中华烧伤与创面修复杂志

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Objective To observe the influence of the stiffness of three-dimensionally bioprinted extracellular matrix analogue on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into skin appendage cells. Methods (1) Sodium alginate of 1 g and 4 g gelatin, 3 g sodium alginate and 8 g gelatin were mixed respectively, and the two mixtures were dissolved in 100 mL ultra-pure water respectively to prepare two sodium alginate-gelatin composite hydrogels, named 1A4G hydrogel and 3A8G hydrogel, which were used in the subsequent experiments. The morphology of the two hydrogels at room temperature, after condensation for 15-30 min at 4 ℃ (the same condensation condition below), after condensation and cross-linking with 25 g/L calcium chloride solution (the same cross-linking condition below), and after condensation and three-dimensional printing with a three-dimensional bioprinter (the same three-dimensional printer below) and cross-linking were observed respectively. Young′s modulus (stiffness) of the two kinds of hydrogels was measured by Young′s modulus tester after condensation and cross-linking (n=3). Two kinds of hydrogels were cross-linked and freeze-dried, and their pore structure was observed by scanning electron microscope. Two hydrogels were cross-linked and freeze-dried, and the porosity was detected by anhydrous ethanol replacement method (n=3). (2) BMSCs were isolated from femur and tibia of 20 C57BL/6 mice (no limitation with sex, born 7 days) and cultured, and the second passage of cells was used for further test. The BMSCs single cell suspension (1.0×10⁷ /mL) was mixed with 1A4G hydrogel and 3A8G hydrogel respectively at 1∶9 volume ratio to prepare BMSCs-loaded 1A4G hydrogel and BMSCs-loaded 3A8G hydrogel for three-dimensional printing. One construct was printed with 1 mL cell-loaded hydrogel (the same dosage for printing below). Mesenchymal stem cells (MSCs) specific medium was added after cross-linking, and the printed constructs were divided into 1A4G group and 3A8G group according to the hydrogel. One construct of each group cultured for 7 days was tested with live/dead kit to count the live cells and dead cells in 50-fold field of view. Nine printed constructs from each of the two groups were taken, and BMSCs of nine wells (1.0×10⁶ per well) cultured with 2 mL MSCs specific medium were set as two-dimensional culture group. After 1, 3, 5 day (s) of culture, three printed constructs from 1A4G group and 3A8G group respectively and three wells of cells from two-dimensional culture group were taken to detect the absorbance value in culture medium by cell counting kit 8, denoting the cell proliferation activity. (3) BMSCs-loaded 1A4G hydrogel and BMSCs-loaded 3A8G hydrogel of 10 mL respectively were prepared as in experiment (2), which were respectively mixed with 0.5 mL plantar dermis homogenate extracted from 10 C57BL/6 mice of 1 day postnatal with unknown sex, then three-dimensionally printed, cross-linked, cultured with MSCs specific medium for 3 days and then changed to sweat gland specific medium. The printed constructs were divided into 1A4G group and 3A8G group according to their hydrogel. After 7 days of culture with sweat gland specific medium, the expressions of epithelial cell surface markers cytokeratin-5 (CK5) and CK14, sweat gland cell surface markers CK18 and Na⁺ /K⁺ -ATPase (NKA), and hair follicle cell surface markers CK17 and alkaline phosphatase (ALP) at protein level in cells of printed constructs in the two groups were detected by immunofluorescence method. The expressions of CK5, CK14, CK18, NKA (detecting ATP1a1), CK17, and ALP at mRNA level in cells of printed constructs in the two groups were detected with real-time fluorescent quantitative reverse transcription polymerase chain reaction (n=3). Data were statistically analyzed with independent sample t test, Fisher′s exact probability test, analysis of variance for factorial design, and Bonferroni method. Results (1) Compared with that of 3A8G hydrogel, 1A4G hydrogel had lower viscosity and better fluidity at room temperature. Both kinds of hydrogels were gel-like after condensation, based on which, the shape of cross-linked hydrogels was uniform and regular, with three-dimensional printing and cross-linking made hrdrogels forming solid crisscross cylindrical constructs. The Young′s modulus of 1A4G hydrogel was (52±6) kPa, which was obviously lower than (218±5) kPa of 3A8G hydrogel (t=40.470, P<0.01). The pore structure of the two hydrogels was similar, with all the cross-sections showing porous network structure. The porosity of the two hydrogels was similar (t=0.930, P>0.05). (2) The distribution of live/dead cells between 1A4G group and 3A8G group was similar after 7 days of culture (P>0.05), most of which were live cells. The absorbance value in culture medium of printed constructs among 1A4G group, 3A8G group, and two-dimensional culture group didn′t show statistically significant differences after 1, 3, 5 day (s) of culture (P>0.05). Compared with that after 1 day of culture within each group, the absorbance value in culture medium of printed constructs in 1A4G group and 3A8G group was significantly increased after 3 and 5 days of culture (P<0.05 or P<0.01), and the absorbance value in culture medium of cells in two-dimensional culture group was significantly increased after 5 days of culture (P<0.01). Compared with that after 3 days of culture within each group, the absorbance value in culture medium of printed constructs in 1A4G group and 3A8G group and that of cells in two-dimensional culture group was significantly increased after 5 days of culture (P<0.01). (3) After 7 days of culture with sweat gland specific medium, the CK5, CK14, CK18, NKA, CK17, and ALP were positively expressed at protein level in cells of printed constructs in the two groups. After 7 days of culture with sweat gland specific medium, the expressions of CK5, CK14, CK18, and NKA at mRNA level in cells of printed constructs were close between the two groups (t=0.362, 0.807, 0.223, 1.356, P>0.05); the expressions of CK17 and ALP at mRNA level in cells of printed constructs in 3A8G group were 1.96±0.21 and 55.57±11.49, respectively, which were significantly higher than 1.05±0.42 and 2.01±0.27 in 1A4G group (t=3.333, 8.074, P<0.05 or P<0.01). Conclusions BMSCs cultured three-dimensionally in 1A4G and 3A8G hydrogels tend to differentiate into sweat gland cells, but the BMSCs cultured three-dimensionally in 3A8G hydrogel show a stronger tendency to differentiate into hair follicle cells than the cells cultured in 1A4G hydrogel. It suggests that relatively high stiffness of three-dimensionally bioprinted extracellular matrix analogue facilitates not only differentiation of BMSCs into sweat gland cells, but also their differentiation into hair follicle cells.

Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice

Hao Yi, Yang Qinxin, Wang Qi, Xu Guangchao, Qi Fang, Deng Chengliang, Wei Zairong, Wang Dali

2020, 36(11): 1024-1034. doi: 10.3760/cma.j.cn501120-20200720-00351

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Objective To investigate the feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice. Methods (1) Ten eight-week-old C57BL/6 male mice were collected and full-thickness skin tissue with diameter of 1.0 cm on both sides of the midline of the back was taken with a perforator to make the normal skin tissue homogenate supernatant. At 48 h after the full-thickness skin defect wound was established, the wound tissue within 2 mm from the wound edge was taken to make inflammatory wound tissue homogenate supernatant. Two kinds of tissue homogenate supernatant were taken to adjust the total protein concentration to 1 mg/mL, and the tumor necrosis factor α (TNF-α) content was detected by enzyme-linked immunosorbent assay. The number of sample was 6. (2) The primary passage of human umbilical cord mesenchymal stem cells (hUCMSCs) were collected and cultured to the 3rd passage with the normal exosomes being extracted from the hUCMSCs after cultured for 48 h. Another batch of hUCMSCs in the 3rd passage was collected and stimulated with inflammatory wound tissue homogenate supernatant of 30, 50, and 100 μg/mL total protein and normal skin tissue homogenate supernatant of 30, 50, and 100 μg/mL total protein, respectively. After cultured for 48 h, the exosomes stimulated with normal protein of 30, 50, and 100 μg/mL and exosomes stimulated with inflammatory protein of 30, 50, and 100 μg/mL were extracted. Normal exosomes, exosomes stimulated with 30 μg/mL normal protein, and exosomes stimulated with 30 μg/mL inflammatory protein were collected, the morphology was observed by transmission electron microscope, the particle size was detected by nanoparticle tracking analyzer, and the expressions of CD9 and CD63 were detected by Western blotting. (3) Twenty one-day-old C57BL/6 mice were taken to isolate the primary passage of fibroblasts (Fbs) and the 3rd passage of Fbs, whose morphology was observed under the inverted phase contrast microscope. The Fbs of 3rd passage were collected to observe the expression of vimentin by cell crawling method combined with immunofluorescence method at culture hour (CH) 2. (4) The Fbs of 3rd passage were divided into control group, normal exosome group, 30, 50, 100 μg/mL normal protein stimulating exosome group, and 30, 50, 100 μg/mL inflammatory protein stimulating exosome group according to the random number table, with 4 wells in each group. Cells in control group received no treatment, and cells in the other 7 groups were respectively added with normal exosomes, exosomes stimulated with normal protein of 30, 50, and 100 μg/mL, and exosomes stimulated with inflammatory protein of 30, 50, and 100 μg/mL prepared in experiment (2). The final mass concentration of exosomes was adjusted to 10 μg/mL. The cell viability was detected by cell count kit 8 at CH 48. (5) Two batches of Fbs in the 3rd passage were divided and treated as those in experiment (4), with 4 wells in each group, and the final mass concentration of exosomes was adjusted to 1 and 10 μg/mL, respectively. The cell mobility was detected by cell scratch test at CH 6, 12, and 24. (6) Two batches of the Fbs of 3rd passage were collected, divided, and treated as those in experiment (4) except with no control group, with 3 wells in each group, and the final mass concentration of exosomes was respectively adjusted to 1 and 10 μg/mL. The mRNA expression levels of transforming growth factor β₁ (TGF-β₁), TGF-β₃, and α smooth muscle actin (α-SMA) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction at CH 48. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Bonferroni method. Results (1) The content of TNF-α in inflammatory wound tissue homogenate supernatant of mice was (116±3) pg/mL, significantly higher than (97±5) pg/mL in normal skin tissue homogenate supernatant at post injury hour 48 (t=3.306, P<0.05). (2) Normal exosomes, exosomes stimulated with 30 μg/mL normal protein, and exosomes stimulated with 30 μg/mL inflammatory protein of hUCMSCs showed the typical saucer-like shape. The particle sizes of the three exosomes of hUCMSCs were 30-150 nm, which were all within the normal particle size range of exosome. Three exosomes of hUCMSCs positively expressed CD9 and CD63. (3) The primary passage of cells were clearly defined and showed protruding spindle shape, irregular polygon shape, or slender strip shape. The morphology of the 3rd and the primary passage of cells is similar. At CH 2, vimentin in cells was positively expressed, and the cells were identified as Fbs. (4) At CH 48, the cell viability was (137.4±2.8)% in 30 μg/mL inflammatory protein stimulating exosome group, obviously higher than 100%, (107.5±2.4)%, (113.3±3.2)%, (104.0±2.0)%, and (101.9±1.5)% in control group, normal exosome group, 30 μg/mL normal protein stimulating exosome group, and 50 and 100 μg/mL inflammatory protein stimulating exosome groups, respectively (P<0.01), and cell viability in 30 μg/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 μg/mL normal protein stimulating exosome groups [(103.4±2.2)% and (102.5±1.4)%], respectively (P<0.01). (5) At CH 6, 12, and 24, the mobility rate of cells in 30 μg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, 30 μg/mL normal protein stimulating exosome group, and 50 and 100 μg/mL inflammatory protein stimulating exosome groups, respectively, when the final mass concentrations of exosome was 1 μg/mL (P<0.05) . At CH 12, the mobility rate of cells in 30 μg/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 μg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 1 μg/mL (P<0.05). At CH 6, the mobility rate of cells in 30 μg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group and normal exosome group (P<0.05), and the mobility rate of cells in 30 μg/mL normal protein stimulating exosome group was significantly higher than that in 50 and 100 μg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 μg/mL (P<0.05). At CH 12 and 24, the mobility rate of cells in 30 μg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 μg/mL inflammatory protein stimulating exosome groups (P<0.05), and the mobility rate of cells in 30 μg/mL normal protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 μg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 μg/mL (P<0.05). (6) There were no statistically significant differences in mRNA expression levels of TGF-β₁, TGF-β₃, and α-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 1 μg/mL (F=1.123, 1.537, 1.653, P>0.05). There were no statistically significant differences in mRNA expression levels of TGF-β₁ and α-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 10 μg/mL (F=1.487, 1.308, P>0.05), and mRNA expression level of TGF-β₃ of cells in 50 μg/mL inflammatory protein stimulating exosome group at CH 48 was significantly higher than that in normal exosome group, 50 μg/mL normal protein stimulating exosome group, and 30 and 100 μg/mL inflammatory protein stimulating exosome groups when the final mass concentration of exosome was 10 μg/mL (P<0.05). Conclusions The pretreatment with inflammatory wound tissue homogenate supernatant of mice has no significant effect on the total protein of hUCMSCs exosomes. The hUCMSCs exosomes stimulated by low concentration inflammatory wound tissue homogenate supernatant can significantly promote the proliferation and migration ability of Fbs. The content of inflammatory mediators in the wound tissue homogenate supernatant during the inflammatory phase is extremely low, which may be the reason that the anti-inflammation and tissue repair paracrine effects of mesenchymal stem cell cannot be effectively started.

Clinical effects of combined application of skin-stretching device and vacuum sealing drainage in repairing the diabetic foot wounds

Ji Peng, Zhang Yue, Hu Dahai, Zhang Zhi, Li Xiaoqiang, Tong Lin, Han Juntao, Tao Ke

2020, 36(11): 1035-1039. doi: 10.3760/cma.j.cn501120-20200621-00318

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Objective To investigate the clinical effects of skin-stretching device (hereinafter referred to as stretcher) combined with vacuum sealing drainage (VSD) in repairing diabetic foot wounds. Methods From March 2016 to January 2020, 25 patients with diabetic foot wounds were admitted to the First Affiliated Hospital of Air Force Medical University, including 18 males and 7 females, with age of 40 to 70 years. After debridement, intermittent VSD was performed for 3 to 10 days, with negative pressure value of -10.67 kPa. Then, the wound area was 5.0 cm×3.0 cm to 10.0 cm×7.0 cm. After infection control and detumescence, the wound was treated with stretcher for 3 to 5 days. The wound area after stretching ranged from 5.0 cm×0.3 cm to 10.0 cm×0.5 cm. The wound was closed with full-thickness suture. Two weeks after the suturing operation, the healing grade of the foot wound of patients was observed, and the serious complications such as recurrence of ulcer wound and gangrene on the foot, scar condition of the wound were observed during follow-up. Results Two weeks after the suturing operation, the wounds of 23 patients were healed with grade A. Soft tissue infection ulcer relapsed in 2 patients during the stretch period. After anti-infection, thorough debridement, and VSD, the wounds were healed after another 16 days of stretch treatment. During the follow-up of 3 to 36 months, 23 patients had linear scar left on the stretch wounds, and the skin elasticity, color, sensation was similar to the surrounding normal tissue, and the limb mobility was good, and 2 patients had obvious scar hyperplasia. One patient had recurrence of diabetic foot and serious vascular occlusion and gangrene in the affected limb at follow-up of 10 months, which was treated with amputation of the lower leg. Conclusions The use of stretcher combined with VSD in treating diabetic foot wound can avoid donor site injury, with healed wound achieving similar appearance to adjacent skin and satisfactory repair effects.

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

Wang Tianyuan, Chen Yinchen, Wang Wei, Jiang Dong, Liu Lan, Yang Hui, Wang Aiping

2020, 36(11): 1040-1049. doi: 10.3760/cma.j.cn501120-20191022-00409

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Objective To investigate the mechanism of maggot debridement therapy (MDT) in promoting wound angiogenesis in patients with diabetic foot ulcer (DFU). Methods (1) From June 2018 to June 2019, the patients admitted to Nanjing Junxie Hospital who met the inclusion criteria were recruited, including 12 DFU patients given MDT for three days [6 males and 6 females, aged (56±12) years] and 12 acute trauma patients without diabetes mellitus [6 males and 6 females, aged (53±10) years], who were enrolled into DFU group and non-diabetic trauma group respectively. Before and after application of MDT, the wound characteristics of patients in DFU group were observed and the wound tissue samples were taken. The wound tissue in non-diabetic trauma group was taken at patient′s first visit before debridement. The expression of angiogenesis marker CD31 in the wound tissue of patients in DFU group was detected by immunohistochemistry before and after application of MDT. Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) were used respectively to detect the protein and mRNA expressions of fatty acid synthase (FAS) in wound tissue of patients in DFU group before and after application of MDT and in non-diabetic trauma group before debridement. (2) Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell culture medium containing 10% fetal bovine serum. The 3rd to 6th passages of cells in logarithmic growth phase were used in the following experiments. Excretions/secretions (ES) were extracted from 3-day-old sterile Lucilia sericata larvae for subsequent experiments. Three batches of cells were divided into phosphate buffer solution (PBS) control group, high glucose alone group, high glucose+ 5 μg/mL maggot ES group, and high glucose+ 10 μg/mL maggot ES group, which were treated with PBS, glucose in final molarity concentration of 20 mmol/L, glucose in final molarity concentration of 20 mmol/L+ maggot ES in final mass concentration of 5 μg/mL, and glucose in final molarity concentration of 20 mmol/L+ maggot ES in final mass concentration of 10 μg/mL respectively. The total volume of reagents in each group was the same. After 48 hours of culture, Western blotting, real-time fluorescent quantitative RT-PCR and immunofluorescence method were used to detect the protein and mRNA expressions of FAS in each batch of cells and the expression and localization of FAS protein in cells respectively. The number of samples for mRNA expression was 3. (3) Two batches of cells were divided into small interference RNA (siRNA) alone group, siRNA control+ maggot ES group and siRNA-FAS+ maggot ES group, which were transfected with 100 μmol/L (final molarity concentration) insignificant control siRNA, insignificant control siRNA, and siRNA-FAS for 4-6 h respectively, and then they were routinely cultured for 24 h with PBS added, maggot ES in final mass concentration of 10 μg/mL, and maggot ES in final mass concentration of 10 μg/mL respectively. The total volume of reagents in each group was the same. One batch of cells was used for scratch test, the scratch width was observed at 24 hour after scratching to detect the cell migration ability; one batch of cells was subjected to tube forming experiment, and the formation of cell tubules was observed after 24 hours of culture. The number of samples was 3 in scratch test and tube forming experiments. Data were statistically analyzed with t test, one-way analysis of variance, least significant difference test, analysis of variance for repeated measurement, and Bonferroni method. Results (1) Compared with those before application of MDT, fresh granulation tissue significantly increased and necrotic tissue decreased obviously in wound, and the expression of CD31 significantly increased in wound tissue of patients in DFU group after application of MDT. The expression of FAS protein in wound tissue of patients in DFU group before application of MDT was significantly lower than that in non-diabetic trauma group before debridement, and the expression of FAS protein in wound tissue of patients in DFU group after application of MDT was significantly higher than that before application of MDT. The expression of FAS mRNA in wound tissue of patients in DFU group before application of MDT was 1.00±0.17, which was significantly less than 3.87±1.02 in non-diabetic trauma group before debridement (t=9.808, P<0.01). The expression of FAS mRNA in wound tissue of patients in DFU group after application of MDT was 1.85±0.31, which was significantly higher than that before application of MDT (t=-10.853, P<0.01). (2) After 48 hours of culture, Western blotting detection showed that the expression of FAS protein in cells in high glucose alone group was significantly less than that in PBS control group, and the expressions of FAS protein in cells in high glucose+ 5 μg/mL maggot ES group and high glucose+ 10 μg/mL maggot ES group were significantly higher than the expression in high glucose alone group. Real-time fluorescent quantitative RT-PCR determination showed that the expression of FAS mRNA in cells in high glucose alone group was 0.392±0.073, which was significantly lower than 1.000±0.085 in PBS control group (P<0.01); there was statistically significant difference between the expression of FAS mRNA in cells in high glucose+ 5 μg/mL maggot ES group (0.561±0.047) and that in high glucose+ 10 μg/mL maggot ES group (0.687±0.013) (P<0.05), both of which were significantly higher than the expression in high glucose alone group (P<0.01). The results of immunofluorescence detection showed that FAS protein was mainly located in the cytoplasm of cells in each group, and its expression was similar to that detected by Western blotting. (3) At 24 hour after scratch, the uncured widths of cell scratch in siRNA control+ maggot ES group and siRNA-FAS+ maggot ES group were significantly narrower than the uncured width in siRNA alone control group (P<0.01), and the uncured width of cell scratch in siRNA-FAS+ maggot ES group was significantly wider than that in siRNA control+ maggot ES group (P<0.01). After 24 hours of culture, the numbers of tubules in siRNA+ maggot ES group and siRNA-FAS+ maggot ES group were significantly more than the number in siRNA alone control group (P<0.05 or P<0.01), and the number of tubules in siRNA-FAS+ maggot ES group was obviously less than that in siRNA control+ maggot ES group (P<0.05). Conclusions MDT up-regulates the expression of FAS through maggot ES, which promotes the activity of vascular endothelial cells, thus promoting the wound angiogenesis in patients with DFU.

Effect and mechanism of astaxanthin on acute kidney injury in rats with full-thickness burns

Yu Meirong, Guo Songxue, Jin Ronghua, You Chuangang, Wang Xingang, Han Chunmao

2020, 36(11): 1050-1059. doi: 10.3760/cma.j.cn501120-20200526-00287

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Objective To explore the effect and mechanism of astaxanthin on acute kidney injury in rats with full-thickness burns. Methods Forty-eight male Sprague Dawley rats of 8 to 10 weeks were divided into sham injury group, simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group according to the random number table, with 8 rats in each group. The back skin of rats in sham injury group were immersed in warm water of 20 ℃ for 15 s to simulate burn injury, and the back skin of rats in the other 5 groups were immersed in boiled water of 100 ℃ for 15 s to inflict full-thickness burn of 30% total body surface area. Fluid resuscitation was performed in rats in the 5 groups except of sham injury group immediately and 6 h after injury. At 30 min after injury, the rats in sham injury group and simple burn group were injected with 1 mL/kg normal saline via tail vein, rats in burn+ vehicle group were injected with 1 mL/kg astaxanthin solvent via tail vein, and rats in burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group were respectively injected with 5, 10, 20 mg/kg astaxanthin solution of 5, 10, 20 mg/mL via tail vein. The renal tissue was collected at post injury hour (PIH) 48, and hematoxylin eosin staining was used for histopathological observation and renal tubular injury score. At PIH 48, the venous blood was collected for detecting serum creatinine level through blood biochemical analyzer, and blood urea nitrogen (BUN) level was detected by enzyme-linked immunosorbent assay. The renal tissue was collected to detect the mRNA expressions of myeloperoxidase (MPO), interleukin-1β (IL-1β), and IL-6 by real-time fluorescent quantitative reverse transcription polymerase chain reaction method, and the protein expressions of Toll like receptor 4 (TLR4), phosphorylated nuclear factor kappa B (p-NF-кB) p65, and heme oxygenase 1 (HO-1) were detected by Western blotting. Besides, the expression of HO-1 in renal tissue was detected by immunofluorescence method. Data were statistically analyzed with Kruskal-Wallis H test, Dunn-Sidák correction, one-way analysis of variance, and Bonferroni method. Results (1) At PIH 48, there were no inflammatory cell infiltrating and degeneration or necrosis of cells in renal tissue of rats in sham injury group, and the structure of renal tubules was intact. The renal tubules of burn rats in each group showed injury manifestation of separation between epithelial cell and basement membrane, and vacuole cells and lysate protein aggregation. The injury degree of renal tissue of rats in burn+ high-dose astaxanthin group was obviously decreased compared with that in simple burn group. (2) At PIH 48, compared with that of sham injury group, the renal tubular damage scores of rats in simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ medium-dose astaxanthin group were significantly increased (P<0.05 or P<0.01). Compared with those of simple burn group and burn+ vehicle group, the renal tubular damage scores of rats in burn+ medium-dose astaxanthin group and burn+ high-dose astaxanthin group were significantly decreased (P<0.05 or P<0.01). Compared with that of burn+ low-dose astaxanthin group, the renal tubular damage score of rats in burn+ high-dose astaxanthin group was significantly decreased (P<0.01). (3) At PIH 48, the level of serum creatinine of rats in sham injury group was (2.42±0.06) mg/L, which was significantly lower than (6.11±0.11), (6.48±0.08), (5.79±0.09), (4.03±0.12) mg/L of simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ medium-dose astaxanthin group (P<0.05 or P<0.01). The level of BUN of rats was (21.9±1.3) mmol/L in sham injury group, significantly lower than (32.1±7.4) mmol/L of simple burn group and (30.2±4.8) mmol/L of burn+ vehicle group (P<0.05 or P<0.01). At PIH 48, compared with those of simple burn group and burn+ vehicle group, the levels of serum creatinine and BUN of (16.0±2.9) mmol/L in burn+ medium-dose astaxanthin group, serum creatinine of (3.02±0.08) mg/L and BUN of (14.5±2.9) mmol/L in burn+ high-dose astaxanthin group, and serum creatinine of (22.8±5.5) mmol/L of rats in burn+ low-dose astaxanthin group were significantly decreased (P<0.05 or P<0.01). At PIH 48, compared with those of burn+ low-dose astaxanthin group, the levels of serum creatinine and BUN of burn+ high-dose astaxanthin group and serum creatinine of burn+ medium-dose group were obviously decreased (P<0.05 or P< 0.01). (4) At PIH 48, compared with those of sham injury group, the mRNA expressions of MPO, IL-1β, and IL-6 in renal tissue of rats in simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ medium dose astaxanthin group, and the mRNA expressions of IL-1β and IL-6 in renal tissue of rats in burn+ high-dose astaxanthin group were obviously increased (P<0.01). Compared with those of simple burn group and burn+ vehicle group, the mRNA expressions of MPO, IL-1β, and IL-6 in renal tissue of rats were significantly decreased in burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group (P<0.01). Compared with those of burn+ low-dose astaxanthin group, the mRNA expressions of MPO, IL-1β, and IL-6 in renal tissue of rats were significantly decreased in burn+ medium-dose astaxanthin group and burn+ high-dose astaxanthin group (P<0.01). The mRNA expressions of MPO, IL-1β, and IL-6 in renal tissue of rats in burn+ high-dose astaxanthin group were significantly decreased compared with those of burn+ medium-dose astaxanthin group (P<0.01). (5) At PIH 48 h, compared with those of sham injury group, the protein expressions of TLR4 and p-NF-кB p65 in renal tissue of rats in simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ high-dose astaxanthin group were obviously increased (P<0.01). Compared with those of simple burn group, the protein expressions of TLR4 and p-NF-кB p65 in renal tissue of rats in burn+ low-dose astaxanthin group, burn+ medium dose astaxanthin group, and burn+ high-dose astaxanthin group were significantly decreased (P<0.01). (6) The results of Western blotting combined with immunofluorescence method showed that compared with that of sham injury group, the protein expression of HO-1 in renal tissue of rats in burn+ vehicle group, burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group were significantly increased at PIH 48 (P<0.01), and the protein expression of HO-1 in renal tissue of rats in burn+ medium-dose astaxanthin group and burn+ high-dose astaxanthin group was significantly increased compared with that of simple burn group (P<0.01). Conclusions Astaxanthin can attenuate the structural damage and functional decline of renal tissue and regulate the release of injury-related inflammatory factors, thus to protect the rats from acute kidney injury after burn. The HO-1/TLR4/NF-кB signaling pathway is the main regulatory mechanism of astaxanthin to achieve anti-inflammation-based renoprotection.

Influence of comprehensive incubational measures on the perioperative treatment of extensively burned patients who underwent escharectomy and skin grafting

Gu Lan, Wang Ling, Miao Wen, Cheng Shasha, Dai Jiaojiao

2020, 36(11): 1060-1064. doi: 10.3760/cma.j.cn501120-20191218-00461

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Objective To explore the influence of standardized and comprehensive incubational measures on perioperative treatment of extensively burned patients who underwent escharectomy and skin grafting. Methods From January 2017 to November 2018, 50 patients with extensive burn who underwent escharectomy and skin grafting in the First Affiliated Hospital of Air Force Medical University and met the inclusion criteria of this study, were recruited in this retrospective cohort study. According to the incubational measures at that time, 20 patients (14 males and 6 females, aged (33.5±5.2) years) who received routine incubation during the perioperative period from January to October 2017 were set as routine incubation group, and 30 patients (23 males and 7 females, aged (35.8±1.4) years) who received standardized comprehensive incubational measures during the perioperative period from November 2017 to November 2018 were set as comprehensive incubation group. Their body temperature was controlled mainly in 4 stages: preoperative preparation and transfer from intensive care unit (ICU) to operating room, preoperative preparation in operating room, intraoperative operating room management, as well as postoperative transfer from operating room to ICU. The initial body temperature in operating room and intraoperative hypothermia duration, intraoperative blood loss, postoperative recovery time, postoperative chill, blister, and ulcer, and wound healing rate on post operation day (POD) 10 were recorded and calculated. Data were statistically analyzed with two independent samples t test and chi-square test. Results (1) The initial body temperature in operating room of patients in comprehensive incubation group was (36.3±0.4) ℃, which was significantly higher than (35.6±0.4)℃ in routine incubation group, t=6.658, P<0.01; the intraoperative duration of hypothermia was (205±38) min, which was significantly shorter than (234±42) min in routine incubation group, t=2.564, P<0.05. (2) The intraoperative blood loss of patients in comprehensive incubation group was (323±114) mL, which was significantly less than (490±162) mL in routine incubation group, t=4.272, P<0.01; the postoperative recovery time was (36±8) min, which was significantly shorter than (49±17) min in routine incubation group, t=3.229, P<0.01. (3) The incidence of postoperative chill of patients in comprehensive incubation group was significantly lower than that in routine incubation group (χ²=28.626, P<0.01). The incidences of postoperative blister and ulcer of patients between the 2 groups were close. (4) On POD 10, the wound healing rate of patients in comprehensive incubation group was (78.08±0.06)%, which was significantly higher than (71.03±0.08)% in routine incubation group, t=3.694, P<0.01. Conclusions The standardized and comprehensive incubational measures can effectively improve the initial body temperature of patients entering the operating room, shorten the intraoperative duration of hypothermia, reduce the amount of blood loss and postoperative complications, as well as shorten the postoperative recovery time, thus improve the wound healing rate.

Effect of venous anastomosis of the pedicled digital artery dorsal branch island flap in repairing fingertip or pulp defects of the same finger

Wang Hui, Yang Xiaoxi, Wang Bin, Huo Yongxin, Chang Hong, Yang Shanhui, Li Junran

2020, 36(11): 1065-1069. doi: 10.3760/cma.j.cn501120-20191113-00427

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Abstract:
Objective To explore the effect of venous anastomosis of the pedicled digital artery dorsal branch island flap in repairing fingertip or pulp defects of the same finger. Methods From February 2016 to September 2018, a total of 56 emergency cases (67 fingers) with fingertip or pulp defects in 2-5 fingers were admitted to the Second Hospital of Tangshan (hereinafter referred to as the author′s affiliation), and the prospective research was conducted. The patients were divided into venous anastomosis group of 29 cases (35 fingers) and non-venous anastomosis group of 27 cases (32 fingers) by drawing lots. There were 18 males and 11 females in venous anastomosis group, aged 17 to 62 years, with wound area of 1.6 cm×1.3 cm-3.1 cm×2.4 cm after debridement. There were 17 males and 10 females in non-venous anastomosis group, aged 20 to 59 years, with wound area of 1.7 cm×1.2 cm-3.0 cm×2.4 cm after debridement. According to the location and size of the fingertip or pulp defect of patients in the two groups, the flap was designed on the dorsum of the middle or proximal phalanx of the injured finger. During the operation, the epineurium of dorsal branch of the proper digital nerve or the dorsal digital nerve carried by the flap was anastomosed end-to-end with the stump of proper digital nerve in the wound. One or two superficial vein (s) carried by the flap was/were interrupted end-to-end anastomosed with the superficial veins on the dorsum or palm of the wound surface in venous anastomosis group, and the venous anastomosis was not performed in non-venous anastomosis group. The flap size resected ranged from 1.8 cm×1.5 cm to 3.4 cm×2.6 cm in venous anastomosis group, and that ranged from 1.9 cm×1.4 cm to 3.3 cm×2.6 cm in non-venous anastomosis group. The donor site wounds in the two groups were resurfaced by free full-thickness skin graft harvested from the proximal forearm or the medial side of the upper arm. The blood circulation of the flap of patients in 2 groups was observed after operation. During follow-up after operation, the patients′ satisfaction for the appearance of the flap was evaluated by Michigan Hand Function Questionnaire, the flap color was observed by the chief physician of the Department of Hand Surgery in the author′s affiliation, and the incidence of flap pigmentation was calculated. Data were statistically analyzed with t test and chi-square test. Results All the flaps of patients survived without tension blister in venous anastomosis group after operation. Tension blisters occurred in 6 cases (6 fingers) in non-venous anastomosis group due to venous reflux obstruction, and the flaps survived after removing some sutures of the pedicle and changing dressing. During follow-up of 8-20 months, with an average of 15 months, the patients′ satisfaction score for flap appearance in venous anastomosis group was (4.6±0.5) points, which was obviously higher than (4.3±0.6) points of non-venous anastomosis group (t=2.482, P<0.05). The incidence of flap pigmentation in venous anastomosis group was 9% (3/35), which was significantly lower than 31% (10/32) of non-venous anastomosis group (χ²=5.498, P<0.05). Conclusions The pedicled digital artery dorsal branch island flap with venous anastomosis repairs the same fingertip or pulp defects, resulting in unobstructed venous reflux, low incidence of flap pigmentation, good appearance and high patient satisfaction.

Establishment and test results of an artificial intelligence burn depth recognition model based on convolutional neural network

He Zhiyou, Wang Yuan, Zhang Pihong, Zuo Ke, Liang Pengfei, Zeng Jizhang, Zhou Situo, Guo Le, Huang Mitao, Cui Xu

2020, 36(11): 1070-1074. doi: 10.3760/cma.j.cn501120-20190926-00385

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Objective To establish an artificial intelligence burn depth recognition model based on convolutional neural network, and to test its effectiveness. Methods In this evaluation study on diagnostic test, 484 wound photos of 221 burn patients in Xiangya Hospital of Central South University (hereinafter referred to as the author′s unit) from January 2010 to December 2019 taken within 48 hours after injury which met the inclusion criteria were collected and numbered randomly. The target wounds were delineated by image viewing software, and the burn depth was judged by 3 attending doctors with more than 5-year professional experience in Department of Burns and Plastic Surgery of the author′s unit. After marking the superficial partial-thickness burn, deep partial-thickness burn, or full-thickness burn in different colors, the burn wounds were cut according to 224×224 pixels to obtain 5 637 complete wound images. The image data generator was used to expand images of each burn depth to 10 000 images, after which, images of each burn depth were divided into training set, verification set, and test set according to the ratio of 7.0∶1.5∶1.5. Under Keras 2.2.4 Python 2.8.0 version, the residual network ResNet-50 of convolutional neural network was used to establish the artificial intelligence burn depth recognition model. The training set was input for training, and the verification set was used to adjust and optimize the model. The judging accuracy rate of various burn depths by the established model was tested by the test set, and precision, recall, and F1_score were calculated. The test results were visualized to generate two-dimensional tSNE cloud chart through the dimensionality reduction tool tSNE, and the distribution of various burn depths was observed. According to the sensitivity and specificity of the model for the recognition of 3 kinds of burn depths, the corresponding receiver operator characteristics (ROC) curve was drawn, and the area under the ROC curve was calculated. Results (1) After the testing of the test set, the precisions of the artificial intelligence burn depth recognition model for the recognition of superficial partial-thickness burn, deep partial-thickness burn, or full-thickness burn were 84% (1 095/1 301), 81% (1 215/1 499) and 82% (1 395/1 700) respectively, the recall were 73% (1 095/1 500), 81% (1 215/1 500) and 93% (1 395/1 500) respectively, and the F1_scores were 0.78, 0.81, and 0.87 respectively. (2) tSNE cloud chart showed that there was small overlapping among different burn depths in the test results for the test set of artificial intelligence burn depth recognition model, among which the overlapping between superficial partial-thickness burn and deep partial-thickness burn and that between deep partial-thickness burn and full-thickness burn were relatively more, while the overlapping between superficial partial-thickness burn and full-thickness burn was relatively less. (3) The area under the ROC curve for 3 kinds of burn depths recognized by the artificial intelligence burn depth recognition model was ≥0.94. Conclusions The artificial intelligence burn depth recognition model established by ResNet-50 network can rather accurately identify the burn depth in the early wound photos of burn patients, especially superficial partial-thickness burn and full-thickness burn. It is expected to be used clinically to assist the diagnosis of burn depth and improve the diagnostic accuracy.

One case of atypical septic shock with acute pulmonary edema in a patient with extensive burn