Effects of Panax notoginseng saponins on apoptosis induced by hydrogen peroxide in cultured rabbit bone marrow stromal cells via altering the oxidative stress level and down-regulating caspase-3

Objective： To investigate the effects of Panax notoginseng saponins （PNS） on hydrogen peroxide （H2O2）-induced apoptosis in cultured rabbit bone marrow stromal cells （BMSCs）. Methods： The effects of different concentrations of PNS on proliferation and early osteoblast differentiation of BMSCs were determined by the MTT assay and an alkaline phosphatase （ALP） assay. An optimal effective concentration of PNS was determined and used in subsequent experiments. The cultured BMSCs were divided into three groups： untreated control, H2O2 treated, and PNS pretreatment of H2O2 treated. The oxidative stress level was assessed by superoxide dismutase （SOD） and malondialdehyde （MDA） assays. Flow cytometry was used to determine BMSC apoptosis by staining with annexinV-FITC/propidium iodide （PI）. The activity of caspase-3 enzyme was measured by spectrofluorometry. Results： PNS （0.1g/L） significantly increased both BMSC proliferation rate and ALP activity, while it decreased the indicators of oxidative stress, caspase-3 activity, and the apoptosis rate of BMSCs induced by H2O2.. Conclusion： PNS, acting as a biological antioxidant, had a protective effect on H2O2-induced apoptosis in cultured rabbit BMSCs by decreasing oxidative stress and down-regulating caspase-3.

An injectable self-adaptive polymer as a drug carrier for the treatment of nontraumatic early-stage osteonecrosis of the femoral head

Core decompression(CD)with the elimination of osteonecrotic bone is the most common strategy for treating early-stage nontraumatic osteonecrosis of the femoral head(ONFH).Adjuvant treatments are widely used in combination with CD as suitable methods of therapy.Existing augmentations have to be fabricated in advance.Here,we report a novel injectable glycerin-modified polycaprolactone(GPCL)that can adapt to the shape of the CD cavity.GPCL shows great flowability at 52.6℃.After solidification,its compressive modulus was 120 kPa at body temperature(37℃).This excellent characteristic enables the polymer to provide mechanical support in vivo.In addition,GPCL acts as a carrier of the therapeutic agent zoledronic acid(ZA),demonstrating sustained release into the CD region.ZA-loaded GPCL was injected into ONFH lesions to treat early-stage nontraumatic cases.Compared to that in the CD group,CD+ZA-loaded GPCL injection preserved bone density and increased the collagen level in the femoral head.At the interface between the GPCL and CD tunnel wall,osteogenesis was significantly promoted.In addition,morphological evaluations revealed that the femoral heads in the CD+ZA-GPCL group exhibited improved pressure resistance.These results suggest a strategy effective to preserve the bone density of the femoral head,thus decreasing the possibility of femoral head collapse.This novel injectable polymer has,therefore,considerable potential in clinical applications.

Effects of recombinant adeno-associated viruses expressing human vascular endothelial growth factor 165 on spinal cord injury

Numerous studies have confirmed that vascular endothelial growth factor （VEGF） improves the function of neural cells following spinal cord injury （SCI）. However, some studies have also verified that VEGF cannot significantly induce the increase in vascular density at or surrounding the lesion, and that VEGF therapy exacerbated secondary damage following SCI. Based on the dual effects of VEGF on SCI, we constructed the recombinant adeno-associated viruses （rAAV）-hVEGF165-IRES-human recombinant green fluorescent protein （hrGFP） （AAV-VEGF） and rAAV-IRES-hrGFP （AAV-GFP）. Our results suggested that rAAV expressed hVEGFles, and a low dose of VEGF relieved increased vascular permeability, improved microcirculation in the local spinal cord, lessened spinal cord edema, and decreased neuronal apoptosis. These results verified that the releasing effects of the rAAV virus vector had protective effects on the spinal cord.

Isolation,culture,and purification of olfactory mucosa-derived olfactory ensheathing cells using modified differential attachment with low concentration serum

BACKGROUND：Studies have demonstrated that olfactory mucosa can promote the regeneration and formation of axonal medullary sheath of injured neurons. To date, olfactory ensheathing cells （OECs） utilized in basic and clinical research arise primarily from the olfactory bulb mucosa. However, little is known regarding culture, purification, and biological properties of OECs . OBJECTIVE： To isolate and culture OECs utilized modified, differential attachment in combination with neurotrophic factor 3 （NT3） and low concentration serum to explore an optimal in vitro culture method for OECs.DESIGN, TIME AND SETTING： Single-sample observation was performed at the Medical Experimental Center of Stomatology College, Xi＇an Jiaotong University between March 2006 and December 2007. MATERIALS： Twelve samples from aborted embryos, 4-6 months, were used to isolate OECs; rabbit-anti-human p75NTR and glial fibrillary acidic protein （GFAP） antibody were provided by Sigma, USA. METHODS： The differential time was six hours. This was repeated twice, based on Nash＇s differential attachment. Attached OECs were cultured in DMEM-F12 culture medium containing 10% fetal bovine serum （FBS） or 2.5% FBS and NT3. MAIN OUTCOME MEASURES： OEC morphology was observed, and p75NTR and GFAP immunocyto-chemistry was used for identification and purity detection. RESULTS： Some cells attached after three days in culture. Several cells possessed short neurites with good refractivity. Some shuttle-shaped fibroblasts could be seen. On day six, more cells attached, exhibiting a three-dimensional appearance. Many cells appeared dipolar or tripolar, with slender neurites, and fibroblasts were clustered. On day nine, the number of dipolar or tripolar cell bodies with slender neurites was increased, and fibroblasts were clustered. On day 15, fibroblasts occupied the majority of the bottom of the culture bottle, with several OECs surviving at the upper layer. OECs were positive for P75NTR and GFAP expression, as identified by an immunocytologically stained brown cell body and neurites. However, fibroblasts were P75NTR and GFAP-negative. On day 9, OEC purity reached 81%, and the number of proliferating fibroblasts significantly increased. By the end of day 12, OEC purity was reduced to 56%. CONCLUSION： Modified differential attachment, in combination with low concentration serum and NT3, removes fibroblasts and reduces OEC loss. This is an appropriate method for the isolation and culture of human fetal olfactory mucosa-derived OECs.

Establishment of a recombinant adeno-associated virus expressing hVEGF_(165)

BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION： The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.

An experiment study of osteogenesis of Ad-VEGF165 transfected human bone marrow mesenchymal stem cells in vitro

Objective ：To evaluate the effect of osteogenic potential on human marrow mesenchymal stem cells （hMSCs） transferred with human vascular endothelial growth factor（VEGF） gene by adenovirus, methods：hMSCs were isolated from human marrow, cultured in vitro and randomly divided into 3 groups ：Ad-VEGF165 group： adding 1×10^10 OPU/ml Ad-VEGF in hMSCs culture fluid after incubating 24 hours, changing into ordinary complete culture and continuing culturing; Positive control group： Cultured hMSCs with 1 nmol/L dexamethasone, 10 mmol/L glycerophosphate and 50 mg/L vitamin C ,exchanging this conditioned medium twice a week; blank control group：no special treatment but culturing hMSCs in DMEM.To evaluate osteogenesis competence, Von Kossa＇s staining and a quantitative alkaline phosphates （ALP） activity analysis were performed after 2 weeks treatment. Results：The calcified nodes formed after 2 weeks treatment in Ad-VEGF165 group and Positive control group but not in blank control group. ALP activities in Ad-VEGF165 group ,Positive control group and blank control group were （7.91 ± 0.90）u/L, （8.18 ± 0.76 u/L） and （3.46 ± 0.49）u/L respectively. The differences were no statistical significance between Ad-VEGF165 group and positive control group （P 〉 0.05）, but Ad-VEGF165 group and Positive control group were significantly different with blank control group （P 〈 0.05）. Conclusion：Adenovirus mediated VEGF165 gene can transfect hMSCs and promote osteogenesis of hMSCs.

Construction of recombinant adeno-associated virus vector co-expressing hVEGF_(165) and hBMP_7 gene

Objective： To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165（hVEGF165） and bone morphogenetic protein 7（hBMPT）, measure the virus titer and verify the recombination. Methods：The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid plRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMP7, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results：Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5 ×10^11vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion：Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.

Clinical experimental study of Arnebia Root oil in increasing FGF expression and promoting wound surface healing

Objective：To investigate the effection of Arnebia Root oil on the FGF expression in wound surface and the ability to promote wound surface healing. Methods：24 wound surfaces of patients were divided into two groups. Experimental group was treated by Arnebia Root oil and the control was treated by petrolatum gauze. Histology, histochemistry, electron microscope methods and healing rate measurement were used to show the FGF expression and wound healing process. Results：Endogenous FGF were expressed in both of the groups, in which of the experimental group was higher than that of the control group, the wound surface healing rate of experimental group was also higher and paralleled with FGF expression. Conclusion：Arnebia Root oil has effects to promote FGF expression and enhances wound surface repair. The wound healing mechanism between the action of Arnebia Root oil and function of FGF need further investigating.

高龄股骨颈骨折股骨头置换术的智能术前规划应用

目的探讨基于髋关节置换机器人辅助手术系统的智能术前规划在高龄股骨颈骨折行人工股骨头置换患者人群术中的临床意义。方法回顾性分析西安交通大学第二附属医院骨科自2019年10月至2022年2月完成的创伤后单侧股骨颈骨折行人工股骨头置换患者资料,排除病理性骨折、骨折时间大于4周及严重骨质疏松的病例。所有患者均由同一组手术医生经后外侧入路行人工股骨头置换术,均使用同一种生物固定型假体。收集患者年龄、性别、身体质量指数(BMI)、手术时间、术中出血量、是否进行术前规划,术前规划股骨柄假体型号及术中实际使用股骨柄假体型号等,在手术记录中以及术后X片中明确是否发生术中假体周围骨折,以及假体周围骨折类型等资料。按照是否进行术前规划将所有患者分为两组,利用独立样本t检验,卡方检验比较两组之间的基线资料、手术时间、术中出血量及术中假体周围骨折发生率等。结果共纳入186例患者,其中男性59例,女性127例,年龄(84±4)岁;BMI(23±4)kg/m^(2);手术时间(44±7)min;术中出血量(265±45)ml。有62例患者于术前进行规划,其中56例患者按照术前规划股骨柄型号成功植入股骨柄假体,6例患者术中实际植入假体与术前规划型号不符。186例患者中,发生术中假体周围骨折患者9例,其中术前规划组有1例发生假体周围骨折,非术前规划组有8例发生假体周围骨折。术前规划组的手术时间、手术出血量均低于非规划组(t=10.153、11.412,均为P<0.05);术前规划组术中假体周围骨折发生率低于非规划组(1.6%vs.6.5%),但差异无统计学意义(P=0.227)。结论基于机器人辅助系统的术前规划可以有效降低手术时间及术中出血量,是否能降低术中假体周围骨折的发生率还需要更大样本的研究。