Biomaterials preparation by electrospinning of gelatin and sodium hyaluronate/gelatin nanofibers with non-toxic solvents.

Gelatin (Ge) based fibers have been produced by electrospinning with a non-toxic solvent for preparing membranes usable in maxillofacial surgery. Ge and Ge/sodium hyaluronate (SH) nanofibers were successfully electrospun to produce membranes whose thickness was around 150 to 200µm. The mean fiber diameter reached a maximum of 660nm for Ge fibers and 210nm for Ge/SH fibers. The presence of Ge and SH was confirmed in the membranes by Raman spectroscopy. Ge membranes had low mechanical properties and only small samples of 0.5cm in size could be retrieved from the collector as larger sample tended to tear and break. Ge/SH membranes could be retrieved from the collector slightly easily. Membranes could be handled carefully but in vivo implantation could not be planned due to poor mechanical resistance. Crosslinking by glutaraldehyde vapors reduced the mean porosity of Ge membranes; it totally prevents membranes to be retrieved from the collector. Beta tricalcium phosphate (ß-TCP) particles were added with Ge during electrospinning to increase osseointegration of the membranes and promote bone formation. ß-TCP particles formed agglomerates outside the fibers, and we could not obtain ß-TCP particles inside the Ge fibers due to their low diameter. In general, electrospun membranes lacked reproducibility. Despite the great interest of Ge-based membranes and Ge/ß-TCP membranes, the low mechanical properties of the fibers, the lack of reproducibility and the difficulty to retrieve the membranes from the collector did not allow our biomaterials to be implanted or to be envisaged for industrial production.

Behavior of macrophage and osteoblast cell lines in contact with the ß-TCP biomaterial (beta-tricalcium phosphate).

Beta-tricalcium phosphate (ß-TCP) is a synthetic ceramic used for filling bone defects. It is a good alternative to autologous grafts since it is biocompatible, resorbable and osteoconductive. Previous in vivo studies have shown that macrophages are one of the first cells coming in contact with the biomaterial followed by osteoclasts and osteoblasts that will elaborate new bone packets. Studies have focused on osteoclast morphology and very few of them have investigated the role of macrophages. The aims of this study were to characterize (i) the biomaterial surface; (ii) the in vitro behavior of macrophages (J774.2 and Raw264.7 cells) using the description of cell morphology by scanning electron microscopy (SEM) at 7 and 14 days; (iii) the behavior of osteoblasts (SaOs-2 and MC3T3-E1 cells) seeded at the surface of the biomaterial 24, 48 and 72hours by SEM and confocal microscopy. Cell proliferation was analyzed by MTT assays. Viability and affinity of the macrophages for ß-TCP were found significantly increased after 7 and 14d. MC3T3-E1 cells were anchored and stretched onto the ß-TCP surface as early as 24h with a high proliferation rate (+190%) when compared to the surface of a well plate. SaOs-2 exhibited the same morphological profile at 72h. Proliferation became significantly higher compared to the plastic surface at only 72h (+129%). This study emphasises the importance of choice of the cell line used in exploring the osteoconductive and osteoinductive properties of a biomaterial. Additional studies are needed to analyze differentiation of macrophages into giant multinucleated cells and how the biomaterial surface influences osteoblast differentiation.

In vivo erosion of orthopedic screws prepared from nacre (mother of pearl).

BACKGROUND: Biodegradable biomaterials have been proposed to prepare orthopedic devices. Nacre is a natural aragonitic material made of calcium carbonate and is bioerodible. WORKING HYPOTHESIS: We postulated that nacre is biodegradable without provoking bone erosion and favors bone apposition. MATERIAL AND METHODS: We prepared orthopedic screws from nacre of the giant oyster Pinctada maxima. Threaded screws (3.5mm diameter) were implanted in 6 ewes in the upper tibial metaphysis (3 to 4 screws per animal). Their trajectory was transcortical and intramedullary to the opposite cortex. Animals were kept for 3months (n=2) and 6 months (n=4). They did not develop local inflammation. Before euthanasia, they received a double calcein labeling. Bone samples were analyzed by X-ray nanotomography and histology after embedding in poly(methyl methacrylate). The fractal dimension of the screw profiles (measured by the box-counting method) was used to quantify surface erosion. RESULTS: 3D nanotomography showed a gradual erosion of the threads, which was confirmed by a decreased fractal dimension. Histologically, multinucleated cells (non-osteoclastic appearance) were visible at the surface of the screws. No ruffled border was seen in these cells but they had extensions creeping in the organic matter between the aragonite tablets. Bone apposition was noted in the transcortical path of the screws with limited osteoconduction at the endosteum. Mineralization rate was increased in these zones composed of woven bone in contact with the nacre. DISCUSSION AND CONCLUSION: Screws prepared from nacre have the advantage of an in vivo resorbability by macrophage-derived cells and an osteoconductive apposition in contact with the material without triggering a local inflammatory reaction.

Factors associated with an increased risk of vertebral fracture in monoclonal gammopathies of undetermined significance.

Monoclonal gammopathies of undetermined significance (MGUS) have been shown to be associated with an increased risk of fractures. This study describes prospectively the bone status of MGUS patients and determines the factors associated with vertebral fracture. We included prospectively 201 patients with MGUS, incidentally discovered, and with no known history of osteoporosis: mean age 66.6±12.5 years, 48.3% women, 51.7% immunoglobulin G (IgG), 33.3% IgM and 10.4% IgA. Light chain was kappa in 64.2% patients. All patients had spinal radiographs and bone mineral density measurement in addition to gammopathy assessment. At least one prevalent non-traumatic vertebral fracture was discovered in 18.4% patients and equally distributed between men and women. Fractured patients were older, had a lower bone density and had also more frequently a lambda light chain isotype. Compared with patients with κ light chain, the odds ratio of being fractured for patients with λ light chain was 4.32 (95% confidence interval 1.80-11.16; P=0.002). These results suggest a high prevalence of non-traumatic vertebral fractures in MGUS associated with lambda light chain isotype and not only explained by low bone density.

The use of animal models in multiple myeloma.

In myeloma, the understanding of the tissular, cellular and molecular mechanisms of the interactions between tumor plasma cells and bone cells have progressed from in vitro and in vivo studies. However none of the known animal models of myeloma reproduce exactly the human form of the disease. There are currently three types of animal models: (1) injection of pristane oil in BALB/c mice leads to intraperitoneal plasmacytomas but without bone marrow colonization and osteolysis; (2) injection of malignant plasma cell lines in immunodeficient mice SCID or NOD/SCID; the use of the SCID-hu or SCID-rab model allows the use of fresh plasma cells obtained from MM patients; (3) injection of allogeneic malignant plasma cells (5T2MM, 5T33) in the C57BL/KalwRij mouse induces bone marrow proliferation and osteolytic lesions. These cells did not grow in vitro and can be propagated by injection of plasma cells isolated from bone marrow of a mouse at end stage of the disease into young recipient mice. The 5TGM1 is a subclone of 5T33MM cells and can grow in vitro. Among the different models, the 5TMM models and SCID-hu/SCID-rab models were extensively used to test pathophysiological hypotheses and to assess anti-osteoclastic, anti-osteoblastic or anti-tumor therapies in myeloma. In the present review, we report the different types of animal models of MM and describe their interests and limitations.

Comparison between quantitative X-ray imaging, dual energy X-ray absorptiometry and microCT in the assessment of bone mineral density in disuse-induced bone loss.

OBJECTIVES: We recently introduced a new methodology called quantitative X-ray imaging (qXRI) to investigate bone mineral density in isolated rodent bones. The aims of the present study were to compare DXA and microCT with qXRI in a rat model of disuse osteoporosis. METHODS: Fourteen Copenhagen rats were injected with a single dose of botulinum toxin (BTX - 2 UI) in the right Mus quadriceps femoris. The left hindlimb serves as control. Areal BMD and vBMD were determined with a Hologic Discovery-W device and a Skyscan 1172 microcomputed tomograph (microCT). Absorbing material density (AMD) was determined on digitized X-ray images obtained with a Faxitron M020 device. RESULTS: All three methods highlighted significant lower values for aBMD, vBMD and AMD in trabecular and cortical bone in the BTX-injected side. In trabecular bone, aBMD, vBMD and AMD were significantly correlated with BV/TV. In cortical bone, only aBMD and vBMD were significantly correlated with cortical bone mass On the other hand, only AMD was significantly correlated with the mechanical parameters bending strength and bending modulus. CONCLUSIONS: qXRI is a rapid and cheap method to assess trabecular bone mass in isolated rodent bones and can be used as a surrogate for the densitometry of small animals.

Disuse induced by botulinum toxin affects the bone marrow expression profile of bone genes leading to a rapid bone loss.

OBJECTIVES: Molecular events occurring in the bone marrow microenvironment of an immobilized mouse limb after Botulinum toxin (BTX) injection haven't been characterized. BTX injection induces a localized disuse in which the tissue events have well been characterized. METHODS: BTX injection was performed in the right quadriceps; saline injection in the left side was used as control. Mice were sacrificed at 0, 7, 14, 21 and 28 days; tibias were used for microCT analysis; bone marrow from femurs for RT-PCR analysis. RESULTS: MicroCT revealed bone loss and microarchitectural damages on the immobilized side as from 7d; cortical area tended to be lower on the immobilized limb at 28d. Gene expression of formation factors was altered as from 7 days post-BTX: alkaline phosphatase, Tgfß1, Lrp5, Sfrp2. Only Sfrp2 and Lrp5 were maintained altered until 28d. Expression of Dkk1 increased from 21d and represented a late inhibitor of formation. Gene expression of resorption markers increased as from 7d (Rankl, Tracp, Il1α, Il1ß and Il6) and was maintained until 28d for Tracp and Il6. CONCLUSION: A localized disuse induces rapid modifications in the bone marrow gene expression leading to bone loss due to an early decrease of formation associated with an increase in resorption.

Bone status in a mouse model of genetic hemochromatosis.

UNLABELLED: Genetic hemochromatosis is a cause of osteoporosis; mechanisms leading to iron-related bone loss are not fully characterized. We assessed the bone phenotype of HFE (-/-) male mice, a mouse model of hemochromatosis. They had a phenotype of osteoporosis with low bone mass and alteration of the bone microarchitecture. INTRODUCTION: Genetic hemochromatosis is a cause of osteoporosis. However, the mechanisms leading to iron-related bone loss are not fully characterized. Recent human data have not supported the hypothesis of hypogonadism involvement. The direct role of iron on bone metabolism has been suggested. METHODS: Our aim was to assess the bone phenotype of HFE (-/-) male mice, a mouse model of human hemochromatosis, by using microcomputed tomography and histomorphometry. HFE (-/-) animals were sacrificed at 6 and 12 months and compared to controls. RESULTS: There was a significant increase in hepatic iron concentration and bone iron content in HFE (-/-) mice. No detectable Perls' staining was found in the controls' trabeculae. Trabecular bone volume (BV/TV) was significantly lower in HFE (-/-) mice at 6 and 12 months compared to the corresponding wild-type mice: 9.88 ± 0.82% vs 12.82 ± 0.61% (p = 0.009) and 7.18 ± 0.68% vs 10.4 ± 0.86% (p = 0.015), respectively. In addition, there was an impairment of the bone microarchitecture in HFE (-/-) mice. Finally, we found a significant increase in the osteoclast number in HFE (-/-) mice: 382.5 ± 36.75 vs 273.4 ± 20.95 ¢/mm(2) (p = 0.004) at 6 months and 363.6 ± 22.35 vs 230.8 ± 18.7 ¢/mm(2) (p = 0.001) at 12 months in HFE (-/-) mice vs controls. CONCLUSION: Our data show that HFE (-/-) male mice develop a phenotype of osteoporosis with low bone mass and alteration of the microarchitecture. They suggest that there is a relationship between bone iron overload and the increase of the osteoclast number in these mice. These findings are in accordance with clinical observations in humans exhibiting genetic hemochromatosis and support a role of excess iron in relation to genetic hemochromatosis in the development of osteoporosis in humans.

Strontium ranelate decreases the incidence of new caudal vertebral fractures in a growing mouse model with spontaneous fractures by improving bone microarchitecture.

UNLABELLED: Young mice over-expressing Runx2 fail to gain bone relative to wild type mice with growth and present spontaneous fractures. It allows, for the first time in rodents, direct assessment of anti-fracture efficacy of strontium ranelate which was able to decrease caudal vertebrae fracture incidence through an improvement of trabecular and cortical architecture. INTRODUCTION: The aim was to investigate whether strontium ranelate was able to decrease fracture incidence in mice over-expressing Runx2, model of severe developmental osteopenia associated with spontaneous vertebral fractures. METHODS: Transgenic mice and their wild type littermates were treated by oral route with strontium ranelate or vehicle for 9 weeks. Caudal fracture incidence was assessed by repeated X-rays, resistance to compressive loading by biochemical tests, and bone microarchitecture by histomorphometry. RESULTS: Transgenic mice receiving strontium ranelate had significantly fewer new fractures occurring during the 9 weeks of the study (-60%, p < 0.05). In lumbar vertebrae, strontium ranelate improves resistance to compressive loading (higher ultimate force to failure, +120%, p < 0.05) and trabecular microarchitecture (higher bone volume and trabecular number, lower trabecular separation, +60%, +50%, -39%, p < 0.05) as well as cortical thickness (+17%, p < 0.05). In tibiae, marrow cavity cross-section area and equivalent diameter were lower (-39%, -21%, p < 0.05). The strontium level in plasma and bone was in the same range as the values measured in treated postmenopausal women. CONCLUSIONS: This model allows, for the first time, direct assessment of anti-fracture efficacy of strontium ranelate treatment in rodents. In these transgenic mice, strontium ranelate was able to decrease caudal vertebral fracture incidence through an improvement of trabecular and cortical architecture.

Comparison of histomorphometric descriptors of bone architecture with dual-energy X-ray absorptiometry for assessing bone loss in the orchidectomized rat.

We have compared different methods for measuring bone loss in the orchidectomized (ORX) rat model of male osteoporosis: densitometry (DXA), ash weight, anatomic bone indices, histomorphometry and two-dimensional trabecular architecture analysis. Forty-eight male Wistar rats were studied at 2, 4, 8 and 16 weeks (four groups). In each group, 6 rats were ORX and 6 sham-operated were used as control. DXA was performed on the whole body, tibia and femur. Histomorphometry was performed on the secondary spongiosa of the tibia: trabecular bone volume (BV/TV) and trabecular characteristics (number, separation and thickness) were measured. Architecture analysis comprised strut identification, star volume of the marrow spaces and trabeculae, Euler-Poincaré number (E) and Kolmogorov fractal dimension (Dk). Bone mineral densities of the whole body, tibia and femur were reduced at 16 weeks in the ORX group. BV/TV was significantly decreased in the ORX group from the fourth week. Differences in the sensitivity of the architectural methods were found. There were no differences in trabecular thickness nor in trabecular star volume between ORX and controls even after 16 weeks. E became different at 8 weeks. Trabecular number, node count, star volume of the marrow spaces and trabecular separation became significantly different at 4 weeks Dk was modified after 2 weeks (p < 0.05 at 2 weeks, p < 0.001 from 4 weeks). In the ORX model, Dk appeared the most potent descriptor of trabecular bone disorganization by revealing the earliest changes at the network level.

Bone microarchitecture and bone fragility in men: DXA and histomorphometry in humans and in the orchidectomized rat model.

In men, the risk of fragility fractures increases as bone mineral declines but there is an overlap in the bone mineral density (BMD) measurements between patients with and those without fractures. Biomechanical competence of trabecular (Tb) bone depends on the amount of bone and on microarchitecture. We have developed new histomorphometric methods for evaluating microarchitecture on histological sections. These methods were used in the orchidectomized male rat (ORX--a model of hypogonadism-induced osteoporosis) and on transiliac bone biopsies performed in male osteoporotic patients. ORX rats were studied at 2, 4, 8, and 16 weeks post-ORX. Bone mineral content (BMC) was reduced at 16 weeks. Trabecular bone volume (BV/TV) was significantly decreased from the 4th week. Differences in the sensitivity of the methods were found. Fractal dimension was modified as early as 2 weeks and appeared the most potent descriptor of Tb disorganization. The architectural changes in this model mimic those observed in hypogonadic men. We examined the relationships among BMD, micro-architecture, and vertebral fracture in 108 men with lumbar osteopenia (T-score <-2.5). At least one vertebral fracture was observed in 62 patients and none in 46 patients. After adjusting for age, body mass index (BMI), and BMD, there was no significant difference between the two groups in BV/TV, Tb.Th, and Star volume. In contrast, the mean values of ICI and Tb.Sp were significantly higher whereas Tb.N and nodes were lower in patients with vertebral fracture. Logistic regression analysis showed that ICI, strut analysis, and Tb.N were significant predictors of the presence of vertebral fracture: odds ratios for an alteration of I SD ranged from 1.7 for nodes to 3.2 for ICI. These results strongly suggest that bone Tb microarchitecture is a major and independent determinant of vertebral fracture in men with osteoporosis.

Lean, fat and bone masses are influenced by orchidectomy in the rat. A densitometric X-ray absorptiometric study.

In man, hypogonadism is a risk factor for osteoporosis. Orchidectomy (ORX) in the rat leads to an imbalance between resorption and formation resulting in bone loss. We have measured whole body weight, lean and fat mass, whole bone mass (BMC) in the ORX rat model by dual X-ray densitometry (DXA). Forty-eight male Wistar rats (18-19 weeks old) were studied at 2, 4, 8 and 16 weeks. In each group, 6 rats were ORX and 6 sham-operated were used as control. DXA was performed on the whole body and isolated tibia. The whole body weight of the ORX animals became significantly decreased only at 16 weeks. Whole body BMC was reduced from 8 weeks in the ORX group. The most striking result was a net decrease in lean mass that reached -15.7% at 16 weeks. On the other hand, fat mass remained unchanged during the time series in the ORX animals.

Comparison insight dual X-ray absorptiometry (DXA), histomorphometry, ash weight, and morphometric indices for bone evaluation in an animal model (the orchidectomized rat) of male osteoporosis.

We have compared the measurements obtained by different methods: dual energy X-ray absorptiometry (DXA), histomorphometry, ash weight, and two morphometric indices (robusticity and bone weight/bone length index) in the orchidectomized (ORX) rat model of male osteoporosis. We examined 144 male wistar rats: 48 sham-operated, 48 ORX, and 48 ORX-treated with a bisphosphonate (risedronate) 2 or 10 mg/kg/day, 5 days per week. Rats were sacrificed at 2, 4, 8, or 16 weeks after the beginning of the study. DXA was performed on a Hologic QDR 2000 on the whole body, whole tibia, and tibial metaphysis. Bone volumes (C.BV/C.TV, and BV/TV) were measured by histomorphometry on the proximal tibial. A significant correlation was obtained between weight measured by DXA and scale (r = 0.993, P <0.000001). However, DXA underestimated weight by 0.3%. This discrepancy was dependent on the rat's weight. The weight bone length (WL) index was linearly correlated with BMD (r = 0.86), BMC (r = 0.96), and ash weight (r = 0.97). Correlation with robusticity was lower than with the WL index. A significant correlation was found between BMC of the metaphyseal region and the bone volumes but this explained only 27% of the variance; correlation with BMD was poorer (r = 0.40). BMC and ash weight were highly correlated (r = 0.992, P <0.000001). However, DXA overestimated BMC by 11% and the overestimation was found to be clearly dependent on the net mineral content of the bone.