Cytotoxicity and proliferative effects of Iodoform-containing root canal-filling material on RAW 264.7 macrophage and RKO epithelial cell lines.

OBJECTIVE: The present study investigated the effect of the Iodoform-containing root canal filling material on the viability of cultured macrophages and epithelial cells, and on cytokine secretion. DESIGN: The effect of Endoflas F.S. on the proliferation of a RAW 264.7 macrophage cell line and on a RKO epithelial cell line, and on the production of tumour necrosis factor alpha (TNFα) from macrophages was examined. Cell vitality was evaluated using a colourimetric XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) assay. The presence of cytokines was determined by two-site enzyme-linked immunosorbent assay (ELISA). RESULTS: Direct exposure of Endoflas F.S. and its media, up to a dilution of 1/8, decreased the viability of macrophages and epithelial cells by ∼70% compared to control media (P<0.05). Media dilution from 1/16 to 1/1024 demonstrated a proliferative effect, increasing cell viability by about 60% compared to media without Iodoform-containing root canal filling material. CONCLUSIONS: Direct and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation.

The effect of surface processing of titanium implants on the behavior of human osteoblast-like Saos-2 cells.

BACKGROUND: The surface qualities of dental implants appear to modulate osteoblasts' growth and differentiation, affecting bone healing. During manufacturing of implants, the surface quality is affected by industrial processes. PURPOSE: To examine the effect of manufacturing procedures on the growth and differentiation of human osteoblast-like cells, Saos-2. MATERIALS AND METHODS: Saos-2 cells were cultured on titanium (Ti) disks. Cell growth was examined using the XTT assay, and cell differentiation was tested by alkaline phosphatase (ALP) activity and osteocalcin secretion. The following variables were examined: roughening of the surface by sandblasting and acid-etching, aging of the acid used for etching, fluoride modification of the surface, and the type of the packaging material. RESULTS: An inverse relationship was noted between Saos-2 growth and ALP activity on the tested surfaces. Roughening of the surface tended to decrease cell proliferation and to increase differentiation. Immersion of up to 200 cycles in acid decreased proliferation and increased differentiation. Cells grown on fluoride-modified surfaces exhibited more ALP activity as compared to the unmodified surfaces. No difference was noted between the three packaging materials tested. CONCLUSIONS: The data suggests that industrial processes may affect the behavior of osteoblast-like cells around titanium implants and should be monitored carefully by bioassays.

Strain-dependent activation of the mouse immune response is correlated with Porphyromonas gingivalis-induced experimental periodontitis.

AIMS: To evaluate the effect of oral infection with three Porphyromonas gingivalis strains on alveolar bone loss (ABL) and its correlation with the mouse immune response. MATERIALS AND METHODS: Mice were orally infected with P. gingivalis strains 381, 33277 and 53977. After 42 days, maxillae were analysed for ABL using micro-computed tomography and the serum for anti-P.gingivalis IgG1 and IgG2a levels. The cytokine response to P. gingivalis was tested using the subcutaneous chamber model. RESULTS: The P. gingivalis 53977-infected group showed the highest ABL, which was significantly different from all other groups (p<0.001). In addition, the humoral response to P. gingivalis 53977 was significantly lower than the response to P. gingivalis 381 and 33277 (p< or =0.01). The IgG2a/IgG1 ratio was higher in the P. gingivalis 33277-infected group (1.6) compared with the P. gingivalis 381-infected group (0.51). Four days post-infection, interleukin (IL)-1beta levels remained significantly higher in the P. gingivalis 53977-infected group only (1198.2+/-260.0, p<0.05), while IL-4 levels remained significantly higher in the P. gingivalis 381-infected group (265.8+/-131.6, p<0.05). CONCLUSIONS: The high levels of ABL induced by P. gingivalis 53977 were inversely correlated with the humoral response to this bacterium. In addition, ABL was correlated with an elevated pro-inflammatory response.

Effect of a niobium-containing titanium alloy on osteoblast behavior in culture.

BACKGROUND: The chemistry of titanium is a key factor in determining implant-tissue interactions. Reports that a vanadium-based titanium alloy (Ti-6Al-4V) exhibits some cytotoxicity led to a search for an alternative. AIM: The aim of the present study was to investigate the behavior of human osteoblast-like cells (Saos-2) cultured on Ti-6Al-4V or Ti-6Al-7Nb disks with a rough or a machined surface. RESULTS: In all four groups, the cells proliferated rapidly between days 1 and 3, and then plateaued. On day 1 of culture, the highest proliferation rate was of cells cultured on disks containing Nb with a machined surface. On day 7, there was no significant difference in cell density on all the tested surfaces. Alkaline phosphatase (ALP) activity was lower on the machined surfaces, regardless of the material used, suggesting that cells on rough surfaces exhibit a more mature phenotype. On day 3, cells cultured on rough disks made of Ti-6Al-7Nb showed the highest ALP activity; the lowest activity was observed on the machined Ti-6Al-4V surface. The highest level of osteocalcin (day 7) was found in the cells cultured on rough Ti-6Al-7Nb disks. Also, higher levels of transforming growth factor (TGFbeta) were noted for cells cultured on the rough Ti-6Al-7Nb disks, suggesting that the Nb-containing alloy supports more rapid maturation of the osteoblast. CONCLUSIONS: The results of the present study suggest that according to our cell culture preclinical model, Ti-6Al-7Nb may replace the Ti-6Al-4V alloy as an implant material.

Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response.

AIM: To compare the effect of oral infection with Porphyromonas gingivalis or Fusobacterium nucleatum versus infection with both bacteria on mouse periodontal tissues, and to characterize the inflammatory response. MATERIALS AND METHODS: Mice were orally infected with P. gingivalis, F. nucleatum or both. At 42 days post-infection, alveolar bone loss was quantified using micro-computerized tomography. Tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta levels induced by the infection were quantified using the subcutaneous chamber model. RESULTS: Mice orally infected with F. nucleatum/P. gingivalis exhibited significantly more bone loss compared with that of mono-infected and sham-infected mice. F. nucleatum/P. gingivalis infection also increased the levels of TNF-alpha and IL1beta compared with the levels found in the mono-infected groups. CONCLUSIONS: Polymicrobial infection with P. gingivalis/F. nucleatum aggravates alveolar bone loss and induces a stronger inflammatory response compared with that observed upon infection with either bacterium alone. The results suggest that oral infection of mice with a mixture of P. gingivalis and F. nucleatum may be superior to mono-infection models of experimental periodontitis.

Behavior of two osteoblast-like cell lines cultured on machined or rough titanium surfaces.

BACKGROUND: Two osteosarcoma-derived cell lines have been extensively used to investigate the biological events occurring on titanium surfaces: MG63 and Saos-2. However, the behavior of the two lines on different titanium surfaces has never been compared. AIM: The aim of the present study was to compare the behavior of MG63 and Saos-2 cells on two different titanium surfaces, machined and rough (sandblasting and acid-etched). We compared cell proliferation and morphology, alkaline phosphatase (ALP) activity and secretion of osteocalcin (OC). RESULTS: The most pronounced difference between the two cell lines was that ALP activity in the Saos-2 cells was 10-fold higher than in the MG63 cells. The proliferation rate of the MG63 cells was much higher than that of the Saos-2 cells at all the tested cell concentrations. MG-63 cells, but not Saos-2 cells, grown on rough surface titanium proliferated more rapidly than cells grown on machined surfaces. Morphological analysis revealed that Saos-2 cells and cells grown on the rougher surface, displayed a more mature phenotype. The level of OC secreted by the Saos-2 cells, but not the MG63 cells, were higher on the rough surface than on the machined surface. CONCLUSIONS: This study shows that Saos-2 cells exhibit a more mature osteoblast phenotype, compared with that of MG63 cells, rendering them a good candidate for an in vitro model of osseointegration.

Inflammatory response to chlorhexidine, minocycline HCl and doxycycline HCl in an in vivo mouse model.

AIM: To examine the effect of locally delivered antimicrobial drugs on the inflammatory response in an in vivo mouse chamber model. MATERIAL AND METHODS: Two weeks following chamber implantation, 24 BALB/c mice, in the experimental group, were given an intra-chamber challenge of heat-killed Porphyromonas gingivalis, followed immediately by injection of the specific antimicrobial drug: 2000 microg/ml chlorhexidine (CHX); 1500 microg/ml minocycline HCl;and 1500 microg/ml doxycycline HCl (concentrations achieved in the periodontal pocket with commercial controlled-release delivery systems). A second group of 24 animals received only the antimicrobial treatment without P. gingivalis challenge. Intra-chamber exudates were sampled at 2 and 24 h following the challenge, and leucocytes, TNFalpha, IFNgamma and IL-10 were evaluated. RESULTS: At 2 h, minocycline HCl induced high levels of IL-10, TNFalpha and IFNgamma, while CHX reduced the levels of TNFalpha and IFNgamma. By 24 h, these responses were attenuated. Following bacterial challenge, the antibacterial agents attenuated the inflammatory process, each in its own fashion. CONCLUSIONS: Antibacterial agents applied locally have the ability to induce an inflammatory response. They also modify the inflammatory response to P. gingivalis independent of their antimicrobial effect. CHX and doxycycline HCl appear to have the most marked anti-inflammatory effect.

TCR zeta down-regulation under chronic inflammation is mediated by myeloid suppressor cells differentially distributed between various lymphatic organs.

T cell AgR zeta chain down-regulation associated with T cell dysfunction has been described in cancer, infectious, and autoimmune diseases. We have previously shown that chronic inflammation is mandatory for the induction of an immunosuppressive environment leading to this phenomenon. To identify the key immunosuppressive components, we used an in vivo mouse model exhibiting chronic inflammation-induced immunosuppression. Herein, we demonstrate that: 1) under chronic inflammation secondary lymphatic organs display various immunological milieus; zeta chain down-regulation and T cell dysfunction are induced in the spleen, peripheral blood, and bone marrow, but not in lymph nodes, correlating with elevated levels of Gr1(+)Mac-1(+) myeloid suppressor cells (MSC); 2) MSC are responsible for the induction of such an immunosuppression under both normal and inflammatory conditions; and 3) normal T cells administered into mice exhibiting an immunosuppressive environment down-regulate their zeta expression. Such an environment is anticipated to limit the success of immunotherapeutic strategies based on vaccination and T cell transfer, which are currently under investigation for immunotherapy of cancer.

The effect of immunization on the response to P. gingivalis infection in mice is adjuvant-dependent.

AIM: Studies on vaccines against the periodontal pathogen Porphyromonas gingivalis have produced conflicting results, but no consideration has been given to the role of different adjuvants in these vaccines. We have previously shown that an intra-chamber challenge with heat-killed P. gingivalis was modified by immunization with different adjuvants. This study tested the hypothesis that different adjuvants in P. gingivalis vaccines would differentially modify the host response to a live P. gingivalis infection. RESULTS: Using P. gingivalis-infected subcutaneous chambers in mice, we show that vaccination with P. gingivalis in alum attenuated the pro-inflammatory cytokine levels at the site of infection, while the vaccine containing incomplete Freund's adjuvant did the opposite. Although both vaccines induced a similar humoral IgG response, P. gingivalis-induced abscesses were significantly smaller in the alum-adjuvant group. CONCLUSIONS: The results suggest that the immune response and the resultant protection to a P. gingivalis infection, in P. gingivalis-vaccinated mice, are adjuvant-dependent.

Reduced expression of gamma interferon in serum and marked lymphoid depletion induced by Porphyromonas gingivalis increase murine morbidity and mortality due to cytomegalovirus infection.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent of severe forms of periodontal disease. Although periodontal disease is considered a localized disease, accumulating evidence indicates that it may lead to a predisposition to a decline in immunocompetence. Human cytomegalovirus (CMV) commonly infects all human populations without producing significant clinical symptoms. Immunocompromised patients usually develop a primary or reactivated CMV infection, which is associated with high rates of morbidity and mortality. The aim of this study was to determine whether P. gingivalis increases animal susceptibility to CMV infection. Mice were inoculated with CMV and infected locally with P. gingivalis 3 days after the virus inoculation. Mortality rates were monitored, and traces of viral DNA and bacterial infection were detected systemically by using real-time PCR. Local and systemic cytokine secretion was measured, and histological sections were used to assess the pathological state of infected organs. P. gingivalis- and CMV-coinfected mice showed dramatically higher mortality rates than mice infected with P. gingivalis or CMV only. Although the organs of coinfected mice exhibited decreased viral titers, distinct necrosis and tissue damage were more evident in the livers and spleens of these mice than in those of mice infected with CMV only. Furthermore, systemic gamma interferon levels were decreased in coinfected mice, and marked lymphoid depletion was observed in their necrotic organs. In parallel control Escherichia coli-CMV coinfection experiments, the mortality and pathological results were the same as those found in mice infected with CMV only. Our results suggest a specific influence of P. gingivalis on the mouse immune response, causing increased susceptibility to CMV infection.

Sustained exposure to bacterial antigen induces interferon-gamma-dependent T cell receptor zeta down-regulation and impaired T cell function.

T cell antigen receptor zeta chain down-regulation and impaired in vitro T cell function have been described in cancer and autoimmune and infectious diseases. However, the immunological basis for this phenomenon is unknown. Sustained exposure to antigen and chronic systemic inflammation, factors shared by the various pathologies, might account for this phenomenon. We developed an in vivo experimental system that mimics these conditions and show that sustained exposure of mice to bacterial antigens was sufficient to induce T cell antigen receptor zeta chain down-regulation and impair T cell function, provided an interferon-gamma-dependent T helper type 1 immune response developed. This indicates zeta chain down-regulation could be a physiological response that attenuates an exacerbated immune response. However, it can act as a 'double-edged sword', impairing immune responses to chronic diseases.