预防硬膜外瘢痕粘连的实验研究

目的通过动物实验观察几丁糖对预防椎板切除术后硬膜瘢痕粘连的作用。方法24只成年山羊行腰2腰4短节段椎弓根钉系统固定及取自体髂骨行后外侧植骨融合术,腰3水平全椎板切除,切除口约3cm×1.5cm,腰3硬膜外分别放置几丁糖或空白对照各12只。于术后24周进行大体形态、组织学观察,并进行硬膜外瘢痕粘连程度分级。结果几丁糖组硬膜与瘢痕无明显粘连,空白对照组硬膜与瘢痕紧密粘连。结论几丁糖能有效地预防椎板切除术后硬膜外瘢痕粘连。     

载报告基因壳聚糖纳米粒制剂体外转染活性考察

目的 考察壳聚糖纳米粒体外基因转染活性及壳聚糖纳米粒经表面修饰的体外转染活性变化。方法 用复凝聚法制备纳米粒，透射电镜观察粒子形态，体外基因转染实验评价纳米粒的体外转染活性，用倒置荧光显微镜观察和流式细胞仪测定转染结果。通过考察递送不同剂量的基因和转染后不同时间的转染效率，寻找本递送系统较好的转染条件。用纳米粒表面连接PEG以及半乳糖基白蛋白，对纳米粒进行修饰，通过体外转染实验，评价表面修饰对其转染活性的影响。结果 未经表面修饰的载基因纳米粒能够转染人胚胎肾细胞(HEK293)和肝癌细胞(HepG2)，但转染效率仍不如脂质体转染试剂，且在转染实验后约72h较高，递送基因较佳的剂量是4μg，在以上两种细胞中，转染效率也不同。经PEG表面修饰的纳米粒仍保持纳米粒的体外转染活性。连接半乳糖基牛血清白蛋白的纳米粒转染活性却反而略有下降。结论 壳聚糖纳米粒能将基因递送到细胞内，并且报告基因能在细胞内表达。因此，可以用作基因递送的载体系统，值得进一步研究。经PEG表面修饰和冷冻干燥处理,保持生物活性．为基因药物制剂化的可能性提供了证据。对于靶向配基的选择．宜继续进行筛选。     

Effectiveness of tissue engineered based platelet gel embedded chitosan scaffold on experimentally induced critical sized segmental bone defect model in rat

BackgroundHealing and regeneration of large bone defects are a challenging problem for reconstructive orthopedic surgeons.PurposeThis study investigated the effectiveness of chitosan scaffold (CS), platelet gel (PG) and their combination (CS-PG) on healing process of an experimentally induced critical sized segmental bone defect model in rat.MethodsFifty bilateral defects were created in the mid diaphysis of the radial bones of 25 Sprague-Dawley rats. The animals were randomly divided into five equal groups. The bone defects were either left untreated or treated with corticomedullary autograft, CS, PG or CS-PG. Plain radiographs were provided from the radial bones on weeks 2, 5, and 8 after injury. In addition, clinical examinations were done for the healing radial bones. The animals were euthanized after 8 weeks of injury, and their harvested samples were evaluated by gross morphology, histopathology, scanning electron microscopy, CT-scan, and biomechanical testing.ResultsCompared with the defect group, the PG and autograft treated bone defects had significantly superior radiological scored values, bone volume and biomechanical performance which had positive correlation with their superior gross pathological, histopathological and ultra-structural features. Compared with the untreated defects, the PG and CS-PG treated defects showed significantly superior structural and functional properties so that PG had the highest value. In addition, CS had low value in bone regeneration. Although combination of CS and PG improved the healing efficacy of the CS, this strategy reduced the ability of PG to increase osteoconduction and osteoinduction during bone regeneration.ConclusionApplication of PG alone enhanced bone healing and can be regarded as a promising option for bone tissue engineering in clinical settings. Chitosan was not effective in bone reconstruction surgery and further investigations should be conducted to find a suitable carrier for PG.     

Repair of bone defect in femoral condyle using microencapsulated chitosan, nanohydroxyapatite/collagen and poly(L-lactide)-based microsphere-scaffold delivery system

Bone repair ability of microencapsulated chitosan, nanohydroxyapatite/collagen (nHAC), and poly(L-lactide) (PLLA)-based microsphere-scaffold delivery system was investigated in present research, with nHAC/PLLA composite scaffold as a control. Chitosan microspheres (CMs) encapsulated with bone morphogenetic protein-2-derived synthetic peptide were incorporated into nHAC and PLLA-based matrix via a thermally induced phase separation method, in which dioxane was used as the solvent for PLLA. Compared with the rapid release from CMs, the synthetic peptide was delivered from CMs/nHAC/PLLA microsphere-scaffold composite in a temporally controlled manner, depending on the degradation of both incorporated CMs and PLLA matrix. MC3T3-E1 osteoblastic cells were seeded into nHAC/PLLA and CMs/nHAC/PLLA scaffolds, respectively, and in vitro cytocompatibility was tested by scanning electron microscopy and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results indicated that, with the appearance of CMs in microsphere-scaffold composite, the osteoblasts exhibit better morphology and proliferation ability. In vivo tissue compatibility was evaluated by transplanting the scaffolds into rabbit femoral condyles with a defect 6 mm in diameter. After implanting for 4, 8, and 12 weeks, respectively, radiographic and histological observation revealed that the CMs/nHAC/PLLA composite can accelerate the regeneration of cancellous bone defect as compared with the nHAC/PLLA scaffold. The results demonstrated that the CMs/nHAC/PLLA possesses better biocompatibility, which should be attributed to both the incorporated chitosan component and the encapsulated bioactive synthetic peptide. The promising CMs/nHAC/PLLA microsphere-scaffold composite can be used as delivery system for multiple bioactive factors or as inductive implant scaffold for bone regeneration.     

透明质酸修饰壳聚糖/pDNA纳米微球的制备及体外转染软骨细胞

目的 以透明质酸(HA)修饰基因载体壳聚糖(CS)纳米微球,制作一种新型的HA/CS/pDNA基因转染系统,观察其结构特征及体外对软骨细胞的转染能力.方法 将不同比例的HA和CS与负载增强型绿色荧光蛋白基因(EGFP)的质粒DNA(pDNA)以复凝聚法制成纳米微球,以扫描电镜检测纳米微球形态,Zeta电位粒度分析仪测定其粒径、表面电位;凝胶电泳阻滞实验观察CS和pDNA的结合力;体外转染兔关节软骨细胞,以流式细胞仪及荧光显微镜检测转染效率.结果 HA/CS/pDNA纳米微球多呈球形,粒径为(223±51)nm,表面Zeta电位为(17.4±6.1)mV,可有效保护pDNA免受 DNaseⅠ的降解.体外转染实验证明HA/CS/pDNA纳米微球能介导pEGFP转染软骨细胞并在细胞内表达绿色荧光蛋白,转染率达(15.450±0.404)%,比裸pDNA组和CS/pDNA组有更高的转染效率(P<0.05).结论 复凝聚法制备的HA/CS/pDNA纳米微球是一种有效的新型非病毒基因转染系统,对软骨细胞有着潜在的靶向基因转染能力.

Abstract：

Objective To prepare hyaluronic acid (HA)-modified chitosan (CS)/pDNA (HA/CS/pDNA) nanoparticles as novel gene vectors, study their structural characteristics and gene transfection efficiency for chondrocytes in vitro in rabbits. Methods The HA/CS/pDNA nanoparticles were prepared by a DNA (pDNA), which load enhanced green fluorescent protein (EGFP) gene. The morphology of the nanoparticles was observed under the transmission electron microscopy. The sizes and zeta-potentials of the nanoparticles were measured by a Marven-nano laser diffractometer. The binding of pDNA was evaluated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed with the chondrocytes of rabbits. The gene transfection efficiency was measured by using flow cytometry and under the fluorescence microscopy. Results HA/CS/pDNA nanoparticles were mainly spherical, with an average size of (223±51) nm, and zeta-potential of (17.4±6.1) mV. The agarose gel electrophoresis analysis confirmed that they could effectively protect pDNA from degradation against DNase I. Gene transfection in vitro proved that HA/CS/pDNA nanoparticles could be efficiently transfected into chondrocytes of rabbits, the expression of green fluorescent proteins was observed under the fluorescent microscopy, and the transfection efficiency reached as high as (15.450±0.404)%, significantly higher than that of the naked pDNA or the CS/pDNA nanoparticles (P<0.05). Conclusion HA/CS/pDNA nanoparticles were an effective novel non-viral gene transfer vector, which possessed the potential targeting transfection ability on chondrocytes.     

几丁糖在兔角膜碱烧伤中的抗氧化损伤作用

目的 探讨20g/L医用几丁糖在角膜碱烧伤中对脂质过氧化损伤、炎症反应的影响。方法 以15只新西兰大白兔制作兔角膜碱烧伤模型，设立几丁糖治疗组（右眼）和对照组（左眼）。于烧伤后第7，14，28天各取两组5只兔的角膜和房水检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量，并观察角膜组织病理学变化。结果 医用几丁糖治疗组较对照组同期SOD活性高，MDA生成量少，角膜炎症反应轻。结论 几丁糖在角膜碱烧伤急性期治疗中具有较强的抗脂质过氧化损伤、抑制炎症及促进愈合的作用。     

FK506-壳聚糖膜与体外培养雪旺细胞生物相容性的研究

目的 探讨FK506-壳聚糖膜片与培养的雪旺细胞生物相容性.方法 将生长状况和纯度较好的第3代雪旺细胞,分别传代接种于含有FK506-壳聚糖膜片、单纯壳聚糖膜片的培养皿中培养,以盖玻片作为对照组.计算3组雪旺细胞倍增时间、雪旺细胞纯度;用MTT法比较不同膜片上雪旺细胞的生长活力,绘制生长曲线;S-100免疫荧光染色.结果 雪旺细胞倍增时间对照组为5.9 d,单纯壳聚糖膜片及FK506-壳聚糖膜片组均为4.0 d;雪旺细胞纯度分别为80%、89%,93%;雪旺细胞增殖情况以FK506-壳聚糖组最佳,单纯壳聚糖组次之,对照组最差;各组雪旺细胞均呈S-100阳性,FK506-壳聚糖组与单纯壳聚糖组雪旺细胞排列呈典型的漩涡状或栅栏状.结论 FK506-壳聚糖膜片具有更显著的组织相容性与生物功能性,并保持了雪旺细胞的生物学特性.     

重组人骨形态发生蛋白-2壳聚糖纳米微球的制备及检测

目的 制备负载重组人骨形态发生蛋白-2(rhBMP-2)壳聚糖纳米微球,并检测其粒径、形态、降解及药理特性,以评估壳聚糖纳米微球作为rhBMP-2缓释载体的可行性.方法 以壳聚糖为原料、三聚磷酸钠为交联剂,通过离子交联法制备负载rhBMP-2壳聚糖纳米微球,应用透视电镜观察微球的形态、激光粒径,分析其粒径分布、溶菌酶降解,了解降解特性.通过酶联免疫吸附实验(ELISA)检测rhBMP-2壳聚糖微球的载药率、包封率和释药规律.结果 离子交联法制备的壳聚糖纳米微球,平均粒径大小为230nm,成球性较好,包封率和载药率分别为(66.867±4.575)％、(33.437±2.290)μg/mg;体外释药试验rhBMP-2可以从壳聚糖纳米微球中缓慢释放,释放行为符合双向动力学规律,整个释放过程可达30 d.结论 离子交联法可成功制备壳聚糖纳米微球并具有缓释rhBMP-2的能力,为进一步应用于骨组织工程研究提供实验依据.     

壳聚糖微球转染人IL-1Ra与TGF-β1基因治疗兔骨关节炎

目的 探讨壳聚糖微球转染人IL-1Ra与TGF-β1基因治疗兔膝关节早期骨关节炎的方法与效果.方法 制备分别包裹IL-1Ra质粒DNA和TGF-β1质粒DNA的壳聚糖微球缓释系统,分组向兔膝关节早期骨关节炎模型关节腔注射含IL-1Ra基因和(或)TGF-β1基因的壳聚糖微球、不含上述基因的壳聚糖溶液,定期处死,使用ELISA检测关节腔灌洗液的IL-1Ra、TGF-β1浓度,取关节标本Mankin评分、苏木精-伊红(HE)染色、番红O染色及免疫组化检测.结果 经过60 d观察,壳聚糖微球转染基因组的IL-1Ra与TGF-β1的基因可持续表达,双基因组的软骨标本从外观、染色观察,损伤程度轻于单基因组.双基因组的Mankin评分明显低于单基因组(P＜0.05),单基因组的Mankin评分明显低于空白组(P＜0.05).结论 关节腔注射壳聚糖微球转染IL-1Ra与TGF-β1基因能抑制软骨的退变和促进软骨的修复.

Abstract：

Objective To explore the method and effect of transinfection of rabbit early knee osteoarthritis models via chitosan microsphere with gene of recombined human IL-1Ra gene and TGF-β1 gene. Methods Chitosan microspheres with plasmids of IL-1Ra gene and TGF-β1 gene, and rabbit early knee osteoarthritis models were prepared. Rabbits in different groups had intra-articular injections of chitosan microsphere containing IL-1Ra gene and / or TGF-β1 gene, and chitosan solution as control group before being executed regularly and randomly. The joint specimens were evaluated by HE staining, lycopene red O staining and immunohistochemical analysis and Mankin's score. ELISA was used for detection of IL-IRa and TGF-β1 concentration of articular cavity fluid in each group. Results The control group was consistent with the pathological changes of early OA. In co-transinfection group, judging from the appearance and staining of cartilage,the OA damage of the specimens was less serious than other groups'. Its Mankin's score was significantly lower than single-gene transinfection group (P ＜ 0.05), and the latters Mankin's score were significantly lower than control group (P ＜ 0.05). Conclusion Intra-articular injection of chitosan microspheres containing both IL-1Ra gene and TGF-β1 gene could inhibit the degeneration of cartilage and promote cartilage repair.     

关节假体表面壳聚糖季铵盐涂层的抗感染性能研究

目的 研制一种抑制细菌黏附和生物膜形成,同时具有良好骨生物活性的材料,用于关节假体表面涂层,以降低人工关节置换术后的感染率.方法 合成6%,18%和44%取代度的壳聚糖季铵盐,利用金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌和表皮葡萄球菌检测其抗菌性能和对生物膜形成的影响,利用骨髓间充质干细胞检测其骨生物活性.共价交联法在钛金属表面制备壳聚糖季铵盐涂层.SD大鼠股骨下端髓腔内注射表皮葡萄球菌后分组植入涂层和未涂层钛棒,通过观察术后体温和体重变化,并进行血常规、X线摄片、微生物学及组织学检测分析涂层的抗感染性能.结果 壳聚糖季铵盐的抗菌活性随着其取代度的升高而增加;取代度为18%的壳聚糖季铵盐对于干细胞具有良好的生物相容性;而取代度为44%的壳聚糖季铵盐具有一定的细胞毒性.动物实验表明,取代度为18%的壳聚糖季铵盐涂层能有效防止表皮葡萄球菌引起的骨内感染.结论 中等取代度（18%）壳聚糖季铵盐兼具较强的抑菌能力和较好的骨生物活性,具有作为关节假体表面抗感染涂层的应用前景.