Fluocinolone Acetonide Is a Potent Synergistic Factor of TGF‐β3–Associated Chondrogenesis of Bone Marrow–Derived Mesenchymal Stem Cells for Articular Surface Regeneration

Articular cartilage repair remains a challenging problem. Based on a high‐throughput screening and functional analysis, we found that fluocinolone acetonide (FA) in combination with transforming growth factor beta 3 (TGF‐β3) strongly potentiated chondrogenic differentiation of human bone marrow–derived mesenchymal stem cells (hBMSCs). In an in vivo cartilage defect model in knee joints of immunocompromised mice, transplantation of FA/TGF‐β3–treated hBMSCs could completely repair the articular surface. Analysis of the intracellular pathways revealed that FA enhanced TGF‐β3–induced phosphorylation of Smad2 and Smad3. Additionally, we performed a pathway array and found that FA activates the mTORC1/AKT pathway. Chemical inhibition of mTORC1 with rapamycin substantially suppressed FA effect, and inhibition of AKT completely repressed chondrogenesis of hBMSCs. Inhibition of glucocorticoid receptor with mifepristone also suppressed FA effect, suggesting that FA involves binding to the glucocorticoid receptor. Comparative analysis with other glucocorticoids (triamcinolone acetonide [TA] and dexamethasone [DEX]) revealed the unique ability of FA to repair articular cartilage surgical defects. Analysis of intracellular pathways showed that the mTORC1/AKT pathway and the glucocorticoid receptor was highly activated with FA and TA, but to a lesser extent with DEX. Collectively, these results show a unique ability of FA to enhance TGF‐β3–associated chondrogenesis, and suggest that the FA/TGF‐β3 combination may be used as major inducer of chondrogenesis in vitro. Additionally, FA/TGF‐β3 could be potentially applied in a clinical setting to increase the efficiency of regenerative approaches based on chondrogenic differentiation of stem cells. © 2015 American Society for Bone and Mineral Research.     

靳三针疗法对窒息脑瘫幼鼠神经干细胞增殖与分化的实验研究

[目的]观察靳三针疗法对脑瘫大鼠神经干细胞(NSCs)增殖与分化的影响.[方法]选用SD新生大鼠,随机分为假手术组、模型组、靳三针疗法治疗组(治疗组,针刺百会、颞Ⅰ针、曲池、内关、足三里、涌泉),模型组与治疗组均采用结扎单侧颈总动脉法复制脑瘫大鼠模型,其中治疗组于手术后24 h开始针刺,共14 d.各组于造模后15 d处死大鼠制备脑组织提取液.另取健康SD新生大鼠,取大脑海马部位,分离、扩增NSCs,采用免疫细胞化学法检测NSCs特征性标记Nestin与Brdu表达,以鉴定培养的细胞及体外分化能力.培养细胞分为正常对照组,低、高浓度组(分别加入100、300μL/mL.的治疗组脑组织提取液),培养1、3、5 d后,采用免疫细胞化学法和免疫荧光法检测各组神经元样细胞和星形胶质样细胞的特征性标志物神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP)的表达,观察各组NSCs分化情况.采用流式细胞术检测各组NF、GFAP阳性细胞数,观察脑组织提取液促进NF、GFAP表达的时效和量效关系.[结果]扩增后的细胞球Nestin与Brdu均呈阳性,证实体外培养的细胞为NSCs,并处于分裂增殖旺盛期.细胞培养1、3、5 d后,NF、GFAP染色均呈阳性,且正常对照组阳性细胞少,低、高浓度组明显增多,呈浓度依赖性,表明NSCs已分化为神经元细胞和星形胶质细胞.同一时间段内低、高浓度组NF、GFAP阳性细胞数均较正常对照组显著增高(P<0.05或P<0.01),高浓度组在培养1、3、5 d后,NF、GFAP阳性细胞数也显著增高(P<0.05或P<0.01),表明治疗组脑组织提取液可促进NF、GFAP表达,且呈一定的量效和时效关系.[结论]靳三针疗法治疗脑瘫可能是通过促进NSCs的增殖和定向分化,从而达到修复脑损伤的目的.     

鱼腥草离体再生及农杆菌介导的遗传转化

【目的】建立鱼腥草离体再生系统和农杆菌介导的遗传转化方法。【方法】以鱼腥草叶片为外植体置于不同激素配比的MS培养基上,进行愈伤组织诱导和分化再生以根癌农杆菌EHA105／pcAMBIA1305．2进行转化,将外源GUS（β-葡萄糖醛糖苷酶）基因、潮霉素抗性基因转入鱼腥草,以组织化学染色法检测GUS活性,以聚合酶链反应（PCR）法鉴定基因组中的GUS基因。【结果】MS培养基中添加0．1mg／L α-夺乙酸（NAA）、2．0mg／L 6-苄基腺嘌呤（6-BA）、0．5mg/L呋喃氨基嘌呤（KT）适于以鱼腥草叶片为外植体进行离体再生,出芽率达96．7％。潮霉素作为筛选试剂,使用浓度为2．5mg／L;头孢霉素作为抑菌剂,使用浓度为250mg／L。经感染的外植体中GUS反应呈阳性的占75．0％,8周统计抗性芽比率5．5％,PCR分析表明GUS基因已整合到鱼腥草基因组中。【结论】通过鱼腥草离体培养条件的研究及根癌农杆菌介导的遗传转化,获得了较高的离体再生率,实现了鱼腥草的遗传转化。     

Pre-Pyloric Neural Electrical Stimulation Produces Cholinergically-Mediated Reverse Peristalsis in the Acute Canine Model of Microprocessor-Invoked Gastric Motility for the Treatment of Obesity

Background: Gastric Electrical Stimulation (GES) has been suggested as a new tool for the treatment of obesity. Among the known methods for GES, only Neural Gastric Electrical Stimulation (NGES) provides direct control of contractility without utilizing the spontaneously existing gastric electrical activity as an intermediary. However, only one of the established GES techniques, gastric pacing, has been described to produce retrograde peristalsis for delaying gastric emptying. The aim of this study was to explore the possibility of producing retrograde peristalsis using either single electrode set or dual electrode set NGES. Methods: 8 anesthetized dogs underwent laparotomy and implantation of 2 circumferential electrode sets approximately 3 cm and 7 cm proximal to the pylorus, respectively. Single-set and dual-set NGES sessions were repeatedly administered using a custom-designed implantable neurostimulator. Gastric motility patterns were captured using 3 force transducers implanted on the anterior gastric wall along the gastric axis. Motility indices and velocities were employed to quantify the produced contractile patterns. Results: Both single-set and dual-set NGES produced circumferential lumen-occluding contractions in the vicinity of the electrode sets. The invoked contractions propagated proximally in a retrograde fashion. The propagation scope was different depending on the number of electrode sets used. Different velocities of the invoked retrograde contractions associated with single- and dual-set NGES were observed and quantified. Contractility patterns reflected by the normalized motility indices were very similar regardless of the electrode stimulation technique. Conclusion: Pre-pyloric NGES can produce controlled retrograde peristalsis and serve as another avenue for the treatment of obesity.     

电针,神经生长因子影响周围神经再生形态与功能恢复的比较分析

采用神经原纤维镀银、髓鞘染色和前肢运动能力评价相结合的方法 ,动态观察电针、神经生长因子 (NGF)对豚鼠臂丛前索神经损伤再生的影响。结果表明 ,电针、NGF均能够加快轴突再生速度 ,NGF治疗组再生神经有髓纤维密度、轴突直径、髓鞘厚度显著优于电针组和自然恢复组 ,接近健侧水平 ;而臂丛前索运动功能指数 (BPFI)恢复却以电针组最优 ,NGF组与自然恢复组接近。     

电针治疗实验性猫脊髓损伤的组织化学及免疫组织化学法研究

用Ａｌｌｅｎ氏致伤法损伤成年猫腰１节段脊髓，伤后动物出现截瘫。随机分为两组：①电针组，伤后４小时给以电针治疗；②对照组，不作任何治疗。损伤后３天和７天，酸性磷酸酶组织化学检测，电针组该酶的含量均高于对照组，两组间存在非常明显的差异。神经中丝免疫单克隆抗体检测，伤后２周、４周电针组神经纤维再生的数量明显多于对照组。该结果提示电针促进损伤脊髓机能恢复的机理可能是促进损伤脊髓内神经的再生。     

Bio-oss骨粉在根管充填中的应用研究

目的评价Bio-oss骨粉在根尖吸收、根尖区溢脓患牙病例的根管充填中应用的效果。方法选取80例根尖吸收、根尖区溢脓的患牙,根管预备后拍X光片,分别用传统根管充填材料以及用Bio-oss骨粉糊剂进行根管充填,观察其疗效,随访。结果应用Bio-oss骨粉糊剂作为根管充填材料的患者X光片示根尖有新生骨样组织生长,根尖区阴影减少,临床症状消失;传统材料充填的患者X光片示根尖区阴影无明显改变,治疗后复发率高。结论Bio-oss骨粉在根尖吸收、根尖区溢脓患牙病例根管充填中应用,可取得较好的临床效果。     

Chitosan,Gelatin and Poly(L-Lysine) Polyelectrolyte-Based Scaffolds and Films for Neural Tissue Engineering

Biomaterial implants are a promising strategy to replace neural tissue that is lost after traumatic nerve damage. Chitosan (Ch) is a suitable material for nerve implantation when it is used at a minimum amount of 2% (w/v). The goal of this study was to determine the best mixture of 2% Ch with gelatin (G) and poly(L-lysine) (PLL) for use in neural tissue engineering. Using different physicochemical approaches we showed that all mixtures formed polyelectrolyte complexes with distinct electrostatic interactions between their compounds. This gave rise to different gel morphologies, among which Ch + G exhibited a significantly smaller pore size, unlike Ch + G + PLL. However, thermal resistance to degradation and the wettability of the Ch-based films were not affected. Additionally, these differences affected glial cells growth in long-term (14 days) cultures performed on Ch-based films. Astrocytes and olfactory ensheathing cells proliferated on G and Ch + G films which induced both flattened and spindle cell morphologies. Meanwhile, cortical and hippocampal neurons were similarly viable in all studied films and significantly lower than those observed in controls. Lastly, neurites from dorsal root ganglia extended the most on Ch + G films. These results show that a Ch + G mixture is a promising candidate for use in neural tissue engineering.     

The behavior of neural stem cells on biodegradable synthetic polymers

The biocompatibility of polymer scaffolds as neural stem cell transplantation matrices has not yet been studied extensively. In this study, we evaluated the biocompatibility of various biodegradable polymers for neural stem cells. The biocompatibility tests were performed by culturing hippocampal progenitor cells (HiB5) on films of poly(lactic-co-glycolic acid) (PLGA), poly(L-lactide-co-ε-caprolactone) (PLCL) and poly(L-lactic acid) (PLLA) or in the presence of extracts from these polymers. Specifically, the viability, mitochondrial metabolic activity, proliferation, apoptosis and neurite out-growth of HiB5 cells were examined in biocompatibility tests. Among the tested polymers, PLGA performed best with respect to cell viability, mitochondrial metabolic activity and apoptotic activity. Compared to the other polymers, PLLA showed the worst results in all categories evaluated. PLGA also showed favorable results for neurite out-growth of HiB5 cells. The results of this study demonstrate the promising biocompatibility of PLGA as a scaffold for neural stem cell transplantation for nerve regeneration.     

Biocompatibility of platinum-metallized silicone rubber: in vivo and in vitro evaluation

Silicone rubber is commonly used for biomedical applications, including implanted cuff electrodes for both recording and stimulation of peripheral nerves. This study was undertaken to evaluate the consequences of a new platinum metallization method on the biocompatibility of silicone rubber cuff electrodes. This method was introduced in order to allow the manufacture of spiral nerve cuff electrodes with a large number of contacts. The metallization process, implying silicone coating with poly(methyl methacrylate) (PMMA), its activation by an excimer laser and subsequent electroless metal deposition, led to a new surface microtexture. The neutral red cytotoxicity assay procedure was first applied in vitro on BALB/c 3T3 fibroblasts in order to analyze the cellular response elicited by the studied material. An in vivo assay was then performed to investigate the tissue reaction after chronic subcutaneous implantation of the metallized material. Results demonstrate that silicone rubber biocompatibility is not altered by the new platinum metallization method.