Gingival fibroblasts prevent BMP-mediated osteoblastic differentiation.

OBJECTIVES: The inhibitory action of the superficial gingival connective tissues may limit the regenerative potential of alveolar bone in periodontal therapy or dental implant applications. The aims of this study were to investigate the hypothesis that gingival fibroblasts (GF) can inhibit bone morphogenetic protein (BMP)-induced osteoblastic differentiation, to determine their expression of BMP inhibitors, and finally to determine whether reduction of these inhibitors can relieve suppression of osteoblastic differentiation. METHODS: Gingival fibroblasts were co-cultured either directly or indirectly with calvarial osteoblasts to assess alkaline phosphatase inhibitory activity, a marker of osteoblastic differentiation. To test total BMP-inhibitory activity of rat GF, conditioned media (GFCM) were collected from cultures. ROS 17/2.8 osteoblastic cells were stimulated with BMP2, together with GFCM. Inhibitor expression was tested using RT-qPCR, Western blotting and in situ hybridization. Removal of inhibitors was carried out using immunoprecipitation beads. RESULTS: Co-culture experiments showed GF-secreted factors that inhibit BMP-stimulated ALP activity. 10 ng/ml BMP2 increased alkaline phosphatase expression in ROS cells by 41%. GFCM blocked BMP activity which was equivalent to the activity of 100 ng/ml Noggin, a well-described BMP inhibitor. Cultured gingival fibroblasts constitutively expressed BMP antagonist genes from the same subfamily, Grem1, Grem2 and Nbl1 and the Wnt inhibitor Sfrp1. Gremlin1 (6.7 × reference gene expression) had highest levels of basal expression. ISH analysis showed Gremlin1 expression was restricted to the inner half of the gingival lamina propria and the PDL. Removal of Gremlin1 protein from GFCM eliminated the inhibitory effect of GFCM on ALP activity in ROS cells. Subsequent addition of recombinant Gremlin1 restored the inhibitory activity. CONCLUSIONS: Factors secreted by gingival fibroblasts inhibit BMP-induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues. These inhibitors, particularly Gremlin1, may limit coronal alveolar bone regenerative potential during oral and periodontal surgery.

Gene expression profiles of mandible reveal features of both calvarial and ulnar bones in the adult rat.

OBJECTIVES: Limb and mandibular alveolar bone of the mandible are susceptible to disuse osteopenia, whilst skull and mandibular basal bone appear to resist excessive generalised bone loss. We wanted to compare the site-specific transcriptome of anatomically and functionally distinct bones to confirm the composite nature of the mandible at the molecular level. METHODS: Gene expression profiles were obtained for the mandible, ulna, and calvaria of adult male rats using Affymetrix Rat Genome 230 2.0 GeneChips. Ingenuity Pathways Assist generated association maps, and RGD database software identified site-specific pathways. RESULTS: The majority of expressed transcripts (84%) are common to all three sites. The mandible expressed 873 transcripts in common with ulna but not calvaria, and 1014 transcripts in common with calvaria but not ulna. Transcripts in these groups were excluded if they showed significant differential expression (>2-fold) and the remaining mapped genes were filtered for those related to modulation of gene transcription. Analysis of these genes revealed common pathways shared by the mandible and ulna, or mandible and calvaria, which were not shared by the calvaria and ulna. CONCLUSIONS: There were relatively few differences in the expression of genes responsible for the bone formation process per se in different functional skeletal sites. Differential transcription factor expression suggests that it is the regulation of bone formation and not the mechanism of bone formation itself that differs between the skeletal sites. CLINICAL SIGNIFICANCE: The mandible has areas both prone to, and resistant to, resorption whilst skull and limb bone differ in their susceptibilities to osteopenia. This report reveals that the mandible shares some genetic pathways in common with calvaria and others in common with ulna. Study of these pathways could identify novel treatment strategies for bone preservation.

Adult rat bones maintain distinct regionalized expression of markers associated with their development.

The incidence of limb bone fracture and subsequent morbidity and mortality due to excessive bone loss is increasing in the progressively ageing populations of both men and women. In contrast to bone loss in the weight-bearing limb, bone mass in the protective skull vault is maintained. One explanation for this could be anatomically diverse bone matrix characteristics generated by heterogeneous osteoblast populations. We have tested the hypothesis that adult bones demonstrate site-specific characteristics, and report differences at the organ, cell and transcriptome levels. Limb bones contain greater amounts of polysulphated glycosaminoglycan stained with Alcian Blue and have significantly higher osteocyte densities than skull bone. Site-specific patterns persist in cultured adult bone-derived cells both phenotypically (proliferation rate, response to estrogen and cell volumes), and at the level of specific gene expression (collagen triple helix repeat containing 1, reelin and ras-like and estrogen-regulated growth inhibitor). Based on genome-wide mRNA expression and cluster analysis, we demonstrate that bones and cultured adult bone-derived cells segregate according to site of derivation. We also find the differential expression of genes associated with embryological development (Skull: Zic, Dlx, Irx, Twist1 and Cart1; Limb: Hox, Shox2, and Tbx genes) in both adult bones and isolated adult bone-derived cells. Together, these site-specific differences support the view that, analogous to different muscle types (cardiac, smooth and skeletal), skull and limb bones represent separate classes of bone. We assign these differences, not to mode of primary ossification, but to the embryological cell lineage; the basis and implications of this division are discussed.

Interaction of adrenomedullin and calcitonin gene-related peptide with the periodontal pathogen Porphyromonas gingivalis.

The nature of the interaction between Porphyromonas gingivalis and the multifunctional peptides adrenomedullin and calcitonin gene-related peptide (CGRP) was investigated. Growth of P. gingivalis was not inhibited in the presence of either of these peptides [minimal inhibitory concentration (MIC)>250 microg mL(-1)]. The ability of the arginine- and lysine-specific proteases from P. gingivalis to breakdown these peptides was investigated. Adrenomedullin and CGRP were incubated with culture supernatants from wild-type and protease gene knockout strains. No significant effect on antimicrobial activity against the indicator organism Escherichia coli BUE55 was found (MIC=6.25 microg mL(-1) in all cases). The role of anionic components on the surface of P. gingivalis, which may alter binding of these cationic peptides, was also investigated in relation to adrenomedullin. Growth of gene knockout strains lacking surface polysaccharide and capsule components was not inhibited (MIC>250 microg mL(-1)). It is suggested that a lack of sensitivity to adrenomedullin and CGRP may enable P. gingivalis to persist in the oral cavity and cause disease.

Polymorphisms of TLR4 but not CD14 are associated with a decreased risk of aggressive periodontitis.

OBJECTIVE: To study whether there is an association between the frequency of functional polymorphisms in the toll-like receptor 4 (TLR4) and cluster differentiation 14 (CD14) genes and periodontitis. METHODOLOGY: Genotyping for the TLR4 single-nucleotide polymorphisms (SNPs) Asp299Gly, Thr399Ile and the CD14 SNPs -159 and -1359 was completed for subjects with periodontal disease compared with control subjects. Two disease populations were investigated: 73 subjects with aggressive periodontitis (AgP; 28 males, 45 females) and 95 males with chronic periodontitis (CP). The TLR4 and CD14 polymorphisms were determined using SNaPshot primer extension with capillary electrophoresis. Comparison of allele and genotype frequencies for each polymorphism was by Fisher's exact test or chi2 analysis. RESULTS: The TLR4 Asp299Gly genotype was present in a significantly (p=0.026) lower proportion of AgP subjects (5.5%) compared with control subjects (16.3%). The unadjusted odds ratio for the Asp299Gly genotype to be associated with AgP was 0.30, 95% confidence interval 0.10-0.91. No differences were found in the prevalence of the TLR4 Asp299Gly genotype in men with CP (18.9%) compared with an age-matched control group with no evidence of periodontitis (17%). In addition, there was no difference in the distribution of the CD14 polymorphisms in either the AgP or CP populations studied compared with controls. CONCLUSION: It is concluded that in West European Caucasians, the Asp299Gly TLR4 gene polymorphism is associated with a decreased risk of AgP but not CP. Promoter polymorphisms of the CD14 gene, however, did not influence susceptibility to inflammatory periodontitis in the population cohorts studied.

Antimicrobial activity does not predict cytokine response to adrenomedullin or its shortened derivatives.

The aim of this study was to investigate cytokine release from oral keratinocytes and fibroblasts in response to AM and shortened derivatives previously characterised in terms of their antimicrobial activities. Cells were incubated with AM or its fragments (residues 1-12, 1-21, 13-52, 16-21, 16-52, 22-52, 26-52, and 34-52), and culture supernatants collected after 1, 2, 4, 8, and 24 hours. A time-dependant increase in production of interleukin1-alpha and interleukin 1-beta from keratinocytes in response to all peptides was demonstrated. However, exposure to fragments compared to whole AM resulted in reduced production of these cytokines (60% mean reduction at 24 hours, P<.001). No consistent differences were shown between the cytokine response elicited by antimicrobial and nonantimicrobial fragments. The production of interleukin-6 and interleukin-8 did not change significantly with time or peptide used. Fibroblast cells were relatively unresponsive to all treatments. This study demonstrates that antimicrobial activity does not predict cytokine response to adrenomedullin or its shortened derivatives.

Relationship between IL-1A polymorphisms and gingival overgrowth in renal transplant recipients receiving Cyclosporin A.

AIM: Levels of interleukin-1alpha (IL-1alpha) are elevated in periodontal inflammation. IL-1A gene polymorphisms are associated with inflammatory diseases. This study aimed to investigate IL-1A gene polymorphism in Cyclosporin A (CsA)-treated renal transplant patients and investigate the association between this polymorphism and gingival crevicular fluid (GCF) levels of several cytokines. MATERIALS AND METHODS: Fifty-one renal transplant patients on CsA treatment (25 with and 26 without gingival overgrowth) and 29 healthy controls were recruited for the study. Demographic, pharmacological and periodontal parameters were recorded and gingival overgrowth was assessed. RESULTS: Multiple regression analysis showed that genotype was significantly associated with gingival overgrowth (p=0.02). Carriage of the IL-1A (-889) T allele was strongly protective [95% confidence interval (CI): 0.046-0.77], although not significantly associated with IL-1alpha protein levels in GCF. IL-1alpha, IL-1beta and IL-8, but not IL-6, were detected in GCF of CsA-treated patients, but none of them was significantly associated with gingival overgrowth. CONCLUSIONS: This study is the first to associate a gene polymorphism as a risk factor for CsA-induced gingival overgrowth in renal transplant patients, demonstrating that IL-1A polymorphism might alter individual susceptibility to CsA. However, there was no association between GCF cytokine levels and the presence of gingival overgrowth or patient IL-1A genotype.

Prognostic factors in the treatment of generalized aggressive periodontitis: I. Clinical features and initial outcome.

AIMS: The aims of this study were to investigate prognostic factors for initial response to non-surgical periodontal treatment for generalized aggressive periodontitis. METHODS: Seventy-nine patients with generalized aggressive periodontitis were included in this prospective follow-up intervention study. Patients' clinical and demographic parameters were collected at baseline and 10 weeks following a standard course of treatment (four visits of non-surgical root surface debridement together with OHI as required). The relationship between clinical variables and treatment outcome were analysed at site-specific level by chi(2) analysis and for patient-specific variables by logistic regression. RESULTS: In general, there was a good response to the treatment provided. In deep sites the mean pocket depth reduction was 2.11+/-2.01 mm. Site-specific analysis showed that the presence of plaque had a small but significant predictive effect on outcome (odds ratio 1.4). Sites on teeth with grade II/III mobility showed a significantly reduced response to treatment. Twenty-five patients were classified as "non-responders". Current smoking was strongly associated with non-responding patients (odds ratio 3.8) in a logistic regression model; plaque, baseline bleeding and initial pocket depth were not significantly associated with treatment outcomes. CONCLUSIONS: Overall, the results emphasize the importance of smoking as a negative prognostic factor, and suggest that treatment outcomes may be determined by a wide range of different determinants requiring further study.

Prognostic factors in the treatment of generalized aggressive periodontitis: II. Effects of smoking on initial outcome.

AIMS: The aim of this study was to investigate the effects of smoking on the response to non-surgical treatment for aggressive periodontitis. METHODS: Seventy-nine patients with generalized aggressive periodontitis were included in the study; 20 were smokers. All patients received a course of non-surgical periodontal therapy and outcomes assessed 10 weeks post-operatively. Non-responding patients were designated if they had 30% or more non-responding deep sites. RESULTS: At baseline, bleeding scores were lower in smokers. There was no difference in baseline plaque, pocket depth (PD), recession or clinical attachment levels (CALs); when sites were selected by equal levels of CAL, increased recession was seen in smokers. Outcomes were poorer in smokers (mean PD change 1.75+/-0.56 versus 2.23+/-0.87 mm). The odds ratio for 30% of sites not responding in smokers was 2.9; for 40% non-responding it was 5.9. Smoking altered the distribution of site-specific responses to increase specifically the number of non-responding sites. There was no significant difference in responses between ex-smokers and never-smokers. CONCLUSIONS: The results demonstrate that smoking is a major risk factor for poor response to initial treatment and emphasize the importance of smoking cessation in periodontal therapy.

Bone morphogenetic protein-2 induces expression of murine zinc finger transcription factor ZNF450.

The bone morphogenetic protein-2 (BMP-2) is a potent secreted factor that promotes osteoblast differentiation during development. Exposure to BMP-2 is sufficient to cause a lasting change in cell fate presumably by activating specific target genes. To identify genes downstream of BMP-2 we treated the murine pluripotent embryonic cell line, C3H10T1/2 that can be induced to form an osteoblastic phenotype, with 100 ng/ml BMP-2 for 24 h. Using suppression subtractive hybridisation we found the novel zinc finger transcription factor, ZNF450 was upregulated. The single-copy ZNF450 gene spans 15.6 kb on chromosome 10B1 and consists of seven exons, the first of which is untranslated. The open reading frame encodes a 710 reside protein. Analysis of the protein sequence reveals a highly conserved amino-terminal BTB/POZ dimerisation domain, an AT-hook motif, and eight C2H2 zinc fingers. Library screening identified a second mRNA isoform encoding a short protein isoform with one zinc finger. Using reverse transcriptase-real time PCR to measure mRNA expression we found that ZNF450, Runx2/Cbfa-1, and Sp7/osterix were induced by BMP-2 after 4 h in C2C12 myoblast cells. Treatment of C2C12 cells with BMP-2 causes a shift from a myoblastic to osteoblastic phenotype. ZNF450 was upregulated three to fivefold after 24 h in C3H10T1/2 cells and required 100 ng/ml BMP-2. Expression of the 3 kb major transcript was highest in liver, testis, and kidney. However, ZNF450 mRNA was found also in a wide range of adult tissues. The consistent induction of ZNF450 by BMP-2 after 4 h in three murine pluripotent cell lines suggests that ZNF450 may play a role in the BMP-2 signalling pathway.

In vitro behavior of osteoblastic cells cultured in the presence of pseudowollastonite ceramic.

Pseudowollastonite ceramic (psW) is a bioactive ceramic that binds to bone when implanted in vivo and may be useful for the treatment of skeletal defects. However, there have been no studies that examined the interaction between psW and osteoblastic cells in vitro. This study investigated the suitability of psW as a substratum for cell attachment and the ability of the material to effect osteoblasts at a distance from the material surface. Fetal rat calvarial cells were plated onto the ceramic and examined by scanning electron microscopy. The findings reported show that cells attached and proliferated on the surface to the ceramic. Attachment by cells to the material can be enhanced by preincubation of psW in serum or media containing fibronectin. The adhesion of cells can be inhibited by addition of GRGDS peptides suggesting that adhesion to psW is mediated by integrin binding to adsorbed proteins. To study the effects of psW at a distance, cells were cultured in the presence but not in direct contact with the material. Subsequent changes in proliferation, alkaline phosphatase expression, and bone nodule formation were assessed. Cells grown in wells containing psW demonstrated an increase in both the rate and total numbers of bone nodules formed, although there were no differences in proliferation or alkaline phosphatase expression. Overall, these results suggest that psW is biocompatible and osteoconductive.

The association between neutrophil numbers and interleukin-1alpha concentrations in gingival crevicular fluid of smokers and non-smokers with periodontal disease.

OBJECTIVES: To test whether neutrophil numbers are directly correlated with interleukin-1alpha (IL-1alpha) concentrations in gingival crevicular fluid (GCF) of patients with periodontitis, and to investigate the effects of smoking on these parameters. MATERIALS AND METHODS: A total of 99 GCF samples from 33 patients (14 smokers) suffering from severe chronic periodontitis were collected using Durapore filter strips. Polymorphonuclear leucocyte (PMN) numbers were counted using a Coulter cell counter and IL-1alpha levels were determined by ELISA. Total GCF protein was measured by Bio-Rad assay as a surrogate measure of GCF volume. RESULTS: Mean IL-1alpha concentrations were significantly reduced in smokers compared with non-smokers (non-smokers: 3.29+/-2.02 pg/microg protein, smokers 1.59+/-1.13 pg/microg protein). There was no association between PMN numbers and IL-1alpha concentrations found when analysed either by site or by patient. PMN numbers were not significantly different between the two groups (non-smokers: 1.16 x 10(6)+/-1.04 x 10(6); smokers: 7.30 x 10(5)+/-8.07 x 10(5)). Smoking did not affect mean total protein concentration of samples. CONCLUSIONS: Smoking significantly decreased IL-1alpha concentrations in GCF without affecting GCF volume sampled. The lack of association between IL-1alpha concentration and neutrophil numbers suggests that the reduced IL-1alpha concentrations seen in smokers is independent of any possible effect of smoking on neutrophil chemotaxis, and further suggests that smoking may directly inhibit IL-1alpha production.