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.

Fatty acid-binding protein 4 downregulation drives calcification in the development of kidney stone disease.

Nephrolithiasis is a significant source of morbidity, and its incidence has increased significantly over the last decades. This rise has been attributed to concurrent increasing rates of obesity, associated with a 3-time risk of developing NL. To date, the mechanism by which obesity is linked to stone formation has not been elucidated. We aimed to utilize a transcriptomics approach to discover the missing link between these two epidemic diseases. We investigated gene expression profiling of nephrolithiasis patients by two RNA-sequencing approaches: comparison between renal papilla tissue with and without the presence of calcified Randall's plaques (RP), and comparison between the papilla, medulla, and cortex regions from within a single recurrent stone forming kidney. Results were overlaid between differently expressed genes found in the patient cohort and in the severely lithogenic kidney to identify common genes. Overlay of these two RNA-sequencing datasets demonstrated there is impairment of lipid metabolism in renal papilla tissue containing RP linked to downregulation of fatty acid binding protein (FABP) 4. Immunohistochemistry of human kidney specimens and microarray analysis of renal tissue from a nephrolithiasis mouse model confirmed that FABP4 downregulation is associated with renal stone formation. In a FABP4 knockout mouse model, FABP4 deficiency resulted in development of both renal and urinary crystals. Our study revealed that FABP4 plays an important, previously unrecognized role in kidney stone formation, providing a feasible mechanism to explain the link between nephrolithiasis and metabolic syndrome.

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.

Beginnings of nephrolithiasis: insights into the past, present and future of Randall's plaque formation research.

PURPOSE OF REVIEW: Kidney stones form as a result of heterogeneous nucleation on a calcium phosphate lesion in the renal papilla known as Randall's plaque. Stone disease has plagued humans for millennia with relatively little progress made in the realm of prevention. An understanding of the historical aspects of research into Randall's plaque is necessary to interpret novel correlative imaging discoveries. Focus for the past several decades has been on the distal papillary tip, and the overlooked Anderson-Carr-Randall progression is revitalized with novel supporting evidence. RECENT FINDINGS: Novel correlative techniques of three-dimensional micro-XCT imaging combined with electron and light microscopy techniques have revealed that the earliest mineralization event in the papilla is a distinct event that occurs proximal to the region where Randall's plaque has traditionally been identified. SUMMARY: The history of Randall's plaque research and the Anderson-Carr-Randall progression is reviewed. Proximal intratubular mineral deposits in normal and Randall's plaque affected papillae may be a target for future therapeutic interventions for nephrolithiasis. Further collaboration between nephrologists and urologists is necessary to cure this debilitating disease.

Novel insights into renal mineralization and stone formation through advanced imaging modalities.

Purpose/Aim: The most common kidney stone composed of calcium oxalate forms on interstitial calcium phosphate mineral known as a Randall's plaque (RP). Due to limited information about events leading to the initial deposition of nanometer size interstitial calcium phosphate pre-clusters, there continues to be a debate on the initial site of calcium phosphate deposition and factors leading to stone formation. MATERIALS AND METHODS: High-resolution X-ray micro-computed tomography (CT), and light and electron microscopy techniques were used to characterize human renal pyramids and five representative kidney stones with identifiable stems. Mineral densities of mineralized aggregates within these specimens were correlated with micro- and ultra-structures as seen using light and electron microscopy techniques. RESULTS: The earliest detectable biominerals in the human renal papilla were proximal intratubular plate-like calcium phosphate deposits. Unoccluded tubules in stems connected to calcium phosphate stones were observed by electron microscope and X-ray micro-CT. These tubules were similar in diameter (30-100 µm) and shape to those observed in the distal regions of the renal papilla. CONCLUSIONS: Observations were patterned through a novel and unified theory of stepwise-architecture guided biomineralization (a combination of smaller structures leading to a larger but similar structural framework). A plausible stepwise progression in renal biomineralization is proposed; proximal intratubular calcium phosphate deposits can lead to interstitial yet calcium phosphate rich RP and mature into a stem on which a calcium oxalate stone grows within the collecting system of a kidney.

The origins of urinary stone disease: upstream mineral formations initiate downstream Randall's plaque.

OBJECTIVES: To describe a new hypothesis for the initial events leading to urinary stones. A biomechanical perspective on Randall's plaque formation through form and function relationships is applied to functional units within the kidney, we have termed the 'medullo-papillary complex' - a dynamic relationship between intratubular and interstitial mineral aggregates. METHODS: A complete MEDLINE search was performed to examine the existing literature on the anatomical and physiological relationships in the renal medulla and papilla. Sectioned human renal medulla with papilla from radical nephrectomy specimens were imaged using a high resolution micro X-ray computed tomography. The location, distribution, and density of mineral aggregates within the medullo-papillary complex were identified. RESULTS: Mineral aggregates were seen proximally in all specimens within the outer medulla of the medullary complex and were intratubular. Distal interstitial mineralisation at the papillary tip corresponding to Randall's plaque was not seen until a threshold of proximal mineralisation was observed. Mineral density measurements suggest varied chemical compositions between the proximal intratubular (330 mg/cm3 ) and distal interstitial (270 mg/cm3 ) deposits. A review of the literature revealed distinct anatomical compartments and gradients across the medullo-papillary complex that supports the empirical observations that proximal mineralisation triggers distal Randall's plaque formation. CONCLUSION: The early stone event is initiated by intratubular mineralisation of the renal medullary tissue leading to the interstitial mineralisation that is observed as Randall's plaque. We base this novel hypothesis on a multiscale biomechanics perspective involving form and function relationships, and empirical observations. Additional studies are needed to validate this hypothesis.

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.

Organic Matrix Derived from Host-Microbe Interplay Contributes to Pathological Renal Biomineralization.

Matrix stones are a rare form of kidney stones. They feature a high percentage of hydrogel-like organic matter, and their formation is closely associated with urinary tract infections. Herein, comprehensive materials and biochemical approaches were taken to map the organic-inorganic interface and gather insights into the host-microbe interplay in pathological renal biomineralization. Surgically extracted soft and slimy matrix stones were examined using micro-X-ray computed tomography and various microspectroscopy techniques. Higher-mineral-density laminae were positive for calcium-bound Alizarin red. Lower-mineral-density laminae revealed periodic acid-Schiff-positive organic filamentous networks of varied thickness. These organic filamentous networks, which featured a high polysaccharide content, were enriched with zinc, carbon, and sulfur elements. Neutrophil extracellular traps (NETs) along with immune response-related proteins, including calprotectin, myeloperoxidase, CD63, and CD86, also were identified in the filamentous networks. Expressions of NETs and upregulation of polysaccharide-rich mucin secretion are proposed as a part of the host immune defense to "trap" pathogens. These host-microbe derived organic matrices can facilitate heterogeneous nucleation and precipitation of inorganic particulates, resulting in macroscale aggregates known as "matrix stones". These insights into the plausible aggregation of constituents through host-microbe interplay underscore the unique "double-edged sword" effect of the host immune response to pathogens and the resulting renal biominerals.

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.

Structural and chemical heterogeneities of primary hyperoxaluria kidney stones from pediatric patients.

OBJECTIVE: Calcium oxalate stones are the most common type among stone-forming patients and in some cases result from predisposed genetic conditions. In this work, we examined the differences in structure and chemical composition between oxalate stones from patients from three groups: 1) pediatric patients that were genetically predisposed (primary hyperoxaluria) to form stones (PPH); 2) control pediatric patients that did not have such genetic predisposition (PN-PH); 3) adult patients that formed oxalate stones without the genetic predisposition (A-CaOx). A variety of instrumental analyses were conducted to identify physicochemical properties of stones characteristic of predisposed pediatric (PPH), pediatric hyperoxaluria (PN-PH), and adult (A-CaOx) patient populations. METHODS: Genetic variants of 16 stone-forming patients were determined using whole-exome gene sequencing. Components of stones from PPH (n = 6), PN-PH (n = 5), and A-CaOx (n = 5) groups were identified using Fourier transform infrared (FTIR) spectroscopy. Stone morphology and density were evaluated using high resolution X-ray computed tomography (micro-XCT). Stone microstructure and elemental composition were mapped with scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy, respectively. RESULTS: Calcium oxalate bipyramidal crystals were found on stones from all groups. Stones from PPH patients with PH types I and II were composed of calcium oxalate monohydrate (COM) with relatively uniform mineral density (1224 ± 277 mg/cc) and distinct smooth surfaces. By contrast, micro-spherical calcium phosphate particles were found only on PN-PH stones, which also showed a broader range of mineral densities (1266 ± 342 mg/cc). Stones from the PN-PH group also contained phosphorus (P), which was absent in NP-PH stones. A-CaOx stones were of significantly lower mineral density (645 ± 237 mg/cc) than pediatric stones and were more heterogeneous in their elemental composition. CONCLUSION: Unique structural and compositional characteristics were identified in stones from pediatric patients with primary hyperoxaluria. These include the absence of phosphorus, a narrower mineral density distribution, and a uniform elemental composition compared to stones from pediatric patients without the genetic predisposition. Thus, characterization of stones at the macro- and micro-scales in combination with genetic testing of patients can provide insights and accurate diagnosis to develop a treatment plan for effective patient care.

Structure and elemental composition of Ceftriaxone induced pediatric nephrolithiasis.

Ceftriaxone is a widely used antibiotic because to its broad-spectrum gram-negative coverage, safety, and biological half life (5-9 h) permit dose once-daily administration. It is specifically used in pediatric patients in developing countries. Ceftriaxone forms insoluble sludge/stone when combined with calcium in the urinary system. In this study, Ceftriaxone induced sludge/stones from pediatric patients were collected to identify its microstructure and composition to gather insights into the mechanism of Ceftriaxone induced sludge/stone formation. The results illustrated that Ceftriaxone induced stones formed rapidly following antibiotic administration. Ceftriaxone calcium salt crystals could easily be broken with minimal intervention. However, Ceftriaxone combined with calcium phosphate formed an insoluble stone aggregate.

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.

High-Throughput Screening: today's biochemical and cell-based approaches.

High-throughput screening (HTS) provides starting chemical matter in the adventure of developing a new drug. In this review, we survey several HTS methods used today for hit identification, organized in two main flavors: biochemical and cell-based assays. Biochemical assays discussed include fluorescence polarization and anisotropy, FRET, TR-FRET, and fluorescence lifetime analysis. Binding-based methods are also surveyed, including NMR, SPR, mass spectrometry, and DSF. On the other hand, cell-based assays discussed include viability, reporter gene, second messenger, and high-throughput microscopy assays. We devote some emphasis to high-content screening, which is becoming very popular. An advisable stage after hit discovery using phenotypic screens is target deconvolution, and we provide an overview of current chemical proteomics, in silico, and chemical genetics tools. Emphasis is made on recent CRISPR/dCas-based screens. Lastly, we illustrate some of the considerations that inform the choice of HTS methods and point to some areas with potential interest for future research.

Design of drug-like hepsin inhibitors against prostate cancer and kidney stones.

Hepsin, a transmembrane serine protease abundant in renal endothelial cells, is a promising therapeutic target against several cancers, particularly prostate cancer. It is involved in the release and polymerization of uromodulin in the urine, which plays a role in kidney stone formation. In this work, we design new potential hepsin inhibitors for high activity, improved specificity towards hepsin, and promising ADMET properties. The ligands were developed in silico through a novel hierarchical pipeline. This pipeline explicitly accounts for off-target binding to the related serine proteases matriptase and HGFA (human hepatocyte growth factor activator). We completed the pipeline incorporating ADMET properties of the candidate inhibitors into custom multi-objective optimization functions. The ligands designed show excellent prospects for targeting hepsin via the blood stream and the urine and thus enable key experimental studies. The computational pipeline proposed is remarkably cost-efficient and can be easily adapted for designing inhibitors against new drug targets.

Physicochemical and biochemical spatiotemporal maps of a mouse penis.

Spatiotemporal mechanobiology resulting in penile pathologies continues to be investigated using small scale animals models such as mice. However, species-dependent functional biomechanics of a mouse penis, is not known. In this study, spatial mapping of a mechanosensitive transcription factor, scleraxis (Scx), at ages 4, 5, 6 weeks, and 1 year were generated to identify mechanoactive regions within penile tissues. Reconstructed volumes of baculum collected using micro X-ray computed tomography illustrated significantly increased baculum length with decreased porosity, and increased mineral density (p < 0.05) with age. The bony-baculum was held centrally in the Scx positive corpus cavernosum glandis (CCG), indicating mechanoactivity within the struts in a 6 week old mouse. The struts also were stained positive for fibrillar proteins including collagen and elastin, and globular proteins including protein gene product 9.5, and α-smooth muscle actin. The corpus cavernosum penis (CCP) contained significantly (p < 0.05) more collagen than CCG within the same penis, and both regions contained blood vessels with equivalent innervation at any given age. Comparison of volumes of flaccid and erect penile forms revealed functional characteristics of the CCP. Results of this study provided insights into biomechanical function of the CCG; in that, it is a high-pressure chamber that stiffens the penis and is similar to the human corpus cavernosum.