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

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

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

Botulinum toxin in masticatory muscles of the adult rat induces bone loss at the condyle and alveolar regions of the mandible associated with a bone proliferation at a muscle enthesis

In man, botulinum toxin type A (BTX) is injected in masticatory muscles for several indications such as trismus, bruxism, or masseter hypertrophy. Bone changes in the mandible following BTX injections in adult animal have therefore became a subject of interest. The aim of this study was to analyze condylar and alveolar bone changes following BTX unilateral injections in masseter and temporal muscles in adult rats. Mature male rats (n=15) were randomized into 2 groups: control (CTRL; n=6) and BTX group (n=9). Rats of the BTX group received a single injection of BTX into right masseter and temporal muscles. Rats of the CTRL group were similarly injected with saline solution. Rats were sacrificed 4weeks after injections. Masticatory muscles examination and microcomputed tomography (microCT) were performed. A significant difference of weight was found between the 2 groups at weeks 2, 3 and 4 (p<0.05). Atrophy of the right masseter and temporal muscles was observed in all BTX rats. MicroCT analysis showed significant bone loss in the right alveolar and condylar areas in BTX rats. Decrease in bone volume reached -20% for right alveolar bone and -35% for right condylar bone. A hypertrophic bone metaplasia at the digastric muscle enthesis was found on every right hemimandible in the BTX group and none in the CTRL group. BTX injection in masticatory muscles leads to a significant and major mandible bone loss. These alterations can represent a risk factor for fractures in human. The occurrence of a hypertrophic bone metaplasia at the Mus Digastricus enthesis may constitute an etiological factor for tori

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

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

Maxillary Sinus Lift with Beta-Tricalcium Phosphate (β-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 (β-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 β-TCP were osseointegrated and covered with mineralized bone; osteoid tissue was also filling the inner porosity. Macrophages having engulfed numerous β-TCP grains were observed in marrow spaces. β-TCP was identified by nanoCT as osseointegrated particles and as granules in the cytoplasm of macrophages. Raman microspectroscopy permitted to compare the spectra of β-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 β-TCP were found in osseointegrated β-TCP granules and in the grains phagocytized by the macrophages. The hydration degree was maximal for β-TCP phagocytized by macrophages. Raman microspectroscopy associated with nanoCT is a powerful tool in the analysis of the biomaterial degradation and osseointegration

Thiazolidinediones cause compaction of nuclear heterochromatin in the pluripotent mesenchymal cell line C3H10T1/2 when inducing an adipogenic phenotype.

OBJECTIVE: To characterize the nuclear changes induced in vitro by thiazolidinediones (TZDs) in a murine pluripotent mesenchymal cell line. STUDY DESIGN: The C3H10T1/2 cell line, which can differentiate either in osteoblast or in adipocyte, was cultured in the presence of pioglitazone (5 microM) or rosiglitazone (0.5 microM) for 6, 8 and 9 days (D). Quantitative real-time polymerase chain reaction analysis evaluated the expression of key genes of the adipocytic or osteoblastic differentiation (PPARgamma[peroxisome proliferatoractivated receptor gamma], Runx2 [runt-related transcription factor 2] and alkaline phosphatase). Cells were stained with Oil Red O for lipids, and chromatin was counter-stained with hematoxylin. Cells were photographed at x 1,000 magnification and analyzed with texture analysis software. Nuclear area, mean gray level and run-length parameters were calculated. RESULTS: PPARgamma was significantly expressed from D6 (normalized ratio > 7) in TZD groups (ratio >27 at D9). No significant differences were found for either Runx2 or alkaline phosphatase expression versus control at D6 or D9. Cells cultured with TZDs began to differentiate into adipocytes with numerous lipid droplets which appeared at D6. Nuclear area decreased suddenly at D6 for both TZDs, and the mean gray level increased. Run-length parameters changed significantly due to chromatin compaction. CONCLUSION: TZDs provoked differentiation of C3H10T1/2 into adipocytes, leading to inactivation of genes that were highly compacted into heterochromatin

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. β-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 (β-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