Rat bone marrow cell response to titanium and titanium alloy with different surface roughness

In general, cell response is affected by both chemical composition and surface roughness of implant materials. The aim of this study was to evaluate the effect of titanium (Ti) chemical composition and surface roughness on the response of rat bone marrow cells, examining cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on both commercially pure titanium (cpTi) and titanium-6-aluminum-4-vanadium alloy (Ti-A) discs with four different average roughnesses (Ra). For attachment evaluation, cells were cultured for 2 h. After 14 days, cell proliferation, total protein content, and ALP activity were evaluated. Bone-like nodule formation was evaluated after 21 days. Data were compared by anova and Duncan's multiple range test when appropriate. Cell attachment and total protein content were affected by neither Ti chemical composition (P = 0.201, and P = 0.639, respectively) or surface roughness (P = 0.972, and P = 0.660, respectively). Proliferation, ALP activity, and bone-like nodule formation were affected only by Ti chemical composition (P = 0.0001, P = 0.064, and P = 0.0001, respectively). These results suggest that cpTi would optimize osteoblastic differentiation by rat bone marrow cells, including reduced cell proliferation, and increased ALP activity and bone-like nodule formation, while surface roughness, within the Ra parameters used, would not affect significantly the rat bone marrow cell response.     

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.     

Nitric acid passivation does not affect in vitro biocompatibility of titanium

PURPOSE: In general, both chemical composition and surface features of implants affect cell response. The aim of this study was to evaluate the effect of titanium (Ti) passivation on the response of rat bone marrow cells, considering cell attachment, cell morphology, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bonelike nodule formation. MATERIALS AND METHODS: Cells were cultured on both commercially pure titanium (cpTi) and titanium-aluminium-vanadium alloy (Ti-6Al-4V) discs, either passivated or not. For attachment evaluation, cells were cultured for 4 and 24 hours. Cell morphology was evaluated after 4 days. After 7, 14, and 21 days, cell proliferation, total protein content, and ALP activity were evaluated. Bonelike nodule formation was evaluated after 21 days. Data were compared by analysis of variance and the Duncan multiple range test. RESULTS: Cell attachment, cell morphology, cell proliferation, total protein content, ALP activity, and bonelike nodule formation all were unaffected by Ti composition or passivation. DISCUSSION AND CONCLUSION: Although the protocol for passivation used here could interfere with the pattern of ions released from Ti-6Al-4V and cpTi surfaces, the present study did not show any effect of this surface treatment on in vitro biocompatibility of Ti as evaluated by osteoblast attachment, proliferation, and differentiation.     

Osteoblastic differentiation of cultured rat bone marrow cells on hydroxyapatite with different surface topography

Hydroxyapatite (HA) has been used in orthopedic, dental, and maxillofacial surgery as a bone substitute. OBJECTIVE: The aim of this investigation was to study the effect of surface topography produced by the presence of microporosity on the response of the rat bone marrow cells, evaluating: cell attachment, proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. METHODS: Cells were cultured on HA discs manufactured by a combination of uniaxial powder pressing and different sintering conditions, with different percentage of microporosity (<5%-HA5, 15%-HA15, and 30%-HA30). For attachment evaluation, cells were cultured for 2 h. Proliferation was evaluated after 7 and 14 days. After 14 days, total protein content and ALP activity were measured. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by ANOVA and Duncan's multiple range test when appropriate. RESULTS: Cell attachment was not affected by surface topography (p=0.37). Proliferation (p=0.001), total protein content (p=0.039), ALP activity (p=0.050), and bone-like nodule formation (p=0.00001) were all significantly decreased by the most irregular surface (HA30). SIGNIFICANCE. These results suggest that initial cell events were not affected by the surface topography of the HA. However, intermediary and final events such as proliferation, protein synthesis, ALP activity, and bone-like nodule formation favored surfaces with a more regular topography, such as that presents in HA with 15% or less of microporosity.     

In vitro osteogenesis induced by cells derived from sites submitted to sinus grafting with anorganic bovine bone

OBJECTIVES: This study evaluated key parameters of the in vitro osteogenesis induced by osteoblastic cells obtained from sites submitted to sinus grafting with anorganic bovine bone (ABB) in comparison with cells derived from bone sites of the same patients. MATERIALS AND METHODS: In three patients, the augmentation of maxillary sinus was carried out using ABB (Bio-Oss). After at least 6 months, during the surgical intervention for titanium implants placement, biopsies were taken from these areas using trephine burs (grafted group). Bone fragments, of the same patients, from sites that had not received graft were also obtained with trephine burs and used as a control group. Osteoblastic cells were obtained from grafted and control groups by enzymatic digestion and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured in 24-well culture plates. Cell adhesion was evaluated at 24 h. For proliferation and viability assay, cells were cultured for 1, 3, 7, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 3, 7, 10, 14, 17, and 21 days. Cultures were stained with Alizarin red S at 21 days, for detection of mineralized matrix. Data were compared by Student's t-test. RESULTS: Cell adhesion and viability were not affected by cell source (P>0.05). Total protein content was greater (P<0.05) for grafted group. Cell proliferation, ALP activity, and bone-like nodule formation were all greater (P<0.05) for the control group. CONCLUSION: Taken together, these results indicate that the in vivo long-term contact of cells with ABB downregulates the expression of osteoblast phenotype and consequently the in vitro osteogenesis.     

Corrosion resistance and biocompatibility of a new porous surface for titanium implants

Alterations of the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study was to investigate the biocompatibility of cpTi when submitted to a new, porous titanium, surface treatment (porous Ti). Five types of surface treatments, namely sintered microspheres porous titanium (porous Ti), titanium plasma spray (TPS), hydroxyapatite (HA), sandblasted and acid etched (SBAE), and resorbable blast medium, sandblasted with hydroxyapatite (RBM) were made. In the experimental methods, the corrosion potentials were measured over time, and then a linear sweep voltammetric analysis measured the polarization resistances and corrosion currents. For biocompatibility evaluation, MG63 osteoblast-like cells were used. Cell morphology, cell proliferation, total protein content, and alkaline phosphatase (ALP) activity were evaluated after 2 h, and after 2, 4 and 7 d. Porous Ti and SBAE showed a better corrosion resistance, with a weak corrosion current and a high polarization resistance, than the other surfaces. Cell attachment, cell morphology, cell proliferation, and ALP synthesis were influenced by the surface treatments, with a significant increase observed of the activity of osteoblast cells on the porous coating (porous Ti). Based on these results, it is suggested that the porous Ti surface has a significantly better biocompatibility than the other surface treatments and an excellent electrochemical performance.     

Development of the osteoblast phenotype of serial cell subcultures from human bone marrow

Bone marrow cells have been used for testing biocompatibility of bone substitute materials that would be applied in maxillofacial and orthopedic surgeries. However, it remains unclear whether cells in serial subcultures retain the ability to differentiate into osteoblasts. The purpose of this study was to compare the development of osteoblast phenotype of serially passaged cells from human bone marrow. Cells from first to third passage were cultured (2x10(4) cells/well) in supplemented culture medium. Cells were incubated at 37 masculineC in a humidified atmosphere of 5% CO2 and 95% air. Cell attachment was assessed at 4 and 24 h. At 7, 14 and 21 days, cell proliferation, cell viability, total protein content and alkaline phosphatase (ALP) activity were evaluated. Bone-like formation was evaluated at 14 and 21 days. Data were compared by two-way ANOVA and Duncan's multiple range test. Cell attachment, cell viability and total protein content were not affected by serial subcultures. However, serial subcultures did interfered negatively with osteoblast differentiation as shown by osteoblast parameters observed in second and third subcultures, such as continuous cell proliferation, lower ALP activity and bone-like formation in comparison to first subculture. Therefore, it is important to evaluate cell ability to growth and differentiate before selecting the cell population for studies that investigate the biocompatibility of materials to replace bone tissue.     

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     

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.     

Effect of titanium surface on secretion of IL1β and TGFβ1 by mononuclear cells

Mononuclear cells play an important role in the modulation of healing. The characteristics of implant surface topography may alter the production of signaling molecules such as cytokines. The aim of this in vitro study was to evaluate the effects of commercially available titanium surface treatments on both cell viability and the secretion of the antagonist cytokines, IL1β and TGFβ1. Human mononuclear cells were cultured on 10 mm diameter commercially pure titanium (cpTi) disks that were prepared using a turning procedure (control = machined surface) and either acid etched or bio-anodized for 1-7 days. Adhered cells were investigated with respect to cell viability using an MTT assay, and cytokine production was verified using an ELISA assay. The results indicate that surface characteristics did not alter the cell viability at days 1 and 4, although the machined surface presented the highest absorbance values at day 7 (p = 0.0084). Cell viability was reduced throughout the time course for all analyzed surfaces (p < 0.05). On day 4, IL1β levels were significantly higher on bio-anodized compared to acid etched surfaces (p = 0.0097). TGFβ1 did not show differences among the surfaces at days 1 and 4. The responses of non-stimulated mononuclear cells to titanium surfaces suggest only modest effects of the surface treatment and roughness on pro-inflammatory cytokine (IL1β) release.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

淫羊藿素对 SD 大鼠骨髓基质细胞增殖与成骨分化的影响

目的：研究淫羊藿素（ICT）对体外培养 SD 大鼠骨髓基质细胞（rBMSCs）增殖与成骨分化的影响。方法：体外分离培养 rBMSCs,传代至第4代作多向分化鉴定。分别以10－9、10－8、10－7、10－6、10－5 mol／L ICT 刺激 rBMSCs 后3、6、9 d,分别用 cck-8及碱性磷酸酶 ALP 试剂盒检测 rBMSCs 的增殖及 ALP 活性;10－9 mol／L ICT 处理 rBMSCs 后21 d 作茜素红（AR）染色以判断钙结节的形成。结果：原代培养的 rBMSCs 贴壁生长、呈梭形,能多向分化;ICT 明显抑制了 rBMSCs 的增殖;但增高其 ALP 活性、钙结节形成。结论：ICT 以剂量依赖方式抑制 rBMSCs 的增殖但促进其分化和矿化。     

Osteoblast differentiation of human bone marrow cells under continuous and discontinuous treatment with dexamethasone

Dexamethasone (Dex) has been shown to induce osteoblast differentiation in several cell culture systems. This study investigated the effect of continuous and discontinuous treatment with Dex on osteoblast differentiation of human bone marrow stromal cells (BMSC). Primary culture and first passage were cultured in media with or without Dex 10(-7) M. During the culture period, cells were incubated at 37 degrees C in humidified atmosphere of 5% CO2 and 95% air. At 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity and bone-like formation were evaluated. Data were compared by two-way analysis of variance. Dex did not affect cell viability and total protein content, but reduced cell number. ALP activity and bone-like formation increased when only first passage or both primary culture and first passage were treated with Dex, in comparison to the groups that did not have contact with Dex after first passage. The results of this study indicate that, for human BMSC, continuous presence of Dex did not appear to be required for development of the osteoblast phenotype, but Dex must be present after first passage to allow osteoblast differentiation expressed by reduced cell proliferation and increased ALP activity and bone-like formation.     

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.     

兔骨髓基质细胞在不同纯钛表面的早期粘附

目的：比较兔骨髓基质细胞在3种不同化学蚀刻纯钛表面的早期粘附情况。方法：分别用HNO3、热H2SO4／H2O2、热H2SO4／HCl处理纯钛片30min。采用扫描电子显微镜、X射线光电子能谱对试样表面形貌及成分进行分析。取兔骨髓基质细胞接种于钛片表面，培养30、60、120min，采用荧光显微镜和四唑盐比色法对细胞粘附进行观察和分析。结果：HNO3组表面形貌光滑，平整；H2SO4／HCl、H2SO4／H2O2组表面粗糙。3组钛片表面的主要成分为钛、氧和碳。细胞在H2SO4／HCl、HNO3组表面粘附伸展良好，在H2SO4／H2O2组表面伸展较差。结论：细胞在H2SO4／H2O2组表面的粘附不及H2SO4／HCl组，甚至不如HNO3组。     

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。     

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.     

Determining optimal surface roughness of TiO(2) blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone

In the complex process of bone formation at the implant-tissue interface, implant surface roughness is an important factor modulating osteoblastic function. In this study, primary cultures of osteoblast-like cells, derived from human mandibular bone, were used. The aim was to examine the effect of varying surface roughness of titanium implant material on cellular attachment, proliferation and differentiation. A recognized method of increasing surface roughness and enlarging the surface area of titanium implants is by blasting with titanium dioxide particles: the four specimen types in the study comprised surfaces which were machine-turned only, or blasted after turning, with 63-90 microm, 106-180 microm, or 180-300 microm TiO(2) particles, respectively. The specimens were analyzed by scanning electron microscopy and confocal laser scanning. The turned samples had the smoothest surfaces: average height deviation (S(a)) of 0.20 microm. The roughest were those blasted with 180-300 microm particles, S(a) value 1.38 microm. Blasting with intermediate particle sizes yielded S(a) values of 0.72 microm and 1.30 microm, respectively. Cell profile areas were measured using a semiautomatic interactive image analyzer. Figures were expressed as percentage of attachment. DNA synthesis was estimated by measuring the amount of [(3)H]-thymidine incorporation into trichloroacetic acid (TCA) insoluble cell precipitates. The specific activity of alkaline phosphatase was assayed using p-nitrophenylphosphate as a substrate. The ability of the cells to synthesize osteocalcin was investigated in serum-free culture medium using the ELSA-OST-NAT immunoradiometric kit. After 3 h of culture, the percentage of cellular attachment did not differ significantly between specimens blasted with 180-300 micromparticles and the turned specimens. All blasted surfaces showed significantly higher [(3)H]-thymidine incorporation than the turned surfaces (P<0.05), with the highest on the surfaces blasted with 180-300 microm particles. Osteocalcin synthesis by the cells in response to stimulation by 1,25(OH)2D3, was also significantly greater (P<0.05) on the surfaces blasted with TiO(2) particles. However, analysis of alkaline phosphatase activity disclosed no significant differences among the four surface modifications. It is concluded that in this cellular model, the proliferation and differentiation of cells derived from human mandibular bone is enhanced by surface roughness of the titanium implant. However, increasing the size of the blasting particles to 300 microm does not further increase the initial attachment of the cells compared to turned surfaces and those blasted with 63-90 microm particles.