[Influence of pH value on tube formation of human dermal microvascular endothelial cells and its molecular mechanism].

Objective: To explore the influence of pH value on tube formation of human dermal microvascular endothelial cells (HDMECs) and study its molecular mechanism, so as to provide theoretical basis for the study of promoting angiogenesis in the process of wound healing. Methods: The experimental study methods were applied. HDMECs of 4 or 5 passages in the logarithmic growth phase were collected for experiments. Culture mediums with pH values of 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8 were prepared, and the cells were adaptively cultured (the same culture method below) for 24 h before further experiments being carried out. After another 36 h of culture, the relative fluorescence value of cytoplasmic pH value was measured by flow cytometry, and the correlation analysis between the relative fluorescence value of cytoplasmic pH value and the medium pH value was carried out. After another 1.5, 2.5, 3.5, 4.5, and 5.5 days of culture, the cell proliferation activity was detected with cell counting kit 8. OrisTM cell migration detection kit was used to detect the remaining area of cell migration at 0 (immediately), 24, and 48 h after removing the cell seeding stopper. Three-dimensional stromal gel cell tube formation experiment was carried out to detect the lumen diameter of tube formed by cells after another 48 h of culture. The protein expressions of phosphorylation sites 473 and 308 of protein kinase B (Akt) were detected by Western blotting after another 48 h of culture. The sample number was 3. Data were statistically analyzed with Pearson correlation analysis, one-way analysis of variance, analysis of variance for factorial design, analysis of variance for repeated measurement, and Bonferroni correction. Results: After another 36 h of culture, the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 6.8-7.8 mediums were significantly higher than the level in pH 6.4 medium (P<0.05); compared with those in pH 6.6-7.0 mediums, the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 7.4-7.8 mediums were significantly increased (P<0.05), and the relative fluorescence value of cytoplasmic pH value of cells cultured in pH 6.6 medium was significantly lower than that in pH 7.0 or 7.2 mediun (with P values all <0.05); the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 7.6 and 7.8 mediums were significantly higher than those in pH 7.2 and 7.4 mediums (P<0.05). The relative fluorescence value of cytoplasmic pH value was significantly positively correlated with the medium pH value (r=0.99, P<0.05). The proliferation activity was similar among cells cultured in 8 mediums of different pH values for another 1.5 days (P>0.05). After another 2.5 days of culture, the proliferation activity of cells cultured in pH 6.4-6.8 mediums was significantly decreased compared with that in pH 7.6 medium (P<0.05). After another 3.5 days of culture, the proliferation activity of cells cultured in pH 7.0-7.8 mediums was significantly higher than that in pH 6.4-6.8 mediums (P<0.05); compared with that in pH 7.6 medium, the proliferation activity of cells cultured in pH 7.0-7.4 and 7.8 mediums was significantly decreased (P<0.05). After another 4.5 or 5.5 days of culture, the proliferation activity of cells cultured in pH 6.8-7.8 mediums was significantly higher than that in pH 6.4 medium (P<0.05); compared with that in pH 6.6 and 6.8 mediums, the proliferation activity of cells cultured in pH 7.0-7.8 mediums was significantly increased (P<0.05). After another 4.5 days of culture, the proliferation activity of cells cultured in pH 7.6 medium was significantly higher than that in pH 7.0 medium (P<0.05). After another 5.5 days of culture, the proliferation activity of cells cultured in pH 7.2-7.6 mediums was significantly increased compared with that in pH 7.0 medium (P<0.05); the proliferation activity of cells cultured in pH 7.2 and 7.4 mediums was significantly lower than that in pH 7.6 medium (with P values all <0.05) but significantly higher than that in pH 7.6 medium (with P values all <0.05). Immediately after removing the cell seeding stopper, the remaining migration areas were similar among cells cultured in 8 mediums of different pH values (P>0.05). At 24 h after removing the cell seeding stopper, the remaining migration areas of cells cultured in pH 6.6-7.8 mediums were significantly smaller than the area in pH 6.4 medium (P<0.05); compared with those in pH 6.6 and 6.8 mediums, the remaining migration areas of cells cultured in pH 7.0 to 7.6 mediums were significantly reduced (P<0.05). At 48 h after removing the cell seeding stopper, compared with those in pH 6.4 and 6.6 mediums, the remaining migration areas of cells cultured in pH 7.0-7.8 mediums were significantly reduced (P<0.05); the remaining migration areas of cells cultured in pH 7.2 and 7.4 mediums were significantly smaller than those in pH 6.8, 7.0, and 7.8 mediums (P<0.05) but significantly larger than the area in pH 7.6 medium (P<0.05); the remaining migration area of cells cultured in pH 7.6 medium was significantly smaller than that in pH 6.8 or 7.8 medium (with P values all <0.05). After another 48 h of culture, the lumen diameters of tubes formed by cells cultured in pH 7.0, 7.2, 7.4, 7.6, and 7.8 mediums were (5.0±0.5), (7.6±0.9), (8.5±0.7), (11.0±0.8), and (5.3±0.8) µm, respectively, which were significantly longer than (2.8±0.8) µm in pH 6.4 medium (P<0.05); the lumen diameters of tubes formed by cells cultured in pH 6.6 ((4.2±0.3) µm), 6.8 ((4.5±0.6) µm), 7.0, and 7.8 mediums were significantly shorter than the diameter in pH 7.6 medium (P<0.05). After another 48 h of culture, compared with those in pH 6.4 and 6.6 mediums, the protein expressions of Akt phosphorylation sites 473 and 308 of cells cultured in pH 6.8 to 7.8 mediums were significantly increased (P<0.05). Moreover, the protein expression of Akt phosphorylation site 308 of cells cultured in pH 6.6 medium was significantly higher than that in pH 6.4 medium (P<0.05); compared with the expression in pH 6.8 medium, the protein expressions of Akt phosphorylation site 473 of cells cultured in pH 7.0 and 7.4-7.8 mediums were significantly increased (P<0.05); compared with the expression in pH 7.6 medium, the protein expressions of Akt phosphorylation site 473 of cells cultured in pH 7.0-7.4 and 7.8 mediums were significantly decreased (P<0.05); compared with the expression in pH 7.8 medium, the protein expressions of Akt phosphorylation site 308 of cells cultured in pH 7.0 to 7.6 mediums were significantly increased (P<0.05). Conclusions: pH value can regulate the lumen diameter of HDMEC-formed capillaries, which is closely related to the activation of Akt. 7.2-7.6 is the appropriate pH value for constructing tissue engineered capillaries.

[Clinical application of three-dimensional printed preformed titanium mesh combined with free latissimus dorsi muscle flap in the treatment of squamous cell carcinoma with skull defect in the vertex].

Objective: To explore the clinical effects of three-dimensional printed preformed titanium mesh combined with latissimus dorsi muscle flap free transplantation in the treatment of wounds with skull defect after radical surgery of squamous cell carcinoma in the vertex. Methods: A retrospective observational study was conducted. From January 2010 to December 2019, 5 patients with squamous cell carcinoma in the vertex accompanied with skull invasion who met the inclusion criteria were admitted to the Department of Burns and Plastic Surgery of the Second Affiliated Hospital of Air Force Medical University, including four males and one female, aged 50 to 65 years. The original lesion areas ranged from 5 cm×4 cm to 15 cm×8 cm. The titanium mesh was prefabricated via three-dimensional technic based on the result the scope of skull resection predicted with computerized tomography three-dimensional reconstruction before surgery. During the first stage, the soft tissue defect area of scalp (8 cm×7 cm to 18 cm×11 cm) after tumor enlargement resection was repaired with the preformed titanium mesh, and the titanium mesh was covered with latissimus dorsi muscle flap, with area of 10 cm×9 cm to 20 cm×13 cm. The thoracodorsal artery/vein was anastomosed with the superficial temporal artery/vein on one side. The muscle ends in the donor site were sutured together or performed with transfixion, and then the skin on the back were covered back to the donor site. On the 10th day after the first-stage surgery, the second-stage surgery was performed. The thin intermediate thickness skin graft was taken from the anterolateral thigh to cover the latissimus dorsi muscle flap. The duration and intraoperative blood loss of first-stage surgery were recorded. The postoperative muscle flap survival after the first-stage surgery and skin graft survival after the second-stage surgery was observed. The occurrence of complications, head appearance, and recurrence of tumor were followed up. Results: The average first-stage surgery duration of patients was 12.1 h, and the intraoperative blood loss was not more than 1 200 mL. The muscle flaps in the first-stage surgery and the skin grafts in the second-stage surgery all survived well. During the follow-up of 6-18 months, no complications such as exposure of titanium mesh or infection occurred, with good shape in the recipient sites in the vertex, and no recurrence of tumor. Conclusions: Three-dimensional printed preformed titanium mesh combined with latissimus dorsi muscle flap free transplantation and intermediate thickness skin graft cover is an effective and reliable method for repairing the wound with skull defect after extended resection of squamous cell carcinoma in the vertex. This method can cover the wound effectively as well as promote both recipient and donor sites to obtain good function and appearance.

Simvastatin induces osteogenic differentiation of MSCs via Wnt/ß-catenin pathway to promote fracture healing.

OBJECTIVE: This study was designed to investigate whether Simvastatin could facilitate osteogenic differentiation of rat marrow mesenchymal stem cells (MSCs) by modulating the Wnt/ß-catenin pathway, thus promoting fracture healing. MATERIALS AND METHODS: MSCs were isolated from rat bone marrow specimens and their purity was identified. The third generation of MSCs was cultured in osteoinduction medium containing simvastatin of gradient concentration, and the highest dose of simvastatin that did not cause cell proliferation was determined by the result of the CCK8 assay. The effects of simvastatin on osteogenic differentiation of MSCs were evaluated by ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific gene expression. Finally, Wnt pathway antagonist DKK1 and ß-catenin disturbing agent were added to MSCs to detect the ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific genes of MSCs respectively, and to evaluate whether simvastatin promoted osteogenic differentiation of MSCs by activating Wnt/ß-catenin pathway. RESULTS: After osteoinduction, simvastatin of 0.3 nmol/L was found to be the highest dose that did not induce the proliferation of MSCs. After treated with 0.3 nmol/L simvastatin for 7 days, the ALP activity of cells and the number of cell calcified nodules significantly increased. Meanwhile, the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were clearly up-regulated. However, when the MSCs were treated with DKK1 for 7 days, the ALP activity and the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were found decreased. Simvastatin markedly up-regulated the expression of the ß-catenin protein, while transfection of ß-catenin shRNA inhibited the expression of osteoblast-related genes including ALP, Runx2, OCN, and OPN. CONCLUSIONS: Simvastatin can promote the differentiation of rat MSCs into osteoblast-like cells, and its mechanism may be related to the Wnt/ß-catenin pathway.

Circulating periostin levels increase in association with bone density loss and healing progression during the early phase of hip fracture in Chinese older women.

The present study shows that hip fracture women had higher serum periostin (sPostn) levels. The elevation of sPostn is associated with bone density loss, yet fracture itself may even increase sPostn levels during early healing phase. INTRODUCTION: The study aims to quantify the associations of sPostn levels with bone density loss and the possible effect on the fracture healing. METHODS: This study enrolled 261 older women with osteoporotic hip fracture and 106 age-matched women without fracture serving as controls. Clinical features, bone mineral density (BMD), and bone turnover markers including sPostn level were measured after fracture within 2 days. Follow-up sPostn levels during 1 year after 2 days were available for 128 patients. RESULTS: Initial levels of sPostn after fracture were significantly higher in patients than controls. sPostn was correlated with BMD of femoral neck (r = -0.529, P < 0.001), ß-isomerized C-terminal crosslinking of type I collagen (ß-CTX) (r = 0.403, P = 0.008), and N-terminal procollagen of type I collagen (PINP) (r = 0.236, P = 0.042) in the entire cohort. After multivariate analysis, sPostn remained as an independent risk factor for femoral neck BMD, which explained 19.1% of the variance in BMD. sPostn sampled within 7 days after fracture were acutely increasing from day 2 and then decreased and maintained at slightly high levels at 360 days. The percentage changes of sPostn positively correlated with the variation in ß-CTX (r = 0.396, P = 0.002) and PINP (r = 0.180, P = 0.033) at day 7 after fracture. CONCLUSIONS: High sPostn levels were an independent predictor of femoral neck BMD in older women presenting with an acute hip fracture. Increased sPostn levels during early healing phase may imply that Postn play a role in bone repair.

Post-earthquake outbreak of insecticide-associated conjunctivitis in a primary school of Lixian district, China: An epidemiological investigation.

OBJECTIVES: To describe the time course and characteristics of an insecticide-associated incident and highlight potential risks from similar outbreaks that may occur in other areas to enhance the preparedness of emergency physicians and other healthcare providers to deal with the sequelae of these events. STUDY DESIGN: Outbreak investigation METHODS: From 5 to 8 August 2008, an outbreak investigation was carried out among the affected primary school located in the refugee camp area of Lixian district, Sichuan, China. Affected pupils, parents, teachers and doctors working in the local medical stations were visited. Clinical checking, clinical treatment, epidemiological investigation and environmental investigation were undertaken. RESULTS: In total, 138 individuals were diagnosed with acute conjunctivitis, characterized by inflammation of the conjunctiva and intense ocular symptoms such as redness, itching and mucus discharge. According to the results, all risk factors (i.e. drinking water, indoor air and the materials used in camp classrooms) were excluded except insecticide exposure. The characteristics of symptoms, distribution of cases and records of irregular insecticide spraying support the assumption that the conjunctivitis outbreak was associated with synthetic pyrethroid alphacypermethrin exposure. CONCLUSION: The results suggest that non-standard operating procedures in pest control could lead to disease incidents. Medical rescue teams should receive training and education in preventive techniques.