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.

Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur.

Osteoporosis is characterized by a bone loss associated to an increased bone marrow adiposity; however, it is still unclear what kind of lipids are involved. Therefore, the main purpose of this study was to see if there is any local bone lipid changes related to osteoporosis, by using the ovariectomy-induced osteoporosis (OVX) rat model. Female SD rats (operated at 6 months of age for skeletal maturity) were divided in control SHAM and OVX groups (n = 6/group) and maintained for 9 month post-surgery. Lipids were analyzed in two compartments of femoral diaphyses: bone marrow (BM) and mineralized tissue (MT), by chromatographic methods. As expected, osteoporotic femurs had a larger BM mass associated with a two-fold increase of lipid content. The MT had a similar lipid enrichment, indicating that adiposity affected the mineral part as well. The main lipids concerned were triglycerides, sphingomyelin, phosphatidylcholine and phosphatidylserine in BM, and triglycerides and cholesterol esters in MT. The increase of both energy-storage and membrane-associated lipids in BM suggested that cell number and/or size was enhanced to allow more triglyceride storage. Interestingly, in MT of osteoporotic femurs, sphingomyelin was decreased, suggesting that its catabolism could be linked to osteoporosis. In both femoral compartments, fatty acid profiles were enriched in 14:0 and 16:1, lowered in 18:0 and 20:4 n-6, and two-fold higher stearoyl-CoA desaturase indexes (16:1/16:0 and 18:1/18:0 ratios), suggesting an increased de novo lipogenesis in osteoporotic femurs. Thus, the present study is first to report local changes of individual lipids in rat osteoporotic femurs and suggests that osteoporosis is a pathologic condition associated with an enhanced de novo lipogenesis. Further studies will be needed to better understand the consequences of these lipid changes in osteoporotic bones.

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.

A Novel microCT Method for Bone and Marrow Adipose Tissue Alignment Identifies Key Differences Between Mandible and Tibia in Rats.

Bone homeostasis is influenced by the bone marrow adipose tissue (BMAT). BMAT distribution varies from one anatomical location in the skeleton to another. We developed an advanced microfocus computed tomography imaging and analysis protocol that allows accurate alignment of both the BMAT distribution and bone micro-architecture as well as calculation of the distance of the BMAT adipocytes from the bone surface. Using this protocol, we detected a different spatial BMAT distribution between the rat tibia and mandible: in the proximal metaphysis of the tibia a large amount of BMAT (~ 20% of the total BMAT) was located close to the bone surface (< 20 µm), whereas in the alveolar ridge ~ 30% of the total BMAT was located between 40 and 60 µm from the bone surface. In the alveolar ridge of rats, the trabecular bone volume was 48.3% higher compared to the proximal metaphysis of the tibia (p < 0.0001) and the percentage of adiposity determined to the relative marrow volume was lower (1.5%) compared to the proximal metaphysis of the tibia (9%, p = 0.0002). Interestingly, in the tibia a negative correlation was found between the percentage of adiposity in the total volume and the trabecular thickness (r =- 0.74, p = 0.037). The present study highlights that in comparison to tibial proximal metaphysis, the mandibular bone exhibits a massive trabecular network and a low BMAT content with almost no contact with the bone surface. These findings are of great interest because of the importance of the fat-bone interaction and its potential relevance to several resorptive bone diseases.

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.

Characterization of bone diagenesis by histology in forensic contexts: a human taphonomic study.

The diagenesis of a bone in the postmortem period causes an identifiable deterioration in histology. This degradation is characterized by a collagenous alteration, which can be observed very early. In order to develop a method for determining a postmortem interval for medico-legal use, two ribs collected from six human bodies were studied prospectively over 2 years. Each bone was studied after staining with Sirius red to demonstrate the degradation of collagen as a function of time. This study demonstrated a time-based bone alteration characterized by the architectural degradation of the lamellar bone, without any microbial influence in this postmortem period. The staining was carried out by using Sirius red and correlated this alteration with a collagenic degradation by chemical hydrolysis owing to the affinity of this dye to the amino acids lysine, hydroxylysine, and arginine. Our work asserts that human bone samples that were studied in a controlled environment and analyzed for 24 months underwent a diagenetic trajectory whose main element was collagen hydrolysis.

Bone Samples Extracted from Embalmed Subjects Are Not Appropriate for the Assessment of Bone Quality at the Molecular Level Using Raman Spectroscopy.

Bone samples extracted from embalmed cadavers are commonly used as controls in the study of bone. The effects of embalmment on the molecular composition of bone are unknown. The objective of this study was to determine the effect of embalmment on the molecular composition and structure of bone, as evaluated by Raman spectroscopy. Bone samples of femoral heads from five embalmed donors and five fresh-frozen donors were compared using Raman microspectroscopy with DuoScan technology. Physicochemical parameters simultaneously describing the organic and mineral phases of bone were compared using the Mann-Whitney U test. Partial least squares discriminant analysis (PLS-DA) was used to determine specific Raman spectral features of each group. Study of the mineral phase showed a 15% reduction of the mineral-to-matrix ratio (p < 0.001), an 8% decrease of type B carbonate substitution (p < 0.001), and a 2% increase in crystallinity (p < 0.001) in the embalmed donors group compared to those of the fresh donors group. Regarding the organic phase of bone, the hydroxyproline-to-proline ratio was increased by 18% in the embalmed group (p < 0.001), with no variation in both the relative proteoglycan content (GAG/CH3) (p = 0.08) and collagen maturity (p = 0.57). PLS-DA showed that the embalmed group was characterized mainly by peaks assigned to hydroxyproline, lipids, and collagen. Embalmment induces significant modifications of the molecular composition of bone. Bone samples from embalmed subjects should be avoided as controls for Raman spectroscopy studies. Preservation procedures performed prior to bone sampling should be reported in studies using human cadaver samples.

Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation.

BACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages. In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation. RESULTS: Gene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage. CONCLUSION: This model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes.

Comparison of two-dimensional fast Raman imaging versus point-by-point acquisition mode for human bone characterization.

Recent technical developments gave rise to a new technology for two-dimensional fast Raman imaging: the DuoScan averaging mode (DS-Avg). This technology allows the acquisition of a Raman spectrum over a rastered macro spot. The aim of this study was to evaluate the interest of the DS-Avg applied on trabecular human bone. The evaluation was based on the comparison of the DS-Avg versus the point-by-point mapping mode in real usage conditions. The signal-to-noise ratio, the spectral difference, and the physicochemical parameters were estimated for comparison of the efficiency of both modes. Principal component analysis was performed to explore the capacity of both modes to detect compositional variations. Results showed that the DS-Avg spectrum was equivalent to the average spectrum of individual spectra acquired with the point-by-point mode for the same sample area. The physicochemical parameters can be also determined from DS-Avg acquisition. The DS-Avg combined with an objective ×50 allows a drastic decrease of the acquisition time, but the information about the micrometric composition is lost. The combination of the DS-Avg with an objective ×100 is a good compromise between acquisition time and resolution. The DS-Avg is a useful technology for imaging mineral and organic phases of bones and for assessing their spatial distribution on large samples. The point-by-point imaging mode is more appropriate to assess the heterogeneous composition of bone within the micrometer scale. For the first time, this study compares the DuoScan averaging mode to the point-by-point imaging mode on a trabecular human bone.

Reproducibility of 1H MR spectroscopy of hip bone marrow at 3 Tesla.

PURPOSE: To assess the reproducibility of fat content (FC) values in five different anatomical areas of proximal femur by the means of (1)H 3 Tesla MR spectroscopy and to evaluate if any statistical difference exists when comparing right side to left side FC values in the same individual. MATERIALS AND METHODS: Thirty-three volunteers underwent unilateral (1)H MR spectroscopy of the hip. From 1 to 4 weeks later, they repeat the MR examination of the same hip to assess the reproducibility of the technique. Fifteen other volunteers underwent a bilateral (1)H MR hip spectroscopy to compare right and left side FC values. RESULTS: The reproducibility of (1)H MR spectroscopy was high in all the anatomic areas considered, ranging from 0.90 to 0.98. No statistically significant difference was found when the fat content values on the right side were compared to those on the left side. (P > 0.16). CONCLUSION: The 3 Tesla (1)H MR spectroscopy of hip bone marrow permitted highly reproducible fat content values in all the five anatomic areas examined. No statistical significant difference existed when comparing fat content values of the right side to those of the left side in the same individual.

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.

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.

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.

Laser preconditioning on cranial bone site: analysis of morphological vascular parameters.

BACKGROUND AND OBJECTIVES: Bone vascularization is a key factor in the bone healing process following X-ray irradiation. Preserving the vascular network from X-ray-induced injury is a relevant approach in the promotion of bone healing. Previously, we developed a protocol of laser preconditioning (810 nm diode laser, 36 J/cm²) prior to X-ray radiation (18.75 Gy) which protects the bone vascular network from deleterious effects of X-ray radiation. The aim of this present work is to characterize the effects of laser preconditioning on the bone through a morphological analysis of vascular parameters. MATERIALS AND METHODS: Digital images of the vascular plexus were taken through an optical bone chamber which was implanted onto the calvaria of rabbits. Bespoke software was used for the quantification of the vessels (classified in four groups according to their diameter), vessel length, and number of nodes at weeks 0, 4, and 8. Twenty rabbits were divided into four groups: control group #1 (n = 5); laser group #2 (n = 5). X-ray radiation group #3 (n = 5), laser preconditioning 24 hours prior to X-ray radiation group #4 (n = 5). RESULTS: The bone vascular network was stable for groups #1 and #2. Statistical analysis showed a significant reduction of each observed vascular parameter for groups #3 and #4. In the laser preconditioned group #4 the loss was less marked than in the X-ray group #3, especially for large vessels (diameter >50 µm). DISCUSSION AND CONCLUSION: We provide in vivo microcirculatory evidence to support the concept of laser preconditioning of bone. A computer-based semi-automatic system is described to quantify superficial bone vascular network parameters that had been treated by laser preconditioning prior to X-ray radiation. Laser preconditioning significantly attenuates the deletion of the superficial bone vascular network irradiated by X-ray, especially concerning large diameter vessels.

T1-weighted MRI images accurately represent the volume and surface of architectural mineral damage of osteonecrosis of the femoral head: Comparison with high-resolution computed tomography.

The potency of magnetic resonance imaging (MRI) to measure the exact extent of osteonecrosis of the femoral head (ONFH) remains uncertain. The objective of this study was to determine if the volume of necrosis assessed with MRI accurately reflects the volume of architectural mineral alterations in osteonecrosis of the femoral head by comparison with high-resolution microfocus X-ray computed tomography (HR-µCT). Fourteen male patients aged 53 years [46.2;59.0] suffering from ONFH were prospectively enrolled to undergo preoperative MRI and ex vivo analysis using HR-µCT. The necrotic zone on T1-weighted MRI scans was defined as total necrosis (delimited by the low-signal peripheral band) or dark necrosis (low-signal lesions only). The HR-µCT scans delimited outer necrosis and inner necrosis by including or excluding the sclerotic zone. The intra-class correlation coefficient (ICC) was calculated to compare the agreement of surface areas and volumes of necrosis measurements with the two techniques. There was an overall excellent agreement between MRI dark necrosis volume and HR-µCT outer necrosis volume (ICC=0.91[0.54;0.98]) while the MRI total necrosis volume showed poor agreement with both HR-µCT delimitations of necrosis volume. For surface area, agreement between MRI dark necrosis and HR-µCT delimitations was good for inner necrosis (ICC=0.70[0.21;0.9]) and moderate for outer necrosis (ICC=0.58[0.07;0.85]). This study demonstrates that measurement of the MRI lesions provides a reliable assessment of the extent of ONFH-related architectural damage.

A preliminary investigation into the effects of X-ray radiation on superficial cranial vascularization.

Radiation therapy (RT) is an established treatment modality for malignant neoplasms. RT induces tissue damage that may lead to osteoradionecrosis in more severe cases. Suitable animal models to study RT-induced changes in membranous craniofacial bone are currently not available. The aim of this study was therefore to quantify RT-induced changes in cranial microcirculation using a newly developed calvaria chamber model and to relate these changes to RT-induced histological damage. New Zealand white rabbits received a total radiation dose of 18.75 Gy through the calvaria chamber, and the number of vessels, the vessel length density (VLD), and angiogenic sprouting were quantified on a weekly basis during a 12-week period. At the end of 12 weeks, the RT-treated (n = 5) or control (n = 5) calvarias were biopsied for histopathological analysis. RT resulted in a steep reduction in the number of vessels and the VLD during the first 3 weeks, particularly in larger-diameter vessels, followed by a flat stabilization/remodeling phase in the subsequent 9 weeks that never restored to baseline values. Histomorphometric analysis revealed a high degree of osteocytic depletion, prominent hypocellularity in the lacunae and intraosseous vasculature, enlarged and nonconcentric Haversian systems, and a severely disorganized bone matrix in the RT-treated calvarias. Despite the prevalence of some angiogenic potential, the RT-induced effects in the early phase persisted in the intermediate to late phase, which may have contributed to the poor recovery of the RT-treated bone.

Brief Report From the 4th International Meeting on Bone Marrow Adiposity (BMA2018).

The 4th International Meeting on Bone Marrow Adiposity (BMA2018) was hosted at the premises of the Regional Government of Hauts de France in Lille, from August 29th to August 31st 2018. This congress brought together physicians and scientists working on rheumatology and bone biology, oncology, hematology, endocrinology, and metabolic diseases, all interested in bone marrow adiposity. They shared their opinions, hypothesis, and original results. Six invited keynotes were given by S. Badr, B.C.J. van der Eerden, M.J. Moreno Aliaga, O. Naveiras, C.J. Rosen, and A.V. Schwartz. Twenty-one short talks were also given. This report briefly summarizes the scientific content of the meeting and the progress of the working groups of the BMA Society (http://bma-society.org/).

Marrow adiposity and bone: Review of clinical implications.

There is growing interest in the relationship between bone marrow fat (BMF) and skeletal health. Progress in clinical studies of BMF and skeletal health has been greatly enhanced by recent technical advances in our ability to measure BMF non-invasively. Magnetic resonance imagery (MRI) with or without spectroscopy is currently the standard technique for evaluating BMF content and composition in humans. This review focuses on clinical studies of marrow fat and its relationship with bone. The amount of marrow fat is associated with bone mineral density (BMD). Several studies have reported a significant negative association between marrow fat content and BMD in both healthy and osteoporotic populations. There may also be a relationship between marrow fat and fracture (mostly vertebral fracture), but data are scarce and further studies are needed. Furthermore, a few studies suggest that a lower proportion of unsaturated lipids in vertebral BMF may be associated with reduced BMD and greater prevalence of fracture. Marrow fat might be influenced by metabolic diseases associated with bone loss and fractures, such as diabetes mellitus, obesity and anorexia nervosa. An intriguing aspect of bariatric (weight loss) surgery is that it induces bone loss and fractures, but with different impacts on marrow fat depending on diabetic status. In daily practice, the usefulness for clinicians of assessing marrow fat using MRI is still limited. However, the perspectives are exciting, particularly in terms of improving the diagnosis and management of osteoporosis. Further studies are needed to better understand the regulators involved in the marrow fat-bone relationship and the links between marrow fat, other fat depots and energy metabolism.

Bone disorders associated with diabetes mellitus and its treatments.

Both type 1 and type 2 diabetes mellitus are associated with bone disorders, albeit via different mechanisms. Early studies in patients with type 1 diabetes suggested a 10-fold increase in the hip fracture risk compared to non-diabetic controls. Meta-analyses published more recently indicate a somewhat smaller risk increase, with odds ratios of 6 to 7. Diminished bone mineral density is among the contributors to the increased fracture risk. Both types of diabetes are associated with decreased bone strength related to low bone turnover. The multiple and interconnected pathophysiological mechanisms underlying the bone disorders seen in type 1 diabetes include insulin deficiency, accumulation of advanced glycation end products, bone microarchitecture alterations, changes in bone marrow fat content, low-grade inflammation, and osteocyte dysfunction. The bone alterations are less severe in type 2 diabetes. Odds ratios for hip fractures have ranged across studies from 1.2 to 1.7, and bone mineral density is higher than in non-diabetic controls. The odds ratio is about 1.2 for all bone fragility fractures combined. The pathophysiological mechanisms are complex, particularly as obesity is very common in patients with type 2 diabetes and is itself associated with an increased risk of fractures at specific sites (humerus, tibia, and ankle). The main mechanisms underlying the bone fragility are an increase in the risk of falls, sarcopenia, disorders of carbohydrate metabolism, vitamin D deficiency, and alterations in cortical bone microarchitecture and bone matrix. The medications used to treat both types of diabetes do not seem to play a major role. Nevertheless, thiazolidinediones and, to a lesser extent, sodium-glucose cotransporter inhibitors may have adverse effects on bone, whereas metformin may have beneficial effects. For the most part, the standard management of bone fragility applies to patients with diabetes. However, emphasis should be placed on preventing falls, which are particularly common in this population. Finally, there is some evidence to suggest that anti-fracture treatments are similarly effective in patients with and without diabetes.