RESUMEN
BACKGROUND AND OBJECTIVE: The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal tissues and may contribute to periodontitis progression. TNF-α stimulates formation and activity of osteoclasts, the cells that are recruited in periodontitis, that cause alveolar bone degradation and subsequent tooth loss. We previously showed that TNF-α is elevated in co-cultures of periodontal ligament fibroblast (PDLF) and peripheral blood mononuclear cells (PBMC). Hence, TNF-α could be a determining factor in osteoclast formation in these cultures, as osteoclasts are formed despite the fact that prototypical osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand is outnumbered at least 100-fold by its inhibitor osteoprotegerin in these cultures. MATERIAL AND METHODS: To assess the role of TNF-α in periodontitis-associated osteoclast formation in vitro, osteoclast formation was analyzed in the presence of the anti-TNF-α therapeutic agent infliximab in two culture systems: (i) PBMC in co-culture with PDLFs from controls and patients with periodontitis, or (ii) with PBMC only. PDLFs from control and patients with periodontitis were exposed to infliximab, PBMCs were added and the formation of osteoclast-like cells was assessed. RESULTS: TNF-α was highest levels in supernatants at 7 d in co-cultures and declined at 14 and 21 d. TNF-α was undetectable in cultures that received infliximab. The formation and activity of osteoclasts in co-cultures was not affected by infliximab. In contrast, infliximab in cultures of only PBMC significantly reduced the formation of osteoclasts. This reduction was accompanied by a decreased number and size of cell clusters, a step that precedes the formation of osteoclasts. TNF-α was again undetectable in the supernatant of infliximab-treated cultures, but was detectable at similar levels in cell lysates of control and infliximab-treated PBMC cultures. CONCLUSION: Our study shows that the contribution of TNF-α to osteoclast formation is cell system dependent. It contributes to PBMC-induced osteoclast formation, possibly by establishing stronger cell-cell interactions that precede osteoclast formation.
Asunto(s)
Osteoclastos , Proteínas Portadoras , Diferenciación Celular , Fibroblastos , Humanos , Infliximab , Leucocitos Mononucleares , Glicoproteínas de Membrana , Ligamento Periodontal , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Factor de Necrosis Tumoral alfaRESUMEN
AIM: To investigate the cytotoxicity of a modified salt solution (MSS) and evaluate the antimicrobial properties of MSS on in vitro biofilm models. METHODOLOGY: In a metabolic assay, fibroblasts derived from periodontal ligaments (PDL) of human extracted teeth were cultured and challenged with MSS or controls. Then, in active attachment biofilm models, the efficacy of MSS in the presence of dentine powder and in eliminating mature biofilms was investigated. In the dentine assay, a biofilm of Enterococcus faecalis was employed. For the final assay, microorganisms were retrieved from infected root canals and cultured to produce biofilms. After the treatments with MSS or the controls, the biofilms were collected, serially diluted and plated. The colony-forming units were counted. One-way anova was used to analyse the differences between the groups. A P < 0.05 was considered significant. RESULTS: The PDL fibroblasts remained metabolically active after challenges with MSS. Dentine powder did not alter the efficacy of MSS (P > 0.05). In endodontic biofilms, the culturable bacteria were equally reduced by MSS, 2% chlorhexidine (CHX) or 2% sodium hypochlorite (NaOCl) (P > 0.05). CONCLUSIONS: Modified salt solution is noncytotoxic in vitro and has good antimicrobial properties equal to CHX and NaOCl. Although the results are promising, ex vivo and in vivo studies are needed before its use as an interappointment root canal dressing can be considered.
Asunto(s)
Antiinfecciosos Locales/farmacología , Biopelículas/efectos de los fármacos , Cavidad Pulpar/efectos de los fármacos , Cavidad Pulpar/microbiología , Dentina/efectos de los fármacos , Desinfección/métodos , Irrigantes del Conducto Radicular/farmacología , Sales de Tetrazolio/farmacología , Células Cultivadas , Clorhexidina/farmacología , Recuento de Colonia Microbiana , Enterococcus faecalis , Fibroblastos , Humanos , Técnicas In Vitro , Hipoclorito de Sodio/farmacología , SolucionesRESUMEN
BACKGROUND AND OBJECTIVE: To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we compared the pro-inflammatory and matrix-degrading responses of gingival and granulation tissue fibroblasts from periodontally healthy controls, peri-implantitis, and periodontitis lesions to an in vitro challenge with Porphyromonas gingivalis. METHODS: Fibroblasts from periodontally healthy, peri-implantitis and periodontitis donors were challenged with viable P. gingivalis. The inflammatory reactions of fibroblasts were analyzed before and after 6 h P. gingivalis challenge, and 2.5 and 18 h after removal of the challenge. Gene expression and induction of pro-inflammatory mediators, and matrix metalloproteinases (MMPs) were assessed by real-time polymerase chain reaction. Protein expression was measured by enzyme-linked immunosorbent assay. RESULTS: Non-challenged fibroblasts from peri-implantitis and periodontitis lesions expressed higher levels of interleukin (IL)-1ß, IL-8, and monocyte chemotactic protein (MCP)-1 than fibroblasts from periodontally healthy individuals. The P. gingivalis challenge induced expression of IL-1ß, IL-8, IL-6, MCP-1, and MMP-1 in periodontitis and peri-implantitis fibroblasts, but not in fibroblasts from periodontally healthy individuals. MMP-8 expression was higher in non-challenged peri-implantitis fibroblasts than in fibroblasts from periodontally healthy individuals. However, the P. gingivalis challenge downregulated MMP-8 gene expression in peri-implantitis fibroblasts. After removal of the P. gingivalis challenge, peri-implantitis fibroblasts sustained higher induction of IL-1ß, MCP-1, and MMP-1 compared to periodontitis fibroblasts. CONCLUSIONS: Fibroblasts from peri-implantitis and periodontitis lesions gave a more pronounced inflammatory response to the P. gingivalis challenge than fibroblasts from healthy donors. They may therefore be involved in the development of inflammation in peri-implantitis and periodontitis. Moreover, the sustained upregulation of inflammatory mediators and MMP-1 in peri-implantitis fibroblasts may play a role in the pathogenesis of peri-implantitis.
Asunto(s)
Citocinas/análisis , Encía/microbiología , Metaloproteinasas de la Matriz/análisis , Periimplantitis/microbiología , Porphyromonas gingivalis/inmunología , Técnicas de Cultivo de Célula , Células Cultivadas , Quimiocina CCL2/análisis , Periodontitis Crónica/enzimología , Periodontitis Crónica/inmunología , Periodontitis Crónica/microbiología , Femenino , Fibroblastos/enzimología , Fibroblastos/inmunología , Fibroblastos/microbiología , Encía/enzimología , Encía/inmunología , Tejido de Granulación/enzimología , Tejido de Granulación/inmunología , Tejido de Granulación/microbiología , Humanos , Mediadores de Inflamación/análisis , Interleucina-1beta/análisis , Interleucina-6/análisis , Interleucina-8/análisis , Masculino , Metaloproteinasa 1 de la Matriz/análisis , Metaloproteinasa 8 de la Matriz/análisis , Persona de Mediana Edad , Periimplantitis/enzimología , Periimplantitis/inmunología , Porphyromonas gingivalis/enzimología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Tiempo , Regulación hacia ArribaRESUMEN
In periodontitis, tissue damage results mainly from aberrant host responses to oral microorganisms. Fibroblasts can play an important role in this. Gingival fibroblasts do not develop tolerance against the lipopolysaccharide of Porphyromonas gingivalis, a keystone pathogen in periodontitis, which may partly explain the persistence of inflammation. However, besides lipopolysaccharide, live P. gingivalis possess numerous virulence traits to impair host-responses. We hypothesized that fibroblast-responsiveness to a bacterial challenge could be affected by live P. gingivalis. We investigated if inflammatory responses of gingival fibroblasts to P. gingivalis were altered, when the fibroblasts had encountered P. gingivalis previously. On consecutive days, primary human gingival fibroblasts were challenged twice for 6 h with live P. gingivalis, or fibroblasts were preincubated for 24 h with a lower concentration of live P. gingivalis and re-challenged for 6 h with a higher concentration. As the P. gingivalis capsule and proteases are involved in modulating host responses, we used encapsulated P. gingivalis W83 and a non-encapsulated mutant, and P. gingivalis ATCC33277 and a lys-gingipain and arg-gingipain mutant, to challenge fibroblasts. With all P. gingivalis-strains, interleukin-8 and monocyte chemoattractant protein-1 responses to the second challenge were less strong in fibroblasts that had been challenged with P. gingivalis before. These lower responses might correspond with higher interleukin-1 receptor agonist expression. Fibroblast responses to a second challenge were not influenced by 24 h preincubation. Reduced chemokine responses after consecutive potent P. gingivalis challenges indicate that gingival fibroblast responsiveness is affected by a previous bacterial encounter. In periodontitis, such reduced chemokine responses may impair chemotaxis and clearance of oral microorganisms, thereby leading to prolonged inflammatory responses and tissue damage.
Asunto(s)
Fibroblastos/microbiología , Encía/microbiología , Periodontitis/inmunología , Periodontitis/microbiología , Porphyromonas gingivalis/patogenicidad , Adhesinas Bacterianas/inmunología , Adulto , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Quimiocinas/inmunología , Quimiotaxis/inmunología , Cisteína Endopeptidasas/inmunología , Femenino , Fibroblastos/inmunología , Cisteína-Endopeptidasas Gingipaínas , Encía/citología , Encía/inmunología , Gingivitis/inmunología , Gingivitis/microbiología , Humanos , Interleucina-1beta/biosíntesis , Interleucina-1beta/inmunología , Interleucina-6/biosíntesis , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-8/biosíntesis , Interleucina-8/genética , Interleucina-8/inmunología , Masculino , Persona de Mediana Edad , Porphyromonas gingivalis/inmunología , Estadísticas no Paramétricas , Factores de VirulenciaRESUMEN
BACKGROUND AND OBJECTIVE: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. MATERIAL AND METHODS: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n=14) and healthy control subjects (n=8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll-like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor-κB1 and its putative inhibitor NF-κB inhibitor-like protein1, and of interleukin-1ß, interleukin-6, interleukin-8, tumour necrosis factor-α, monocyte chemotactic protein-1 and regulated upon activation, normal T-cel expressed, and secreted, were assessed by real-time PCR. RESULTS: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture-positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis-negative persons. CONCLUSION: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis-positive donors are more responsive to an in vitro P. gingivalis challenge.
Asunto(s)
Fibroblastos/microbiología , Encía/patología , Ligamento Periodontal/patología , Periodontitis/patología , Porphyromonas gingivalis/inmunología , Proteínas Adaptadoras Transductoras de Señales , Células Cultivadas , Quimiocina CCL2/análisis , Placa Dental/microbiología , Femenino , Fibroblastos/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Interleucina-1beta/análisis , Interleucina-6/análisis , Interleucina-8/análisis , Receptores de Lipopolisacáridos/análisis , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Persona de Mediana Edad , FN-kappa B/análisis , Periodontitis/inmunología , Linfocitos T/inmunología , Receptor Toll-Like 1/análisis , Receptor Toll-Like 2/análisis , Receptor Toll-Like 4/análisis , Receptor Toll-Like 6/análisis , Receptor Toll-Like 7/análisis , Receptor Toll-Like 9/análisis , Factor de Necrosis Tumoral alfa/análisisRESUMEN
BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. MATERIAL AND METHODS: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real-time PCR, and protein expression was analyzed using ELISA. RESULTS: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted (RANTES). Macrophage colony-stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL-6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. CONCLUSION: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.