Cytokine and chemokine response of bone cells after dentin challenge in vitro

OBJECTIVE: The aim of this study was to characterize the effects of dentin extracts on cytokine, chemokine and nitric oxide (NO) production by primary rat bone cells. STUDY DESIGN: Osteoblastic bone marrow cultures were exposed to particulate (D-part), non-particulate (D-n-part) and demineralized dentin extracts and evaluated for proliferative activity, cell morphology, alkaline phosphatase activity and bone-like nodule formation. Cytokine production was assessed by enzyme-linked immunosorbent assay and NO release by the Griess method. RESULTS: The dentin extracts did not affect osteoblast numbering. Conversely, they up regulated in a dose-dependent manner the production by the osteoblasts of the pro-inflammatory interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, IL-6, cytokine-induced neutrophil chemoattractant-1, and of the anti-inflammatory cytokine, IL-10. The NO production was stimulated only by D-n-part. CONCLUSION: These results demonstrate that dentin induces the production of inflammatory cytokines by osteoblasts and suggest that pro-resorptive pathways might be stimulated when dentin molecules come into contact with bone cells during pathological processes associated with dentin and bone matrix dissolution.     

Expression of human beta-defensins-1 and -2 peptides in unresolved chronic periodontitis

BACKGROUND: Human beta-defensins (hBDs) are antimicrobial peptides which contribute to host innate immunity by disrupting the membrane integrity of a broad spectrum of microorganisms. OBJECTIVES: This study aimed to determine the expression profiles of hBD-1 and -2 peptides in gingiva and to assess the possible relations of these antimicrobial peptides with periodontal health and disease. METHODS: Seven periodontally healthy subjects and 22 patients with unresolved chronic periodontitis were recruited and the gingival biopsies collected consisted of healthy tissues from the healthy subjects (HT-C); periodontal pocket tissues (PoT) and inflamed connective tissues (ICT) from the base of pocket, i.e. granulation tissues, as well as clinically healthy tissues (HT-P) from the adjacent clinically healthy sites from the patients. The expression of hBD-1 and -2 peptides was detected by immunohistochemistry and quantitatively analyzed with a computerized image processing system. RESULTS: Both hBD-1 and -2 peptides were detected in all periodontally healthy subjects, while hBD-1 was detected in all patients and hBD-2 was found in most of the patients. Their expression was mainly confined to the granular and spinous layers of gingival epithelium, in which hBD-1 was detected in both intercellular spaces and cytoplasm, whereas hBD-2 was mainly observed in the cytoplasm. HT-C expressed significantly higher levels of hBD-2 than HT-P (p < 0.05). Within the patients, both defensins were up-regulated significantly in PoT as compared with the adjacent HT-P (p < 0.05). CONCLUSIONS: The present study showed that hBD-1 and -2 were frequently expressed in the granular and spinous layers of gingival epithelia and their expression may be associated with periodontal health and disease.     

Differences in innate immune responses upon stimulation with gram-positive and gram-negative bacteria

BACKGROUND AND OBJECTIVES: Host recognition pathways for gram-negative and gram-positive bacteria comprise pattern recognition receptors among which Toll-like receptors (TLRs) play a pivotal role. TLRs share common signaling pathways yet exhibit specificity as well. Periodontal disease is initiated and maintained in the first line by gram-negative but also gram-positive bacterial infection of the gingival sulcus. To date only limited information is available on whether gram-positive and gram-negative bacteria induce different host responses (strength or quality). MATERIALS AND METHODS: To elucidate these differential effects we focused on proinflammatory cytokine releases by assessing ex vivo stimulation of whole blood with heat-killed gram-negative and gram-positive bacteria and thereof derived microbial products associated with distinct TLRs. Tumor necrosis factor-alpha and interleukin-8 release were measured in the supernatants by enzyme-linked immunosorbent assay. In addition, innate immune responses of peritoneal macrophages from mice lacking TLR2 and TLR4 were tested. RESULTS: We observed that gram-negative and gram-positive species induced distinct patterns of cytokine production. Gram-negative species produced higher amounts of tumor necrosis factor-alpha while gram-positive species released higher amounts of the chemokine interleukin-8. Data from TLR knockout mice and TLR-transfected HEK cells revealed a somehow specific role of TLR4 and TLR2 for the recognition of gram-negative and gram-positive bacteria, respectively, an observation that goes along with the dominant recognition of the respective pathogen associated molecular patterns lipopolysaccharide and lipoteichoic acid. CONCLUSIONS: The results show that gram-negative and gram-positive bacterial species induce different patterns of immunoregulatory activity, which might be the result of activation of different TLRs.     

Immunohistochemical study on expression of alpha-defensin and beta-defensin-2 in human buccal epithelia with candidiasis

OBJECTIVES AND DESIGN: It has been previously reported that alpha-defensin (HNPs) and beta-defensin-2 (HBD-2) peptides with antifungal and cytotoxic activities can be detected in oral carcinomas and the saliva of patients with oral carcinomas. The present study investigated the presence of HNPs and HBD-2 in oral epithelia with candidiasis. MATERIALS AND METHODS: Tissue sections (4 microm) were prepared from biopsy and surgically removed specimens diagnosed as oral candidiasis (n = 10). The sections were examined immunohistochemically with antibodies directed against HNPs and HBD-2. RESULTS: Tissue sections of oral candidiasis were immunostained with antidefensin antibodies. Neutrophils in the inflamed lamina propria were positively immunostained with anti-HNPs antibody. The cytoplasm of cells in the upper spinous layer, in the lower spinous layer and in the parakeratinized layer of buccal epithelia with candidiasis was immunostained intensely with anti-HBD-2 antibody. In contrast, the expression of HBD-2 in the normal spinous layer was much weaker than that in oral candidiasis. No signals of HNPs were found in normal buccal epithelium. CONCLUSION: Buccal specimens from individuals with oral candidiasis show greater levels of expression of both HNPs and HBD-2. There might be a dual protection manner by defensins against fungal inflammation in infected buccal epithelia locally. Generally, HBD-2 signals have been found everywhere in the buccal epithelium; however, in an infected area, the signal intensity of HBD-2 has increased. HNPs signals have not been found in the normal buccal epithelium; however, HNPs signals have increased when the infection occurred.     

Loss of human β-defensin 1, 2, and 3 expression in oral squamous cell carcinoma

Introduction:  Human β-defensins (HBDs) are cationic, antimicrobial peptides produced by epithelial cells and involved in various aspects of the innate and acquired immune responses. They are expressed by oral tissues as constitutive and inducible genes. Recently, single nucleotide polymorphisms (SNPs) of β-defensins have been correlated with increased susceptibility to certain diseases. Studies have reported altered expression of β-defensins in cancers suggesting their involvement in carcinogenesis. The purpose of this study was to evaluate the regulation of HBD-1 (also published as DEFB1), HBD-2 (DEFB4) and HBD-3 (DEFB103A) ( http://www.genenames.org/index.html ) and HBD-1 SNPs in oral squamous cell carcinoma cell lines (OSCC) and healthy gingival keratinocytes.
Methods:  β-defensin expression was quantitatively assessed using real-time polymerase chain reactions in OSCC and control cell lines after exposure to interleukin-1β, tumor necrosis factor-α, and interferon-γ. Control data were obtained in a previous study. DNA from 19 OSCC cell lines and 44 control subjects were extracted and the HBD-1 region spanning the 5' untranslated region to the first intron was sequenced and analysed for SNP identification and distribution.
Results:  HBD-1 and HBD-2 basal messenger RNA expression were significantly lower in OSCC. In addition, the ability to be induced was significantly reduced in OSCC for all three β-defensins. Four HBD-1 SNPs were differentially distributed between cancer and control populations. Genotype distribution at the HBD-1 locus also suggested loss of heterozygosity in OSCC.
Conclusions:  The genetic variation observed in OSCC compared with that in control cell lines may account for differences in β-defensin expression. These results suggest a putative role for β-defensins in carcinogenesis and indicate that β-defensins may be useful markers of OSCC.     

Effect of compressive force on the expression of inflammatory cytokines and their receptors in osteoblastic Saos-2 cells

OBJECTIVE: In orthodontic tooth movement, some cytokines released from periodontal ligament fibroblasts and alveolar bone osteoblasts on the pressure side can alter the normal processes of bone remodelling, resulting in physiological bone resorption. We examined the effect of compressive force and interleukin (IL)-1 type I receptor antagonist (IL-1ra) on the expression of inflammatory cytokines that promote osteoclast formation, as well as on their receptors, in osteoblastic Saos-2 cells. DESIGN: The cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum with or without continuous compressive force (0.5-3.0 g/cm(2)) and/or IL-1ra for up to 24h. The gene expression levels of the cytokines and their receptors were estimated by determining mRNA levels using real-time PCR; the protein levels were determined using ELISA or immunohistochemical staining. RESULTS: The expression of IL-1beta, IL-1 receptor, IL-6, IL-6 receptor, IL-8 receptor, IL-11 and tumor necrosis factor-alpha (TNFalpha) increased depending on the strength and duration of the compressive force, whereas the expression of IL-8, IL-11 receptor and TNFalpha receptor did not change with the application of compressive force. The expression of cytokines and their receptors produced by 3.0 g/cm(2) of compressive force decreased with the simultaneous addition of IL-1ra and the decrease was remarkable in IL-8 receptor, IL-11 and TNFalpha. CONCLUSION: These results indicate that mechanical stress induces the production of inflammatory cytokines and their receptors in osteoblasts and the phenomenon is enhanced by the autocrine action of IL-1beta, which is increased in amount by mechanical stress.     

Facilitated diffusio1n by iontophoresis of vasoactive agents to the rat incisor pulp

The use of iontophoresis for facilitated diffusion of vasoactive agents into the dental pulp was investigated in lower incisor teeth of anaesthetized rats. Acetylcholine, carbachol and noradrenaline were iontophoresed with anodal and sodium nitroprusside with cathodal direct current through a superficial dentin exposure. Pulpal blood flow was measured with laser Doppler flowmetry. Current intensities below 100 μA of both polarities, using sodium chloride as a medium, caused no or minor afferent nerve-induced vasodilation, but excited sympathetic fibres of the pulp in a current-dependent manner. The current threshold for facilitated diffusion of acetylcholine was about 20 μA. The vascular responses to the cholinergic and noradrenergic drugs appeared within a minute after the onset of current and they were abolished by systemic administration of atropine and phenoxy benzamine, respectively. Iontophoresis of acetylcholine (40–100 μA for 20–120 s) caused a 3-fold increase of pulpal blood flow which was not dose-dependent; carbachol provoked a high-magnitude, long-lasting vasodilation and so did sodium nitroprusside. Noradrenaline caused a long-lasting vasoconstriction. In denervated rats iontophoresis of carbachol had effects similar to those seen in intact animals. None of the drugs used locally had any effect on systemic blood pressure. The results of this study indicate that iontophoresis can be used for delivery of vasoactive agents from an exposed dentin surface into the pulp in sufficient quantity to elicit drug-specific local vascular responses without causing systemic vascular effects.     

In vitro study by fluorescence microscopy and microradiography of tetracycline-tooth interaction

Abstract – The administration of tetracycline to the growing child may cause discoloration and hypoplasia in developing teeth. Whether tetracycline may also cause changes in fully mineralized enamel and dentin, is still a matter of discussion. The present investigation examines the in vitro reaction between tetracycline chloride and enamel and dentin of extracted human teeth. Specimens were immersed in 0.5 mg/ml to 20 mg/ml aqueous tetracycline solutions. After 24 hr the specimens were sectioned and studied by fluorescence microscopy and microradiography. The tetracycline solutions, which were all very acidic, were found to cause 1) demineralization of enamel and dentin, 2) incorporation of fluorescent material in enamel and, notably, dentin; and 3) formation of yellow, rhombohedral crystals on dental surfaces, especially in the more concentrated solutions. No fluorescence was seen in totally demineralized dentin. The findings indicate that the incorporation of tetracycline into enamel and dentin is caused by physicochemical processes that may take place regardless of the developmental stage of the mineralized tissues.