新型有序纳米介孔生物活性玻璃的不同浓度浸提液对成骨细胞影响的研究

目的评价新型有序纳米介孔生物活性玻璃（80SMBG）的浸提浓度对成骨细胞的影响。方法采用大鼠成骨细胞体外培养,按细胞增殖度法,取0.25%、0.5%、1%、2%、4%共五种浓度的80SMBG浸提培养液进行检测,分别于3、4、5天用倒置显微镜观察细胞生长情况;用四唑盐（MTT）比色法计数计算细胞相对增殖度,用6级毒性分类法评级;用偶氮偶联法（PNPP）染色,分析碱性磷酸酶活性的变化。结果 80SMBG浸提液在高浓度时轻度抑制细胞增殖,促进细胞分化;低浓度促进细胞增殖,轻度抑制细胞分化。结论 80SMBG的体外生物活性较高,对体液微环境的影响轻微,细胞相容性好,作为骨组织修复替代材料或骨组织工程支架材料具有较高的研究和应用价值。     

新型有序纳米介孔生物活性玻璃在模拟体液中的表面生物活性研究

目的评价新型行序纳米介孔生物活性玻璃（80SMBG）的体外生物活性及其对体液环境的影响。方法以Novabone为对照,利用模拟人体体液体外浸泡实验（SBF）的方法和扫描电镜（SEM）、等离子发射光谱仪（ICP）pH仪等技术,比较80SMBG与Novabone体外生成类似于天然骨中无机矿物的羟基磷灰石（HA）的能力,分析SBF中各离子浓度、pH值的变化情况。结果80SMBG在SBF中浸泡4h表面即有羟基磷灰石（HA）生成,浸泡1～4hSBF中Ca、P、Si的离子浓度迅速达到峰值,8h后逐渐降低直至稳定,pH仅在7．40～7．65范围内变化。结论80SMBG的体外生物活性更高,对体液微环境的影响更轻微,作为骨组织修复替代材料或骨组织工程支架材料具有较高的研究和应用价值。     

医用新型Mg-Li-Ca合金材料体外生物相容性及生物活性评价

背景:新型Mg-Li-Ca合金是否具有生物相容性和生物活性,目前还未被证实。目的:通过体外实验评价新型医用Mg-Li-Ca合金的组织相容性及生物活性,初步探讨其作为医用植入材料的可行性。方法:分别采用Mg-Li-Ca合金浸提液、纯Mg浸提液、AZ31B合金浸提液与α-MEM培养液培养第3代小鼠胚胎成骨细胞,培养1,3,5 d,采用MTT法检测细胞A值并计算相对增殖率;培养5,7 d,使用碱性磷酸酶试剂盒检测细胞碱性磷酸酶活性。将第3代小鼠胚胎成骨细胞与Mg-Li-Ca合金、纯Mg、AZ31B合金分别共培养于24孔板,培养1 d后扫描电镜观察材料表面黏附及增殖情况。结果与结论:Mg-Li-Ca合金、纯Mg和AZ31合金对成骨细胞的细胞毒性为Ⅰ级,无细胞毒性,且Mg-Li-Ca合金对细胞毒性明显小于纯Mg和AZ31B合金,对成骨细胞生长增殖无显著影响,呈现出良好的生物相容性。Mg-Li-Ca合金和纯Mg对成骨细胞正常合成碱性磷酸酶无影响,具有良好的生物活性;AZ31B对成骨细胞正常合成碱性磷酸酶有显著影响。成骨细胞可在Mg-Li-Ca合金、纯Mg和AZ31合金表面正常黏附生长。表明新型Mg-Li-Ca合金有望成为新型骨科植入材料。     

纳米羟基磷灰石/细菌纤维素复合材料及其降解物的细胞相容性评价

背景:新型复合材料纳米羟基磷灰石/细菌纤维素是一种极具应用前景的骨组织工程材料,而骨组织工程材料要求其本身及其降解产物具有良好的细胞相容性,实验在传统的MTT法评价细胞相容性的基础上,进一步应用流式细胞术的方法从DAN合成周期的角度进行评价。目的:评价新型纳米复合材料纳米羟基磷灰石/细菌纤维素及其酶降解产物的细胞相容性。方法:应用体外细胞培养法,观察纳米羟基磷灰石/细菌纤维素复合材料及其降解物对成骨细胞形态学的影响,同时采用MTT比色法评价纳米羟基磷灰石/细菌纤维素及其降解物对成骨细胞生长和增殖的影响,并尝试用流式细胞仪检测材料作用于细胞后细胞周期时相的变化,从而在分子水平上评价材料对细胞增殖的影响。结果与结论:纳米羟基磷灰石/细菌纤维素复合材料及其降解物对成骨细胞的形态无明显影响,对细胞生长和增殖无明显抑制作用。MTT细胞毒性试验显示原材料及其降解物的细胞增殖率均在80%以上,细胞毒性均为1级,材料对培养细胞无明显细胞毒性。流式细胞仪检测结果显示材料与细胞接触后能降低G0/G1期细胞比例,增加S,G2/M期细胞比例,能增加成骨细胞DNA的合成,促进成骨细胞生长和组织修复。提示纳米羟基磷灰石/细菌纤维素复合材料细胞相容性良好,是一种安全的、很有应用前景的骨组织工程支架材料。     

成骨细胞在生物活性材料中黏附性能研究进展

成骨细胞与骨基质材料间的相互作用是骨组织工程研究的主要领域,其中细胞与材料的黏附是基础,细胞必须与材料发生适当的黏附,才能进行迁移、增殖和分化。综述了与成骨细胞黏附有关的蛋白质、生物活性复合材料的表面特征和表面修饰对成骨细胞黏附性能的影响,为骨组织工程的研究提供一定的依据,尤其为组织工程新材料的研制提供参考。     

梯度掺锶羟基磷灰石骨水泥的体外细胞生物学性能

背景：锶掺入羟基磷灰石骨水泥后可以改善材料的结晶性和相容性,但产生的细胞毒性以及对细胞表面黏附、增殖和表达的影响还需要深入研究。 目的：观察梯度掺锶羟基磷灰石骨水泥的体外细胞生物学性能。 方法：制备4组羟基磷灰石骨水泥试样,分别编为0%,1%,5%和10%掺锶羟基磷灰石骨水泥,每组取6个平行试样紫外线照射灭菌3 h备用。浸提递质为含体积分数10%胎牛血清的DMEM培养液。按照掺锶羟基磷灰石骨水泥粉末质量∶浸提液=1 g∶10 mL比例配制。选用L-929成纤维细胞和幼兔成骨细胞,对成骨细胞进行鉴定。扫描电镜观察幼兔成骨细胞在梯度掺锶羟基磷灰石骨水泥表面的黏附和增殖情况,细胞的表达情况采用碱性磷酸酶活性检测法检测。 结果与结论：①不同掺锶量羟基磷灰石骨水泥的细胞毒性评级均为0或1级,各试样的细胞毒性与其掺锶量、作用时间、浸提液浓度有一定关联性。②适量锶元素的加入可以促进成骨细胞的黏附、增殖及表达,改善材料的溶解动力学,提高其生物降解性,更加符合临床要求。但目前尚缺乏有关的远期实验结果。     

温敏性壳聚糖水凝胶对大鼠成骨细胞的毒性

背景：温敏性壳聚糖与多种细胞相容性良好,是组织工程中不可多得的优良载体,但其对成骨细胞毒性研究相对缺乏。 目的：验证温敏性壳聚糖水凝胶对成骨细胞的毒性。 方法：成骨细胞在温敏性壳聚糖水凝胶中进行培养,显微镜下观察细胞形态及扩增情况,同时,SD大鼠成骨细胞在不同浓度的温敏性壳聚糖水凝胶浸提液中体外培养24,48,72,96 h,MTT法测定细胞相对增殖率,判断细胞毒性的级别。 结果与结论：SD大鼠成骨细胞在温敏性壳聚糖水凝胶中培养24 h内镜下观察呈圆形,48 h后开始伸出触角并扩增;温敏性壳聚糖水凝胶浸提液中培养的各组细胞在不同时间点相对增殖率在92%~112%之间,各浓度的温敏性壳聚糖水凝胶材料浸提液的细胞毒性均为0级或1级,完全符合生物材料的安全评价标准。     

生物活性玻璃对鼠成骨细胞体外增殖的形态学研究

通过在培养液内加入生物活性玻璃,观察生物活性玻璃对成骨细胞增殖的影响。将生物活性玻璃晶体溶于DMEM培养液内,测定其中的Na、Ca、P、Si 4种离子的浓度。以对照组培养液和实验组培养液分别培养鼠成骨细胞,观察成骨细胞生长形态,测定成骨细胞生长指数并绘制生长曲线,动态观察细胞内甲肷的形态,观察细胞超微结构。结果表明:Si离子的浓度在实验培养液内是对照培养液的26.12%(P<0.01),Ca是88.03%(P<0.01),P是73.23%(P<0.01),Na的浓度无差异(P>0.05)。大体细胞形态:开始2天内实验组细胞胞体较对照组大,随时间延长,实验组成骨细胞比对照组密度高。动态观察细胞内甲肷:实验组较对照组成骨细胞增殖活跃,其定量指标即细胞生长指数在培养第2天,即出现明显的差异(P<0.01),随时间延长差异越明显,两条生长曲线呈分离趋势。细胞超微结构:实验组成骨细胞内比对照组有较多的粗面内质网、线粒体和核糖体。说明生物活性玻璃可促进成骨细胞增殖。     

钴合金和钛合金的细胞毒性研究

对植入材料磨损态的Co-29Cr-5Mo(CCM)和Ti-6Al-4V(Ti64)的细胞毒性进行研究。与固溶态相比,在完全培养液中,通过噻唑蓝(MTT)法研究两种合金在不同浸提浓度、不同浸提时间下的材料浸提液对小鼠成纤维细胞(L929)的毒性及影响机制。扫描电子显微镜(SEM)观察腐蚀前后材料表面的变化,ICP发射光谱仪(ICP-MS)检测CCM和Ti64合金中主要金属离子的释放,电化学工作站(CHI760E)表征两种磨损态合金在完全培养液中的腐蚀情况。MTT实验结果表明:磨损态的两种合金随材料浸提浓度和时间的增大,细胞的相对增殖率(RGR)均减小,其中CCM的RGR由98.36%减小至73.28%,细胞毒性由0级升至2级;Ti64的RGR减小至85.86%,细胞毒性升至1级。ICP-MS结果表明,磨损态合金有害离子的释放量随着浸提时间不断上升,浸提至168 h时,磨损态CCM合金Co和Cr释放量分别高达1 249.7和293.9 μg/L;磨损态Ti64合金Al和V释放量分别为30.7和19.7 μg/L。CCM的Tafel曲线和SEM分析表明,在完全培养液中,CCM表面化学位较高的棱、尖角以及微米级沟壑位置发生了缝隙腐蚀,造成金属离子的释放,并对L929细胞的生长和分裂产生抑制作用。     

生物活性玻璃对成骨细胞Ⅰ型胶原和骨钙素基因表达的影响

背景：在成骨细胞培养液中加入生物活性玻璃离子,对成骨细胞的细胞因子分泌是正相关还是负相关？ 目的：观察生物活性玻璃对成骨细胞Ⅰ型胶原和骨钙素基因表达的影响,以进一步探讨生物活性玻璃促进成骨细胞增殖的可能机制。 方法：以DMEM完全培养液作为对照组培养液,以加入生物活性玻璃离子液的DMEM完全培养液作为实验组培养液,取第2代乳鼠颅骨源性成骨细胞,用两种培养液分别培养4 d进行实验观察。 结果与结论：鼠成骨细胞2种生长因子的表达量实验组与对照组之比为：Ⅰ型胶原=3.376、骨钙素=2.687,两者与内参基因相对量两两比较差异有显著性意义(P < 0.05)。生物活性玻璃促进成骨细胞对Ⅰ型胶原、骨钙素的基因表达,有利于骨修复过程中成骨细胞周围基质的形成和矿化,从而促进骨愈合。 关键词：生物活性玻璃;成骨细胞;细胞因子;Ⅰ型胶原;骨钙素 doi:10.3969/j.issn.1673-8225.2012.08.002     

MBG/PLGA composite microspheres with prolonged drug release

Mesoporous bioactive glass (MBG) and composite microspheres with MBG particles embedded in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) matrix have been prepared and used to load gentamicin (GS). The in vitro drug release experiments from both MBG and composite microspheres were conducted in distilled water and phosphate buffered saline (PBS) solution at 37 degrees C for more than 30 days. In both water and PBS, GS release from the MBG was very fast with about 60 wt % of the loaded drug released in the first 24 h, and more than 80 wt % released in two days. MBG/PLGA composite microspheres showed an initial release of about 33 wt % in the first day, and 48 wt % in 2 days, and a subsequent sustained release lasting for more than 4 weeks in PBS. MBG/PLGA composite microspheres may be used as an alternative drug release system, especially as a bone void filler for bone repair due to their combined advantages of sustained release of antibiotics and apatite-forming ability.     

Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins

The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione S-transferase-tagged recombinant proteins in an Escherichia coli system. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.     

血糖控制水平对COPD伴2型糖尿病患者 肺功能及其预后的影响

目的 血糖控制水平对慢性阻塞性肺病（COPD）伴2型糖尿病（T2DM）患者肺功能及其预后的影响。方法 选择2017年1月~2019年3月在我院呼吸内科接受治疗的COPD合并T2DM患者167例,根据患者住院期间血糖水平（MBG）不同分为MBG正常组108例和MBG高水平组59例;采用FEV1、FEV1/FVC、FVC、6 min步行试验（6MWT）和mMRC呼吸困难表评分评估两组患者肺功能,世界卫生组织生活质量量表（WHOQOL-100）评估患者生活质量,比较两组超敏C反应蛋白（hs-CRP）、纤维蛋白原（FIB）、动脉氧分压（PO2）、血浆D-二聚体（D-D）、动脉二氧化碳分压（PCO2）、临床疗效及不良反应发生情况。结果 ①本研究最终共纳入167例患者,MBG正常组108例,MBG高水平组59例;②治疗后,两组FEV1、FEV1/ FVC、FVC、6MWT均较治疗前升高,mMRC呼吸困难表评分较治疗前下降,且MBG正常组较MBG高水平组改善明显,差异均有统计学意义（P＜0.05）;③两组FIB、PO2、D-D均较治疗前升高,hs-CRP、PCO2下降,且MBG正常组FIB、PO2、D-D、hs-CRP及PCO2改善程度优于MBG高水平组,差异均有统计学意义（P＜0.05）;④MBG正常组治疗总有效率为81.48%,高于MBG高水平组的57.63%,差异有统计学意义（P＜0.05）;MBG高水平组并发症发生率为35.59%,高于MBG正常组的14.81%,差异有统计学意义（P＜0.05）;⑤MBG正常组生活质量评分高于MBG高水平组,差异均有统计学意义（P＜0.05）。结论 COPD合并T2DM患者血糖控制水平与其治疗效果、生存质量及并发症发生率密切相关,血糖控制不佳易导致COPD疗效和患者生存质量降低、并发症发生风险升高。     

介孔生物玻璃对人骨髓间充质干细胞早期黏附增殖行为的影响*

目的制备具有高度有序介孔结构的介孔生物玻璃,研究人骨髓间充质干细胞（hBMSCs）在其表面黏附、铺展、增殖等早期细胞生物学行为。方法通过溶胶凝胶法制备介孔生物玻璃（MBG）;接种人骨髓间充质干细胞（hBMSCs）于不同组材料表面共培养,采用四唑盐比色法（MTT）分析其对细胞增殖的影响,绘制增殖曲线;使用DAPI染核,在荧光显微镜下观测材料表面的细胞粘附情况;利用扫描电子显微镜（SEM）直接观察细胞在材料表面的铺展形态。结果在4h和12h,MBG能够显著促进hBMSCs在其表面的黏附;同时,第1~7d,MBG表面的细胞增殖数量显著多于没有介孔结构的普通生物玻璃（NBG）表面的细胞增殖数;在24h内的荧光显微镜观察下,MBG表面在早期有更多的hBMSCs黏附;在第1、4、7d SEM的观察下,MBG表面的细胞能够更均匀的分布并覆盖材料。结论具有有序介孔结构的介孔生物玻璃能够促进人骨髓间充质干细胞在其表面的早期黏附、铺展和增殖,具有更好的生物活性。     

Interaction of an ordered mesoporous bioactive glass with osteoblasts,fibroblasts and lymphocytes,demonstrating its biocompatibility as a potential bone graft material

Ordered mesoporous 85SiO₂–10CaO–5P₂O₅ bioactive glass (MBG85) is an excellent candidate as a graft for bone tissue regeneration, owing to its excellent textured properties, structural characteristics and crystalline apatite rate formation. To assess MBG85 biocompatibility, different parameters have been evaluated (cell morphology, size/complexity, proliferation, viability, cell cycle, reactive oxygen species content, lactate dehydrogenase release) using human Saos-2 osteoblasts after treatment with either MBG85 extracts or 1% MBG85 directly added to cells. The osteoblast response to MBG85 was compared with L929 fibroblast behaviour after the same treatment. The high cell viability observed and the absence of signs of cell damage in both cell types demonstrates MBG85 biocompatibility. Only a cytostatic effect was observed through the reduction of cell proliferation, related with the initial Ca elution, whereas Si leaching did not result into any negative effect. In vitro lymphocytic proliferation analysis was also carried out with SR.D10 clone after treatment with either MBG85 extracts or culture supernatants of L929 fibroblasts previously treated with 1% MBG85 (cell-conditioned extracts). The absence of modification of in vitro T-cell response underlines the biocompatibility of MBG85 and its potential application in the field of bone and dental grafting.     

Delivery of dimethyloxallyl glycine in mesoporous bioactive glass scaffolds to improve angiogenesis and osteogenesis of human bone marrow stromal cells

Development of hypoxia-mimicking bone tissue engineering scaffolds is of great importance in stimulating angiogenesis for bone regeneration. Dimethyloxallyl glycine (DMOG) is a cell-permeable, competitive inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH), which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression. The aim of this study was to develop hypoxia-mimicking scaffolds by delivering DMOG in mesoporous bioactive glass (MBG) scaffolds and to investigate whether the delivery of DMOG could induce a hypoxic microenvironment for human bone marrow stromal cells (hBMSC). MBG scaffolds with varied mesoporous structures (e.g. surface area and mesopore volume) were prepared by controlling the contents of mesopore-template agent. The composition, large-pore microstructure and mesoporous properties of MBG scaffolds were characterized. The effect of mesoporous properties on the loading and release of DMOG in MBG scaffolds was investigated. The effects of DMOG delivery on the cell morphology, cell viability, HIF-1α stabilization, vascular endothelial growth factor (VEGF) secretion and bone-related gene expression (alkaline phosphatase, ALP; osteocalcin, OCN; and osteopontin, OPN) of hBMSC in MBG scaffolds were systematically investigated. The results showed that the loading and release of DMOG in MBG scaffolds can be efficiently controlled by regulating their mesoporous properties via the addition of different contents of mesopore-template agent. DMOG delivery in MBG scaffolds had no cytotoxic effect on the viability of hBMSC. DMOG delivery significantly induced HIF-1α stabilization, VEGF secretion and bone-related gene expression of hBMSC in MBG scaffolds in which DMOG counteracted the effect of HIF-PH and stabilized HIF-1α expression under normoxic condition. Furthermore, it was found that MBG scaffolds with slow DMOG release significantly enhanced the expression of bone-related genes more than those with instant DMOG release. The results suggest that the controllable delivery of DMOG in MBG scaffolds can mimic a hypoxic microenvironment, which not only improves the angiogenic capacity of hBMSC, but also enhances their osteogenic differentiation.     

Detection of antiviral activity of monoclonal antibodies, specific to Marburg virus proteins

Monoclonal antibodies (MAbs) specific to Marburg virus (MBG), Popp strain, have been previously produced and characterized by indirect ELISA. Protein specificity of MAbs was determined by immunoblotting with SDS-PAGE proteins of MBG: one to NP, four to VP40, and protein specificity of one antibody was not detected. The effect of MAb binding to protein epitopes on viral functions was investigated in vitro and in vivo. None of antibodies neutralized the virus in the neutralization test in vitro, but MAb 5G9.G11 and 5G8.H5 specific to MBG VP40 protein were active in antibody-dependent complement mediated lysis of virus-infected cells. In vivo these antibodies (5G9.G11 and 5G8.H5) protected guinea pigs from lethal MBG infection after passive inoculation. Studies of biological activity and analysis of epitope specificity of MAb-antiVP40 by competitive ELISA showed that 2 of 7 epitopes of VP40 protein of MBG induce the production of protective antibodies. Hence, MAbs 5G9.G11 and 5G8.H5 reacting with MBG VP40 protein caused lysis of virus infected cells in the presence of the complement in vitro and protected guinea pigs from MBG infection by passive inoculation.     

The effect of mesoporous bioactive glass on the physiochemical,biological and drug-release properties of poly(dl-lactide-co-glycolide) films

Poly(lactide-co-glycolide) (PLGA) has been widely used for bone tissue regeneration. However, it lacks hydrophilicity, bioactivity and sufficient mechanical strength and its acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Mesoporous bioactive glass (MBG) with highly ordered structure (pore size 2–50 nm) possesses higher bioactivity than non-mesoporous bioactive glass (BG). The aim of this study is to investigate the effect of MBG on the mechanical strength, in vitro degradation, bioactivity, cellular response and drug release of PLGA films and optimize their physicochemical, biological and drug-delivery properties for bone tissue engineering application. The surface and inner microstructure, mechanical strength and surface hydrophilicity of MBG/PLGA and BG/PLGA films were tested. Results indicated that MBG or BG was uniformly dispersed in the PLGA films. The incorporation of MBG into PLGA films significantly improved their tensile strength, modulus and surface hydrophilicity. MBG/PLGA resulted in an enhanced mechanical strength, in vitro degradation (water absorbance, weight loss and ions release), apatite-formation ability and pH stability in simulated body fluids (SBF), compared to BG/PLGA. MBG/PLGA and BG/PLGA films enhanced human osteoblastic-like cells (HOBs) attachment, spreading and proliferation compared to PLGA. HOBs differentiation was significantly upregulated when cells were cultured on 30 MBG/PLGA for 14 days, compared to 30 BG/PLGA. MBG/PLGA enhanced the accumulative release of dexamethazone (DEX) at early stages (0–200 h) compared to BG/PLGA, however, after 200 h, DEX-release rates for MBG/PLGA was slower than that of BG/PLGA. The contents of MBG in PLGA films can control the amount of DEX released. Taken together, MBG/PLGA films possessed excellent physicochemical, biological and drug-release properties, indicating their potential application for bone tissue engineering by designing 3D scaffolds according to their corresponding compositions.