兔成骨细胞与纳米氧化锆强韧化高孔隙率磷酸钙人工骨细胞支架生物相容性研究

目的 研究成骨细胞与纳米氧化锆强韧化高孔隙率磷酸钙人工骨细胞支架在体外培养条件下的生物相容性。方法 将成骨细胞株置于含体积分数为10％胎牛血清的DMEM培养基中培养，传代后改用含地塞米松、β-甘油磷酸钠和维生素C的条件培养基培养，分为纳米支架复合细胞组和单纯细胞组，不同时间用倒置相差显微镜、HE染色光镜及扫描电镜观察。MTT法进行细胞增殖测定，并进行细胞微量蛋白含量检测和碱性磷酸酶的定量检测。结果 成骨细胞体外培养时复合或不复合纳米支架均生长良好，表现出典型的细胞形态特征和生物学特性，纳米支架利于细胞的贴附、生长与增殖，并对细胞的功能无不良影响。结论 纳米支架是较理想的骨组织工程支架材料，成骨细胞复合纳米支架用于骨缺损的修复，具有广阔的临床应用前景。     

活性氧在铁过载影响成骨细胞生物活性中的作用

目的 观察铁过载对人成骨细胞（hFOB1.19）生物活性的影响,同时观察活性氧在这一实验变化过程中的作用。 方法 体外培养成骨细胞,一组运用200μmol/L枸橼酸铁铵（FAC）干预,一组运用2.5mmol/L抗氧化剂N-乙酰半胱氨酸（NAC）干预,一组NAC预处理1h后运用相同浓度FAC干预,一组为正常对照;细胞培养48h后,流式细胞仪检测各组细胞内活性氧(ROS)的水平;CCK-8法检测各组细胞活力;RT-PCR法检测各组细胞OPG、BGP和COL1 mRNA表达的变化;碱性磷酸酶活性试剂盒检测各组细胞碱性磷酸酶活性。 结果 不同干预组成骨细胞内活性氧含量差异显著不同（P<0.05）,FAC组显著高于对照组,FAC+NAC组低于FAC组、高于NAC组;各组间活性氧含量的变化与成骨细胞活力、OPG、BGP和COL1 mRNA表达光密度比值、碱性磷酸酶活性呈负相关性,组间比较有统计学差异（P<0.05）。 结论 铁过载降低成骨细胞生物活性可能与铁过载增加成骨细胞内活性氧水平有关。     

成骨细胞株与强韧化纳米人工骨支架联合培养实验研究

目的 :观察成骨细胞株 ( 3T3 E1)在新型纳米氧化锆强韧化高孔隙率人工骨支架 (下文简称人工骨支架 )材料上生长情况。方法 :成骨细胞株 ( 3T3 -E1)与人工骨支架联合培养 ,通过光镜观察和细胞计数的方法了解成骨细胞与支架结合能力 ,扫描电镜观察细胞生长的情况。结果 :新型强韧化纳米人工骨材料与建株成骨细胞有良好的结合能力 ,成骨细胞株 ( 3T3 E1)在人工骨材料上生长良好。结论 :纳米骨支架是较理想的骨组织工程支架材料 ,成骨细胞复合支架用于骨缺损的修复 ,具有广阔的临床应用前景。     

p44/42和p38 MAPKs在骨髓间充质干细胞向成骨细胞分化中发挥不同的功能

目的 观察p44／42MAPK、p38MAPK通路在维生素C(Vit C)、β-磷酸甘油(β-GP)诱导骨髓间充质干细胞(BMSCs)向成骨细胞分化过程中的作用。方法 用[^3H]-甲基胸腺嘧啶掺入率法反映细胞增殖情况；洲定碱性磷酸酶活性与钙沉积积反映细胞向成骨细胞分化状态；有Western-blotting法反映MAPK的表达情况。结果 与溶剂对照组相比．在促成骨细胞分化剂Vit C，β-GP作用下，骨髓间充质干细胞(BMSCs)p44／42MAPK、p38MAPK通路均提前5d激活。p44／42MAPK通路阻断剂(PID8059)明显减少[^3H]-甲基胸腺嘧啶掺入率，抑制BMSCs的增殖；而p38MAPK通路的阻断剂(SB203580)则显著降低ALP活性及钙沉积量．抑制BMSCs向成骨细胞的分化。结论 p44／42MAPK通路在BMSCs的增殖过程中起苇要作用，而p38MAPK通路可能与BMSCs向成骨细胞分化调节有关。     

淫羊藿苷纳米微粒对大鼠成骨细胞成熟与矿化的影响

目的观察淫羊藿苷纳米微粒(ICA-NS)对出生48 h内的大鼠乳鼠颅骨成骨细胞成熟与矿化的作用。方法显微镜观察淫羊藿苷纳米微粒对成骨细胞形态的影响; Hoechst 3342/PI双染色法检测淫羊藿苷纳米微粒对成骨细胞的毒性反应;碱性磷酸酶检测试剂盒检测成骨细胞中碱性磷酸酶(ALP)活性;茜苏红染料对淫羊藿苷纳米微粒处理过的细胞进行染色,观察钙化结节的数量和面积;检测成骨细胞中与成骨相关的特异性蛋白表达情况。结果与淫羊藿苷组相比,淫羊藿苷纳米微粒组细胞形态无明显变化,且Hoechst 3342/PI双染色法结果进一步显示淫羊藿苷纳米微粒对成骨细胞无明显毒副作用;碱性磷酸酶活性测定结果显示淫羊藿苷纳米微粒显著提高了成骨细胞中碱性磷酸酶水平,而且淫羊藿苷纳米微粒组茜苏红钙化结节染色面积明显增大,颜色显著加深,成骨性相关蛋白的表达量也显著高于对照组。结论淫羊藿苷纳米微粒能显著促进成骨细胞的矿化与成熟,且对成骨细胞无明显毒副作用。     

地塞米松在人骨髓基质细胞诱导成骨细胞中的作用

目的 通过对人骨髓基质细胞体外培养及检测，研究地塞米松在骨髓基质细胞体外增生并向成骨细胞定向分化过程中的应用。方 法骨折内固定术扩髓前收集髓腔内骨髓进行原代和传代培养，传代后改用条件培养基（含地塞米松）和基础培养基（不含地塞米松）分别进行培养，应用组织化学及von Kossa方法检测细胞碱性磷酸酶和细胞外基质矿化程度；半定量RT-PCR方法检测Cbfal mRNA和Osterix mRNA在细胞培养过程中不同时间点的表达。并观测地塞米松对上述成骨细胞相关基因表达的影响。结果 条件培养基组细胞Cbfal mRNA和Osterix mRNA的表达峰值高于基础培养基组细胞，并且峰值出现的时间提前。结论 地塞米松通过促进Cbfal mRNA和Osterix mRNA的表达而促进骨髓基质细胞向成骨细胞分化。     

性激素对大鼠成骨细胞增殖及功能的影响

目的　观察、比较 17β 雌二醇 (E2 )、孕酮 (P)及二者联合应用对体外培养的大鼠成骨细胞 (ROB)增殖及成骨功能的影响。　方法　分离培养新生ROB ,分为培养液中不含激素 (对照组 )、含E2 10 -8mol/L(E2 组 )、含P10 -2 mol/L(P组 )、含E2 10 -8mol/L +P10 -7mol/L(E2 +P组 ) 4组。用 3 (4,5 二甲基 2 噻唑 ) 2 ,5 二苯基四氮溴唑盐 (MTT)法评价细胞增殖能力 ,逆转录 聚合酶链反应 (RT PCR)方法测定I型胶原 (α2 )链Col1mRNA含量 ,放射免疫法 (RIA)测定无血清培养液中I型原胶原羧基端延长肽 (P1CP) ,2 氨基 2 甲基丙醇法测碱性磷酸酶 (AKP) ,VonKossa染色法显示细胞层钙结节 ,用原子吸收仪测定钙含量。　结果　与对照组比较 ,P组细胞增殖能力显著增加 (P <0 .0 1) ;P及E2 +P组Col 1mRNA、P1CP、AKP、钙结节、钙原子含量均显著增加 (P <0 .0 1) ;E2 组Col1mRNA及P1CP均显著增加 (P <0 .0 1) ,但E2 组AKP、钙结节面积及钙原子含量均无显著差异(P >0 .0 5 )。E2 及E2 +P组对细胞增殖无显著影响 (P >0 .0 5 )。　结论　P刺激体外培养的ROB增殖 ,P及E2 +P增加ROB的骨胶原合成及钙化功能 ,而单纯E2 增加ROB骨胶原的合成 ,但不影响细胞增殖及钙化能力。     

Cdc42在人乳腺癌细胞MCF-7阿霉素敏感株和耐药株中的表达

目的探讨细胞分裂周期蛋白42(Cdc42)在乳腺癌细胞MCF-7阿霉素敏感株和耐药株MCF-7/Adr中表达的变化以及对细胞耐药性的影响。方法将Cdc42 siRNA转染MCF-7/Adr细胞株后,用RT-PCR和Weastern Blot方法检测MCF-7组,MCF-7/Adr组,MCF-7/Adr siRNA干扰组细胞Cdc42的转录以及蛋白的表达水平;采用四甲基偶氮唑蓝(MTT)法测定siRNA处理后阿霉素(ADM)对MCF-7/Adr细胞的杀伤作用;用荧光分光光度计测定细胞内ADM药物浓度。结果 MCF-7/Adr细胞中Cdc42 mRNA及蛋白表达量显著高于MCF-7细胞(P0.05);siRNA干扰后Cdc42表达受到明显抑制(P0.05),并显著提高细胞内ADM的浓度(P0.05)。结论特异性siRNA能明显抑制Cdc42 mR-NA及蛋白表达,并逆转细胞耐药性。     

酪氨酸激酶途径在成骨细胞增殖及c-fos表达中的作用

目的探讨酪氨酸激酶信息传递途径在成骨细胞增殖及ｃ－ｆｏｓ表达中的作用。方法采用阶段性酶消化法分离拳头新生大鼠颅盖骨居骨细胞,用ＭＴＴ法观察成骨细胞增殖,用免疫组化ＳＰ法结合计算机图像处理系统检测成骨细胞ｃ－ｆｏｓ表达。结果酪氨酸激酶抑制剂Ｇｅｎｉｓｔｅｉｎ和ＨｅｒｂｉｍｙｃｉｎＡ抑制成骨细胞增殖,且两者均部分阻断血清刺激的成骨细胞ｃ－ｆｏｓ表达。结论血清刺激和维持成骨细胞增殖及诱导ｃ－ｆｏｓ表达均     

New insight into the mechanism of hip prosthesis loosening: effect of titanium debris size on osteoblast function.

The incidence of rheumatoid arthritis and osteoarthritis is on the rise due to our expanding elderly population. Total joint arthroplasty is the most successful, prevalent treatment modality for these and other degenerative hip conditions. Despite the wide array of prosthetic devices commercially available, hip prostheses share a common problem with a gradual and then accelerating loss of bone tissue and bone-implant interface integrity, followed by implant instability and loosening. Implant failure is largely the result of inevitable wear of the device and generation of wear debris. To provide information for the development of improved prosthetic wear characteristics, we examined the effects of size-separated titanium particles on bone forming cell populations. We demonstrate unequivocally that particle size is a critical factor in the function, proliferation, and viability of bone-forming osteoblasts in vitro. In addition, we have elucidated the time-dependent distribution of the phagocytosed particles within the osteoblast, indicating an accumulation of particles in the perinuclear area of the affected cells. The report finds that particle size is a critical factor in changes in the bone formation-related functions of osteoblasts exposed to simulate wear debris, and that 1.5-4 microm titanium particles have the greatest effect on osteoblast proliferation and viability in vitro. The size of titanium particles generated through wear of a prosthetic device may be an important consideration in the development of superior implant technology.     

Insulin-like growth factor-I diminishes the activation status and expression of the small GTPase Cdc42 in articular chondrocytes.

Insulin-like growth factor-I (IGF-I) is an important anabolic growth factor in the maintenance of articular cartilage phenotypic expression. Chondrocyte morphology is also tightly linked to phenotype. The small G-protein Cdc42 plays a key role in regulation of cell morphology and phenotypic expression in several cell types and, we show here, in articular chondrocytes. The purpose of these studies was to investigate possible links between the intracellular signaling pathways of IGF-I and Cdc42 in articular chondrocytes. Treatment of chondrocytes with IGF-I resulted in a rapid and sustained decrease in the activation state (decreased GTP-bound) of Cdc42. Nucleotide exchange and hydrolysis experiments suggest that the decreased activation occurs through increased hydrolysis. Transient expression of dominant-negative Cdc42(T17N) allowed for enhanced expression of normal chondrocyte phenotype as determined by increased mRNA expression of collagen type II (Coll II) with decreased matrix metalloproteinase-3 (MMP-3) expression. The results of these studies suggest a novel link between IGF-I and Cdc42 signaling pathways. Further, an additional mechanism for the regulation of chondrocyte phenotype is defined through the IGF-I induced down-regulation of Cdc42 activation.     

Signaling through the small G-protein Cdc42 is involved in insulin-like growth factor-I resistance in aging articular chondrocytes.

During aging, chondrocytes become unresponsive to insulin-like growth factor-I (IGF-I). This study examined the role of Cdc42 (cell-division-cycle 42) in IGF-I signaling during aging. Experiments were performed using cartilage and chondrocytes isolated from horses ages 1 day-25 years. Northern analysis was used to examine expression of the small GTPases Cdc42, Rac, and RhoA. Western analysis was utilized to assess total Cdc42 (GTP + GDP-bound); active, GTP-Cdc42 was assessed using a pulldown assay with Western analysis. GTP-Cdc42 was also measured following IGF-I treatment. Gene expression for Cdc42 and Rac were decreased in mature samples, but there was no difference in total Cdc42 (GTP + GDP-bound) protein expression due to age. GTP-Cdc42 was significantly greater in prepubescent samples compared to other age groups. IGF-I diminished the GTP-bound state of Cdc42 in prepubescent chondrocytes; however, this effect was lost during aging. No differences in results were observed due to sample type; that is, cartilage tissues versus isolated chondrocytes. These studies suggest that loss of IGF-I-mediated regulation of Cdc42 activation may be a mechanism for the chondrocyte unresponsive state during aging. Further, the activation state of Cdc42, measured in native and IGF-I-treated cartilage tissue for the first time, is similar to that of isolated chondrocytes, indicating that the activation state of small G-proteins is not affected by isolation of chondrocytes from the extracellular matrix. Continued studies will identify the upstream regulators of Cdc42, which will further elucidate the molecular mechanism of IGF-I resistance during aging thereby providing insight into targeted strategies for age-related osteoarthritis.     

Interleukin‐1α, ‐6, and ‐8 decrease Cdc42 activity resulting in loss of articular chondrocyte phenotype

Small GTPase proteins mediate changes in cellular morphology and other cellular functions. The aim of this study was to examine signaling of the small GTPase Cdc42 by stimulating chondrocytes grown in monolayer with long‐ (96 h) or short‐ (2 and 30 min) term exposure to interleukin‐1α (IL‐1α), IL‐6, or IL‐8. Quantitative PCR was used to determine changes in collagen type IIB (COL2A1), aggrecan (AGG), and matrix metalloproteinase‐13 (MMP‐13) gene expression after prolonged cytokine exposure. Effects of short‐term treatment with IL‐α, IL‐6, or IL‐8 on endogenous GTP‐bound Cdc42 levels were assessed using an affinity assay, and on actin filament organization using confocal microscopy. Cytokine treatments significantly decreased COL2A1 and AGG expression and increased MMP‐13 expression. Short exposure to IL‐1α, IL‐6, or IL‐8 decreased endogenous GTP‐Cdc42 and increased stress fibers, which were reversed with cytochalasin D treatment. These results show that IL‐mediated Cdc42 signaling modifies chondrocyte phenotype and morphology. This may lend insight into the altered chondrocyte phenotype in catabolic conditions such as osteoarthritis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:246–251, 2012     

齿科种植体钛的耐磨损性质探讨

目的：探讨齿科种植体材料钛的耐磨损性及其对种植体牙周维护的远期影响。方法：应用Martens划痕实验与超声洁牙机磨损实验对钛板进行耐磨损性检测。结果：Martens划痕实验与超声洁牙机磨损实验结果显示其在不同的荷重下均会对钛板造成不同程度的划痕及磨损。讨论：采用常规牙周维护方法可能会对钛种植体造成磨损,可能加重种植体表面上牙石及菌斑的沉积与结合,对钛种植体的远期预后产生不良影响。     

阿仑磷酸钠对成骨细胞增殖、分泌和成骨功能的影响

目的　观察阿仑膦酸钠对人成骨细胞功能的影响 ,探讨使用此药防治人工关节无菌性松动的可行性。方法　采用体外培养成骨细胞的方法 ,观察不同浓度的阿仑膦酸钠 (1× 10 -11、1× 10 -9、1× 10 -7、1× 10 -5mol/L)对成骨细胞的增殖、碱性膦酸酶活性、分泌骨钙素及成骨能力的影响。结果　阿仑膦酸钠浓度达 1× 10 -5mol/L时抑制成骨细胞增殖 ;浓度为 1× 10 -7、1× 10 -5mol/L时增强成骨细胞的碱性膦酸酶活性和骨钙素分泌 ;对其成骨能力 ,浓度在 1× 10 -11、1× 10 -9mol/L时具有刺激作用 ,1× 10 -5mol/L有抑制作用 ,中间浓度 (1× 10 -7mol/L)无影响。结论　阿仑膦酸钠低浓度时有利于成骨细胞的成骨 ;局部使用有可能成为防治人工关节无菌性松动的方法之一。     

Silver oxide‐containing hydroxyapatite coating supports osteoblast function and enhances implant anchorage strength in rat femur

Antibacterial silver with hydroxyapatite (Ag–HA) is a promising coating material for imparting antibacterial properties to implants. We previously reported that 3% (w/w) silver with HA (3% Ag–HA) has both antibacterial activity and osteoconductivity. In this study, we investigated the effects of Ag–HA on the in vitro osteoblast function and the in vivo anchorage strength and osteoconductivity of implants. Production of the osteoblast marker alkaline phosphatase, but not cytotoxicity, was observed in cells of the osteoblast cell line MC3T3‐E1 cultured on the 3% Ag–HA‐coated surface. These results were similar to those observed with silver‐free HA coating. In contrast, a significant high level of cytotoxicity was observed when the cells were cultured on a 50% Ag–HA‐coated surface. The anchorage strength of implants inserted into the femur of Sprague–Dawley (SD) rats was enhanced by coating the implants with 3% Ag–HA. On the 3% Ag–HA‐coated surface, both metaphyseal and diaphyseal areas were largely covered with new bone and had adequate osteoconductivity. These results suggest that 3% Ag–HA, like conventional HA, promotes osteogenesis by supporting osteoblast viability and function and thereby contributes to sufficient anchorage strength of implants. Application of 3% Ag–HA, which combines the osteoconductivity of HA and the antibacterial activity of silver, to prosthetic joints will help prevent postoperative infections. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1391–1397, 2015.     

钛合金板置入在颅骨缺损修复中的应用

目的:探讨钛合金板置入在颅骨缺损修复中的应用。方法:对12例颅骨缺损的患者先进行三维CT检查,设计个性化的钛合金板置入以修复颅骨缺损。结果:本组患者均愈合良好,无感染、钛板外露及头皮血运障碍,避免了再次手术。结论:应用钛合金板修复颅骨缺损,具有良好的生物学力度,抗击性强,并发症少,生物相容性好等优点,可获得较好的治疗效果。     

The influence of bone allograft processing on osteoblast attachment and function.

In order to assess the influence of eight different sterilisation and disinfection methods for bone allografts on adhesion, proliferation, and differentiation of human bone marrow stromal cells (BMSC), cells were grown in culture and then plated onto pieces of human bone allografts. Following processing methods were tested: autoclavation (AUT), low-temperature-plasma sterilisation of demineralised allografts (D-LTP), ethylene oxide sterilisation (EtO), fresh frozen bone (FFB), 80 degrees C-thermodisinfection (80 degrees C), gamma-irradiation (Gamma), chemical solvent disinfection (CSD), and Barrycidal-disinfection (BAR). The seeding efficiency was determined after one hour to detect the number of attached cells before mitosis started. The cell viability was determined after 3, 7, and 21 days. Tests to confirm the osteoblastic differentiation included histochemical alkaline phosphatase staining and RT-PCR for osteocalcin. Human BMSC showed greatest attachment affinities for D-LTP-, 80 degrees C-, and CSD-allografts, whereas less cells were found attached to AUT-, EtO-, FFB-, Gamma-, and BAR-probes. Cell viability assays at day 3 revealed highest proliferation rates within the FFB- and 80 degrees C-groups, whereas after 21 days most viable cells were found in D-LTP-, 80 degrees C-, CSD-, and Gamma-groups. BAR-treatment showed a considerably toxic effect and therefore was excluded from all further experiments. Highest AP-activity and gene expression of osteocalcin were detected in the D-LTP-group in comparison with all other groups. In summary, our results demonstrate that cell adhesion, final population, and function of BMSC are influenced by different disinfection and sterilisation methods. Therefore, processing-related alterations of BMSC-function may be important for the success of bone grafting. The experimental setup used in the present work may be useful for further optimisation of bone allograft processing.