Effect of grooved titanium substratum on human osteoblastic cell growth

Various surface treatments have been developed to increase the clinical performance of titanium-based implants. Many in vitro tests have been carried out on substrates with varied surface topography for a complete understanding of osteoblasts. In previous research, we made the observation that surface roughness must be taken into account, not only in terms of amplitude but also in terms of organization. In this study, we tested the adhesion and proliferation of human primary osteoblasts on grooved titanium surfaces with various amplitudes and organizations of topography. The roughness was described at a scale above (macro-roughness) or below (micro-roughness) the cell size. We observed better orientation and proliferation of human osteoblasts on surfaces with a micro-roughness characterized by a lower Order (parameter describing the organization of topography) and by a higher Ra and Rz (parameters describing the amplitude of topography). It appears that cultured human osteoblasts prefer surfaces with relatively high micro-roughness amplitude and with a low level of repeatability.     

Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-(epsilon-caprolactone) surfaces

This study was aimed to investigate whether the activation of poly-(epsilon-caprolactone) (PCL) surface by low-energy irradiation and/or the biofunctionalization by absorption of arginine-glycine-aspartic sequences (RGD), can modify the expression of integrins closely related to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization, the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5 x 10(15)He(+)/cm(2) (10 keV energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover, irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was observed only on irr-PCL (surface coverage: 7.0 pmol/cm(2)). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed alpha1, alpha2, alpha3, alpha5, beta1, and alphaVbeta3; after 24h, cells cultured on tissue-plastic expressed high levels of beta1 and alphaVbeta3, while alpha1 showed a low intensity and alpha2, alpha3, and alpha5 were negative. beta1 and alphaVbeta3 were selected to evaluate the interaction between cells and PCL samples. The beta1 expression was higher in hOB cultured on irr-PCL than on the other samples. A significant increase in alphaVbeta3 expression was observed only in irr-PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to improve the osteoconductivity of selected materials.     

The influence of surface roughness of titanium on beta1- and beta3-integrin adhesion and the organization of fibronectin in human osteoblastic cells

Mechanisms of cell adhesion and extracellular matrix formation are primary processes in the interaction with the material surface of an implant which are controlled by integrin receptors. The aim of our study was to find out whether beta1- and beta3-integrins of osteoblastic cells sense the surface topography of titanium, and if structural alterations of integrin adhesions were involved in the organization of fibronectin. Pure titanium surfaces were modified by polishing (P), machining (NT), blasting with glass spheres (GB), and blasting with corundum particles (CB) resulting in increasing roughness. Confocal microscopic investigations revealed fibrillar adhesions of beta1- and alpha5-integrins on P, NT, and GB, but on CB with its sharp edges these integrin subunits did not form fibrillar adhesions. beta3 generally appeared in focal adhesions. We observed aligned fibrillar structures of fibronectin on NT not only on the basal site but interestingly, also on the apical cell surface. In contrast, on CB, fibronectin appeared apically clustered. We suggest that this alignment of fibronectin fibrils depends on the directed actin cytoskeleton and in particular, on the capability of the beta1-integrins to form fibrillar adhesions, which is affected by the surface roughness of titanium.     

Effect of biomaterial surface properties on fibronectin-alpha5beta1 integrin interaction and cellular attachment

The ability of fibronectin (Fn) to mediate cell adhesion through binding to alpha(5)beta(1) integrins is dependent on the conditions of its adsorption to the surface. Using a model system of alkylsilane SAMs with different functional groups (X=OH, COOH, NH(2) and CH(3)) and an erythroleukemia cell line expressing a single integrin (alpha(5)beta(1)), the effect of surface properties on the cellular adhesion with adsorbed Fn layers was investigated. (125)I-labeled Fn, a modified biochemical cross-linking/extraction technique and a spinning disc apparatus were combined to quantify the Fn adsorption, integrin binding and adhesion strength, respectively. This methodology allows for a binding equilibrium analysis that more closely reflects cellular adhesion found in stable tissue constructs in vivo. Differences in detachment strength and integrin binding were explained in terms of changes in the adhesion constant (psi, related to affinity) and binding efficiency of the adsorbed Fn for the alpha(5)beta(1) integrins (CH(3) approximately NH(2)相似文献   

Topography effects of pure titanium substrates on human osteoblast long-term adhesion

Classically various treatments are applied to increase the roughness of titanium implants and improve their integration in the tissues. Many in vitro studies have been performed to better understand the mechanisms underlying the adhesion of cells on materials. Frequently, the adhesion is related to the attachment of cells during the first hours of contact with the substrate. For several years, our objective has been to develop experimental methods to evaluate the long-term adhesion of human osteoblasts from some hours to several weeks in order to model in vitro a tissue-like interface. This culture model allows for the formation over 21 days of a complex osteoblast/extracellular matrix/material interface. We recently developed a new parameter called adhesion power (AP) to evaluate this long-term adhesion. In this study, our objective is to check its efficiency in discriminating the long-term adhesion of human osteoblasts on pure titanium substrates with seven different surface morphologies obtained by electro-erosion, sandblasting, polishing, acid-etching and machine-tooling. By scanning electron microscopy, we observed that the human osteoblasts did spread more intimately on surface with low roughness amplitude than on rough ones. However, the AP was higher on rough isotropic surfaces obtained by electro-erosion, sandblasting or acid-etching and lower on smoother surfaces obtained by polishing and machine-tooling. We demonstrated that the AP was pertinent for evaluating human osteoblast's long-term adhesion on pure titanium surfaces with various roughness parameters. Its correlation with the order parameter, which describes the organization of the roughness, confirmed once more that human osteoblasts are more sensitive to the organization and morphology of the roughness than to its amplitude.     

Quantitative characterization of mineralized silk film remodeling during long-term osteoblast–osteoclast co-culture

The goal of this study was to explore quantitative assessments of mineralized silk protein biomaterial films by co-cultures of human mesenchymal stem cell-derived osteoblasts and human acute monocytic leukemia cell line-derived osteoclasts during long-term culture (8–32 weeks). The remodeled films were quantitatively assessed using three different techniques during this extended cultivation to provide more comprehensive insight into the impact of co-cultures on surface remodeling. Scanning electron microscopy (SEM) with three dimensional surface reconstructions was used to quantitatively determine various surface morphological features and measures of roughness indicative of remodeling by the cells. Additionally, reconstructed surfaces were converted to depth images for Fourier analysis to quantify the potential fractal organization of biomineralization. The long-term remodeled films were also imaged using confocal reflectance microscopy and micro-computed tomography (micro-CT) to further quantify morphological changes. Films remodeled in co-culture demonstrated increased roughness parameters, fractal organization, and volume compared to films remodeled by osteoblasts alone. The combination of these techniques to quantify remodeling of mineralized protein films shows promise for quantifying processes related to mineralized surfaces.     

The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro.

The adhesion, orientation and proliferation of human gingival epithelial cells and human maxillar osteoblast-like cells in primary and secondary culture were studied on glossy polished, sandblasted and plasma-sprayed titanium surfaces by scanning electron microscopy and in thin sections. The primary cultured explants of human gingival epithelial cells attached, spread and proliferated on all titanium surfaces with the greatest extension on the polished and the smallest extension on plasma-sprayed surfaces. In secondary suspension cultures of gingival keratinocytes, attachment spreading and growth was only observed on polished and plasma-sprayed surfaces, but not on sandblasted surfaces. Moreover, the attachment of these cells depended on the seeding concentration as well as on the coating with fetal calf serum. Cells on polished surfaces developed an extremely flat cell shape, but on sandblasted and plasma-sprayed surfaces a more cuboidal shape. In contrast human maxillar osteoblasts seeded as secondary suspension cultures attached very well to all three differently textured titanium surfaces and showed identical growth patterns independent of the titanium surface structure. These findings suggest that cell morphology, orientation, proliferation and adhesion of human gingival epithelial cells in primary or secondary culture are dependent on the texture of the titanium surface whereas no such differences were observed for maxillar osteoblast-like cells. In conclusion, the soft tissue integration and response is more influenced by the surface texture than the process of osseointegration.     

Integrin beta1 silencing in osteoblasts alters substrate-dependent responses to 1,25-dihydroxy vitamin D3

Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. The observations that beta1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and that it is regulated by 1alpha,25(OH)2D3 in a surface-dependent manner, suggest that beta1 may play a role in mediating osteoblast response. To test this hypothesis, we silenced beta1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the beta1-silenced osteoblasts to surface microtopography and 1alpha,25(OH)2D3. To better understand the role of beta1, MG63 cells were also treated with two different monoclonal antibodies to human beta1 to block ligand binding. beta1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, beta1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)2D3. Anti beta1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z caused dose-dependent decreases in cell number and alkaline phosphatase and an increase in osteocalcin. Effects of 1alpha,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)2D3. The results also show that beta1 mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)2D3.     

Effects of adhesion molecules on the behavior of osteoblast-like cells and normal human fibroblasts on different titanium surfaces

This study examined the influences of titanium (Ti) discs with similar surface roughnesses (R(a) values), but with different topographies and chemical compositions, on the adhesion, spreading, and the alkaline phosphatase (ALP) activity of osteoblast-like cells and normal human fibroblasts. The presence of adhesion molecules on the Ti surfaces and their effects on cell activity were also investigated. Two types of Ti discs were prepared. One kind was a mechanically polished Ti disc, and the other type was a disc obtained by the heating of hydroxyapatite (HA) dip-coated Ti. Scanning electron microscopy, optical interferometry, and scanning Auger electron spectroscopy were used to examine the surface morphology, roughness, and chemical composition, respectively, of the superficial Ti layer. The two types of Ti discs had different topographies and chemical compositions, but had similar R(a) values. The cells on both surface types had similar behaviors and ALP activities. A biological evaluation of the surface-modified Ti discs showed that the type I collagen coating was functionally active in terms of cell spreading in both types of Ti discs. In the mechanically polished Ti discs, fibronectin was functionally active in the normal human fibroblasts, but not in the osteoblast-like cells. Cell adhesion was slightly better on the heat-treated HA dip-coated Ti discs, but not on the mechanically polished Ti discs. Type I collagen and fibronectin mediated the adhesion and spreading of osteoblast-like cells through alpha2beta1 integrin and alpha5beta1 integrin, respectively. These results suggest that type I collagen might be a good candidate for the biochemical modification of Ti surfaces, particularly those surfaces obtained by heating of HA dip-coated Ti.     

Importance of integrin beta1-mediated cell adhesion on biodegradable polymers under serum depletion in mesenchymal stem cells and chondrocytes

To evaluate the predominant mechanism of chondrogenic cell [mesenchymal stem cells (MSCs) and chondrocytes] adhesion under serum free conditions, we measured the surface roughness and wettability of poly(lactic acid:polyglycolic acid=75:25) (PLGA), poly(lactic acid) (PLA), and poly(-epsilon-caprolactone) (PCL)-coated glass plates. Also to evaluate the biological reactions involved in cell-polymer interactions, integrin beta1, one of the cell adhesion molecules, was blocked with monoclonal antibody. In cell attachment test, MSCs and chondrocytes adhesion to synthetic polymers in 1h were very low and ranged from 2.8% to 8.0%. In present study, the correlation between attachment rate and surface roughness, contact angle, or integrin beta1 blocking on PLGA, PLA and PCL-coated plates could not be proved. However, we found that L-arginine-coated PLA highly increased the attachment rates of MSCs (30.2%) and of chondrocytes (26%), whereas integrin beta1 blocking significantly decreased these attachment rates to 5.6% and 7.4%, respectively, suggesting that increased cell adhesion to L-arginine-coated plates is mediated by integrin beta1.In this study, we showed that polymer characteristics such as roughness and wettability did not play an important role in cell adhesion under serum free conditions, because there was no significant difference according to polymer characteristics, whereas biological interactions mediated by integrin beta1 were critical during the early period of cell adhesion. The results suggest that L-arginine could be useful for facilitating early cell adhesion to synthetic polymers in cartilage tissue engineering.     

Engineering integrin-specific surfaces with a triple-helical collagen-mimetic peptide

Integrin-mediated cell adhesion to extracellular matrix proteins anchors cells and triggers signals that direct cell function. The integrin alpha(2)beta(1) recognizes the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif in residues 502-507 of the alpha(1)(I) chain of type I collagen. Integrin recognition is entirely dependent on the triple-helical conformation of the ligand similar to that of native collagen. This study focuses on engineering alpha(2)beta(1)-specific bioadhesive surfaces by immobilizing a triple-helical collagen-mimetic peptide incorporating the GFOGER binding sequence onto model nonadhesive substrates. Circular dichroism spectroscopy verified that this peptide adopts a stable triple-helical conformation in solution. Passively adsorbed GFOGER-peptide exhibited dose-dependent HT1080 cell adhesion and spreading comparable to that observed on type I collagen. Subsequent antibody blocking conditions verified the involvement of integrin alpha(2)beta(1) in these adhesion events. Focal adhesion formation was observed by immunofluorescent staining for alpha(2)beta(1) and vinculin on MC3T3-E1 cells. Model functionalized surfaces then were engineered using three complementary peptide-tethering schemes. These peptide-functionalized substrates supported alpha(2)beta(1)-mediated cell adhesion and focal adhesion assembly. Our results suggest that this peptide is active in an immobilized conformation and may be applied as a surface modification agent to promote alpha(2)beta(1)-specific cell adhesion. Engineering surfaces that specifically target certain integrin-ligand interactions and signaling cascades provides a biomolecular strategy for optimizing cellular responses in biomaterials and tissue engineering applications.     

Collagen type I-coating of Ti6Al4V promotes adhesion of osteoblasts

The initial contact of osteoblasts with implant surfaces is an important event for osseointegration of implants. Osseointegration of Ti6Al4V may be improved by precoating of its surface with collagen type I. In this study, the adhesion of rat calvarial osteoblasts to uncoated and collagen type I-coated titanium alloy was investigated over a period of 24 h. Collagen type I-coating accelerates initial adhesion of osteoblasts in the presence of fetal calf serum. One hour after plating, no differences in the percentage of adherent cells between the surfaces investigated were found. Adhesion of osteoblasts to uncoated surfaces was reduced by the GRGDSP peptide by about 70%, whereas adhesion to collagen type I-coated surfaces remained unaffected by treatment of the cells with the peptide. Cell adhesion to coated materials was reduced by about 80% by anti-integrin beta1 antibody. The integrin beta1 antibody did not influence the adhesion to uncoated titanium alloy. The results suggest that osteoblasts adhere to collagen type I-coated materials via integrin beta1 but not by interacting with RGD peptides, whereas adhesion to uncoated titanium alloy is mediated by RGD sequences but not via integrin beta1. Fibronectin does not seem to be involved in the adhesion of osteoblasts to either coated or uncoated titanium alloy.     

电参数对钛微弧氧化膜层理化性能及细胞相容性的影响

目的:研究纯钛微弧氧化电参数（脉冲宽度及脉冲频率）的改变对膜层表面形貌、理化性能及成骨细胞生物行为的影响。 方法:对喷砂酸蚀钛片进行微弧氧化处理,通过脉宽及频率的变化,研究其对膜层的表面形貌、摩擦性能、耐腐蚀性等影响,分析得出不同表面形貌所对应的参数条件（脉冲频率400 Hz、脉冲宽度60 μs微弧氧化组为A组,脉冲频率500 Hz、脉冲宽度60 μs微弧氧化组为B组,脉冲频率500 Hz、脉冲宽度75 μs的微弧氧化组为C组）,进一步比较在该参数下的微弧氧化膜的耐腐蚀性及成骨细胞在其表面的粘附、增殖情况及细胞形态。 结果:电镜下3组试件表面均形成粗糙多孔的膜层,且A组纯钛微弧氧化膜表面粗糙度最高;C组所制备的微弧氧化膜层耐腐蚀性能及耐磨性优于A、B两组。细胞粘附及增殖实验A、B、C各组之间差异有统计学意义（P<0.05）,实验表明A组表面成骨细胞粘附及增殖均优于B、C两组。 结论:脉冲宽度及频率变化对微弧氧化膜层表面生物活性及理化性能均有影响,当脉冲频率400 Hz、脉冲宽度60 μs时,膜层表面的细胞生长最佳。     

Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion

Integrin-mediated cell adhesion to proteins adsorbed onto synthetic surfaces anchors cells and triggers signals that direct cell function. In the case of fibronectin (Fn), adsorption onto substrates of varying properties alters its conformation/structure and its ability to support cell adhesion. In the present study, self-assembled monolayers (SAMs) of alkanethiols on gold were used as model surfaces to investigate the effects of surface chemistry on Fn adsorption, integrin binding, and cell adhesion. SAMs presenting terminal CH(3), OH, COOH, and NH(2) functionalities modulated adsorbed Fn conformation as determined through differences in the binding affinities of monoclonal antibodies raised against the central cell-binding domain (OH > COOH = NH(2) > CH(3)). Binding of alpha(5)beta(1) integrin to adsorbed Fn was controlled by SAM surface chemistry in a manner consistent with antibody binding (OH > COOH = NH(2) > CH(3)), whereas alpha(V) integrin binding followed the trend: COOH > OH = NH(2) = CH(3), demonstrating alpha(5)beta(1) integrin specificity for Fn adsorbed onto the NH(2) and OH SAMs. Cell adhesion strength to Fn-coated SAMs correlated with alpha(5)beta(1) integrin binding (OH > COOH = NH(2) > CH(3)), and experiments with function-perturbing antibodies demonstrated that this receptor provides the dominant adhesion mechanism in this cell model. This work establishes an experimental framework to analyze adhesive mechanisms controlling cell-surface interactions and provides a general strategy of surface-directed control of adsorbed protein activity to manipulate cell function in biomaterial and biotechnological applications.     

Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries

Biomaterial surface properties modulate protein adsorption and cell adhesion to elicit diverse cellular responses in biomedical and biotechnological applications. We used alkanethiol self-assembled monolayers presenting well-defined chemistries (OH, CH(3), NH(2), and COOH) to analyze the effects of surface chemistry on myoblast proliferation and differentiation. Surfaces were pre-coated with equivalent densities of fibronectin. C2C12 skeletal myoblasts exhibited surface-dependent differences in cell proliferation (COOH = NH(2) > CH(3) = OH). Myogenin and troponin T gene expression levels were up-regulated on CH(3) and OH surfaces compared to other chemistries. Furthermore, immunostaining for sarcomeric myosin revealed surface chemistry-dependent differences in myogenic differentiation following the pattern OH > CH(3) > NH(2) = COOH. Immunostaining analyses of integrin subunits demonstrated surface chemistry-dependent differences in integrin binding to adsorbed fibronectin. OH and CH(3) surfaces supported selective binding of alpha(5)beta(1) integrin while the COOH and NH(2) functionalities displayed binding of both alpha(5)beta(1) and alpha(V)beta(3) Myogenic differentiation correlated with differences in integrin binding; surface chemistries that supported selective binding of alpha(5)beta(1) displayed enhanced differentiation. Finally, blocking beta(1), but not beta(3), integrins inhibited differentiation, implicating specific integrins in the differentiation process. These results demonstrate that surface chemistry modulates myoblast proliferation and differentiation via differences in integrin binding to adsorbed fibronectin.     

Short-term effects of adhesion peptides on the responses of preosteoblasts to pBMP-9

Adhesion peptides are currently used to enhance the interactions of osteoblasts with biomaterials. However, little is known about the effects of adhesion peptides on cell responses to growth factors, especially the bone morphogenetic proteins (BMPs). We used adhesion peptides Ac-CGGNGERPRGDTYRAY-NH(2) (pRGD), derived from bone sialoprotein, and Ac-CGGDGEA-NH(2) (pDGEA), derived from collagen, which interact with alpha(v)beta(3) and alpha(2)beta(1) integrins, respectively. We analyzed the effects of pRGD- and pDGEA-coated polystyrene (PS) on the responses of murine MC3T3-E1 preosteoblasts to a peptide derived from human BMP-9 (pBMP-9) in serum-free medium. After 1h, pRGD favoured interactions with alpha(v) while pDGEA bound beta(1) integrin subunits. Adding pBMP-9 (400 ng/mL) increased the amount of alpha(v) integrin subunits in cell membranes on pRGD-coated PS, but had no effect on beta(1) integrin subunits. Only on this substratum, collagen type I mRNA was enhanced and the addition of pBMP-9 promoted the early cell differentiation, increasing their alkaline phosphatase (ALP) activity within 24 h. These cells also organized beta(1) integrin subunits at their focal adhesion points. Inhibiting alpha(2)beta(1) integrins by pDGEA pre-treatment decreased this ALP activity. It is therefore important to understand the impact of adhesion peptides on the early cell responses to growth factors in order to improve biomimetic materials.     

Response of normal female human osteoblasts (NHOst) to 17beta-estradiol is modulated by implant surface morphology

Titanium (Ti) surfaces with rough microtopographies enhance osteogenic differentiation, local factor production, and response to osteogenic agents in vitro and increase pullout strength of dental implants in vivo. Estrogens regulate bone formation, resorption, and remodeling in females and may be important in implant success. Here, we tested the hypothesis that estrogen modulates osteoblast response to implant surface morphology. Primary female human osteoblasts were cultured to confluence on three Ti surfaces (pretreatment, PT - R(a) 0.60 microm; sandblasted and acid-etched, SLA - R(a) 3.97 microm; and Ti plasma-sprayed, TPS - R(a) 5.21 microm) and treated for 24 h with 10(-7) or 10(-8) M 17beta-estradiol (E(2)). Cell number decreased with increasing surface roughness, but was not sensitive to E(2). Alkaline phosphatase specific activity of isolated cells and cell layer lysates was lower on rough surfaces. E(2) increased both parameters on smooth surfaces, whereas on rough surfaces, the stimulatory effect of E(2) on alkaline phosphatase was evident only when measuring cell layer lysates. Osteocalcin levels were higher in the conditioned media of cells grown on rough surfaces; E(2) had no effect in cultures on the plastic surfaces, but increased osteocalcin production on all Ti surfaces. TGF-beta1 and PGE(2) production was increased on rough surfaces, and E(2) augmented this effect in a synergistic manner; on smooth surfaces, there was no change in production with E(2). The response of osteoblasts to surface topography was modulated by E(2). On smooth surfaces, E(2) affected only alkaline phosphatase, but on rough surfaces, E(2) increased levels of osteocalcin, TGF-beta1, and PGE(2). These results show that normal adult human female osteoblasts are sensitive to surface microtopography and that E(2) can alter this response.     

Integrin alpha(5) controls osteoblastic proliferation and differentiation responses to titanium substrates presenting different roughness characteristics in a roughness independent manner

Integrin alpha(5)beta(1) regulates osteoblast proliferation and differentiation on smooth synthetic surfaces presenting different chemistries, but it is not known whether this integrin controls osteoblast behavior on surfaces that have micron-scale rough topographies. We cultured MG63 human osteoblast-like cells on titanium substrates with three different roughness characteristics: chemically polished (PT), grit blasted and acid etched with a complex topography consisting of 20-100 mum craters and 0.5-2 mum micropits (SLA), and plasma-sprayed Ti with irregular projections (TPS). Cells spread well on PT but displayed a smaller footprint on SLA or TPS. Nuclei were larger on PT as well. alpha(5)beta(1) binding and FAK phosphorylation were greater on the rougher surfaces (TPS > SLA > PT). Antibodies against the alpha(5)beta(1) binding site on fibronectin had no effect on cell number at 3 days, but [(3)H]-thymidine incorporation was increased, suggesting that binding to fibronectin was necessary for cell cycle regulation. Antibodies to the alpha(5) subunit reduced cell number at 3 days on PT and TPS and reduced DNA synthesis on all substrates in a surface microstructure-independent manner. At 7 days, cell numbers were reduced on PT, and DNA synthesis was reduced by 50% on all surfaces. At 7 days, anti-alpha(5) antibodies caused a partial reduction in alkaline phosphatase enzyme activity on all surfaces, but this effect was independent of surface microstructure. These results indicate that surface micron-scale topography modulates alpha(5)beta(1) integrin binding and FAK activation. Signaling via alpha(5)-dependent mechanisms is required for DNA synthesis and regulation of alkaline phosphatase, but this effect is independent of surface microstructure.     

Expression of cell adhesion complexes in epithelial cells seeded on biomaterial surfaces

Clinical studies indicate that soft tissue responses around dental implants vary, depending on the material used. It is therefore also possible that there are differences in how epithelial cells attach to various biomaterial surfaces. We studied the adhesion of cultured epithelial cells to five different dental material surfaces and to glass. The efficacy of adhesion was evaluated by using scanning electron microscopy (SEM) and immunofluorescence microscopy (IF) with antibodies to vinculin and alpha(6)beta(4) integrin, two cell surface molecules that are functional in epithelial cell adhesion. Our results indicate that epithelial cells adhere and spread more avidly on metallic surfaces (titanium, Ti(6)Al(4)V titanium alloy, dental gold alloy) than on ceramic surfaces (dental porcelain, aluminum oxide). As revealed by SEM, cells on metallic surfaces had a flattened morphology and formed multicellular islands. On porcelain and aluminum oxide most cells were round and adhesion occurred as single cells. Surface coverage was over twofold on metallic surfaces as compared to ceramic surfaces. IF of cells grown on metallic surfaces revealed vinculin in well-organized focal contacts and alpha(6)beta(4) integrin in punctate patterns typical of prehemidesmosomes. On porcelain and aluminum oxide surfaces the cells were mostly round and showed less well-organized adhesion complexes. Our results indicate that smooth metallic biomaterial surfaces are optimal for epithelial cell adhesion and spreading. These findings may have clinical implications in the design of transgingival implant structures.     

Nanostructured niobium oxide coatings influence osteoblast adhesion

The interaction of osteoblasts was correlated to the roughness of nanosized surface structures of Nb(2)O(5) coatings on polished CP titanium grade 2. Nb(2)O(5) sol-gel coatings were selected as a model surface to study the interaction of osteoblasts with nanosized surface structures. The surface roughness was quantified by determination of the average surface finish (Ra number) by means of atomic force microscopy. Surface topographies with Ra = 7, 15, and 40 nm were adjusted by means of the annealing process parameters (time and temperature) within a sol-gel coating procedure. The observed osteoblast migration was fastest on smooth surfaces with Ra = 7 nm. The adhesion strength, spreading area, and collagen-I synthesis showed the best results on an intermediate roughness of Ra = 15 nm. The surface roughness of Ra = 40 nm was rather peaked and reduced the speed of cell reactions belonging to the adhesion process.