Effect of bioactive glass air-abrasion on the wettability and osteoblast proliferation on sandblasted and acid-etched titanium surfaces

The aim of this study was to evaluate the hydrophilicity, surface free energy, and proliferation and viability of human osteoblast-like MC3T3-E1 cells on sandblasted and acid-etched titanium surfaces after air-abrasion with 45S5 bioactive glass, zinc-containing bioactive glass, or inert glass. Sandblasted and acid-etched titanium discs were subjected to air-abrasion with 45S5 bioactive glass, experimental bioactive glass (Zn4), or inert glass. Water contact angles and surface free energy were evaluated. The surfaces were studied with preosteoblastic MC3T3-E1 cells. Air-abrasion with either type of glass significantly enhanced the hydrophilicity and surface free energy of the sandblasted and acid-etched titanium discs. The MC3T3-E1 cell number was higher for substrates air-abraded with Zn4 bioactive glass and similar to that observed on borosilicate coverslips (controls). Confocal laser scanning microscopy images showed that MC3T3-E1 cells did not spread as extensively on the sandblasted and acid-etched and bioactive glass-abraded surfaces as they did on control surfaces. However, for 45S5- and Zn4-treated samples, the cells spread most at the 24 h time point and changed their morphology to more spindle-like when cultured further. Air-abrasion with bioactive glass and inert glass was shown to have a significant effect on the wettability and surface free energy of the surfaces under investigation. Osteoblast cell proliferation on sandblasted and acid-etched titanium discs was enhanced by air-abrasion with 45S5 bioactive glass and experimental Zn4 bioactive glass compared with air-abrasion with inert glass or no air-abrasion.     

Surface analysis of machined versus sandblasted and acid-etched titanium implants

Initially, implant surface analyses were performed on 10 machined implants and on 10 sandblasted and acid-etched implants. Subsequently, sandblasted and acid-etched implant cytotoxicity (using L929 mouse fibroblasts), morphologic differences between cells (osteoblast-like cells MG63) adhering to the machined implant surfaces, and cell anchorage to sandblasted and acid-etched implant surfaces were evaluated. Results indicated that acid etching with 1% hydrofluoric acid/30% nitric acid after sandblasting eliminated residual alumina particles. The average roughness (Ra) of sandblasted and acid-etched surfaces was about 2.15 microns. Cytotoxicity tests showed that sandblasted and acid-etched implants had non-cytotoxic cellular effects and appeared to be biocompatible. Scanning electron microscopic examination showed that the surface roughness produced by sandblasting and acid etching could affect cell adhesion mechanisms. Osteoblast-like cells adhering to the machined implants presented a very flat configuration, while the same cells adhering to the sandblasted and acid-etched surfaces showed an irregular morphology and many pseudopodi. These morphologic irregularities could improve initial cell anchorage, providing better osseointegration for sandblasted and acid-etched implants.     

Immunolocalization of fibronectin and vitronectin receptors in human gingival fibroblasts spreading on titanium surfaces

The adhesion and spreading of human gingival fibroblasls on glass and differently processed titanium surfaces was studied by immunolocalization of vinculin and the alpha and beta subunits of the fibronectin (α₅β₁) and vitronectin (α_vβ3) receptors. Vinculin-containing focal contacts were present both at 4 and 24 h of spreading in cells grown on glass or electropolished or etched titanium surfaces but not in cells spreading on sandblasted titanium surfaces. Immunostaining for the α5 and β1 subunits of the fibronectin receptor showed only a diffuse membrane fluorescence after 4 h of cell spreading irrespective of the growth surface. The α_v and β₃ subunits of the vitronectin receptor were at this stage detected in focal contacts in cells spreading on glass or electropolished or etched titanium surfaces. In cells spreading on sandblasted titanium surfaces, however, the vitronectin receptor had only a diffuse distribution. In cells that had been allowed to spread for 24 h on glass or electropolished or etched titanium surfaces the α₅ and β₁ integrin subunits were either diffusely distributed or showed a localization typical of extracellular matrix contacts. The α_v and β₃ integrin subunits were, as earlier, localized to typical focal contacts in cells grown on glass or electropolished or etched titanium surfaces. Cells attached to sandblasted titanium surfaces still expressed all the integrin subunits only diffusely. The results show that the surface texture of the substratum can affect the expression of integrin subunits in human gingival fibroblasts. As evidenced by the recruitment of integrin subunits to focal and extracellular matrix contacts, smooth or finely grooved titanium surfaces appear to be optimal in supporting the attachment of human gingival fibroblasts.     

The effect of titanium surface roughening on protein absorption, cell attachment, and cell spreading

PURPOSE: The purpose of this study was to compare properties of roughened and polished titanium with respect to their ability to attach to cells and bind to protein as well as their cell spreading behavior. MATERIALS AND METHODS: Three different titanium surface treatments were compared for their ability to support cell attachment and spreading: sandblasted and acid-etched, resorbable blast media, and machine-polished titanium. The surface of the materials was characterized for surface roughness, surface energy, and surface chemistry. Osteoblast-like MG-63 cells were tested for in vitro attachment and spreading in the presence of serum proteins. Cell attachment was assessed by direct counting, dye binding, and microculture titanium assays. Cell spreading was determined by measuring area/cell in phalloidin-AlexaFluor 488 stained cells. Absorption of bovine serum albumin was determined by assay. RESULTS: Scanning electron micrography and x-ray diffractometry confirmed increased surface roughness of the roughened materials. All 3 materials had similar albumin binding kinetics. Three different methods confirmed that roughened surfaces enhance early cell attachment to titanium in the presence of serum. Cells spread better on smoother machined surfaces than on the roughened surfaces. CONCLUSION: Roughened titanium surfaces exhibited better early cell attachment than smooth surfaces in the presence of serum. The cells attached to roughened titanium were less spread than those attached to machined titanium. Although albumin binding was not different for roughened surfaces, it is possible that roughened surfaces preferentially bound to serum adhesive proteins to promote early cell attachment.     

Immunolocalization of proteins specific for adhaerens junctions in human gingival epithelial cells grown on differently processed titanium surfaces

The localization of desmoplakins 1 and 2 (DP 1&2), components of desmosomes, vinculin, and actin, was studied in gingival epithelial cells grown on cell culture glass and on titanium plates with various surface topography. The results showed that epithelial cells attached and spread more readily on smooth than on rough, sandblasted titanium surfaces. Moreover, the cells appeared to develop more granular DP 1&2 immunoreactivity at their ventral surfaces when grown on smooth or etched titanium as compared to glass. In cells grown on sandblasted titanium surfaces, DP 1&2-specific immunoreactivity was primarily located at cell-cell contacts. Cells grown on smooth titanium surfaces harbored a fine network of actin filaments with apparent cell-to-cell organization. Vinculin was confined to cell-cell contact areas. No vinculin-containing focal adhesions could be detected, suggesting that the cells adhere either by means of close contacts, extracellular matrix contacts, or by means of hemidesmosomes. The findings suggest that smooth of finely grooved titanium surfaces could be optimal in maintaining the adhesion and specialized phenotype of gingival epithelial cells.     

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目的 探讨应用等离子体电解氧化(plasma electrolytic oxidation,PEO)技术处理钛表面后对成骨细胞(人成骨肉瘤Saos-2细胞)早期增殖与分化的影响.方法 应用PEO技术在纯钛表面制备多孔氧化钛陶瓷膜(PEO组),并以钛表面机械抛光处理(机械抛光组)及喷砂酸蚀处理(喷砂酸蚀组)作为对照,应用场发射扫描电镜、粗糙度测试仪分析表面形貌.将Saos-2细胞接种于3组样品表面,通过对样品表面细胞形态、细胞增殖及碱性磷酸酶(ALP)活性的检测,分析3组样品时Saos-2早期生物学行为的影响.结果 应用PEO法可以在纯钛表面形成多孔结构.Saos-2在3组样品表面的黏附与增殖差异无统计学意义(P＞0.05);PEO组的ALP活性高于机械抛光组与喷砂酸蚀组(P＜0.05).结论 PEO处理后的多孔钛表面能促进Saos-2的早期分化功能.     

The effects of Er:YAG laser treatment on titanium surface profile and osteoblastic cell activity: an in vitro study

Background: Laser light has been proposed as a tool to decontaminate the surface of endosseous implants. The effects of this maneuver on the interactions between cells and surface, however, are poorly known. The goal of the present study is to investigate osteoblast growth and differentiation on three commercially available surfaces untreated or after irradiation by erbium‐doped:yttrium, aluminum, and garnet (Er:YAG) laser at two levels: 150 and 200 mJ/pulse at 10 Hz. Methods: Human osteoblastic Saos‐2 cells were plated on machined, sandblasted and acid‐etched titanium, or titanium plasma‐sprayed disks. The effects of lasing were observed with a scanning electronic microscope, and cell viability was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Moreover, we measured the production of the osteoblast‐specific protein osteocalcin and of osteoprotegerin in the supernatants by immunoenzymatic assays. Results: Although no visible changes were observed on machined or titanium plasma‐sprayed disk samples at the tested levels, titanium peaks on sandblasted and acid‐etched titanium disks appeared fused as a consequence of laser irradiation. Interestingly, cell proliferation was slower on irradiated titanium at both intensities on all the surfaces. Cell differentiation, as assessed by osteocalcin production, was generally unaffected by laser treatment, whereas the production of osteoprotegerin was decreased on all the surfaces irradiated at the intensity of 200 mJ/10Hz. Conclusions: These results indicate that Er:YAG laser at energy levels used in this study can alter the surface profile of titanium implants and these changes may negatively affect the viability and the activity of osteoblastic cells. Therefore, Er:YAG lasers should be used with caution on titanium surfaces.     

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.     

Behavior of two osteoblast-like cell lines cultured on machined or rough titanium surfaces

Background: Two osteosarcoma-derived cell lines have been extensively used to investigate the biological events occurring on titanium surfaces: MG63 and Saos-2. However, the behavior of the two lines on different titanium surfaces has never been compared.
Aim: The aim of the present study was to compare the behavior of MG63 and Saos-2 cells on two different titanium surfaces, machined and rough (sandblasting and acid-etched). We compared cell proliferation and morphology, alkaline phosphatase (ALP) activity and secretion of osteocalcin (OC).
Results: The most pronounced difference between the two cell lines was that ALP activity in the Saos-2 cells was 10-fold higher than in the MG63 cells. The proliferation rate of the MG63 cells was much higher than that of the Saos-2 cells at all the tested cell concentrations. MG-63 cells, but not Saos-2 cells, grown on rough surface titanium proliferated more rapidly than cells grown on machined surfaces. Morphological analysis revealed that Saos-2 cells and cells grown on the rougher surface, displayed a more mature phenotype. The level of OC secreted by the Saos-2 cells, but not the MG63 cells, were higher on the rough surface than on the machined surface.
Conclusions: This study shows that Saos-2 cells exhibit a more mature osteoblast phenotype, compared with that of MG63 cells, rendering them a good candidate for an in vitro model of osseointegration.     

Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces

Objective: The aim of this study is to analyze the morphology and proliferation of human osteoblastic cells in vitro on five commercially available titanium surfaces. Materials and methods: Human primary cells of the osteoblastic lineage were obtained from bone explants. The cells were plated on polished (T1), machined (T2), sand‐blasted/acid‐etched (T3), sand‐blasted/acid‐etched, modified with hydrogen peroxide rinse (T4), and plasma‐sprayed titanium (T5) disks. Cell morphology was studied after 6, 24, 72 h, 7 and 14 days of culture by scanning electron microscopy. The formation and distribution of focal adhesions was investigated by immunocytochemical staining at 3, 6 and 24 h. Cell growth was measured by an MTT assay after 3, 7 and 9 days of culture. Moreover, the production of osteocalcin and osteoprotegerin (OPG) was evaluated in the supernatants by ELISA. Results: Morphological analysis revealed that substrate topography profoundly affected cells' shape and their anchoring structures. Large lamellipodia were formed on polished and machined surfaces, while thin filopodia were more frequently observed on T3 and T4 samples. Moreover, cells formed stronger focal adhesions on T3 and T4 surfaces, and cell proliferation was higher on rough surfaces. Osteocalcin production was higher on the T4 surface, whereas OPG steadily increased on every surface. Conclusions: Taken together, these data show that all the surfaces allowed cell attachment, adhesion and proliferation, but T4 and T5 surfaces appeared to be a better substrate for the adhesion, proliferation and differentiation of cells of the osteoblastic lineage. To cite this article:
Passeri G, Cacchioli A, Ravanetti F, Galli C, Elezi E, Macaluso GM. Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces.
Clin. Oral Impl. Res. 21 , 2010; 756–765.
doi: 10.1111/j.1600‐0501.2009.01906.x     

Influence of surface treatments developed for oral implants on the physical and biological properties of titanium. (II) Adsorption isotherms and biological activity of immobilized fibronectin.

The influence of titanium surface properties on in vitro adsorption isotherms of tibronectin, promotion of Staph;lococcus aureus adhesion, and binding of a monoclonal antibody to the cell‐binding domain of fibronectin was examined. Treatments producing different surface roughness were applied to a single side of commercially pure titanium coverslips, which was either mechanically polished (P), or polished and then acid attacked with H₂SO₄/HCI (PA), or sandblasted and then acid attacked (SLA), whereas the untreated side was blocked by an albumin coating layer. Incubation of the coverslips with concentrations of soluble ³H‐labelled fibronectin increasing from 1 to 16 μg/ml led to the saturation of all surfaces with immobilized protein from 4 to 16 μg/ml. Promotion of S. aureus adhesion by fibronectin adsorbed on all surfaces and binding of the monoclonal antibody to its cell‐binding domain was to some extent proportional to the amount of immobilized protein but also showed some minor differences between surfaces. More important material‐related differences were observed when fibronectin adsorption isotherms were expressed as a function of the effective, roughness‐corrected surface area, yielding amounts of immobilized fibronectin on the rough PA and SLA titanium surfaces which were 50% lower than those adsorbed on either smooth P or polymethylmethacrylate coverslips used as controls. In conclusion, surface treatments increasing the surface roughness of titanium do not increase, but may partly decrease in vitro adsorption of fibronectin. Despite adsorbing different amounts of fibronectin, both rough and smooth titanium surfaces promote normal expression of 2 major functional domains of this protein.     

Histologic evaluation of human bone integration on machined and sandblasted acid-etched titanium surfaces in type IV bone

The aim of this preliminary study was to evaluate the influence of a sandblasted acid-etched surface on bone-implant contact percentage (BIC%) as well as the bone density in the threads area (BD%) in type 4 bone after 2 months of unloaded healing. Five subjects (mean age = 42.6 years) received 2 microimplants each during conventional implant surgery in the posterior maxilla. The microimplants with commercially pure titanium surface (machined) and sandblasted acid-etched surface served as the control and test surfaces, respectively. After a healing period of 2 months, the microimplants and the surrounding tissue were removed and prepared for ground sectioning and histomorphometric analysis. One microimplant with a machined surface was found to be clinically unstable at the time of retrieval. Histometric evaluation indicated mean BIC% was 20.66+/-14.54% and 40.08+/- 9.89% for machined and sandblasted acid-etched surfaces, respectively (P=.03). The BD% was 26.33 +/-19.92% for machined surface and 54.84+/-22.77% for sandblasted acid-etched surface (P=.015). Within the limits of this study, the data suggest that the sandblasted acid-etched implant surface presented a higher percentage of bone-implant contact compared with machined surfaces, under unloaded conditions in posterior maxilla after a healing period of 2 months.     

微弧氧化钛表面对成骨细胞形态及细胞骨架的影响

目的 研究微弧氧化钛表面对成骨细胞形态及细胞骨架的影响。方法 将直径15 mm、厚度1 mm的纯钛片根据表面处理方法不同分为4组：机械打磨（G）组、喷砂（SB）组、打磨微弧氧化（GMAO）组和喷砂微弧氧化（SBMAO）组。采用扫描电子显微镜（SEM）、激光共聚焦显微镜（LSCM）研究钛片表面成骨细胞生长情况及细胞骨架的改变。结果 成骨细胞接种12 h后,各组细胞均沿钛片表面铺展开,且GMAO组和SBMAO组细胞覆盖于火山口状微孔上。各组肌动蛋白纤维清晰可见,GMAO组和SBMAO组肌动蛋白纤维平行排列,汇聚成束伸向微孔内。结论 微弧氧化后的钛表面可以影响成骨细胞铺展的形态及细胞骨架的排列。     

Response of osteoblast‐like SAOS‐2 cells to zirconia ceramics with different surface topographies

Objectives: Zirconia is a suitable biomaterial for use in medicine (stomatology, orthopaedics) due to its good biocompatibility and outstanding mechanical properties. This study compares the effect of (i) zirconia to the widely used titanium and (ii) zirconia with two different surface topographies (sandblasted and sandblasted/etched) on the adhesion, proliferation and differentiation of SAOS‐2 osteoblasts. Methods: SAOS‐2 cells were cultured on either sandblasted or sandblasted/etched zirconia and compared with sandblasted/etched titanium. 2 and 24 h after plating, cell morphology was investigated by scanning electron microscope (SEM) and fluorescence imaging. At 24 and 48 h, cell number‐relevant parameters were determined. Alkaline phosphatase (ALP) activity and mineral accumulation were measured at days 8, 11, 15 and day 22 of culture, respectively. Results: SEM and fluorescence images revealed a faster spreading as well as higher number of adherent cells after 24 h incubation on zirconia compared with titanium. Also, the cellular metabolic activity after 24 h and the proliferation rate after 48 h is higher with zirconia compared with titanium. Zirconia had a more pronounced effect compared with titanium on the differentiation of SAOS‐2 cells: ALP activity, an early differentiation marker increased earlier and mineralization, a late differentiation marker was increased. Only minor differences were found between zirconia with two different surface topographies; etched zirconia promoted slightly greater the differentiation of SAOS‐2 cells. Conclusions: These data indicate that zirconia mediates a pronounced stronger effect on the adhesion, proliferation and differentiation compared with titanium; and that topographical differences of zirconia have minor effects on osteoblast biology. To cite this article:
Hempel U, Hefti T, Kalbacova M, Wolf‐Brandstetter C, Dieter P, Schlottig F. Response of osteoblast‐like SAOS‐2 cells to zirconia ceramics with different surface topographies.
Clin. Oral Impl. Res. 21 , 2010; 174–181.
doi: 10.1111/j.1600‐0501.2009.01797.x     

In vitro osteogenesis on a microstructured titanium surface with additional submicron-scale topography

The present study aimed to evaluate key parameters of in vitro osteogenesis on (1) commercially pure titanium (cpTi) discs with 20-200-microm-scale microtopography patterned with additional micron- and submicron-scale topography (0.5-20 microm; Plus surface, Dentsply Friadent), (2) control cpTi discs with 20-200-microm-scale microtopography (DPS, Deep Profile Surface, Dentsply Friadent), and (3) a machined surface. Using calvaria-derived osteogenic cultures, the following parameters were assessed: cell adhesion and spreading, growth curve and cell viability, alkaline phosphatase (ALP) activity and total protein content, immunolocalization of fibronectin, bone sialoprotein (BSP) and osteopontin (OPN), and bone-like tissue formation. The results showed no major differences between surfaces in terms of cell adhesion, growth curve, cell viability (days 4 and 11), ALP activity, or total protein content (days 11 and 17). At day 11, cultures grown on Plus exhibited small, well-defined nodular areas of calcified matrix, which were only rarely observed on DPS and absent on the machined surface. Such areas were larger at day 17 and were not associated with the typical mineralized bone-like nodules (with BSP- and OPN-positive osteoblastic cells on top). At day 17, the total mineralized area was significantly larger on DPS than on a Plus or machined surface (DPS>Plus>machined; Kruskal-Wallis test, P<0.05). Direct fluorescence allowed the straightforward observation of higher amounts of apoptotic bodies associated with mineralized nodules for Plus. The results suggested the occurrence of an additional, early pattern of matrix mineralization mostly for the Plus microstructured surface, which did not necessarily translate into larger bone-like tissue formation in vitro.     

电解蚀刻法处理的钛及钛合金表面的对比研究

目的 通过成骨细胞的体外培养,初步探讨钛及钛合金微-纳米三维形貌对成骨细胞生物学行为的影响。方法采用电解蚀刻法在纯钛及钛合金表面构建出不同尺寸的微-纳米三维形貌,并观察其三维结构表面对成骨细胞黏附、增殖、细胞形态、碱性磷酸酶（ALP）活性的影响。结果在成骨细胞的黏附和增殖方面,纯钛组和钛合金组表面均高于纯钛机械抛光组。纯钛组表面细胞胞体饱满,伸出大量伪足,并可见大量功能颗粒。ALP活性显著高于钛合金和纯钛机械抛光组表面。结论通过电解蚀刻法在纯钛和钛合金表面可形成不同直径和深度的碗形巢样及纳米结构;两个表面即30~50 μm和5~8 μm的表面和光滑表面相比,都明显促进了细胞的附着;30~50 μm的纯钛表面更有利于促进细胞的增殖和分化。     

Bone healing around titanium and titanium nitride-coated dental implants with three surfaces: an experimental study in rats

Background: Titanium nitride (TiN) has been used in many fields as a coating of surgical instruments, with the purpose of creating materials more resistant to wear and corrosion and also reducing adhesion. Purpose: The aim of this study was to evaluate the biocompatibility of TiN‐coated dental implants. Materials and Methods: Forty‐five rats were used in this study. One hundred eighty 2 mm 2 mm implants (P.H.I., San Vittore Olona, Milano, Italy) were used. The implants were divided into the following three groups: Group 1 (n= 60):30 machined and 30 machined coated with TiN Group 2(n= 60):30 sandblasted and 30 sandblasted coated with TiN Group 3 (n= 60):30 titanium plasma sprayed, 30 titanium plasma sprayed and coated with TiN Four implants were placed in each rat, two implants coated with TiN on the right tibia and two uncoated implants on the left. The animals were killed after 5, 10, 20, 30, or 60 days. Another 18 implants were used for surface roughness analysis. Results: The present study showed that the healing around the TiN‐coated implants was similar to that observed around the uncoated surfaces. Conclusions: TiN coating demonstrated a good biocompatibility, did not have untoward effects on the periimplant bone formation, and did not change the surface roughness values.