Evaluation of topical methylene blue nanoemulsion for wound healing in diabetic mice.

CONTEXT: Diabetic wounds (DW) are a complication of diabetes and slow wound healing is the main manifestation. Methylene blue (MB) has been shown to exhibit therapeutic effects on diabetes-related diseases. OBJECTIVE: To investigate the mechanisms of action of MB-nanoemulsion (NE) in the treatment of DW. MATERIALS AND METHODS: The concentration of MB-NE used in the in vivo and in vitro experiments was 0.1 mg/mL. Streptozocin-induced diabetic mice were used as models. The mice were separated into nondiabetic, diabetic, MB-NE treated, and NE-treated groups. Intervention of high glucose-induced human umbilical vein endothelial cells using MB-NE. The mechanism by which MB-NE promotes DW healing is investigated by combining histological analysis, immunofluorescence analysis, TUNEL and ROS assays and western blotting. RESULTS: In diabetic mice, the MB-NE accelerated DW healing (p < 0.05), promoted the expression of endothelial cell markers (α-SMA, CD31 and VEGF) (p < 0.05), and reduced TUNEL levels. In vitro, MB accelerated the migration rate of cells (p < 0.05); promoted the expression of CD31, VEGF, anti-apoptotic protein Bcl2 (p < 0.05) and decreased the expression of the pro-apoptotic proteins cleaved caspase-3 and Bax (p < 0.05). MB upregulated the expression of Nrf2, catalase, HO-1 and SOD2 (p < 0.05). In addition, MB reduced the immunofluorescence intensity of TUNEL and ROS in cells and reduced apoptosis. The therapeutic effect of MB was attenuated after treatment with an Nrf2 inhibitor (ML385). DISCUSSION AND CONCLUSION: This study provides a foundation for the application of MB-NE in the treatment of DW.

Deep sternal wound infection and pectoralis major muscle flap reconstruction: A single-center 20-year retrospective study.

Background: One of the most drastic complications of median sternal incision is deep sternal wound infection (DSWI), as it can lead to prolonged hospitalization, increased expected costs, re-entry into the ICU and even reoperation. Since the pectoralis major muscle flap (PMMF) technique was proposed in the 1980s, it has been widely used for sternal reconstruction after debridement. Although numerous studies on DSWI have been conducted over the years, the literature on DSWI in Chinese population remains limited. The purpose of this study was to investigate the clinical characteristics of DSWI in patients and the clinical effect of the PMMF at our institution. Methods: This study retrospectively analyzed all 14,250 consecutive patients who underwent cardiac surgery in the Department of Cardiothoracic Surgery of Drum Tower Hospital from 2001 to 2020. Ultimately, 134 patients were diagnosed with DSWI.,31 of whom had recently undergone radical debridement and transposition of the PMMF in the cardiothoracic surgery or burns and plastic surgery departments because of DSWIs, while the remaining patients had undergone conservative treatment or other methods of dressing debridement. Results: In total, 9,824 patients were enrolled in the study between 2001 and 2020, of whom 134 met the DSWI criteria and 9690 served as controls. Body mass index (OR = 1.08; P = 0.02; 95% CI, 1.01∼1.16) and repeat sternotomy (OR = 5.93; P < 0.01; 95% CI, 2.88∼12.25) were important risk factors for DSWI. Of the 134 patients with DSWI, 31 underwent the PMMF technique, and the remaining 103 served as controls. There were significant differences in coronary artery bypass grafting (CABG) (P < 0.01), valve replacement (P = 0.04) and repeat sternotomy (P < 0.01) between the case group and the control group. The postoperative extubation time (P < 0.001), ICU time (P < 0.001), total hospitalization time (P < 0.001) and postoperative hospitalization time (P < 0.001) in the PMMF group were significantly lower than those in the control group. The results of multivariate regression analysis showed that PMMF surgery was an important protective factor for the postoperative survival of DSWI patients (OR = 0.12; P = 0.04; 95% CI, 0.01∼0.90). Conclusions: Staphylococcus aureus was the most common bacteria causing DSWI, which was associated with BMI and reoperation, and can be validly treated with PMMF.

[Experimental study on the effect of vascular endothelial growth factor 165 gene on vascularization of dermal substitute].

OBJECTIVE: To investigate the effect of vascular endothelial growth factor 165 (VEGF 165) gene on vascularization of dermal substitute in vivo. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in M199 medium containing FBS in the volume fraction of 10% (briefly called complete medium). (1) HUVECs were divided into non-transfection group (without transfection), empty vector group [transfected with pIRES2-enhanced green fluorescent protein (EGFP) plasmid], and VEGF plasmid group (transfected with pIRES2-EGFP-VEGF plasmid) according to the random number table, with 6 wells in each group. At post transfection hour (PTH) 24, the expression of green fluorescent protein (GFP) in each group was observed under inverted phase contrast fluorescence microscope, and the expression rate of GFP was detected with flow cytometer. Cells in non-transfection group were tested with the same methods as listed above. The cells in stable transfection in empty vector group and VEGF plasmid group were sifted by neomycin. The mRNA and protein expression levels of VEGF 165 in cells and the protein amount of VEGF 165 in the supernatant of cell culture medium in 3 groups were respectively determined by real-time fluorescent quantitation PCR, Western blotting, and enzyme-linked immunosorbent assay. (2) Forty-eight male nude mice were divided into 4 groups according to the random number table, with 12 mice in each group. Mice in saline group were subcutaneously implanted with dermal substitutes which had been cultured in saline for 2 days on both sides of back (the same site below); mice in medium group were subcutaneously implanted with dermal substitutes which had been cultured in complete medium for 2 days; mice in non-transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in complete medium with non-transfected HUVECs for 2 days; mice in transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in complete medium with HUVECs stably transfected with VEGF plasmid for 2 days. The dermal substitutes in every group were taken out on post operation day (POD) 3, 7, 14, and 21. Distributions of microvessels and HUVECs in dermal substitutes were observed by immunohistochemical staining, and the microvessel number was counted on POD 14; the expression level of VEGF 165 protein in dermal substitutes was determined by Western blotting. The experiments were all done in triplicate. Data were processed with one-way analysis of variance and LSD method. RESULTS: (1) Obvious green fluorescence was only observed in the two groups with transfected cells at PTH 24. Expression rates of GFP in the cells of non-transfection group, empty vector group, and VEGF plasmid group were respectively 0, (85.2 ± 3.2) %, and (93.1 ± 2.4) %. In the non-transfection group, empty vector group, and VEGF plasmid group, the relative expression amounts of VEGF 165 mRNA were respectively 1, 1.05 ± 0.09, and 3.02 ± 0.13 (F = 5.28, P < 0.05); the relative expression amounts of VEGF 165 protein were respectively 0.78 ± 0.16, 0.76 ± 0.13, and 1.92 ± 0.18 (F = 7.62, P < 0.05); the protein quantity of VEGF 165 in cell supernatant was respectively (62.4 ± 2.7), (73.1 ± 3.8), (117.5 ± 3.1) pg/mL (F = 15.08, P < 0.05). The mRNA and protein levels of VEGF 165 and VEGF 165 protein amount in supernatant were significantly higher in VEGF plasmid group than in the other two groups, with P values all below 0.05. (2) The number of HUVECs in dermal substitutes of transfected cells group was significantly higher than that of the other three groups on POD 14. The numbers of microvessels of dermal substitutes on POD 14 in saline group, medium group, non-transfected cells group, transfected cells group were respectively 4.2 ± 1.1, 5.2 ± 1.1, 6.6 ± 0.9, 13.8 ± 0.8 per 200 times visual field (F = 17.96, P < 0.01). The microvessel number in transfected cells group was significantly higher than that of the other three groups, with P values all below 0.05. The relative expression ratio of VEGF 165 protein of dermal substitutes in transfected cells group was significantly higher than that in saline group as of POD 7. On POD 14 and 21, the relative expression ratios of VEGF 165 proteins in non-transfected cells group (1.652 ± 0.086, 2.152 ± 0.062) and transfected cells group (2.403 ± 0.091, 2.879 ± 0.047) were significantly higher than those of saline group (1.299 ± 0.027, 1.362 ± 0.103), with P values all below 0.05. And the index level of transfected cells group was significantly higher than that in non-transfected cells group (with P values below 0.05). The VEGF 165 protein content in dermal substitutes increased with time extension in all groups. CONCLUSIONS: Transfection of VEGF 165 gene in HUVEC could effectively facilitate vascularization of dermal substitutes in vivo by high expression of VEGF 165 protein.