Objective:To construct curved microgrooves with gradient surface roughness on polycaprolactone (PCL) members by regulating alkali etching time and to investigate the synergistic effect of surface roughness and curved microgrooves on the in vitro osteogenic differentiation of mouse pre-osteoblasts (MC3T3-E1), aiming to determine the optimal PCL surface modification strategy. Methods:Soft lithography and melt-casting techniques were used to fabricate PCL membranes with regularly arranged curved microgrooves (CMP). Alkali etching was performed for 24, 48, and 72 h. Groups: smooth PCL (control), CMP (curved microgrooves only), CMP-24 h, CMP-48 h, CMP-72 h (CMP etched for 24, 48, 72 h, respectively). Surface physicochemical properties were characterized: surface morphology was observed by scanning electron microscopy (SEM), surface roughness was measured by atomic force microscopy (AFM), and surface hydrophilicity was evaluated by contact angle measurement. MC3T3-E1 cells were cultured in vitro. Cell adhesion, proliferation, and osteogenic differentiation were assessed using cell counting (CCK-8), immunofluorescence staining, alkaline phosphatase (ALP) and Alizarin red staining with quantification. The mRNA expression levels of osteogenesis-related genes [ALP, collagen type Ⅰ (COL-1), Runt-related transcription factor 2 (RUNX-2), osteocalcin (OCN), osteopontin (OPN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). Results:Curved microgrooves were successfully fabricated on PCL membranes. Alkali treatment improved surface hydrophilicity and increased roughness. The CMP-72 h group exhibited the best hydrophilicity (contact angle: 33.2°±5.5°), with significant differences compared to all other groups (all P<0.05). The CMP-72 h group showed the highest roughness [(59.966±4.729) nm], which was significantly different from all other groups (all P<0.05). CCK-8 results on day 5 showed that both curved microgrooves and roughness promoted cell proliferation: CMP-24 h (0.292±0.003) and CMP-72 h (0.383±0.004) were significantly higher than those in the smooth group (0.270±0.005) (all P<0.05). Immunofluorescence staining revealed that curved microgrooves induced significant contact guidance of cells; this effect weakened with increasing etching time. ALP and Alizarin red staining indicated the deepest osteogenic staining in the CMP-48 h group. Both ALP activity (0.013 021±0.000 032) and Alizarin red quantification (0.290±0.003) were highest in the CMP-48 h group, significantly different from all other groups (all P<0.05). RUNX-2 expression in CMP-24 h and CMP-48 h groups (1.845±0.087 and 1.837±0.027, respectively) was significantly higher than in other groups (all P<0.05), with no significant difference between these two groups ( P>0.05). CMP-48 h group exhibited the highest mRNA expression of all osteogenic genes tested, specifically ALP (2.194±0.028), COL-1 (1.983±0.024), OCN (7.644±0.156), and OPN (2.648±0.031), all significantly greater than other groups (all P<0.05). Conclusions:Both curved microgrooves and surface roughness modification enhance the in vitro osteogenic differentiation of cells on PCL membranes. Among the tested strategies, alkali etching of curved microgrooves for 48 hours (CMP-48h) provided the optimal enhancement of osteogenic capability for MC3T3-E1 cells and represented a promising surface modification strategy for future PCL membranes.

Objective:To construct curved microgrooves with gradient surface roughness on polycaprolactone (PCL) members by regulating alkali etching time and to investigate the synergistic effect of surface roughness and curved microgrooves on the in vitro osteogenic differentiation of mouse pre-osteoblasts (MC3T3-E1), aiming to determine the optimal PCL surface modification strategy. Methods:Soft lithography and melt-casting techniques were used to fabricate PCL membranes with regularly arranged curved microgrooves (CMP). Alkali etching was performed for 24, 48, and 72 h. Groups: smooth PCL (control), CMP (curved microgrooves only), CMP-24 h, CMP-48 h, CMP-72 h (CMP etched for 24, 48, 72 h, respectively). Surface physicochemical properties were characterized: surface morphology was observed by scanning electron microscopy (SEM), surface roughness was measured by atomic force microscopy (AFM), and surface hydrophilicity was evaluated by contact angle measurement. MC3T3-E1 cells were cultured in vitro. Cell adhesion, proliferation, and osteogenic differentiation were assessed using cell counting (CCK-8), immunofluorescence staining, alkaline phosphatase (ALP) and Alizarin red staining with quantification. The mRNA expression levels of osteogenesis-related genes [ALP, collagen type Ⅰ (COL-1), Runt-related transcription factor 2 (RUNX-2), osteocalcin (OCN), osteopontin (OPN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). Results:Curved microgrooves were successfully fabricated on PCL membranes. Alkali treatment improved surface hydrophilicity and increased roughness. The CMP-72 h group exhibited the best hydrophilicity (contact angle: 33.2°±5.5°), with significant differences compared to all other groups (all P<0.05). The CMP-72 h group showed the highest roughness [(59.966±4.729) nm], which was significantly different from all other groups (all P<0.05). CCK-8 results on day 5 showed that both curved microgrooves and roughness promoted cell proliferation: CMP-24 h (0.292±0.003) and CMP-72 h (0.383±0.004) were significantly higher than those in the smooth group (0.270±0.005) (all P<0.05). Immunofluorescence staining revealed that curved microgrooves induced significant contact guidance of cells; this effect weakened with increasing etching time. ALP and Alizarin red staining indicated the deepest osteogenic staining in the CMP-48 h group. Both ALP activity (0.013 021±0.000 032) and Alizarin red quantification (0.290±0.003) were highest in the CMP-48 h group, significantly different from all other groups (all P<0.05). RUNX-2 expression in CMP-24 h and CMP-48 h groups (1.845±0.087 and 1.837±0.027, respectively) was significantly higher than in other groups (all P<0.05), with no significant difference between these two groups ( P>0.05). CMP-48 h group exhibited the highest mRNA expression of all osteogenic genes tested, specifically ALP (2.194±0.028), COL-1 (1.983±0.024), OCN (7.644±0.156), and OPN (2.648±0.031), all significantly greater than other groups (all P<0.05). Conclusions:Both curved microgrooves and surface roughness modification enhance the in vitro osteogenic differentiation of cells on PCL membranes. Among the tested strategies, alkali etching of curved microgrooves for 48 hours (CMP-48h) provided the optimal enhancement of osteogenic capability for MC3T3-E1 cells and represented a promising surface modification strategy for future PCL membranes.

Objective:To establish training system for postoperative delirium (POD) assessment and evaluate the efficacy of training for anesthesia nurses.Methods:Sixteen nurse anesthetists of both sexes in our hospital were selected and received the systemic training for POD assessment.The training system included questionnaire survey, theoretical teaching, simulated visit, clinical observation, independent evaluation, centralized question-answering, evaluation of efficacy and random inspection.The level of POD knowledge tests were performed before the training and at the end of the fourth week of independent evaluation, respectively.At week 1 and 4 of independent evaluation, the diagnostic rate of POD and sensitivity and specificity of the assessment were calculated, and Kappa consistency analysis was used to assess the consistency between anesthesia nurses and training group in diagnosis of POD.In the first week of the third month after the end of training, the evaluation results were randomly inspected, the POD diagnosis rate was calculated between the anesthesia nurses and the training group, and the consistency analysis was conducted.Results:Compared with the scores of POD knowledge questionnaire and sensitivity of the assessment of the anesthesia nurses in the first week of training, the scores were significantly increased ( P<0.05), and no significant change was found in the POD diagnosis rate in the fourth week of training ( P>0.05). Compared with the training group, the diagnosis rate of POD of anesthesia nurses was significantly decreased in the first week of training ( P<0.05), and no significant change was found at the fourth week of training ( P>0.05). In the first and fourth weeks of training, the Kappa value of anesthesia nurses and the training group was 0.676 and 0.954 ( P<0.001), respectively.In the first week of the third month after the end of training, the Kappa value between anesthesia nurses and the training group in diagnosis of POD was 0.862 ( P<0.05). Conclusion:The training system of POD assessment has been successfully established, and the standardized anesthesia nurses training of POD has been achieved with good results.

Objective To investigate the high-risk factors of stroke in Anqing area, and to analyze the value of standard treatment for the intervention of high-risk population for stroke. Methods A total of 3 062 permanent residents over 40 years of age in Anqing were surveyed by a questionnaire for the high-risk population of stroke. Physical examination was carried out for people at a high risk of stroke. The physical examination included general physical examination, laboratory tests, and carotid artery color Doppler ultrasound examination. People at a high risk of stroke were investigated on whether or not they received standardized symptomatic treatment and prevention interventions. Results Hypertension or taking antihypertensive drugs accounted for the highest risk factors of stroke in Anqing area, followed by smoking and seldom physical exercise. The abnormal rates of body mass index, blood pressure, contents of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL) , homocysteine (HYC), and blood glucose (GLU), as well as intimal thickening, plaque and carotid artery stenosis in people at high risk of stroke receiving standardized treatment were significantly lower than those in people receiving no standardized treatment, and the difference was statistically significant (P<0.05). The standard treatment was an independent factor influencing the body mass index, blood pressure, TC, TG, LDL, HDL, HYC, GLU, intimal thickening, plaque and carotid artery stenosis (P<0.05). Conclusion Hypertension accounted for the highest proportion of high-risk factors for stroke in Anqing area. Standardized symptomatic treatment can effectively control the physical indicators, laboratory test indicators and carotid artery state of stroke high-risk populations, which can play a positive role in the prevention of stroke.

Objective To observe the effects ofLiuwei Dihuang Decoction on cAMP and PDE3B in adipose tissues of rats with type 2 diabetes;To explore its mechanism.MethodsTotally 80 SD rats were randomly divided into two groups:control group and molding group. Rats in the molding group established the type 2 diabetic models by feeding high sugar and fat diet combined with intraperitoneal injection of Streptozotocin. The successful modeling rats were divided into model group, Rosiglitazone group, andLiuwei Dihuang Decoction group. Administration groups were given relevant medicine for gavage, while model group and blank group were given the same amount of normal saline for 30 days. FBG levels were detected, and cAMP and INS levels were detected by ELISA. The protein expressions of PDE3B in adipose tissues were determined by Western blot. The mRNA expressions of PDE3B in adipose tissues were detected by RT-PCR.Results Compared with the model group, the FBG and INS levels in type 2 diabetic rats were reduced in Rosiglitazone group andLiuwei DihuangDecoction group (P<0.05,P<0.01). InLiuwei Dihuang Decoction group, the protein and mRNA expressions of PDE3B in the adipose tissues were higher, and cAMP levels were lower than the model group (P<0.01).ConclusionLiuwei Dihuang Decoction can increase PDE3B expression, and decrease cAMP level, which may be one of the mechanisms for improving insulin resistance in type 2 diabetic rats.

BACKGROUND:Expanded polytetrafluoroethylene is a kind of porous polymer materials which is commonly used as clinical implants, and it has good biocompatibility, and is not easy to deformation or metamorphism. There is no existence of inflammation absorption reaction, and it al ows the cel migration and tissue ingrowth.
OBJECTIVE:To study the biocompatibility of expanded polytetrafluoroethylene scaffold and human adipose-derived stem cel s.
METHODS:The passage 4 human adipose-derived stem cel s were co-cultured with expanded polytetrafluoroethylene scaffold in vitro. The morphology and function of cel s adhered to the scaffold were observed by inverted phase contrast microscope, and cel adhesive rates and proliferation rates were also calculated by MTT assay.
RESULTS AND CONCLUSION:The inoculated cel s were round and bright, distributed on the surface of scaffolds uniformly, with good cel viability. After 3 hours a large number of adherent cel s were observed from the micrograph;after 24 hours there were a smal amount of short-spindle adipose-derived stem cel s. After cultured for 3 days, the short fusiform or polygon cel s could be seen clearly. After cultured for 7 days, the number of cel s increased significantly, few cel s fel off from the scaffold, and cel adhesion rate was up to an average of 95.7%. Meanwhile, the cel s revealed normal splitting proliferation rate. These findings indicate that human adipose-derived stem cel s are able to attach, grow and proliferate wel on the scaffold. Expanded polytetrafluoroethylene reveals excel ent cel ular compatibility and can be used as a vehicle for adipose tissue engineering.

Objective To investigate the expression and purification I278T-mutant human cystathionineβsynthase(CBS) in E . coli .Methods Site-directed mutagenesis by overlap extension using the polymerase chain reaction (PCR) was employed to construct mutant plasmids pGEX4T-1-CBS(I278T) ,which was induced and expressed in a medium containing 3% ethanol ,purified by affinity chromatography to obtain mutated CBS (I278T) protein .The activity ,UV-visible absorption spectroscopy ,protein particle size and Zeta potential of the purified protein were measured .Results Plasmid pGEX4T-1-CBS(I278T) was successfully constructed .The yield ,the specific activity and activity recovery of purified mutant CBS (I278T ) protein were 2 .3 mg/L ,21 .4 U/mg and 22 .6% .S-adenosylmethionine(AdoMet) with final concentration of 1 mmol/L showed no activation toward mutant CBS (I278T) protein .Ac-cording to UV-visible absorption spectroscopy analysis ,purified mutant CBS(I278T) had characteristic absorption peaks at 429 nm and 550 nm for heme-binding proteins .Protein average particle size was 7 .5 -10 .1 nm ,mainly in the form of tetramers ,and Zeta potential was - 16 .3 mV .Conclusion The methods of expression ,purification and identification of I278T-mutant human cystathionineβsynthase in E .coli were successfully established .

BACKGROUND:The establishment of a good blood supply is a key mechanism for successful implantation of engineered tissues. OBJECTIVE:To observe the effect of basic fibroblast growth factor on the migration of human adipose-derived stem cells via implanting the human adipose-derived stem cells and sodium hyaluronate composite graft at the subcutaneous site of BALB/C mice, in order to explore an optimal scheme for soft tissue reconstruction. METHODS:Human adipose-derived stem cells were isolated from the adipose tissue of healthy cosmetic patients which received liposuction, and the cells were subcultured. Then 5×109/L passage 3 cellsuspension labeled by cm-dil was prepared. The working solution containing 2 mg/L basic fibroblast growth factor was prepared. Composite tissue al-lografts which were the mixtures of 0.25 mL sodium hyaluronate, 0.2 mL cellsuspension and 0.05 mL working solution or DMEM were implanted into the subcutaneous site of both sides of the mouse back. Specimens were taken at 6 weeks after operation and were evaluated histological y after hematoxylin-eosin and vascular immunofluorescent staining. RESULTS AND CONCLUSION:No necrosis, liquefaction, nodular tissue or gel remained in operated position. The hematoxylin-eosin staining showed the main components of the specimens were the adipose tissue and the loose connective tissue. The immunofluorescence staining showed the overlaps between the cm-dil fluorescence from human adipose-derived stem cells and the FITC fluorescence from the vascular endothelium in the experimental group were more than those in the control group (P<0.05). Basic fibroblast growth factor promotes the migration and the differentiation of human adipose-derived stem cells in the sodium hyaluronate scaffold into vascular endothelium.

Objective To study the effect of the exogenous recombinant human FGF-basic with different concentrations upon inducing human adipose-derived stem cells (hASCs) to differentiate into adipose cells,and the optimum concentration of exogenous rh-bFGF by experimental research.Methods hASCs were isolated and extracted by enzymatic digestion from the liposuction aspirate.hASCs using adipogenic supplement were divided into experimental group and blank group:the experimental group of adipogenic supplement was divided into adding the exogenous rh-bFGF 10 ng/ml,20 ng/ml and 40 ng/ml,the blank group of adipogenic supplement was cultured without exogenous rh-bFGF.MTT method was used to detect the adipocytes proliferation.The oil red O staining was used in the qualitative analysis on the time of newly forming adipocyte cells.Western blot was used to detect the effects of rh-bFGF on the expression of lipid droplets surface protein CIDEC at different stages during the culture.Results The experimental group could obviously shorten the period of inducing hASCs to differentiate into adioicytes,and promote the proliferation of adipocytes.The formation rate and the proliferation of adipocytes in the group adding 40 ng/ml rh-bFGF were superior to those in the experimental group else and blank group.The average time of the newly formed lipid droplets by adding 40 ng/ml rh-bFGFwas (11.5±1.9)h.The average absorbance of cell proliferation by adding 40ml rh-bFGF was 0.52 ±0.10.The CIDEC expression quantity of adding 40 ng/ml rh-bFGF group was also superior to that in the experimental group and blank group.Conclusions rh-bFGF in hASCs adipogenic supplement could promote the proliferation of adipocytes and dramatically accelerates the program of hASCs differentiating to adipocytes,in which the optimum concentration of rh-bFGF is 40ng/ml.