A new ex vivo human model of osteoarthritis cartilage calcification.

OBJECTIVE: Cartilage pathologic calcification is a hallmark of osteoarthritis (OA). Here, we aimed to describe a new ex vivo human model to study the progression of cartilage calcification. METHOD: Cartilage explants (n = 11), as well as primary chondrocytes (n = 3), were obtained from OA patients undergoing knee replacement. Explants and chondrocytes were cultured in control (NT) or calcification (CM) medium (supplemented with ascorbic acid and ß-glycerophosphate). Calcification was evaluated by micro-CT scan at day 0 and 21 in explants, and by Alizarin red staining in chondrocyte monolayers. Raman spectrometry allowed characterization of the crystal type. Interleukin-6 (IL-6) secretion in explant and cell supernatants was measured by ELISA. Finally, matrix degradation was evaluated by Safranin-O staining of explant sections and by glycosaminoglycans (GAG) release in supernatants. RESULTS: Micro-CT scan showed calcifications in all explants at baseline (day 0), which in the CM group increased significantly in number and size after 21 days compared with the NT group. Raman spectrometry revealed that crystals were exclusively basic calcium phosphate crystals (carbonated hydroxyapatite) both in NT and CM. IL-6 secretion was significantly increased in calcifying conditions. Finally, CM significantly increased cartilage catabolism as assessed by decreased Safranin-O staining of tissue explants and increased GAG release in supernatants. CM effects (enhanced calcification, IL-6 secretion and proteoglycans turn-over) were recapitulated in vitro in OA chondrocytes. CONCLUSIONS: We have described a new ex vivo human model of cartilage calcification that can summurize the triad of events seen during osteoarthritis progression, i.e. calcification, inflammation, and cartilage degradation. This model will allow the identification of new anti-calcification compounds.

Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.

Bone Molecular Modifications Induced by Diagenesis Followed-Up for 12 Months.

After death, diagenesis takes place. Numerous processes occur concomitantly, which makes it difficult to identify the diagenetic processes. The diagenetic processes refer to all processes (chemical or physical) that modify the skeletal remains. These processes are highly variable depending on the environmental factors (weather, temperature, age, sex, etc.), especially in the early stages. Numerous studies have evaluated bone diagenetic processes over long timescales (~millions of years), but fewer have been done over short timescales (between days and thousands of years). The objective of the study is to assess the early stages of diagenetic processes by Raman microspectroscopy over 12 months. The mineral and organic matrix modifications are monitored through physicochemical parameters. Ribs from six humans were buried in soil. The modifications of bone composition were followed by Raman spectroscopy each month. The decrease in the mineral/organic ratio and carbonate type-B content and the increase in crystallinity reveal that minerals undergo dissolution-recrystallization. The decrease in collagen cross-linking indicates that collagen hydrolysis induces the fragmentation of collagen fibres over 12 months.

Material and nanomechanical properties of bone structural units of cortical and trabecular iliac bone tissues from untreated postmenopausal osteoporotic women.

The differences in bone nanomechanical properties between cortical (Ct) and trabecular (Tb) bone remain uncertain, whereas knowing the respective contribution of each compartment is critical to understand the origin of bone strength. Our purpose was to compare bone mechanical and intrinsic properties of Ct and Tb compartments, at the bone structural unit (BSU) level, in iliac bone taken from a homogeneous untreated human population. Among 60 PMMA-embedded transiliac bone biopsies from untreated postmenopausal osteoporotic women (64 ± 7 year-old), >2000 BSUs were analysed by nanoindentation in physiological wet conditions [indentation modulus (elasticity), hardness, dissipated energy], by Fourier transform infrared (FTIRM) and Raman microspectroscopy (mineral and organic characteristics), and by X-ray microradiography (degree of mineralization of bone, DMB). BSUs were categorized based on tissue age, osteonal (Ost) and interstitial (Int) tissues location and bone compartments (Ct and Tb). Indentation modulus was higher in Ct than in Tb BSUs, both in Ost and Int. dissipated energy was higher in Ct than Tb, in Int BSUs. Hardness was not different between Ct and Tb BSUs. In Ost or Int BSUs, mineral maturity (conversion of non-apatitic into apatitic phosphates) was higher in Ct than in Tb, as well as for collagen maturity (Ost). Mineral content assessed as mineral/matrix (FTIRM and Raman) or as DMB, was lower in Ct than in Tb. Crystallinity (FTIRM) was similar in BSUs from Ct and Tb, and slightly lower in Ct than in Tb when measured by Raman, indicating that the crystal size/perfection was quite similar between Ct and Tb BSUs. The differences found between Ost and Int tissues were much higher than the difference found between Ct and Tb for all those bone material properties. Multiple regression analysis showed that Indentation modulus and dissipated energy were mainly explained by mineral maturity in Ct and by collagen maturity in Tb, and hardness by mineral content in both Ct and Tb. In conclusion, in untreated human iliac bone, Ct and Tb BSUs exhibit different characteristics. Ct BSUs have higher indentation modulus, dissipated energy (Int), mineral and organic maturities than Tb BSUs, without difference in hardness. Although those differences are relatively small compared to those found between Ost and Int BSUs, they may influence bone strength at macroscale.

The extracellular matrix of human bone marrow adipocytes and glucose concentration differentially alter mineralization quality without impairing osteoblastogenesis.

Bone marrow adipocytes (BMAds) accrue in various states of osteoporosis and interfere with bone remodeling through the secretion of various factors. However, involvement of the extracellular matrix (ECM) produced by BMAds in the impairment of bone marrow mesenchymal stromal cell (BM-MSC) osteoblastogenesis has received little attention. In type 2 diabetes (T2D), skeletal fragility is associated with several changes in bone quality that are incompletely understood, and BMAd quantity increases in relationship to poor glycemic control. Considering their altered phenotype in this pathophysiological context, we aimed to determine the contribution of the ECM of mature BMAds to osteoblastogenesis and mineralization quality in the context of chronic hyperglycemia. Human BM-MSCs were differentiated for 21 days in adipogenic medium containing either a normoglycemic (LG, 5.5 mM) or a high glucose concentration (HG, 25 mM). The ECM laid down by BMAds were devitalized through cell removal to examine their impact on the proliferation and differentiation of BM-MSCs toward osteoblastogenesis in LG and HG conditions. Compared to control plates, both adipocyte ECMs promoted cell adhesion and proliferation. As shown by the unmodified RUNX2 and osteocalcin mRNA levels, BM-MSC commitment in osteoblastogenesis was hampered by neither the hyperglycemic condition nor the adipocyte matrices. However, adipocyte ECMs or HG condition altered the mineralization phase with perturbed expression levels of type 1 collagen, MGP and osteopontin. Despite higher ALP activity, mineralization levels per cell were decreased for osteoblasts grown on adipocyte ECMs compared to controls. Raman spectrometry revealed that culturing on adipocyte matrices specifically prevents type-B carbonate substitution and favors collagen crosslinking, in contrast to exposure to HG concentration alone. Moreover, the mineral to organic ratio was disrupted according to the presence of adipocyte ECM and the glucose concentration used for adipocyte or osteoblast culture. HG concentration and adipocyte ECM lead to different defects in mineralization quality, recapitulating contradictory changes reported in T2D osteoporosis. Our study shows that ECMs from BMAds do not impair osteoblastogenesis but alter both the quantity and quality of mineralization partly in a glucose concentration-dependent manner. This finding sheds light on the involvement of BMAds, which should be considered in the compromised bone quality of T2D and osteoporosis patients more generally.

Critical aspects of Raman spectroscopy as a tool for postmortem interval estimation.

The estimation of the postmortem interval (PMI) from skeletal remains represents a challenging task in forensic science. PMI is often influenced by extrinsic factors (humidity, dryness, scavengers, etc.) and intrinsic factors (age, sex, pathology, way of life, medical treatments, etc.). Raman spectroscopy combined with multivariate data analysis represents a promising tool for forensic anthropologists. Despite all the advantages of the technique, Raman spectra of skeletal remains are influenced by these extrinsic and intrinsic factors, which impairs precision and reproducibility. Both parameters have to reach a high level of confidence when such spectroscopy is used as a way to predict PMI. As a consequence, advanced multivariate data analysis is necessary to quantify the effect of all factors to improve the estimation of the PMI. The objective of this work is to evaluate the effect of intrinsic and extrinsic factors on the Raman spectra of skeletal remains. We designed a protocol close to a real-world scenario. We used ANOVA-simultaneous component analysis (ASCA) to unmix and quantify the effect of 1 intrinsic (source body) and 1 extrinsic (burial time) factors on the Raman spectra. In our model, the burial time was found to generate the highest variability after the source body. ASCA showed that the variability due to the burial time has 2 mixed contributions. Seasonal variations are the first contribution. The second contribution is attributed to diagenesis. A decrease in the mineral bands and an increase in the organic bands are observed. The source body was also found to contribute to the variability in Raman spectra. ASCA showed that the source body induces variability related to the composition of bones. This quantification cannot be assessed by basic chemometrics methods such as PCA. The results of this study highlighted the need to use an advanced chemometric data analysis tool (like ASCA) combined with Raman spectroscopy to estimate the postmortem interval.

Severity Level and Duration of Energy Deficit in Mice Affect Bone Phenotype and Bone Marrow Stromal Cell Differentiation Capacity.

Anorexia nervosa is known to induce changes in bone parameters and an increase in bone marrow adiposity (BMA) that depend on the duration and seriousness of the disease. Previous studies have found that bone loss is associated with BMA accumulation. Sirtuin of type 1 (Sirt1), a histone deacetylase that is partly regulated by energy balance, was shown to have pro-osteoblastogenic and anti-adipogenic effects. To study the effects of the severity and duration of energy deficits related to bone loss, a mouse model of separation-based anorexia (SBA) was established. We recently demonstrated that moderate body weight loss (18%) 8-week SBA protocol in mice resulted in an increase in BMA, bone loss, and a significant reduction in Sirt1 expression in bone marrow stromal cells (BMSCs) extracted from SBA mice. We hypothesised that Sirt1 deficit in BMSCs is associated with bone and BMA alterations and could potentially depend on the severity of weight loss and the length of SBA protocol. We studied bone parameters, BMA, BMSC differentiation capacity, and Sirt1 expression after induction of 4 different levels of body weight loss (0%,12%,18%,24%), after 4 or 10 weeks of the SBA protocol. Our results demonstrated that 10 week SBA protocols associated with body weight loss (12%, 18%, 24%) induced a significant decrease in bone parameters without any increase in BMA. BMSCs extracted from 12% and 18% SBA groups showed a significant decrease in Sirt1 mRNA levels before and after co-differentiation. For these two groups, decrease in Sirt1 was associated with a significant increase in the mRNA level of adipogenic markers and a reduction of osteoblastogenesis. Inducing an 18% body weight loss, we tested a short SBA protocol (4-week). We demonstrated that a 4-week SBA protocol caused a significant decrease in Tb.Th only, without change in other bone parameters, BMA, Sirt1 expression, or differentiation capacity of BMSCs. In conclusion, this study showed, for the first time, that the duration and severity of energy deficits are critical for changes in bone parameters, BMSC differentiation, and Sirt1 expression. Furthermore, we showed that in this context, Sirt1 expression could impact BMSC differentiation with further effects on bone phenotype.

Bone matrix quality in paired iliac bone biopsies from postmenopausal women treated for 12 months with strontium ranelate or alendronate.

Bone quality is altered mainly by osteoporosis, which is treated with modulators of bone quality. Knowledge of their mechanisms of action is crucial to understand their effects on bone quality. The goal of our study was to compare the action of alendronate (ALN) and strontium ranelate (SrRan) on the determinants of bone quality. The investigation was performed on over 60 paired human iliac biopsies. Paired samples correspond to biopsies obtained from the same patient, one before treatment (baseline) and one after 12 months of treatment, in postmenopausal women with osteoporosis. Vibrational spectroscopy (Raman and FTIRM) and nanoindentation were used to evaluate the effect of both drugs on bone quality at the ultrastructural level. Outcomes measured by vibrational spectroscopy and nanoindentation are sensitive to bone age. New bone packets are distinguished from old bone packets. Thus, the effect of bone age is distinguished from the treatment effect. Both drugs modify the mineral and organic composition in new and old bone in different fashions after 12 months of administration. The new bone formed during ALN administration is characterized by an increased mineral content, carbonation and apatite crystal size/perfection compared to baseline. Post-translational modifications of collagen are observed through an increase in the hydroxyproline/proline ratio in new bone. The proteoglycan content is also increased in new bone. SrRan directly modulates bone quality through its physicochemical actions, independent of an effect on bone remodeling. Strontium cations are captured by the hydrated layer of the mineral matrix. The mineral matrix formed during SrRan administration has a lower carbonate content and crystallinity after 12 months than at baseline. Strontium might create bonds (crosslinks) with collagen and noncollagenous proteins in new and old bone. The nanomechanical properties of bone were not modified with either ALN or SrRan, probably due to the short duration of administration. Our results show that ALN and SrRan have differential effects on bone quality in relation to their mechanism of action.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Dual-energy computed-tomography-based discrimination between basic calcium phosphate and calcium pyrophosphate crystal deposition in vivo.

BACKGROUND: Dual-energy computed tomography (DECT) is being considered as a non-invasive diagnostic and characterization tool in calcium crystal-associated arthropathies. Our objective was to assess the potential of DECT in distinguishing between basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) crystal deposition in and around joints in vivo. METHODS: A total of 13 patients with calcific periarthritis and 11 patients with crystal-proven CPPD were recruited prospectively to undergo DECT scans. Samples harvested from BCP and CPP calcification types were analyzed using Raman spectroscopy and validated against synthetic crystals. Regions of interest were placed in BCP and CPP calcifications, and the following DECT attenuation parameters were obtained: CT numbers (HU) at 80 and 140 kV, dual-energy index (DEI), electron density (Rho), and effective atomic number (Z eff). These DECT attenuation parameters were compared and validated against crystal calibration phantoms at two known equal concentrations. Receiver operating characteristic (ROC) curves were plotted to determine the highest accuracy thresholds for DEI and Z eff. RESULTS: Raman spectroscopy enabled chemical fingerprinting of BCP and CPP crystals both in vitro and in vivo. DECT was able to distinguish between HA and CPP in crystal calibration phantoms at two known equal concentrations, most notably by DEI (200 mg/cm3: 0.037 ± 0 versus 0.034 ± 0, p = 0.008) and Z eff (200 mg /cm3: 9.4 ± 0 versus 9.3 ± 0, p = 0.01) analysis. Likewise, BCP calcifications had significantly higher DEI (0.041 ± 0.005 versus 0.034 ± 0.005, p = 0.008) and Z eff (9.5 ± 0.2 versus 9.3 ± 0.2, p = 0.03) than CPP crystal deposits with comparable CT numbers in patients. With an area under the ROC curve of 0.83 [best threshold value = 0.0 39, sensitivity = 90. 9% (81.8, 97. 7%), specificity = 64.6% (50.0, 64. 6%)], DEI was the best parameter in distinguishing between BCP and CPP crystal depositions. CONCLUSION: DECT can help distinguish between crystal-proven BCP and CPP calcification types in vivo and, thus, aid in the diagnosis of challenging clinical cases, and in the characterization of CPP and BCP crystal deposition occurring in osteoarthritis.

Short-term high-dose zoledronic acid enhances crystallinity in mandibular alveolar bone in rats.

Owing to its antiresorptive properties, zoledronic acid (ZOL) is commonly used in the management of benign as well as malignant bone diseases. This molecule targets sites where bone is actively remodeling, and high concentrations have been reported in the jaw. The purpose of this study was to investigate whether treatment of male rats with ZOL, at a dosage equivalent to that used for antitumor treatment, impacts the short-term qualitative properties of mandibular bone independent of bone remodeling. Thirty rats were randomly assigned to treatment either with ZOL or with serum-vehicle (control) (weekly injections: 100 µg kg-1 for 6 wk, n = 15 per group). Using the tetracycline double-labeling technique, remodeled bone areas, corresponding to the preferential site of bisphosphonate binding, were found in the alveolar bone along the alveolar bone proper. The composition of bone in these areas was characterized using Raman microspectroscopy and compared with adjacent, non-remodeled, older bone. The ZOL-treated group exhibited higher crystallinity in the remodeled bone areas (+2%), reflecting an early maturation of the apatite mineral after ZOL injection. Our findings highlight a direct and rapid effect of clinically relevant anti-tumoral ZOL doses on the qualitative properties of mandibular bone, especially on mineral crystallinity in the vicinity of the teeth, namely, the alveolar bone proper.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.

Mandibular bone is protected against microarchitectural alterations and bone marrow adipose conversion in ovariectomized rats.

Osteoporosis is a disease that leads to a loss of bone mass and to alterations in the bone microarchitecture that occur in a site-specific manner; however it remains controversial in the jaw. The involvement of bone marrow adipose tissue (BMAT) in the bone metabolism has been suggested in several physiopathological contexts, such as in aging and osteoporosis. To test whether the BMAT content is related to mandibular bone loss, this study aimed to investigate the potential correlations between the trabecular bone microarchitecture on one hand and BMAT content and its spatial distribution in relation to bone surface on the other hand during aging and ovariectomy (OVX) during a long-term follow-up in a mature rat model. No age-related microarchitectural or BMAT changes were observed in the mandible. The OVX-induced bone loss was three-fold lower in the mandible than in the tibia and was observed only in the alveolar bone (not in the condyle). We also report a delayed increase in the mandibular BMAT content that remained 4-6-fold lower compared to tibia. This low BMAT content in the mandible was located at a distance from the trabecular bone surface (only 5% in contact with the bone surface versus 87% in the tibia). These findings highlight a specific mandibular response to OVX, in particular fewer microarchitectural alterations compared to that in the tibia. For the latter, the trabecular bone thickness and surface were correlated with the BMAT content. Oral functions may have a protective effect on the mandibular BMAT conversion in an OVX context.

Impaired mineral quality in dentin in X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a skeletal disorder arising from mutations in the PHEX gene, transmitted in most cases as an X-linked dominant trait. PHEX deficiency leads to renal phosphate wasting and hypophosphatemia, as well as impaired mineralization of bone and dentin, resulting in severe skeletal and dental complications. Dentin mineralization defects appear as characteristic, large interglobular spaces resulting from the lack of fusion of calculospherites in the circumpulpal region during the mineralization process. Here, we examined changes in the composition and structure of dentin using Raman spectroscopy on XLH human teeth, and using transmission electron microscopy on the dentin of Hyp mice (the murine model of XLH). The dentin of patients with XLH showed changes in the quality of the apatitic mineral, with greater carbonate substitution and lower crystallinity compared to the dentin of age-matched control teeth. In addition, ultrastructural analysis by transmission electron microscopy revealed a major disorganization of the peri- and intertubular structure of the dentin, with odontoblast processes residing within an unmineralized matrix sheath in the Hyp mouse. Taken together, these results indicate that like for bone and tooth cementum, there are impaired mineral quality and matrix changes in XLH dentin reflecting high sensitivity to systemic serum phosphate levels and possibly other local changes in the dentin matrix.

Unraveling the compromised biomechanical performance of type 2 diabetes- and Roux-en-Y gastric bypass bone by linking mechanical-structural and physico-chemical properties.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder associated with obesity and hyperglycemia. Roux-en-Y gastric bypass (RYGB) surgery is a common treatment for severely obese patients and T2DM. Both RYGB and T2DM are linked to increased skeletal fragility, though the exact mechanisms are poorly understood. Our aim was to characterize the structural, mechanical and compositional properties of bones from diet-induced obese and RYGB-treated obese (bypass) mice to elucidate which the exact factors are contributing to the increased skeletal fragility. To achieve this, a combinatory approach including microfocus X-ray computed tomography, 3-point bending, finite element modeling and Raman spectroscopy, was used. Compared to aged-matched lean controls, the obese mice displayed decreased cortical thickness, trabecular bone loss, decreased stiffness and increased Young's modulus. For the bypass mice, these alterations were even more pronounced, and additionally they showed low mineral-to-matrix ratio in the cortical endosteal area. Accumulation of the advanced glycation end-product (AGE) pentosidine was found in the cortex of obese and bypass groups and this accumulation was correlated with an increased Young's modulus. In conclusion, we found that the increased fracture risk in T2DM- and post-RYGB bones is mainly driven by accumulation of AGEs and macro-structural alterations, generating biomechanical dysfunctionality.

Influence of collecting substrate on the Raman imaging of micron-sized particles.

The influence of six common substrates on the Raman imaging of micron-sized inorganic aerosol particles was examined. Laboratory-generated single-component particles of calcite (CaCO3) and mixed particles of calcite (CaCO3), nitratine (NaNO3), hematite (Fe2O3) and anglesite (PbSO4) were deposited by cascade impaction on Ag, In, Si, SiO2, microscope slide and TEM-grid substrates. The spectral contribution of substrates to Raman images of the deposited particles was evaluated by Multivariate Curve Resolution. The shape and intensity of the substrate spectra affect the effectiveness capability of the spectral deconvolution. The substrates were characterized and compared with respect to their effect on the reconstruction of Raman images of aerosol particles. The TEM-grid substrate yielded spatially stable sample measurements with a homogeneous spectral contribution, satisfactory Raman map reconstruction and the potential for application in other techniques (e.g., SEM-EDX).

Simultaneous three-dimensional visualization of mineralized and soft skeletal tissues by a novel microCT contrast agent with polyoxometalate structure.

Biological tissues have a complex and heterogeneous 3D structure, which is only partially revealed by standard histomorphometry in 2D. We here present a novel chemical compound for contrast-enhanced microfocus computed tomography (CE-CT), a Hafnium-based Wells-Dawson polyoxometalate (Hf-POM), which allows simultaneous 3D visualization of mineralized and non-mineralized skeletal tissues, such as mineralized bone and bone marrow vasculature and adipocytes. We validated the novel contrast agent, which has a neutral pH in solution, by detailed comparison with (immuno)histology on murine long bones as blueprint, and showed that Hf-POM-based CE-CT can be used for virtual 3D histology. Furthermore, we quantified the 3D structure of the different skeletal tissues, as well as their spatial relation to each other, during aging and diet-induced obesity. We discovered, based on a single CE-CT dataset per sample, clear differences between the groups in bone structure, vascular network organization, characteristics of the adipose tissue and proximity of the different tissues to each other. These findings highlight the complementarity and added value of Hf-POM-based CE-CT compared to standard histomorphometry. As this novel technology provides a detailed 3D simultaneous representation of the structural organization of mineralized bone and bone marrow vasculature and adipose tissue, it will enable to improve insight in the interactions between these three tissues in several bone pathologies and to evaluate the in vivo performance of biomaterials for skeletal regeneration.

Tissue-specific mineralization defects in the periodontium of the Hyp mouse model of X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a dento-osseous disorder caused by inactivating mutations in the PHEX gene, leading to renal phosphate wasting and hypophosphatemia, and impaired mineralization of bones and teeth. In the oral cavity, recent reports suggest a higher susceptibility of XLH patients to periodontitis, where patients present with impaired tooth cementum - a bone-like tissue involved in tooth attachment to the jaw bones and post-eruption tooth positioning - and a higher frequency of intrabony defects. In the present study, the pathobiology of alveolar bone and tooth cementum was investigated in the Hyp mouse, the murine analog of XLH. PHEX deficiency in XLH/Hyp dramatically alters the periodontal phenotype, with hypoplasia of tooth root cementum associated with a lack of periodontal ligament attachment and the presence of an immature apatitic mineral phase of all periodontal mineralized tissues. Challenging the Hyp periodontium in two surgical experimental models - ligature-induced periodontal breakdown and repair, and a model of tooth movement adaptation inducing cementum formation - we show that bone and cementum formation, and their healing, are altered. Bone and cementum mineralization appear similarly disturbed, where hypomineralized pericellular matrix surrounds cells, and where the protein osteopontin (OPN, a mineralization inhibitor) accumulates in a tissue-specific manner, most notably in the perilacunar matrix surrounding osteocytes. Although the pathobiology is different between XLH/Hyp bone and cementum, our results show a major XLH phenotype in oral mineralized tissues consistent with variations in patient susceptibility to periodontal disorders.

Correction: New Insights on the Composition and the Structure of the Acellular Extrinsic Fiber Cementum by Raman Analysis.

[This corrects the article DOI: 10.1371/journal.pone.0167316.].