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 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.     

不同钛表面形貌对人牙龈成纤维细胞生物学行为的影响

目的:良好的种植体颈部软组织封闭是种植体远期成功的重要因素之一。生物封闭的的主要决定因素之一是种植体颈部的表面形貌。本实验的目的是通过比较三种不同的纯钛表面形貌结构对人牙龈成纤维细胞的生物学行为的影响,寻找一种有利于牙龈胶原纤维附着的较理想的种植体颈部表面形貌,为种植体颈部设计提供指导。方法:本研究采用机械加工处理、电化学腐蚀、电化学腐蚀加酸蚀的方法在纯钛金属表面形成三组不同的表面形貌结构;用激光共聚焦显微镜检测其表面粗糙度;扫描电镜观察其表面微观形态并用X射线能谱色散谱仪检测其表面成分;将其与人牙龈成纤维细胞共同培养,MTT法检测表面细胞的增殖情况;细胞计数仪进行表面细胞计数;扫描电镜观察表面细胞的形状及排列。结果:机械加工组表面较光滑,呈浅的等向排列的微沟纹,表面粗糙度Sa为0.8783±0.2578μm;电化学腐蚀组表面略粗糙,呈圆形或椭圆形浅碟状凹,直径约10～15μm,分布均匀,凹内含有散在的小孔,小孔的直径约为1-5μm,表面粗糙度Sa为1.7530±0.3711μm;电化学腐蚀加酸蚀组表面略粗糙,呈圆形或椭圆形浅碟状凹,直径约10～15μm,分布均匀,凹内含有散在的小孔,小孔的直径约为1-5μm,表面见均匀的半球形纳米突起形成,直径约50～100nm,表面粗糙度Sa为1.6763±0.3440μm。表面成分检测显示三组均未有任何污染物在钛表面。牙龈成纤维细胞在各组钛片表面生长良好,形态正常,各组钛片对于细胞没有明显的毒性,生长曲线与正常细胞的生长曲线规律一致。机械加工组表面细胞沿着材料表面微沟纹平行排列,细胞扁平,伸展较差,细胞伸出的伪足较短;电化学腐蚀组表面细胞自由分布,细胞丰满,伸展良好,细胞伸出的伪足较长,有的伪足伸入到邻近凹内;电化学腐蚀加酸蚀组表面细胞丰满,伸展良好,伸出大量伪足,伪足很长,呈细丝状或带状,可伸入到相隔较远的凹孔内,呈悬空样,也可相互交织成网状。结论:电化学腐蚀加酸蚀可在纯钛表面形成均匀分布的直径为10-20μm的凹及直径为1-5μm的孔,凹及孔的表面均匀布满了直径为50-100nm的半球状突起。电化学腐蚀加酸蚀表面促成纤维细胞粘附作用最强。本实验中形成的微米凹、孔形态及半球形纳米形态均有利于成纤维细胞的粘附。电化学腐蚀加酸蚀方法形成的表面是一种较为理想的种植体颈部表面,可以为种植体颈部设计提供指导。     

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.     

Effects of implant microtopography on osteoblast cell attachment

PURPOSE: The overall aim of this project was to study osteoblast cell attachment on titanium surfaces with varying surface roughness. MATERIALS AND METHODS: Commercially pure titanium surfaces were prepared by polishing through 600-grit sandpaper, sandblasting, or sandblasting followed by acid etching to produce surfaces of varying roughness, as determined by scanning electron microscopy and atomic force microscopy. In vitro cell attachment of MC3T3-E1 osteoblasts was performed on the prepared surfaces in both serum-containing and serum-free media conditions. RESULTS: Cell attachment was directly related to the average surface roughness, with the highest levels of cell attachment observed on sandblasted and sandblasted-acidetched surfaces. Similar patterns of cell attachment were observed when serum-free conditions were employed. CONCLUSIONS: Combined surface analytical and cell/molecular biological techniques are powerful tools to broaden our understanding of biological events occurring at the implant-tissue interface. Data acquired from these in vitro techniques provide a translational application to in vivo clinical models leading to the next generation of dental implants.     

Attachment of human marrow stromal cells to titanium surfaces

The attachment of human bone marrow stromal cells to titanium alloy (Ti6Al4V) surfaces was investigated. Titanium disks were polished and modified by surface roughening and by passivation in nitric add. Cell attachment to titanium surfaces and tissue culture plastic (TCP) was determined by tetrazolium bromide (MTT) assay at 2, 6, 24, and 48 hours after seeding. Cell proliferation was determined by thymidine incorporation. Attachment on titanium surfaces was 75.6% to 94.9% of attachment on TCP control. The difference between cell attachment on the TCP compared with smooth or rough titanium was statistically significant (P < .05). However, no statistically significant difference was found between attachment to TCP and passivated titanium. Cell proliferation on titanium surfaces after 24 hours was approximately 70% of proliferation on TCP. There was a statistically significant difference (P < .05) between proliferation on tissue culture and smooth and passivated titanium but not on rough titanium. These results indicate that titanium provides a surface that is conducive to cell attachment and that passivating titanium improves cell attachment, approaching levels seen with TCP, a surface specifically developed to enhance cell attachment. Increasing surface roughness results in improved cell proliferation on titanium.     

The effects of scaling titanium implant surfaces with metal and plastic instruments on cell attachment

This study examined the ability of tissue culture fibroblasts to attach and colonize on the surface of pure titanium dental implants following instrumentation of the implant surface with curettes of dissimilar composition. Pure titanium dental implants were scaled with a plastic, titanium-alloy, or stainless steel curette and then immersed in a cell suspension of 3T3 fibroblasts. Counts of attached cells were made at 24 and 72 hours; the implants were then processed for scanning electron microscopy (SEM). At 24 hours, only surfaces scaled with a stainless steel curette showed a significant reduction in number of attached cells relative to untreated control surfaces. At 72 hours, both stainless steel and titanium-alloy curette instrumented surfaces showed significantly fewer attached cells than untreated control surfaces, with the greatest reduction in cell attachment observed on the stainless steel curette instrumented surfaces. SEM observations showed that fibroblasts on stainless steel instrumented surfaces tended to show a somewhat rounded morphology and a relatively reduced degree of spreading: while fibroblasts on untreated control, plastic, or titanium-alloy instrumented surfaces showed a well-spread, polygonal morphology, more typical of fibroblasts in favorable culture conditions. To the extent that such observations of cell attachment and morphology are indicative of in vivo biocompatibility, these findings could have clinical implications for the proper maintenance of titanium dental implants.     

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.     

Effect of magnetic field on the fibronectin adsorption, cell attachment and proliferation on titanium surface

OBJECTIVES: We studied the effect of various static magnetic fields (SMFs) on the adsorption of specific recombinant fibronectin (FN) peptide (hFNIII9-10) on the titanium surface. Furthermore, the responses of human osteosarcoma TE-85 cells in the SMF were observed. MATERIAL AND METHODS: Various magnetic fields--1, 2, 3, 5, 7, 10 mT--were established by controlling the distance from Nd-Fe-B magnet to the disks. For FN adsorption experiment, machined titanium disks were incubated in 1 microM hFNIII9-10 at 37 degrees C overnight under magnetic field. The adsorbed hFNIII9-10 was measured as optical density (OD). For attachment study, TE-85 cells were incubated for 2 h on the hFNIII9-10 coated machined titanium disks and OD values were measured. As for proliferation study, titanium disks were incubated for 48 h after washing unattached cells in 2 h. The amount of proliferated TE-85 cell was also measured as OD value. Attachments of TE-85 cells under various intensities of magnetic field were observed using a scanning electron microscope. RESULTS: The amount of adsorbed hFNIII9-10 showed no significant difference between control (0 mT) and six experimental groups (1, 2, 3, 5, 7, 10 mT). However, TE-85 cells attached significantly higher in groups of 1, 2, 5, 10 mT than in control group (P=0). Cell attachment in groups of 3, 7 mT showed no significant difference with that of control group. TE-85 cells were observed to attach through filopodia. Especially in 1 mT, flattened cells were predominant. In proliferation assay, 1 mT stimulated TE-85 cells showed significantly higher proliferation than those in 2, 3 and 7 mT (P=0). CONCLUSION: Magnetic fields under 10 mT did not influence FN adsorption on the titanium surface. However, a significant effect was found on cell attachment and proliferation.     

The effect of titanium surface roughness on the adhesion of monocytes and their secretion of TNF-alpha and PGE2

Dental implant surfaces are important in determining the tissue/surface interaction. One of the first cells to adhere to the implant surface is the monocyte. This study examines the effect of surface roughness on monocyte adhesion and cytokine secretion. Monocyte adherence to titanium discs of 4 different degrees of surface roughness and plastic surfaces was assayed. Blood mononuclear cells were incubated for 1.5 h in 16 mm culture wells into which titanium discs had been placed. Non-adherent cells were washed off and the numbers of remaining adherent monocyte determined by DNA quantification. TNF-alpha and PGE2 secretion in media from overnight cultures of attached monocytes stimulated with lipopolysaccharide (LPS) was quantified using ELISA and RIA, respectively. Monocyte adherence to rough titanium surfaces was greater than to turned titanium surfaces, while the lowest adherence was to the plastic surface. No significant differences in adherence to 250, 75 or 25 microm blasted surfaces could be detected. The number of adherent monocytes increased with time, with maximum adhesion after 2 h of incubation. Incubation of monocytes adherent to titanium surfaces resulted in a decrease of less than 30% in their numbers over 7 days, whereas cells attached to plastic surfaces decreased to non-detectable numbers after 48 h. Porphyromonas gingivalis LPS stimulation upregulated TNF-alpha and PGE2 secretion into the media. The LPS-induced TNF-alpha and PGE2 secretion was independent of the titanium surface roughness, however the lowest amounts of TNF-alpha and PGE2 were secreted from cells attached to plastic surfaces. The results of this study indicate that the number of monocytes attached to blasted titanium surfaces is significantly greater than to machined titanium surfaces. PGE2 and TNF-alpha secretion is less influenced by titanium surface roughness.     

纯钛表面阳极氧化电压对成骨细胞早期黏附及伸展的影响

目的:通过成骨细胞离体培养试验,观察不同阳极氧化电压对成骨细胞早期黏附及伸展的影响。方法:分别在电压为140V、200V及260V,电流密度为70A/m2等条件下,在0.03mol/L甘油磷酸钙(Ca-GP)和0.15mol/L醋酸钙混合电解液中,对纯钛样本表面进行阳极氧化处理。对阳极氧化后样本的平均粗糙度进行测量,并在其表面进行人成骨细胞培养,对早期细胞黏附及伸展等情况进行研究。采用SPSS13.0forWindows统计分析软件中的单因素方差分析进行统计学处理。结果:纯钛表面平均粗糙度为0.17μm,经阳极氧化后,随电压升高,平均粗糙度分别为0.23μm、0.26μm及0.33μm,统计学分析表明有显著性差异(P<0.05)。经过2h细胞培养后,阳极氧化处理后,纯钛表面细胞骨架发生形态学改变,且随阳极氧化电压的增高,细胞形态不规则呈现升高的趋势。统计分析结果表明,260V电压阳极氧化组,细胞黏附数显著高于对照组(P<0.05)。结论:阳极氧化处理可改善纯钛表面特性,成骨细胞在其表面的黏附及伸展等早期细胞行为受阳极氧化电压的影响。     

成骨细胞在微弧氧化处理后纯钛表面的附着、增殖及ALP活性

目的 :对表面微弧氧化 (microarcoxidation ,MAO)处理后的纯钛材料进行成骨细胞生物相容性检测 ,评价改进的MAO工艺应用于钛植入材料表面处理的可能性。方法 :纯钛材料经过 2种MAO处理后 (MAO 1和MAO 2工艺 ) ,采用MC 3T3细胞系对不同时间点成骨细胞在材料表面的附着率、生长增殖情况以及ALP活性进行检测 ,以未经处理光滑纯钛表面作为对照 ,以SPSS对实验结果进行统计分析。结果 :早期 (0 .5h、1h)细胞附着率差异有统计学意义 ,MAO 1组 >MAO 2组 >纯钛组 ;2h后 ,MAO 1组与MAO 2组无差异 ,2组细胞附着率都显著高于纯钛组。细胞增殖及ALP活性测试中 ,MAO 1处理组在各时间点都显著高于另外 2组。结论 :MAO处理后的纯钛对成骨细胞的生物相容性优于未处理组 ,改进的MAO 1处理工艺较一般工艺可以更有效提高成骨细胞的早期粘附、增殖及ALP活性。     

两种生长因子对人牙周膜成纤维细胞在钛金属表面附着和生长的影响

目的：研究两种生长因子对牙周膜成纤维细胞在然金属表面附着和生长的影响。方法：将纯钛、钛75试件入在12孔培养板内，取生长良好的第五代人牙周膜成纤维细胞（PDLF）接种在试件表面，分别在接种后4h、12h、24h、72h进行贴壁细胞地数。结果：接种后4h、12h、24h、72h、bFGF组纯钛、钛75表面细胞附着数与空白对照组的差异均有显著性（P＜0．05），rhBMP－2组纯钛、然75表面细胞附着数在初期（24h）与空白对组无显著性差异（P＞0．05）。72h时与空白对照组差异有显著性（P＜0．05），表明bFGF促进细胞附着和生长作用显著，而rhBMP－2促进细胞生长作用较促附着作用明显。结果：PDLF在钛金属表面的附着和生长可被生长因子所增强，但不同的生长因子对细胞附着和生长的生物学效应不尽相同。     

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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.     

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.     

Response of rat bone marrow cells to commercially pure titanium submitted to different surface treatments

OBJECTIVES: Alterations in the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study is to investigate the biocompatibility of cpTi submitted to different surface treatments. METHODS: The cpTi surfaces were prepared so that machined and blasted surfaces, either acid etched or not, were compared using rat bone marrow cells cultured to differentiated into osteoblast. For attachment evaluation, cells were cultured for 4 and 24h. Cell morphology was evaluated after 3 days. After 7, 14, and 21 days cell proliferation was evaluated. Total protein content and alkaline phosphatase (ALP) activity were evaluated after 14 and 21 days. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by analysis of variance. RESULTS: Cell attachment, cell morphology, cell proliferation, and ALP activity were not affected by surface treatments. Total protein content was reduced by blasted and acid etched surface. Bone-like nodule formation was significantly reduced by blasted, acid etched, and a combination of both blasted and acid etched surfaces. CONCLUSIONS: Based on these results, it can be suggested that cpTi surfaces that were submitted only to machining treatment favor the final event of osteoblastic differentiation of the rat bone marrow cells, evidenced by increased bone-like nodule formation.     

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.     

Cell attachment activity of cementum proteins and mechanism of endotoxin inhibition.

Cementum occupies a unique anatomical location where soft connective tissues of the periodontium are attached to root surfaces. Cell attachment properties of proteins present in cementum were studied. Human and bovine cementum were extracted with 0.5 mol/L CH3COOH followed by 4 mol/L guanidine, and proteins were separated by ion-exchange chromatography and SDS-polyacrylamide gel electrophoresis. Cells were labeled with radioactive amino acids and added to tissue-culture plastic plates incubated with cementum proteins, and attachment was measured. Results showed that cementum proteins promoted the attachment of smooth muscle cells, endothelial cells, and fibroblasts, but not epithelial cells. Fibroblasts attached more efficiently than other cell types, and they manifested spreading with re-organization of actin filaments. No attachment occurred to plates incubated with endotoxin from A. actinomycetemcomitans. Fewer fibroblasts attached to plates treated with cementum proteins in the presence of endotoxin, but cells pre-treated with endotoxin attached normally. Attachment was not inhibited when plates were incubated first with attachment proteins and then with endotoxin; however, it was decreased when endotoxin or bovine serum albumin preceded cementum proteins. Cementum proteins with Mr 68,000, 61,000, 55,000, and 36,000 (p68, p61, p55, and p36, respectively) manifested attachment activity, while protein(s) with Mr 23,000-24,000 did not. Western blots revealed that guanidine extracts contained three bands cross-reacting with anti-bovine sialoprotein-II antibody, but the p61, p55, and p36 were negative. We conclude that cementum contains bovine sialoprotein-II and at least four other fibroblast attachment proteins, and that they do not support epithelial cell attachment.(ABSTRACT TRUNCATED AT 250 WORDS)