Oral streptococci subvert the host innate immune response through hydrogen peroxide.

In periodontal health, oral streptococci constitute up to 80% of the plaque biofilm. Yet, destructive inflammatory events of the periodontium are rare. This observation suggests that oral streptococci may possess mechanisms to co-exist with the host. However, the mechanisms employed by oral streptococci to modulate the innate immune response have not been well studied. One of the key virulence factors produced by oral streptococci is hydrogen peroxide (H2O2). In mammalian cells, H2O2 triggers the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key pathway mediating antioxidant defence. This study aimed to determine (1) if H2O2 producing oral streptococci activated the Nrf2 pathway in macrophages, and (2) if the activation of Nrf2 influenced the innate immune response. We found that oral streptococci downregulated the innate immune response in a H2O2 dependent manner through the activation of the Nrf2. The activation of the Nrf2 signalling pathway led to the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB), the key transcription factor regulating pro-inflammatory response. This study showed for the first time that oral streptococci are unlikely passive bystanders but could play an active role in the maintenance of periodontal health by preventing overt inflammation.

Correlation between microbial host factors and caries among older adults.

BACKGROUND: There is little knowledge about factors which may affect oral health among older adults. The objective of this study was to determine the relationship between Streptococcus mutans (MS) and Lactobacilli (LB) counts and caries among older adults. METHODS: In this community-based observation study, 141 participants aged 60 years and above were recruited from the west district of Singapore. Alongside the clinical examination, saliva samples were collected to determine Streptococcus mutans (MS) and Lactobacilli (LB) counts, as well as to record salivary flow rate and buffering capacity of saliva. RESULTS: Of the 141 participants, 63.8% were female and 94.3% were of Chinese ethnicity. The mean DMFT was 11.08 (s.d. 8.27). 9.9% of participants had at least one decayed tooth, 52.5% had minimum one missing tooth and 86.5% had at least one filled tooth. 67.4% had MS counts of ≥ 105 while LB counts were ≥ 105 for 48.2%. 83.7% had normal salivary flow or hypersalivation (> = 1 mL/min), the buffering capacity of the saliva was alkaline in 61% of the participants. Multivariate analysis showed that participants who had high MS counts were less likely to have a DMFT < 12 [OR (95% CI), 0.29 (0.11-0.77)] whereas participants who had high LB counts were less likely to have a DMFT ≤ 14 [OR (95% CI), 0.45 (0.20-1.002)]. CONCLUSION: Our study showed a positive correlation between MS and LB counts and caries experience in older adults. The mean DMFT was on the low side in our sample despite having a relatively high MS count. This suggests that there are many other factors which vary according to host environment, physiological and biological conditions that may affect MS and LB counts in the oral cavity. CLINICAL RELEVANCE: Our study supports the knowledge that the aetiology of dental caries among older adults is a complex process and it would be wrong to consider caries as a same problem with the same solution for all age groups.

Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 ameliorates chemotherapy-induced oral mucositis.

Oral mucositis (OM) is a common complication of cancer therapy, however OM management remains unsatisfactory. There is a growing interest in the therapeutic potential of probiotics in OM due to positive findings of its use in intestinal mucositis. This study aimed to determine the efficacy and safety of the probiotic combination Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 strains in chemotherapy-induced OM. Mice were divided into 4 groups. PBS/water and PBS/LR groups comprised of mice injected with PBS intraperitoneally (i.p.), and were given water or the mixture of L. reuteri (LR) DSM 17938 and ATCC PTA 5289 in water respectively. The 5-FU/water and 5-FU/LR groups comprised of mice injected with 5-FU i.p., and were given water or L. reuteri DSM 17938 and ATCC PTA 5289 in water respectively. Histopathological analysis revealed that the oral epithelia of the 5-FU/water and 5-FU/LR groups were thinner compared to PBS/water and PBS/LR groups. However, epithelial damage was significantly reduced in the 5-FU/LR compared to 5-FU/water group. Additionally, the 5-FU/LR group showed reduced oxidative stress and inflammation in the oral mucosa. We further showed that L. reuteri reduced oxidative stress through the nuclear factor E2-related factor-2 (Nrf-2) signalling. There was no evidence of translocation of L. reuteri systemically. This study demonstrated for the first time that L. reuteri protected oral mucosa against damage induced by chemotherapy.

Pterostilbene complexed with cyclodextrin exerts antimicrobial and anti-inflammatory effects.

Resveratrol (RES) is a natural polyphenol with potential as an adjunctive therapeutic modality for periodontitis. However, its inferior pharmacokinetics and toxicity concerns about its commonly used solvent dimethyl sulfoxide (DMSO) hinder translation to clinical applicability. Our study aimed to investigate the comparative antimicrobial properties of RES and its analogues (pterostilbene [PTS], oxyresveratrol [OXY] and piceatannol [PIC]), utilizing 2-hydroxypropyl-ß-cyclodextrin (HPßCD) as a solubiliser, which has a well-documented safety profile and FDA approval. These properties were investigated against Fusobacterium nucleatum, a key periodontal pathogen. PTS demonstrated the most potent antibacterial effects in HPßCD, with MIC > 60-fold lower than that of RES, OXY and PIC. In addition, PTS inhibited F. nucleatum biofilm formation. PTS exerted antimicrobial effects by eliciting leakage of cellular contents, leading to loss of bacterial cell viability. PTS also conferred immunomodulatory effects on F. nucleatum-challenged macrophages via upregulation of antioxidant pathways and inhibition of NF-κB activation. Given the superior antimicrobial potency of PTS against F. nucleatum compared to RES and other analogues, and coupled with its immunomodulatory properties, PTS complexed with HPßCD holds promise as a candidate nutraceutical for the adjunctive treatment of periodontitis.

Antibiotics Used in Regenerative Endodontics Modify Immune Response of Macrophages to Bacterial Infection.

INTRODUCTION: Ciprofloxacin, amoxicillin, and metronidazole are antibiotics used in regenerative endodontic therapy (RET). Although their antimicrobial properties are well-documented, there is a lack of information on the effects of these antibiotics on the immune response by host macrophages and periapical healing. Thus, this study had 2 objectives: (1) to determine the immune response of macrophages to bacterial infection in response to the combination of ciprofloxacin or amoxicillin and metronidazole and (2) using conditioned media produced by these macrophages to simulate the periapical microenvironment, to determine the impact on the expression of extracellular matrix (ECM) components by periodontal fibroblasts. METHODS: Macrophages were treated with ciprofloxacin and metronidazole or amoxicillin and metronidazole at 10-1000 µg/mL. The treated macrophages were exposed to lipopolysaccharide, and the pro- and anti-inflammatory cytokines produced were quantified with enzyme-linked immunosorbent assay. Periodontal fibroblasts were treated with conditioned media from these treated macrophages, and the expression of ECM genes was determined by quantitative polymerase chain reaction. RESULTS: Lipopolysaccharides elicited the production of proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha by macrophages, but this was suppressed by ciprofloxacin and metronidazole. Moreover, only conditioned media from macrophages treated with ciprofloxacin and metronidazole rescued microbial-induced down-regulation of ECM genes by periodontal fibroblasts. Specifically, ciprofloxacin was the antibiotic responsible for these observations. In contrast, these effects were not observed with amoxicillin and metronidazole. CONCLUSIONS: Apart from disinfection of the root canal system, the combination of ciprofloxacin and metronidazole also exerts an immunomodulatory effect, which may aid in periapical healing.

The Danger Signal Extracellular ATP Is an Inducer of Fusobacterium nucleatum Biofilm Dispersal.

Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP) is an example of a danger associated molecular pattern (DAMP) employed by mammalian cells to elicit inflammatory and damage healing responses. Although, the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors such as eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also showed higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a host danger signaling molecule to detach from biofilms. Our results further showed that dispersed F. nucleatum possessed distinct virulence characteristics compared to their biofilm and planktonic counterparts.

Cysteamine Enhances Biofilm Eradication Efficacy of Calcium Hydroxide.

INTRODUCTION: Calcium hydroxide (Ca[OH]2) is a widely used interappointment dressing, but its antibacterial property is compromised by dentin. Hence, the addition of chlorhexidine (CHX) with Ca(OH)2 has been proposed. However, the antimicrobial efficacy of this mixture compared with Ca(OH)2 alone is currently still debatable. Cysteamine is a mucolytic agent used to reduce the viscosity of mucus through the disruption of proteins, which are also important components of the extracellular matrix of biofilms. The aims of this study were to determine the efficacy of cysteamine alone and in combination with Ca(OH)2 to eradicate Enterococcus faecalis biofilm compared with CHX with Ca(OH)2, and to determine if this effect is affected by dentin. METHODS: The biofilm eradication efficacies of Ca(OH)2 alone and with cysteamine were determined using 7-day E. faecalis biofilm cultured on dentin discs and compared with Ca(OH)2 with 2% CHX. The effects of dentin on the efficacies of Ca(OH)2 alone and with either cysteamine or CHX were examined. RESULTS: Cysteamine alone completely abolished E. faecalis biofilm at 200 mg/mL. The combination of Ca(OH)2 with either cysteamine at 10 mg/mL or 2% CHX completely obliterated E. faecalis biofilm. Cysteamine with Ca(OH)2 completely eradicated E. faecalis biofilm despite preincubation with dentin, whereas CHX with Ca(OH)2 was less effective. CONCLUSIONS: Cysteamine effectively eliminated E. faecalis biofilm and showed synergistic effects in combination with Ca(OH)2, which were unaffected by dentin. Hence, our findings support the use of cysteamine as a potential adjunct to Ca(OH)2 as an interappointment dressing.

Reduced Glutathione Mediates Resistance to H2S Toxicity in Oral Streptococci.

Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in disease. A key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H2S) abundantly as a by-product of anaerobic metabolism. So far, H2S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H2S, the potential effects of H2S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H2S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of Streptococcus mitis and Streptococcus oralis were inhibited by H2S. However, H2S did not significantly affect the growth of Streptococcus gordonii or Streptococcus sanguinis. The differential susceptibility of oral streptococci to H2S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H2S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H2S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora.

Isolation of alkaline-tolerant bacteria from primary infected root canals.

INTRODUCTION: Alkaline-tolerant bacteria in primary infected root canals could have enhanced survival capacity against antimicrobials commonly used in root canal treatment. The aims of this study were to isolate and characterize alkaline-tolerant bacteria before endodontic treatment (S1), after chemomechanical root canal preparation (S2), and after calcium hydroxide dressing (S3). METHODS: Bacteriologic samples were obtained from 43 primary infected root canals. Samples were inoculated into culture media at a pH of 9 and incubated anaerobically. The identities of bacterial isolates were determined by 16S ribosomal RNA sequencing. RESULTS: All S1 samples were culture positive, with 70% harboring bacteria tolerating a pH of 9. Gram-positive bacteria Pseudoramibacter alactolyticus and Streptococcus spp were the most frequently isolated strains with a prevalence of 54%. Of 13 culture-positive S2 samples, 8 isolates tolerated a pH of 9, namely Streptococcus sanguinis, Enterococcus faecalis, Enterobacter cancerogenus, Streptococcus oralis, and Fusobacterium nucleatum. Seven of these 8 isolates (88%) were correspondingly isolated at S1. All 3 culture-positive S3 samples tolerated a pH of 9, namely S. sanguinis and E. faecalis, which were also isolated in the corresponding S1 and S2 samples. CONCLUSIONS: We showed that the presence of alkaline-tolerant Streptococcus and Enterococcus spp in primary infected root canals could lead to their persistence during and after root canal treatment and could pose a challenge to current treatment efficacy.

Rapid method for the detection of root canal bacteria in endodontic therapy.

INTRODUCTION: Complete eradication of microorganisms is essential for successful root canal therapy. However, current methods to evaluate persistent bacteria after therapy are not widely practiced. Adenosine triphosphate (ATP) is an indicator of viable cells. The bioluminescence-based ATP assay is easy to perform, and results can be obtained in a clinically relevant time frame of 5 minutes. The aims of this study were to evaluate the sensitivity of the ATP detection method and the specificity of this assay for viable cells and to compare the ATP and culture methods from root canal samples of patients undergoing endodontic treatment. METHODS: The sensitivity of the ATP assay was determined using bacterial species commonly isolated from root canals. Bacteria were treated with sodium hypochlorite; after which, culture plating and the ATP assay were performed. Forty-three root canal samples before (S1) and after (S2) instrumentation and 36 samples after the removal of calcium hydroxide dressing (S3) were collected from patients undergoing root canal treatment and subjected to ATP assay and anaerobic culture. RESULTS: The sensitivity of the ATP assay was determined to be between 10 and 100 bacterial cells. This method of detection also correlated well with the presence of viable bacteria. The ATP readings obtained allowed clear segregation of anaerobic culture-positive and -negative samples obtained from infected root canals of patients. CONCLUSIONS: The ATP detection method can be used as a rapid tool to determine the presence of viable bacteria during root canal therapy. This method may be potentially useful as an adjunct to root canal treatment.

Effects of Epigallocatechin gallate against Enterococcus faecalis biofilm and virulence.

OBJECTIVE: Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea (Camellia sinesis) has been shown to exert antimicrobial effects on numerous bacterial pathogens. However its efficacy against Enterococcus faecalis biofilm, which is associated with persistent root canal infection is unknown. The aims of this study were to investigate the effects of EGCG against E. faecalis biofilm and virulence. DESIGN: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG on E. faecalis were determined. The efficacy of EGCG on E. faecalis biofilms was tested by exposing 7-day old E. faecalis biofilm to EGCG. Flow cytometry analysis of hydroxyphenyl fluorescein (HPF) labelled E. faecalis was used to determine if EGCG induced intracellular hydroxyl radical formation. Co-treatment of EGCG with the iron chelator 2,2-dipyridyl (DIP) was carried out to determine if hydroxyl radical generated through Fenton reaction played a role in EGCG-mediated killing of E. faecalis. Furthermore, the effects of EGCG on the expression of virulence genes in E. faecalis were evaluated by quantitative polymerase chain reaction. RESULTS: EGCG exhibited a MIC and MBC of 5 µg/mL and 20 µg/mL respectively and effectively eradicated E. faecalis biofilms. EGCG induced the formation of hydroxyl radicals in E. faecalis. The addition of DIP protected E. faecalis against EGCG-mediated antibacterial effects. At sub-MIC, EGCG induced significant down-regulation of E. faecalis virulence genes. CONCLUSIONS: EGCG is an effective antimicrobial agent against both the planktonic and biofilm forms of E. faecalis, inhibiting bacterial growth and suppressing the expression of specific genes related to virulence and biofilm formation. The antimicrobial action of EGCG on E. faecalis occurred through the generation of hydroxyl radical.

Human embryonic stem cells derived keratinocyte as an in vitro research model for the study of immune response.

BACKGROUND: The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. METHODS: The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. RESULTS: The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. CONCLUSION: Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.

Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria.

Individuals with type 2 diabetes are at increased risk of acquiring melioidosis, a disease caused by Burkholderia pseudomallei infection. Although up to half of melioidosis patients have underlying diabetes, the mechanisms involved in this increased susceptibility are unknown. We found that B. pseudomallei-infected PBMCs from diabetic patients were impaired in IL-12p70 production, which resulted in decreased IFN-γ induction and poor bacterial killing. The defect was specific to the IL-12-IFN-γ axis. Defective IL-12 production was also observed during Mycobacterium tuberculosis infection, in which diabetes is likewise known to be a strong risk factor. In contrast, IL-12 production in diabetic cells was not affected upon Salmonella enterica infection or in response to TLR2, -3, -4, and -5 ligands. Poor IL-12 production correlated with a deficiency in intracellular reduced glutathione (GSH) concentrations in diabetic patients. Addition of GSH or N-acetylcysteine to PBMCs selectively restored IL-12 and IFN-γ production and improved bacterial killing. Furthermore, the depletion of GSH in mice led to increased susceptibility to melioidosis, reduced production of IL-12p70, and poorer disease outcome. Our data thus establish a link between GSH deficiency in diabetes and increased susceptibility to melioidosis that may open up new therapeutic avenues to protect diabetic patients against some intracellular bacterial pathogens.

N-acetylcysteine inhibits growth and eradicates biofilm of Enterococcus faecalis.

INTRODUCTION: The aims of this study were to evaluate the antibacterial and biofilm eradication efficacies of N-acetylcysteine (NAC) on Enterococcus faecalis. METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of NAC on E. faecalis were determined. In addition, the ability of dentin powder to neutralize the antibacterial activity of NAC was examined. Calcium hydroxide, a commonly used intracanal medicament, was included as a comparison. The efficacy of NAC on E. faecalis biofilms was tested by exposure of 21-day old E. faecalis biofilms to NAC. RESULTS: NAC was most bactericidal at pH 11 with MIC and MBC of 1.56 mg/mL and 12.5 mg/mL, respectively. Although preincubation of calcium hydroxide with dentin powder abolished its antibacterial effects, NAC completely killed E. faecalis regardless of dentin powder preincubation. In addition, prolonged incubation of NAC with dentin powder (up to 3 weeks) did not significantly reduce its antibacterial activity on E. faecalis. Furthermore, NAC also effectively eradicated E. faecalis biofilms. CONCLUSIONS: NAC was bactericidal against both the planktonic and biofilm forms of E. faecalis. This antibacterial property of NAC was unaffected by the presence of dentin.

Signaling pathways mediating beta3-adrenergic receptor-induced production of interleukin-6 in adipocytes.

The beta(3)-adrenergic receptor (beta(3)AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of beta(3)AR activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the beta(3)AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific beta(3)AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of beta(3)ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the G(s)-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-kappaB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block beta(3)AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the beta(3)AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the beta(3)AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.

Beta2 adrenergic receptor activation stimulates pro-inflammatory cytokine production in macrophages via PKA- and NF-kappaB-independent mechanisms.

Activation of the beta(2) adrenergic receptor (beta(2)AR) located on macrophages has been reported to possess anti-inflammatory properties, inhibiting nuclear factor kappaB (NF-kappaB) activation and cytokine production induced by pro-inflammatory stimuli. Here, we show that activation of the beta(2)AR in the absence of pro-inflammatory stimuli produced up to an 80- and 8-fold increase in IL-1beta and IL-6 transcripts, respectively, in the RAW 264.7 murine macrophage cell line. This increase in mRNA expression was accompanied by a significant increase in IL-1beta and IL-6 protein production. Pre-treatment of RAW cells with pharmacological inhibitors of protein kinase A (PKA) or NF-kappaB pathway failed to block this cytokine increase. Instead, the beta(2)AR-mediated increase in cytokines required activation of both the B-raf-ERK1/2 and p38 pathways. Treatment of RAW cells with the exchange protein directly activated by cAMP (EPAC) agonist also resulted in the up-regulation of IL-1beta and IL-6 transcripts. Examination of the main transcription factors downstream of the ERK1/2 and p38 signaling revealed that beta(2)AR activation resulted in the stimulation of CRE-, but not C/EBPbeta-, ETS-, or NF-kappaB-dependent transcription. Western blot analysis further showed that among the transcription factors which recognize the CRE-binding site, ATF-1 and ATF-2 but not CREB proteins were phosphorylated in an ERK1/2- and p38-dependent manner. Collectively, these results demonstrate that beta(2)ARs possess pro-inflammatory properties and that their activation leads to IL-1beta and IL-6 production through ERK1/2- and p38-dependent activation of ATF-1 and ATF-2 transcription factors.

The role of titanium surface topography on J774A.1 macrophage inflammatory cytokines and nitric oxide production.

A role for monocyte/macrophage modulation of wound healing at endosseous implants is proposed. The modification of the endosseous implant surface topography can alter cell adhesion and resultant cell behavior. The aim of this study was to define the effect of increased cpTitanium surface topography on adherent J744A.1 macrophage phenotype in culture. The J744A.1 cells were cultured on 20mm diameter cpTitanium disks prepared with smooth and grit-blasted/acid rough surface topographies for 24-72 h. Following culture in growth media with or without lipopolysaccharide (LPS), total RNA was isolated and real-time polymerase chain reaction (PCR) was used to measure the steady-state levels of the pro-inflammatory cytokines interleukin 1-beta (IL-1beta) and interleukin 6 (IL-6) and the anti-inflammatory cytokine interleukin-10 (IL-10). Additional evidence of pro-inflammatory signaling was sought by measurement of cellular nitric oxide (NO) production. In the absence of LPS, IL-1beta levels were increased on grit-blasted/acid rough surfaces during the first 48 h. In contrast, IL-6 levels were reduced on the grit-blasted/acid rough surfaces. When cultures were treated with LPS, high levels of IL-1beta and IL-6 expression were measured, irrespective of surface topography. The responses of J744A.1 cells to surface and superimposed LPS stimulation suggest only modest effects of the modeled endosseous implant surface on adherent cell pro-inflammatory cytokine expression and NO signaling.