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.

Effect of alpha tocopherol acetate in Walker 256/B cells-induced oxidative damage in a rat model of breast cancer skeletal metastases.

The pathophysiological changes and the oxidative-antioxidative status were evaluated in the bone microenvironment of rat inoculated with Walker 256/B mammary gland carcinoma cells, and used alpha-tocopherol acetate (ATA) as a countermeasure. Walker 256/B cells were injected into the right femora of aged male rats. Animals were randomized into three groups: 12 rats were injected with saline (control group); 14 rats were injected with Walker 256/B cells (5x10(4)) in the medullar cavity (W256 group); 14 rats were inoculated with Walker 256/B cells and treated with ATA (45mg/kg BW) (W256+ATA group). After 20 days, rats were euthanized and the femurs were radiographed. Micro architectural parameters were measured by microcomputed tomography and histology. Serum, bone and bone marrow were evaluated for oxidative damage. In parallel, cell cultures were done in the presence of ATA and ROS were measured by fluorescence; apoptotic cells were determined in parallel. W256 groups had osteolytic damages with marked resorption of cortical and trabecular bone. W256+ATA animals presented marked osteosclerotic areas associated with tumor necrosis areas inside the bone cavity. Levels of lipid peroxidation and protein oxidation were found to increase in W256 rats; a significant reduction in SOD and GSH-p activities was also observed. W256+ATA group had significantly reduced oxidative damage, but not reversed back to the control levels. The present study shows that Walker 256/B cells induce skeletal metastases associated with oxidative damage in the bone microenvironment. ATA reduced the oxidative stress damage, enhanced osteosclerosis and tumor cell apoptosis both in vitro and in vivo.

In vitro kinetic study of growth and mineralization of osteoblast-like cells (Saos-2) on titanium surface coated with a RGD functionalized bisphosphonate.

Osteoconduction and osseointegration are the critical stages for implantation success. Peptides containing RGD (Arg-Gly-Asp) adhesive sequence are known to promote cell adhesion and consequently to favor osseointegration of medical devices. In this study, RGD peptides were coupled to a bisphosphonate used as an anchor system and chemically adsorbed on polished titanium discs. Two different concentrations, 10(-10) mol/L (RGD 10(-10)) and 10(-4) mol/L (RGD 10(-4)) were compared to non coated discs (RGD 0). Adhesion, spreading, and mineralization of osteoblast-like cells (Saos-2) were assessed. Mineralization kinetic was done at 3, 6, 10, 14, and 18 days of culture; the extent of mineral deposits was quantified by image analysis. Histogram repartitions of nuclear area, characterizing cell spreading, showed a shift to higher values in cells cultured on RGD coated titanium disks. Mineralization started at day 3 in the three groups, but had a faster development in the RGD 10(-10) group from day 6 to day 18 compared to RGD 0 and RGD 10(-4). At day 18, the percentage of mineralized area was significantly higher for RGD 10(-10) compared to RGD 0 (p < 0.05). In the present study, this new method was found suitable to anchor RGD containing species on titanium: this favored adhesion and spreading of osteoblast-like cells and mineralization compared to noncoated titanium.

Bone grafts cultured with bone marrow stromal cells for the repair of critical bone defects: an experimental study in mice.

Tissue engineering of autologous bone combined with osteoprogenitor cells is a suitable strategy for filling large bone defects. The aim of this study was to evaluate the osteogenicity of a xenogenic bone graft cultured with allogenic bone marrow stromal cells (BMSC) in a mouse critical size craniotomy. Bovine trabecular bone grafts were made free of bone marrow cells or debris and were delipidated. BMSC were harvested from C57BL/6-Tg(ACTbEGFP)1Osb/J mice (GFP+ cells) and were cultured 14 days on bone grafts in control or osteogenic medium. Engineered grafts were implanted in calvarial defect in C57BL/6 mice. Four groups were studied: graft with BMSC differentiated in osteoblasts (G-Ob), graft with BMSC (G-BMSC), graft without cells (G) and no graft. Calvariae were studied 2 and 8 weeks after implantation by radiographic and histomorphometric analyses. G group: the bone ingrowth was limited to the edges of the defect. The center of the graft was filled by a fibrovascular connective tissue. G-BMSC or G-Ob groups: bone formation occurred early in the center of the defect and did not increase between 2 and 8 weeks; the newly formed woven bone was partially replaced by lamellar bone. The preoperative osteoblastic differentiation of BMSC did not allow faster and better bone regeneration. After 2 weeks, GFP+ cells were observed around the grafted bone but no GFP+ osteocyte was present in the newly formed bone. No GFP+ cell was noted after 8 weeks. However, pre-implantation culture of the biomaterial with allogenic BMSC greatly enhanced the bone regeneration.

Chemical structure of methylmethacrylate-2-[2',3',5'-triiodobenzoyl]oxoethyl methacrylate copolymer, radio-opacity, in vitro and in vivo biocompatibility.

The properties of copolymers (physical, chemical, biocompatibility, etc.) depend on their chemical structure and microstructural characteristics. We have prepared radio-opaque polymers based on the copolymers of methyl methacrylate (MMA) and 2-[2',3',5'-triiodobenzoyl]oxoethyl methacrylate (TIBOM). The copolymerization reaction between TIBOM and MMA showed that the reactivity ratios were r(1)=0.00029 and r(2)=1.2146. The composition diagram is typical for a practically non-homopolymerizable monomer (TIBOM) and a very reactive monomer (MMA). The copolymers were analyzed on an X-ray microcomputed tomograph and they proved to be radio-opaque even at low concentrations of TIBOM. The biocompatibility was tested both in vitro (with J774.2 macrophage and SaOS-2 osteoblast like cells) and in vivo in the rat. These materials were found to be non-toxic and were well tolerated by the organism. These combined results led to the suggestion that this type of polymer could be used as dental or bone cements in place of barium or zirconium particles, which are usually added to provide X-ray opacity.

Orchidectomy models of osteoporosis.

Long considered a disease of post-menopausal women, osteoporosis is increasingly being recognized among the growing population of elderly men. Androgen deficiency may be associated with an increase of bone resorption in elderly men and so, with remodeling imbalance and fracture risk. It is firmly established that androgen withdrawal induced by orchidectomy (ORX) results in decreased bone mass in animal models especially in rodents. The mature rat is the model of choice. Skeletal effects of ORX in rats have been studied at the tissular and cellular level. It induces a decrease of BMD and BV/TV with microarchitecture alterations due to an increased bone remodeling. The present chapter focuses on the ORX surgery in rats and mice.

Effects of risedronate in a rat model of osteopenia due to orchidectomy and disuse: densitometric, histomorphometric and microtomographic studies.

Dual energy X-ray absorptiometry (DXA), histomorphometry and X-ray microtomography (microCT) were used to assess effects of risedronate and testosterone in a combined rat model of orchidectomy (ORX) and local paralysis induced by botulinum neurotoxin (BTX). Four groups of mature rats were studied for 1 month: SHAM operated; ORX and right hindlimb immobilization (BTX); ORX+BTX+risedronate or testosterone. Changes in bone and body composition were measured by DXA (BMC, lean and fat mass), histomorphometry (BV/TV2D, Tb.Th and microarchitectural parameters) and microCT (BV/TV3D, SMI and cortical parameters). ORX and BTX had additive effects on bone loss since differences were maximized on the immobilized bone. The decrease in BMC on the tibial metaphysis reached -33.6% vs. -11.3% in the non-immobilized limb. BV/TV and Tb.N decreased and Tb.Sp increased in both hindlimbs whereas Tb.Th was significantly lower only in the immobilized limb. Decrease of tibial cortical area and thickness was greater in the immobilized limb. Risedronate prevented BMC, BV/TV and architecture loss but not reduction in Tb.Th. Cortical bone was preserved only in the non-immobilized limb. Testosterone was unable to prevent trabecular and cortical bone loss, but it prevents loss of whole body lean mass. In conclusion, ORX and BTX resulted in additive effects on bone loss. Risedronate had protective effects on trabecular bone loss but was less effective on cortical bone.

Absence of renal lesions in C57BL/KaLwRij mice with advanced myeloma due to 5T2MM cells.

Renal failure is one of the main complications in multiple myeloma (MM) and histopathological lesions are due to light chains accumulation in the kidney. The 5T2MM mouse model closely mimics osteolytic lesions observed in clinics. We studied the occurrence of pathological changes in the kidney of mice inoculated with 5T2MM myeloma cells. No renal lesions due to light chain deposition were observed after histological, immunological staining and dosage of creatinine in serum and urine. PTH levels decreased in 5T2MM mice, confirming the absence of secondary hyperparathyroidism. Osteolytic lesions appear to be the unique consequence of 5T2MM cells inoculation.

Mandibular bone loss in an animal model of male osteoporosis (orchidectomized rat): a radiographic and densitometric study.

In humans, hypogonadism is associated with osteoporosis and can be studied by densitometry (DXA) on the vertebrae or long bones. There is some controversy about the relationships between bone loss in these sites and in the mandible. Osteoporosis has been suggested as a risk factor for dental problems. In the rat, orchidectomy (ORX) is associated with an increased bone resorption resulting in bone loss. We have studied the time effects of ORX on the alveolar bone in the rat. Forty-eight male Wistar rats were divided into four groups and studied over 2, 4, 8 and 16 weeks. In each group, six rats were ORX and six sham-operated (SHAM) animals were used as control. The mandible of each rat was dissected. Numeric radiographs, centered on the molar region, were obtained. Bone loss was observed qualitatively at 16 weeks in ORX animals. Quantitative modifications were confirmed by texture analysis of numeric radiographs using the run-length technique. The bone mineral content (BMC) and density (BMD) of the hemimandible and in a region centered on the molars were measured by DXA. The coefficient of variation (CV) for BMC was poor on the whole bone and no differences could be observed even at 16 weeks. For BMC of the molar region, the CV was improved and significant bone loss occurred in the ORX group at 16 weeks ( P<0.016). This study confirms that in the male rat, the reduction of sex hormones induced by ORX is associated with a decrease in bone mass in the mandible.

Selection of a highly aggressive myeloma cell line by an altered bone microenvironment in the C57BL/KaLwRij mouse.

In multiple myeloma (MM), bone marrow microenvironment has an important role for the survival and growth of plasma cells. We previously showed that a high bone turnover, induced by ovariectomy, increased MM cells growth in the 5T2MM model. The present study characterized a new plasma cell line (5THL) which was isolated from 5T2MM mice previously ovariectomized. Cells were propagated unchanged in normal C57BL/KaLwRij mice during six generations. 5THL was compared to the original 5T2MM phenotype. Paraproteinemia was detected 6 weeks post injection in 5THL mice and after 8 weeks in 5T2MM mice. All 5THL mice developed a hind-limb paralysis after 10 weeks. 5T2MM mice were euthanized at 16 weeks, due to a more progressive development of the disease. In 5THL mice, osteolytic lesions were observed after 8 weeks and severe bone destruction was evidenced at 10 weeks. In 5T2MM mice, minimal lesions were observed only after 10 weeks. Like in 5T2MM mice, no extra osseous lesions were observed in 5THL mice. The 5THL MM model closely mimics human myeloma with higher and faster bone aggressiveness. This new aggressive cell line, with a preserved phenotype, was selected by an altered microenvironment due to an increased bone turnover.

A tumour necrosis factor alpha autocrine loop contributes to proliferation and nuclear factor-kappaB activation of Theileria parva-transformed B cells.

Theileria infection of bovine leucocytes induces uncontrolled proliferation and a transformed phenotype comparable to tumour cells. Infected cells have many characteristics of activated leucocytes and use autocrine loops to augment proliferation. We have shown previously that, in infected B cells, PI3-K controls a granulocyte-macrophage colony-stimulating factor (GM-CSF) autocrine loop to increase both proliferation and activation of the activator protein 1 (AP-1) transcription factor. We show here that the same infected B cells also use a tumour necrosis factor (TNF) alpha autocrine loop that again contributes to proliferation and augments nuclear factor (NF)-kappaB activation. Interestingly, both pharmacological inhibition of TNF synthesis and neutralizing anti-TNF antibodies lead to a reduction in proliferation and a 50% drop in NF-kappaB activation, without inducing apoptosis.

Increased bone remodeling due to ovariectomy dramatically increases tumoral growth in the 5T2 multiple myeloma mouse model.

Bone destruction is the main clinical consequence of multiple myeloma (MM). It is known that a "vicious circle" exists between bone and myeloma cells with plasma cells stimulating bone cells, which in return stimulate the neoplastic growth. We hypothesized that an accelerated bone remodeling induced by ovariectomy (OVX) could provide a more fertile environment for the myeloma cell growth. Ovariectomy performed on C57BL/KaLwRij mice resulted in reduced trabecular bone volume and increased osteoclast number. To test our hypothesis, we ovariectomized C57BL/KaLwRij mice one week before injection of 5T2MM myeloma cells. A non-OVX 5T2MM group was used as control. Ovariectomy increased the tumor growth and induced an earlier development of osteolytic lesions. OVX 5T2MM mice were euthanized at 10 weeks, instead of 16 weeks, for the non-OVX 5T2MM, mice because of the accelerated development of disease in OVX animals. Treatment of OVX 5T2MM mice with pamidronate reduced osteolysis but had little effect on the time development of the tumor. This combined model (ovariectomy + 5T2MM) confirmed the interdependence of bone and myeloma cells and could explain the some sudden burden of indolent MM into aggressive MM in humans.