Guillaume Falgayrac

New Method for Raman Investigation of the Orientation of Collagen Fibrils and Crystallites in the Haversian System of Bone

Knowledge of the organization of the components of bone is of primary importance in understanding how this tissue responds to stresses and provides a starting point for the design and development of biomaterials. Bone structure has been the subject of numerous studies. The mineralized fiber arrangement in cortical bone is either a twisted or orthogonal plywood structure. Both mineral models coexist in compact bone. Raman polarized spectroscopy offers definite advantages in the study of biological samples, enabling the simultaneous analysis of mineral and organic components and the determination of molecular orientation through the polarization properties of the Raman scattering. In this study, we used the Raman polarization approach to simultaneously investigate the orientation of collagen fibrils and apatite crystals in human cortical bone. Raman bands ratios were monitored as a function of sample orientation. Specific ratios were chosen—such as ν3 PO4/ν1 PO4, amide III (1271 cm−1)/amide III (1243 cm−1), and amide I/amide III (1243 cm−1)—due to their sensitivity to apatite-crystal and collagen-fibril orientation. Based on this original approach, spatial changes were monitored as a function of distance from the Haversian canal. The results revealed simultaneous tilting in intra-lamellar collagen-fibril and mineral crystal orientations. These results are consistent with a twisted plywood organization in the Haversian bone structure at the lamellar level. But at molecular level, the co-alignment of the collagen fibrils and the apatite crystal is observed in the innermost lamellae and becomes gradually less ordered as the distance from the Haversian canal increases. This work highlights the interest of Raman spectroscopy for the multiscale investigation of bone structure.

Molecular interactions between zoledronic acid and bone: An in vitro Raman microspectroscopic study

The aim of this study was to investigate molecular interactions between a bisphosphonate (BP), zoledronic acid, and bone tissue by the use of Raman microspectroscopy. In this way, samples of hydroxyapatite (HA), as a bone model, and Wistar rat femurs were soaking in zoledronic acid solutions. Sample surfaces were studied by Environmental Scanning Electron Microscopy and Raman spectroscopy. The amount of zoledronic acid incorporated onto the samples and the inorganic phosphate released in solution were determined by (31)P NMR spectroscopy. Total carbonate content in solution was evaluated by inorganic carbon analyser. After impregnation new Raman bands with frequencies close to characteristic peaks of zoledronic acid (in particular phosphate moieties and imidazole ring of the R2 side-chain) were observed on both types of samples. Physico-chemical parameters of the bone were also significantly modified (P<0.0001). The mineral to organic ratio and the carbonate to phosphate ratio decreased and the crystallinity increased. Released inorganic phosphate and carbonate were detected in the solutions. The Raman shift of the bands corresponding to the phosphate groups and the imidazole ring of the BP highlight their implication in the binding to the mineral. The detection of released inorganic phosphate and carbonate in solution, the modifications of the mineral to phosphate ratio and the carbonate to phosphate ratio reveal that BP decrease the amount of inorganic phosphate and limit the dissolution of bone mineral. The increase of the crystallinity after BP binding shows a re-organisation of the lattice with a higher symmetry. Thus, it seems that zoledronic acid has an important contribution on the increase of crystallinity. The use of Raman spectrometry brings new and complementary information on the impact of zoledronic acid on bone composition at molecular level. Raman spectrometry could help to understand by which way BPs improve bone strength and decrease fracture risk.

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

BackgroundOsteoblasts 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.ResultsGene 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.ConclusionThis 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.

No anti-angiogenic effect of clinical dosing regimens of a single zoledronic acid injection in an experimental bone healing site

INTRODUCTION An anti-angiogenic effect of bisphosphonates has been reported in different experimental models. Zoledronic acid is currently administered in osteoporotic patients as a single 5 mg injection once a year and its vascular effect in bone has not been yet evaluated. MATERIALS AND METHODS The vascular dose effect of a single injection of zoledronic acid was evaluated on healing vascularization developed under a bone chamber implanted on the calvaria of 30 rats. After 3 weeks of healing, the rats were randomized into 3 groups receiving an injection of either physiologic saline solution (PSS) or zoledronic acid tested at 120 microg/kg, the equivalent of a 5 mg dose of zoledronic acid in humans (Z120), and 400 microg/kg, a supra-pharmacologic dose (Z400). A longitudinal follow-up of the healing vascular network was carried out at days (D) 1, 3, 6, 9, 12, 15 and 28 after injection by intravital imaging. Variations in vascular density, total length of the vascular network and mean diameter of vascular network branches were determined by image analysis (Aphelion software). RESULTS A decrease was observed in both vascular density and total length of the network in control and treated groups (time effect). No difference in variation in vascular density was observed between the PSS group and the Z120 group at any time point (p=NS). A trend to a higher decrease in vascular density was noted between D12 and D15 in the Z400 group. A significant decrease in total length was noted at D15 in the Z400 group (p=0.03) compared to the PSS group, whereas no change was noted in rats treated with 120 microg/kg compared to PSS rats on any of the follow-up days (p=0.2). No variation in mean diameter of vascular network branches was noted in any of the three groups at any of the follow-up days (p=0.53). CONCLUSION A single injection of clinically relevant dosing regimens of zoledronic acid may not have a notable impact on vascularization in bone sites. The anti-angiogenic effect of bisphosphonates seems to express itself, in our model, at higher doses than those used in patients treated for osteoporosis.

Region specific Raman spectroscopy analysis of the femoral head reveals that trabecular bone is unlikely to contribute to non-traumatic osteonecrosis

Non-traumatic osteonecrosis (ON) of the femoral head is a common disease affecting a young population as the peak age of diagnosis is in the 40 s. The natural history of non-traumatic ON leads to a collapse of the femoral head requiring prosthetic replacement in a 60% of cases. Although trabecular bone involvement in the collapse is suspected, the underlying modifications induced at a molecular level have not been explored in humans. Here, we examine changes in the molecular composition and structure of bone as evaluated by Raman spectroscopy in human end-stage ON. Comparing samples from femoral heads harvested from 11 patients and 11 cadaveric controls, we show that the mineral and organic chemical composition of trabecular bone in ON is not modified apart from age-related differences. We also show that the molecular composition in the necrotic part of the femoral head is not different from the composition of the remaining ‘healthy’ trabecular bone of the femoral head. These findings support that quality of trabecular bone is not modified during ON despite extensive bone marrow necrosis and osteocyte death observed even in the ‘healthy’ zones on histological examination.

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

Acellular extrinsic fiber cementum is a mineralized tissue that covers the cervical half of the tooth root surface. It contains mainly extrinsic or Sharpey’s fibers that run perpendicular to the root surface to anchor the tooth via the periodontal ligament. Acellular cementum is continuously and slowly produced throughout life and exhibits an alternating bright and dark pattern under light microscopy. However, although a better understanding of the structural background of acellular cementum is relevant to many fields, such as cementochronology, periodontology and tissue engineering, acellular cementum remains rarely studied and poorly understood. In this work, we studied the acellular cementum at the incremental line scale of five human mandibular canines using polarized Raman spectroscopy. We provided Raman imaging analysis and polarized acquisitions as a function of the angular orientation of the sample. The results showed that mineral crystals were always parallel to collagen fibrils, and at a larger scale, we proposed an organizational model in which we found radial collagen fibers, “orthogonal” to the cementum surface, and “non-orthogonal” fibers, which consist of branching and bending radial fibers. Concerning the alternating pattern, we observed that the dark lines corresponded to smaller, more mineralized and probably more organized bands, which is consistent with the zoological assumption that incremental lines are produced during a winter rest period of acellular cementum growth.

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.

Mica Dust and Pneumoconiosis Example of a Pure Occupational Exposure in a Muscovite Milling Unit

Objective: We present pulmonary disorders of four employees who were exposed to high concentration of pure mica dust in a muscovite milling unit. Method: All cases underwent traditional examinations with a dual-energy chest computed tomographic scan. An analysis of exhaled breath condensate by Raman microspectrometry and of mineralogical content of a lung biopsy was performed for one case. Results: All cases showed bilateral micronodular ground glass opacities and mediastinal and hilar hyperdense lymph nodes consistent with the nodal sequestration of mineral particles. Histological analysis showed giant cell granulomas without typical silicotic nodule with high concentration of birefringent particles consistent with mica. Mica particles found in the exhaled breath condensate were identical to particles in ambient air at the company. Conclusion: Occupational exposure to mica dust is responsible for diffuse infiltrative lung disease by overload processes.

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.