EFFECTS OF APATITE CERAMICS ON CELL GROWTHS AND DNA SYNTHESES IN HUMAN OSTEOBLAST LIKE CELL

Sintered strontium hydroxyapatite (Sr-HAP) and hydroxapatite (HAP) werekinds or apatite ceramics as substitutes of bone tissues.They should be safety to human body.Sr-HAP and HAP were co-cultured with human osteoblast like cells for 2, 4,and 6 days invitro. Effect of Sr-HAP and HAP on cell growth numbers with thymidine incorporationtest. In order to estimated cytotoxicities of materials,relative cell growth rate (RGR) werecalculated from data and score of cytotoxicties were assaied. Results showed cell growth numbers and rate of 3H-thymidine incorporation in Sr-HAP and HAP groups were the same asthat of blank control group. Values were increased with the increase of co-cultured time.There were no inhibition of cell growth and DNA syntheses in human osteoblast like cells. Accoding to RGR and score of cytotoxicity degree,all values were qurlified with sdandard ofbiomaterials. Results suggested these qurlified with sdandard or biomaterials. Results suggested these apatite ceramics had no obvious cytotoxicities.

Effects of Anastrozole Combined with Shuganjiangu Decoction on Osteoblast-like Cell Proliferation, Differentiation and OPG/RANKL mRNA Expression

Objective： To investigate the effects of anastrozole combined with Shuganjiangu decoction on osteoblast-like cells. Methods： Human osteoblast-like cells MG-63 were cultured and divided into four groups control, anastrozole, Shuganjiangu decoction （SGJGD）, and anastrozole combined with SGJGD. Cell proliferation was investigated by M-IF assay. Alkaline phosphatase （ALP） and osteocalcin, the indicators of cell differentiation, were evaluated by p-nitrophenyl- phosphate method and radioimmunoassay, respectively. Gene expressions of ALP, osteocalcin, osteoprotegerin （OPG） and receptor activator of nuclear factor kappa B ligand （RANKL） were examined by real-time PCR. Results： As evidenced by MTT assay, cell proliferation of MG-63 was inhibited by anastrozole, but stimulated with treatment of SGJGD alone and combined with anastrozole （P〈O.01）. Compared with control group, ALP activity was increased by the treatment of SGJGD alone and combined with anastrozole （P〈0.01）. Also, osteocalcin secretion was enhanced with the treatment of SGJGD single and combination with anastrozole （P〈O.05）. In the real-time PCR assay, gene expressions of ALP and osteocalcin were significantly increased （P〈0.01 for ALP, P〈0.05 for osteocalcin） by the treatment of SGJGD and anastrozole combined with SGJGD, but the expression of RANKL was decreased （P〈O.05）. Moreover, anastrozole combined with SGJGD upregulated gene expression of OPG （P〈O.01）. Conclusion： SGJGD may alleviate the injury effects of anastrozole on MG-63 cells through adjusting bone formation and resorption indicators.

Implication of Receptor Activator of NF-κB Ligand in Wnt/β-Catenin Pathway Promoting Osteoblast-like Cell Differentiation

Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly in-creased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.

SCANNING ELECTRON MICROSCOPIC STUDY OF FETAL CHICKEN CALVARIAL OSTEOBLAST-LIKE CELLS CULTURED IN VITRO

Three types of osteoblast-like cells with different cnfigurations could be ob-tained through culturing fetal chicken calvaria in vitro. They were spindle-shaped cells,globular cells, and polygonal or squamous cells. With passage of culture time, there werechanges in configuration so that the spindle-shaped cells and the globular cells turnedgradually into squamous cells, in quantity which increased greatly to produce confluenceand multi-layer formation of cells, and in function as evidenced by emergence ofintracytoplasmic granules, reflecting collagen synthesis.

EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON GROWTH OF ISOLATED CELLS FROM EMBRYONIC CHICKEN FRONTAL BONE CULTURED IN VITRO (A HISTOCHEMICAL STUDY) Ⅱ.THE DEVELOPMENT AND MATURATION OF OSTEOBLAST-LIKE CELLS

The isolated osteoblast-like cells from embryonic chicken frontal bone werecultured in vitro and histochemical methods adopted to observe the effect of RadixSalviac Miltiorrhizae (RSM) on proliferation, differentiation, and osteogenic capacity ofthese cells. It was found that: 1. The mitosis and proliferation of the osteoblast-like cellscould be accelerated by RSM, resulting in increased density of the cells in RSM groupas compared with the control. 2. After 48 h, the pseudopodia stretched out and drew backactively in osteoblast-like cells in RSM group. Small particles produced in the cells weresecreted through exocytosis to the extracellular medium. However, in the control group,the capacity to form and secrete these particles was limited. These particles showed posi-tive Alcian blue staining in Alcian blue-Sirius red reaction, so they were acidmucopolysaccharide particles. 3. The osteoblast-like cells could secrete vesicular particles 3micra in diameter. These vesicular particles could be stained with Alcian blue in earlystage, then they could be stained with Sirius red, and finally by Alizarin red S. Thesevesicular particles could aggregate and fuse around the cell colonies, forming bonenodules and bone flakes. The quantity and volume of the bone nodules and flakes inRSM group were larger than in the control group. 4. The bone nodules and flakes couldbe labeled vitally with tetracycline, and show strong yellow fluorescence under thefluorescence microscope. Therefore, these substances were the newly formed bone sub-stances.

Cell Proliferation Ability of Mouse Fibroblast-Like Cells and Osteoblast-Like Cells on a Ti-6Al-4V Alloy Film Produced by Selective Laser Melting

Successful regeneration of tissues and organs relies on the application of suitable substrates or scaffolds in scaffold-based regenerative medicine. In this study, Ti-6Al-4V alloy films (Ti alloy film) were produced using a three-dimensional printing technique called Selective Laser Melting (SLM), which is one of the metal additive manufacturing techniques. The thickness of produced Ti alloy film was approximately 250 μm. The laser-irradiated surface of Ti alloy film had a relatively smooth yet porous surface. The non-irradiated surface was also porous but also retained a lot of partially melted Ti-6Al-4V powder. Cell proliferation ability of mouse fibroblast-like cells (L929 cells) and mouse osteoblast-like cells (MC3T3-E1 cells) on both the surfaces of Ti alloy film was examined using WST assay. Both L929 and MC3T3-E1 cells underwent cell proliferation during the culture period. These results indicate that selective laser melting is suitable for producing a cell-compatible Ti-6Al-4V alloy film for biomaterials applications.

乳酸对成骨样细胞成骨表型及碱性磷酸酶基因表达的影响研究

目的明确聚乳酸降解终产物乳酸对细胞成骨表型和基因表达的影响机理,为聚乳酸支架的精确设计提供参考意义。方法配制乳酸浓度为8、16、22、27 mmol/L的培养基,将不含乳酸培养基作为对照组,检测培养基的pH和渗透压。利用培养基培养大鼠成骨样细胞Ros17/2.8,观察细胞形态,测定细胞Ⅰ型胶原、碱性磷酸酶(alkaline phosphatase,ALP)活性和骨钙素含量,盐酸四环素染色钙结节并定量分析。采用实时定量PCR法检测细胞ALP mRNA表达。结果与对照组相比,随着乳酸浓度增加,培养基pH下降,渗透压升高,成骨样细胞形态改变,数量减少。低浓度乳酸组(8、16 mmol/L)的Ⅰ型胶原量都显著高于对照组,其余组与对照组相比无显著差异。含乳酸组的细胞ALP活性都低于对照组,并且乳酸浓度越高,细胞ALP活性越低。含乳酸组的骨钙素量与对照组之间没有显著差异。随着乳酸浓度增加,含乳酸组细胞所形成的钙结节阳性面积减少,都低于对照组。低浓度乳酸组(8、16 mmol/L)的细胞ALP mRNA表达量都低于对照组。结论聚乳酸降解终产物乳酸可抑制细胞ALP mRNA的表达,降低ALP活性。随着乳酸浓度增加,乳酸抑制作用增强,最终降低细胞的成骨矿化能力。此外,乳酸不会抑制细胞分泌I型胶原和骨钙素。

黄芪甲苷和柚皮苷对成骨样细胞增殖迁移及成骨分化的影响

目的:观察黄芪甲苷和柚皮苷两种中药单体对人成骨样细胞MG-63增殖、迁移和成骨分化的影响。方法:应用CCK-8法观察两种单体对MG-63细胞增殖的影响;应用Transwell小室观察两种中药单体对MG-63细胞迁移的影响;应用茜素红染色法观察两种中药单体对细胞成骨分化的影响,应用Western Blot和qRT-PCR检测两种中药单体对成骨相关标志物I型胶原COL-I和骨钙素BGP mRNA与蛋白表达的影响。结果:黄芪甲苷和柚皮苷均能明显促进MG-63细胞增殖(P<0.01),柚皮苷的作用强于黄芪甲苷(P<0.05);黄芪甲苷和柚皮苷均能促进MG-63细胞的迁移(P<0.01),黄芪甲苷对细胞迁移能力的作用要强于柚皮苷(P<0.01);黄芪甲苷和柚皮苷均具有促进MG-63细胞成骨分化的作用,二者比较未见明显差异;黄芪甲苷和柚皮苷均能提高COL-I的mRNA和蛋白表达水平(P<0.05),均不影响BGP的表达水平,两种药物比较未见明显差异。结论:本研究中黄芪甲苷与柚皮苷对成骨样细胞增殖迁移和成骨均有促进作用,但柚皮苷促进细胞增殖作用明显,黄芪甲苷促进细胞迁移作用明显,两种药物对细胞成骨分化的影响未见明显差异。

伴破骨细胞样巨细胞的胰腺未分化癌的CT及MRI表现(附5例报道及文献复习)

目的:分析伴破骨细胞样巨细胞的胰腺未分化癌的CT及MRI特征,加深对该病的认识,以提高诊断准确率。方法:回顾性分析2019年6月—2022年11月于浙江大学医学院附属第一医院经手术病理证实的伴破骨细胞样巨细胞的胰腺未分化癌5例患者的临床影像资料并文献回顾。5例患者均行腹部CT及MRI扫描。结果:5例患者中3例为女性,2例为男性,年龄44~77岁,平均66岁;4例病灶位于胰腺头颈部,1例位于胰尾部。肿瘤最大径为1.7~9.6 cm。CT平扫3例呈囊实性,2例呈实性,1例内见钙化。增强扫描后病灶实性成分或边缘、分隔延迟强化。肿瘤实性部分在T_(1)WI上呈稍低信号,T_(2)WI呈高信号,DWI呈高信号,增强扫描呈延迟强化。结论:伴破骨细胞样巨细胞的胰腺未分化癌的影像学表现具有一定特征性,可为临床诊断及术前评估提供重要依据。

Effects of Pulsed Electric Fields on DNA Synthesis in an Osteoblast-Like Cell Line (UMR-106)

WTFZ] The study of the bioeffects of electromagnetic fields (EMFs) is an important national task in biological physics. Using EMFs to treat bone diseases involves electrical technology, biology, and medicine. But the effects of EMFs are still controversial and the mechanisms are not yet clear. Therefore, more effect is needed to detect the effects at the cellular and molecular levels. This paper investigates the effects of low-energy, low-frequency pulsed capacitively coupled electric fields (PCCEFs) on DNA synthesis in UMR-106 osteoblast-like cells. The equipment can generate 25250Hz frequency, 0300V amplitude and 0.2ms pulse width signal. DNA synthesis is judged by the uptake of 3 H-thymidine ( 3 H-TdR). The results showed that the response of UMR-106 cells to electric field exposure are characterized by: (a) a frequency window for increased DNA synthesis, with a peak near 125Hz; (b) decreased synthesis with increasing electric intensity with repression at 100V/cm and 25Hz.[

In Vitro Biocompatibility of MC3T3-E1 Osteoblast-like Cells on Arg-Gly-Asp Acid Peptides Immobilized Graphite-like Carbon Coating on Carbon/Carbon Composites

Carbon/carbon （C/C） composites were deposited with graphite-like carbon （GLC） coating, and then, Arg-Gly- Asp acid （RGD） peptides were successfully immobilized onto the functionalized GLC coating. GLC coating was utilized to prevent carbon particles releasing and create a uniform surface condition for C/C composites. RGD peptides were utilized to improve biocompatibility of GLC coating. Surface chemical characterizations of functionalized GLC coating were detected by contact angle measurement, X-ray photoelectron spectroscopy and Raman spectra. Optical morphology of GLC coatings was observed by confocal laser scanning microscopy. In vitro biological performance was determined using samples seeded with MC3T3-E1 osteoblast-like cells and cultured for 1 week. Surface characterizations and morphological analysis indicated that C/C composites were covered by a dense and uniform GLC coating. Contact angle of GLC coating was reduced to 27.2° when it was functionalized by H202 oxidation at 40 ℃ for 1 h. In vitro cytological test showed that the RGD peptides immobilized GLC coating had a significant improvement in biocompatibility. It was suggested that RGD peptides provided GLC coating with a bioactive surface to improve cell adhesion and proliferation on C/C composites.

成骨生长肽对大鼠颅盖骨成骨细胞样细胞增殖和分化的影响

目的研究成骨生长肽(OGP)在体外对成骨细胞样细胞增殖分化的影响。方法在体外培养的新生大鼠颅盖骨成骨细胞样细胞中加入不同浓度的OGP(10-11mol/L^10-7mol/L),用MTT法检测细胞增殖,酶联免疫法检测细胞内碱性磷酸酶(ALP)的表达,RT-PCR法检测核心结合因子1(Cbfa1)mRNA的表达。结果OGP对成骨细胞样细胞增殖有促进作用(P<0.05),最大效应浓度为10-9mol/L;能增加细胞内ALP活性(P<0.05),最大效应浓度为10-8mol/L;可促进Cbfa1mRNA的表达(P<0.05),其最佳促进表达浓度为10-8mol/L,Cbfa1mRNA与β-actinmRNA像素值比值为0.89±0.02。结论OGP可促进成骨细胞样细胞增殖,增强Cbfa1mRNA的表达水平。

淫羊藿总黄酮对体外培养的人成骨样细胞增殖和骨形成功能的影响

目的 :研究淫羊藿总黄酮对人成骨样细胞增殖和骨形成功能的影响。方法 :以人成骨样细胞OS 732为实验材料 ,进行细胞生长曲线的绘制、MTT测定、形态学观察、碱性磷酸酶 (ALP)测定和染色等。结果 :d3时 ,与对照组相比 ,加药组的细胞数目明显增加 ;d6时加药组的细胞数目仍多于对照组。MTT测定得到了类似的结果。加药后d4 ,加药组与空白对照组相比细胞数目多、形态大而规则 ,染色质深而致密 ,核质比小。ALP染色结果显示 ,加药组细胞ALP颗粒染色明显深于对照组 ,并且ALP颗粒在胞浆中的分布均匀。ALP的定量测定表明加药组ALP水平明显高于对照组。结论 :淫羊藿总黄酮对体外培养的人成骨样细胞OS 732具有促增殖和分化作用 ,并表现出浓度依赖性。

地塞米松诱导原代培养的成骨样细胞凋亡的分子细胞学研究

目的 :通过干扰体外培养的成骨样细胞 ,探索糖皮质激素所致成骨样细胞凋亡的分子细胞机制。 方法 :地塞米松诱导原代培养并传代的小鼠成骨样细胞 ,应用噻唑蓝 (MTT)法检测细胞生存能力 ,流式细胞仪检测细胞凋亡 ,比色法检测半胱天冬酶Caspase 3的活性以及EMSA检测核因子KappaB(NF κB)活性。 结果 :地塞米松使成骨样细胞存活数量下降 ,Caspase 3活性升高 ,NF κB活性下降。 结论 :地塞米松对原代未转化的成骨细胞有诱导凋亡的作用 ,并且是Caspase 3依赖性的 ,但NF κB似乎不参与该过程。

氟化钠对成骨样细胞功能表达影响的研究

目的 探讨氟对成骨样细胞功能表达的影响。方法 用细胞生长曲线和四唑盐比色 (MTT)法测定细胞增殖 ,对硝基苯基磷酸二钠盐比色 (PNPP)法检测碱性磷酸酶 (ALP)活性及放免法测定骨钙素分泌量 ,研究不同浓度 ( 10 -7mol/L～ 10 -3 mol/L)氟化钠 (NaF)对成骨样细胞UMR10 6增殖和分化功能的影响。结果 NaF对UMR10 6细胞增殖及早期分化呈双向调节作用 ,主要表现为低浓度促进 ,高浓度抑制。染氟一定时间后 ,与对照组相比 10 -7mol/L、 10 -6mol/L、 10 -5mol/LNaF组细胞增殖及ALP活性均增加 (P <0 0 5 ) ,而 10 -4mol/L、 10 -3 mol/LNaF组则表现为抑制细胞增殖及ALP活性 (P <0 0 5 )。但是 ,NaF对UMR10 6细胞晚期分化呈单一作用 ,上述不同浓度的NaF均可促进骨钙素分泌 ,与对照组相比有统计学意义 (P <0 0 5 )。而且 ,随NaF浓度升高骨钙素分泌量增加。结论 NaF对成骨样细胞的增殖呈双向调节 ,但对其分化因暴露时间和浓度不同而呈多样性。

杜仲对成骨样细胞增殖的作用

杜仲的水提液及水提液的不同萃取物和UMR1 0 6成骨样细胞体外共同培养 ,用MTT法检测细胞增殖 ,结果水提液在 0 4mg/ml培养 48小时具有最强的促进细胞增殖作用 ,其乙酸乙酯萃取物和正丁醇萃取物对细胞增殖有一定促进作用 ,但无显著性差异 ,而乙酸乙酯和正丁醇萃取后的剩余水层仍保留与原水提液相似的促进细胞增殖作用。表明杜仲中极性大部位可能含有直接作用于成骨细胞的活性成分。

成骨样细胞体外培养法筛选杜仲叶防治骨质疏松症的药效成份

目的 探讨杜仲叶提取成份对体外培养的成骨样细胞代谢功能的影响,筛选杜仲叶防治骨质疏松的药效成份。方法 从杜仲叶提取分离出4个化合物,以不同浓度(10-4g/L、10-6g/L、10-8g/L)加入体外培养的MG-63成骨细胞株中,并设空白组和对照组。培养4 d后,MTT比色法测定细胞增殖,对硝基磷酸盐法测定培养基中ALP活性。结果 细胞增殖数量与对照组比较,成份I 10-6g/L浓度组、Ⅱ、Ⅲ10-8g/L浓度组和成份Ⅳ10-4g/L、Ⅲ10-8g/L浓度组显著提高(P<0.05);成份Ⅱ、Ⅲ的10-4g/L、10-6g/L浓度组和成份Ⅳ10-6g/L浓度组极显著提高(P<0.01)。ALP活性与对照组比较,成份I 10-6g/L浓度组、成份Ⅱ10-4g/L、10-8g/L浓度组、成份Ⅲ10-8g/L浓度组和成份Ⅳ的10-4g/L、10-8g/L浓度组有显著性增强;成份Ⅱ10-6g/L浓度组和成份Ⅲ的10-4g/L、10-6g/L浓度组均极显著增强(P<0.01)。结论 4个成份均有一定的促进成骨细胞增殖、调节成骨细胞代谢的作用,以Ⅱ和Ⅲ效果明显。

不同流动剪切力调节大鼠成骨样细胞增殖的体外研究

目的 从细胞水平探索机械应力刺激调节骨改建的机制 ,并探讨促进骨改建的最适生理应力值。方法 对体外培养的大鼠成骨样细胞施加一系列不同大小的流动剪切力 ,利用流式细胞技术衡量其细胞的分裂增殖能力。结果 大鼠成骨样细胞受到流动剪切力后 ,细胞与流动方向一致的长轴被拉长 ,细胞沿流动方向排列。随着剪切力的增加 ,成骨样细胞的增殖指数 (PI)不断增加 ,当剪切力为 12 dyn/cm2 时 ,PI值与对照组相比有显著性差异 (P<0 .0 5 )。随剪切力的继续增加 ,PI值开始下降 ,当剪切力大于 14dyn/cm2 时 ,PI值与对照组相比显著下降 (P <0 .0 1)。结论 机械应力对骨改建的调节作用与成骨细胞受到应力诱导产生的流动剪切力的刺激有关 ,低剪切力对细胞的增殖影响不大 ,中等大小的剪切力 (12 dyn/cm2 )明显刺激细胞的增殖 ,高剪切力将抑制细胞的增殖。

几种生药的提取部位对成骨样细胞的增殖作用

考察了石斛、骨碎补、地骨皮、槲寄生的不同提取部位对成骨样细胞UMR10 6的增殖作用。将 4种生药的水提液、乙醇提取液及其经树脂柱洗脱后的不同提取部位与UMR10 6成骨样细胞共同体外培养 ,用MTT法检测各部位对细胞的增殖作用。结果表明 :除石斛的活性部位在 6 0 %、90 %的醇层外 ,骨碎补、地骨皮、槲寄生的活性部位集中在水层、30 %的醇层。

生长因子网络调节对骨形成作用的研究Ⅴ.不同生长因子交互应用对人胚成骨样细胞增殖及分化的影响

目的：考察将转化生长因子β（ＴＧＦ－β）、胰岛素样生长因子Ⅱ（ＩＧＦ－Ⅱ）、碱性成纤维细胞生长因子（ｂＦＧＦ）及血小板衍生性生长因子（ＰＤＧＦ）４种生长因子两两交互应用对人胚成骨样细胞增殖与分化的影响。方法：在不同的实验间期检测成骨样细胞３Ｈ－胸腺嘧啶脱氧核苷、３Ｈ－脯氨酸的掺入量和碱性磷酸酶的含量。结果：ＰＤＧＦ、ｂＦＧＦ、ＩＧＦ－Ⅱ和ＴＧＦ－β４种生长因子交互联合应用，均对促进体外培养的人胚成骨样细胞的增殖和分化功能具有协同作用。结论：具有协同作用的生长因子联合应用，可提高其促进骨形成的生物效应。