Effect of biomimetic deposition on anodized titanium surfaces

Surface energy and hydrophilicity of implant surfaces have been known to play an important role in subsequent cellular responses on the implant surface. The aim of the present study was to evaluate the effects of biomimetic deposition of anodized surfaces on surface wettability, surface energy, and osteoblast responses. Ti discs with 2 different surface topographies (machined and anodized) were immersed in Hanks' balanced salt solution (HBSS) and modified simulated body fluid (SBF) solution for 2 weeks at physiologic conditions of 37 °C, initial pH of 7.4, and p(CO(2)) of 0.05 atm. Scanning electron microscopic (SEM) observation and energy-dispersive spectroscopic (EDS) microanalysis showed the deposition of calcium phosphate (CaP) onto anodized Ti surfaces immersed in modified SBF. Surface energy, surface wettability, and osteoblast responses, including cell attachment capacity, cell proliferation rate, and cell differentiation level, significantly increased on anodized Ti surfaces immersed in modified SBF. The effects of biomimetic deposition with modified SBF on physiochemical surface characteristics and cell biological responses were greater on anodized surfaces than on machined surfaces. These results indicate that biomimetic deposition with effective SBF may enhance the interaction between anodized Ti surfaces and their biological environment, consequently improving bone healing of dental Ti implants.     

In vitro reaction of human osteoblasts on alumina-toughened zirconia

Objectives: Alumina toughening enhances the mechanical properties of zirconia ceramics but the biocompatibility of this material has rarely been addressed. In this study, we examined the osteoblast response to alumina-toughened zirconia (ATZ) with different surface topographies.
Material and methods: Human osteoblasts isolated from maxillary biopsies of four patients were cultured and seeded onto disks of the following substrates: ATZ with a machined surface, airborne-particle abraded ATZ, airborne-particle abraded and acid etched ATZ. Airborne-particle abraded and acid etched titanium (SLA) and polystyrene disks served as a reference control. The surface topography of the various substrates was characterized by profilometry ( R _a, R _p−v) and scanning electron microscopy (SEM). Cell proliferation, cell-covered surface area, alkaline phophatase (ALP) and osteocalcin production were determined. The cell morphology was analyzed on SEM images.
Results: The surface roughness of ATZ was increased by airborne-particle abrasion, but with the R _a and R _p−v values showing significantly lower values compared with SLA titanium (Mann-Whitney U-test P <0.05). The proliferation assay revealed no statistically significant differences between the ATZ substrates, SLA titanium and polystyrene (Kruskal–Wallis test, P >0.05). All substrates were densely covered by osteoblasts. ALP and osteocalcin production was similar on the examined surfaces. Cell morphology analysis revealed flat-spread osteoblasts with cellular extensions on all substrates.
Conclusions: These results indicate that ATZ may be a viable substrate for the growth and differentiation of human osteoblasts. Surface modification of ATZ by airborne-particle abrasion alone or in combination with acid etching seems not to interfere with the growth and differentiation of the osteoblasts.     

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.     

Altered cell motility and attachment with titanium surface modifications

Background: Titanium implants are widely used in dentistry to replace lost teeth. Various surface modifications have been used to improve implant retention and osseointegration. This study is designed to compare the ability of three titanium surfaces to promote cell attachment and cell motility of cells relevant to periodontal tissues. Methods: Three clinically relevant surfaces were tested: 1) machined titanium; 2) a titanium surface roughened through acid etching (dual thermal‐etched titanium [DTET]); and 3) a titanium surface roughened with nanometer‐scale calcium phosphate deposition (nanoscale calcium phosphate–impregnated titanium [NCPIT]). Cell attachment and migration were examined for four cell types: rat osteosarcoma cells, human osteoblasts, and gingival and periodontal ligament (PDL) fibroblasts. Results: All four cell types attached to each of the three titanium surfaces equally by 2 hours, and the PDL and gingival fibroblasts generally displayed less attachment than the osteosarcoma cells and osteoblasts. The cells displayed differential motility and long‐term attachment to each of the titanium surfaces. Osteosarcoma cells displayed preferential motility on NCPIT, whereas PDL fibroblasts were more motile on machined titanium, and gingival fibroblasts moved more rapidly on both DTET and NCPIT. Osteoblasts displayed little motility on any of the titanium surfaces and lost viability on NCPIT after 24 hours. Gingival fibroblasts lost attachment to machined titanium. Conclusions: Periodontal cells displayed differential motility and long‐term attachment to titanium surfaces. Selective modification of titanium surface properties in various regions of an implant may be useful in guiding specific cell populations to specific locations where they might best aid in osseointegration and soft tissue remodeling.     

不同表面处理方法对CAD/CAM氧化锆种植体表面显微形貌的影响研究

目的：：研究采用不同表面处理方法对CAD/CAM氧化锆种植体表面显微形貌特征及粗糙度的影响。方法：通过CAD/CAM技术加工氧化锆圆盘与一段式氧化锆种植体( Y-TZP, WIELAND),根据表面处理方式分为终烧结表面、喷砂表面及喷砂加热酸蚀处理表面;标准对照组选用BEGO钛种植体表面。各组圆盘试件及种植体用扫描电子显微镜及Keyence 3D激光显微形貌测量显微镜进行表面显微形貌观察与测量。采用单因素方差分析比较各组统计学差异。结果：各组CAD/CAM氧化锆试件表面显微形貌观察显示,喷砂后表面出现边缘锐利的凹坑及沟槽;喷砂加热酸蚀处理后,氧化锆表面可见纳米级的微小孔隙及沟纹。氧化锆种植体粗糙度测量结果显示：终烧结组的表面粗糙度值(Ra=0.69μm)显著低于其他3组(P<0.001),喷砂组Ra值(Ra=1.30μm)显著低于喷砂加热酸蚀组(Ra=1.49μm)及BEGO钛种植体组(Ra=1.57μm)(P<0.01),而喷砂加热酸蚀组与BEGO钛种植体组则无显著差异(P=0.196)。结论：CAD/CAM氧化锆试件终烧结后喷砂或喷砂加热酸蚀处理均可获得较为理想的表面粗糙度,热酸蚀处理能够改变氧化锆表面的纳米级微观结构。     

喷砂酸蚀对钛铌锆锡合金表面成骨细胞粘附、增殖和分化的影响

目的:研究喷砂酸蚀(SLA)对钛及钛铌锆锡合金(Ti-24Nb-4Zr-7.9Sn,TNZS)表面形貌的影响,观察合金的形貌学特征,评价其生物相容性。方法:将试样分为钛机械打磨并抛光组(Ti组),钛铌锆锡机械打磨并抛光组(TNZS组),钛喷砂酸蚀组(Ti-SLA组)和钛铌锆锡喷砂酸蚀组(TNZS-SLA组),共4组。通过扫描电镜观察各组试样的表面形貌,3D激光共聚焦显微镜和接触角测量仪测量各组试样表面的粗糙度与亲水性。接种MC3T3-E1小鼠前成骨细胞于各组试样表面,检测细胞在试样表面的粘附、增殖与矿化的能力,评估其生物相容性。结果:SLA处理后在材料表面形成纳米级及微米级的凹坑,产生均匀分布的粗糙结构,经过处理后材料仍保持亲水性。细胞在TNZS组上短期粘附明显高于其它组(P<0.05),TNZS-SLA组细胞增殖、分化能力均明显高于其它组(P<0.05)。结论:喷砂酸蚀后材料表面相对于光滑材料表面能更有效的促进成骨细胞在其表面增殖、分化,经喷砂酸蚀的钛铌锆锡合金具有良好的细胞相容性。     

Magnesium vs. machined surfaced titanium - osteoblast and osteoclast differentiation

PURPOSE

This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source.

MATERIALS AND METHODS

40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs).

RESULTS

MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM.

CONCLUSION

Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.     

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??Objective    To compare the effects of pure titanium and conventional steel ultrasonic scaler tips on titanium surface and evaluate the adhesion strength of human periodontal ligament cells??hPDLCs??on the titanium surface treated by the two different tips. Methods    Totally 60 smooth titanium discs were used in this research. The discs were randomly divided into five groups??each of which contained 12 discs. Ti10 group??discs were scaled for 10s with pure titanium tip??Ti30 group??discs were scaled for 30s with pure titanium tip??Cv10 group??discs were scaled for 10s with conventional steel tip??Cv30 group??discs were scaled for 30s with conventional steel tip. Control group??discs were not scaled. After the treatment according to the protocols??the morphology and roughness of the discs were evaluated by scanning electron microscope??SEM??and profilometer separately. In order to test the biocompatibility of the treated titanium discs??hPDLCs were co-cultured with those discs for 12 hours??and the cell toxicity of treated discs and the adhesion strength of hPDLCs on titanium discs was calculated with CCK-8 method. Results       SEM results showed that Ti10 group had less morphological changes than Cv10 group and this change was highly consistent with the profilometer results which also showed that the roughness of titanium at Ti10 group was significantly lower than Cv10 group??P < 0.05??. However??if the scaling time increased to 30 seconds??there were no statistical difference among groups. There were no significant difference among groups for the adhesion strength of hPDLCs after being treated by different scaler tips and no toxicity was found. Conclusion    Pure titanium ultrasonic scaler tips are better than conventional ultrasonic scaler tip with respect to the protection of titanium surface??although there were no significant differences in the promotion of cell adhesion.     

Behavior of CAL72 osteoblast-like cells cultured on zirconia ceramics with different surface topographies

OBJECTIVES: Because of its inherent strength, biocompatibility, and tooth-like color, zirconia ceramics have the potential to become an alternative to titanium as dental implant material. This study aimed at investigating the osteoblastic response to yttrium-stabilized tetragonal zirconia polycrystal (Y-TZP) with different surface topographies. METHODS: CAL72 osteoblast-like cells were cultured on machined (TZP-m), airborne particle abraded (TZP-s), and airborne particle abraded and acid-etched Y-TZP (TZP-sa) surfaces. Polystyrene and airborne particle abraded with large grit and acid-etched (SLA) titanium served as a reference control. The surface topography was examined by scanning electron microscopy (SEM) and profilometry. At culture days 3, 6, and 12, cell proliferation, at day 12 cell morphology, and cell-covered surface area were determined. RESULTS: The surface roughness of Y-TZP was increased by airborne particle abrasion and additionally by acid etching. No statistically significant differences were found between average roughness (R(a)) and maximum peak-to-valley height (R(p-v)) values of airborne particle abraded and acid-etched Y-TZP and SLA titanium. Whereas the cell proliferation assay revealed statistically significant greater values at day 3 for surface-treated Y-TZP and polystyrene cultures as compared with machined Y-TZP, no differences between the Y-TZP groups, SLA titanium, and polystyrene were observed at culture days 6 and 12. CONCLUSIONS: Cell morphology and cell-covered surface area were not affected by the type of substrate. The results suggest that roughened Y-TZP is an appropriate substrate for the proliferation and spreading of osteoblastic cells.     

The Effect of Surface Processing of Titanium Implants on the Behavior of Human Osteoblast‐Like Saos‐2 Cells

Background: The surface qualities of dental implants appear to modulate osteoblasts’ growth and differentiation, affecting bone healing. During manufacturing of implants, the surface quality is affected by industrial processes. Purpose: To examine the effect of manufacturing procedures on the growth and differentiation of human osteoblast‐like cells, Saos‐2. Materials and Methods: Saos‐2 cells were cultured on titanium (Ti) disks. Cell growth was examined using the XTT assay, and cell differentiation was tested by alkaline phosphatase (ALP) activity and osteocalcin secretion. The following variables were examined: roughening of the surface by sandblasting and acid‐etching, aging of the acid used for etching, fluoride modification of the surface, and the type of the packaging material. Results: An inverse relationship was noted between Saos‐2 growth and ALP activity on the tested surfaces. Roughening of the surface tended to decrease cell proliferation and to increase differentiation. Immersion of up to 200 cycles in acid decreased proliferation and increased differentiation. Cells grown on fluoride‐modified surfaces exhibited more ALP activity as compared to the unmodified surfaces. No difference was noted between the three packaging materials tested. Conclusions: The data suggests that industrial processes may affect the behavior of osteoblast‐like cells around titanium implants and should be monitored carefully by bioassays.     

Response of rat bone marrow cells to differently roughened titanium discs

The purpose of the present in vitro study was to examine the effect of surface roughness on the behaviour of osteoblast-like cells. Rat bone marrow (RBM) cells were cultured on commercially pure titanium discs. The discs were used as machined (Ti M) or ground with 4000 (Ti 4000) or 320 (Ti 320) grit paper. Proliferation rate and alkaline phosphatase activity were determined, and morphology of the cells was studied with scanning electron microscopy (SEM). Besides, fluorescent markers, energy dispersive spectroscopy (EDS), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) were used to obtain quantitative and compositional information about the produced calcified extracellular matrix (ECM). Results demonstrated after 2 days of incubation no significant difference in the percentage of attached cells to all substrates. At 5 days, Ti 320 surfaces showed significantly lower (P < 0.05) cell attachment percentages compared with Ti M and Ti 4000 surfaces. At 8 days, Ti 320 surfaces showed significantly more (P < 0.05) cell attachment than the other surfaces. The Ti 4000 surfaces showed after 8 days significantly (P < 0.05) higher alkaline phosphatase activity compared to both other surfaces. At 15 days of incubation, the alkaline phosphatase activity on Ti 4000 substrates was significantly lower (P < 0.05) than on the other substrates. No significant difference in mineralized ECM formation was observed on the ground substrate compared to the machined substrates. Physicochemical analysis confirmed the apatite-like nature of the deposited ECM on all substrates. On the basis of these findings, we concluded that our in vitro study could not clearly confirm the effect of surface roughness on the proliferation, differentiation and calcification of rat bone marrow cells.     

钛种植体基台的表面粗糙度与细菌粘附

目的了解临床使用的4种种植系统基台的表面粗糙度与细菌粘附的关系.方法用Talysurf/6p-120型表面形貌仪测定4种基台的表面粗糙度.采用体外粘附实验,了解变形链球菌和粘性放线菌在钛金属片上的粘附量受表面粗糙度影响的状况.结果 4种种植基台表面粗糙度的Ra值分别为0.155 6、0.207 3、0.381 1 和0.697 6 μm.变形链球菌在表面粗糙度为0.108 8 μm试片上的粘附量与0.452 8 μm和1.273 8 μm试片上的粘附量差异具有显著性.粘性放线菌在0.108 8 μm和0.221 9 μm试片上的粘附量与1.273 8 μm试片上的粘附量差异具有显著性.结论轮廓算术平均偏差(Ra)＜0.4 μm、10个峰谷高度平均值(Rz)＜3.4 μm是种植体基台的较为理想的粗糙度范围.     

Focal adhesion contact formation by fibroblasts cultured on surface-modified dental implants: an in vitro study

A major consideration in designing dental implants is to create a surface that provides strong attachment of the implant to bone, connective tissue and epithelium. The aim of the present study was to examine the influence of different treatments of titanium (Ti) implant surfaces on focal adhesion contact (FAC) formation in fibroblast cultures. Human gingival fibroblasts were cultured on glass sheets and polished Ti discs with different surface coatings (applied by physical vapor deposition (PVD): Ti, titanium nitride (TiN), zirconium nitride (ZrN)) or on Ti discs with different surface topographies. For characterization of all surfaces, modified estimation of surface roughness and spacing parameter was carried out using a contact stylus profilometer. Contact angle measurements were carried out to calculate surface energy. Fibroblasts were prepared for transmission electron microscopy at day 3 after seeding, and the number of FACs and the ratio FAC/cellular cross-sections was determined at a length of 300 microm in ultrathin sections. To visualize the extracellular fibronectin and vitronectin molecules and the intracellular actin and vinculin in FAC areas, immunogold labeling was performed. The results revealed a strong correlation between the number of FACs and the surface roughness. The highest number of FACs and the majority of the immunogold-labeled intra- and extracellular matrix molecules were counted on surfaces with the lowest surface roughness: glass sheets coated with either Ti, TiN or ZrN (roughness average=0.03-0.1 microm). These surfaces appear to favor cellular attachment of human gingival fibroblasts and moreover in previous studies the hard coatings have been shown to reduce bacterial adhesion.     

微纳复合结构对成纤维细胞黏附增殖的影响

目的：探讨经不同方法处理后的纯钛表面对成纤维细胞黏附增殖的影响。方法：将36个试件分平均为3组：机械抛光组（A组）;喷砂酸蚀组（B组）;喷砂酸蚀碱热组（C组）,每组均12个试件。通过扫描电子显微镜（SEM）观察分析3组试件表面微观结构和细胞在试件表面的铺展情况,激光共聚焦显微镜（CLSM）检测各试件表面的粗糙度;运用CCK-8试剂盒在450 nm波长下检测各试件对成纤维细胞（ L929）黏附与增殖的吸光度值（ OD值）。结果：A组表面光滑,试件表面成纤维细胞骨架大多呈梭形铺展,伸展较差;B组和C组表面粗糙,且C组表面可见微纳复合结构,试件表面成纤维细胞骨架呈三维空间向铺展,表面黏附成纤维细胞数量明显多于A组和B组。观察第1,3,5 d试件表面细胞增殖情况,可见粗糙表面较光滑表面更利于成纤维细胞的增殖。结论：喷砂酸蚀碱热方法处理后的纯钛表面形成微米-纳米复合孔洞,表面活性好,促进成纤维细胞早期黏附及表面铺展,且不抑制细胞的增殖。     

Spreading of epithelial cells on machined and sandblasted titanium surfaces: an in vitro study

BACKGROUND: The purpose of this investigation was to determine the influence of the surface structure of dental implants on epithelial cell spreading and growth in vitro. Cell morphology on machined and sandblasted titanium surfaces was investigated. METHODS: A total of 10 machined and 10 sandblasted discs and 10 glass coverslips were used for the present study. Samples were analyzed using scanning electron microscopy (SEM) and the cell spreading area was determined using a video image analysis system. RESULTS: After 24 hours incubation, keratinocytes grown on sandblasted titanium samples displayed numerous, long, and branched or dendritic filopodia closely adapted to the surface roughness. Filopodia varied from 3 to 12 microm in length and 0.1 to 0.3 microm in width. Cells cultured on a machined surface did not present such cytoplasmic extensions and displayed a round morphology. Keratinocytes seeded on glass coverslips were flat and edged by filopodia (maximum length 7 to 8 microm) on the spreading site of the cluster. Though cell morphology is comparable with that observed on sandblasted specimens, cytoplasmic extensions suggestive of strong adhesion and spreading attitude were less pronounced. CONCLUSION: These results indicate that sandblasted surfaces are the optimal substrata for epithelial cell adhesion and spreading.     

MinTBP-1-PRGDN双靶向融合肽对成骨细胞粘附影响的比较研究

目的：以胎牛血清（Fetal bovine serum,FBS）涂层为标准对照,研究minTBP-1-PRGDN双靶向融合肽对成骨细胞粘附的影响。方法：对商用纯钛进行minTBP-1-PRGDN双靶向融合多肽涂层,以无涂层的钛表面及胎牛血清涂层的钛表面分别作为空白对照和阳性对照,比较不同涂层处理对成骨细胞附着及伸展的影响。结果：成骨细胞粘附1h后,minTBP-1-PRGDN融合肽涂层的钛片上的细胞附着数量最多,FBS涂层表面次之,无涂层的钛片表面细胞数量最少、且与minTBP-1-PRGDN组比较有统计学差异（P〈0.05）;经形态计量学分析：FBS组细胞伸展面积最大,minTBP-1-PRGDN组次之,无涂层组细胞伸展最小。结论：minTBP-1-PRGDN融合肽涂层能提高成骨细胞在钛表面的附着和伸展能力。     

Physico-chemical characterization of the surface of 9 dental implants with 3 different surface treatments

There are many surface treatments applied to dental implants. The aim of the present investigation is to compare the physicochemical characteristics of titanium dental implant surfaces with different surface treatments. 9 dental implants from the same batch were divided in 3 groups and received 3 different surface treatments: machined, acid etched and a new chemical surface treatment called Avantblast. Scanning electron microscopy and confocal microscopy were used to image the treated surfaces, and energy-dispersive spectrometry and X-ray photoelectron spectrometry to provide a chemical characterization of the surfaces. RESULTS: The acid etched and chemical etched surfaces had an increased roughness over the machined one. Surface chemical composition had differences between processes, as the surface with the new treatment presented a reduced level of impurities and increased thickness of the titanium oxide layer. CONCLUSIONS: Surface roughness of titanium dental implants and thickness of the titanium oxide layer can be increased with a suitable surface treatment.     

微弧氧化钛基种植材料对成骨细胞早期黏附的影响

目的：检测纯钛种植体材料微弧氧化（microarc oxidation，MAO）表面改性后的成骨细胞生物相容性，探讨微弧氧化技术在钛种植体表面改性中的价值。方法：在纯钛种植体材料表面用微弧氧化法制备羟基磷灰石陶瓷薄膜，将MAO改性钛种植体材料作为实验组Ⅰ，将纯钛表面阳极氧化改性处理的种植体材料作为实验组Ⅱ．并设立对照组Ⅰ（纯钛种植体材料）和对照组Ⅱ（即细胞直接生长在培养板上），分别进行扫描电镜（SEM）、能谱分析（EDS）、X射线衍射图谱分析（XRD）等检测，比较成骨细胞的黏附水平，对数据采用SPSS11．0统计软件包进行单因素方差分析。结果：MAO改性后生成粗糙、多孔的陶瓷薄膜层，与处理前电解液成分相比，钙磷比无显著改变。MAO改性钛组黏附的细胞密度显著高于其他组（P〈0．01），而对照组Ⅰ、Ⅱ的细胞密度无显著差异（P〉0．05）。结论：与阳极氧化表面改性材料相比，该钛基微弧氧化薄膜层能够显著促进成骨细胞的附着，具有良好的细胞相容性。