成骨细胞与镁合金表面硅酸盐-磷酸盐复合涂层的体外相容性

目的 评价成骨细胞在镁合金表面硅酸盐磷酸盐复合涂层上的生长状况,探讨镁合金表面硅酸盐磷酸盐复合涂层与成骨细胞的生物相容性。 方法 用胶原酶消化法分离培养大鼠成骨细胞,并用免疫荧光和茜素红染色法进行鉴定;然后将第4代大鼠的成骨细胞接种于材料表面,用扫描电镜观察成骨细胞在不同表面上黏附形态的变化,同时制备材料浸提液,用浸提液法培养细胞,并通过四甲基偶氮唑盐（MTT）法和碱性磷酸酶（ALP）活性法检测成骨细胞的增殖和分化情况。 结果 将成骨细胞与材料进行复合培养后,扫描电镜下可见,涂层组的成骨细胞生长活力旺盛、形态饱满、分布均匀,而单纯镁合金组未见细胞生长;MTT和ALP活性分析结果显示,硅含量为1%~5%涂层对大鼠成骨细胞的体外生长、增殖和分化的促进作用最佳。 结论 镁合金表面硅酸盐磷酸盐涂层与成骨细胞具有良好的相容性,在体外培养的环境下,有利于细胞的生长、黏附、增殖和分化。     

镁合金表面含硅涂层对成骨细胞的黏附、形态和细胞周期的影响

目的:评价成骨细胞在镁合金表面含硅涂层上的生长状况,为研究镁合金表面含硅涂层与成骨细胞的生物相容性提供实验依据.方法:用胶原酶消化法分离培养大鼠成骨细胞,并用免疫荧光和碱性磷酸酶染色法进行鉴定;然后将处于对数生长期的大鼠成骨细胞接种于材料表面,用扫描电镜观察成骨细胞在不同表面上粘附形态的变化,同时制备材料浸提液,用浸提...     

掺锶聚磷酸钙共培养成骨细胞与内皮细胞:行为和功能蛋白的变化

背景:前期实验发现掺锶的聚磷酸钙材料对单独培养内皮细胞或成骨细胞的行为有显著促进作用。目的:观察掺锶聚磷酸钙对共培养下成骨细胞与脐静脉内皮细胞行为和功能蛋白表达的影响。方法:取第3代人脐静脉内皮细胞与人成骨肉瘤细胞MG63以2∶1的浓度比接种于24孔板中,然后再分别加入掺锶聚磷酸钙、聚磷酸钙与羟基磷灰石,共培养7 d后。采用ELISA法检测细胞血管内皮生长因子、碱性成纤维细胞生长因子蛋白的表达,采用MTT法检测细胞活性。结果与结论:与聚磷酸钙与羟基磷灰石相比,在掺锶聚磷酸钙表面生长的细胞呈现更加良好的形态,细胞融合生长形成单层覆盖在材料表面,并且在材料表面有一定的跨度生长,说明掺锶聚磷酸钙材料能促进内皮细胞在其上黏附、伸展,有利于细胞的生长和增殖。掺锶聚磷酸钙组细胞分泌的血管内皮生长因子、碱性成纤维细胞生长因子明显高于聚磷酸钙组和羟基磷灰石组(P0.05),说明掺锶聚磷酸钙可上调血管内皮生长因子、碱性成纤维细胞生长因子蛋白的表达。     

掺锶聚磷酸钙的细胞相容性

背景:研究显示,以可降解生物陶瓷聚磷酸钙为基材,加入微量锶元素,通过调控材料结构如孔径、孔隙率等性能,可制备出有足够力学强度并可控降解的掺锶聚磷酸钙。目的:观察成骨细胞与掺锶聚磷酸钙的细胞相容性。方法:将兔成骨细胞与不同含锶质量分数(0～100%)的掺锶聚磷酸钙体外复合培养,进行形态学和功能测定。MTT法检测掺锶聚磷酸钙中成骨细胞的活力,并测定成骨细胞碱性磷酸酶活性。结果与结论:在倒置显微镜、扫描电子显微镜和激光共聚焦显微镜下观察到成骨细胞在掺锶聚磷酸钙的表面和孔隙内大量黏附、增殖和生长,并且分泌细胞外基质及细胞表面的大量微绒毛,显示成骨细胞在掺锶聚磷酸钙材料上生长形态良好;同时通过MTT和碱性磷酸酶测定表明细胞功能良好,其中含锶1%掺锶聚磷酸钙细胞增殖活性及碱性磷酸酶活性最高,对成骨细胞无毒性,具有良好的细胞相容性。     

纯钛种植体表面微弧氧化涂层的生物相容性

背景：各种纯钛种植体表面微弧氧化涂层效果不尽相同。 目的：观察3种不同微弧氧化涂层种植体钛片对小鼠成骨细胞的细胞增殖、碱性磷酸酶活性和β1-integrin的基因表达水平的影响。 方法：采用国际常用小鼠成骨细胞系(MC3T3-E1),3种不同涂层钛片作为影响因素,纯钛作为对照,采用MTT法和电镜法观察细胞附着和细胞增殖,PNPP法测定碱性磷酸酶的活性,RT-PCR法检测β1-integrin在小鼠成骨细胞中的表达。 结果与结论：MTT值、碱性磷酸酶值、β1-integrin的基因表达水平和电镜观察均显示含钙、磷、镁、锌元素的二氧化钛涂层钛片生物相容性最好,含钙磷盐的二氧化钛涂层钛片次之,二氧化钛涂层钛片最差。小鼠成骨细胞在其多孔,含有钙、磷、镁、锌元素表面的黏附及增殖最优。     

双相磷酸钙涂覆羟基磷灰石多孔支架的制备及性能

背景：目前的人工骨大多只能制备成小体积的填充用材料,大段负重骨及大块结构性植骨材料仍面临缺乏理想骨缺损修复材料和材料成型过程难以加工调控两大难题。目的：制备与人体大段负重骨形貌结构相似且兼具一定力学性能、优良生物相容性和部分可降解性的多孔羟基磷灰石/双相磷酸钙涂层复合支架,评估其性能。方法：采用水热合成法结合喷雾干燥技术分别制备羟基磷灰石粉体、羟基磷灰石晶须和β-磷酸三钙粉体,评估羟基磷灰石粉体、β-磷酸三钙粉体浸提液的细胞毒性。在羟基磷灰石粉体中添加不同含量(5%,10%,15%)的晶须,借助3D打印技术制备成多孔骨支架,设置不同的烧结工艺,通过正交实验筛选力学性能、孔隙率等最优的晶须含量与烧结工艺组合,检测最优组合支架的流体力学。采用浸渍提拉法将双相磷酸钙涂层涂覆在多孔骨支架上,体外评估双相磷酸钙涂层中β-磷酸三钙粉体的降解性能。结果与结论：(1)MTT实验显示,羟基磷灰石粉体、β-磷酸三钙粉体无明显的细胞毒性;(2)正交实验结果显示,影响多孔支架抗压强度最主要的因素为烧结温度,其次是晶须含量,最后是烧结时间;各因素组合中的最优组合为晶须含量10%、烧结温度1 300℃、烧结时间3...     

新工艺制备纳米钛酸钙涂层及生物相容性评价

目的 :研究新型纳米钛酸钙(CaTiO_3)涂层钛合金材料的生物相容性。方法 :将钛板、羟基磷灰石涂层钛板和纳米CaTiO_3涂层钛板分为钛板组、涂层组、纳米组(各60例)。通过扫描电镜、X射线衍射对3组材料进行分析。将各组材料与成骨细胞(MC3T3-E1)共培养,通过免疫荧光染色、MTT法、碱性磷酸酶(ALP)含量测定评估材料表面细胞的存活、增殖及分化情况;通过电镜检测材料表面成骨细胞的钙化。结果 :涂层组、纳米组比钛板组有更高的活细胞数量、MTT值、ALP含量,有更好的细胞结构形态和钙化,而涂层组、纳米组无差异。结论 :该新型纳米CaTiO_3涂层材料有良好的生物相容性,为其将来临床植入体内提供了一定的实验依据。     

SENP3对大鼠成骨细胞端粒酶活性及端粒长度的影响

目的:研究sentrin特异性蛋白酶3(SENP3)对大鼠成骨细胞端粒酶活性及端粒长度的影响。方法:首先0.2 mmol/L H_2O_2处理体外培养的大鼠成骨细胞后,Western blotting法检测SENP3及特异性蛋白1(Sp1)的表达。pc DNA3.0-SENP3转染成骨细胞,分别于24 h、48 h、72 h后采用四甲基偶氮唑蓝(MTT)法检测细胞活力的变化。转染48 h后,Western blotting法检测Sp1和端粒酶逆转录酶(TERT)的表达,PCR-TRAP法及PCR法检测端粒酶活性及端粒长度;ELISA检测上清中碱性磷酸酶(ALP)和骨桥蛋白(OPN)的含量;放射免疫法(RIA)检测骨钙蛋白(OCN)的含量。最后将pc DNA3.0-SENP3与siRNA-Sp1共转染成骨细胞,并检测以上指标。结果:H_2O_2处理成骨细胞后,SENP3和Sp1的表达显著上升。pc DNA3.0-SENP3转染成骨细胞后,Sp1和TERT的表达显著上升,细胞活力、ALP、OPN及OCN含量也都显著上升;端粒酶活性显著增加及端粒长度缩短显著延缓。而当pc DNA3.0-SENP3与siRNA-Sp1共转染成骨细胞后,细胞活力,ALP、OPN及OCN含量,端粒酶活性及端粒长度均未发生显著变化。结论:SENP1通过上调Sp1的表达促进TERT的表达,增加端粒酶活性上升及延缓端粒长度缩短,从而增强成骨细胞增殖能力。     

脱细胞天然骨基质与诱导性成骨细胞的体外相容性

背景：前期工作表明TritonX-100处理的脱细胞骨基质已满足组织学和免疫学方面的修复要求。如果细胞能在材料表面很好地生长,将利于进一步进行体内动物实验。 目的：采用细胞培养法在体外评估脱细胞骨基质与诱导后成骨细胞的生物相容性。 方法：第3代骨髓基质干细胞经成骨诱导分化培养液诱导分化为成骨细胞,接种于TritonX-100处理的脱细胞骨基质及羟基磷灰石表面,检测成骨细胞的碱性磷酸酶表达并用扫描电镜观察材料表面的细胞生长情况。 结果与结论：碱性磷酸酶活性分析均表明,TritonX-100处理的脱细胞骨基质在培养48 h之后比羟基磷灰石更利于诱导成骨细胞生长;扫描电镜下可见,成骨细胞在脱细胞骨基质表面呈现立体生长方式,细胞呈球形,并且聚集成簇。体外实验结果显示成骨细胞与脱细胞天然骨基质有较好的生物相容性。     

茶多酚对脂多糖刺激下成骨细胞增殖分化的影响

背景：研究表明,茶多酚在牙周炎等疾病的研究中能发挥抗炎、抗氧化作用。目的：探讨茶多酚在脂多糖刺激下对小鼠成骨细胞增殖分化及氧化应激的影响。方法：体外培养小鼠成骨细胞MC3T3-E1,分4组处理：对照组常规培养;脂多糖组加入10μg/mL脂多糖,茶多酚组加入1μg/mL茶多酚,联合组加入10μg/mL脂多糖+1μg/mL茶多酚。采用MTT法检测细胞增殖情况,碱性磷酸酶试剂盒检测细胞碱性磷酸酶活性,偶氮偶联法检测细胞碱性磷酸酶染色程度,钙检测试剂盒检测细胞钙沉积量,超氧化物试剂盒检测细胞超氧化物含量,丙二醛试剂盒检测细胞丙二醛含量。结果与结论：(1)与对照组相比,脂多糖组成骨细胞增殖活性、碱性磷酸酶活性、钙沉积量降低(P <0.05),碱性磷酸酶染色程度降低(P <0.01),超氧化物和丙二醛含量增加(P <0.05);茶多酚组成骨细胞增殖活性、碱性磷酸酶活性、钙沉积量升高(P <0.05),碱性磷酸酶染色程度提高(P <0.01),超氧化物和丙二醛含量减少(P <0.05);(2)与脂多糖组相比,联合组成骨细胞增殖活性、钙沉积量升高(P <0.0...     

Interaction of Sr-doped hydroxyapatite nanocrystals with osteoclast and osteoblast-like cells

This article reports the effect of strontium incorporation into hydroxyapatite nanocrystals on bone cells response. Hydroxyapatite nanocrystals were synthesized at strontium contents of 0, 1, 3, 7 atom %. Strontium incorporation for calcium is confirmed by the linear increase of the unit cell parameters of hydroxyapatite, in agreement with the different ionic radii of the two ions. Moreover, strontium substitution slightly affects hydroxyapatite structural order and the shape of the nanocrystals. Osteoblast-like MG63 cells cultured on the nanocrystals display good proliferation and increased values of the differentiation parameters. In particular, when cultured on samples with Sr concentration in the range 3-7 atom %, osteoblasts display increased values of ALP activity, collagen type I, and osteocalcin production. Moreover, the osteoclast number on all the Sr-doped samples is significantly smaller than on hydroxyapatite, and it decreases on increasing strontium content. The data indicate that strontium stimulates osteoblast activity and exerts its inhibitory effect on osteoclast proliferation even when incorporated into hydroxyapatite.     

Human osteoblast response to silicon-substituted hydroxyapatite

Human osteoblasts were cultured on hydroxyapatite (HA), 0.8 wt % silicon substituted hydroxyapatite (Si-HA) and 1.5 wt % Si-HA discs. The influence of these substrates on cell behaviour in vitro was assessed by measuring total protein in the cell lysate and the production of several phenotypic markers: collagen type I (COL I), alkaline phosphatase (ALP), osteocalcin (OC), and the formation of bone mineral. After 7 days, beta-glycerophosphate and physiological levels of hydrocortisone were added to the culture medium to stimulate cell differentiation and mineral production. There was a significantly higher production of ALP on 1.5 wt % Si-HA at day 7 following which, the addition of hydrocortisone promoted the differentiation of cells on the other two substrates. Hydrocortisone addition also decreased the production of OC. During the period, when hydrocortisone was present, no significant difference in behavior was seen between cells on Si-HA and HA; however, following removal of hydrocortisone, cells responded to 0.8 wt % Si-HA with a significant increase in protein production. Using fluorescence microscopy, nodular structures labeled with tetracycline were observed on the surface of all substrates after 21 days. These structures were deposited on areas of high cell density but were not related to the presence or level of silicon in the substrate. These results indicate that human osteoblasts are affected by the presence of silicon in the HA substrate and that the timing of these effects may be dependent upon the level of silicon substitution.     

碳化硅对成骨细胞增殖和分化的影响

目的研究碳化硅(SiC)的体外生物相容性,为探索SiC作为骨缺损修复材料的可行性奠定基础。方法实验组为实质SiC,对照组为致密羟基磷灰石(HA)。将取自大鼠胎鼠颅骨的成骨细胞分别接种于2组材料表面。分别采用扫描电子显微镜、四甲基偶氮唑盐(MTT)法、碱性磷酸酶(ALP)含量测定技术、流式细胞仪检测细胞周期,观察分析两种材料表面上的细胞的增殖和分化能力。结果扫描电子显微镜显示:原代成骨细胞在两种材料表面伸展良好,细胞在材料表面伸出很多伪足,并且在材料表面可见明显的细胞外基质沉积。两组材料上细胞的MTT值从1、3、5、7 d随观察时间逐渐升高,并且各个时间点实质SiC均高于致密HA,两者之间的差异有统计学意义(P<0.05)。两组材料上细胞的ALP含量从1、3、5、7 d随观察时间逐渐升高;在1、3 d时,致密HA高于实质SiC,在5、7 d时,致密HA低于实质SiC,但是两组之间的差异均无统计学意义(P>0.05)。1、3、5 d两组材料的细胞增值指数(PI)值逐渐升高,高峰出现在第5天,第7天时下降,两组材料在各个时间点的PI均接近,差异无统计学意义(P>0.05)。结论实质SiC具有与致密HA相似的良好的细胞相容性。     

Characterization of titanium surfaces with calcium and phosphate and osteoblast adhesion

The titanium surfaces containing calcium, phosphate ions and the carbonate apatite were characterized. The effect of surface chemistry on the initial rabbit osteoblast response on these surfaces was investigated. The cell count and alkaline phosphatase (ALP) specific activity assay were used for biochemical analyses. Scanning electron microscopy was used for morphology observation and in particular X-ray photoelectron spectroscopy (XPS) for surface chemistry characterization. The number of cells adhering to the apatite coating surface was the maximum, the number of cells on the surface containing calcium without phosphate ions was higher than that containing phosphate without calcium, and the number on the unmodified titanium surface was the least. The osteoblasts cultured on the apatite surface exhibited the highest ALP specific activity, next were the ones on the surface containing solely calcium, the lowest were on the unmodified titanium surface. On the substrate surfaces removed of adhered cells, the order of nitrogen amounts detected by XPS was consistent with ones of ALP specific activity and cell number, except for the unmodified titanium surface. For the substrate surfaces removed of adhered osteoblasts, XPS analysis showed that calcium and phosphorous amounts decreased during cell adhesion. After cell culture the Ca2p binding energy (BE) values for apatite coating and the surface containing solely calcium were similar to those of the two surfaces adsorbed bovine serum albumin (BSA). The P2p BE values for the surfaces containing phosphate ions, including the apatite coating and the surface containing solely phosphate ions, showed the same change. But after cell culture the decrease of the P2p BE value for the coating surface was larger than the one for the surface containing solely phosphate ions. Considering the bovine serum albumin adsorption on the same samples, these results indicated that calcium ions on titanium surfaces play a more important role than phosphate ions in initial interactions among culture medium, osteoblasts and titanium surfaces. On the apatite coating surface, calcium ions are active sites for osteoblast adhesion, while calcium and phosphate ions co-exist on titanium surfaces, the former promotes the osteoblast adhesion onto the phosphate sites on titanium surfaces. The cell adhesion was a complicated biological and chemical process relating to surface several elements similar to protein adsorption.     

Synthesis of a novel b-tricalcium phosphate/hydroxyapatite biphasic calcium phosphate containing niobium ions and evaluation of its osteogenic properties

To promote the osteogenic properties of osteoblasts, we synthesized a hydroxyapatite (HAp) with β-tricalcium phosphate (β-TCP) biphasic calcium phosphate containing Nb ions (NbTCP/HAp). NbTCP/HAp was prepared by annealing precipitates obtained by coprecipitation of an aqueous solution of Ca(NO₃)₂ and a mixture of (NH₄)₂HPO₄ and aqueous Nb solution. The precipitates can be regarded as a calcium-deficient HAp, the PO₄ sites of which are partly occupied by Nb ions. NbTCP/HAp was successfully synthesized by thermal decomposition of the precipitates. NbTCP/HAp enhanced the calcification of normal human osteoblasts (NHOst), and the amount of calcified tissue increased in proportion to the Nb ion concentration in the NbTCP/HAp. The alkaline phosphatase (ALP) activity of NHOst was also enhanced by NbTCP/HAp. Because Nb ions significantly enhance the ALP activity of NHOst, calcification by NbTCP/HAp is considered to be due to enhancement of ALP activity induced by Nb ions dissolved from NbTCP/HAp. These results indicate that NbTCP/HAp can be an effective bone repair material.     

不同浓度地塞米松体外对人成骨细胞和破骨细胞分化的影响

目的: 探讨地塞米松(DEX)体外对人成骨细胞(OBs)和破骨细胞(OCs)分化的影响。方法: 以DEX为主要成分的诱导剂体外刺激正常人骨髓间充质干细胞(MSCs),茜素红染色及碱性磷酸酶(ALP)定量法检测OBs间钙结节形成及OBs中ALP水平;改变DEX浓度后,再检测ALP水平以及用Western blotting检测OBs中磷酸化GSK-3β蛋白的表达;外源性核因子κB受体活化因子配基(RANKL)刺激人骨髓单个核细胞(BMMs)14 d后,用抗酒石酸酸性磷酸酶染色鉴定OCs形成情况、Western blotting检测BMMs中RANKL下游的信号途径;不同浓度DEX刺激MSCs 7 d后,用ELISA和Western blotting检测培养液中可溶性RANKL(sRANKL)水平及MSCs中RANKL蛋白表达。结果: 含0.1 μmol/L DEX的诱导剂刺激MSCs后可有钙结节形成,在DEX 0.001-0.1 μmol/L浓度范围内,OBs中ALP水平逐渐增高、磷酸化GSK-3β蛋白表达逐渐增强,当DEX浓度进一步增加后(0.1-10 μmol/L), ALP水平和磷酸化GSK-3β蛋白表达反而下降;外源性RANKL可以诱导BMMs分化为OCs,且BMMs中磷酸化IκBα、SPAK/JNK、p38 MAPK、p44/42 MAPK表达增强;在0.001-10 μmol/L浓度范围内,DEX刺激MSCs后,培养液中 sRANKL水平和MSCs中RANKL蛋白表达逐渐增高,且sRANKL水平和DEX浓度呈正相关(r=0.821,P<0.05)。结论: 在低浓度(0.001-0.1 μmol/L)范围内,DEX既促进OBs分化,又可促进OCs分化。在高浓度(0.1-10 μmol/L)范围内,仍能促进OCs分化,但失去了其对OBs的分化作用。     

Dissolution control and cellular responses of calcium phosphate coatings on zirconia porous scaffold

Different types of calcium phosphates [hydroxyapatite (HA), fluorapatite (FA), tricalcium phosphate (TCP), and their composites (HA + FA, HA + TCP)] were coated on a zirconia (ZrO(2)) porous scaffold using a powder slurry method. The ZrO(2) porous scaffold was intended for a load-bearing implant, and the apatite layers were coated to improve osteoconductivity. The insertion of an FA intermediate layer between the coating layer and ZrO(2) scaffold effectively suppressed the reaction between the calcium phosphate and ZrO(2) and maintained the coating layer at the initial powder composition. The obtained coating layer, of a thickness of approximately 30 microm, was relatively microporous and firmly adherent to the ZrO(2) scaffold. Dissolution tests in physiological solution showed typical differences depending on the coating layers, with the dissolution rate increasing in the order TCP > HA + TCP > HA > HA + FA > FA. This result suggests the functional coating of the calcium phosphates in view of tailoring the solubility. Osteoblast-like cells, MG63 and HOS, responded similarly in terms of cell growth, morphology, and proliferation rate regardless of the coating types, indicating favorable and comparable cell viability. However, the alkaline phosphatase (ALP) activity of the cells on the pure HA and HA composite coatings (HA + FA and HA + TCP) expressed at higher levels compared to those on pure FA and pure TCP coatings for both MG63 and HOS cells, suggesting a selective cell activity depending on the coating types. All the calcium phosphate-coated-ZrO(2) scaffolds showed higher ALP levels compared to pure ZrO(2) scaffold.     

Practice of a testing bench to study the effects of cyclic stretching on osteoblast-orthopaedic ceramic interactions

A new experimental method has been used to study the behaviour of human osteoblasts cultured on bioceramics subjected to mechanical strains. The ceramics were alumina, hydroxyapatite (HA) and a duplex system composed of hydroxyapatite-covered alumina. The system applied 400 microdeformations for a 6-h period with a cycle frequency of 0.5 Hz to osteoblasts growing on ceramic-covered disks. The effects of strains on short-term cell viability, cell growth, alkaline phosphatase (ALP) activity, and collagen biosynthesis were assessed. When possible, the parameters (lactate dehydrogenase) were studied along the experiment in samples of the culture medium, in the other cases by comparison of stretched and unstretched cultures on the same ceramics with the same cell line. In relationship with the coating, mechanical strains resulted in a decrease in DNA corresponding to cell number, an LDH release during straining, an unchanged (alumina) or decreased (HA and duplex) ALP activity, a decrease (HA and duplex) of collagen and total protein synthesis or an increase of it (alumina). The stress-producing device and its associated protocol are shown to be suitable for investigating the behaviour of cells, cultured on biomaterials subjected to mechanical strain.