Effects of fluoride-modified titanium surfaces on osteoblast proliferation and gene expression

PURPOSE: The objective of this study was to test the hypothesis that fluoride-modified titanium surfaces would enhance osteoblast differentiation. Osteoblast growth on a moderately rough etched fluoride-modified titanium surface (alteration in cellular differentiation) was compared to osteoblast growth on the same surface grit-blasted with titanium dioxide. The potential role of nanometer-level alterations on cell shape and subsequent differentiation was then compared. MATERIALS AND METHODS: Human embryonic palatal mesenchymal (HEPM) cultures were incubated on the respective surfaces for 1, 3, and 7 days, followed by analysis for cell proliferation, alkaline phosphatase (ALP) -specific activity, and mRNA steady-state expression for bone-related genes (ALP, type I collagen, osteocalcin, bone sialoprotein [BSP] II, Cbfa1, and osterix) by real-time polymerase chain reaction (PCR). RESULTS: The different surfaces did not alter the mRNA expression for ALP, type I collagen, osterix, osteocalcin, or BSP II. However, Cbfa1 expression on the fluoride-modified titanium surface was significantly higher (P < .001) at 1 week. The number of cells on this surface was 20% lower than the number of cells on the surface TiO2-blasted with 25-microm particles but not significantly different from the number of cells on the surface TiO2-blasted with 125-microm particles. Cells grown on all the titanium surfaces expressed similar levels of ALP activity. CONCLUSIONS: The results indicated that a fluoride-modified surface topography, in synergy with surface roughness, may have a greater influence on the level of expression of Cbfa1 (a key regulator for osteogenesis) than the unmodified titanium surfaces studied.     

钛片表面粗糙度和氧化膜对成骨细胞增殖和分化的影响

目的：研究钛片表面粗糙度和氧化膜对成骨细胞增殖和分化的影响,为种植体表面处理提供理论依据。方法：采用粒度分别为108～130 μm(S1)、216～301 μm(S2)和356～411 μm(S3)的二氧化钛颗粒对纯钛钛片表面进行喷砂处理,钛浆喷涂(titanium-sprayed plasma, TPS)表面处理组由Straumman 公司提供,600目砂纸打磨组(S0)作为对照组,在钛片表面进行成骨细胞培养。采用表面轮廓测量仪测量其表面粗糙度,电子探针(electron microprobe)测定钛片表面氧化膜结构。分别在1、3、5及7 d时,采用四锉盐比色(MTT)法检测不同处理表面对成骨细胞增殖(OD值)的影响;通过碱性磷酸酶活性(ALP)及骨钙素分泌(OC)检测比较不同处理的表面对成骨细胞分化的影响。采用SPSS12.0软件包对数据进行单因素方差分析。结果：S0 、S1 、S2 、S3 和TPS组表面粗糙度由0.372 μm至5.239 μm递增;喷砂组钛片表面氧化膜结构完整、连续;粗糙表面比光滑表面更利于成骨细胞增殖和分化。喷砂表面粗糙度越高,越利于成骨细胞增殖和分化。S3组成骨细胞增殖和分化优于TPS组。结论：表面粗糙度较高的喷砂表面,更利于成骨细胞增殖和分化。     

Effect of three distinct treatments of titanium surface on osteoblast attachment, proliferation, and differentiation

Cell-titanium interactions are crucial to the clinical success of bone and dental implants. The physico-chemical characteristics of the substrates surface influence osteoblast proliferation, differentiation, and activity as well. The osteoblast behavior was analyzed on three different titanium surfaces: ground with an abrasive 600 grit SiC paper, blasted with alumina particles (65 microm diameter) and alumina blasted followed by a double chemical etch (4% HF+4% HF/8% H2O2). Scanning electron microscopy (SEM) and profilometry showed distinct microtopographies. Ground samples showed parallel-groove orientation. The Al2O3-blasted surface presented the roughest microtopography with aluminum-rich particles incrusted in the titanium surface. Osteoblasts cells from femora of Balb/c mice were seeded onto the substrates tested. Cell morphology and initial attachment were evaluated by SEM. Osteoblasts adhered to and spread on all samples tested. However, on rough surfaces, osteoblasts did not spread completely and acquired a polygonal morphology. Besides, the cell proliferation rate was diminished at the beginning of incubation on rough surfaces. Our results suggest a delay, rather than an impairment, in osteoblast viability and alkaline phosphatase activity when cells are cultured on rough surfaces, inducing a distinct osteoblast phenotype, rather than blocking its activity. At least in the culture conditions used in this work, alumina particles did not affect osteoblast behavior.     

Effect of surface roughness of titanium dental implant placed under periosteum on gene expression of bone morphogenic markers in rat

Various bone matrix proteins are produced during the process of osteogenesis. Many previous studies suggested that the topography of an implant surface might affect the expression of osteoblast-mediated cytokines. However, these earlier studies were performed using in vitro cell culture. This study investigated the influence of the surface topography of a titanium implant placed under the periosteum on the gene expression of bone morphogenic markers in rat. Six custom-made implants with a rough upper surface and 6 custom-made implants with a smooth machined upper surface were placed subcrestally with the upper surface facing up in the femurs of 6 adult male rats. Five rats were sacrificed 7 days after the implant placement, and the periosteum above the embedded implant was obtained and analyzed by quantitative real-time RT-PCR for the target genes: alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OCN). The other rat was sacrificed at day 7, and both implants and the surrounding tissue were embedded in paraffin. For light microscopic observations, paraffin sections were stained with toluidine blue. Gene expression of ALP, BSP and OCN at the rough surface implant was significantly higher than that at the smooth machined surface implant. At day 7, both types of implant were covered with soft tissue, but a lower number of cells stained with toluidine blue was observed on the machined surface compared with on the rough surface. It is considered that rough surfaces may stimulate osteoblasts, and that ALP activity is increased indirectly. Furthermore, the two other markers were also increased by the rough surface in vivo, and different distributions of cellular and extracellular components on the upper surface of the implants were observed at day 7. These results suggest that a rough surface implant under the periosteum promotes higher gene expression of ALP, BSP and OCN in rat.     

Different titanium surface treatment influences human mandibular osteoblast response

BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.     

In vitro preliminary study of osteoblast response to surface roughness of titanium discs and topical application of melatonin

Objectives: To observe human osteoblast behavior cultured in vitro on titanium discs (Ti) in relation to surface roughness and melatonin application. Study Design: Human osteoblasts (MG-63) were cultured on 60 Ti6Al4V discs divided into three groups: Group I: discs treated with dual acid etching; Group II dual acid etching and blasting with calcium phosphate particles; Group III (control) machined discs. Surface roughness and topography of the discs were examined with scanning electron microscope (SEM) and confocal laser scanning electron microscope( CLSM). Osteoblast adhesion, proliferation and cell morphology were determined by means of fluorescence microscopy with Image-Pro Plus software and SEM. Results: Group II presented the roughest discs, while the least rough were Group III. Cell adhesion was greatest in Group II. The addition of melatonin improved cell proliferation. Conclusions: 1. Surface treatments (dual acid etching, calcium phosphate impaction) increase surface roughness in comparison with machined titanium. 2. Greater surface roughness tends to favor cell adhesion after 24-hour cell culture. 3. The addition of melatonin tends to favor osteoblast proliferation. Key words:Osteoblasts, titanium, roughness, melatonin, dental implants, osseointegration.     

Inhibition of cyclooxygenase by indomethacin modulates osteoblast response to titanium surface roughness in a time-dependent manner

Prostaglandin E2 (PGE2) and transforming growth factor-beta 1 (TGF-beta 1) production are increased in cultures of osteoblasts grown on rough surfaces and prostaglandins are involved in osteoblast response to surface roughness. In the present study, we examined the effect of inhibiting cyclooxygenase on this response. MG63 osteoblast-like cells were cultured on cpTi disks with Ra values of 0.60 micron (PT), 3.97 microns (SLA), and 5.21 microns (TPS) in the presence or absence of 10(-7) M indomethacin. Treatment was begun on days 1, 2, 3, or 4 after seeding, and all cultures were harvested on day 5. Indomethacin decreased PGE2 release by the cells to less than 50% of basal levels when the cells were cultured on plastic. Cell number decreased with increasing surface roughness and indomethacin treatment abrogated the surface roughness effect over time. Alkaline phosphatase specific activity (ALP) increased with surface roughness; after one day with indomethacin, ALP was decreased on smooth surfaces, but increased on rough surfaces. Over time, ALP decreased on all surfaces examined and remained greater than plastic only in cultures on TPS. Indomethacin also caused a time-dependent decrease in osteocalcin production on rough surfaces, eventually abrogating the increases due to surface roughness, but had no effect on osteocalcin production on smooth surfaces. TGF-beta 1 levels in the cell layer and media were sensitive to surface roughness; on rougher surfaces, TGF-beta 1 shifted from the media to the matrix. Indomethacin reduced TGF-beta 1 levels over time, but the surface roughness effect was still evident at 4 days. This indicates that prostaglandin production mediates the effects of surface roughness, since indomethacin causes a time-dependent abrogation of the response, but has no effect on proliferation, osteocalcin release, or TGF-beta 1 levels on smooth surfaces. Indomethacin's effect was not immediate, suggesting that clinical protocols could be designed that would reduce inflammation without preventing osteoblastic differentiation. The effect of indomethacin was not complete, since TGF-beta 1 and ALP remained elevated on rough surfaces, suggesting that pathways or factors other than prostanoids are involved. TGF-beta 1 is preferentially stored in the matrix, acting on the cells through autocrine signaling, and may contribute to ALP even in the presence of indomethacin. These results demonstrate the importance of local factors in the autocrine regulation of osteogenesis and the potential for factors released in response to surface morphology to act in a paracrine manner.     

钛片表面粗糙度和氧化膜对成骨细胞黏附影响的体外研究

目的 研究钛片表面粗糙度和氧化膜对成骨细胞黏附的影响,为选择临床种植体的表面处理方法提供依据.方法 250片纯钛钛片分为5组.采用直径分别为108～130 μm(S<,1>组)、216～301 μm(S<,2>组)、356～411 μm(S<,3>组)TiO<,2>砂对钛片表面进行喷砂处理:另外一组钛片采用钛浆喷涂(T...     

In vitro study of human osteoblast proliferation and morphology on orthodontic mini-implants

Objective:To evaluate the proliferation and morphology of human osteoblasts cultured on two brands of mini-implants after 24, 48, and 72 hours, in addition to the chemical composition found on their surface.Materials and Methods:Two brands of mini-implant (Morelli and Neodent) were evaluated; polystyrene was used as a control group (n  =  3). Osteoblasts were cultured on the surface of sterilized mini-implants in a CO₂ incubator at different time periods (24, 48, and 72 hours). Osteoblast proliferation was quantified by scanning electron microscopy using up to 5000× magnification, and cell morphology was analyzed by a single observer. For the chemical analysis, spectroscopy X-ray fluorescence was used to identify and quantify chemical components on the surface of the mini-implants.Results:Two-way ANOVA showed no significant interaction between the factors studied (P  =  0.686). A Tukey test revealed no significant difference in osteoblast proliferation between the mini-implants at all studied periods; however, a difference in cell proliferation was detected between the Neodent and the control group (P  =  .025). For all groups, time had a direct and positive effect on osteoblast proliferation (P < .001). The significant elements present in both brands of mini-implants were titanium, aluminum, vanadium, and iron.Conclusions:Osteoblast proliferation was present on the mini-implants studied, which increased over time; however, no significant difference between brands was observed. No difference was seen between the mini-implants evaluated in terms of chemical composition. Cell adhesion after 72 hours suggests that areas of bone remodeling can be achieved, thus initiating the process of mini-implant anchorage.     

Effect of cpTi surface roughness on human bone marrow cell attachment,proliferation, and differentiation

There is general agreement that rough surfaces improve both biologic and biomechanical responses to titanium (Ti) implants. The aim of this investigation was to study the effect of Ti surface roughness on the response of human bone marrow cell culture evaluating: cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on commercially pure titanium (cpTi) discs with fourdifferent average roughnesses (Ra). For attachment evaluation, cells were cultured for 4 h. After 21 days, cell proliferation, total protein content, and ALP activity were evaluated. For bone-like nodule formation, cells were cultured for 28 days. Data were compared by ANOVA and Duncan's multiple range test. Cell attachment was not affected by surface roughness. For cells cultured on Ti with Ra ranging from 0.80 microm to 1.90 microm, proliferation was reduced while total protein content, and ALP activity were increased. There was a non-statistically significant increase of bone-like nodule formation on a surface with Ra near 0.80 microm. These results suggest that for Ti an Ra ranging from 0.80 microm to 1.90 microm would optimize both intermediary and final cellular responses but not affect the initial response, and a smoother surface would not favor any evaluated response.     

Influence of bisphosphonates on the osteoblast RANKL and OPG gene expression in vitro

Bisphosphonates are widely used in the clinical treatment of bone diseases with increased bone resorption. In terms of side effects, they are widely known to be associated with osteonecrosis of the jaw (BONJ). The objective of this study was to evaluate the effect of bisphosphonates on the gene expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in vitro. Nitrogen-containing and non-nitrogen containing bisphosphonates have been compared. Human osteoblasts were stimulated with zoledronate and ibandronate at concentrations of 5 × 10⁻⁵ M, 5 × 10⁻⁶ M, and 5 × 10⁻⁷ M over the experimental period of 14 days. Furthermore, the hOB cell lines were stimulated by clodronate at concentrations of 5 × 10⁻³ M, 5 × 10⁻⁵ M, and 5 × 10⁻⁶ M. At each point in time, the gene expression levels of RANKL and OPG were quantified by real-time RT-PCR. The results showed a moderate enhancement of OPG gene expression whereas RANKL gene expression was strongly increased by nitrogen-containing bisphosphonates reaching a maximum after 14 days at high concentrations of 5 × 10⁻⁵ M. Lower concentrations did not enhance the RANKL and OPG expression considerably. The non-nitrogen-containing bisphosphonate clodronate, however, effected OPG and RANKL gene expression much less, even at higher concentrations of 5 × 10⁻³ M. The above-mentioned data suggest an enhanced RANKL/OPG gene expression after stimulation by bisphosphonates. Interestingly, clodronate might have little influence on osteoblast/osteoclast interaction with respect to OPG and RANKL gene expression.     

气流喷砂对牙面粗糙度的影响

目的　探讨使用气流喷砂技术对牙面粗糙度的影响。方法　光波干涉显微镜观察 2 4颗离体牙在使用气流喷砂后牙面粗糙度的变化 ,并研究磨光糊剂磨光对减轻其粗糙度的作用。结果　气流喷砂和超声洁治一样都会使牙面糊糙度显著增加 (P <0 .0 1 ) ,进一步磨光可使牙面粗糙度显著下降 (P <0 .0 1 )。结论　临床上使用气流喷砂后 ,需进一步磨光来增加牙面光洁度     

Influence of titanium surface roughness on attachment of Streptococcus sanguis: an in vitro study

The purpose of the present study was to investigate the efficacy of the decontamination protocol for bacterial removal in titanium surfaces with three different levels of roughness using a high-pressure sodium bicarbonate device for 1 minute under aseptic conditions. Group 1 was composed of 10 as-machined titanium sheets and Groups 2 and 3 of titanium sheets blasted with aluminum oxide (Al2O3, alumina) particles with different diameters: Group 2 was blasted with 65-microm particles and Group 3 with 250-microm particles. The titanium specimens were sterilized and incubated in tubes containing a suspension of Streptococcus sanguis. The colony-forming units were counted before and after the application of the decontamination protocol. The arithmetic mean roughness (R(a)) per group was: Group 1, 0.17 microm +/- 0.01; Group 2, 1.14 microm +/- 0.15; and Group 3, 3.17 microm +/- 0.23. After the contamination period, Group 1 remained with 49 x 10(3) bacterial cells, and the bacterial concentrations of Groups 2 and 3 were 11 x 10(4) and 35 x 10(5), respectively. After the application of the decontamination protocol, no viable bacteria were detected. With the increase of the surface roughness, an exponential increase in bacterial cells was observed. The results showed that the decontamination protocol treatment with a high-pressure sodium bicarbonate device efficiently removed all bacterial cells in all surfaces tested. This indicates that high-pressure sodium bicarbonate spray should be used in the maintenance phase of implant treatment.     

喷砂酸蚀复合MAO纯钛表面对成骨细胞粘附的影响

目的:研究喷砂酸蚀(sandblast-acid etch,SA)复合微弧氧化(micro arc oxidation,MAO)纯钛表面对成骨细胞粘附的影响,探讨SA与MAO复合处理技术在钛种植体表面改性中的价值.材料和方法:将纯钛按表面处理方法的不同分为4组:MAO-SA组为250μm直径Al2O3颗粒喷砂HF酸蚀后MAO处理,MAO组为单纯MAO处理,MAO-HT组为MAO处理后水热处理8h,SM组为光滑组.通过扫描电镜、激光共聚焦显微镜观察、Bradford蛋白定量法、四唑盐比色法和实时荧光定量PCR反应检测分析各表面对蛋白吸附、成骨细胞形态和骨架改建、粘附水平、粘附强度以及整合素表达的影响.数据采用SPSS16.0进行方差分析.结果:MAO-SA表面促进纤连蛋白的后期吸附,有利于成骨细胞粘附和骨架改建,使成骨细胞较早表现出良好的分泌功能形态,增强细胞粘附强度,显著上调成骨细胞整合素αv亚基的mRNA表达水平,但对β1亚基的表达无促进作用.讨论:对纯钛表面进行SA和MAO复合处理,获得独特的表面微形貌,提高粗糙度,从而促进细胞外基质蛋白吸附和成骨细胞粘附.结论:MAO-SA表面显著促进成骨细胞的粘附,具有良好的生物相容性.     

表面粗糙度对水与唾液在纯钛及PMMA上润湿作用的影响

目的:评价全口义齿基托材料及其表面粗糙度对义齿固位的影响。方法:测定水与唾液在表面粗糙度不同的纯钛和聚甲基丙烯酸甲酯(PMMA)上的前进接触角和后退接触角,通过两者的差值θh反映水与唾液对它们的润湿效果。结果:1、随着表面粗糙度增加,唾液在纯钛上的θh逐渐降低,但当Ra≥1.0,Rz≥1.5时,θh突然增大;而PMMA组,当Ra<1.2,Rz<2时,粗糙度对θh无明显影响,当Ra≥1.2,Rz≥2时θh也显著增加,多数情况下,唾液在相同粗糙度的纯钛上的θh大于在PMMA上的θh。2、水在光滑的PMMA和纯钛上的前进接触角比唾液的大。结论:唾液对纯钛的润湿效果,可以通过高度磨光或形成较为粗糙的表面(Ra≥1.0,Rz≥1.5)得到提高,多数情况下这种润湿效果好于PMMA。提示从润湿效果看,对于不能高度研磨和抛光的义齿组织面,全口义齿上颌腭托选择纯钛并形成较为粗糙的表面可能更有利于固位。