Impact of local steroid or statin treatment of experimental temporomandibular joint arthritis on bone growth in young rats.

INTRODUCTION: Juvenile idiopathic arthritis in temporomandibular joints (TMJs) is often treated with intra-articular steroid injections, which can inhibit condylar growth. The purpose of this study was to compare simvastatin (a cholesterol-lowering drug that reduces TMJ inflammation) with the steroid triamcinolone hexacetonide in experimental TMJ arthritis. METHODS: Joint inflammation was induced by injecting complete Freund's adjuvant (CFA) into the TMJs of 40 growing Sprague Dawley rats; 4 other rats were left untreated. In the same intra-articular injection, one of the following was applied: (1) 0.5 mg of simvastatin in ethanol carrier, (2) ethanol carrier alone, (3) 0.15 mg of triamcinolone hexacetonide, (4) 0.5 mg of simvastatin and 0.15 mg of triamcinolone hexacetonide, or (5) nothing additional to the CFA. The animals were killed 28 days later, and their mandibles were evaluated morphometrically and with microcomputed tomography. RESULTS: The analysis showed that the TMJs subjected to CFA alone had decreased ramus height compared with those with no treatment (P <0.05). Groups that had injections containing the steroid overall had decreases in weight, ramus height, and bone surface density when compared with the CFA-alone group (P <0.0001). Groups that had injections containing simvastatin, however, had overall increases in weight (P <0.0001), ramus height (P <0.0001), condylar width (P <0.05), condylar bone surface density (P <0.05), and bone volume (P <0.0001) compared with the groups receiving the steroid injections, and they were not different from the healthy (no treatment) group. CONCLUSIONS: Treatment of experimentally induced arthritis in TMJs with intra-articular simvastatin preserved normal condylar bone growth.

The effect of local simvastatin delivery strategies on mandibular bone formation in vivo.

Systemic simvastatin is known to reduce cholesterol and stimulate modest bone formation, but local surgical placement in polylactic acid domes causes robust bone formation and local swelling. A less invasive and more flexible injection protocol was studied to evaluate the bone-inducing effects compared to surgical implantation. Bone formation rate, short- and long-term bone augmentation histology, and mechanical properties were evaluated to characterize the new bone in a rat bilateral mandible model (test and control sides in same animal). Results demonstrated that multiple (3) injections of 0.5 mg simvastatin effectively reduced soft tissue swelling while preserving bone growth (60% increase of bone width at 24 days) compared to simvastatin dome placement (43% increase at 24 days). Compared to controls, bone formation rate was significantly higher on the simvastatin side, especially in the dome. Three-point bending tests revealed higher maximum force to fracture and stiffness at 24 days with simvastatin injections. Long-term evaluation showed that 55% of maximum new bone formed 24 days post-injection was retained at 90 days.

Injectable simvastatin in periodontal defects and alveolar ridges: pilot studies.

BACKGROUND: Topical injection of simvastatin in methylcellulose gel was shown to stimulate bone growth and inflammation over mouse calvaria and in rat mandible models. The purpose of these pilot studies was to evaluate the potential of locally injected simvastatin in human-sized periodontal defects. METHODS: Chronic periodontal defects were created bilaterally in seven 1-year-old beagle dogs: 3-walled intrabony defects distal of the mandibular second premolar and mesial of the fourth premolar and Class II furcation defects at the buccal furcation of the mandibular first molars. The edentulous space distal to the mandibular canine was left undisturbed. After 16 weeks of healing, defect sites were treated with scaling and root planing, and mandible sides were randomly selected to receive three weekly injections of 0.5 mg simvastatin in 30 microl methylcellulose gel and contralateral gel alone (n=3) or 2.0 mg simvastatin/methylcellulose gel and contralateral gel alone (n=4). Two months following drug application, block sections, including teeth and surrounding tissues, and submandibular lymph nodes were obtained for histomorphometric analysis. RESULTS: Two trends were noted in this pilot study: buccal edentulous ridge thickness was 29% greater with simvastatin, 0.5 mg, compared to gel alone (P=0.0845), and the simvastatin groups had bone-height loss in interproximal intrabony and furcation defects, but the length of new cementum in the interproximal intrabony defects was greater with simvastatin, 0.5 mg (0.35+/-0.14 mm), compared to gel alone (0.06+/-0.15 mm; P=0.069). No new cementum was found in furcations. CONCLUSIONS: Multiple injections of simvastatin are not appropriate for the treatment of intrabony or furcation defects. However, this approach shows potential to augment bone thickness in closed alveolar environments.

Subgingival Microbiome Colonization and Cytokine Production during Early Dental Implant Healing.

Little is known about longitudinal development of the peri-implant subgingival microbiome and cytokine production as a new sulcus forms after dental implant placement. Therefore, the purpose of this observational study was to evaluate simultaneous longitudinal changes in the oral microbiome and cytokine production in the developing peri-implant sulcus compared to control natural teeth. Four and 12 weeks after implant placement and abutment connection, a dental implant and a natural tooth were sampled in 25 patients for subgingival plaque and gingival crevicular fluid (GCF [around teeth] and peri-implant crevicular fluid [PICF] around implants). DNA from plaque samples was extracted and sequenced using Illumina-based 16S rRNA sequencing. GCF and PICF samples were analyzed using a customized Milliplex human cytokine and chemokine magnetic bead panel. Beta diversity analysis revealed that natural teeth and implants had similar subgingival microbiomes, while teeth had greater alpha diversity than implants. At the genus level, however, few differences were noted between teeth and dental implants over 12 weeks. Specifically, Actinomyces and Selenomonas were significantly elevated around teeth versus dental implants at both 4 weeks and 12 weeks, while Corynebacterium and Campylobacter were significantly elevated only at 4 weeks around teeth. The only difference between PICF and GCF biomarkers was significantly elevated granulocyte-macrophage colony-stimulating factor levels around teeth versus dental implants at the 4-week visit. The subgingival microbiome and cytokine production were similar between teeth and implants during early healing, suggesting that these profiles are driven by the patient following dental implant placement and are not determined by anatomical niche. IMPORTANCE Dental implants are a common treatment option offered to patients for tooth replacement. However, little is known regarding initial colonization of the subgingival microbiome and simultaneous longitudinal cytokine production in humans during the early healing phase following implant placement. We report findings from an in vivo study that assessed initial colonization of the subgingival microbiome and concomitant early cytokine production in a newly formed anatomical space, namely, an implant sulcus. This approach may be useful in future interventional studies to influence dental implant success. Our data showed that the subgingival microbiome and cytokine profile were similar for control natural teeth and dental implants at both 4 and 12 weeks after implant placement. These data suggest that these profiles are driven by the patient and not by anatomical location (i.e., tooth versus dental implant).

Effect of Simvastatin Prodrug on Experimental Periodontitis.

BACKGROUND: Local application of statins has shown potential in preventing and regenerating bone loss associated with experimental periodontitis. This study evaluates the effect of a novel simvastatin (SIM) prodrug (capable of delivering high doses to periodontitis inflammatory lesion and cells) on experimental periodontitis bone loss and inflammation. METHODS: Forty mature female Sprague Dawley rats were subjected to ligature-induced experimental periodontitis between maxillary first and second molars (M1-M2). Equal groups were treated with three weekly doses of: 1) prodrug carrier alone (mPEG); 2) 0.5 mg SIM dose equivalent in carrier (SIM/SIM-mPEG); 3) 1.0 mg SIM/SIM-mPEG; 4) 1.5 mg SIM/SIM-mPEG; or 5) ligature alone. Contralateral molars served as unmanipulated controls. Four weeks after initiation of periodontitis, animals were euthanized, the M1-M2 interproximal was evaluated with microcomputed tomography and histology, and data were analyzed with one-way analysis of variance. RESULTS: Ligature alone caused a mean bone loss of 1.01 ± 0.06 mm from the cemento-enamel junction, whereas all doses of SIM/SIM-mPEG reduced bone loss, especially 1.5 mg SIM/SIM-mPEG (0.68 ± 0.05 mm, P <0.001), which was not statistically different from contralateral control (0.47 ± 0.06 mm). A dose of 1.5 mg SIM/SIM-mPEG also reduced percentage of neutrophils compared with carrier alone (2.0% ± 1.0% versus 5.7% ± 1.1%; P <0.05), and increased amount of uninflamed connective tissue in the M1-M2 interproximal area (65.2% ± 3.3% versus 46.3% ± 3.3%; P <0.001). The mPEG carrier alone did not have bone-sparing or anti-inflammatory properties. CONCLUSION: Multiple local 1.5-mg doses of a macromolecular SIM prodrug decreases amount of experimental periodontitis bone loss and inflammation in rats.

Local simvastatin effects on mandibular bone growth and inflammation.

BACKGROUND: Simvastatin has been shown to increase bone growth when applied topically to murine bone; however, it causes considerable soft tissue inflammation at high doses (2.2 mg), making future clinical use problematic. This study evaluated the effect of lower simvastatin doses and cyclooxygenase (COX) synthase inhibitors on tissue inflammation and bone growth in rats and gene expression in mice. METHODS: Adult female rats were untreated or treated with a single dose of 0.1, 0.5, 1.0, 1.5, or 2.2 mg simvastatin in methylcellulose gel in a polylactic acid membrane (SIM) on the lateral aspect of the mandible. The contralateral mandible side was implanted with methylcellulose gel/polylactic acid membrane alone (GEL), and five rats in each dose pairing were evaluated histomorphometrically after 3, 7, and 24 days. Subsequent rats were similarly treated with 0.5 mg simvastatin (optimal dose) and daily intraperitoneal injections of COX-2 inhibitor (NS-398; 1 mg/kg x 7 days; N = 16), general COX inhibitor (indomethacin; 1 mg/kg x 7 days; N = 16), or no inhibitor (N = 10) and evaluated histomorphometrically after 7 or 24 days by analysis of variance (ANOVA). Gene arrays were also used to evaluate osteogenic gene expression from 0.5 mg simvastatin in murine calvaria (N = 12). RESULTS: There was a 45% increase in bone area with 0.5 mg simvastatin versus gel control (P <0.001; similar to the 2.2-mg dose), and clinical swelling was reduced compared to the high simvastatin dose (P <0.05). The 0.1-mg simvastatin dose failed to stimulate significant bone growth. NS-398 and indomethacin reduced inflammation and bone growth. Simvastatin significantly upregulated procollagen, fibronectin, and matrix metalloproteinase-13 genes. CONCLUSION: Reducing the simvastatin dose from 2.2 to 0.5 mg reduced inflammation to a more clinically acceptable level without sacrificing bone-growth potential, but COX-associated inflammation appears to be necessary for in vivo bone growth.

Alveolar bone loss is associated with circulating anti-citrullinated protein antibody (ACPA) in patients with rheumatoid arthritis.

BACKGROUND: This study examines: 1) alveolar bone loss (ABL), a hallmark of periodontitis, in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) patients versus control patients with osteoarthritis (OA); and 2) the association of ABL with RA disease activity and ACPA concentrations, including multiple antigen-specific ACPA. METHODS: This multicenter case-control study includes 617 patients diagnosed with RA (n = 287) or OA (n = 330). Panoramic radiographs were taken; patients were categorized into low, moderate, or high tertiles based on mean percentage ABL. Serum ACPA was measured using second-generation anticyclic citrullinated peptide enzyme-linked immunosorbent assay and a multiplex platform to assess distinct antigen-specific ACPA. A generalized linear mixed model for binary data was used to compare stratified ABL in RA versus OA patients. Associations of moderate and high ABL (versus low) with RA disease activity and severity measures were examined using multivariate regression. Antigen-specific ACPA responses were compared among ABL tertiles using significance analysis of microarrays. RESULTS: ACPA-positive patients with RA had a significantly higher mean percentage of sites with ABL >20% compared with patients with OA (P = 0.03). After multivariate adjustment, greater ABL was significantly associated with higher serum ACPA concentration (P = 0.004), 28-joint Disease Activity Score (P = 0.023), health assessment questionnaire disability (P = 0.05), tender joint count (P = 0.02) and joint space narrowing scores (P = 0.05) among patients with RA. ACPAs targeting citrullinated vimentin and histone were significantly higher in moderate and high ABL groups versus low, regardless of smoking status (q <0.1%). CONCLUSIONS: Greater ABL was associated with higher ACPA, consistent with findings at articular sites. ACPA targeting could provide novel insight into important linkages between RA and periodontitis.

Impact of increased occlusal contact, interleukin-1 genotype, and periodontitis severity on gingival crevicular fluid IL-1beta levels.

BACKGROUND: The study of occlusal therapy in humans with periodontitis is problematic due to potential irreversible bone loss in control subjects. The hypothesis of this pilot investigation was that increased interocclusal contact initiated by short-term occlusal splint disuse would increase tooth mobility and the bone-resorptive cytokine interleukin (IL)-1beta in gingival crevicular fluid (GCF), comparable to IL-1-positive genotype and increased periodontitis severity. METHODS: Nineteen non-smoking chronic periodontitis patients using nocturnal occlusal splints and undergoing periodontal maintenance in a private practice were evaluated at five time points: 24 hours after continuous splint use; 1, 2, and 3 days after no occlusal splint use; and 14 days after resumption of customary nighttime splint use. Subjects were evaluated to confirm that the plaque index and gingival index were < or = 1.0, and to categorize past periodontitis (moderate or severe) and IL-1 genotype (1A +4845 plus IL-1B +3954). Test sites on two anterior teeth vulnerable to occlusal trauma were sampled for mobility, GCF IL-1beta, and IL-1 receptor antagonist (ra). RESULTS: Tooth mobility remained low during the 3-day period when patients were not wearing their occlusal appliance. GCF IL-1beta decreased after not wearing the appliances (P = 0.016), especially at 48 hours. At this time, genotype-positive subjects had higher levels of GCF IL-1beta/IL-1ra than genotype-negative subjects (P = 0.045), and patients who had experienced severe periodontitis had higher IL-1beta levels than moderate periodontitis subjects (P = 0.004). CONCLUSIONS: These findings suggest that short-term discontinuation of occlusal splint therapy in non-smoking periodontitis patients undergoing periodontal maintenance does not result in potential signs of early occlusal trauma (increasing mobility or GCF IL-1beta). Longer-term studies may be needed to determine appropriate therapy applications for periodontitis-susceptible patients with definable occlusal discrepancies and/or parafunction.

Effects of simvastatin gels on murine calvarial bone.

BACKGROUND: The cholesterol-lowering drug simvastatin has been shown to stimulate murine calvarial bone growth after multiple injections. The purpose of this study was to test if similar bone stimulation could be induced by 2 single-dose drug delivery systems appropriate to periodontal therapy. METHODS: ICR Swiss mice were treated with the following protocols: 1) injection of methylcellulose gel alone, subcutaneously over the calvarium (INJ-GEL; n = 8); 2) injection of gel with simvastatin (INJ-SIM; 2.2 mg, n = 16); 3) polylactide membrane (PLA) containing gel alone implanted over calvarium (MEM-GEL; n = 10); 4) implanted PLA membrane containing gel and simvastatin (MEM-SIM; n = 10); and 5) untreated mice (n = 12). Animals were sacrificed after 22 or 44 days, calvaria decalcified and stained with hematoxylin and eosin, and images digitized and measured for bone thickness and area. Data were compared using analysis of variance. RESULTS: INJ-SIM stimulated a 53% (P = 0.02) increase at the thickest point of calvarial bone, while MEM-SIM caused a highly significant (P < or = 0.0005) increase in bone thickness (159% to 172%) and bone area (144% to 180%) compared to gel controls. Simvastatin gels caused soft tissue inflammation, which appeared to be related to bone increases. If INJ-SIM animals showing leakage of gel and/or no inflammation were excluded from analysis, INJ-SIM resulted in more bone (58% to 83%) than gel controls. An insignificant amount of SIM-stimulated bone was lost over the long term (44 days). CONCLUSIONS: A single, high dose of simvastatin gel can stimulate murine cranial bone apposition, particularly when delivered under an occlusive membrane. Both approaches should be investigated further for possible development for periodontal therapy.

Impact of local and systemic alendronate on simvastatin-induced new bone around periodontal defects.

BACKGROUND: Simvastatin has been shown to stimulate new bone growth on rat mandibles, but much of the bone is lost over time. The purpose of this study is to evaluate the impact of a locally or systemically applied antiresorptive agent (alendronate) on simvastatin-induced bone formation in and adjacent to a rat periodontal defect. METHODS: Fenestration defects were created over mandibular molar roots in 65 mature female Sprague-Dawley rats. Two weeks later, animals were divided into eight groups of eight to nine rats, and three weekly injections around the defect were applied: 1) 0.5 mg simvastatin in ethanol (SIM-EtOH); 2) 0.5 mg simvastatin in alendronate-cyclodextrin conjugate (SIM-ALN-CD); 3) EtOH alone; 4) ALN-CD alone; or 5) no injections. Twenty-four animals were evaluated for new bone width around the defect 21 days after the last injections (short-term) and 41 rats were followed for 48 days (long-term). Three SIM-EtOH groups of long-term rats also were subjected to 2 weeks of daily systemic ALN or saline either during or 3 to 4 weeks after SIM-EtOH injections. Decalcified, hematoxylin-and-eosin-stained cross-sections of the defect area were analyzed for new bone width and groups were compared using mixed-model analyses of variance. RESULTS: All groups showed nearly 100% bone fill, with no differences among the short-term groups. However, in the long-term animals, two-fold to three-fold more new bone width (≤ 0.004) was seen around the periphery of the defect with the use of systemic ALN after SIM-EtOH injections (0.93 ± 0.12 and 0.78 ± 0.11 mm with early and late systemic ALN, respectively) compared to local SIM/ALN-CD preparations (0.32 ± 0.10 mm) or short-term SIM-EtOH injections (0.35 ± 0.10 mm). No significant new cementum formation or ankylosis was noted. CONCLUSION: The use of a short course of systemic ALN during the healing period after bone anabolic SIM injections has the potential to enhance local bone augmentation.

Inflammatory mediator release following bone grafting in humans: a pilot study.

AIM: The aim of this pilot study was to track markers of periodontal inflammation and bone resorption associated with decalcified freeze-dried bone allografts. MATERIAL AND METHODS: Eleven subjects completed standardized treatment of intrabony defects > or =3 mm with allografts. Gingival crevicular fluid was collected from the defect site and an adjacent interproximal site within the surgical field at baseline, 2, 4, and 8 weeks post-operatively, and analysed for biochemical markers of inflammation/bone resorption. Probing depth, recession, bleeding on probing, plaque, and 6-month radiographic bone height change were measured. RESULTS: Both prostaglandin E(2) (p=0.007) and bone-specific type 1 collagen (p=0.01) increased in crevicular fluid after 2 weeks in the bone graft sites. Matrix metalloproteinase-9 levels remained constant over time. There were positive correlations between prostaglandin levels during the first 8 weeks and bone height change over 6 months. CONCLUSIONS: Periodontal bone grafts stimulate an inflammatory response during the first 2 weeks post-operatively, and the potential negative effects of inhibiting prostaglandins post-operatively should be investigated further.