Periodontal research contributions to basic sciences: From cell communication and host-parasite interactions to inflammation and bone biology

The periodontium comprises all structures surrounding the teeth, including gingiva, root cementum, periodontal ligament and alveolar bone. Those tissues aim to protect and support the teeth and are challenged by a residing microbiota that leads to subclinical inflammation even in physiological conditions. Periodontitis, a prevalent multicausal inflammatory and destructive disease, develops as a result from complex host-parasite interactions. This unique physiologic and pathologic scenario enables the development of research methods which allows conclusions beyond the simple understanding of periodontal homeostasis. The aim of this viewpoint was to explore potential contributions of periodontal research to a wide array of basic science specialties, such as cell and molecular biology, microbiology, immunology, endocrinology, rheumatology, among others.     

Time-dependent mechanical behaviour of the periodontal ligament

The process of tooth displacement in response to orthodontic forces is thought to be induced by the stresses and strains in the periodontium. The mechanical force on the tooth is transmitted to the alveolar bone through a layer of soft connective tissue, the periodontal ligament. Stress and/or strain distribution in this layer must be derived from mathematical models, such as the finite element method, because it cannot be measured directly in a non-destructive way. The material behaviour of the constituent tissues is required as an input for such a model. The purpose of this study was to determine the time-dependent mechanical behaviour of the periodontal ligament due to orthodontic loading of a tooth. Therefore, in vivo experiments were performed on beagle dogs. The experimental configuration was simulated in a finite element model to estimate the poroelastic material properties for the periodontal ligament. The experiments showed a two-step response: an instantaneous displacement of 14.10 +/- 3.21 microns within 4 s and a more gradual (creep) displacement reaching a maximum of 60.00 +/- 9.92 microns after 5 h. This response fitted excellently in the finite element model when 21 per cent of the ligament volume was assigned a permeability of 1.0 x 10(-14) m4/N s, the remaining 97 per cent was assigned a permeability of 2.5 x 10(-17) m4/N s. A tissue elastic modulus of 0.015 +/- 0.001 MPa was estimated. Our results indicate that fluid compartments within the periodontal ligament play an important role in the transmission and damping of forces acting on teeth.     

The role of periodontal disease in atherosclerotic cardiovascular disease

Atherosclerotic cardiovascular disease (ASCVD) includes a group of disorders of the heart and blood vessels and accounts for major morbidity and premature death worldwide. Periodontitis is a chronic inflammatory disease with the gradual destruction of supporting tissues around the teeth, including gingiva, periodontal ligament, alveolar bone, and cementum. Periodontitis has been found to potentially increase the risk of ASCVD. Generally, oral microorganisms and inflammation are the major factors for periodontitis to the incidence of ASCVD. Recently, evidence has shown that the loss of masticatory function is another important factor of periodontitis to the incidence of ASCVD. In this review, we illustrate the recent finding of the relationship between periodontitis and ASCVD, from a microscale perspective-oral microorganisms, inflammation, and tooth loss. With the high prevalence of periodontitis, it is important to add oral therapy as a regular ASCVD prevention strategy. Regular dental visits could be a helpful strategy for ASCVD patients or general medical practitioners.     

Caiman periodontium as an intermediate between basal vertebrate ankylosis-type attachment and mammalian "true" periodontium

The teeth of many fish, amphibia, and reptiles are attached to the alveolar bone via ankylosis. In contrast, mammalian periodontia are characterized by a gomphosis, an attachment of the tooth root in the alveolar bone socket via periodontal ligament fibers. Among the reptiles, the crocodilians are the only group featuring a gomphosis-type connection between tooth root and alveolar bone, while in other reptiles tooth-root and jawbone are connected via ankylosis. The purpose of the present study was to compare several key features of the crocodilian periodontium with those of the mammalian and noncrocodilian reptile periodontium. As experimental models for our study we chose the periodontium of newborn geckos (Hemidacylus turcicus), juvenile caimans (Caiman crocodilus crocodilus), and 10-day-postnatal Swiss-Webster mice (Mus musculus) as representative models for noncrocodilian reptiles, crocodilian reptiles, and mammals. The caiman periodontium emerged as an intermediary between the mineral-free mouse ligament and the mineralized gecko ankylosis-type attachment. Caiman ligament fibers were less organized than mouse ligament fibers but featured distinct fasciae surrounding ligament fiber bundles. Caiman Hertwig's epithelial root sheath (HERS) was similarly perforated as mouse HERS and distinctly different from the continuous gecko HERS. Both caiman and mouse HERS covered the entire tooth root length, while in the gecko HERS was limited to the coronal portion of the root, allowing for cementoid-mediated ankylosis at the apical tip of the root. We interpret our data to indicate distinct differences in mineral distribution, periodontal ligament fiber organization, and HERS distribution between noncrocodilian reptiles, crocodilian reptiles, and mammals. Mineral deposits in the caiman ligament may reflect an evolutionary position of the caiman periodontium between ankylosis and gomphosis.     

Natural frequency analysis of tooth stability under various simulated types and degrees of alveolar vertical bone loss

The aim of this study was to test natural teeth stability under various simulated types and degrees of alveolar vertical bone loss, as well as to assess the role that the surrounding bone played for maintaining tooth stability. A three-dimensional finite element model of the human maxillary central incisor with surrounding tissue, including periodontal ligament, enamel, dentin, pulp, and alveolar bone, was established. One side and multiple vertical bone loss were simulated by means of decreasing the surrounding bone level apically from the cemento-enamel junction in 1 mm steps incrementally downward for 10 mm. Natural frequency values of the incisor model with various types and degrees of bone loss were then calculated. The results showed that, with one-sided bone resorption, the model with labial bone loss had the lowest natural frequency decreasing rates (8.2 per cent). On the other hand, in cases of multiple bone loss, vertical bone resorption at the mesial and distal sides had more negative effects on tooth stability compared to vertical bone losses on facial and lingual sides. These findings suggest that the natural frequency method may be a useful, auxiliary clinical tool for diagnosis of vertical periodontal diseases.     

Age-dependent changes of the periodontal ligament in rats

Even after the end of the natural tooth eruption, there is a continuous renewal of the periodontal collagenous fiber system, depending on functional demands. The aim of this study was to analyse the age-dependent changes and regional differences of the collagen renewal rate of the periodontal ligament in healthy rats. The study was performed by autoradiography of the molars of rats aged 1, 8, and 18 months, where collagen was labelled by intravenously applied 3H-proline. After an 8-hour incorporation period, the animals were killed. For comparative examinations, molar roots were subdivided into cervical, middle, and apical thirds. Structural and quantitative analyses were performed by light microscopy and autoradiography, using an image-analysing computer-assisted operating unit that determined the 3H-proline-labelled collagen by photometry based on extinction measurement. With increasing age of the animals, the number of silver grains (3H-proline-blackened collagen) was reduced and the quantitative evaluation indicated a reduction of 3H-proline in the periodontal ligament. The lowest level of 3H-proline activities was observed in the middle, and the highest level in the apical root third, independent of age. All preparations revealed condensations of silver grains, which were located in the region of the periodontal ligament adjacent to the alveolar bone, but did not reveal any preferred position with regard to the dental topography. With progressive age, the uptake of 3H-proline in the periodontal ligament was reduced by about 20 to 30%, a result that corresponds to a decrease in collagenous fiber production. Collagen was mainly formed in the apical and cervical root third, starting from the alveolar bone side, presumably in response to functional strain.     

Resin luting materials: Tissue response in dog's teeth

The aim of this study was to evaluate radiographically and histologically the pulpal and periapical response to self‐adhesive (Rely X? Unicem) and self‐etching and self‐curing (Multilink^®) resin‐based luting materials in deep cavities in dogs' teeth. Deep class V cavities (0.5‐mm–thick dentin) were prepared in 60 canine premolars and the following materials were applied on cavity floor: Groups I/V—RelyX? Unicem; Groups II/VI—Multilink^®; Groups III/VII—zinc phosphate cement (control) and; Groups IV/VIII—gutta‐percha (control). Cavities were restored with silver amalgam. Animals were euthanized after 10 days (groups I–IV) and 90 days (groups V–VIII). Tooth/bone blocks were radiographed and processed for histopathological evaluation of pulp and periapical tissue response to the materials. All materials presented similar histopathological features and radiographic findings at both periods. The pulp tissue was intact. The apical and periapical regions and periodontal ligament thickness were normal. No inflammatory cells, resorption of mineralized tissue (dentin, cementum, and alveolar bone) or bacteria were observed. The lamina dura was intact and no areas of periapical bone rarefaction or internal/external root resorption were observed radiographically. It can be concluded that Rely X? Unicem and Multilink^® caused no adverse tissue reactions and may be indicated for cementation of indirect restorations in deep dentin cavities without pulp exposure. Microsc. Res. Tech. 78:1098–1103, 2015. © 2015 Wiley Periodicals, Inc.     

Exosomes derived from osteoclasts under compression stress inhibit osteoblast differentiation

Orthodontic tooth movement is triggered by orthodontic force loading on the periodontal ligament and is achieved by alveolar bone remodeling, which is regulated by intimate crosstalk between osteoclastogenesis and osteoblast differentiation. Whether the communication between osteoclasts and osteoblasts is influenced by orthodontic compression stress requires further clarification. In this study, osteoclasts were differentiated for 10 days. On day 4 of differentiation, the number of pre-osteoclasts peaked, as determined by the increased expression of RANK and the number of multinucleated cells. After 24 h of compression stress loading, on day 4, the number of osteoclasts increased, and the optimal magnitude of stress to promote osteoclastogenesis was determined as 1 g/cm2. Moreover, the results of RNA-sequencing analysis showed that the miRNA expression profile changed markedly after compression loading and that many of the altered miRNAs were associated with cell communication functions. A series of indirect co-culture experiments showed an inhibitory effect of osteoclasts on osteoblast differentiation, especially after compression. Next, we added osteoclast-derived exosomes to hPDLSCs during osteoblast differentiation. Exosomes derived from osteoclasts under compression (cEXO) showed a greater inhibitory effect on osteoblast differentiation, compared to exosomes from osteoclasts without compression (EXO). Therefore, we analyzed differentially expressed miRNAs associated with bone development functions in exosomes: miR-223-5p and miR-181a-5p were downregulated, whereas miR-133a-3p, miR-203a-3p, miR-106a-5p, and miR-331-3p were upregulated; these altered expressions may explain the enhanced inhibitory effect of compression stress.     

The relation between structural,rugometric and fractal characteristics of hard dental tissues at micro and nano levels

Human tooth exhibits a structure of a mixture of inorganic hydroxyapatite nanocrystals and organic phases. The aim of this study is to investigate different tissues of human canine teeth surface along with the micro structure parameters of each tissue. X‐ray diffraction (XRD) is used to study the amorphous or crystalline nature of each tissue with different mineral compositions and crystalline structures where the highest crystalline quality is related to enamel. The surfaces are also examined by energy‐dispersive X‐ray spectrometry. Moreover, crystalline quality factor is carried out to estimate the crystallinity of the tissues. Also, based on the basic Scherrer equation, the Williamson–Hall equation is applied to extend the formula for the XRD. Enamel and cementum tissues of a typical human tooth, which look similar, are composed of a large variety of wide lines with different widths through Raman spectra analysis. In addition, the applied scanning electron microscopy extracts similar morphology for all tissues with round granular structures which are denser in the cementum. Atomic force microscopy is finally used for investigation of micro‐morphologies of the different tissues and the results are compared with the fractal analysis which ends to the bifractal and anisotropic nature of enamel and cementum along with monofractal and isotropic nature of dentin.     

Periodontal regeneration: a challenge for the tissue engineer?

Periodontitis affects around 15 per cent of human adult populations. While periodontal treatment aimed at removing the bacterial cause of the disease is generally very successful, the ability predictably to regenerate the damaged tissues remains a major unmet objective for new treatment strategies. Existing treatments include the use of space-maintaining barrier membranes (guided tissue regeneration), use of graft materials, and application of bioactive molecules to induce regeneration, but their overall effects are relatively modest and restricted in application. The periodontal ligament is rich in mesenchymal stem cells, and the understanding of the signalling molecules that may regulate their differentation has increased enormously in recent years. Applying these principles for the development of new tissue engineering strategies for periodontal regeneration will require further work to determine the efficacy of current experimental preclinical treatments, including pharmacological application of growth factors such as bone morphogenetic proteins (BMPs) or Wnts, use of autologous stem cell reimplantation strategies, and development of improved biomaterial scaffolds. This article describes the background to this problem, addresses the current status of periodontal regeneration, including the background biology, and discusses the potential for some of these experimental therapies to achieve the goal of clinically predictable periodontal regeneration.     

Mechanobiology of bone healing and regeneration: in vivo models

Mechanical boundary conditions are well known to influence the regeneration of bone and mechanobiology is the study of how mechanical or physical stimuli regulate biological processes. In vivo models have been applied over many years to investigate the effects of mechanics on bone healing. Early models have focused on the influence of mechanical stability on healing outcome, with an interest in parameters such as the magnitude of interfragmentary movement, the rate and timing of application of micromotion and the number of loading cycles. As measurement techniques have been refined, there has been a shift in orders of magnitude from investigations targeted at the organ level to those targeted at the tissue level and beyond. An understanding of how mechanics influences tissue differentiation during repair and regeneration crucially requires spatial and temporal knowledge of both the local mechanical environment in the healing tissue and a characterization of the tissues formed over the course of regeneration. Owing to limitations in the techniques available to measure the local mechanical conditions during repair directly, simulation approaches, such as the finite element method, are an integral part of the mechanobiologist's toolkit, while histology remains the gold standard in the characterization of the tissue formed. However, with rapid advances occurring in imaging modalities and methods to characterize tissue properties, new opportunities exist to better understand the role of mechanics in the biology of bone regeneration. Combined with developments in molecular biology, mechanobiology has the potential to offer exciting, new regenerative treatments for bone healing.     

Scanning electron microscopic observation of microvasculature in periodontium.

In this study, vascular resin cast models in the periodontium of beagle dogs were prepared and three-dimensional observation of the relationship between the gingiva and periodontal ligament (PDL) vascular network was performed. After the perfusion of Ringer's solution and fixative, synthetic resin was injected from the inferior alveolar arteries. Soft tissue was digested by proteinase solution and specimens were examined under scanning electron microscope (SEM). The gingival vascular network (GVN) in the region facing the teeth consisted of sulcular and junctional epithelium. The vascular network of the sulcular epithelium (SE) had a renal glomerulus-like form and the junctional epithelium (JE) consisted of squamous mesh. The gingival sulcular fluid exudated from the vascular network directly beneath the JE, and leukocytes permeated from the vascular network beneath the epithelium. Thus, we considered that the GVN performs an important function in the protection against the inflammation. Periodontal ligament had a polygonal mesh vascular network that was anastomosed to the venous plexus of alveolar bone through Volkmann's canals (VC). When occlusal force was applied, the blood in the periodontal vessels flowed out through VC into the bone marrow, and when the force was removed, it flowed backward into the PDL. This blood transfer acted as an absorber against occlusal force. Our findings suggest that the blood vessels of the gingiva perform an important function in defending against inflammation, while the blood vessels of the PDL play a key role in absorbing occlusal force.     

Comparative study of decalcification versus nondecalcification for histological evaluation of one‐wall periodontal intrabony defects in dogs

Biological reactions between biomaterials and surrounding tissues, analyzed by histology, may be important predictors of clinical healing pattern and selection of slide preparation techniques requires a careful consideration regarding sample properties. In this study, we compared histology of bone specimens prepared with or without decalcification and performed histological and histomorphometrical assessments. For the histological evaluation, one‐wall intrabony defects were created around the mandibular molars of six adult dogs, filled with biphasic calcium phosphate, synthetic bone graft material/recombinant human bone morphogenic protein‐2, and healing pattern was histologically evaluated at 4 and 12 weeks. New bone formation in 5 × 4 × 4 mm defects and the length of new cementum, connective tissue attachments around the teeth and number of osteoclasts were measured by histomorphometric analysis. After decalcification, new cementum was easily observed and was significantly increased at week 4. In nondecalcified samples, significantly increased connective tissue attachments were seen at week 12. After 12 weeks, the number of countable multinucleated osteoclasts was significantly increased by 62% in nondecalcified versus calcified tissue sections (P = 0.030). Histomorphometric results may be significantly affected by histological preparation method and therefore, selecting the most appropriate histological preparation method is essential for reliable diagnosis and evaluation of bony samples in studies analyzing tissue regeneration. Microsc. Res. Tech. 78:94–104, 2015. © 2014 Wiley Periodicals, Inc.     

Morphological study and stress analysis of Korean Mandibular second premolar

Tooth morphology has an important role in esthetical characteristics, functional performance and physiological action. Mandibular second premolars among teeth were taken for a close investigation with a micro-CT for a nondestructive measurement. Thus, the shape of the tooth was classified into a few partitions and measured by setting the reference axis to express internal and external morphology numerically. About a thousand tomographic images were obtained for each specimen and were reconstructed to a three-dimensional form. The lengths and widths of each dental tissue were measured from the three-dimensional form. In this tooth type, the width of dentine is comparatively larger than that of enamel, and buccal tissue of the crown is thicker than lingual tissue. Hence, buccal tissue could afford to withstand against exterior impact or stimulus. Finite element models were drawn up, applied with an ordinary occlusal force and analyzed to get the stress distribution. Thus it was shown that the irregular feature of the tooth is not useful only to masticating and pronouncing as well known, but that it is also suitable for protecting inner tissue by dispersing stress and delivering proper pressure to periodontal tissue to continue a physiological action. These results could be used for development of dental implants and clinical trials.     

Serial section reconstruction using a computer graphics system: Applications to intracellular structures in yeast cells and to the periodontal structure of dogs' teeth

A computer graphics system for reconstruction from serial section micrographs was applied to intracellular details of a yeast target cell (Saccharomyces cerevisiae cell) induced by the α factor mating pheromone and was also applied to a periodontal structure of a dog tooth moved orthodontically. In the former, intracellular organelles and a distribution of vesicles could be clearly observed through the cell membrane using the transparent display method in which the smoothing of the reconstructed outer cell membrane surface by computer processing was applied to the transparent display. In the latter case, by cutting through a reconstructed dog tooth and its periodontal tissues, labiolingual and mesiodistal cut surfaces of the tooth and of adjacent alveolar bone could be observed with fine details (232 sections were used).     

外摆线型单螺杆泵螺杆-衬套副的运动特性分析

通过分析外摆线型单螺杆泵螺杆-衬套副的相对运动,发现螺杆-衬套副的固定接触点出现在衬套骨线上,衬套可采用金属材料,螺杆可在金属基体表面黏接橡胶外套;用瞬心法推导出齿凸接触点和齿凹接触点处相对滑动速度的计算公式,并用MATLAB生成速度曲线,用SolidWorks建立螺杆泵实体模型,用SolidWorks Motion进行运动仿真,对比速度仿真值与速度公式曲线可知,拟合度很高,最大偏差率仅为-0.101%;将速度曲线与啮合状态进行对应,发现齿凹接触点处相对滑动速度的最大值发生在螺杆齿凸中点与衬套齿凹中点相接触处,齿凸接触点处相对滑动速度最大值发生在螺杆齿凸中点与衬套齿凸中点相接触处。     

Differentiation and functions of osteoclasts and odontoclasts in mineralized tissue resorption

The differentiation and functions of osteoclasts (OC) are regulated by osteoblast-derived factors such as receptor activator of NFKB ligand (RANKL) that stimulates OC formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. In examination of the preosteoclast (pOC) culture, pOCs formed without any additives expressed tartrate-resistant acid phosphatase (TRAP), but showed little resorptive activity. pOC treated with RANKL became TRAP-positive OC, which expressed intense vacuolar-type H(+)-ATPase and exhibited prominent resorptive activity. Such effects of RANKL on pOC were completely inhibited by addition of OPG. OPG inhibited ruffled border formation in mature OC and reduced their resorptive activity, and also induced apoptosis of some OC. Although OPG administration significantly reduced trabecular bone loss in the femurs of ovariectomized (OVX) mice, the number of TRAP-positive OC in OPG-administered OVX mice was not significantly decreased. Rather, OPG administration caused the disappearance of ruffled borders and decreased H(+)-ATPase expression in most OC. OPG deficiency causes severe osteoporosis. We also examined RANKL localization and OC induction in periodontal ligament (PDL) during experimental movement of incisors in OPG-deficient mice. Compared to wild-type OPG (+/+) littermates, after force application, TRAP-positive OC were markedly increased in the PDL and alveolar bone was severely destroyed in OPG-deficient mice. In both wild-type and OPG-deficient mice, RANKL expression in osteoblasts and fibroblasts became stronger by force application. These in vitro and in vivo studies suggest that RANKL and OPG are important regulators of not only the terminal differentiation of OC but also their resorptive function. To determine resorptive functions of OC, we further examined the effects of specific inhibitors of H(+)-ATPase, bafilomycin A1, and lysosomal cysteine proteinases (cathepsins), E-64, on the ultrastructure, expression of these enzymes and resorptive functions of cultured OC. In bafilomycin A1-treated cultures, OC lacked ruffled borders, and H(+)-ATPase expression and resorptive activity were significantly diminished. E-64 treatment did not affect the ultrastructure and the expression of enzyme molecules in OC, but significantly reduced resorption lacuna formation, by inhibition of cathepsin activity. Lastly, we examined the expression of H(+)-ATPase, cathepsin K, and matrix metalloproteinase-9 in odontoclasts (OdC) during physiological root resorption in human deciduous teeth, and found that there were no differences in the expression of these molecules between OC and OdC. RANKL was also detected in stromal cells located on resorbing dentine surfaces. This suggests that there is a common mechanism in cellular resorption of mineralized tissues such as bone and teeth.     

Regeneration of periodontal Ruffini endings in adults and neonates

We reviewed the regeneration of periodontal Ruffini endings, primary mechanoreceptors in the periodontal ligament, following injury to the inferior alveolar nerve (IAN) in adult and neonatal rats. Morphologically, mature Ruffini endings are characterized by an extensive arborization of axonal terminals and association with specialized Schwann cells, called lamellar or terminal Schwann cells. Following injury to IAN in the adult, the periodontal Ruffini endings of the rat lower incisor ligament regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells migrate into regions where they are never found under normal conditions. The development of periodontal Ruffini endings of the rat incisor is closely associated with the eruption of the teeth; the morphology and distribution of the terminal Schwann cells became almost identical to those in adults during postnatal days 15-18 (PN 15-18d) when the first molars appear in the oral cavity, while the axonal elements showed extensive ramification around PN 28d when the functional occlusion commences. When the IAN was injured in neonates, the regeneration of periodontal Ruffini endings was delayed compared with the adults. The migration of terminal Schwann cells is also observed following IAN injury, after which the distribution of terminal Schwann cells became almost identical to that of the adults, i.e., PN 14d. Since the interaction between axon and Schwann cell is important during regeneration and development, further studies are required to elucidate its molecular mechanism during the regeneration as well as the development of the periodontal Ruffini endings.