An in vivo method for measuring the adsorption of plasma proteins to titanium in humans.

A novel method of collecting in vivo plasma proteins of humans from osteotomies prepared during insertion of an oral implant is described. A rod containing a collecting portion with a predetermined surface is introduced into the osteomy, removed, and transferred for enzyme-linked immunosorbent assay analysis. Two experiments were used to examine the feasibility of the method. In the first, titanium (Ti) rods with different roughness were exposed for 10 min to the blood. Blasted and acid-etched surfaces adsorbed four times more and acid-etched surfaces adosorbed two times more plasma proteins as compared to machined surfaces. In the second experiment, blasted and acid-etched rods were wetted for 10 s prior to the insertion. The adsorption for fibronectin, albumin, fibrinogen, and IgG was enhanced significantly compared with nonwetted rods. These results are discussed in the light of previous methods used in studies on adsorption. Thus, use of the collecting instrument enables aspects of human plasma-implant interface to be studied in a more realistic manner.

Adsorption of human salivary proteins to titanium powder. I. Adsorption of human salivary albumin.

Titanium (Ti) is among the most widely used implant materials in dentistry today. The success of Ti implants is associated with their interactions with the surrounding tissues and biological fluids. In the present study, the adsorption of salivary proteins to Ti and the effect of calcium (Ca) on this process were investigated. Untreated and Ca-treated Ti powders were suspended in human clarified whole saliva. After incubation, the supernatant fluid was collected for protein analysis. The powders were then washed and resuspended in EDTA to desorb proteins from Ti surfaces. Sodium dodecylsulphate polyacrylamide gel electrophoresis and Bradford protein assay were conducted to determine the concentration and type of proteins that adsorbed onto Ti surfaces. The presence of Ca ions enhanced the adsorption of salivary proteins to Ti. A 66 kDa protein, identified by immunoblotting as albumin, was found as the main adsorbed salivary protein. Adsorption of albumin to Ti pretreated with Ca was significantly greater than to native Ti. The Ca-dependent adsorption process was reversed by EDTA. The data suggest that salivary albumin is one of the main constituents of a salivary biofilm formed on Ti dental implants and its adsorption to Ti surfaces is Ca-dependent. The presence of albumin on Ti dental implants may affect plaque accumulation on the implants and the biocompatibility of Ti implants.

Effect of amine and stannous fluoride on human neutrophil functions in vitro.

Amine fluoride (AmF)- and stannous fluoride (SnF2)-containing products were found to have a therapeutic effect on gingivitis and periodontitis. This effect was suggested to correlate with the antibacterial activity of the fluoride compounds. However, their effect on inflammatory cell function can also play a role in the therapeutic effect on gingival inflammation. The present study was designed to test the effects of AmF, SnF2, and an AmF/SnF2 combination on the function of human peripheral blood neutrophils, as compared with effects of chlorhexidine and salicylic acid. Neutrophils were isolated from human blood by ficoll centrifugation followed by dextran sedimentation. The neutrophils were pre-incubated with AmF, SnF2, or AmF/SnF2, followed by stimulation with fMLP. Cell vitality was verified by trypan-blue exclusion (> 95% vitality at all tested concentrations). Superoxide production was measured by cytochrome C reduction and the enzymatic activity of lysozyme and beta-glucoronidase by optical density measurement of substrate conversion. The results showed that AmF, SnF2, or AmF/SnF2 enhanced by two- to three-fold the superoxide release from fMLP-stimulated human neutrophils. Furthermore, the effective concentration of the AmF/SnF2 combination was several-fold lower than that of AmF or SnF2 alone (10 nM for AmF, 0.5 microM for SnF2, and 3 pM for SnF2/AmF). On the other hand, chlorhexidine and salicylic acid were found to reduce superoxide production by the cells. All the tested compounds had no effect on granular enzyme release by the stimulated neutrophils. The results suggest that AmF and SnF2 enhance the oxygen-dependent antibacterial activity of neutrophils. This effect may contribute to a more efficient elimination of bacteria from the periodontal environment, resulting in improvement in gingival health.

Sustained release device containing metronidazole for periodontal use.

The purpose of this study was to develop a sustained release device containing metronidazole for insertion within periodontal pockets and to examine the release kinetics in vitro and in vivo. Cast films of ethyl cellulose with or without polyethylene glycol, containing metronidazole, were prepared and exhibited sustained release. Release rate of metronidazole from the film was measured by means of a UV spectrophotometer, and kinetics of release in vitro was found to conform to Higuchi's diffusional model. The microbiological results proved that embedding metronidazole in ethyl cellulose film does not inhibit the biological activity. The release kinetics in vivo correlated with in vitro results, exhibiting a sustained release of metronidazole over a period of three days from 30% metronidazole with polyethylene glycol or 40% metronidazole in ethyl cellulose chloroform cast. This study demonstrates that, by embedding metronidazole in ethyl cellulose, it is possible to obtain sustained release of the drug within the periodontal pocket for three days.

The effect of previous diet on plaque pH response to different foods.

In this study we investigated the effect of previous diet on the response of plaque pH to three test foods. The study population consisted of 11 dental students. Plaque pH was measured, by means of the touch electrode method, on the first two upper molars. The study was conducted at two sessions, one week apart. Subjects followed a 48-hour high-sugar diet before the first test session and a 48-hour low-sugar diet before the second test session. During both 48-hour periods, students refrained from all oral hygiene practices and fluoride utilization. At each session, three foods were ingested at one-hour intervals: cola, beer, and chocolate bar. pH measurements at baseline and at selected times after food ingestion were recorded and analyzed. Multivariate analysis of variance revealed significant independent effects of food, previous diet, and their interaction on plaque pH. After the same foods were ingested, plaque pH response after a previous high-sugar diet was significantly more acidic than after a previous low-sugar diet.

Anticariogenic activity of licorice and glycyrrhizine I: Inhibition of in vitro plaque formation by Streptococcus mutans.

The effect of licorice and its active sweet component glycyrrhizin was tested on the growth and adherence to glass of the cariogenic Streptococcus mutans. Neither licorice nor glycyrrhizin promoted growth or induced plaque formation. In the presence of sucrose, glycyrrhizin did not affect bacterial growth, but the adherence (plaque formation) was markedly inhibited. At 0.5-1% glycyrrhizin, inhibition was almost complete. These results support our previous suggestions that glycyrrhizin might serve as an efficient vehicle for topical oral medications.

Effect of human leukocyte extracts and gingival exudate on periodontopathic bacteria.

The effect of leukocyte hydrolytic enzymes on periodontopathic bacteria was examined in vitro. A frozen and thawed extract of human peripheral blood leukocytes (LE) and human gingival crevicular exudate (GE) were shown to be able to cause the release of 50% of the radioactivity from a leukotoxic strain of Actinobacillus actinomycetemcomitans (Aa Y4), labeled by 14C. A nonleukotoxic strain (Aa 653) was shown to be more susceptible to both LE and GE, up to 68% of the total radioactivity was solubilized by LE at pH 7.4. Both bacterial strains were found to be resistant to the activity of lysozyme, but highly susceptible to lysolecithin and mixtures of lysolecithin and lysozyme or LE. Capnocytophaga sputigena strain 4 was also found to be partially susceptible to the effect of LE and GE. The possible role of leukocyte hydrolytic enzymes in bacteriolysis and release of bacterial products in relation to periodontal disease is discussed.

Ultrastructural studies of the effect of human leukocyte extracts on periodontopathic bacteria.

Human gingival crevicular exudate (GE) and leukocyte extracts (LE) were previously shown to be able to release the bulk of radioactivity from a variety of 14C-labeled oral bacteria. The present study demonstrates that the release of radiolabel by these enzymatic agents from two periodontopathic bacteria (Actinobacillus actinomycetemcomitans strain Y4 and Capnocytophaga sputigena strain 4) is accompanied by both cell wall and cytoplasmatic damage. The possible role of bacterial products released by lysosomal enzymes, present in the gingival crevice, is discussed.

Bacteriolytic activity of human gingival exudate.

We investigated the bacteriolytic activity of gingival crevicular fluid (CF) on 14C-labeled Streptococcus faecalis, Streptococcus mutans, Staphylococcus aureus, and on whole dental plaque. CF was collected from 100 healthy donors pooled and centrifuged at 200 g. CF supernate and a frozen and thawed extract of the pellet were interacted with the different bacterial strains, while Streptococcus faecalis and Staphylococcus aureus released 60% and 75% of the radioactive label, only 38% of it was solubilized from Streptococcus mutans, following their incubation with the CF supernate. The findings agreed with results obtained by interacting bacteria with a frozen and thawed lysate of human peripheral blood leukocytes. On the other hand, extracts from frozen and thawed CF pellet were inactive. Further, lipoteichoic acid and lipopolysaccharide were released by CF from Gram-positive and Gram-negative bacteria, respectively. The role of bacteriolytic factors, present in CF, as a result of the interaction between microorganisms and leukocytes at inflammatory sites is discussed.

Phagocytosis and binding via complement receptors by salivary polymorphonuclear leukocytes. Modulation by saliva and gingival exudate.

The ability of oral polymorphonuclear leukocytes (PMNs) to phagocytose Candida albicans cells and bind Salmonella typhi via complement receptors was investigated. A significantly higher percent of oral PMNs could phagocytose and bind via complement receptors as compared to peripheral blood PMNs. While treatment of peripheral blood PMNs with the donor's saliva caused an increase in the number of complement-receptor bearing cells, as well as a partial increase in phagocytosis, PMNs treated with gingival crevicular fluid (CF) showed a decrease both in phagocytosis and binding. The complexity of environmental conditions and factors, and its role in PMN functions in inflammatory sites is discussed.

The use of sustained release delivery of chlorhexidine for the maintenance of periodontal pockets: 2-year clinical trial.

The release of chlorhexidine from an ethyl cellulose-based dosage form (SRD) has been shown to be effective in the reduction of the flora associated with periodontal pockets as well as in reducing probing depths. In this study, treatment with this dosage form was compared to routine maintenance therapy (RMT) in a 2-year, split mouth clinical trial. Ten patients with 84 pockets greater than or equal to 5 mm who had not received any periodontal therapy or systemic antibiotics over the last 6 months were included. The patients all received a full mouth scaling and root planing together with through oral hygiene instruction. Two months later (baseline) plaque index (P1I), bleeding on probing (BOP), pocket probing depth (PD), and attachment levels (AL) were assessed at all selected sites. Pockets on the control side then received RMT while the experimental pockets were treated with the SRD only. Treatment was repeated every 3 months for 2 years. The SRD treatment resulted in an improvement of greater than or equal to 3 mm in PD of at least 1 pocket in 8 of 10 patients, while RMT resulted in a similar improvement in only 1 of 10 patients (P = 0.012). Similarly a gain of attachment of greater than or equal to 3 mm was found in at least 1 SRD pocket in 8 of 10 patients. The RMT resulted in a similar improvement in only 2 patients (P = 0.012).(ABSTRACT TRUNCATED AT 250 WORDS)

HLA A9 and B15 are associated with the generalized form, but not the localized form, of early-onset periodontal diseases.

HLA proteins are genetically determined, and account in part for individual immune response. Several studies have been performed seeking an association between HLA antigens and various forms of periodontitis with no conclusive results. The aim of the present study was to determine the frequency of HLA antigens of patients suffering from the localized (LJP) and the generalized (SGP) forms of early-onset periodontitis (EOP). Twenty-six EOP patients from the same ethnic group were studied in comparison to 113 race-matched controls. The EOP group included 11 LJP and 15 SGP patients. HLA-A9 and B15 antigens were found to be significantly elevated in the patient group. These differences were found to be due to the high frequency of A9 and B15 antigens in the SGP patients, with the LJP patient group showing no significant difference from the control group. The results are in agreement with previous studies in which A9 and B15 were found to be associated with EOP. However, previous studies did not differentiate between the localized and the generalized form of EOP. These results support the hypothesis that the generalized and the localized forms of EOP are under different genetic control.

A new degradable controlled release device for treatment of periodontal disease: in vitro release study.

The substantivity of a drug in the periodontal pocket is an important factor determining its effect on the subgingival flora. Therefore, one of the predominant factors in the development of a sustained release delivery device is the ability to control the rate of release of the drug. Previous studies have demonstrated the advantages of the local sustained release of chlorhexidine from nondegradable devices in the treatment of periodontal diseases. The aim of this study was to develop a degradable sustained release device composed of a cross-linked protein containing chlorhexidine as the therapeutic agent. The in vitro release profile of chlorhexidine from the degradable films was altered by the amount of chlorhexidine incorporated into the film, by the cross-link density of the polymer, and by the chlorhexidine salt used. The chlorhexidine in the final pharmaceutical preparation did not lose its antibacterial activity as was shown in an in vitro antibacterial test. This work demonstrates that the release of chlorhexidine from a degradable delivery system and the degradation of the matrix can be controlled by variation in the formulation. This presents a new dental drug delivery system that can be used as an adjunct in the treatment of periodontal diseases in the future. These studies enable us to choose the pharmaceutical formulations for clinical trials to be conducted testing the efficacy of this treatment modality.

The secretion of PGE2, IL-1 beta, IL-6, and TNF alpha by adherent mononuclear cells from early onset periodontitis patients.

The secretion of prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6) by adherent mononuclear cells (AMNC) from 28 patients with early-onset periodontitis was studied. The early onset-periodontitis patients consisted of 12 patients with localized juvenile periodontitis (LJP) and 16 patients with severe generalized periodontitis (SGP). The AMNC responses to different concentrations of lipopolysaccharide (LPS) (E. coli) were determined in these 28 patients and compared to 14 healthy controls. Mediator levels in the supernatant were measured using radioimmunoassays for PGE2, IL-1 beta, and IL-6 determination and an enzyme linked immunosorbent assay for TNF alpha levels. The mean age of the patients was 19.9 years for the LJP group, 30.4 years for SGP, and 28.0 years for the controls. The mean number of teeth per patient with attachment loss of > 6 mm was 4.75 in the LJP patients and 17.3 in the SGP group. In the absence of LPS, LJP AMNC secreted significantly more PGE2 than unstimulated control or SGP AMNC, while similar baseline amounts of IL-1 beta, IL-6, and TNF alpha were secreted by AMNC from the 3 patient groups. LPS stimulation resulted in the dose-dependent secretion of significantly higher levels of PGE2 by LJP AMNC compared to SGP AMNC which in turn secreted significantly more than controls. TNF alpha secretion by LJP monocytes was significantly greater than the SGP and the control groups while IL-1 beta secretion by the SGP AMNC was depressed compared to the other two patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Periodontitis associated with Papillon-Lefèvre syndrome.

The predominant subgingival microflora, host immune response, and genetic history of a 14-year-old girl with Papillon-Lefèvre Syndrome (PLS) are reported. The patient had high counts of Actinobacillus actinomycetemcomitans and surface translocating bacteria. She had significantly raised levels of antibodies to five of the bacterial species studied with the levels to A. actinomycetemcomitans remaining high after antibiotic therapy. The polymorphonuclear leukocytes (PMN) also released significantly increased amounts of O2 compared to controls. The data presented support a role for A. actinomycetemcomitans and PMN dysfunction in the pathogenesis of PLS.

Sequential manifestation of different forms of early-onset periodontitis. A case report.

Pathogenic bacteria constitute the primary extrinsic agent in the etiology of early onset periodontitis. However, the risk of developing periodontal disease is not equal for all individuals, suggesting host factors are involved in determining an individual's disease susceptibility. In this report, a case of an otherwise healthy female, who exhibited prepubertal periodontitis (PPP) at age 10, juvenile periodontitis (JP) at age 13, and rapidly progressive periodontitis (RPP) at age 29 years, is presented. Microbial, immunological, and genetic features of the case are presented. PPP, JP, and RPP are considered distinct disease entities, albeit with similar pathology and pathogenesis, yet all were manifest sequentially in the same individual. This report presents the idea that certain individuals are predisposed to early-onset periodontal diseases and the early identification of risk factors is important in the management of these individuals.

Whole genomic DNA probe for detection of Porphyromonas endodontalis.

The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled using the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphyromonas gingivalis, Porphyromonas asaccharolytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x 10(4) cells.

Dental caries and its determinants among recent immigrants from rural Ethiopia.

Adults (35-45 yr old) and children (5 and 12 yr old) recently arrived in Israel from rural areas in Ethiopia were examined. Caries levels were low: e.g. DMFT = 0.31 among 12-yr-olds, and 1.27 amongst adults. The 5-yr-olds were 86.8% caries-free, while 12-yr-olds were 81.8% and adults 54% caries-free respectively. According to interview data, the diet in Ethiopia had been based on local agricultural products and was almost sugar-free. The mean total count of salivary bacteria, as determined on blood agar, was 3.4 x 10(8); mean count of Streptococcus viridans, on mitis salivarius, was 6.7 x 10(7); and mean count of S. mutans, as determined on mitis salivarius with bacitracin, was 1.7 x 10(7). These levels were all high and were not significantly different from a control group of 20 Israelis. The mean number of lactobacilli, on Rogosa agar, was 2.75 x 10(4), which was significantly higher than among the controls (3.6 x 10(3). Salivary pH levels were generally similar between the Ethiopian group and the controls. Salivary flow was significantly higher for the Ethiopians (1.93 ml/min) than for controls (1.16 ml/min). Low levels of caries in this population can be attributed to an almost sugar-free diet and high salivary flow, but not to the composition of oral microflora.

Inflammatory cells and bacteria in pericoronal exudates from acute pericoronitis.

The present work is one in a series of studies carried out to verify the relationship between bacteria and gingival tissues in pericoronitis. Exudates from 6 cases of acute pericoronitis were examined by light and electron microscopy, including ultrathin sections and negative staining. While bacterial phagocytosis was prevalent in all the exudates studied, spirochetes, which were the predominant microorganisms, were not observed being phagocytized by PMNs or macrophages. The presence of spirochetes in pericoronitis as compared with acute necrotizing ulcerative gingivitis is discussed.

Bacterial colonization of the parotid duct in xerostomia.

Salivary flow rate and bacterial colonization of the parotid main duct were investigated in 17 patients referred for sialography and in 10 healthy controls. Cultures in the controls were negative whilst the patient group revealed bacterial colonization of the main duct, especially in those with the lowest flow rates. The implications of this finding in patients at high risk of endocarditis who will be undergoing sialography are discussed.