The use of animal models in multiple myeloma

Among biomaterials used for filling bone defects, beta-tricalcium phosphate (β-TCP) is suitable in non-bearing bones, particularly in dental implantology, oral and maxillofacial surgery. When β-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 β-TCP granules. Granules were prepared with different concentrations of β-TCP powder in slurry (10, 11, 15, 18, 21, and 25 g of β-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-β-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 β-TCP granules; the difference between hydroxyapatite and β-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

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

Beta-tricalcium phosphate (β-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 β-TCP were found significantly increased after 7 and 14d. MC3T3-E1 cells were anchored and stretched onto the β-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

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\u27t 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, Tgfβ1, 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, Il1β 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

The effects of Mad2 over-expression on murine lung tumorigenesis

Sixty-eight percent of human solid tumors are aneuploid, which is classically associated with poor patient prognosis. Several mouse models disturbing the spindle assembly checkpoint (SAC) have been developed to study the consequences of chromosome instability (CIN) and aneuploidy in vivo. Current knowledge suggests that aneuploidy can promote tumorigenesis or act as a tumor suppressor. Mad2 is found over-expressed in human tumors, and Mad2 over-expression in mice induces the development of aneuploid tumors and facilitates KrasG12D lung tumor relapse. A proposed mechanism of tumor acceleration by CIN is the facilitation of tumor suppressor loss of heterozygosity. In how far Mad2 over-expression influences Kras driven lung tumorigenesis in a p53 heterozygous background, remains hitherto unclear. In this thesis, I show that Mad2 over-expression increases p53(+/–);KrasG12D mice survival by delaying tumor initiation and progression. Different tumor populations (expressing low, intermediate and high levels of Mad2) have co-evolved from an original population of Mad2-expressing type 2 pneumocytes. My data suggest that high Mad2-expressing lung nodules are selected against during early tumorigenesis and are mainly composed of instable aneuploid cells. Using time-lapse microscopy on mouse embryonic fibroblasts, I analyzed the effect of Mad2 over-expression in the context of p53 heterozygosity. Upon Mad2 over- expression, the inactivation of one copy of p53 rescued mitotic cell death by inducing mitotic slippage and polyploid cells. In vivo, high Mad2 levels impaired S phase entry in tumor cells. Moreover, p53(+/– )KM high nodules strongly induced p21 in a p53-dependent manner. This data suggests that one copy of p53 can induce G1 cell cycle arrest in tumors. Although Mad2 over- expression generates aneuploidy, it does not accelerate p53 loss of heterozygosity (LOH), since Mad2 down-regulation occurs prior to LOH. Importantly, Mad2 over- expression together with p53 heterozygosity also delayed EFGRL858R-induced lung cancer

Human macrophages and osteoclasts resorb β-tricalcium phosphate in vitro but not mouse macrophages

β-TCP is a resorbable bony biomaterial but its biodegradation mechanisms in vivo remains unclear. Osteoclast can resorb β-TCP but a role for macrophages has also been suggested by in vivo studies. However no in vitro study has clearly evidenced the action of macrophages in the resorption process. We prepared flat β-TCP tablets with a smooth surface to investigate the in vitro capability of murine (RAW 264.7) and human macrophage cells (PBMCs) to resorb the biomaterial. In parallel, these cells were differentiated into multinucleated osteoclasts with M-CSF and RANK-L. The action of these cells was evaluated by scanning electron microscopy and Raman microspectroscopy after a 21 day culture on the tablets. Human macrophages and osteoclasts derived from PBMCs appeared able to resorb β-TCP by forming resorption pits at the surface of the flat tablets. RAW macrophages were unable to resorb β-TCP but they exhibited this possibility when they have been differentiated into osteoclasts. These cells can engulf β-TCP grains in their cytoplasm as evidenced by light and TEM microscopy with production of carbonic anhydrase (revealed by the immunogold technique in TEM). The resorbed areas were characterized by severe degradation of the grains showing speckled and stick-like aspects indicating a chemical corrosion. The effect was maximal at the grain boundaries which have a slightly different chemical composition. Changes in the Raman spectrum were observed between the resorbed and un-resorbed β-TCP suggesting crystal modifications. In contrast, un-differentiated murine macrophages were not able to chemically attack β-TCP and no resorption pit was observed. RAW cell is not a representative model of the macrophage-biomaterial interactions that occur in human. This in vitro study evidences that both human osteoclasts and macrophages represent active cell populations capable to resorb β-TCP

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

Vector analysis of porosity evidences bone loss at the epiphysis in the BTX rat model of disuse osteoporosis

Introduction Botulinum toxin (BTX) injected in a muscle causes paralysis with a subsequent bone loss. It represents a model of disuse osteoporosis. Although bone loss has been regularly evaluated at the metaphysis of long bones, little is known concerning the bone changes occurring in the epiphysis. Animals and methods Ten Copenhagen male rats received a single BTX injection in the Mus quadriceps femoris on the right side and unilateral paralysis developed in the following days. Animals were euthanized after 28 days; femur and tibia were harvested and analyzed by microCT. Vector analysis of porosity was applied to the 2D sections and produced a frontal image with mapping in pseudo-colors. This allows quantitative analysis at the epiphysis and metaphysis. “Hot spot” were evidenced and indicated bone loss. Quantitative analysis of these images was done by decomposition of the R, G and B planes and deriving the ratio of R + G pixels on the whole pixel number. Results At the metaphysis, this ratio was correlated with measurement of the bone volume obtained by microCT. At the epiphysis, which has a complex shape in 3D, the method easily identified the bone loss. Discussion Paralysis of a unilateral quadriceps induces bone loss at the metaphysis of the long bones. However, the epiphysis, having a reduced bone remodeling is also concerned by disuse. MicroCT analysis of this part of the bones is difficult due to its complex shape in 3D. Vector analysis is a new and robust method to quantify bone loss in such complex areas

Altered bone microarchitecture and gene expression profile due to calcium deficiency in a mouse model of myeloma

It is not clear why patients with an indolent form of multiple myeloma (MM) develop into an aggressive form with poor prognostic. We investigated the effect of a dietary calcium deficiency on tumor growth, osteolysis and gene expression in the 5T2MM murine model. Two groups of C57BL/KaLwRij mice received 5T2MM cells and started a diet with normal (0.8%; "normal-Ca-MM") or low calcium content (0.05%; "low-Ca-MM"). Two control groups (without 5T2MM cells) received either a normal or low calcium diet (normal-Ca and low-Ca groups). Tumor growth, osteolysis and marrow gene expression of the Wnt pathway, RANKL and MIP-1α were monitored at 6, 8 and 10 weeks (w) after cell injection. In low-Ca mice, serum level of PTH was higher after 10w; microCT showed trabecular bone loss and decrease of cortical thickness at the tibia. A higher M-protein level was evidenced at 10w and 4 mice developed paraplegia at 8/9w in low-Ca-MM group only. Numerous cortical perforations of the tibia were observed in MM groups with a marked decrease in cortical thickness in low-Ca-MM. At 6w, osteoclast number from the endosteum was significantly higher in low-Ca-MM compared to normal-Ca MM. This observation was not found at 8 and 10w. MicroCT of the lumbar vertebrae showed dramatic bone destruction in the low-Ca-MM group. qPCR revealed no difference in RANKL expression whereas differences were obtained in the expression of Lrp5/Lrp6 and MIP-1α from 6w. A low calcium diet induced higher bone destruction in the tibia and vertebra associated with an earlier decrease in bone formation level and a higher increase in bone resorption level at early time in the MM development

The cathepsin K inhibitor AAE581 induces morphological changes in osteoclasts of treated patients

Inhibitors of Cathepsin K (Cat-K) are recognized as an interesting way to inhibit osteoclast (OC) activity. OCs from patients treated with the anticathepsin-K inhibitor AAE581 (balicatib) were found enlarged. They contained numerous vacuoles filled with tartrate resistant acid phosphatase (TRAcP), an intracellular enzyme that terminates the degradation of collagen internalized in OC transcytotic vesicles. In a phase 2 clinical study, 675 patients with postmenopausal osteoporosis received the Cat-K inhibitor AAE581 at 0, 5, 10, 25, or 50 mg/D during 1 year. Eleven patients had a transiliac bone biopsy, studied undecalcified. Histoenzymatic detection of TRAcP was used to identify and count OC number. The histomorphometrist was not aware of the randomization of patients at the time of analysis. OC were unstained in one patient because of a failure in the fixation protocol, but easily observable in the 10 remaining patients. Whatever the received dose, treated patients exhibited a characteristic aspect of the OC cytoplasm which appeared filled of deeply-stained brown vacuoles, making cells looking like bunches of grape. These round vacuoles, evidenced on TRAcP-stained sections, were due to the accumulation of intracytoplasmic TRAcP. This led to a moderate enlargement of the OC size when compared to a series of control osteoporotic patients. AAE581 did not induce OC apoptosis at any dosage but it modified OC morphology. Cat-K inhibition (inhibiting the extracellular collagen breakdown) is associated with a compensatory accumulation of intracellular TRAcP that could not be used to complete protein degradation. TRAcP is also known to be degraded by Cat-K. Microsc. Res. Tech., 2010

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