Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology

Surface implant modifications have been shown to have a relevant importance in modifying cell response. Expression profiling by DNA microarray is a new molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) on new implant surface (nanoPORE, Out-Link, Sweden and Martina, Due Carrare, Padova, Italy), several genes whose expressions were significantly down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) vesicular transport (c) apoptosis and cell cycle regulation. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of an implant surface. They can be relevant to better understand the molecular mechanism of implant osseointegration and as a model for comparing other materials.     

Biological responses in osteoblast-like cell line according to thin layer hydroxyapatite coatings on anodized titanium

Several features of the implant surface, such as roughness, topography and composition play a relevant role in implant integration with bone. This study was conducted in order to determine the effects of various thin layer hydroxyapatite (HA) coatings on anodized Ti surfaces on the biological responses of a human osteoblast-like cell line (MG63). MG63 cells were cultured on 100 nm HA (100 nm HA coating on anodized surface), 500-700 nm HA (500-700 nm HA coating on anodized surface), 1 mum HA (1 mum HA coating on anodized surface) and anodize (non-HA coating on anodized surface) Ti. The morphology of these cells was assessed by scanning electron microscopy (SEM). The cDNAs prepared from the total RNAs of the MG63 were hybridized into a human cDNA microarray (1152 elements). The appearances of the surfaces observed by SEM were different on each of the four dental substrate types. MG63 cells cultured on 100 nm HA, 1 mum HA and anodize exhibited cell-matrix interactions. It was 500-700 nm HA surface showing cell-cell interaction. In the expression of genes involved in osseointegration, several genes, including bone morphogenetic protein 2, latent transforming growth factor beta binding protein 1, catenin (cadherin-associated protein), integrin, PDGFRB and GDF-1 growth differentiation factor 1 were up-regulated on the different surfaces. Several genes, including fibroblast growth factor receptor 3, fibroblast growth factor 12 and CD4 were down-regulated on the different surfaces. The attachment and expression of key osteogenic regulatory genes were enhanced by the surface morphology of the dental materials used.     

The Th1/Th2 immune-type response of the recurrent aphthous ulceration analyzed by cDNA microarray

BACKGROUND: The reduced ability to activate oral tolerance plays a role in the pathogenesis of some gastrointestinal inflammatory diseases. This activation may reflect a preferential reduction of a T-helper (Th)2- or Th3-type response. In recurrent aphthous ulceration (RAU), genetic and environmental factors may contribute to low tolerance, permitting a cytotoxic reaction against the oral epithelium. The cytokine profile has not permitted the definition of RAU as resulting from enhanced Th1 or Th2 responses. A cDNA microarray study would allow the identification of differentially expressed genes and provide a basis for classification of the immune response. METHODS: The cDNA from 29 samples of aphthae and from 11 samples of normal mucosa from aphthae-free volunteers were hybridized on microarray membranes with 1176 genes. RESULTS: Forty-one differentially expressed genes were identified, and a higher expression level of the Th1 gene cluster in RAU was found. CONCLUSIONS: Microarrays permitted us definition of the gene expression profile of the lesion and identify an increased Th1 activity in RAU lesions.     

Analysis of gene and protein expression in healthy and carious tooth pulp with cDNA microarray and two-dimensional gel electrophoresis

Complementary DNA (cDNA) microarray and two-dimensional (2-D) gel electrophoresis, combined with mass spectrometry, enable simultaneous analysis of expression patterns of thousands of genes, but their use in pulp biology has been limited. Here we compared gene and protein expression of pulp tissues from sound and carious human teeth using cDNA microarray and 2-D gel electrophoresis to evaluate their usefulness in pulp biology research and to identify the genes with changes in carious teeth. The cDNA microarray revealed several differentially expressed genes and genes with a high expression in both tissues. These genes have various functions, e.g. effects on vascular and nerve structures, inflammation, and cell differentiation. Variability between cDNA hybridizations indicates that the overall gene expression pattern may vary significantly between individual teeth. The 2-D gel electrophoresis revealed no change between healthy and diseased tissue. The identification of 96 proteins in the pulp tissue revealed none of the gene products with corresponding high/different mRNA expression in cDNA microarray. Interestingly, we detected also a hypothetical protein (putative nucleoside diphosphate kinase), and present therefore the first evidence for the existence of this protein. Even though the methods reveal potentially important gene expression, they may currently have only limited value in in vivo pulp biology research.     

Identification of genes related to mechanical stress in human periodontal ligament cells using microarray analysis

BACKGROUND AND OBJECTIVE: Differential expression of genes in human periodontal ligament (PDL) under mechanical stress, such as orthodontic force, is thought to be involved in the remodeling of PDL cells and periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress. MATERIAL AND METHODS: We employed microarray analysis to assess, in a comprehensive manner, the gene expression profiles in PDL cells compressed by a static force using an in vitro three-dimensional culture system. Six genes were selected and validated by quantitative real-time polymerase chain reaction analysis, consistent with the microarray data. RESULTS: The microarray data revealed that 108 of 30,000 genes tested were differentially expressed by mechanical force loading. Among them, 85 genes were up-regulated by mechanical stress, while 23 genes were down-regulated, judging by the thresholds of a two-fold increase/decrease compared with the controls. Thirty-two of the up-regulated and eight of the down-regulated genes, well-characterized in protein function, were involved in numerous biological processes including cell communication, cell signaling, cell cycle, stress response, and calcium release. However, several genes differentially expressed in our microarray data have not been well defined as stress-response molecules. CONCLUSION: Our microarray is the first to show the gene profile in PDL cells caused by mechanical stress; however, further studies to clarify the physiological function of these molecules in PDL cells are required.     

氟-羟基磷灰石对骨肉瘤MG63细胞生物学性能的影响

目的 评价氟取代比例为20％的氟-羟基磷灰石(fluor-hydroxyapatite,FHA)对骨肉瘤MG63细胞增殖和成骨分化的影响,以期为FHA的临床应用提供依据.方法 采用化学沉淀法合成FHA,扫描电镜、X射线衍射仪、傅里叶红外光谱仪观察结构并表征.设置FHA组(培养基中加入FHA)、羟基磷灰石(hydroxyapatite,HA)组(培养基中加入HA)和空白对照组(单纯培养基),每组样本量为3,共培养骨肉瘤MG63细胞.甲基噻唑基四唑法检测3组细胞增殖能力,检测3组细胞碱性磷酸酶(alkaline phosphatese,ALP)活性.反转录PCR检测3组细胞成骨分化相关基因(Ⅰ型胶原、ALP、骨钙素和核心结合因子)mRNA表达情况.结果 FHA的X射线衍射图谱主晶相与HA标准图谱一致.培养3d后FHA组细胞增殖(A值为1.87±0.06)显著高于空白对照组(1.25±0.02)(P＜0.05),FHA组与HA组(1.84±0.03)差异无统计学意义(P＞0.05).培养3d后FHA组ALP活性(4.62±0.09)显著高于空白对照组(1.92±0.05)(P＜0.05).与空白对照组相比,FHA上调细胞Ⅰ型胶原、ALP、骨钙素mRNA表达,下调核心结合因子mRNA表达.结论 FHA对MG63细胞增殖和成骨分化相关基因mRNA的表达有促进作用,具有良好的生物相容性.     

Effect of modifications of dual acid-etched implant surfaces on peri-implant bone formation. Part I: organic coatings

Objective: The aim of the present study was to test the hypothesis that peri-implant bone formation can be improved by modifying dual acid-etched (DAE) implant surfaces using organic coatings that enhance cell adhesion and osteogenic differentiation.
Material and methods: Ten adult female foxhounds received experimental titanium implants in the mandible 3 months after removal of all premolar teeth. Six types of implants were evaluated in each animal: (i) implants with a machined surface (MS), (ii) implants with a DAE surface topography, (iii) implants with an acid-etched surface coated with RGD peptides, (iv) implants with an acid-etched surface coated with collagen I, (v) implants with an acid-etched surface coated with collagen I and chondroitin sulphate (CS), (vi) implants with an acid-etched surface coated with collagen I and CS and recombinant human bone morphogenetic protein-2. Peri-implant bone regeneration was assessed by histomorphometry after 1 and 3 months in five dogs each by measuring bone implant contact (BIC) and the bone volume density (BVD) of the newly formed peri-implant bone.
Results: After 1 month, mean BIC was significantly higher in the coated implants group than in the MS group. There was no significant difference when mean BIC in the DAE group was compared with implants with any of the organic coatings, but the difference was significant when compared with the MS implants. Differences in mean BVD value did not reach significance between any of the surfaces. After 3 months, the same held true for the mean BIC of all the groups except for Coll I. Mean volume density of the newly formed bone was higher in all the surface modifications, albeit without statistical significance.
Conclusions: It is concluded that with the exception of Coll I, the tested organic surface coatings on DAE surfaces did not improve peri-implant bone formation when compared with the DAE surfaces but enhanced BIC when compared with the MSs.     

Effect of modifications of dual acid-etched implant surfaces on periimplant bone formation. Part II: calcium phosphate coatings

Abstract: The aim of the present study was to test the hypothesis that calcium phosphate coatings of dual acid-etched surfaces (DAEs) can improve periimplant bone regeneration. Ten adult female foxhounds received experimental titanium screw implants in the mandible 3 months after removal of all premolar teeth. Five types of surface states were evaluated in each animal: (i) implants with a machined surface (MS) (Control 1); (ii) implants with a DAE (Control 2); (iii) implants with a DAE coated with collagen I (Control 3); (iv) implants with a DAE with mineralized collagen I; and (v) implants with a DAE with a hydroxylapatite (HA) coating. Periimplant bone regeneration was assessed by histomorphometry after 1 and 3 months in five dogs each by measuring bone implant contact (BIC) and the volume density of the newly formed periimplant bone (BVD). After 1 month, mean BIC of experimental implants did not differ significantly from implants with DAE and collagen-coated surfaces, but was significantly higher than the MS implants. BVD was enhanced significantly only in implants with mineralized collagen coating compared with DAE and collagen-coated controls. After 3 months, the mean values of BIC had increased significantly in the group of implants with HA and mineralized collagen coating but were not significantly different from implants with DAE and collagen-coated surfaces. The same held true for the mean BVD values. In conclusion, the present study could not verify the hypothesis that calcium phosphate coatings of DAEs in the present form enhanced periimplant bone formation compared with the DAE surface alone.     

Effect of osteoblast cell culture on the bone implant contact

Abstract

Purpose. The aim of this study is to acquire an ideal bone implant contact under the cover of osteogenic effect of osteoblasts derived from Mesenchymal Stem Cells (MSCs). Materials and methods. Thirty dental implants were used for this study. Implants were placed in sheep mandibles and defects were created 4 mm coronally in the dental implants. These defects were filled with Platelet Rich Plasma (PRP) in one group and with PRP + Osteoblast Cell Culture (OCC) in another group. No procedure was conducted on the control group defects (empty defect group). Eight weeks later, osseointegration was investigated with Bone Implant Contact (BIC) measurements histomorphologically. Data were checked statistically. Results. The variation of BIC rates between Empty Defect Group and PRP groups was significant (p <0.05). The BIC rate of the PRP group was higher than that of the Empty Defect Group. The variation of BIC rates between Empty Defect Group and PRP + OCC groups was significant (p <0.05). The BIC rate of the PRP + OCC group was higher than that of the Empty Defect Group. The variation of BIC rates between PRP and PRP + MSC groups was significant (p<0.05). The BIC rate of the PRP + OCC group was higher than that of the PRP group. At the end of the 8-week healing period, it was observed that the percentage of BIC was highest in the PRP + OCC group. Conclusions. Implant–bone connection was better in the OCC?PRP group compared with the PRP group and the empty defect group. The use of OCC-PRP combination was effective on healing. The BIC value was increased significantly by OCC.     

A systematic review of the influence of different titanium surfaces on proliferation, differentiation and protein synthesis of osteoblast-like MG63 cells

OBJECTIVES: Titanium is the standard material for dental and orthopaedical implants. The good biocompatibility has been proven in many experimental and clinical investigations. Different titanium topographies were tested in vitro using different cell culture models. The aim of this systematic review was to evaluate and summarize the medical/dental literature to assess on which kind of titanium surface structure the osteoblast-like osteosarcoma cells MG63 show the best proliferation and differentiation rate, and the best protein synthesis. METHODS: A systematic search was carried out using different on-line databases (PubMed, Web of Science, Cochrane Library, International Poster Journal), supplemented by handsearch in selected journals and by examination of the bibliographies of the identified articles. Inclusion and exclusion criterias were applied when considering relevant articles. Studies which met the inclusion criteria were included and data extraction was undertaken by one reviewer. RESULTS: The search yielded 348 references. Nine articles referring to nine different studies were relevant to our question. Additionally 8 less relevant articles were identified. It was found that regularly textured surfaces of pure titanium with R(a) values (average roughness) of around 4 mum are well-accepted by MG63 cells. CONCLUSIONS: The surfaces and culture conditions vary widely. Therefore it is still difficult to recommend one particular surface. It seems that there are no differences in cell proliferation and differentiation on surfaces treated by blasting and etching. Standardization in fabrication and size of the different test surfaces as well as homogeneity in culture times and plating densities should be aspects for future research.     

基因芯片技术在牙髓生物学研究中的应用进展

基因芯片作为一种高通量、快速和平行核酸序列测定及定量分析技术已经广泛地应用于分子生物学领域。牙髓基因组学的研究将有利于加强人们对牙髓生理和病理机制的认识,从而最终应用于临床预防、诊断和治疗等,因此,本文就基因芯片技术及其与牙髓干细胞相关的基因、牙髓老龄化改变相关的基因和龋病时牙髓相关的基因等研究进展作一综述。     

玉屏风口服液对口腔扁平苔藓差异表达基因干预的初步探讨

目的 应用基因芯片技术及最新公共数据库,观察玉屏风口服液作用后口腔扁平苔藓(oral lichen planus,OLP)组织中基因表达谱改变。方法 分离纯化正常口腔黏膜组织、口腔扁平苔藓病变组织和服用玉屏风口服液后扁平苔藓病变组织mRNA,制备表达谱探针,混合后用BiostarH-40s型基因芯片杂交,用ScanArray 4000荧光扫描仪扫描芯片荧光信号图像,利用GenePix Pro 3.0软件分析检测玉屏风口服液作用口腔扁平苔藓组织中的差异表达基因,并进行筛选。结果 服用玉屏风口服液后,病变组织有5条基因表达上调,3条基因表达下调,功能以转录基因为主。结论 玉屏风口服液对口腔扁平苔藓的差异表达基因具有干预作用,干预基因以转录基因为主。?     

Responses of periodontal ligament stem cells on various titanium surfaces

Oral Diseases (2011) 17 , 320–327 Objective: Periodontal ligament has been reported to have adult stem cells (PDLSCs) which are responsible to regenerate the alveolar bone tissue after tooth is removed from its socket. Also PDLSCs may be the stem cells responsible for the osseointegration of titanium implants after installing the implant immediately in the fresh extracted socket. Here we tested cellular responses of PDLSCs on the various titanium surfaces to verify this notion. Materials and methods: Titanium disc were prepared for the different surface textures; smooth machined, blasted with 75 and 125 μm Al₂O₃ particles, and anodized. PDLSCs were cultured on these titanium discs and tested their proliferation and gene expressions of osteocalcin, osteopontin, type I collagen, and GAPDH. Results: Proliferation of PDLSCs was higher on the rough surface blasted with 75 μm Al₂O₃ particles. Osteocalcin expression was increased on the Al₂O₃ particle treated‐surface regardless of its particle size. Type I collagen expression was generally decreased with time in 6 days culture. Conclusions: In this experiment, it was shown that cultured PDLSCs proliferate in higher rate on the rough surface especially at the 75 μm Al₂O₃ particle treated surface than other surfaces. Also, osteocalcin was highly expressed on the rough surfaces treated with 75 μm and 125 μm Al₂O₃ particles.     

富血小板血浆对人成骨样细胞MG63增殖能力的影响

目的研究富血小板血浆（PRP）对人成骨样细胞MG63增殖能力的影响,探讨PRP的生物学功能。方法采集健康志愿者的静脉血,两次离心法制备PRP,氯化钙加人凝血酶激活后离心提取PRP萃取液。碱性磷酸酶（ALP）染色检测不同体积分数PRP（0、1%、2%、3%）对MG63分化活性的影响,碘化丙啶（PI）荧光染色观察PRP作用下MG63细胞形态的变化,免疫细胞化学检测PRP对MG63表达转化生长因子-β（TGF-β）的影响,扫描电子显微镜（SEM）观察PRP对MG63在人工骨材料表面生长情况的影响,CCK-8法检测PRP对MG63增殖活性的影响,流式细胞术（FCM）检测经PRP培养后不同时间MG63的细胞周期,逆转录聚合酶链反应（RT-PCR）检测PRP作用下MG63中Ⅰ型胶原（Col-Ⅰ）mRNA的表达量。结果ALP检测见3%PRP组ALP阳性细胞染色最深,并随体积分数的增加染色强度增加,且PRP组细胞均出现不同程度的脱片现象。PI荧光染色见PRP组细胞核荧光染色较对照组增强。免疫细胞化学检测见3%PRP组TGF-β表达量最高（P＜0.05）。SEM观察：PRP组材料表面MG63密集,生长状态良好。CCK-8法测定细胞增殖活性,显示4.8%PRP组吸光度A450 nm值高于对照组（P＜0.05）。FCM检测表明：PRP组第2天S期细胞百分比高于对照组（P＜0.05）;第10天PRP组G0/G1期细胞百分比高于对照组（P＜0.05）;除第6天外,PRP组G2/M期细胞百分比均高于对照组。RT-PCR结果表明：PRP组MG63的Col-ⅠmRNA表达量较对照组明显上调。结论适宜体积分数的PRP对MG63的增殖、迁移和相关蛋白、基因的表达有促进作用,PRP具有统一、协调细胞生物学行为的作用。     

氧化应激对人成骨样细胞MG63活性的影响

目的 建立人成骨样细胞氧化应激模型,研究氧化应激对人成骨样细胞形态和增殖活力的影响.方法 用不同浓度黄嘌呤/黄嘌呤氧化酶(xanthine/xanthine oxidase,X/XO)的酶促反应产生的超氧阴离子自由基(O2-˙)刺激人成骨样细胞MG63,建立成骨细胞内氧化应激模型,用X/XO加黄嘌呤氧化酶抑制剂羟嘌呤醇研究其对该氧化应激模型的逆转作用.运用氧化敏感性荧光探针2'7'-二氯荧光黄双乙酸盐结合流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)的生成,用倒置相差显微镜和MTT实验观察研究氧化应激对成骨细胞形态及增殖活力的影响.结果 X/XO处理MG63细胞后,细胞形态发生明显破坏;在相同处理时间下,X/XO浓度越高,细胞内ROS荧光强度值越高,吸光光密度(opitical density,OD)值越低,差异具有统计学意义(P<0.05),且X/XO加羟嘌呤醇联合处理组的细胞内ROS平均荧光强度较相同浓度X/XO单独处理组明显降低(P<0.05).在相同X/XO处理浓度下,随着处理时间的延长,细胞内ROS平均荧光强度逐渐增强,OD值明显降低,120 min时细胞内ROS平均荧光强度比对照组增加了345%.24 h时OD值为相同时间对照组的22.9%.结论 X/XO可导致人成骨样细胞氧化应激损伤,破坏人成骨样细胞的形态,抑制人成骨样细胞的增殖活力.X/XO抑制剂羟嘌呤醇可逆转X/XO引起的人成骨样细胞氧化损伤.