Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM: The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS: We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS: Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 µM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION: Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Dapagliflozin and Liraglutide Therapies Rapidly Enhanced Bone Material Properties and Matrix Biomechanics at Bone Formation Site in a Type 2 Diabetic Mouse Model.

The aim of this study is to compare head-to-head the effects of dapagliflozin and liraglutide on bone strength and bone material properties in a pre-clinical model of diabetes-obesity. Combined low-dose streptozotocin and high fat feeding were employed in mice to promote obesity, insulin resistance, and hyperglycaemia. Mice were administered daily for 28 days with saline vehicle, 1 mg/kg dapagliflozin or 25 nmol/kg liraglutide. Bone strength was assessed by three-point bending and nanoindentation. Bone material properties were investigated by Fourier transform infrared microspectroscopy/imaging. Although diabetic controls presented with dramatic reductions in mechanical strength, no deterioration of bone microarchitecture was apparent. At the tissue level, significant alterations in phosphate/amide ratio, carbonate/phosphate ratio, tissue water content, crystal size index, collagen maturity and collagen glycation were observed and linked to alteration of matrix biomechanics. Dapagliflozin and liraglutide failed to improve bone strength by 3-point bending or bone microarchitecture during the 28-day-treatment period. At bone formation site, dapagliflozin enhanced phosphate/amide ratio, mineral maturity, and reduced tissue water content, crystal size index, and collagen glycation. Liraglutide had significant effects on phosphate/amide ratio, tissue water content, crystal size index, mature collagen crosslinks, collagen maturity, and collagen glycation. At bone formation site, both drugs modulated matrix biomechanics. This study highlighted that these two molecules are effective in improving bone material properties and modulating matrix biomechanics at bone formation site. This study also highlighted that the resulting effects on bone material properties are not identical between dapagliflozin and liraglutide and not only mediated by lower blood glucose.

Sclerostin-Antibody Treatment Decreases Fracture Rates in Axial Skeleton and Improves the Skeletal Phenotype in Growing oim/oim Mice.

In osteogenesis imperfecta (OI), vertebrae brittleness causes thorax deformations and leads to cardiopulmonary failure. As sclerostin-neutralizing antibodies increase bone mass and strength in animal models of osteoporosis, their administration in two murine models of severe OI enhanced the strength of vertebrae in growing female Crtap-/- mice but not in growing male Col1a1Jrt/+ mice. However, these two studies ignored the impact of antibodies on spine growth, fracture rates, and compressive mechanical properties. Here, we conducted a randomized controlled trial in oim/oim mice, an established model of human severe OI type III due to a mutation in Col1a2. Five-week-old female WT and oim/oim mice received either PBS or sclerostin antibody (Scl-Ab) for 9 weeks. Analyses included radiography, histomorphometry, pQCT, microcomputed tomography, and biomechanical testing. Though it did not modify vertebral axial growth, Scl-Ab treatment markedly reduced the fracture prevalence in the pelvis and caudal vertebrae, enhanced osteoblast activity (L4), increased cervico-sacral spine BMD, and improved the lumbosacral spine bone cross-sectional area. Scl-Ab did not impact vertebral height and body size but enhanced the cortical thickness and trabecular bone volume significantly in the two Scl-Ab groups. At lumbar vertebrae and tibial metaphysis, the absolute increase in cortical and trabecular bone mass was higher in Scl-Ab WT than in Scl-Ab oim/oim. The effects on trabecular bone mass were mainly due to changes in trabecular number at vertebrae and in trabecular thickness at metaphyses. Additionally, Scl-Ab did not restore a standard trabecular network, but improved bone compressive ultimate load with more robust effects at vertebrae than at metaphysis. Overall, Scl-Ab treatment may be beneficial for reducing vertebral fractures and spine deformities in patients with severe OI.

Sitagliptin Alters Bone Composition in High-Fat-Fed Mice.

Type 2 diabetes mellitus is recognized as a significant risk factor for fragility of bone. Among the newer anti-diabetic agents, dipeptidyl peptidase-4 inhibitors (DPP4i) have been reported to decrease the occurrence of bone fractures although the reason is unclear. The main aim of this study was to evaluate the impact of sitagliptin treatment on tissue bone strength and compositional parameters in the high-fat-fed mouse model. Male NIH swiss mice were allowed free access to high-fat diet for 150 days to induce chronic hyperglycemia and insulin resistance. Sitagliptin was administered once daily for 3 weeks. High-fat-fed mice administered with saline were used as controls. Bone strength was assessed at the organ and tissue level by three-point bending and nanoindentation, respectively. Bone microarchitecture was investigated by microcomputed tomography and bone composition was evaluated by Fourier transform infrared imaging and quantitative backscattered electron imaging. Administration of sitagliptin increased non-fasting insulin, improved glucose tolerance and increased insulin sensitivity. This was associated with clear ameliorations in bone strength at the organ and tissue level. No changes in trabecular or cortical microarchitectures were observed. On the other hand, higher values of Camean, Caturn, collagen maturity, mineral/matrix ratio, mineral maturity and crystal size index were evidenced after sitagliptin treatment. Correlation analysis significantly linked the modifications of bone strength to changes in bone compositional parameters. These results bring new light on the mode of action of sitagliptin on bone physiology and demonstrate a benefit of DPP4i.

Maxillary sinus floor elevation using Beta-Tricalcium-Phosphate (beta-TCP) or natural bone: same inflammatory response.

Sinus elevation is a common procedure to increase bone volume in the atrophic maxilla to allow placement of dental implants. Autogenous bone is the gold standard but is limited in quantity and causes morbidity at the donor site. ß-TCP is a synthetic biomaterial commonly used in that purpose. It appears to induce a poor inflammatory response. This study aimed to evaluate the degree of edema of the sinus mucosa after sinus lift surgery according to the type of biomaterial. Forty sinuses (20 patients) were included retrospectively and divided into 2 groups according to the biomaterial that was used: synthetic biomaterial (BTCP group), natural bone (BONE group). A control group (CTRL group) was constituted by the non-grafted maxillary sinuses. Twelve measurements per sinus were realized on pre- and post-operative computed tomography and averaged to provide the sinus membrane thickness value (SM.Th). SM.Th was thicker post-operatively in the BTCP and BONE groups in comparison with the CTRL group and in comparison with pre-operative measurements. No difference was found post operatively between the BTCP and BONE groups. We found that a synthetic biomaterial (ß-TCP) induced the same degree of edema, and thus of inflammation, as natural bone. It constitutes therefore an interesting alternative to autogenous bone for maxillary sinus lifts.

Giant cells and osteoclasts present in bone grafted with nacre differ by nuclear cytometry evaluated by texture analysis.

Nacre (mother of pearl) is a natural biomaterial used to prepare orthopedic devices. We have implanted screws and plates made with nacre in five sheeps. Bone were harvested after two months and embedded in poly(methyl methacrylate). Blocks were saws and the thick slabs were grinded, polished and surface stained. Sections were photographed at an ×1000 magnification. Giant cells were found in contact with nacre in eroded areas and true osteoclasts were found at distance in the neighboring bone in Howship lacunae. A texture analysis of the nuclei of giant cells and osteoclasts was done using the run-length method of the MaZda freeware. The size of the nuclei was reduced in osteoclast and their mean gray level appeared reduced. Texture analysis revealed that chromatin had a completely different pattern in giant cells when compared to osteoclasts. Giant cells had a fine repartition of the chromatin with large clear areas around prominent nucleoli. On the contrary, osteoclast nuclei had chromatin blocks evenly dispersed in the nuclei. This reflects the different origin of these cells expressing different functions.

Long-Term Quantitative Evaluation of Muscle and Bone Wasting Induced by Botulinum Toxin in Mice Using Microcomputed Tomography.

Muscle and bone masses are highly correlated and muscles impose large loads on bone. Muscle wasting that accompanies bone loss has been poorly investigated. 21 female mice were spread into seven groups. At day 0, 18 mice received Botulinum toxin (BTX) injection in the quadriceps muscle to induce paralysis of the right hind limb; the left contralateral side was used as control. Mice were sacrificed at 7, 14, 21, 28, 56 and 90 days post-injection. A remaining group was sacrificed at day 0. Trabecular bone volume was determined by microcomputed tomography (microCT) at the distal femur and tibia proximal metaphyses on both sides. Limbs were immersed in an HgCl2 solution allowing muscle visualization by microCT. On 2D sections, the cross-sectional areas and form-factors were measured for the quadriceps at mid-thigh and gastrocnemius at mid-leg and these muscles were dissected and weighed. Bone volume decreased in the paralysed side. Bone loss was maximal at 56 days followed by recuperation at 90 days. The cross-sectional areas of gastrocnemius and quadriceps were significantly lower in the paralysed limb from 7 days; the decrease was maximum at 21 days for the gastrocnemius and 28 days for the quadriceps. No difference in form-factors was found between the two limbs. Similar results were obtained with the anatomical method and significant correlations were obtained between the two methods. Quantitative analysis of muscle loss and recovery was possible by microCT after using a metallic contrast agent. Loss of bone secondary to muscle wastage induced by BTX and recovery showed a parallel evolution for bone and muscles.

The contribution of Micro-CT to the evaluation of trabecular bone at the posterior part of the auricular surface in men.

AIM: Using multi-slice computed tomography (MSCT), Barrier et al. described the disappearance at the posterior auricular surface of a "central line" (CL) and "juxtalinear cells" (JLCs) belonging to a trabecular bundle, and a trabecular density gradient around the CL that decreased with age. The aim of our study was to use micro-CT to test these findings, referring to the concept of Ascadi and Nemeskeri. METHODOLOGY: The coxal bones of fifteen males were used; age was known. CLs were identified on MSCT-sections using Barrier's method (64 detectors, 0.6 mm slice thickness, 0.1 mm overlap) with two different software programs (Synapse®, Amira®). Then, CLs were researched on microCT slices (pixel size: 36 µm). Three volumes of interest were defined (around, above, and below CL), and 3D morphometric parameters of the trabecular microarchitecture (particularly BV/TV and DA) were calculated. Two-tailed statistical analyses were performed attempting to correlate these parameters with age at death. RESULTS: CLs and JLCs were observed on micro-CT slices, but with moderate agreement between both imaging techniques. Their presence was not correlated with the age of the subjects. Around the CL, BV/TV decreased significantly with age; DA was negatively correlated with BV/TV and had a tendency to increase with age. Between areas above and below the CL, there was a BV/TV gradient and both BV/TVs decreased in parallel with age. CONCLUSION: Our findings regarding the contribution of micro-CT to the evaluation of trabecular bone could be a promising research approach for application in a larger study population.

Bone mineralization and vascularization in bisphosphonate-related osteonecrosis of the jaw: an experimental study in the rat.

OBJECTIVES: Pathogenesis of bisphosphonate-related osteonecrosis of the jaws (BRONJ) is not fully explained. An antiangiogenic effect of bisphosphonates (BPs) or an altered bone quality have been advocated. The aims of the present study were to analyze alveolar mandibular vascularization and bone quality in rats with BRONJ. MATERIALS AND METHODS: Thirty-eight Sprague-Dawley rats were randomized into two groups: zoledronic acid (ZA), n = 27, and control (CTRL) n = 11. The ZA group received a weekly IV injection of ZA (100 µg/kg) during 10 weeks. The CTRL group received saline. After 6 weeks, extraction of the right mandibular molars was performed. Rats were sacrificed after 14 weeks. Microtomography characterized bone lesions and vascularization after injection of a radio-opaque material. Raman microspectroscopy evaluated bone mineralization. RESULTS: Fifty-five percent of ZA rats presented bone exposure and signs of BRONJ. None sign was found at the left hemimandible in the ZA group and in the CTRL group. Vascular density appeared significantly increased in the right hemimandibles of the CTRL group compared to the left hemimandibles. Vascularization was reduced in the ZA group. A significantly increased of the mineral-to-amide ratio was found in the alveolar bone of ZA rats by Raman microspectroscopy. CONCLUSIONS: In a rat model of BRONJ, microtomography evidenced osteonecrosis in BRONJ. Raman spectroscopy showed an increased mineralization. Vascularization after tooth extraction was impaired by ZA. CLINICAL RELEVANCE: Prolonged BP administration caused an increase in the mineralization and a quantitative reduction of the vascularization in the alveolar bone; both factors might be involved concomitantly in the BRONJ pathophysiology.

Hypodynamia Alters Bone Quality and Trabecular Microarchitecture.

Disuse induces a rapid bone loss in humans and animals; hypodynamia/sedentarity is now recognized as a risk factor for osteoporosis. Hypodynamia also decreases bone mass but its effects are largely unknown and only few animal models have been described. Hypodynamic chicken is recognized as a suitable model of bone loss but the effects on the quality have not been fully explored. We have used ten chickens bred in a large enclosure (FREE group); ten others were confined in small cages with little space to move around (HYPO group). They were sacrificed at 53 days and femurs were evaluated by microcomputed tomography (microCT) and nanoindentation. Sections (4 µm thick) were analyzed by Fourier Transform InfraRed Microspectroscopy (FTIR) to see the effects on mineralization and collagen and quantitative backscattered electron imaging (qBEI) to image the mineral of the bone matrix. Trabecular bone volume and microarchitecture were significantly altered in the HYPO group. FTIR showed a significant reduction of the mineral-to-matrix ratio in the HYPO group associated with an increase in the carbonate content and an increase in crystallinity (calculated as the area ratio of subbands located at 1020 and 1030 cm-1) indicating a poor quality of the mineral. Collagen maturity (calculated as the area ratio of subbands located at 1660 and 1690 cm-1) was significantly reduced in the HYPO group. Reduced biomechanical properties were observed at the tissue level. Confined chicken represents a new model for the study of hypodynamia because bone changes are not created by a surgical lesion or a traumatic method. Animals have a reduced bone mass and present with an altered bone matrix quality which is less mineralized and whose collagen contains less crosslinks than in control chicken.

Maxillary Sinus Lift with Beta-Tricalcium Phosphate (ß-TCP) in Edentulous Patients: A Nanotomographic and Raman Study.

Sinus lift elevation restores bone mass at the maxilla in edentulate patients before the placement of dental implants. It consists of opening the lateral side of the sinus and grafting beta-tricalcium phosphate granules (ß-TCP) under the olfactory membrane. Bone biopsies were obtained in five patients after 60 weeks. They were embedded undecalcified in poly(methyl methacrylate) (pMMA); blocks were analyzed by nanocomputed tomography (nanoCT); specific areas were studied by Raman microspectroscopy. Remnants of ß-TCP were osseointegrated and covered with mineralized bone; osteoid tissue was also filling the inner porosity. Macrophages having engulfed numerous ß-TCP grains were observed in marrow spaces. ß-TCP was identified by nanoCT as osseointegrated particles and as granules in the cytoplasm of macrophages. Raman microspectroscopy permitted to compare the spectra of ß-TCP and bone in different areas. The ratio of the ~820 cm-1 band of pMMA (-CH2 groups) on the ν1 phosphate band at 960 cm-1 reflected tissue hydration because water was substituted by MMA during histological processing. In bone, the ratio of the ~960 cm-1 phosphate to the amide 1 band and the ratio ν2 phosphate band by the 1240-1250 amide III band reflect the mineralization degree. Specific bands of ß-TCP were found in osseointegrated ß-TCP granules and in the grains phagocytized by the macrophages. The hydration degree was maximal for ß-TCP phagocytized by macrophages. Raman microspectroscopy associated with nanoCT is a powerful tool in the analysis of the biomaterial degradation and osseointegration.

Tooth Extraction Locally Stimulates Proliferation of Multiple Myeloma in a Patient with Mandibular Localizations.

OBJECTIVES: Multiple myeloma (MM) is characterized by the occurrence of osteolytic lesions. MM treatment usually involves antiresorptive drugs (mainly bisphosphonates). CASE REPORT: A patient with an MM presented osteolytic lesions of the mandible. Extraction of teeth 45 and 46 was performed 5 years after the diagnosis of periodontitis. Four months later, osteonecrosis of the jaw (ONJ) was diagnosed at the extraction site. X-ray showed an extension of osteolytic lesions on the right side, close to the extraction site, without modification of the lesions on the left side. Two months later, a curettage was performed because of a painful bone sequestration. X-ray showed an extension of the osteolytic lesions on the right side. RESULTS: Histological analysis found a vascularized plasmacytoma of the soft tissues around the ONJ. Analysis of the bone showed mixed lesions with osteonecrotic areas and living bone resorbed by active osteoclasts surrounding a plasmacytoma. The surface area of the osteolytic foci has considerably increased only close to the extraction site. CONCLUSIONS: Tooth extraction triggered an ONJ associated with bisphosphonate treatment. However, it also seemed to induce a considerable proliferation of plasma cells at the extraction site; we hypothesize that it is due to the increase in bone remodeling related to the surgical trauma.

Nanocomposite particles with improved microstructure for 3D culture systems and bone regeneration.

Nano-apatite and gelatin-alginate hydrogel microparticles have been prepared by a one-step synthesis combined with electrostatic bead generation, for the reconstruction of bone defects. Based on the analysis of bone composition, architecture and embryonic intramembranous ossification, a bio-inspired fabrication has been developed. Accordingly, the mineral phase has been in situ synthesized, calcifying the hydrogel matrix while the latter was crosslinked, finally generating microparticles that can assemble into a bone defect to ensure interconnected pores. Although nano-apatite-biopolymer composites have been widely investigated, microstructural optimization to provide improved distribution and stability of the mineral is rarely achieved. The optimization of the developed method progressively resulted in two types of formulations (15P and 7.5P), with 15 and 7.5 (wt%) phosphate content in the initial precursor. The osteolytic potential was investigated using differentiated macrophages. A commercially available calcium phosphate bone graft substitute (Eurocer 400) was incorporated into the hydrogel, and the obtained composites were in vitro tested for comparison. The cytocompatibility of the microparticles was studied with mouse osteoblast-like cell line MC3T3-E1. Results indicated the best in vitro performance have been obtained for the sample loaded with 7.5P. Preliminary evaluation of biocompatibility into a critical size (3 mm) defect in rabbits showed that 7.5P nanocomposite is associated with newly formed bone in the proximity of the microparticles, after 28 days.

Asymmetric bone remodeling in mandibular and maxillary tori.

OBJECTIVES: Tori are frequent paucisymptomatic bony outgrowths of the oral cavity in three locations: torus palatinus (TP), mandibularis (TM), and maxillaris (TMax). Their usually described histological characteristics are unspecific: normal cortical bone with more or less trabecular bone. The aim of this study was to describe tori's specific morphological and histomorphometric characteristics. MATERIALS AND METHODS: Histological characteristics in a series of 18 tori collected after surgical removal were analyzed. Microcomputed tomography provided a 3D analysis. Mineral apposition rate (MAR) was measured after double tetracycline labeling. Osteoid tissue was identified by Goldner's trichrome and osteoclasts by the tartrate resistant acid phosphatase identification in undecalcified sections. Iron and aluminum were detected by histochemical staining methods. Osteoid thickness and MAR were determined at the outer surface of the torus and in the Haversian canals. RESULTS: Tori appeared made of lamellar Haversian bone in 16/18 cases. Osteoid thickness did not differ between the outer surface and within the canals. An asymmetric bone remodeling was observed in the Haversian canals of 15 tori: osteoid seams were deposited on the side close to the free torus surface, and Howship's lacunae with numerous osteoclasts were observed on the opposite side. A high MAR was found at the surface of the tori and within the canals. There was no iron or aluminum deposit. CONCLUSIONS: Tori may be characterized by a specific asymmetric bone remodeling which seems to determine their shape. CLINICAL RELEVANCE: This finding could constitute a specific histological feature allowing to differentiate tori from exostoses. Graphical abstract.

Stable Incretin Mimetics Counter Rapid Deterioration of Bone Quality in Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus is associated with a high risk for bone fractures. Although bone mass is reduced, bone quality is also dramatically altered in this disorder. However, recent evidences suggest a beneficial effect of the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) pathways on bone quality. The aims of the present study were to conduct a comprehensive investigation of bone strength at the organ and tissue level; and to ascertain whether enzyme resistant GIP or GLP-1 mimetic could be beneficial in preventing bone fragility in type 1 diabetes mellitus. Streptozotocin-treated mice were used as a model of type 1 diabetes mellitus. Control and streptozotocin-diabetic animals were treated for 21 days with an enzymatic-resistant GIP peptide ([D-Ala(2) ]GIP) or with liraglutide (each at 25 nmol/kg bw, ip). Bone quality was assessed at the organ and tissue level by microCT, qXRI, 3-point bending, qBEI, nanoindentation, and Fourier-transform infrared microspectroscopy. [D-Ala2]GIP and liraglutide treatment did prevent loss of whole bone strength and cortical microstructure in the STZ-injected mice. However, tissue material properties were significantly improved in STZ-injected animals following treatment with [D-Ala2]GIP or liraglutide. Treatment of STZ-diabetic mice with [D-Ala(2) ]GIP or liraglutide was capable of significantly preventing deterioration of the quality of the bone matrix. Further studies are required to further elucidate the molecular mechanisms involved and to validate whether these findings can be translated to human patients.

Porosity imaged by a vector projection algorithm correlates with fractal dimension measured on 3D models obtained by microCT.

Porosity is an important factor to consider in a large variety of materials. Porosity can be visualized in bone or 3D synthetic biomaterials by microcomputed tomography (microCT). Blocks of porous poly(2-hydroxyethyl methacrylate) were prepared with polystyrene beads of different diameter (500, 850, 1160 and 1560 µm) and analysed by microCT. On each 2D binarized microCT section, pixels of the pores which belong to the same image column received the same pseudo-colour according to a look up table. The same colour was applied on the same column of a frontal plane image which was constructed line by line from all images of the microCT stack. The fractal dimension Df of the frontal plane image was measured as well as the descriptors of the 3D models (porosity, 3D fractal dimension D3D, thickness, density and separation of material walls. Porosity, thickness Df and D3D increased with the size of the porogen beads. A linear correlation was observed between Df and D3D. This method provides quantitative and qualitative analysis of porosity on a single frontal plane image of a porous object.

Analysis of ß-tricalcium phosphate granules prepared with different formulations by nano-computed tomography and scanning electron microscopy.

Among biomaterials used for filling bone defects, beta-tricalcium phosphate (ß-TCP) is suitable in non-bearing bones, particularly in dental implantology, oral and maxillofacial surgery. When ß-TCP granules are placed in a bone defect, they occupy the void 3D volume. Little is known about the 3D arrangement of the granules, which depends on the nature and size of the granules. The aim of this study was to examine the 3D architecture of porous ß-TCP granules. Granules were prepared with different concentrations of ß-TCP powder in slurry (10, 11, 15, 18, 21, and 25 g of ß-TCP powder in distilled water). Granules were prepared by the polyurethane foam method. They were analyzed by nano-computed tomography (nanoCT) and compared with scanning electron microscopy (SEM). Commercial granules of hydroxyapatite-ß-TCP prepared by the same methodology were also used. The outer and inner architectures of the granules were shown by nanoCT which evidenced macroporosity, internal porosity and microporosity between the sintered grains. Macroporosity was reduced at high concentration and conversely, numerous concave surfaces were observed. Internal porosity, related to the sublimation of the polyurethane foam, was present in all the granules. Microporosity at the grain joints was evidenced by SEM and on 2D nanoCT sections. Granules presented a heterogeneous aspect due to the different mineralization degree of the sintered powder grains in the ß-TCP granules; the difference between hydroxyapatite and ß-TCP was also evidenced. NanoCT is an interesting method to analyze the fine morphology of biomaterials with a resolution close to synchrotron and better than microcomputed tomography.

The peroxisome proliferator-activated receptor γ agonist pioglitazone preserves bone microarchitecture in experimental arthritis by reducing the interleukin-17-dependent osteoclastogenic pathway.

OBJECTIVE: To investigate the effect of pioglitazone on inflammation-induced bone loss and changes in bone microarchitecture in rats with adjuvant-induced arthritis (AIA), focusing on the contribution of interleukin-17 (IL-17) and the balance of RANKL and osteoprotegerin (OPG). METHODS: Male Lewis rats sensitized with Freund's complete adjuvant were treated orally for 21 days with 30 mg/kg/day of pioglitazone or vehicle. Arthritis severity was evaluated by clinical and histologic examination. Bone mineral density (BMD) was assessed by dual x-ray absorptiometry. The therapeutic effect of pioglitazone on changes of the bone architecture was determined by micro-computed tomography (micro-CT). Levels of RANKL, OPG, and IL-17 were determined by serum immunoassay and by synovial tissue immunohistochemistry. Messenger RNA for IL-17 and retinoic acid receptor-related orphan nuclear receptor γt (RORγt) was evaluated by quantitative reverse transcription-polymerase chain reaction and IL-17 promoter activity by gene-reporter assay. RESULTS: Micro-CT analysis revealed that pioglitazone treatment reduced arthritis severity and bone erosion scores and increased BMD in comparison to vehicle treatment. Cortical bone thickness was preserved, although the major beneficial effect of pioglitazone was on indices of the trabeculae, especially trabecular separation. Pioglitazone reduced the ratio of RANKL to OPG, in both the serum and the inflamed synovium. Circulating levels of IL-17 were significantly reduced by pioglitazone treatment, as were the percentages of IL-17-positive cells, mainly polymorphonuclear cells, in the inflamed synovium. Induction of IL-17 was strictly dependent on the binding of RORγt to IL-17 promoter, and lentiviral overexpression of peroxisome proliferator-activated receptor γ (PPARγ) reduced the expression of RORγt. CONCLUSION: Pioglitazone decreased the level of inflammatory bone destruction and protected the bone microarchitecture in rats with AIA by controlling the circulating and local expression of IL-17, with a subsequent decrease in the RANKL-to-OPG ratio. Along with the inhibition of RORγt expression after PPARγ overexpression, these findings provide evidence of the major contribution of reduced IL-17/RANKL-dependent osteoclastogenesis.