成骨细胞和成纤维细胞黏附及增殖:不同宽度表面均一平行沟槽的影响

背景:生物相容性材料表面的微结构可影响真核细胞的黏附和增殖。目的:观察纯钛表面的微结构对成骨细胞和成纤维细胞黏附及增殖的影响,探索优化种植体体部及穿龈部位的材料形貌特征。方法:在光滑的钛片表面设计并制备深度为2μm,宽度分别为100,50,20,10,5μm的5种均一平行微米沟槽,以光滑钛片作为对照,测定样品的粗糙度和亲水接触角。在体外对不同形貌的钛片分别进行成骨细胞MG63和成纤维细胞L929复合培养。结果与结论:微米沟槽钛片的亲水性和粗糙度较光滑钛片均有所增加。不同尺度的沟槽对不同细胞黏附和增殖的影响不同,均一平行沟槽结构普遍促进成纤维细胞的黏附和成骨细胞的增殖(P0.05);而当沟槽的尺寸接近细胞的形态和大小时对细胞行为有负面影响。     

rhBMP-7对小鼠骨髓间充质干细胞向成骨细胞分化的影响

目的:观察重组人骨形成蛋白-7(rhBMP-7)对小鼠骨髓间充质干细胞(BMSCs)向成骨细胞分化的影响。方法:采用密度梯度离心法分离骨髓,体外培养扩增,获得小鼠骨髓间充质干细胞,贴壁培养至第3代,向培养液中加入rhBMP-7,培养5 d后进行碱性磷酸酶(ALP)染色,ALP活性与骨钙素(OC)含量检测,并用RT-PCR法分析成骨细胞标志基因骨钙素(OC)与Ⅰ型胶原(Col-Ⅰ)表达情况,Western blot法检测Ⅰ型胶原(Col-Ⅰ)蛋白的分泌量。结果:rhBMP-7诱导组的骨髓间充质干细胞的ALP染色强度、ALP活性及OC含量明显升高,OC与Col-Ⅰ的mRNA表达水平以及Col-Ⅰ的蛋白表达水平均明显提高。结论:rhBMP-7可促进体外培养的小鼠BMSCs向成骨细胞方向分化。     

表面矿化修饰煅烧骨/P24活性多肽仿生骨材料对MC3T3-E1细胞黏附、增殖和向成骨方向分化的影响

本研究目的是评价表面矿化修饰煅烧骨/P24活性多肽复合仿生骨材料对小鼠MC3T3-E1细胞黏附、增殖和诱导成骨分化的影响。实验分三组:A组为表面矿化修饰煅烧骨加骨形态发生蛋白-2(BMP-2)活性多肽P24修饰的复合仿生骨材料,B组为表面矿化修饰煅烧骨材料,C组为单纯的煅烧骨材料。三组材料在细胞实验前行电镜扫描观察。然后将小鼠MC3T3-E1细胞分别种植在三种材料表面,测定细胞的黏附率,MTT法检测细胞体外增殖活性,同时通过碱性磷酸酶(ALP)染色和ALP活性检测,了解小鼠MC3T3-E1细胞成骨分化情况。电镜观察显示三组材料均保留了天然的孔隙结构,孔径为200~850μm。细胞黏附率和MTT实验显示,A组MC3T3-E1细胞在材料表面的黏附性能和增殖能力明显高于B组和C组,B组则高于C组,组间差异均有统计学意义(P0.05)。ALP染色和ALP活性检测显示:A组细胞ALP活性明显高于B组和C组,B组则高于C组,组间差异均有统计学意义(P0.05)。研究表明表面矿化修饰煅烧骨/P24活性多肽仿生骨材料能促进小鼠MC3T3-E1细胞在骨基质材料表面的黏附、增殖与分化,并能较好地保持细胞的形态。     

TiO_2纳米管对DPT在成骨细胞中表达的影响及机制研究

目的研究不同管径TiO_2纳米管修饰的钛材料与成骨细胞在体外复合培养,观察其对成骨细胞的粘附、增殖和分化的影响,并探讨可能的机制,为纳米材料的临床应用提供理论依据。方法将骨植入材料金属物质分成四组,其中三组经处理后分别内置有50 nm、100 nm、CDW图案的纳米管,以未经任何处理的钛片作为对照组,将MG-63成骨细胞分别接种于内置有50 nm、100 nm、CDW图案的纳米管与对照组钛片的6孔板中,采用MTT实验对四组材料复合培养MG-63的细胞活力进行评估;应用碱性磷酸酶(alkline phosphatase,ALP)试剂盒检测四组细胞碱性磷酸酶活性的改变;荧光共聚焦检测皮肤桥蛋白(DPT)的表达情况;通过Western blot实验检测DPT、黏着斑激酶,粘着斑激酶(Focal Adhesion Kinase,FAK)、骨桥蛋白(osteopontin,OPN)蛋白的表达变化。结果MTT检测显示,三种纳米管复合培养MG-63活性比对照组钛片复合培养的MG-63活性增强,培养的第3d,5d,7d MG-63细胞活性增强显著,呈时间依赖性。与钛片复合培养组相比,50 nm、100 nm、CDW图案的纳米管复合培养组ALP、DPT、FAK及OPN表达水平增加。结论50 nm、100 nm、CDW图案的纳米管复合培养更能促进成骨细胞的生长与铺展,具有良好的生物相容性,其机制可能与增加ALP的表达水平和激活DPT/FAK信号通路相关。     

聚醚醚酮/58S生物活性玻璃骨植入材料的成骨活性

目的 探讨聚醚醚酮/58S生物活性玻璃(PEEK/58S)骨植入材料的成骨活性.方法 MG-63成骨细胞在PEEK和PEEK/58S表面培养一段时间后,扫描电镜(SEM)观察MG-63成骨细胞的黏附铺展状态;CCK-8法检测MG-63成骨细胞的增殖活性;碱性磷酸酶(ALP)活性检测评价MG-63成骨细胞的成骨分化能力;...     

纯钛表面微形态可影响成骨细胞生长及β1整合素表达

背景：目前微弧氧化和渐热处理是常见的两种钛种植体表面化学成分改性方法。 目的：观察纯钛表面微形态对成骨细胞生长的影响。 方法：将原代培养的小鼠成骨细胞与不同表面处理的钛片：纯钛组、微弧氧化组、碱热组、微弧氧化碱热处理组共同培养。 结果与结论：扫描电镜观察微弧氧化碱热处理组表面附着细胞呈扁平的多边形,且有更好的伸展,成骨细胞中β1整合素表达显著高于其他3组(P < 0.05)。提示微弧氧化碱热处理组更有利于成骨细胞的黏附和β1整合素的表达。     

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。     

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。     

密骨打老儿丸含药血清对共育体系中成骨细胞和破骨细胞功能的影响

 目的： 观察密骨打老儿丸（Migu-Dalaoer pill,MDP）含药血清在成骨-破骨细胞共同培养体系中对成骨细胞（osteoblasts,OB）增殖和破骨细胞（osteoclasts,OC）骨吸收功能的影响。方法： 利用分段酶消化法从胎鼠颅骨中分离出OB,取1日龄SD大鼠四肢股骨和胫骨分离培养OC,建立培养上清相通但2种细胞间互不接触的成骨-破骨细胞共育模型。实验分为不同浓度（低、中、高）的MDP含药血清组和对照组进行比较,以细胞增殖（MTT 法）和碱性磷酸酶（alkaline phosphatase,ALP）活性代表OB的成骨活性,以抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase,TRAP）活性和骨吸收陷窝数目代表OC的破骨能力进行测定。结果： 与对照组相比,中、高浓度MDP含药血清在成骨-破骨细胞共同培养体系中6和7 d 能显著提高OB数目和 ALP 活性（P<0.01）。与对照组相比,中、高浓度MDP含药血清在成骨-破骨细胞共同培养体系中6和7 d 能显著降低OC骨吸收陷窝的数目和分泌 TRAP 的活性（P<0.01）。结论： 密骨打老儿丸含药血清在共育体系中能够促进OB增殖和骨形成,同时抑制OC骨吸收功能。     

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.     

抗菌钛合金Ti6Al4V-6Cu表面构建成骨细胞膜片的实验研究

目的:探讨大鼠颌骨成骨细胞在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片的可行性。方法:体外培养大鼠颌骨成骨细胞,采用富含维生素C培养基在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片（细胞膜片组）,并以单纯培养基作对照（对照组）,检测膜片形成过程中碱性磷酸酶（ALP）和成骨相关基因ALP、I型胶原（Col-1）、骨形成蛋白2（BMP-2）的表达情况。结果:采用富含维生素C的培养基连续培养可在抗菌钛合金Ti6Al4V-6Cu表面成功构建成骨细胞膜片,该细胞膜片由多层细胞构成,富含胞外基质。相对于对照组,细胞膜片组的膜片形成过程中成骨细胞ALP活性及成骨相关基因ALP、Col-1、BMP-2的表达均显著增高。结论:在抗菌钛合金Ti6Al4V-6Cu表面可成功构建成骨细胞膜片,有望与Ti6Al4V-6Cu联合应用于引导性骨再生术。     

Increase of BM-MSC proliferation using L-DOPA on titanium surface in vitro

In order to increase biocompatibility, many dental implants have been studied by immobilization of biomolecules on biomaterials. We used l-3,4-dihydroxyphenylalanine (L-DOPA) as a biomolecule for surface-modified titanium. Water contact angles of nontreated titanium discs (negative control), etched titanium discs (positive control), and titanium discs treated with L-DOPA following the etching process (experimental group) were 82.4?±?5.7°, 67.1?±?0.56°, and 44.15?±?0.91°, respectively. Using atomic force microscopy images, we were able to find L-DOPA, which adhered to the titanium surface. The number of human bone marrow mesenchymal stem cells (BM-MSCs) in the experimental group was much higher than that of cells in any other group. Quantification values of amine groups in the positive control and experimental groups were approximately 3 and 7.5?μg, respectively. Therefore, findings from our research suggested the possibility of a causal link between increased L-DOPA content and cell proliferation in BM-MSCs. Moreover, coating of the discs with L-DOPA can result in greater hydrophilicity of the titanium surface and enhancement of cell adhesion and mitochondrial activity.     

Expression of SP7/osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen huoxue decoction北大核心

BACKGROUND: Bushen Huoxue Decoction (BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits. OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase (ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD. METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing (blank control group), Cbfal/RUNX2 gene silencing lentivirus (silencing group), and negative viral vector (negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group. RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity (P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly (P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

微弧氧化陶瓷膜表面骨髓间充质干细胞生物相容性实验

目的观察微弧氧化（micro-arc,MAO）陶瓷膜表面骨髓间充质干细胞（mesenchymal stem cell,MSC）的黏附和增殖生长情况,通过MSC在三组不同表面处理材料表面的黏附和增殖情况评价微弧氧化陶瓷膜表面的生物相容性。方法贴壁法培养大鼠骨髓间充质干细胞,骨诱导培养后,做碱性磷酸酶染色和茜素红钙结节染色,鉴定其成骨潜能。取生长良好的第三代MSC,调整细胞密度为5×10^4/mL,接种到三组钛片表面。在接种细胞后第1、3、5、7天每组分别取出5枚钛片,一个做电镜扫描,另外4个细胞计数。观察MSC在不同材料表面的黏附增殖情况。电镜扫描MAO陶瓷膜表面形貌特征,EDX分析其表面主要元素含量。结果MSC具有良好的成骨特性,在MAO陶瓷膜表面的增殖优于光滑组和喷沙组。电镜下微弧氧化陶瓷膜表面有无数2～10m的微孔,并含有钙、磷成分。结论MAO陶瓷膜具有良好的生物相容性,大鼠骨髓间充质干细胞在其多孔,含有钙、磷元素表面的黏附及增殖均优于其它组。     

Proliferation and differentiation parameters of human osteoblasts on titanium and steel surfaces

Commercially pure titanium (cpTi), titanium alloys, and steel are often used for dental and orthopedic implants. In these applications titanium is considered the "gold standard." However, tissue reactions around titanium implants and the changing trend to leave orthopedic devices in the body have led to a new examination of the preferred material. This in vitro study tested the behavior of osteoblasts on cpTi, Ti-6Al-7Nb, and stainless steel with surface designs similar to clinical implants. After surface characterization by scanning electron microscopy and profilometry, cell proliferation and the differentiation parameters of alkaline phosphatase (ALP) activity and osteocalcin were measured. For all materials tested, the growth curves showed a similar kinetic. On Ti-6Al-7Nb, ALP activity was significantly lower when compared with steel, and cpTi and did not change over the time. ALP activity increased moderately on steel and cpTi. Osteocalcin levels were higher on both titanium materials than on steel. Based on undisturbed cell growth and the relatively high alkaline phosphatase and osteocalcin levels, we suggest that cpTi provides the best biocompatibility with regard to proliferation, in addition to more reliable early and late differentiation markers of human osteoblasts in vitro.     

Differentiation of osteoblasts in three-dimensional culture in processed cancellous bone matrix: quantitative analysis of gene expression based on real-time reverse transcription-polymerase chain reaction

Processed bovine cancellous bone (PBCB) is an attractive material for tissue engineering of bone. It is biocompatible, osteoconductive, nonimmunogenic, and porous and its biomechanical properties are close to those of native bone. In this study, differentiation of primary rat osteoblasts (rOBs) incubated on PBCB was investigated in vitro. rOBs were isolated and expanded in two-dimensional culture. Expanded rOBs were seeded into PBCB disks and cultured either in basal medium (BM) or differentiation medium (DM) containing ascorbic acid, beta-glycerol phosphate, and dexamethasone. Alkaline phosphatase (ALP) activity and RNA expression of ALP, bone sialoprotein (BSP), collagen type I (COL1), osteocalcin (OC), and osteopontin (OPN) were assessed by chemiluminescence assay and quantitative real-time RT-PCR over 14 days. Histologic analysis was performed on day 14. ALP increased over the observation period independent of stimulation. OPN and BSP expression was significantly higher in the DM group whereas COL1 and OC expression was significantly higher in the BM group. Matrix calcification was detectable only in the DM group by von Kossa stain. The observed expression patterns suggest a physiological response of rOBs to the differentiation stimulus. PBCB is a suitable matrix for in vitro differentiation of osteoblasts. Cell-seeded PBCB is a potential osteogenic construct for in vivo application.     

The effects of pulsed electromagnetic field on the functions of osteoblasts on implant surfaces with different topographies

The use of pulsed electromagnetic fields (PEMFs) is a promising approach to promote osteogenesis. However, few studies have reported the effects of this technique on the osseointegration of endosseous implants, especially with regard to different implant topographies. We focused on how the initial interaction between cells and the titanium surface is enhanced by a PEMF and the possible regulatory mechanisms in this study. Rat osteoblasts were cultured on three types of titanium surfaces (Flat, Micro and Nano) under PEMF stimulation or control conditions. Protein adsorption was significantly increased by the PEMF. The number of osteoblasts attached to the surfaces in the PEMF group was substantially greater than that in the control group after 1.5 h incubation. PEMF stimulation oriented the osteoblasts perpendicular to the electromagnetic field lines and increased the number of microfilaments and pseudopodia formed by the osteoblasts. The cell proliferation on the implant surfaces was significantly promoted by the PEMF. Significantly increased extracellular matrix mineralization nodules were observed under PEMF stimulation. The expression of osteogenesis-related genes, including BMP-2, OCN, Col-1,ALP, Runx2 and OSX, were up-regulated on all the surfaces by PEMF stimulation. Our findings suggest that PEMFs enhance the osteoblast compatibility on titanium surfaces but to different extents with regard to implant surface topographies. The use of PEMFs might be a potential adjuvant treatment for improving the osseointegration process.     

Temporal pattern of stimulation of osteoblast-associated genes during mechanically-induced osteogenesis in vivo: early responses of osteocalcin and type I collagen.

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.