Morphological features of cartilage observed during mandibular distraction in rabbits.

Ossification during distraction osteogenesis can be classified as intramembranous or endochondral. It is not known whether cartilage in the distraction gap is transformed into new bone. The aim of this study was to investigate the morphological features of ossification in the transition of cartilage to bone during mandibular distraction osteogenesis in a rabbit model. A cortical osteotomy was performed and custom-made devices were applied. Immediately after surgery, the devices were lengthened by 0.25 mm every 12h for up 10 days, during which time four rabbits were killed at 0, 5 and 10 days and examined using histological staining and immunohistochemical methods. Apoptotic cells were identified by an in-situ detection assay for nuclear DNA fragmentation using a modified TUNEL procedure, with several sections analyzed using software for histomorphometric analysis. The results showed that the amount of cartilage in the distraction gap was significantly decreased. The cartilage had ossified in two ways, termed endochondral ossification and transchondroid bone formation.

SC-19220, a prostaglandin E2 antagonist, inhibits osteoclast formation by 1,25-dihydroxyvitamin D3 in cell cultures.

1,25 Dihydroxy vitamin D3 (1,25(OH)2D3), prostaglandin (PG) E2 and parathyroid hormone (PTH) induce osteoclast formation in cell cultures. Previously, we have shown that SC-19220, an antagonist of the EP1 subtype of PGE receptors, inhibited tartrate-resistant acid phosphatase (TRAP)-positive cell formation by PGE2 and PTH in adherent cell cultures taken from neonatal rats. Since 1,25(OH)2D3 has been shown to induce osteoclast formation through PGE2 synthesis, in this study we have examined the effect of SC-19220 on osteoclast formation induced by 1,25(OH)2D3 in cell cultures by measuring bone resorption as well as TRAP-positive cell formation. SC-19220 inhibited osteoclast formation by 1,25(OH)2D3 as well as by PGE2 in cell cultures. The addition of SC-19220 to the later half but not to the earlier half of the culture inhibited 1,25(OH)2D3-induced formation. In the culture in which hydroxyurea was added in the later half period, SC-19220 inhibited osteoclast formation by 1, 25(OH)2D3. Under these conditions, 17-phenyl PGE1, an EP1 agonist, induced osteoclast formation. Thus, SC-19220 inhibits certain reactions in the later processes of osteoclast formation induced by 1,25(OH)2D3. In addition, SC-19220 also inhibited osteoclast formation induced by interleukin (IL)-11 and IL-6 as well as by PTH. It is suggested that the SC-19220 inhibiting reactions are shared by all the inducers including 1,25(OH)2D3 and are essential for osteoclast formation.

Microbicidal efficacy of ozonated water against Candida albicans adhering to acrylic denture plates.

BACKGROUND/AIMS: Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. We examined the effect of ozonated water on Candida albicans on acrylic denture plate. METHODS: The heat-cured acrylic resins were cultured with C. albicans. After treatment of flowing ozonated water, the number of attached C. albicans was counted. In some experiments, the test samples were treated with ozonated water in combination with ultrasonication. RESULTS: After exposure to flowing ozonated water (2 or 4 mg/l) for 1 min, viable C. albicans cells were nearly nonexistent. The combination of ozonated water and ultrasonication had a strong effect on the viability of C. albicans adhering to the acrylic resin plates. There were no significant differences in antimicrobial activity against C. albicans between plates immersed in ozonated water with ultrasonication and those treated with commercially available denture cleaners. In addition, electron microscopic analysis revealed that small amounts of C. albicans remained on the plate after exposure to flowing ozonated water or immersion in ozonated water with ultrasonication. CONCLUSION: Our results suggest that application of ozonated water may be useful in reducing the number of C. albicans on denture plates.

Tonsillar application of killed Streptococcus mutans induces specific antibodies in rabbit saliva and blood plasma without inducing a cross-reacting antibody to human cardiac muscle.

When Streptococcus mutans cells are injected into the skeletal muscle of rabbits, an antibody against human cardiac muscle, as well as an anti-S. mutans antibody, is induced in blood plasma. Our previous study showed that when sheep erythrocytes are applied to palatine tonsils, an antibody against the applied cells is induced both in blood plasma and saliva. This antibody has no activity against cardiac muscle. It is not clear, however, if S. mutans application to the tonsils evokes an antibody response against cardiac muscle. In this study, we immunized rabbits against S. mutans or Streptococcus sobrinus by tonsillar application or by intramuscular injection every 3 days for 6 weeks. Tonsillar applications of formalin-killed cells of S. mutans induced saliva immunoglobulin A (IgA) and blood plasma IgG to the applied cells. In contrast, intramuscular injection of such cells induced only blood plasma IgG. When the route of immunization was intramuscular injection, antibodies in blood plasma cross-reacted with cardiac muscle. By enzyme-immunohistochemistry and Ouchterlony immunodiffusion tests, no cross-reaction to cardiac muscle was observed with the antibody in saliva or in blood plasma after the tonsillar applications. Western blotting of the S. mutans antigen showed that blood plasma from rabbits injected with S. mutans reacted with antigens of 46, 52, 62, and 85 kDa, while that from rabbits subjected to tonsillar application of S. mutans did not react with these bands. Similar results were obtained for S. sobrinus applications. Thus, tonsillar applications of mutants group streptococci induce antibodies differing in antigen specificity and do not induce any cross-reacting antibody to cardiac muscle.

Streptococcus sobrinus antigens that react to salivary antibodies induced by tonsillar application of formalin-killed S. sobrinus in rabbits.

We previously found that tonsillar application of antigen induces a strong antibody response to Streptococcus sobrinus in saliva and blood plasma. Rabbits immunized against S. sobrinus by tonsillar application were highly resistant to experimental dental caries triggered by oral inoculation of living S. sobrinus organisms with sucrose. In the present study, we examined the reaction of S. sobrinus antigens to the antibodies induced by the tonsillar application of S. sobrinus AHT-k in rabbits and compared them to those antibodies induced by intramuscular injection. In an enzyme-linked immunosorbent assay using ultrasonic fragments from mutans group streptococci, the saliva and blood plasma selectively reacted to S. sobrinus AHT-k (serotype g) and serologically related streptococci (serotypes a, d, and h) in the sixth week after tonsillar application, whereas the blood plasma in the sixth week after intramuscular injection reacted to the unrelated streptococci (serotypes b, c, e, and f) in addition to the aforementioned streptococci. The antibody reactivity induced after tonsillar application was not lost after treatment of the antigen with heat or proteinase digestion, whereas these treatments resulted in a 70% decrease of the antibody reactivity induced by intramuscular injection. The inhibition by haptenic sugars and the decrease in immunoreactivity by heat treatment and proteinase digestion suggested that 80% of the antibodies induced by tonsillar application reacted to saccharides. These saccharide antigens appeared to be involved in a specific reaction with S. sobrinus-specific streptococci and a selective reaction with serologically related streptococci. These antigens are probably involved in anticaries reactions in experimental dental caries.

Tonsillar application of formalin-killed cells of Streptococcus sobrinus reduces experimental dental caries in rabbits.

Living Streptococcus sobrinus cells were orally inoculated into nonimmune rabbits and rabbits immunized with formalin-killed cells of S. sobrinus through tonsillar application to examine the anticaries potential of this method of immunization. The living S. sobrinus cell numbers and the caries areas in the rabbits immunized by tonsillar application decreased to a level one-fifth of that in nonimmune rabbits.

Simultaneous induction of specific immunoglobulin A--producing cells in major and minor salivary glands after tonsillar application of antigen in rabbits.

The immunoglobulin A (IgA)-producing cells in the stroma of major salivary glands are induced by antigenic stimulation of the mucosal immune system. Whether such cells also are induced in minor salivary glands by this stimulation remains to be determined. After application of sheep red blood cells to the palatine tonsils every 3 days for 6 weeks, anti-sheep red blood cell IgA was detected in saliva both by agglutination tests and by enzyme-linked immunosorbent assay. Using enzyme-linked immunospot assay, an increase in the number of anti-sheep red blood cell IgA-producing cells was found in minor as well as in major salivary glands of the sixth week of application; such cells constituted 4.9% to 5.9% of the total number of IgA-producing cells in these tissues. Tonsillar application of whole cells of formalin-killed Streptococcus sobrinus induced anti-S. sobrinus IgA in saliva. The number of anti-S. sobrinus IgA-producing cells in the above glands simultaneously increased over 6 weeks, and reached 5.2-5.6% of the total number of IgA-producing cells.

Evaluation of interleukin 1 as a mucosal adjuvant in immunization with Streptococcus sobrinus cells by tonsillar application in rabbits.

To evaluate interleukin 1 (IL-1) as a mucosal adjuvant in the induction of salivary antibodies to Streptococcus sobrinus, S. sobrinus together with IL-1 was applied through the palatine tonsils of rabbits. IL-1 caused approximately 50 and 100% increases in the antibodies reacting against S. sobrinus fragments in the saliva and blood plasma, respectively, compared to the antibodies in those same fluids after tonsillar applications of S. sobrinus alone. In the case of the addition of IL-1, the antibodies reacting to the protein antigens of S. sobrinus increased in each fluid, without affecting the antibodies reacting to saccharide antigens. Delayed-type hypersensitivity to S. sobrinus, characterized by ear swelling and by an increase in IFN-gamma mRNA in RT-PCR analysis, was found to be induced only in rabbits immunized with IL-1. S. sobrinus protein antigens caused ear swelling as intense as that caused by S. sobrinus fragments. Thus, IL-1 induced an antibody response and cell-mediated immunity mainly reacting to protein antigens of S. sobrinus.

Efficacy of ozone on survival and permeability of oral microorganisms.

In the present study, we examined the effect of ozonated water on oral microorganisms and dental plaque. Almost no microorganisms were detected after being treated with ozonated water (4 mg/l) for 10 s. To estimate the ozonated water-treated Streptococcus mutans, bacterial cells were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Fluorescence microscopic analysis revealed that S. mutans cells were killed instantaneously in ozonated water. Some breakage of ozonated water-treated S. mutans was found by electron microscopy. When the experimental dental plaque was exposed to ozonated water, the number of viable S. mutans remarkably decreased. Ozonated water strongly inhibited the accumulation of experimental dental plaque in vitro. After the dental plaque samples from human subjects were exposed to ozonated water in vitro, almost no viable bacterial cells were detected. These results suggest that ozonated water should be useful in reducing the infections caused by oral microorganisms in dental plaque.

Sheep red blood cell instillation at palatine tonsil effectively induces specific IgA class antibody in saliva in rabbits.

We have shown that the palatine tonsil effectively incorporates exogenous foreign substances instilled at its surface. It is not clear whether antigen-specific IgA can be induced by the instillation. Sheep red blood cells (SRBC) were instilled at the palatine tonsil every three days as the antigen, and the agglutination titer of specific IgA in saliva was examined. Nasal or intragastric administration, which have been shown to induce specific antibody in saliva, were done as control experiments. Anti-SRBC antibody in saliva from the tonsillar instillation group was detected in the second week, and the agglutination titer reached a maximum in the 6th week after instillation. The maximum titers in the tonsillar instillation group and nasal administration group were 16 (P < 0.01, n = 7) and 4 times (P < 0.01, n = 7) higher, respectively, than that in the intragastric administration group. In the tonsillar instillation group, the number of specific antibody- producing cells per 10(5) lymphocytes was the highest in the parotid glands compared with the lymphoid tissues such as the retropharyngeal lymph nodes, nasal mucosa, mesenteric lymph nodes, Peyer's patches, cervical lymph nodes, palatine tonsil and spleen. In the nasal administration group, the number of lymphocytes was the highest in the nasal mucosa. The results indicate that tonsillar instillation was more effective than nasal administration in inducing specific iIgA in saliva.

Preferential degradation of osteoclasts by titanium tetrachloride.

Although titanium alloys are known to be biocompatible with bone tissue after implantation in human beings, the effect of titanium on osteoclasts remains to be studied. We examined the effect of titanium salt on the formation and survival of osteoclasts in cell culture. The addition of 10 microM titanium tetrachloride caused a decrease in the cell number of osteoclast-like cells induced in bone marrow cell cultures taken from mice. The addition of 10 microM titanium tetrachloride caused degradation of the disaggregated osteoclasts taken from neonatal rats and a decrease in bone resorption. Along with the increase in the degradation of osteoclasts, the number of apoptotic cells increased. Titanium tetrachloride dose-dependently decreased the cell number and alkaline phosphatase activity of osteoblastic cell cultures taken from rat calvaria. However, these concentrations were 30-40 times higher than those in the case of osteoclast-like cell formation. These results showed that titanium ions caused a preferential degradation of osteoclasts rather than osteoblasts, most likely by apoptosis.

Detection and rapid increase of salivary antibodies to Staphylococcus lentus, an indigenous bacterium in rabbit saliva, through a single tonsillar application of bacterial cells.

In rabbits, Staphylococcus lentus is one of the major bacteria in saliva and a minor bacteria in jejunum fluids and nasal wash. The presence and induction of naturally occurring antibodies reacting to rabbit indigenous bacteria were studied. In non-immune rabbits, the proportion of anti-S. lentus IgA antibodies to total IgA in the saliva was several times higher than that in the intestinal fluids and the nasal wash. The salivary antibodies were found to have increased 1 week after a single tonsillar application of isolated S. lentus cells but not after a single nasal application or a single intragastric instillation. In addition, the anti-S. lentus antibodies in the saliva highly increased with weekly tonsillar applications of isolated S. lentus but increased only one-fifth with weekly nasal applications of the same cells. These results strongly suggest that the palatine tonsils, which we believe had already been sensitized by S. lentus in the physiological condition, induced naturally occurring antibodies in rabbit saliva.