then the cell survival rate was detected as before. The cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group and treated the same as before. After 30 min of culture, phosphorylation levels of mammalian target of rapamycin complex 1 (mTORC1), p70 ribosomal protein S6 kinase (p70 S6K), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group, and treated correspondingly. At PCH 1, 3, and 6, the expressions of heat shock protein 70 (HSP70) and metallothionein (MT), and the morphology of microtubule were determined with immunofluorescence method. The sample numbers in each index at each time point in each group were all 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference t test, least significant difference test, and Bonferroni correction.  Results  At PCH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (t=4.950, 16.752, 35.484, 34.428, 27.781, P<0.01). Compared within the group at PCH 1, the cell survival rate was significantly decreased in burn serum group at PCH 3, 6, 9, and 12 (P<0.05). Compared within the group at PCH 3, the cell survival rate was significantly decreased in burn serum group at PCH 6, 9, and 12 (P<0.05). Compared within the group at PCH 6 and 9, the cell survival rate was significantly decreased in burn serum group at PCH 12 (P<0.05). There were no statistically significant differences in the cell survival rates in burn serum group between PCH 6 and 9 (P>0.05). Thus PCH 6 was selected as the subsequent intervention time of burn serum. At PCH 6, compared with burn serum alone group, the cell survival rates in burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group were significantly increased (P<0.01). There were no statistically significant differences in cell survival rates between burn serum+12 mmol/L glutamine group and burn serum+16 mmol/L glutamine group (P>0.05). There were no statistically significant differences in cell survival rates in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P>0.05). Thus 12, 16, and 20 mmol/L were selected as the subsequent intervention concentrations of glutamine. After 30 min of culture, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were respectively 1.001±0.042, 0.510±0.024, 0.876±0.022, 0.836±0.074, 0.856±0.041, 1.00±0.11, 0.38±0.09, 0.95±0.13, 0.96±0.13, 0.89±0.24, 1.00±0.07, 0.29±0.08, 0.87±0.27, 0.68±0.08, 0.60±0.21 in normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group. Compared with normal serum group, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were significantly decreased in the other 4 burn serum groups (P<0.01). Compared with those of burn serum alone group, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were significantly increased in the other 3 burn serum groups (P<0.01). The phosphorylation level of 4E-BP1 of cells in burn serum+12 mmol/L glutamine group was significantly higher than the levels in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P<0.05). The expression of MT of cells in burn serum alone group was significantly lower than that in normal serum group at PCH 1 (P<0.05), while the expressions of MT of cells in burn serum alone group were significantly higher than those in normal serum group at the other time points (P<0.05). At PCH 1, 3, and 6, the expressions of HSP70 of cells in burn serum alone group were significantly higher than those in normal serum group (P<0.05), the expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine group were significantly higher than those in burn serum alone group (P<0.05), and the expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group were significantly lower than those in burn serum+12 mmol/L glutamine group (P<0.01). The microtubular structures were intact, displaying grid alinement and uniform staining in cells of normal serum group at PCH 1, 3, and 6. In burn serum alone group, some microtubules showed fracture and irregular grid arrangement at PCH 1; the microtubular structures near the nucleus were clear, while the microtubules at the distal end of the nucleus were blurry at PCH 3; the microtubular structures were blurry at PCH 6. The microtubular damage of cells was alleviated in burn serum+12 mmol/L glutamine group as compared with that in burn serum alone group at each time point of culture. The morphology of microtubules of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group at each time point of culture was similar to that of burn serum alone group.  Conclusions  The burn serum can lead to damages to cardiomyocytes and significant decrease of cell survival rate in rats. Glutamine can exert cell protective function through the regulation of mTOR/p70 S6K/4E-BP1 signaling pathway, thus promoting the expressions of HSP70 and MT and stabilizing the microtubule structures.
Effects of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor complex on avulsion flap with roll compaction in rat
Wu Jinfang, Hong Xudong, Jin Jian, Fei Yanghonghong, Zhang Mengyuan, Si Tingting, Fan Hao, Zhang Xudong
2021, 37(12): 1158-1165. doi: 10.3760/cma.j.cn501120-20200914-00409
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Abstract:
    Objective   To investigate the effect of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor (rTFPI) complex on avulsion flap with roll compaction in rat.    Methods   The experimental methods were adopted. The N-trimethyl chitosan-rTFPI complex solution was prepared by ion cross-linking method. The morphology of the complex was observed by scanning electron microscope, and its diameter was measured. The encapsulation rate of rTFPI in the complex and drug loading rate of the complex was determined and calculated by enzyme-linked immunosorbent assay (ELISA) method (n=3). The concentration of rTFPI in the solution at 0, 10, 30, 45, 60, 90, 120, 240 minutes of storage was measured by ELISA method to observe the release of rTFPI, and its half-life was calculated (n=3). Twenty-four 6-week-old male Sprague-Dawley rats were divided into phosphate buffered saline (PBS) group, N-trimethyl chitosan alone group, rTFPI alone group, and N-trimethyl chitosan-rTFPI group according to the random number table, with 6 rats in each group. The avulsion flaps with roll compaction were prepared on the backs of rats with pedicles located on the line of the bilateral iliac spine and lifted from the surface of the muscle membrane. One injection of corresponding reagents was carried out immediately after in-situ suture and on post operation day (POD) 1, 2, and 3. General changes of the flap were observed on POD 1, 3, and 7. On POD 7, the survival area of the flap was measured and the survival rate of the flap was calculated; the flaps were divided into pedicle, proximal, middle, and distal segments, and the blood perfusion in the proximal, middle, and distal segment tissue of the flap was detected by the laser speckle blood flow imager; tissue samples in the middle of the flap were cut and stained with hematoxylin and eosin to observe the changes in tissue structure and the infiltration of inflammatory cells, and the numbers of embolized blood vessels and new blood vessels per 100 times visual field were counted. Data were statistically analyzed with one-way analysis of variance and least significant difference test.    Results   The N-trimethyl chitosan-rTFPI complex had an irregular spherical structure with a diameter of 150-200 nm. The encapsulation rate of rTFPI in the complex and drug loading rate of the complex were (88.7±2.1)% and (2.83±0.09)%, respectively. The concentration of rTFPI in the solution of the N-trimethyl chitosan-rTFPI complex gradually increased with prolonged storage time, and the release was basically stable at 90 min, with half-life of (651±36) min. On POD 1, the distal parts of flaps of rats in N-trimethyl chitosan alone group darkened significantly. On POD 3, scabs and necrosis were relatively mild on the distal segment of the flaps of rats in rTFPI alone group and N-trimethyl chitosan-rTFPI group as compared with those of the other two groups. On POD 7, the necrosis boundaries of the flaps of rats in each group were clear. On POD 7, the flap survival rates of rats in rTFPI alone group and N-trimethyl chitosan-rTFPI group were (63±7)% and (73±5)%, respectively, which were significantly higher than (41±3)% in PBS group and (52±7)% in N-trimethyl chitosan alone group. Moreover, the flap survival rate of rats in N-trimethyl chitosan-rTFPI group was significantly higher than that in rTFPI alone group (P<0.05). On POD 7, the flaps of rats in each group had blood perfusion; the blood perfusion values in the proximal segment tissue of the rat flaps in N-trimethyl chitosan alone group and the blood perfusion values in the proximal, middle, and distal segment tissue of the rat flaps in rTFPI alone group and N-trimethyl chitosan-rTFPI group were significantly higher than those in PBS group (P<0.05 or P<0.01); the blood perfusion values in the distal segment tissue of the rat flaps in rTFPI alone group and the blood perfusion values in the middle and distal segment tissue of the rat flaps in N-trimethyl chitosan-rTFPI group were significantly higher than those in N-trimethyl chitosan alone group (P<0.05 or P<0.01); the blood perfusion value in the middle segment tissue of the rat flaps in N-trimethyl chitosan-rTFPI group was significantly higher than that in rTFPI alone group (P<0.01). On POD 7, inflammatory cells infiltrated more and cell edema was obvious in the middle segment tissue of the rat flaps in PBS group and N-trimethyl chitosan alone group. Compared with those of the previous two groups, the inflammation degrees in the middle segment tissue of the rat flaps in rTFPI alone group and N-trimethyl chitosan-rTFPI group were significantly milder, the number of embolized blood vessels was significantly decreased (P<0.05 or P<0.01), and the number of new blood vessels was significantly increased (P<0.05 or P<0.01). Compared with that of rTFPI alone group, the number of new blood vessels in the middle segment tissue of the rat flaps in N-trimethyl chitosan-rTFPI group increased significantly (P<0.05).    Conclusions   The effect of sustained release of rTFPI can be achieved by loading rTFPI with N-trimethyl chitosan. Compared with rTFPI alone, the N-trimethyl chitosan-rTFPI complex can further improve the blood perfusion of the avulsion flaps with roll compaction in rat and improve the survival rate of the flap.
Effects of temperature-sensitive hydroxybutyl chitosan hydrogel on wound healing of full-thickness skin defect in rats
Chen Axin, Chen Youbai, Jiang Yufeng, Han Yan
2021, 37(12): 1166-1174. doi: 10.3760/cma.j.cn501120-20200927-00424
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Abstract:
      Objective     To investigate the effects of temperature-sensitive hydroxybutyl chitosan hydrogel on wound healing of full-thickness skin defect in rats.      Methods     The experimental research method was used. Fifty-one no matter male or female Sprague-Dawley rats aged 7-10 weeks were selected, and two round full-thickness skin defect wounds with a diameter of 2 cm were created on the back of each rat at a distance about 1.0 cm to the spine. The rats were divided into temperature-sensitive hydrogel group, gel group, and blank control group according to the random number table, with 17 rats and 34 wounds in each group. Wounds of rats in the first two groups were applied respectively with 0.3 mL temperature-sensitive hydroxybutyl chitosan hydrogel and carboxymethyl chitosan hydrogel immediately after injury, and the wounds of rats in blank control group received no treatment. The wounds of rats in the three groups were all covered with vaseline oil gauze. The states of temperature-sensitive hydroxybutyl chitosan hydrogel in wounds of rats in temperature-sensitive hydrogel group and carboxymethyl chitosan hydrogel in wounds of rats in gel group were observed every day when the dressings were changed, and the difficulty of vaseline oil gauze removal was recorded. On the 3rd, 7th, 10th, 14th, and 21st day after injury, the wound healing of rats in the three groups was observed and the wound healing rates were calculated. On the 3rd, 7th, 10th, 14th, and 21st day after injury, tissue from 4 wounds of 2 rats in each group was collected for the following observation and detection. The infiltration of inflammatory cells, angiogenesis, and re-epithelialization were observed by hematoxylin eosin staining. The regeneration and remodeling of collagen fibers were observed by Masson staining, and the collagen volume fraction was calculated. The expressions of interleukin-6 (IL-6), transforming growth factor β1 (TGF-β1), and matrix metalloproteinase-1 (MMP-1) were detected by enzyme-linked immunosorbent assay method. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Bonferroni test.      Results     The carboxymethyl chitosan gel in wounds of rats in gel group was liquid gel and could flow with the body position, while the temperature-sensitive hydroxybutyl chitosan hydrogel in wounds of rats in temperature-sensitive hydrogel group was solid gel and could not flow with the body position, and the distribution of the latter was more uniform. The vaseline oil gauzes were easily removed in wounds of rats in temperature-sensitive hydrogel group, while the vaseline oil gauzes were difficult to remove in the other two groups. On the 3rd, 7th, 10th, 14th, and 21st day after injury, the wound granulation tissue of rats grew well in temperature-sensitive hydrogel group and gel group, with no obvious infection, and two rats in blank control group died of wound infection on the 3rd and 5th day after injury. On the 7th, 10th, 14th, and 21st day after injury, the wound healing rates of rats in temperature-sensitive hydrogel group and gel group were significantly higher than that in blank control group (P<0.01). On the 10th day after injury, the wound healing rate of rats in temperature-sensitive hydrogel group was significantly higher than that in gel group (P<0.05). A large number of neutrophils and lymphocytes infiltrated into the wounds of rats in the three groups on the 3rd day after injury. The infiltration of inflammatory cells  was gradually reduced and the wound healed gradually in rats of temperature-sensitive hydrogel group and gel group from the 7th to 21st day after injury, and the epidermis and dermis could be seen, without hair follicles and other skin appendages. The wounds of rats in blank control group did not heal completely on 21st day after injury. From the 3rd to 10th day after injury, the newly formed collagen fibers increased gradually in the wounds of rats in the three groups. On the 14th and 21st day after injury, the collagen fibers in the wounds of rats in temperature-sensitive hydrogel group and gel group were denser and more orderly than those in blank control group. On the 10th, 14th, and 21st day after injury, the collagen volume fraction of wounds of rats in temperature-sensitive hydrogel group and gel group was significantly higher than that in blank control group (P<0.01). On the 14th day after injury, the collagen volume fraction of wounds of rats in temperature-sensitive hydrogel group was significantly higher than that in gel group (P<0.01). On the 3rd, 7th, and 10th day after injury, the expressions of IL-6 in wounds of rats in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01), and the expressions of IL-6 in wounds of rats in gel group were significantly lower than those in blank control group (P<0.01). On the 3rd, 7th, and 10th day after injury, the expressions of TGF-β1 in wounds of rats in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01). The expressions of TGF-β1 in wounds of rats in gel group were significantly lower than those in blank control group on the 3rd and 7th day after injury (P<0.01), and the expression of TGF-β1 in wounds of rats in gel group was significantly higher than that in blank control group on the 10th day after injury (P<0.01). On the 14th day after injury, the expression of TGF-β1  in wounds of rats in gel group was significantly higher than that in temperature-sensitive hydrogel group and blank control group (P<0.01). On the 21st day after injury, the expression of TGF-β1 in wounds of rats in temperature-sensitive hydrogel group was significantly lower than that in gel group and blank control group (P<0.01), and the expression of TGF-β1 in wounds of rats in gel group was significantly lower than that in blank control group (P<0.01). On the 7th day after injury, the expression of MMP-1 in wounds of rats in gel group was significantly higher than that in temperature-sensitive hydrogel group and blank control group (P<0.01). On the 10th, 14th, and 21st day after injury, the expressions of MMP-1 in wounds of rats in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01). On the 10th day after injury, the expression of MMP-1 in wounds of rats in gel group was significantly lower than that in blank control group (P<0.01). On the 14th and 21st day after injury, the expressions of MMP-1 in wounds of rats in gel group were significantly higher than those in blank control group (P<0.01).      Conclusions     Temperature-sensitive hydroxybutyl chitosan hydrogel can promote the healing of full-thickness skin defect wounds in rats by increasing the expressions of IL-6, TGF-β1, and MMP-1, regulating the wound healing environment, inhibiting inflammatory reaction, improving the strength of tissue repair, and promoting collagen synthesis or decomposition
The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment
Wei Pei, Xu Zhaorong, Chen Yimin, Chen Xiaodong, Chen Zhaohong
2021, 37(12): 1175-1184. doi: 10.3760/cma.j.cn501120-20210424-00154
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Abstract:
  Objective  To investigate the effect and mechanism of exosomes derived from human amniotic epithelial cells (hAEC-Exos) on the proliferation and migration of HaCaT in high glucose environment.  Methods  The experimental research method was adopted. The amniotic membrane tissue was collected from 10 healthy pregnant women at full term delivery in the Department of Obstetrics and Gynecology of Fujian Medical University Union Hospital from January to June 2019, and the primary human amniotic epithelial cells (hAECs) were isolated. The growth status and morphological changes of the primary hAECs on the 2nd, 4th, and 7th day of culture were observed, and the expressions of the cells surface markers of CD73, CD90, CD29, CD34, and human leukocyte antigen DR (HLA-DR). The 2nd to 4th passages of hAECs were used in the following experiments. The hAEC-Exos were separated by ultracentrifugation method. The HaCaT and hAEC-Exos were co-cultured for 3 h, and the uptake of hAEC-Exos by HaCaT was observed by inverted fluorescence microscopy. The HaCaT were divided into phosphate buffer solution (PBS) group and hAEC-Exos group or dimethyl sulfoxide (DMSO)+PBS group, DMSO+hAEC-Exos group, and LY294002+hAEC-Exos group, which were dealt correspondingly, with 3 wells in each group. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation activity after 0 (immediately), 12, 24, 36, 48, and 60 h of culture. The scratch test was conducted to detect the scratch healing at 0, 24, 48, and 72 h after the scratch, and the scratch healing rate was calculated, respectively. The Transwell experiment was conducted to detect the number of transmembrane cells after 48 h of culture. The Western blotting was used to detect the protein expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), protein kinase B (Akt), and phosphorylated Akt (p-Akt) related to phosphatidylinositol 3-kinase-Akt-mTOR (PI3K-Akt-mTOR) pathway after 24 h of culture. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test.  Results  Most of the primary hAECs were oval and uniform in size on the 2nd day of culture. The hAECs were arranged in a typical cobblestone-like monolayer on the 4th and 7th day of culture. The primary hAECs highly expressed CD73, CD90, and CD29 of mesenchymal stem cell related surface markers, and were with no or low expressions of CD34 and HLA-DR of hematopoietic stem cell related surface markers. After 3 h of culture, hAEC-Exos were successfully endocytosed by HaCaT into the cytoplasm and gathered around the nucleus. After 12, 24, 36, 48, and 60 h of culture, the proliferation activity of HaCaT in hAEC-Exos group was significantly higher than that in PBS group (t=3.691, 10.861, 12.121, 10.531, 14.931, P<0.01). At 24, 48, and 72 h after scratch, the scratch healing rates of HaCaT in PBS group were significantly lower than those in hAEC-Exos group (t=3.342, 6.427, 5.485, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in hAEC-Exos group was significantly more than that in PBS group (t=5.385, P<0.01). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of hAEC-Exos group were significantly higher than those in PBS group (t=4.240, 5.586, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the two groups were similar (P>0.05). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of DMSO+hAEC-Exos group were significantly higher than those in DMSO+PBS group (t=6.155, 8.338, P<0.01) and LY294002+hAEC-Exos group (t=5.030, 3.960, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the three groups were similar (P>0.05). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 12, 24, 36, 48, and 60 h of culture was 0.78±0.05, 1.23±0.07, 1.60±0.09, 1.86±0.09, and 2.03±0.08, which was significantly higher than 0.46±0.04, 0.69±0.07, 0.98±0.08, 1.16±0.08, and 1.26±0.11 in DMSO+PBS group (t=4.376, 7.398, 8.488, 9.766, 10.730, P<0.01). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 24, 36, 48, and 60 h was significantly higher than 0.96±0.09, 1.20±0.08, 1.39±0.08, and 1.55±0.10 in LY294002+hAEC-Exos group (t=3.639, 5.447, 6.605, 6.693, P<0.05 or P<0.01). The scratch healing rates of HaCaT in DMSO+hAEC-Exos group at 24, 48, and 72 h after scratch were significantly higher than those in DMSO+PBS group (t=4.003, 6.349, 7.714, P<0.01) and LY294002+hAEC-Exos group (t=3.805, 4.676, 4.067, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in DMSO+hAEC-Exos group was significantly more than that in DMSO+PBS group and LY294002+hAEC-Exos group, respectively (t=7.464, 1.232, P<0.01).  Conclusions  PI3K-Akt-mTOR pathway can promote the proliferation and migration of HaCaT in high glucose environment by mediating hAEC-Exos.
Weighted gene co-expression network analysis of methylated genes in burn scar tissue
Guo Peng
2021, 37(12): 1185-1190. doi: 10.3760/cma.j.cn501120-20200311-00150
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Abstract:
  Objective  To investigate the methylated genes in burn scar tissue by weighted gene co-expression network analysis (WGCNA), and to discover molecular markers and therapeutic targets of scar formation.  Methods  An observational research method was used. Datasets were downloaded from the National Center for Biotechnology Information Gene Expression Omnibus Database of America. The GSE136906 (n=6) and GSE137134 (n=6) datasets in the same batch were screened out for mRNA sequencing and methylation sequencing respectively, and the dataset GSE108110 (n=9) was incorporated into support vector machine and modeling analysis. The Limma software package was used to identify the differentially expressed genes and differentially methylated genes between scar tissue after burn and normal tissue. WGCNA was used to select the module with strong correlation with clinical features of scar tissue and large number of genes. Functional enrichment analysis of the genes in the module was performed to find genes with abnormal methylation. The receiver operating characteristic (ROC) curve was used to judge diagnostic efficacy of genes with abnormal methylation for scar, and support vector machine (SVM) was used to verify.  Results  A total of 10 modules were identified, and the brown module with large number of genes was highly correlated to burn scar tissue formation. The genes in the brown module were mainly concentrated in "regulation of androgen receptor signaling pathway", "cytokine-cytokine receptor interaction", "positive regulation of insulin secretion", and so on. The model showed 35 genes with abnormal methylation status. The ROC curve (area under the curve>0.9) and SVM modeling (accuracy=93.3%) indicated that CCR2, LMO7, STEAP4, NNAT, and TCF7L2 genes had good diagnostic performance for scar.  Conclusions  CCR2, LMO7, STEAP4, NNAT, and TCF7L2 can be used as potential targets for burn scar treatment.
Case Report
A case of pseudotumor of lower extremity combined with protracted infectious skin ulcer induced by severe hemophilia A
Chen Lan, Xie Weiguo, Yu Gang
2021, 37(12): 1191-1193. doi: 10.3760/cma.j.cn501120-20200801-00366
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Abstract:

On July 11, 2018, a 26 years old male patient with pseudotumor of lower extremity combined with protracted infectious skin ulcer induced by severe hemophilia A was admitted to Tongren Hospital of Wuhan University＆Wuhan Third Hospital. After treatment of supplementary of clotting factors, prothrombin complex, debridement, and vacuum sealing drainage, skin grafting was performed after granulation tissue formation in wounds. After surgery, clotting factor supplement, anti-infection, prevention of hematoma, and so on were performed. The wounds were finally closed with the survival of skin graft, which dramatically improves the patient's quality of life. This case suggests that aggressive and effective surgical treatment can significantly improve the prognosis of patients with severe hemophilia A.
Lecture
Several problems worthy of attention in non-surgical treatment of scar
Liu Yi
2021, 37(12): 1194-1198. doi: 10.3760/cma.j.cn501120-20210705-00235
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Abstract:
Pathologically, scars are divided into physiological scars and pathological scars, and the latter mainly include hyperplastic scars and keloids. Scar treatment includes surgical treatment and non-surgical treatment, with the pathological scars as the major targets in treatment. Until now, there is no treatment with ideal therapeutic effect. Therefore, new therapeutic methods for pathological scars are still being explored at home and abroad. In recent years, some non-surgical therapeutic methods for scars that have received widespread attention have emerged. In this article, several problems worthy of attention including intralesional injection therapy, photoelectric therapy, and rehabilitation robots were discussed.
Reviews
Research advances on the prevention and treatment strategies of burn wound progressive deepening
Li Haisheng, Luo Gaoxing, Yuan Zhiqiang
2021, 37(12): 1199-1204. doi: 10.3760/cma.j.cn501120-20200828-00396
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Abstract:
The progressive deepening of burn wounds is one of the common clinical problems and difficulties in early burn treatment. At the present, it is believed that local ischemia and hypoxia, persistent inflammation, infection, unbalanced local microenvironment, cell necrosis, apoptosis, and autophagy are the main mechanisms of progressive deepening of burn wounds. In recent years, basic and clinical studies have proposed various new strategies for prevention and treatment of progressive deepening of burn wounds, mainly including correct cooling, improving blood perfusion of the wound, early debridement, improving the wound microenvironment, preventing and treating wound infection, reducing wound inflammation, and inhibiting the oxidative stress in the wound. This review focuses on the current progress of prevention and treatment strategies of burn wound progressive deepening, in order to provide references for the treatment of burn wounds.

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