Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal ß-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

Food hardness can regulate orthodontic tooth movement in mice.

BACKGROUND AND OBJECTIVES: Orthodontic treatment is often accompanied with prescription of softer foods to patients. The question to ask is, is this prescribed load regimen congruent with Wolff's law, and does it provide an adequate mechanical stimulus to maintain the functional health of periodontal complex? This question was answered by studying the effects of mice chewing on soft food (SF) and hard food (HF) while undergoing experimental tooth movement (ETM). METHODS: Three-week-old C57BL/6 mice (n = 18) were fed either hard pellet (HF; n = 9) or soft-chow food (SF; n = 9). ETM was performed on mice at 8 weeks of age, and mice were euthanized at 1 min, 2 weeks, and 4 weeks (8, 10, and 12 weeks old, respectively). A logistic regression model was applied to the experimental data to extrapolate the prolonged effects of ETM on the physical features of the dentoalveolar joint (DAJ). RESULTS: By 12 weeks, mice that chewed on SF expressed wider periodontal ligament space than those that chewed on HF. Mice that chewed on SF demonstrated increased alveolar socket roughness with larger alveoli and decreased bone volume fraction but with significantly lower bone mineral density and reduced overall tooth movement. CONCLUSIONS: These altered physical features when contextualized within the DAJ illustrated that (a) the regions farther away from the "site of insult" also undergo significant adaptation, and (b) these adaptations vary between mesial and distal sides of the periodontal complex and topographically differentiate in the direction of the ETM. These insights underpin the main conclusion, in that there is a need to "regulate chewing loads" as a therapeutic dose following ETM to encourage regeneration of periodontal complex as an effective clinical outcome. The discussed multiscale image analyses also can be used on patient cone beam computed tomography data to identify the effectiveness of orthodontic treatment within the realm of masticatory function.

Functional Adaptation of LPS-affected Dentoalveolar Fibrous Joints in Rats.

INTRODUCTION: The functional interplay between cementum of the root and alveolar bone of the socket is tuned by a uniquely positioned 70-80 µm wide fibrous and lubricious ligament in a dentoalveolar joint (DAJ). In this study, structural and biomechanical properties of the DAJ, periodontal ligament space (PDL-space also known as the joint space), alveolar bone of the socket, and cementum of the tooth root that govern the biomechanics of a lipopolysaccharide (LPS)-affected DAJ were mapped both in space and time. METHODS: The hemi-maxillae from 20 rats (4 control at 6 weeks of age, 4 control and 4 LPS-affected at 12 weeks of age, 4 control and 4 LPS-affected at 16 weeks of age) were investigated using a hybrid technique; micro-X-ray computed tomography (5 µm resolution) in combination with biomechanical testing in situ. Temporal variations in bone and cementum volume fractions were evaluated. Trends in mineral apposition rates (MAR) in additional six Sprague Dawley rats (3 controls, 3 LPS-affected) were revealed by transforming spatial fluorochrome signals to functional growth rates (linearity factor - RW) of bone, dentin, and cementum using a fast Fourier transform on fluorochrome signals from 100-µm hemi-maxillae sections. RESULTS: An overall change in LPS-affected DAJ biomechanics (a 2.5-4.5X increase in tooth displacement and 2X tooth rotation at 6 weeks, no increase in displacement and a 7X increase in rotation at 12 weeks; 27% increase in bone effective strain at 6 weeks and 11% at 12 weeks relative to control) was associated with structural changes in the coronal regions of the DAJ (15% increase in PDL-space from 0 to 6 weeks but only 5% from 6 to 12 weeks compared to control). A significant increase (p < 0.05) in PDL-space between ligated and age-matched control was observed. The bone fraction of ligated at 12 weeks was significantly lower than its age-matched control, and no significant differences (p > 0.05) between groups were observed at 6 weeks. Cementum in the apical regions grew faster but nonlinearly (11% and 20% increase in cementum fraction (CF) at 6 and 12 weeks) compared to control. Alveolar bone revealed site-specific nonlinear growth with an overall increase in MAR (108.5 µm/week to 126.7 µm/week after LPS treatment) compared to dentin (28.3 µm/week in control vs. 26.1 µm/week in LPS-affected) and cementum (126.5 µm/week in control vs. 119.9 µm/week in LPS-affected). A significant increase in CF (p < 0.05) in ligated specimens was observed at 6 weeks of age. CONCLUSIONS: Anatomy-specific responses of cementum and bone to the mechano-chemo stimuli, and their collective temporal contribution to observed changes in PDL-space were perpetuated by altered tooth movement. Data highlight the "resilience" of DAJ function through the predominance of nonlinear growth response of cementum, changes in PDL-space, and bone architecture. Despite the significant differences in bone and cementum architectures, data provided insights into the reactionary effects of cementum as a built-in compensatory mechanism to reestablish functional competence of the DAJ. The spatial shifts in architectures of alveolar bone and cementum, and consequently ligament space, highlight adaptations farther away from the site of insult, which also is another novel insight from this study. These adaptations when correlated within the context of joint function (biomechanics) illustrate that they are indeed necessary to sustain DAJ function albeit being pathological.

Biomechanical pathways of dentoalveolar fibrous joints in health and disease.

Spatial and temporal adaptations within periodontal tissues and their interfaces result from functional loads. Functional loads can be physiologic and/or pathologic in nature. The prolonged effect of these loads can alter the overall biomechanics of a dentoalveolar fibrous joint (dentoalveolar joint) by changing the form of the tooth root and its socket. This "sculpting" of the tooth root and alveolar bony socket is a consequence of several mechano-biological changes that occur within the periodontal complex of a load-bearing dentoalveolar joint. These include changes in biochemical expressions, structure, elemental composition, and mechanical properties of alveolar bone, the underlying tissues of the roots of teeth, and their interfaces. These physicochemical changes in tissues continue to prompt mechano-responsive biochemical activities at the attachment sites of periodontal ligament (soft) with bone (hard), and ligament with cementum (hard), which are the entheses of a load-bearing dentoalveolar joint. Forces at soft-hard tissue attachment sites between disparate materials with different stiffness values theoretically generate strain singularities or discontinuities. These discontinuities under prolonged functional loading increase the probability for failure to occur specifically at the enthesial zones. However, in a normal dentoalveolar joint, gradual stiffness gradients exist from ligament to bone, and from ligament to cementum. The gradual transitions in stiffness from softer ligament (lower stiffness) to harder bone or cementum (higher stiffness) or vice versa optimize tissue and interfacial strains. Optimization of tissue and ligament-enthesial physical and chemical properties facilitates transmission of cyclic forces of varying magnitudes and frequencies that collectively maintain the overall biomechanics of a dentoalveolar joint. The objectives of this review are 3-fold: (i) to illustrate physicochemical adaptations at the periodontal ligament entheses of a human periodontal complex affected by subgingival calculus; (ii) to demonstrate how to "program" the hallmarks of periodontitis in small-scale vertebrates in vivo to generate spatiotemporal maps of physicochemical adaptations in a diseased dentoalveolar joint; and (iii) to correlate dentoalveolar joint biomechanics in healthy and diseased states to spatiotemporal maps of physicochemical adaptations within respective periodontal tissues. This interdisciplinary approach demonstrates that physicochemical adaptations within periodontal tissues using the mechanics of materials (tissue mechanics), materials science (tissue composition), and mechano-biology (matrix molecules) can help explain the mechano-adaptation of dentoalveolar joints in normal and diseased functional states. Multiscale biomechanics and mechano-biology approaches can provide insights into the functional competence of a diseased relative to a normal dentoalveolar joint. Insights gathered from interdisciplinary and multiscale biomechanics approaches include the following: (i) physiologic loads related to chewing maintain a balance between mineral-forming and-resorbing biochemical cellular events, resulting in gradual stiffness gradients at the periodontal ligament entheses, and, in turn, sustain the overall biomechanics of a normal "healthy" dentoalveolar joint; (ii) pathologic loads resulting from tissue degradation and physical changes to the periodontal complex promote an abrupt stiffness gradient at the periodontal ligament entheses. The shift from gradual to an abrupt stiffness gradient could prompt a shift in the biochemical cascades, exacerbate mechano-responsive biochemical expressions at periodontal ligament entheses farther away from the site of insult, and culminate in joint degradation; (iii) sustained pathologic function on periodontally diseased joints exacerbates degradation of periodontal ligament entheses providing insights into "rescue therapy", such as the use of an adequate "mechanocal dose" to regain joint function; and (iv) spatiotemporal maps of changes in biochemical expressions, and physicochemical properties of strain-dominated affected sites, including the periodontal ligament entheses, can guide anatomy-specific therapeutics for tissue regeneration and/or disease control with the purpose of regaining dentoalveolar joint function. Modulation of occlusal loads could minimize disease progression and potentially assist in regaining functional attachment of ligament to bone and/or ligament to cementum of the dentoalveolar joint. Elucidating mechanisms that drive the breakdown of the functionally active periodontal complex burdened with microbes will provide the required critical insights into regenerative medicine and/or biomimetic approaches that would facilitate rescue/regain of dentoalveolar joint function.

Microanatomical changes and biomolecular expression at the PDL-entheses during experimental tooth movement.

The novel aspect of this study was to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL)-space within a mechanically activated periodontal complex. The PDL is unique as it is the only ligament with both innervation and vascularization. Maxillary molars in 6-week-old male C57BL/6 mice (N = 5) were experimentally translated for 2 weeks using an elastic spacer. Contralateral teeth were used as controls. Mechanical testing of the periodontal complex of a mouse in situ and imaging using X-ray micro-computed tomography (micro-XCT) illustrated deformations within blood vessels (BV) of the PDL. PDL-bone and PDL-cementum entheses at the widened and narrowed PDL-spaces following experimental tooth movement (ETM) illustrated osterix (OSX), bone sialoprotein (BSP), cluster of differentiation 146 (CD146), and protein gene product 9.5 (PGP9.5), indicating active remodeling at these sites. PGP9.5 positive nerve bundles (NBs) were co-localized with multinucleated cells (MCs), Howship's resorption lacunae, and CD146 positive BVs. Association between nerves and MC was complemented by visualizing the proximity of osmium tetroxide stained NBs with the ultrastructure of MCs by performing scanning transmission electron microscopy. Spatial association of NB with BV, and NB with MC, provided insights into the plausible co-activation of NBs to initiate osteoclastic activity. Resorption of mineral occurred as an attempt to restore PDL-space of the load-bearing complex, specifically at the PDL-entheses. Mapping of anatomy-specific structural elements and their association with regenerative molecules by correlating light and electron micrographs provided insights into the use of these extracellular matrix molecules as plausible targets for pharmacological interventions related to tooth movement. Within the realm of tissue regeneration, modulation of load can reverse naturally occurring mineral formation to experimentally induced resorption, and naturally occurring mineral resorption to experimentally induced formation at the enthesial sites to permit tooth translation.

Removal of SOST or blocking its product sclerostin rescues defects in the periodontitis mouse model.

Understanding periodontal ligament (PDL) biology and developing an effective treatment for bone and PDL damage due to periodontitis have been long-standing aims in dental medicine. Here, we first demonstrated by cell lineage tracing and mineral double-labeling approaches that murine PDL progenitor cells display a 2- and 3-fold higher mineral deposition rate than the periosteum and endosteum at the age of 4 weeks, respectively. We next proved that the pathologic changes in osteocytes (Ocys; changes from a spindle shape to round shape with a >50% reduction in the dendrite number/length, and an increase in SOST) are the key pathologic factors responsible for bone and PDL damage in periostin-null mice (a periodontitis animal model) using a newly developed 3-dimensional FITC-Imaris technique. Importantly, we proved that deleting the Sost gene (a potent inhibitor of WNT signaling) or blocking sclerostin function by using the mAb in this periodontitis model significantly restores bone and PDL defects (n = 4-5; P < 0.05). Together, identification of the key contribution of the PDL in normal alveolar bone formation, the pathologic changes of the Ocys in periodontitis bone loss, and the novel link between sclerostin and Wnt signaling in the PDL will aid future drug development in the treatment of patients with periodontitis.

Contact ratio and adaptations in the maxillary and mandibular dentoalveolar joints in rats and human clinical analogs.

Spatial maps of function-based contact areas and resulting mechanical strains in bones of intact fibrous joints in preclinical small-scale animal models are limited. Functional imaging in situ on intact dentoalveolar fibrous joints (DAJs) in hemimandibles and hemimaxillae harvested from 10 male Sprague-Dawley rats (N = 5 at 12 weeks, N = 5 at 20 weeks) was performed in this study. Physical features including bone volume fraction (BVF), bone pore diameter and pore density, and cementum fraction (CF) of the molars in the maxillary and mandibular joints were evaluated. Biomechanical testing in situ provided estimates of joint stiffness, changes in periodontal ligament spaces (PDL-space) between the molar and bony socket, and thereby localization of contact area in the respective joints. Contact area localization revealed mechanically stressed interradicular and apical regions in the joints. These anatomy-specific contact stresses in maxillary and mandibular joints were correlated with the physical features and resulting strains in interradicular and bony socket compartments. The mandibular joint spaces, in general, were higher than maxillary, and this trend was consistent with age (younger loaded: Mn - 134 ± 55 µm, Mx - 110 ± 47 µm; older loaded: Mn - 122 ± 49 µm, Mx - 105 ± 48 µm). However, a significant decrease (P < 0.05) in mandibular and maxillary joint spaces with age (younger unloaded: Mn - 147 ± 51 µm; Mx - 125 ± 42 µm; older unloaded: Mn - 134 ± 46 µm; Mx - 116 ± 44 µm) was observed. The bone volume fraction (BVF) of mandibular interradicular bone (IR bone) increased significantly with age (P < 0.05) with the percent porosity of coronal mandibular bone lower than its maxillary counterpart. The contact ratio (contact area to total surface area) of maxillary teeth was significantly greater (P < 0.05) than mandibular teeth; both maxillary interradicular and apical contact ratios (IR bone: 41%, 56%; Apical bone: 4%, 12%) increased with age, and were higher than the mandibular (IR bone: 19%, 44%; Apical bone: 1%, 4%) counterpart. Resulting higher but uniform strains in maxillary bone contrasted with lower but higher variance in mandibular strains at a younger age. Anatomy-specific colocalization of physical properties and functional strains in bone provided insights into form-guided adaptive dominance of the maxilla compared to material property-guided adaptive dominance of the mandible. These age-related trends from the preclinical animal model paralleled with age- and tooth position-specific variabilities in mandibular craniofacial bones of adolescent and adult patients following orthodontic treatment.

Standardization of mineral density maps of physiologic and pathologic biominerals in humans using cone-beam CT and micro-CT scanners.

OBJECTIVES: The lack of standardized X-ray imaging remains a challenge for comparative studies on spatial scans acquired from different clinic-specific X-ray scanners. The central objectives of this study are: 1) to delineate mineral density (MD) values, and 2) generate spatial MD maps of various physiologic and pathologic biominerals, and 3) propose a standardization protocol within the safe-operating zone of a CT scanner that underpins normalization of absorbed dose to shape and density of tissues. METHODS: A systematic approach to propose a standardization protocol for CT imaging in vivo included: 1) estimation of pathologic MD ranges by performing a comparative meta-analysis on 2009-2019 data from the PubMed database; 2) calibration of cone-beam CT (CBCT) and micro-CT scanners with phantoms of known mineral densities (0, 250, 500, 750 and 3000 mg/cc) and shapes (cylinders and polyhedrons); 3) scanning craniofacial bones (N = 5) and dental tissues (N = 5), and ectopic minerals from humans (N = 3 each, pulp, salivary gland, kidney and prostrate stones, and penile and vascular plaques); 4) underscoring the effect of shape-factor (surface area-to-volume ratio) on MD of biominerals. RESULTS: Higher MDs of physiologic and pathologic cortical bones (504-1009 mg/cc) compared to trabecular bone (82-212 mg/cc) were observed. An increase in shape-factor increased the CBCT error in MD measurement and revealed that the scanner resolution is dependent on the absorbed dose and shape-factor of detectable features. SIGNIFICANCE: CT scanners should be calibrated with phantoms containing segments of known shape-factors and mineral densities to identify safe-operating zones. The calibrated approach will narrow the gap between length-scale dependent measurements, and will permit spatiotemporal quantitative and reliable detection of pathologies.

Mineralized Peyronie's plaque has a phenotypic resemblance to bone.

Mineralized Peyronie's plaque (MPP) impairs penile function. The association, colocalization, and dynamic interplay between organic and inorganic constituents can provide insights into biomineralization of Peyronie's plaque. Human MPPs (n = 11) were surgically excised, and the organic and inorganic constituents were spatially mapped using multiple high-resolution imaging techniques. Multiscale image analyses resulted in spatial colocalization of elements within a highly porous material with heterogenous composition, lamellae, and osteocytic lacuna-like features with a morphological resemblance to bone. The lower (520 ±â€¯179 mg/cc) and higher (1024 ±â€¯155 mg/cc) mineral density regions were associated with higher (11%) and lower (7%) porosities in MPP. Energy dispersive X-ray and micro-X-ray fluorescent spectroscopic maps in the higher mineral density regions of MPP revealed higher counts of calcium (Ca) and phosphorus (P), and a Ca/P ratio of 1.48 ±â€¯0.06 similar to bone. More importantly, higher counts of zinc (Zn) were localized at the interface between softer (more organic to inorganic ratio) and harder (less organic to inorganic ratio) tissue regions of MPP and adjacent softer matrix, indicating the involvement of Zn-related proteins and/or pathways in the formation of MPP. In particular, dentin matrix protein-1 (DMP-1) was colocalized in a matrix rich in proteoglycans and collagen that contained osteocytic lacuna-like features. This combined materials science and biochemical with correlative microspectroscopic approach provided insights into the plausible cellular and biochemical pathways that incite mineralization of an existing fibrous Peyronie's plaque. STATEMENT OF SIGNIFICANCE: Aberrant human penile mineralization is known as mineralized Peyronie's plaque (MPP) and often results in a loss of form and function. This study focuses on investigating the spatial association of matrix proteins and elemental composition of MPP by colocalizing calcium, phosphorus, and trace metal zinc with dentin matrix protein 1 (DMP-1), acidic proteoglycans, and fibrillar collagen along with the cellular components using high resolution correlative microspectroscopy techniques. Spatial maps provided insights into cellular and biochemical pathways that incite mineralization of fibrous Peyronie's plaque in humans.

Functional adaptation of interradicular alveolar bone to reduced chewing loads on dentoalveolar joints in rats.

OBJECTIVES: The effects of reduced chewing loads on load bearing integrity of interradicular bone (IB) within dentoalveolar joints (DAJ) in rats were investigated. METHODS: Four-week-old Sprague Dawley rats (N = 60) were divided into two groups; rats were either fed normal food, which is hard-pellet food (HF) (N = 30), or soft-powdered chow (SF) (N = 30). Biomechanical testing of intact DAJs and mapping of the resulting mechanical strains within IBs from 8- through 24-week-old rats fed HF or SF were performed. Tension- and compression-based mechanical strain profiles were mapped by correlating digital volumes of IBs at no load with the same IBs under load. Heterogeneity within IB was identified by mapping cement lines and TRAP-positive multinucleated cells using histology, and mechanical properties using nanoindentation technique. RESULTS: Significantly decreased interradicular functional space, IB volume fraction, and elastic modulus of IB in the SF group compared with the HF group were observed, and these trends varied with an increase in age. The elastic modulus values illustrated significant heterogeneity within IB from HF or SF groups. Both compression- and tension-based strains were localized at the coronal portion of the IB and the variation in strain profiles complemented the observed material heterogeneity using histology and nanoindentation. SIGNIFICANCE: Interradicular space and IB material-related mechanoadaptations in a DAJ are optimized to meet soft food related chewing demands. Results provided insights into age-specific regulation of chewing loads as a plausible "therapeutic dose" to reverse adaptations within the periodontal complex as an attempt to regain functional competence of a dynamic DAJ.

Polymicrobial periodontal disease triggers a wide radius of effect and unique virome.

Periodontal disease is a microbially-mediated inflammatory disease of tooth-supporting tissues that leads to bone and tissue loss around teeth. Although bacterially-mediated mechanisms of alveolar bone destruction have been widely studied, the effects of a polymicrobial infection on the periodontal ligament and microbiome/virome have not been well explored. Therefore, the current investigation introduced a new mouse model of periodontal disease to examine the effects of a polymicrobial infection on periodontal ligament (PDL) properties, changes in bone loss, the host immune response, and the microbiome/virome using shotgun sequencing. Periodontal pathogens, namely Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum were used as the polymicrobial oral inoculum in BALB/cByJ mice. The polymicrobial infection triggered significant alveolar bone loss, a heightened antibody response, an elevated cytokine immune response, a significant shift in viral diversity and virome composition, and a widening of the PDL space; the latter two findings have not been previously reported in periodontal disease models. Changes in the PDL space were present at sites far away from the site of insult, indicating that the polymicrobial radius of effect extends beyond the bone loss areas and site of initial infection and wider than previously appreciated. Associations were found between bone loss, specific viral and bacterial species, immune genes, and PDL space changes. These findings may have significant implications for the pathogenesis of periodontal disease and biomechanical properties of the periodontium. This new polymicrobial mouse model of periodontal disease in a common mouse strain is useful for evaluating the features of periodontal disease.

The tooth attachment mechanism defined by structure, chemical composition and mechanical properties of collagen fibers in the periodontium.

In this study, a comparison between structure, chemical composition and mechanical properties of collagen fibers at three regions within a human periodontium, has enabled us to define a novel tooth attachment mechanism. The three regions include, (1) the enthesis region: insertion site of periodontal ligament (PDL) fibers (collagen fibers) into cementum at the root surface, (2) bulk cementum, and (3) the cementum-dentin junction (CDJ). Structurally, continuity in collagen fibers was observed from the enthesis, through bulk cementum and CDJ. At the CDJ the collagen fibers split into individual collagen fibrils and intermingled with the extracellular matrix of mantle dentin. Under wet conditions, the collagen fibers at the three regions exhibited significant swelling suggesting a composition rich in polyanionic molecules such as glycosaminoglycans. Additionally, site-specific indentation illustrated a comparable elastic modulus between collagen fibers at the enthesis (1-3 GPa) and the CDJ (2-4 GPa). However, the elastic modulus of collagen fibers within bulk cementum was higher (4-7 GPa) suggesting presence of extrafibrillar mineral. It is known that the tooth forms a fibrous joint with the alveolar bone, which is termed a gomphosis. Although narrower in width than the PDL space, the hygroscopic CDJ can also be termed as a gomphosis; a fibrous joint between cementum and root dentin capable of accommodating functional loads similar to that between cementum and alveolar bone. From an engineering perspective, it is proposed that a tooth contains two fibrous joints that accommodate the masticatory cyclic loads. These joints are defined by the attachment of dissimilar materials via graded stiffness interfaces, such as: (1) alveolar bone attached to cementum with the PDL; and (2) cementum to root dentin with the CDJ. Thus, through variations in concentrations of basic constituents, distinct regions with characteristic structures and graded properties allow for attachment and the load bearing characteristics of a tooth.

Periodontal ligament entheses and their adaptive role in the context of dentoalveolar joint function.

OBJECTIVE: The dynamic bone-periodontal ligament (PDL)-tooth fibrous joint consists of two adaptive functionally graded interfaces (FGI), the PDL-bone and PDL-cementum that respond to mechanical strain transmitted during mastication. In general, from a materials and mechanics perspective, FGI prevent catastrophic failure during prolonged cyclic loading. This review is a discourse of results gathered from literature to illustrate the dynamic adaptive nature of the fibrous joint in response to physiologic and pathologic simulated functions, and experimental tooth movement. METHODS: Historically, studies have investigated soft to hard tissue transitions through analytical techniques that provided insights into structural, biochemical, and mechanical characterization methods. Experimental approaches included two dimensional to three dimensional advanced in situ imaging and analytical techniques. These techniques allowed mapping and correlation of deformations to physicochemical and mechanobiological changes within volumes of the complex subjected to concentric and eccentric loading regimes respectively. RESULTS: Tooth movement is facilitated by mechanobiological activity at the interfaces of the fibrous joint and generates elastic discontinuities at these interfaces in response to eccentric loading. Both concentric and eccentric loads mediated cellular responses to strains, and prompted self-regulating mineral forming and resorbing zones that in turn altered the functional space of the joint. SIGNIFICANCE: A multiscale biomechanics and mechanobiology approach is important for correlating joint function to tissue-level strain-adaptive properties with overall effects on joint form as related to physiologic and pathologic functions. Elucidating the shift in localization of biomolecules specifically at interfaces during development, function, and therapeutic loading of the joint is critical for developing "functional regeneration and adaptation" strategies with an emphasis on restoring physiologic joint function.

Strain mapping and correlative microscopy of the alveolar bone in a bone-periodontal ligament-tooth fibrous joint.

This study details a method to calculate strains within interradicular alveolar bone using digital volume correlation on X-ray tomograms of intact bone-periodontal ligament-tooth fibrous joints. The effects of loading schemes (concentric and eccentric) and optical magnification on the resulting strain in alveolar bone will be investigated with an intent to correlate deformation gradients with data sets from other complementary techniques. Strain maps will be correlated with structural and site-specific mechanical properties obtained on the same specimen using atomic force microscopy and atomic force microscopy-based nanoindentation technique. Specimens include polydimethylsiloxane as a standard material and intact hemi-mandibles harvested from rats. X-ray tomograms were taken at no-load and loaded conditions using an in situ load cell coupled to a micro X-ray computed tomography unit. Digital volume correlation was used to calculate deformations within alveolar bone. Comparison of strain maps was made as a result of different loading schemes (concentric vs eccentric) and at different magnifications (4× vs 10×). Virtual sections and strain maps from digital volume correlation solutions were aligned with structure and reduced elastic modulus to correlate datasets of the same region within a specimen. Strain distribution between concentrically and eccentrically loaded complexes was different but illustrated a similar range. Strain maps of homogeneous materials (polydimethylsiloxane) resulting from digital volume correlation at different magnifications were similar. However, strain maps of heterogeneous materials at lower and higher magnification differed. The digital volume correlation technique illustrated a dependence on optical magnification specifically for heterogeneous materials such as bone. The results at a higher optical magnification highlight the potential for extracting deformation at higher resolutions. Correlation of data spaces from different complementary techniques is plausible and could provide insights into biological and physicochemical processes that lead to functional adaptation of tissues and joints.

Effect of proteoglycans at interfaces as related to location, architecture, and mechanical cues.

INTRODUCTION: Covalently bound functional GAGs orchestrate tissue mechanics through time-dependent characteristics. OBJECTIVE: The role of specific glycosaminoglycans (GAGs) at the ligament-cementum and cementum-dentin interfaces within a human periodontal complex were examined. Matrix swelling and resistance to compression under health and modeled diseased states was investigated. MATERIALS AND METHODS: The presence of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs at the ligament-cementum and cementum-dentin interfaces in human molars (N=5) was illustrated by using enzymes, atomic force microscopy (AFM), and AFM-based nanoindentation. The change in physical characteristics of modeled diseased states through sequential digestion of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs was investigated. One-way ANOVA tests with P<0.05 were performed to determine significant differences between groups. Additionally, the presence of mineral within the seemingly hygroscopic interfaces was investigated using transmission electron microscopy. RESULTS: Immunohistochemistry (N=3) indicated presence of biglycan and fibromodulin small leucine rich proteoglycans at the interfaces. Digestion of matrices with enzymes confirmed the presence of KS and CS GAGs at the interfaces by illustrating a change in tissue architecture and mechanics. A significant increase in height (nm), decrease in elastic modulus (GPa), and tissue deformation rate (nm/s) of the PDL-C attachment site (215±63-424±94nm; 1.5±0.7-0.4±0.2GPa; 21±7-48±22nm/s), and cementum-dentin interface (122±69-360±159nm; 2.9±1.3-0.7±0.3GPa; 18±4-30±6nm/s) was observed. CONCLUSIONS: The sequential removal of GAGs indicated loss in intricate structural hierarchy of hygroscopic interfaces. From a mechanics perspective, GAGs provide tissue recovery/resilience. The results of this study provide insights into the role of GAGs toward conserved tooth movement in the socket in response to mechanical loads, and modulation of potentially deleterious strain at tissue interfaces.

A Critical Role of TRPM7 As an Ion Channel Protein in Mediating the Mineralization of the Craniofacial Hard Tissues.

Magnesium ion (Mg(2+)) is the fourth most common cation in the human body, and has a crucial role in many physiological functions. Mg(2+) homeostasis is an important contributor to bone development, however, its roles in the development of dental mineralized tissues have not yet been well known. We identified that transient receptor potential cation channel, subfamily M, member 7 (TRPM7), was significantly upregulated in the mature ameloblasts as compared to other ameloblasts through our whole transcript microarray analyses of the ameloblasts. TRPM7, an ion channel for divalent metal cations with an intrinsic serine/threonine protein kinase activity, has been characterized as a key regulator of whole body Mg(2+) homeostasis. Semi-quantitative PCR and immunostaining for TRMP7 confirmed its upregulation during the maturation stage of enamel formation, at which ameloblasts direct rapid mineralization of the enamel matrix. The significantly hypomineralized craniofacial structures, including incisors, molars, and cranial bones were demonstrated by microCT analysis, von Kossa and trichrome staining in Trpm7 (Δkinase∕+) mice. A previously generated heterozygous mouse model with the deletion of the TRPM7 kinase domain. Interestingly, the skeletal phenotype of Trpm7 (Δkinase∕+) mice resembled those found in the tissue-nonspecific alkaline phosphatase (Alpl) KO mice, thus we further examined whether ALPL protein content and alkaline phosphatase (ALPase) activity in ameloblasts, odontoblasts and osteoblasts were affected in those mice. While ALPL protein in Trpm7 (Δkinase∕+) mice remained at the similar level as that in wt mice, ALPase activities in the Trpm7 (Δkinase∕+) mice were almost nonexistent. Supplemented magnesium successfully rescued the activities of ALPase in ameloblasts, odontoblasts and osteoblasts of Trpm7 (Δkinase∕+) mice. These results suggested that TRPM7 is essential for mineralization of enamel as well as dentin and bone by providing sufficient Mg(2+) for the ALPL activity, underlining the key importance of ALPL for biomineralization.

Multiscale biomechanical responses of adapted bone-periodontal ligament-tooth fibrous joints.

Reduced functional loads cause adaptations in organs. In this study, temporal adaptations of bone-ligament-tooth fibrous joints to reduced functional loads were mapped using a holistic approach. Systematic studies were performed to evaluate organ-level and tissue-level adaptations in specimens harvested periodically from rats (N=60) given powder food for 6 months over 8,12,16,20, and 24 weeks. Bone-periodontal ligament (PDL)-tooth fibrous joint adaptation was evaluated by comparing changes in joint stiffness with changes in functional space between the tooth and alveolar bony socket. Adaptations in tissues included mapping changes in the PDL and bone architecture as observed from collagen birefringence, bone hardness and volume fraction in rats fed soft foods (soft diet, SD) compared to those fed hard pellets as a routine diet (hard diet, HD). In situ biomechanical testing on harvested fibrous joints revealed increased stiffness in SD groups (SD:239-605 N/mm) (p<0.05) at 8 and 12 weeks. Increased joint stiffness in early development phase was due to decreased functional space (at 8 weeks change in functional space was -33 µm, at 12 weeks change in functional space was -30 µm) and shifts in tissue quality as highlighted by birefringence, architecture and hardness. These physical changes were not observed in joints that were well into function, that is, in rodents older than 12 weeks of age. Significant adaptations in older groups were highlighted by shifts in bone growth (bone volume fraction 24 weeks: Δ-0.06) and bone hardness (8 weeks: Δ-0.04 GPa, 16 weeks: Δ-0.07 GPa, 24 weeks: Δ-0.06 GPa). The response rate (N/s) of joints to mechanical loads decreased in SD groups. Results from the study showed that joint adaptation depended on age. The initial form-related adaptation (observed change in functional space) can challenge strain-adaptive nature of tissues to meet functional demands with increasing age into adulthood. The coupled effect between functional space in the bone-PDL-tooth complex and strain-adaptive nature of tissues is necessary to accommodate functional demands, and is temporally sensitive despite joint malfunction. From an applied science perspective, we propose that adaptations are registered as functional history in tissues and joints.

Essential role of osterix for tooth root but not crown dentin formation.

Tooth is made of crown and root. It is widely believed that dentin formation in crown and root uses the same regulatory mechanism. However, identification of nuclear factor 1 C (NFIC)'s unique function in determining root but not crown dentin formation challenges the old thinking. In searching for the target molecules downstream of NFIC, we unexpectedly found a sharp reduction of osterix (OSX), the key transcription factor in skeleton formation, in the Nfic knockout (Nfic-KO) tooth root. We then demonstrated a dose-dependent increase of Osx in the odontoblast cell line due to a transient transfection of Nfic expression plasmid. Studies of global and conditional Osx-KO mice revealed no apparent changes in the crown dentin tubules and dentin matrix. However, the OSX conditional KO (cKO) mice (crossed to the 2.3-kb collagen type 1 [Col1]-Cre) displayed an increase in cell proliferation but great decreases in expressions of root dentin matrix proteins (dentin matrix protein 1 [DMP1] and dentin sialophosphoprotein [DSPP]), leading to an inhibition in odontoblast differentiation, and short, thin root dentin with few dentin tubules. Compared to the Nfic-KO tooth, which contains essentially no dentin tubules and remains in a "root-less" status at adult stages, the Osx-cKO root phenotype had partially improved at the late stage, indicating that other factors can compensate for OSX function. Thus, we conclude that OSX, one of the key downstream molecules of NFIC, plays a critical role in root, but not crown, formation.

Biomechanics and strain mapping in bone as related to immediately-loaded dental implants.

The effects of alveolar bone socket geometry and bone-implant contact on implant biomechanics, and resulting strain distributions in bone were investigated. Following extraction of lateral incisors on a cadaver mandible, implants were placed immediately and bone-implant contact area, stability implant biomechanics and bone strain were measured. In situ biomechanical testing coupled with micro X-ray microscopy (µ-XRM) illustrated less stiff bone-implant complexes (701-822 N/mm) compared with bone-periodontal ligament (PDL)-tooth complexes (791-913 N/mm). X-ray tomograms illustrated that the cause of reduced stiffness was due to limited bone-implant contact. Heterogeneous elemental composition of bone was identified by using energy dispersive X-ray spectroscopy (EDS). The novel aspect of this study was the application of a new experimental mechanics method, that is, digital volume correlation, which allowed mapping of strains in volumes of alveolar bone in contact with a loaded implant. The identified surface and subsurface strain concentrations were a manifestation of load transferred to bone through bone-implant contact based on bone-implant geometry, quality of bone, implant placement, and implant design. 3D strain mapping indicated that strain concentrations are not exclusive to the bone-implant contact regions, but also extend into bone not directly in contact with the implant. The implications of the observed strain concentrations are discussed in the context of mechanobiology. Although a plausible explanation of surgical complications for immediate implant treatment is provided, extrapolation of results is only warranted by future systematic studies on more cadaver specimens and/or in vivo models.

Mineral density volume gradients in normal and diseased human tissues.

Clinical computed tomography provides a single mineral density (MD) value for heterogeneous calcified tissues containing early and late stage pathologic formations. The novel aspect of this study is that, it extends current quantitative methods of mapping mineral density gradients to three dimensions, discretizes early and late mineralized stages, identifies elemental distribution in discretized volumes, and correlates measured MD with respective calcium (Ca) to phosphorus (P) and Ca to zinc (Zn) elemental ratios. To accomplish this, MD variations identified using polychromatic radiation from a high resolution micro-computed tomography (micro-CT) benchtop unit were correlated with elemental mapping obtained from a microprobe X-ray fluorescence (XRF) using synchrotron monochromatic radiation. Digital segmentation of tomograms from normal and diseased tissues (N=5 per group; 40-60 year old males) contained significant mineral density variations (enamel: 2820-3095 mg/cc, bone: 570-1415 mg/cc, cementum: 1240-1340 mg/cc, dentin: 1480-1590 mg/cc, cementum affected by periodontitis: 1100-1220 mg/cc, hypomineralized carious dentin: 345-1450 mg/cc, hypermineralized carious dentin: 1815-2740 mg/cc, and dental calculus: 1290-1770 mg/cc). A plausible linear correlation between segmented MD volumes and elemental ratios within these volumes was established, and Ca/P ratios for dentin (1.49), hypomineralized dentin (0.32-0.46), cementum (1.51), and bone (1.68) were observed. Furthermore, varying Ca/Zn ratios were distinguished in adapted compared to normal tissues, such as in bone (855-2765) and in cementum (595-990), highlighting Zn as an influential element in prompting observed adaptive properties. Hence, results provide insights on mineral density gradients with elemental concentrations and elemental footprints that in turn could aid in elucidating mechanistic processes for pathologic formations.