Mark I. Ryder

Oral pathobiont induces systemic inflammation and metabolic changes associated with alteration of gut microbiota

Periodontitis has been implicated as a risk factor for metabolic disorders such as type 2 diabetes, atherosclerotic vascular diseases, and non-alcoholic fatty liver disease. Although bacteremias from dental plaque and/or elevated circulating inflammatory cytokines emanating from the inflamed gingiva are suspected mechanisms linking periodontitis and these diseases, direct evidence is lacking. We hypothesize that disturbances of the gut microbiota by swallowed bacteria induce a metabolic endotoxemia leading metabolic disorders. To investigate this hypothesis, changes in the gut microbiota, insulin and glucose intolerance, and levels of tissue inflammation were analysed in mice after oral administration of Porphyromonas gingivalis, a representative periodontopathogens. Pyrosequencing revealed that the population belonging to Bacteroidales was significantly elevated in P. gingivalis-administered mice which coincided with increases in insulin resistance and systemic inflammation. In P. gingivalis-administered mice blood endotoxin levels tended to be higher, whereas gene expression of tight junction proteins in the ileum was significantly decreased. These results provide a new paradigm for the interrelationship between periodontitis and systemic diseases.

The biomechanical characteristics of the bone-periodontal ligament-cementum complex

The relative motion between the tooth and alveolar bone is facilitated by the soft-hard tissue interfaces which include periodontal ligament-bone (PDL-bone) and periodontal ligament-cementum (PDL-cementum). The soft-hard tissue interfaces are responsible for attachment and are critical to the overall biomechanical efficiency of the bone-tooth complex. In this study, the PDL-bone and PDL-cementum attachment sites in human molars were investigated to identify the structural orientation and integration of the PDL with bone and cementum. These attachment sites were characterized from a combined materials and mechanics perspective and were related to macro-scale function. High resolution complimentary imaging techniques including atomic force microscopy, scanning electron microscopy and micro-scale X-ray computed tomography (Micro XCT) illustrated two distinct orientations of PDL; circumferential-PDL (cir-PDL) and radial-PDL (rad-PDL). Within the PDL-space, the primary orientation of the ligament was radial (rad-PDL) as is well known. Interestingly, circumferential orientation of PDL continuous with rad-PDL was observed adjacent to alveolar bone and cementum. The integration of the cir-PDL was identified by 1-2 microm diameter PDL-inserts or Sharpeys fibers in alveolar bone and cementum. Chemically and biochemically the cir-PDL adjacent to bone and cementum was identified by relatively higher carbon and lower calcium including the localization of small leucine rich proteins responsible for maintaining soft-hard tissue cohesion, stiffness and hygroscopic nature of PDL-bone and PDL-cementum attachment sites. The combined structural and chemical properties provided graded stiffness characteristics of PDL-bone (E(r) range for PDL: 10-50 MPa; bone: 0.2-9.6 GPa) and PDL-cementum (E(r) range for cementum: 1.1-8.3 GPa), which was related to the macro-scale function of the bone-tooth complex.

Structure, chemical composition and mechanical properties of human and rat cementum and its interface with root dentin

This work seeks to establish comparisons of the physical properties of rat and human cementum, root dentin and their interface, including the cementum-dentin junction (CDJ), as a basis for future studies of the entire periodontal complex using rats as animal models. In this study the structure, site-specific chemical composition and mechanical properties of cementum and its interface with root dentin taken from 9- to 12-month-old rats were compared to the physiologically equivalent 40- to 55-year-old human age group using qualitative and quantitative characterization techniques, including histology, atomic force microscopy (AFM), micro-X-ray computed tomography, Raman microspectroscopy and AFM-based nanoindentation. Based on results from this study, cementum taken from the apical third of the respective species can be represented as a woven fabric with radially and circumferentially oriented collagen fibers. In both species the attachment of cementum to root dentin is defined by a stiffness-graded interface (CDJ/cementum-dentin interface). However, it was concluded that cementum and the cementum-dentin interface from a 9- to 12-month-old rat could be more mineralized, resulting in noticeably decreased collagen fiber hydration and significantly higher modulus values under wet conditions for cementum and CDJ (E(rat-cementum)=12.7+/-2.6 GPa; E(rat-CDJ)=11.6+/-3.2 GPa) compared to a 40- to 55-year-old human (E(human-cementum)=3.73+/-1.8 GPa; E(human-CDJ)=1.5+/-0.7 GPa). The resulting data illustrated that the extensions of observations made from animal models to humans should be justified with substantial and equivalent comparison of data across age ranges (life spans) of mammalian species.

Cemental changes in teeth with heavily infected root canals

The prevalence of “pathological granules” in the unexposed cementum of 20 teeth with pulpitis was compared with that of 20 teeth with heavily infected root canals. It was found that 35% of the teeth with microbe-laden root canals exhibited this cemental change. Teeth with pulpitis, but without massive numbers of bacteria in their root canals, did not have this structural change. The results support the suggestion that bacteria play an important role in the formation of pathological granules in cementum and the underlying dentin.

Periodontal disease in HIV/AIDS.

Since the early 1990s, the death rate from AIDS among adults has declined in most developed countries, largely because of newer antiretroviral therapies and improved access to these therapies. In addition, from 2006 to 2011, the total number of new cases of HIV infection worldwide has declined somewhat and has remained relatively constant. Nevertheless, because of the large numbers of existing and new cases of HIV infection, the dental practitioner and other healthcare practitioners will still be required to treat oral and periodontal conditions unique to HIV/AIDS as well as conventional periodontal diseases in HIV-infected adults and children. The oral and periodontal conditions most closely associated with HIV infection include oral candidiasis, oral hairy leukoplakia, Kaposis sarcoma, salivary gland diseases, oral warts, other oral viral infections, linear gingival erythema and necrotizing gingival and periodontal diseases. While the incidence and prevalence of these oral lesions and conditions appear to be declining, in part because of antiretroviral therapy, dental and healthcare practitioners will need to continue to diagnose and treat the more conventional periodontal diseases in these HIV-infected populations. Finding low-cost and easily accessible and acceptable diagnostic and treatment approaches for both the microbiological and the inflammatory aspects of periodontal diseases in these populations are of particular importance, as the systemic spread of the local microbiota and inflammatory products of periodontal diseases may have adverse effects on both the progression of HIV infection and the effectiveness of antiretroviral therapy approaches. Developing and assessing low-cost and accessible diagnostic and treatment approaches to periodontal diseases, particularly in developing countries, will require an internationally coordinated effort to design and conduct standardized clinical trials.

Ammonia as a potential mediator of adult human periodontal infection: inhibition of neutrophil function.

Neutrophils (polymorphonuclear leucocytes) are the principal cell of the host defence system. Consequently, if periodontal pathogen-derived substances in the gingival crevice significantly inhibit their function, they could shift the bacterial-host balance in favour of the bacteria. The hypothesis that ammonia can inhibit neutrophil function was tested. Ammonia was specifically selected because periodontal pathogens produce substantial amounts of ammonia. The findings indicated that ammonia can inhibit neutrophil phagocytosis, degranulation and oxygen metabolism. Ammonia decreased the total number of phagocytosing polymorphonuclear neutrophils (66% of control) and also decreased degranulation (61% of control). Ammonia decreased oxygen metabolism of both resting and stimulated neutrophils (33 and 42% of control, respectively). These observations support the hypothesis that ammonia can inhibit the function of polymorphonuclear leukocytes. They suggest that the presence of ammonia in the gingival crevice may increase the risk of development of periodontal disease.

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system.

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7 ± 0.1 to 0.9 ± 0.2 GPa) and cementum (0.6 ± 0.1 to 0.8 ± 0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.

Nicotine Effects on Neutrophil F-Actin Formation and Calcium Release: Implications for Tobacco Use and Pulmonary Diseases

Alterations in neutrophil functions by tobacco components may play a pivotal role in pulmonary emphysema. This study examined the role of nicotine in altering F-actin formation and calcium (Ca2+) release (two early events in neutrophil motility). The effects of these alterations on the motile function of phagocytosis were also examined. Human peripheral neutrophils from medically healthy nonsmoking subjects were incubated with nicotine at concentrations normally encountered during acute exposure to cigarette smoke (10(-2) to 10(-5) M) and/or the chemotactic peptide FLPEP (10(-7) M). Relative F-actin stain was determined by NBD phallacidin staining followed by flow cytometry. Intracellular Ca2+ was determined by INDO-1 AM loading followed by emission ratio quantitation by fluorometry. Phagocytosis was determined by the % phagocytic cells with carboxylated microspheres. Incubation of neutrophils with varying concentrations of nicotine resulted in a significant elevation of the relative F-actin stain at 30 s at 10(-2) and 10(-3) M (p < .05, ANOVA) and at 30 min at 10(-2) to 10(-4) M (p < 0.05). In time course studies with 10(-7) M FLPEP stimulation, there was a approximately 325% rise in relative F-actin stain at 30-60 s, followed by a gradual decrease to near baseline levels. There was an immediate rise in Ca2+ to approximately 150% over baseline values, followed by a gradual decrease to baseline. By contrast, stimulation with nicotine demonstrated a approximately 105% increase in relative F-actin staining at 10(-2) M (p < .001, ANOVA) and a smaller increase at 10(-3) M, which remained elevated up to 600 s. Intracellular Ca2+ levels also rose in a dose-dependent manner with an increased of 700% over baseline with 10(-2) M nicotine, and remained elevated up to 600 s. Coincubation with both FLPEP and nicotine demonstrated additive effects in relative F-actin staining at both maximal and submaximal concentrations. Preincubation with 10(-2) or 10(-3) M nicotine suppressed the % phagocytic cells by 32% and 16%, respectively (p < .001, ANOVA) with only a 1-4% reduction in cell viability (trypan blue exclusion). The results demonstrate that the concentration of nicotine during acute cigarette exposure can directly stimulate neutrophil F-actin formation and intracellular Ca2+ release by a mechanism different from peptide stimulation. The alteration of these two pivotal neutrophil signaling events by nicotine may in turn alter other neutrophil functions in tobacco-related pulmonary emphysema.

The Adherence to Bone by Cytoplasmic Elements of Osteoclasts

The intimate association observed between osteoclasts and bone has suggested that these cells may be adherent to the bony surface. We investigated this cell-surface relationship in parathyroid hormone-stimulated bone explants following applications of biophysically-induced cell detachment forces. Bone surfaces adjacent to disrupted osteoclasts were examined with transmission electron microscopy for the presence of residual cell membrane elements. Results of this study provide evidence for the adherence of osteoclasts to bony surfaces and implicate elements of both clear zone and ruffled border as cell-membrane bonded sites.

The plastic nature of the human bone–periodontal ligament–tooth fibrous joint

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (μ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by μ-XRF. Interestingly, the hygroscopic 10-30μm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.