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.     

Effects of titanium surfaces blasted with TiO2 particles on the initial attachment of cells derived from human mandibular bone. A scanning electron microscopic and histomorphometric analysis

This study was performed to determine the effect of commercially pure titanium surfaces blasted with TiO2 particles on the biological responses of cells derived from human mandibular bone. The morphology and attachment of those cells were investigated on turned titanium surfaces (control) and surfaces blasted with 45 microns (standard), 45-63 microns, and 63-90 microns TiO2 particles. The surfaces were analyzed in a scanning electron microscope. Based on surface analyses reported elsewhere, the turned samples had the smoothest surfaces and the roughest were those blasted with the largest particles (63-90 microns). The cell profile areas were measured using a semi-automatic interactive image analyzer. The attachment was determined as a ratio of the area of cell profiles and the total micrograph area and was expressed as percentage of attachment. Morphologically, the cells were heterogeneous. In general, the cells had spread well on all titanium surfaces, indicating good attachment to both smooth and rough surfaces. After 1, 3 and 6 h, the percentage of cell attachment did not differ significantly between the surfaces blasted with 63-90 microns and the turned surfaces, but was significantly lower on the surfaces blasted with 45 microns or 45-63 microns particles. After 24 h the surfaces blasted with 63-90 microns particles had a higher rate of cell attachment than all the other surfaces including the controls. It is concluded that attachment and growth of cells originating from human mandibular bone in vitro, are influenced by the micro-texture of the implant surface.     

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.     

Interleukin-1beta induces matrix metalloproteinase-1 expression in cultured human gingival fibroblasts: role of cyclooxygenase-2 and prostaglandin E2

OBJECTIVE: Matrix metalloproteinases (MMPs) degrade extracellular matrices and are responsible for excessive connective tissue breakdown in inflammatory disorders. We investigated the mechanism of MMP-1 expression in human gingival fibroblasts in response to the stimulation with interleukin-1beta (IL-1beta), and the role of inducible-type cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the regulation of MMP-1 expression. MATERIALS AND METHODS: We stimulated cultured human gingival fibroblasts with r(h)IL-1beta, and examined the expression of MMP-1 mRNA and protein by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of indomethacin, dexamethasone, or cycloheximide (CHX) on the IL-1beta-induced expression of MMP-1 was examined. The expression of MMP-1 in gingival fibroblasts stimulated with PGE2 was also examined. RESULTS: IL-1beta stimulated the expressions of mRNA and protein for MMP-1, in cultured fibroblasts, in time- and concentration-dependent manners. Pretreatment of the cells with indomethacin or dexamethasone inhibited the IL-1beta-induced MMP-1 expression. CHX, a protein synthesis inhibitor, also suppressed the MMP-1 expression. IL-1beta also induced COX-2 expression in gingival fibroblasts, and PGE2, a major COX-2 product, was found to enhance MMP-1 expression. CONCLUSION: The IL-1beta-induced MMP-1 expression in gingival fibroblasts may be mediated, at least in part, by COX-2 and its product PGE2.     

Immunolocalization of transforming growth factor beta in rat molars

A prominent cellular event in tooth eruption is the influx of mononuclear cells (monocytes?) into the dental follicle at the onset of eruption. In the mandibular first molar of the rat, this influx of cells reaches its peak at three days postnatally. Because transforming growth factor-beta 1 (TGF-β1) is chemotactic for monocytes, we used immunocytochemical methods to determine its localization in the rat molar during postnatal days 1–4 and day 7. The results indicate that TGF-βl displays both a spatial and temporal localization in the rat molar. It is present in the stellate reticulum (SR) on days 1 and 2 postnatally but is absent in all the subsequent days examined. None of the other soft tissue layers-ameoblasts, stratum intermedium or dental follicle – immunostain for TGF-βl. This localization of TGF-β1 in the SR at a time that just precedes that influx of monocytes into the dental follicle, coupled with the fact that fenestrated capillaries are abundant in the follicle adjacent to the SR, suggests that TGF-βl could play a role in attracting monocytes from the peripheral blood into the follicle.     

Attachment and proliferation of human oral fibroblasts to titanium surfaces blasted with TiO2 particles. A scanning electron microscopic and histomorphometric analysis.

The purpose of this study was to determine the effect of c.p. titanium surfaces blasted with TiO2 particles on the biological responses of human gingival fibroblasts (HGF). Fibroblast morphology and attachment were investigated on turned (control) titanium surfaces and those blasted with 45 microns (standard), 45-63 microns, and 63-90 microns TiO2 particles. The specimens were analyzed using a confocal laser scanner and SEM. The cell profile areas were measured using a semiautomatic interactive image analyser. The figures were expressed as percent of attachment. The turned samples had the smoothest surfaces and the roughest were those blasted with 63-90 microns. All TiO2 blasted specimens had homogeneous surfaces. Cells appeared to flatten, spread and form cellular bridges with the adjacent cells. Fibroblasts on the turned titanium surfaces appeared to follow the direction of the fine irregularities on the surface but tended to spread haphazardly on the blasted surfaces. The attachment assays showed no significant difference in the percentage of fibroblast cell attachment on the standard surfaces compared to the turned surfaces. Both surfaces blasted with 45-63 microns or 63-90 microns had significantly (P < 0.05) lower percentages of cell attachment than the control. The surfaces blasted with 63-90 microns particles had the lowest rate of cell attachment. A significant correlation (P < 0.01) was found between the degree of particle size and attachment of fibroblasts after 1-72 h. It is concluded that surface micro-texture influences the attachment and growth of HGF: surfaces blasted with 45 microns TiO2 do not inhibit fibroblast attachment and smooth or finely grooved surfaces could be conducive to cellular attachment.     

新型钛合金2种不同表面处理对成骨细胞早期附着的影响

目的研究新型低弹性模量钛合金表面及其微弧氧化及耐磨处理后的表面形貌及粗糙度变化,以及不同表面处理的成骨细胞附着与2种涂层的表面形貌变化对细胞早期动力学行为影响特征。方法通过扫描电子显微镜（SEM）观察未处理组不同表面的形貌; 采用SD新生鼠颅骨分离成骨细胞接种在材料表面并进行体外共同培养,MTT法检测30、60、120 min的不同表面的细胞早期附着量;细胞培养2 h后通过激光扫描共聚焦显微镜（CLSM）观察附着细胞的骨架蛋白结构。结果未处理组、耐磨与微弧氧化组材料表面的粗糙度值依次增加;细胞早期附着的吸光度A值随材料表面的粗糙度增加呈上升趋势,各组内3个时间点的细胞附着A值随时间延长而递增;2 h附着细胞骨架蛋白的量在微弧氧化、耐磨处理组表面表达均优于未处理组。结论2种不同涂层处理后新合金表面的形貌及粗糙度发生改变,进而影响到细胞在其表面的早期附着的量及其形态、骨架蛋白的表达。     

Gingival crevicular fluid interleukin-1beta, prostaglandin E2 and periodontal status in a community population

AIM: Interleukin-1 beta (IL-1beta) and prostaglandin E(2) (PGE(2)) are key inflammatory mediators involved in periodontal disease. The purposes of this molecular cross-sectional epidemiological study were to investigate relationships in a community sample between mean concentrations of IL-1beta and PGE(2) in gingival crevicular fluid (GCF) and (1) clinical periodontal signs and (2) risk factors of host inflammatory response and/or periodontal disease. MATERIAL AND METHODS: The sample comprised 6277 community-dwelling adults aged 52-74 years enrolled in the Atherosclerosis Risk in Communities (ARIC) study. IL-1beta and PGE(2) concentrations were measured using enzyme-linked immunosorbent assay. Person-level summary variables were computed for maximum pocket depth (MaxPD), maximum clinical attachment level (MaxCAL) and presence/absence of bleeding on probing (BOP). Mean GCF IL-1beta and PGE(2) concentrations were dependent variables in multiple linear regression models with periodontal measures and covariates as explanatory variables. RESULTS: Both GCF IL-1beta and PGE(2) were positively related to MaxPD and BOP in multiple regression models (p<0.01). Increased levels of IL-1beta and PGE(2) were associated with body mass index >or=30 kg/m(2). CONCLUSION: Higher levels of GCF IL-1beta and PGE(2) were significantly associated with clinical signs of periodontal disease and independently related to patient-based anthropomorphic measures, behaviours and exposures in community-dwelling adults.     

Effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the viability, proliferation and differentiation of osteoblast-like cells cultured on a chemically modified titanium surface

Aim: The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7).
Material and methods: Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed.
Results: Cell viability ( P =0.5516), cell number ( P =0.3485), collagen content ( P =0.1165) and mineralized matrix formation ( P =0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio ( P =0.00001) was affected by treatment and time.
Conclusion: The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.     

Histologic evaluation of the bone integration of TiO(2) blasted and turned titanium microimplants in humans

Twenty-seven patients received 2 microimplants each during implant surgery. One microimplant was blasted with 25 microm sized particles of TiO(2); the other was left as machined i.e. a turned surface. Before insertion the surface topography was characterized with an optical confocal laser profilometer. The surface roughness was greater than standard implants, and was similar for both surface modifications averaging over all parts of the implant i.e. tops, valley and flanks. The mean surface roughness from flank measurements only replicated previously reported findings: i.e. significantly rougher surfaces on blasted implants. After a mean healing period of 6.3 months in the maxillae and 3.9 months in the mandible, the microimplants and surrounding tissue were removed with a trephine burr. The histomorphometrical evaluation demonstrated significantly higher bone-to-implant contact for the blasted implants, inserted in the maxilla or in the mandible. Significantly more bone was found inside the threaded area for the blasted implants in the mandible, but there was no difference for implants positioned in maxillae.     

TiO2喷砂酸蚀处理对钛片表面氧化膜及成骨细胞生长影响的研究

目的研究纯钛钛片经喷砂及喷砂酸蚀处理后,表面氧化膜金相结构和化学成分的变化及对成骨细胞黏附和生长特性的影响。方法将直径为15 mm、厚度为1 mm的纯钛钛片分4组进行表面处理：1）机械打磨组（S0）;2）喷砂组（SB）;3）喷砂酸蚀1组（SLA1）;4）喷砂酸蚀2组（SLA2）。采用电子探针分析仪及X射线衍射仪检测4组钛片表面氧化膜的厚度、化学成分以及金相结构,扫描电镜观察其表面微观形态。而后将成骨细胞培养于4组钛片表面,采用MTT法分析比较4组钛片表面对成骨细胞黏附率以及增殖率的影响。结果与S0组相比,SB、SLA1、SLA2组的粗糙度明显增大（P<0.05）。SB、SLA1、SLA2组间表面平均粗糙度差异无统计学意义（P>0.05）。酸蚀处理使喷砂形成的氧化膜变薄,密度减低,且结构发生改变：原有的金红石型TiO2峰消失,锐钛矿型TiO2减少。在表面平均粗糙度相同条件下,SB组钛片表面氧化膜均匀致密,有利于成骨细胞早期的黏附和增殖。结论喷砂和喷砂酸蚀处理均增加了钛片表面的粗糙度,有利于成骨细胞的黏附和增殖,但酸蚀使TiO2喷砂表面的氧化膜层变薄,在平均粗糙度不变的情况下,单纯喷砂表面成骨细胞的黏附和增殖优于喷砂酸蚀处理表面。     

Effect of three distinct treatments of titanium surface on osteoblast attachment, proliferation, and differentiation

Cell-titanium interactions are crucial to the clinical success of bone and dental implants. The physico-chemical characteristics of the substrates surface influence osteoblast proliferation, differentiation, and activity as well. The osteoblast behavior was analyzed on three different titanium surfaces: ground with an abrasive 600 grit SiC paper, blasted with alumina particles (65 microm diameter) and alumina blasted followed by a double chemical etch (4% HF+4% HF/8% H2O2). Scanning electron microscopy (SEM) and profilometry showed distinct microtopographies. Ground samples showed parallel-groove orientation. The Al2O3-blasted surface presented the roughest microtopography with aluminum-rich particles incrusted in the titanium surface. Osteoblasts cells from femora of Balb/c mice were seeded onto the substrates tested. Cell morphology and initial attachment were evaluated by SEM. Osteoblasts adhered to and spread on all samples tested. However, on rough surfaces, osteoblasts did not spread completely and acquired a polygonal morphology. Besides, the cell proliferation rate was diminished at the beginning of incubation on rough surfaces. Our results suggest a delay, rather than an impairment, in osteoblast viability and alkaline phosphatase activity when cells are cultured on rough surfaces, inducing a distinct osteoblast phenotype, rather than blocking its activity. At least in the culture conditions used in this work, alumina particles did not affect osteoblast behavior.     

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.     

TGF-beta 1 alone and in combination with calcium hydroxide is synergistic to TGF-beta 1 production by osteoblasts in vitro

AIM: To examine the effects of calcium hydroxide (Ca(OH)2), transforming growth factor-beta (TGF-beta 1), and Ca(OH)2/TGF-beta 1 coadministration on TGF-beta 1 and interleukin-6 (IL-6) synthesis by early (subculture 1) and late (subculture 5) osteoblast cultures. METHODOLOGY: Early and late cultures were established using bone cells harvested from 21-day-old fetal rat calvaria. Cell cultures of both early and late osteoblasts were divided into four groups: group 1, control; group 2, cells challenged with Ca(OH)2; group 3, cells challenged with TGF-beta 1; and group 4, cells challenged with Ca(OH)2 and TGF-beta 1 in combination. TGF-beta 1 and IL-6 levels for all groups were determined using ELISA methodology. RESULTS: ANOVA and Tukey HS analyses revealed that osteoblasts of groups 3 and 4 significantly increased (P < 0.001) TGF-beta 1 synthesis in both early and late cultures of osteoblasts. IL-6 was not detected in any of the groups considered in this study. CONCLUSIONS: Exogenous TGF-beta 1 has an autocrine effect on cell cultures of osteoblasts. Administration of TGF-beta 1 alone or in combination with Ca(OH)2 increases the synthesis of TGF-beta 1 in osteoblast cultures. Ca(OH)2 and TGF-beta 1 are compatible when placed in a culture of osteoblasts. Ca(OH)2 provides a favourable environment for the anabolic effects of TGF-beta 1.     

Increased matrix metalloproteinase-2 activity induced byTGF-β1 in duct cells of human salivary gland is associated with the development of cyst formation in vivo

Proteolytic enzyme activity has been shown to be important for cyst formation. In this study, we constructed a cyst-like structure in vivo and analyzed molecular mechanisms involved in the development of the lesion. When SV40-immortalized duct cells of normal human salivary gland (NS-SV-DC) were treated with TGF-βl at a concentration of 1 ng/ml or 5 ng/ml followed by co-inoculation with Matrigel into the backs of nude mice, they formed large cysts containing fluid when 5 ng/ml of TGF-βl was used. Analysis of the fluid demonstrated high MMP activity. Immunohistochemical staining exhibited strong reactivity with anti-MMP-2 antibody in TGF-pl (5 ng/ml)-treated NS-SV-DC. Northern blot analysis indicated that the expression of TGF-β1 and MMP-2 mRNAs in ceils was greatly enhanced by treatment with 5ng/ml TGF-βl. These findings suggest that the in vivo cyst formation by TGF-βl-treated cells is associated with continuous induction of MMP-2 activity.     

Proliferation, differentiation, and protein synthesis of human osteoblast‐like cells (MG63) cultured on previously used titanium surfaces

This study compared osteoblast proliferation, differentiation, and protein synthesis on new and used titanium (Ti) disks to test the hypothesis that cleaning and resterilization of previously used Ti disks does not alter cell response to a particular surface. Ti disks of varying roughness were prepared by one of five different treatment regimens. Standard tissue culture plastic was used as a control. Human osteoblast-like cells (MG63) were cultured on the Ti disks and cell proliferation, cell differentiation, RNA synthesis and matrix production (collagen and noncollagen protein; proteoglycans) measured. After their first use, the disks were cleaned, re-sterilized by autoclaving. and MG63 cells cultured on them as before. At confluence, the same parameters were measured and cell behaviour on new and used disks compared. When confluent cultures of cells on plastic were compared to those cultured on new Ti surfaces, cell number was reduced on the roughest surfaces and equivalent to plastic on the other surfaces. Cell number was further reduced when disks with the roughest surfaces were re-used; no differences in cell number could be discerned after cleaning and re-sterilization. Cell proliferation was inversely related to surface roughness and was less than seen on tissue culture plastic. Re-use of the Ti disks resulted in no change in cell proliferation rate. Alkaline phosphatase specific activity in isolated cells was lowest on the rougher surfaces; no differences between new and used disks were observed. Similarly, enzyme activity in the cell layer was decreased in cultures grown on rougher surfaces, with no effect of prior disk use being noted. RNA synthesis was decreased with respect to plastic in cultures on smoother surfaces and increased on rougher surfaces; prior disk use did not alter RNA synthesis. Collagen production by the cells was decreased on smoother surfaces, but was comparable to tissue culture plastic when grown on rougher surfaces. Non-collagen protein production was unaffected by culture surface and whether or not the disk had been previously used. Proteoglycan synthesis by cells was decreased on all surfaces studied and comparable on both new and used disks. The results of this study indicate that Ti implant surfaces are unaffected by cleaning and resterilization, although rougher surfaces may require more extensive cleaning than smoother ones. This suggests the possibility that implants, in the same patient, could be safely reused. In vivo studies in animals, however, need to be performed before clinical application can be considered.     

Evaluation of the interface between bone and titanium surfaces being blasted by aluminium oxide or bioceramic particles

The surface structure, in particular the surface roughness, and the surface chemistry of titanium implants influence their anchoring in bone. The aim of this study was to analyse metal-bone contact (MBC) after modification of the implant surface, using different materials for blasting. The surface modification of titanium was produced by blasting it with particles made of Al2O3 or bioceramics. The biological effects were then investigated experimentally using 27 rabbits, analysed after 7, 28 and 84 days after the implantation of titanium cylinders treated accordingly. The MBC showed a tendency for more bone after bioceramics were used as a blasting material, compared to Al2O3.     

Autoantibodies to beta1-adrenoceptors in human chronic periodontitis induce overexpression of fibroblast CD40 and trigger prostaglandin E2 generation

Background and Objective: Autoimmune mechanisms may contribute to the pathogenesis of periodontal disease. Autoantibodies with the potential to bind and activate β₁‐adrenoceptors (β₁‐AR) of human gingival fibroblasts were studied to provide evidence of altered humoral immune response in chronic periodontal disease. Material and Methods: Flow cytometry and enzyme‐linked immunosorbent assay using cell culture‐adherent gingival fibroblasts and/or their purified membranes and/or a synthetic peptide corresponding to the second extracellular loop of human β₁‐AR were used to detect serum antibodies. The effects of antibodies from chronic periodontal disease patients on PGE₂ generation and CD40 expression were also tested. Results: Circulating immunoglobulin G (IgG) from chronic periodontal disease patients (but not from normal individuals) interacted with the fibroblast surface, activating β₁‐AR. Atenolol or CGP 20712 (beta 1‐AR antagonists) and β₁ synthetic peptide inhibited the interaction of IgG with β₁‐AR. Immunoglobulin G from chronic periodontal disease patients also displayed agonist‐like activity associated with specific β₁‐AR activation, increasing PGE₂ generation and CD40 overexpression. The corresponding affinity‐purified anti‐β₁‐AR peptide IgG mimicked these effects. Both effects were prevented by inhibition of cyclo‐oxygenase. Conclusion: This article supports the participation of humoral immune alterations in chronic periodontal disease resulting in postsynaptic functional deregulation. Overproduction of proinflammatory mediators (PGE₂ and CD40 expression) is induced as a consequence of antibody–β₁‐AR interaction. The PGE₂–CD40–IgG axis may play a part in the pathophysiological mechanisms underlying the inflammatory process in chronic periodontal disease.     

Interleukin 1, interleukin 6 and transforming growth factor-β production by human gingival mononuclear cells following stimulation with Porphyromonas gingivalis and Fusobacterium nucleatum

Gingival mononuclear cell production of interleukin 1 (IL-1), interleukin 6 (IL-6) and transforming growth factor-β(TGF-β) after stimulation with the putative periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum was investigated. Using an ELISA method, gingival mononuclear cells extracted from 18 adult periodontitis subjects were found to be producing IL-1. However, IL-1 activity could only be detected in 5 out of these 18 cases when tested using a thymocyte proliferation bio-assay, suggesting the presence of IL-1 inhibitors. Depletion of monocytes from peripheral blood cultures resulted in a significant decrease in IL-1 activity following P. gingivalis stimulation while there was no effect in the level of IL-1 activity following stimulation with F. nucleatum. This suggests that P. gingivalis and F. nucleatum stimulate different cell types to produce IL-1. Like IL-1. IL-6 production by gingival mononuclear cells was significantly greater than that produced by the control peripheral blood mononuclear cells. Following P. gingivalis and F. nucleatum stimulation, higher levels of IL-6 could be detected; however, both organisms stimulated similar levels. Intracytoplasmic immunofluorescence staining demonstrated a lower percent TGF-β⁺ cells in bacterial stimulated peripheral blood mononuclear cell cultures compared with cells in medium alone. In the gingival mononuclear cell cultures, the percentage TGF-β⁺ cells peaked at day 1 in F. nucleatum -stimulated, whereas in P. gingivalis -stimulated cultures the peak TGF-β⁺ cells occurred at day 3, again suggesting stimulation of different cell subsets. These results ilustrate that different periodontopathic bacteria may stimulate different cell types to produce cytokines which may have synergistic or antagonistic effects.