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。     

聚乳酸/羟基磷灰石纳米复合物对MC3T3-E1细胞增殖活性的影响

背景:研究表明聚乳酸/羟基磷灰石复合材料与自然骨结构和性能相似,具有骨传导性和良好的生物相容性。目的:观察聚乳酸/羟基磷灰石纳米复合物与成骨细胞株MC3T3-E1的生物相容性。方法:分别采用普通完全培养基(对照组)与聚乳酸/羟基磷灰石纳米复合物浸提液(实验组)培养第3代成骨细胞株MC3T3-E1,培养3,5,7 d,采用CCK-8法检测细胞的吸光度值;在培养第7,14天检测细胞碱性磷酸酶、骨钙素、Ⅰ型胶原、核心结合因子α1/成骨特异性转录因子表达。将聚乳酸/羟基磷灰石纳米复合物与第3代成骨细胞株MC3T3-E1共培养,培养7,14 d细胞骨架染色及扫描电镜观察细胞在支架材料上的形态。结果与结论:随着时间的延长,成骨细胞株MC3T3-E1的吸光度值明显增加,两组细胞吸光度值比较差异无显著性意义。实验组培养第7天细胞碱性磷酸酶活性高于对照组(P0.05),培养第14天细胞碱性磷酸酶、Ⅰ型胶原、核心结合因子α1/成骨特异性转录因子表达高于对照组(P0.05)。种植7 d后细胞在材料上贴附生长,细胞呈多角形;种植14 d后细胞较7 d前数目明显增多,且完全伸展,呈多角形和梭形。表明聚乳酸/羟基磷灰石纳米复合物对成骨细胞有良好的生物相容性,无细胞毒性。     

Vitapex糊剂用于一次性根充术后急症反应的临床观察

目的 比较Vitapex糊剂和氧化锌丁香油糊剂加牙胶尖两种根管充填材料一次性根充后急性反应情况.方法 临床上随机选择247例需要进行根管治疗随机分成实验组与对照组,实验组患牙采用Vitapex糊剂加牙胶尖冷侧压法根充,对照组患牙用氧化锌丁香油糊剂根充,观察术后一周内急性反应的发生情况,用x?检验分析比较.结果 经统计学检验两种术后急性疼痛反应情况,实验组Vitapex糊剂组明显低于对照组氧化锌丁香油糊剂组,两组根充后急性反应比较差异均有统计学意义(P<0.05).结论 Vitapex糊剂加牙胶尖对患牙做一次性根管充填,其术后急性疼痛反应明显减少.     

纯钛表面阳极氧化增强成骨细胞生物活性及护骨素基因的表达

背景:纯钛阳极氧化改性后形成的纳米结构与骨组织具有良好的生物相容性。目的:观察纯钛表面纳米孔结构的形貌和物相构成,以及其对MC3T3-E1小鼠前成骨细胞增殖、黏附等生物学行为和促成骨基因护骨素表达的影响。方法:取纯钛片24份,其中12份仅进行机械抛光,作为对照组;另外12份进行机械抛光后,应用阳极氧化技术在纯钛表面制备纳米孔结构,作为实验组。将小鼠前成骨细胞MC3T3-E1分别接种于两组试件表面,接种7 d后扫描电镜下观察细胞形态,采用MTT法检测细胞增殖情况,绘制生长曲线;同时检测细胞促成骨基因护骨素的表达。结果与结论:阳极氧化后钛片表面形成规格统一的纳米孔结构,但是物相构成并未发生变化。与接种于对照组试件上的成骨细胞相比,实验组试件表面的细胞密度变大,覆盖金属的面积更多,呈现多边形结构,突触向周围移行,可见板状伪足向周围材料伸出;接种第7天时,实验组细胞数目约为对照组的1.4倍,同时纳米孔表面成骨细胞护骨素基因的表达高于对照组(P0.01)。结果表明阳极氧化后形成纳米孔结构的钛片更有利于成骨细胞的黏附、增殖和护骨素基因的表达,进而促进成骨细胞生长,具有良好的生物相容性。     

磁性纳米多孔复合支架材料的细胞相容性研究

目的研究磁性纳米多孔复合(n-HA/PLLA/Fe2O3)材料的细胞相容性,探讨细胞在材料表面黏附、增殖、表达等生物学行为,为其医学应用提供实验依据。方法将大鼠成骨细胞与磁性纳米多孔复合材料共培养,采用CCK-8法检测细胞增殖、扫描电镜观察细胞在材料上的黏附、RT-PCR检测I型胶原和骨钙素基因的表达。结果 CCK-8检测显示实验组磁性纳米多孔复合材料上细胞的增殖与空白对照组没有差异性(0.05);扫描电镜观察到细胞在磁性纳米多孔复合材料的表面和孔隙内大量黏附、增殖和生长,随着共培养时间的增加,材料表面的细胞数量明显增多;RT-PCR显示随着共培养时间的增加,I型胶原基因的表达增强(0.05),骨钙素的表达无明显差异(0.05)。结论磁性纳米多孔复合支架材料适于成骨细胞的黏附、生长和分化,具有良好的细胞相容性。     

三种根充糊剂根尖封闭能力的比较研究

目的 利用葡萄糖定量法检测根管微渗漏模型,评价三种根充糊剂的根尖封闭性能.方法 收集新鲜拔除的人前牙65颗,随机分为A、B、C 三个实验组(每组20颗牙齿)和D组作为对照组(5颗牙齿),A、B、C组分别采用AH-Plus糊剂、新型纳米羟基磷灰石根管充填糊剂(n-HA)、氧化锌丁香油(ZOE)糊剂加牙胶尖侧压充填根管.D组为阴性对照组,用粘蜡充填根管.牙根外表面、根管口及根尖孔处涂二层指甲油.于第7 d、14 d、21 d、28 d用葡萄糖氧化酶法检测从冠方向根方漏出的葡萄糖量.结果 采用重复测量方差分析,发现第7d、14d、21d、28d A、C组,B、C组及C、D组微渗漏值有显著性差异(P<0.01),A、B组微渗漏值无显著性差异(P>0.05).但各组第7 d与第28d微渗漏值比较有显著性差异(P<0.05).结论 AH-Plus糊剂、新型纳米羟基磷灰石根管充填糊剂(n-HA)的根尖封闭性优于氧化锌丁香油ZOE糊剂.而前两种糊剂的根尖封闭性能相当.     

纳米级二氧化锆增韧羟基磷灰石生物复合陶瓷对兔骨髓基质干细胞增殖、分化的影响

背景：课题组拟对纳米级二氧化锆增韧的羟基磷灰石生物陶瓷进行一些初步研究,主要集中在生物力学匹配性,化学稳定性,以及生物相容性实验,其中体外细胞培养实验具有可控性,可重复性,能很好地反映材料的生物相容性。 目的：比较纳米级二氧化锆增韧的羟基磷灰石、纯羟基磷灰石两种材料对兔骨髓基质干细胞增殖、分化的影响。 方法：将骨髓基质干细胞置于含体积分数为20%胎牛血清的DMEM培养基中培养,传代后改用含β-甘油磷酸钠,地塞米松和维生素C的条件培养基培养。取传至第3代的成骨细胞,以1.0×108 L-1浓度接种于放有材料块的细胞培养板中,培养第1~10天倒置相差显微镜观察细胞生长情况,绘制细胞生长曲线,并进行碱性磷酸酶活性检测。培养第6天的细胞和材料复合物用多聚甲醛固定进行扫描电镜观察。 结果与结论：MTT法测得两种材料培养的细胞生长曲线无显著差异。复合培养的兔骨髓基质干细胞能够保持正常分泌碱性磷酸酶的功能。电镜照片也同样证实了两种材料表面均有细胞的附着。说明纳米级二氧化锆增韧的羟基磷灰石、纯羟基磷灰石均不影响成骨细胞增长分化,具有优良的成骨细胞相容性。     

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。     

骨髓间充质干细胞与不同基质修饰的纳米晶胶原基骨体外生物相容性研究

研究大鼠骨髓间充质干细胞(MSCs)诱导培养后与不同基质修饰的纳米晶胶原基骨(nanoHydroxyapatite/collagen,nHAC)的生物相容性,为骨组织工程提供一种新型复合支架材料。SD大鼠MSCs经成骨诱导培养、扩增,进行成骨细胞表征后,种植与支架材料体外复合培养。实验分为4组:实验组A,纤维蛋白(FB)和纤维连接蛋白(FN)修饰的纳米晶胶原基骨((FB FN)-nHAC);实验组B,纤维蛋白修饰的纳米晶胶原基骨(FB-nHAC);实验组C,纤维连接蛋白修饰的纳米晶胶原基骨(FN-nHAC);对照组D,单纯的纳米晶胶原基骨(nHAC)。通过检测支架材料的细胞黏附率、不同时间点(3、7、10、14d)支架材料中细胞数、碱性磷酸酶活性以及扫描电镜观察细胞在材料上的生长状况,比较分析不同支架材料与细胞生物相容性差异。大鼠MSCs经诱导培养14d后,碱性磷酸酶细胞化学染色、I型胶原免疫荧光染色及矿化沉积茜素红染色均为阳性;细胞与支架材料黏附率A组最高为74.4%;支架材料中细胞数量均随培养时间延长而增长,且A组细胞数增加较快,与相同时相点其他各组材料中细胞数差异有显著性(P<0.05);各时相点细胞碱性磷酸酶活性表达A组最高,差异亦有显著性(P<0.05)。电镜观察发现4组材料上均有细胞生长,但A组的细胞生长状况明显好于其他组。大鼠MSCs经成骨诱导培养,可表达成骨细胞表型,(FB FN)-nHAC在体外实验中表现出与细胞优良的生物相容性,可作为较理想的新型复合支架应用于骨组织工程。     

新型壳聚糖基仿生网络复合膜诱导骨髓间充质干细胞定向成骨分化

目的 探讨新型壳聚糖基网络复合膜诱导骨髓间充质干细胞（MSCs）定向成骨分化的可行性.方法 采用仿生学方法,壳聚糖、明胶、果胶按照一定比例制作成新型壳聚糖基仿生网络复合膜.设计4个组：实验组1（复合膜＋常规培养基）,对照组1（常规培养基）,实验组2（复合膜＋成骨诱导（OS）培养基）,对照组2（OS培养基）.通过倒置相差显微镜、四甲基偶氮唑盐（MTT）法、扫描电镜（SEM）检测细胞在复合膜上的生长和增殖情况;通过测定碱性磷酸酶（ALP）活性来评价MSCs在复合膜上的成骨分化能力;通过特殊染色和能谱分析（EDX）来评定钙盐的沉积.结果 MSCs在网络复合膜上贴附、生长良好且增殖旺盛.MTr法检测细胞活力显示实验组吸光度（OD）值与对照组比较无统计学意义（P＞0.05）.SEM观察到细胞在支架材料表面呈聚集生长,分泌大量的细胞外基质,可见散在的结节形成.实验组1的ALP活性明显增高,与实验组2、对照组比较有统计学意义（P＜0.01）.茜素红和Von Kossa染色可见实验组细胞分化后形成的钙化结节;ALP染色可见胞浆内蓝染颗粒;EDX检测到Ca、P沉积.结论 新型壳聚糖基网络复合材料具有良好的生物相容性,在不添加诱导剂的条件下,可以诱导MSCs定向成骨分化.     

In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics.

The biocompatibility of two implantable materials, zirconia and alumina ceramics, was investigated in vitro using human osteoblast cell cultures. The viability of osteoblast cells with the materials was evaluated by the methylthiazole sulfate test that revealed an absence of any cytostatic or cytotoxic effect. Cell proliferation kinetic and total protein synthesis in osteoblasts with zirconia or alumina were similar to that observed in control cells cultured on glass coverslips. Light and scanning electron microscopic examinations showed an intimate contact between osteoblasts and the substrates; well-spread cells were observed on the surfaces of both materials. Adhesion ability and morphological characteristics were preserved in osteoblast cultures with these substrates. Moreover, immunohistochemical staining in osteoblasts with zirconia and alumina showed the capacity of these cells to elaborate the extracellular matrix composed of types I and V collagen, osteocalcin, osteonectin, bone sialoprotein, and cellular fibronectin. Finally, DNA image cytometry and interphase silver-nucleolar organizer regions quantification were applied as complementary biocompatibility tests to detect any changes in DNA synthesis and cell proliferation, respectively. The results showed that neither material altered cell ploidy or cell growth rate in accordance with the absence of any inducing effect on DNA synthesis or proliferation.     

硫唑嘌呤对大鼠骨髓间充质干细胞增殖、细胞周期和凋亡的影响

目的: 研究硫唑嘌呤(AZA)对SD大鼠骨髓间充质干细胞(MSCs)增殖、凋亡和细胞周期的影响,为炎症性肠病的临床治疗提供实验依据。方法: 含10%FBS的低糖DMEM培养基培养大鼠MSCs,50 mg/L、100 mg/L、200 mg/L、300 mg/L的AZA分别作用于大鼠MSCs 24 h、48 h和72 h,用显微镜下直接计数法检测MSCs的增殖情况并绘制生长曲线,用流式细胞术检测MSCs凋亡和细胞周期。结果: MSCs经过3次传代后可纯化。在小于100 mg/L浓度下,AZA对MSCs的增殖、细胞周期与凋亡无显著影响(P>0.05),而AZA在大于200 mg/L浓度下作用72 h后,MSCs生长受到明显抑制,抑制率大于66%,凋亡率明显升高(P<0.05);在300 mg/L浓度下作用72 h时,MSCs的凋亡率则明显降低,而MSCs坏死率明显升高(P<0.05)。在大于200 mg/L的AZA作用下,随着作用时间延长,MSCs进入G₀/G₁期细胞增多,S期细胞减少(P<0.05)。结论: 一定浓度的AZA对大鼠MSCs的增殖、细胞凋亡和细胞周期均有明显的影响。大剂量的AZA会直接造成MSCs死亡。     

Cytotoxicity of partial-stabilized cement

Partial-stabilized cement (PSC) is a kind of modified calcium silicate cement used for root-end surgery. Minor transition metal elements Co, Cr, and Zn were added for enhancing the setting property of to PSC. In our previous study, minor transition metal additions greatly improved the setting property of PSC. However, the concern of metal toxicity was raised, as the material would be used in the human body. In this study, we evaluated the cytotoxicity of PSC in comparison with mineral trioxide aggregate (MTA), which is one of the commercialized materials used for dental root-end filling. Primary osteoblast cell was used as the target cell. Cell proliferation, cytotoxicity, viability, function, and senescence were analyzed. The cytotoxicity of the PSC-Zn group (PSC with Zn addition) was similar to that of MTA. PSC-Zn is not only nontoxic at the cellular level but also has adequate mechanical property, which makes it a potential root-end filling material for apical surgery.     

Improved osteoblast compatibility of medical-grade polyetheretherketone using arc ionplated rutile/anatase titanium dioxide films for spinal implants

Titanium dioxide (TiO(2) ), known to exhibit good biocompatibility, is applied in this study as a thin film formed onto polyetheretherketone (PEEK) substrate, which has been widely used in spinal interbody fusion cages. For successful deposition, an arc ionplating (AIP) technique was applied to deposit TiO(2) at low deposition temperature without damaging PEEK substrate, while providing satisfactory film adhesion. This study systematically investigates the effects of TiO(2) thin film phase composition and surface characteristics, controlled by using different target current and substrate bias, on osteoblast compatibility. Experimental results showed that anatase phase (A-TiO(2) ) and/or rutile phase (R-TiO(2) ) TiO(2) coatings, respectively, can be prepared in appropriate deposition conditions. Overall, the TiO(2) -coated PEEK presented better osteoblast compatibility than the bare PEEK material in terms of cell adhesion, cell proliferation, and cell differentiation abilities, as well as osteogenesis performance (as determined by levels of osteopontin, osteocalcin, and calcium content). Surface roughness and hydrophilicity of the AIP-TiO(2) films were found to be responsible for significant osteoblast cell growth. It is also noticeable that the R-TiO(2) exhibited better osteoblast compatibility than the A-TiO(2) due to the presence of negatively charged hydroxyl groups on R-TiO(2) (110) surface in nature. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2787-2792, 2012.     

表面修饰对羟基磷灰石细胞相容性的影响

探讨精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)多肽表面修饰对羟基磷灰石(hydroxyapatite,HA)细胞相容性的影响。以骨髓基质干细胞(marrow stromal stem cells,MSCs)复合精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石或单纯材料培养制备组织工程骨,观察骨髓基质干细胞的粘附和生长情况,检测细胞活力和碱性磷酸酶(alkaline phosphatase,ALP)活性,流式细胞仪分析细胞周期。结果表明:骨髓基质干细胞在材料表面和孔隙内均可粘附和生长,粘附于RGD多肽修饰羟基磷灰石的细胞活力和碱性磷酸酶活性明显高于未经RGD多肽修饰组(P<0.01,P<0.05)。各组细胞周期未见明显变化,未见异倍体细胞。说明RGD多肽表面修饰对HA材料的细胞相容性有明显的优化作用。     

Osteoblast adhesion on nanophase ceramics.

Osteoblast adhesion on nanophase alumina (Al2O3) and titania (TiO2) was investigated in vitro. Osteoblast adhesion to nanophase alumina and titania in the absence of serum from Dulbecco's modified Eagle medium (DMEM) was significantly (P < 0.01) less than osteoblast adhesion to alumina and titania in the presence of serum. In the presence of 10% fetal bovine serum in DMEM osteoblast adhesion on nanophase alumina (23 nm grain size) and titania (32 nm grain size) was significantly (P < 0.05) greater than on conventional alumina (177 nm grain size) and titania (2.12 microm grain size), respectively, after 1, 2, and 4 h. Further investigation of the dependence of osteoblast adhesion on alumina and titania grain size indicated the presence of a critical grain size for osteoblast adhesion between 49 and 67 nm for alumina and 32 and 56 nm for titania. The present study provides evidence of the ability of nanophase alumina and titania to simulate material characteristics (such as surface grain size) of physiological bone that enhance protein interactions (such as adsorption, configuration, bioactivity, etc.) and subsequent osteoblast adhesion.     

Comparative cell behavior on carbon-coated TiO2 nanotube surfaces for osteoblasts vs. osteo-progenitor cells

Surface engineering approaches that alter the topological chemistry of a substrate could be used as an effective tool for directing cell interactions and their subsequent function. It is well known that the physical environment of nanotopography has positive effects on cell behavior, yet direct comparisons of nanotopographic surface chemistry have not been fully explored. Here we compare TiO(2) nanotubes with carbon-coated TiO(2) nanotubes, probing osteogenic cell behavior, including osteoblast (bone cells) and mesenchymal stem cell (MSC) (osteo-progenitor cells) interactions with the different surface chemistries (TiO(2) vs. carbon). The roles played by the material surface chemistry of the nanotubes did not have an effect on the adhesion, growth or morphology, but had a major influence on the alkaline phosphatase (ALP) activity of osteoblast cells, with the original TiO(2) chemistry having higher ALP levels. In addition, the different chemistries caused different levels of osteogenic differentiation in MSCs; however, it was the carbon-coated TiO(2) nanotubes that had the greater advantage, with higher levels of osteo-differentiation. It was observed in this study that: (a) chemistry plays a role in cell functionality, such as ALP activity and osteogenic protein gene expression (PCR); (b) different cell types may have different chemical preferences for optimal function. The ability to optimize cell behavior using surface chemistry factors has a profound effect on both orthopedic and tissue engineering in general. This study aims to highlight the importance of the chemistry of the carrier material in osteogenic tissue engineering schemes.     

IGF-I and TGF-beta 1 incorporated in a poly(D,L-lactide) implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture

Previous in vivo studies revealed a stimulating effect of locally applied IGF-I and TGF-beta1 released from poly(D,L-lactide)-coated titanium implants on rat and porcine fracture healing. The purpose of the present study was to evaluate the effect of IGF-I (5% w/w) and TGF-beta1 (1% w/w) and the carrier PDLLA on osteoblasts in cell culture to improve the understanding of these growth factors. The well-characterized human osteoblast cell line hFOB 1.19 was used in the study. The implants and cells were cocultured in a noncontact manner. The cells were incubated for 10 days in total, and the implants (n = 6 each group and time point) were added for 1 h, 12 h, 24 h, 2 d, 4 d, or 10 d. To analyze a possible effect of the growth factors or the coating, cell proliferation, metabolism, and differentiation were investigated. As an indicator for differentiation the production of collagen I was chosen. All experimental groups showed comparable cell vitality. No change in the pH of the medium was detectable between the analyzed groups. When the effect of the titanium implant and the PDLLA coating were compared with the control culture, no differences in proliferation, metabolic activity, and collagen I production were detectable. The osteoblasts treated with IGF-I and TGF-beta1 released from PDLLA revealed a significantly enhanced collagen I production with a decrease in proliferation and metabolic activity compared to the other groups. No significant differences in collagen I production were seen due to the incubation time points. None of the experimental groups evoked an immunological response on mouse macrophages. In conclusion, the PDLLA-carrier showed no negative effect on osteoblasts, whereas the incorporated growth factors stimulated osteoblast differentiation.     

Effects of bone morphogenetic protein on neonatal rat calvarial osteoblast-like cells: an in vitro study

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic differentiation and osteogenesis. The aim of this study was to examine the behavior of neonatal rat calvarial osteoblast cells cultured on different concentrations of BMP graft materials. Fifty thousand cells per milliliter were seeded and cultured on graft materials for 24 and 48 h. Different concentrations of BMPs (combination of BMPs numbered from 1 to 14) were supplemented to the medium. To evaluate cellular proliferation and differentiation, specimens were examined for DNA synthesis, alkaline phosphatase (ALP) activity, cell numbers, and viability of the cells. Further, transforming growth factor-beta(1) (TGF-beta(1)) and lactate dehydrogenase (LDH) levels were investigated. Morphological appearance of the specimens at 24 and 48 h of incubation was evaluated using scanning electron microcopy. Evaluations of DNA synthesis, cell count, and cell viability data revealed that a significant difference existed at 24 and 48 h (p < 0.05). The TGF-beta(1) and ALP analysis showed only a significant difference between the groups at the end of 24 h (p < 0.05). Regarding the lactate dehydrogenase activity there was not any significant difference at 24 and 48 h (p > 0.05). No morphological differences were observed in cell morphology on BMP graft material and the control group. These results indicate that BMPs have an inductive effect on osteoblast differentiation and a possible inhibitory effect in the early phases of cell proliferation.     

Enhanced transfection of a bacterial plasmid into hybridoma cells by electroporation: application for the selection of hybrid hybridoma (quadroma) cell lines.

A procedure was investigated to transduce a bacterial plasmid containing a specific drug resistance marker (pSV2-neo), into a hybridoma cell line using electroporation. The effect of several buffers and the form of plasmid DNA (circular or linearized) on the stable transfection frequency were examined. When complete cell culture medium (DMEM) was used as electroporation buffer, we observed a two-fold increase in post-pulse viability and a ten- to thirty-fold increase in the transfection frequency of pSV2-neo, as compared with HEPES buffered 0.15 M sodium chloride. Supplementing DMEM with fetal bovine serum (DMEM + FBS) had some beneficial effect on post-pulse viability of the cells after electroporation, but did not markedly increase stable transfection frequency as compared with DMEM alone. Furthermore, with DMEM + FBS, the intact plasmid was transfected as effectively as linearized PSV2-neo. However, when using HEPES buffered saline, the transfection frequency of pSV2-neo increased two-fold after linearization as compared with intact plasmid. The drug resistance was used successfully as a marker for the selection of hybrid hybridoma (quadroma) cell lines after fusing two different hybridoma cell lines, producing anti-fibrin and anti-plasminogen activator antibodies respectively. The quadroma cells produced bispecific antibodies that are capable of accumulating plasminogen activator on a fibrin surface.