Evaluation of the tissue response to MTA and MBPC: Microscopic analysis of implants in alveolar bone of rats

The aim of this study was to evaluate and compare the quantitative and qualitative inflammatory responses and bone formation potential after implantation of polyethylene tubes filled with a new calcium hydroxide containing sealer (MBPc) and Prolloot mineral trioxide aggregate (MIA). There were 48 Wistar rats divided in three groups: Group I (control group) empty polyethylene tubes were implanted in the extraction site; group II and III, polyethylene tubes were implanted filled with ProRoot mineral trioxide aggregate (MIA) and MBPc, respectively. At 7, 15, and 30 days after tube implantation, the animals were killed, the hemi-maxillas were removed and prepared to light microscopic analyses. The scores obtained were submitted to Kruskal-Wallis statistical test (p < 0.05). Significant differences between the materials were not observed. The results showed that both materials had similar biological response.

Alendronate therapy in cyclosporine-induced alveolar bone loss in rats

Background and Objective: Cyclosporine A is an immunosuppressive drug that is widely used in organ transplant patients as well as to treat a number of autoimmune conditions. Bone loss is reported as a significant side-effect of cyclosporine A use because this can result in serious morbidity of the patients. As we have shown that cyclosporine A-associated bone loss can also affect the alveolar bone, the purpose of this study was to evaluate the effect of the concomitant administration of alendronate on alveolar bone loss in a rat model.Material and Methods: Forty Wistar rats (10 per group) were given cyclosporine A (10 mg/kg, daily), alendronate (0.3 mg/kg, weekly), or both cyclosporine A and alendronate, for 60 d. The control group received daily injections of sterile saline. The expression of proteins associated with bone turnover, including osteocalcin, alkaline phosphatase and tartrate-resistant acid phosphatase (TRAP), and also the calcium levels, were evaluated in the serum. Analysis of the bone volume, alveolar bone surface, the number of osteoblasts per bone surface and the number of osteoclasts per bone surface around the lower first molars was also performed.Results: the results indicate that cyclosporine A treatment was associated with bone resorption, represented by a decrease in the bone volume, alveolar bone surface and the number of osteoblasts per bone surface and by an increase in the number of osteoclasts per bone surface and TRAP-5b. These effects were effectively counteracted by concomitant alendronate administration.Conclusion: It is concluded that concomitant administration of alendronate can prevent cyclosporine A-associated alveolar bone loss.

Inhibition of hydrogen peroxide, nitric oxide and TNF-alpha production in peritoneal macrophages by ethyl acetate fraction from Alchornea glandulosa

The effects of Alchornea glandulosa ethyl acetate fraction (AGF) on hydrogen peroxide (H2O2), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages activated with lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) were investigated. Analysis by thin layer chromatography (TLC) of AGF showed several constituents, including flavonoids, which may have anti-inflammatory activity. Inhibitory effects of the fraction in H2O2 and NO production ranged from 8.59 +/- 7.84% to 70.56 +/- 4.16% and from 16.06 +/- 3.65% to 38.73 +/- 3.90%, respectively. The TNF-alpha production was only partially inhibited in the tested concentrations (12.21 +/- 6.23%-15.16 +/- 0.96%). According to these results, it is suggested that AGF has anti-inflammatory activity. This medicinal plant may have therapeutic potential in the control of inflammatory disorders.

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures - also stained by hematoxylin - were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images - round/ovoid bodies with dense crescent-like chromatin - on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis. (C) 2001 Harcourt Publishers Ltd.

Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro

As demonstrated previously in our laboratory, B-1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage-like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B-1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B-1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild-type mice. Moreover, macrophages from wild-type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1 cells down-regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B-1 cells are one of the main sources of interleukin (IL)-10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL-10 production by cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B-1 cells can influence the effector functions of macrophages in vitro via IL-10 secretion.

EARLY ACTIVATION OF SPLENIC MACROPHAGES BY TUMOR-NECROSIS-FACTOR-ALPHA IS IMPORTANT IN DETERMINING THE OUTCOME OF EXPERIMENTAL HISTOPLASMOSIS IN MICE

Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally. The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells. Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice. Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection. Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion. It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.

EFFECT OF A PERSISTENT INFLAMMATORY PROCESS ON EXPERIMENTAL HETEROTOPIC OSSIFICATION - THE INFLUENCE OF MACROPHAGES

1. We investigated the effect of a persistent carrageenin- or nystatin-induced inflammatory reaction on heterotopic ossification produced by the subcutaneous implant of a demineralized bone matrix in female Swiss mice (25 to 35 g).2. Subcutaneous carrageenin injection (0.3 ml of a 2% solution in saline) into mice induced an inflammatory reaction characterized by a mature granuloma predominantly of macrophages containing particles of the irritant in their cytoplasm and which remained unchanged until the end of the experiment (40th day).3. Subcutaneous nystatin inoculation (30,000 IU in 0.3 ml saline) induced an inflammatory reaction consisting initially of macrophages (4th day) but later turning into an epithelioid granuloma (7th day) consisting predominantly of epithelioid cells and which was present up to the 2 lst day when it was gradually replaced by adipocytes up to the 30th day.4. An intramuscular implant of demineralized bone matrix (DBM, approximately 10 mg) induced the formation of cartilage and bone tissue and of hemopoietic bone marrow (heterotopic ossification) in 100% of the control animals (N = 5). An intramuscular DBM implant in animals that received carrageenin (N = 19) or nystatin (N = 21) induced heterotopic ossification in 100 and 57% (P<0.01)) of the animals, respectively.5. The response to a dorsal subcutaneous DBM implant was essentially negative in control animals (N = 5), whereas implants performed near the site injected with carrageenin (N = 28) or nystatin (N = 31) produced a response in 71 (P <0.01) and 36 % (P<0.01) of the animals, respectively. A DBM implant into the contralateral (control) dorsal subcutaneous tissue of the same animals that received carrageenin (N = 25) or nystatin (N = 29) resulted in heterotopic ossification in 64 (P<0.01) and 7% of the animals, respectively.6. The results suggest that the macrophages present in the mature granuloma induced by carrageenin somehow favored the development of metaplastic plates after subcutaneous DBM implant and that this effect may be systemic since the same response was observed in contralateral subcutaneous tissue.

SUPPORT OF PARACOCCIDIOIDES-BRASILIENSIS MULTIPLICATION BY HUMAN MONOCYTES OR MACROPHAGES - INHIBITION BY ACTIVATED PHAGOCYTES

The interaction of human monocytes or monocyte-derived macrophages and yeast-form Paracoccidioides brasiliensis was studied in vitro. Yeast cells were readily ingested by adherent monocytes or macrophages. Multiplication of P. brasiliensis, measured by growth as colony forming units (cfu) on a supplemented medium with good plating efficiency, was greater in monocyte co-cultures compared to the number of cfu obtained from complete tissue-culture medium (CTCM). Multiplication increased with time in macrophage cocultures, e.g., from two-six-fold in 24 h to nine-fold in 72 h. Microscopic observations indicated that ingested yeast cells multiplied inside macrophages. When monocytes were treated with supernate cytokines (CK) from concanavalin-A-stimulated mononuclear cells, then co-cultured with P. brasiliensis, multiplication was significantly inhibited compared with control monocyte co-cultures. Treatment of macrophages-derived from monocytes by culture in vitro for 3 days-for a further 3 days with CK resulted in maximal inhibition of multiplication over the subsequent 72 h. Similarly, when monocyte-derived macrophages (after culture for 7 days) were treated for 3 days with recombinant human gamma-interferon (IFN; 300 U/ml) or CK they restricted multiplication of P. brasiliensis by 65% and 95%, respectively, compared with control macrophages, Antibody to IFN abrogated the effect of IFN or CK treatment. These findings show that ingested P. brasiliensis can multiply in human monocytes or macrophages and that this multiplication can be restricted by activated monocytes or macrophages.

Macrophages and mast cells control the neutrophil migration induced by dentin proteins

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), the major dentin proteins, have been shown to induce neutrophil migration through release of IL-1beta, TNF-alpha, MIP-2, and KC. However, the sources of these mediators were not determined. Here, the roles of macrophages and mast cells (MC) in dentin-induced neutrophil accumulation were investigated. Peritoneal MC depletion or the enhancement of macrophage population increased DSP- and DPP-induced neutrophil extravasation. Moreover, supernatants from DSP- and DPP-stimulated macrophages caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages was inhibited by dexamethasone or the supernatant of DSP- treated MC. Consistently, dexamethasone and the MC supernatant inhibited the production of IL-1beta, TNF-alpha, and MIP-2 by macrophages. This inhibitory activity of the DSP- stimulated MC was neutralized by anti-IL-4 and anti-IL-10 antibodies. These results indicate that dentin induces the release of the neutrophil chemotactic substance(s) by macrophages, which are down-modulated by MC-derived IL-4 and IL-10.

Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats

Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/ lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in viva. A suitable in viva model for studying these events is the alveolar bone of young rats because it is continuously. Thus, sections of aldehyde fixed alveolar undergoing intense resorption/remodeling bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.

Ultrastructural and histochemical examination of alveolar bone at the pressure areas of rat molars submitted to continuous orthodontic force

It is usually believed that repair in alveolar bone during orthodontic movement occurs after decreasing of force. However, we have recently observed signs of repair in previously resorbed cementum from human teeth exposed to continuous forces. In order to test the hypothesis that bone resorption and deposition occur concomitantly at the pressure areas, a continuous 15 cN force was applied in a buccal direction to upper first molars from eight 2.5-month-old male Wistar rats for 3 d (n=4) and 7 d (n=4). As a control, two additional rats did not have their molars moved. Maxillae were fixed in 2% glutaraldehyde + 2.5% formaldehyde, under microwave irradiation, decalcified in ethylenediaminetetraacetic acid, and processed for transmission electron microscopy. Specimens from one rat from each group were processed for tartrate-resistant acid phosphatase (TRAP) histochemistry. At both the times studied, the alveolar bone surface at the pressure areas showed numerous TRAP-positive osteoclasts, which were apposed to resorption lacunae. In addition, osteoblasts with numerous synthesis organelles were present in the neighboring areas overlying an organic matrix. Thus, this study provides evidence that the application of continuous forces produces concomitant bone resorption and formation at the pressure areas in rat molars.