Bone mass and microarchitecture of irradiated and bone marrow-transplanted mice: influences of the donor strain.

UNLABELLED: Total body irradiation and bone marrow transplantation induced dramatic trabecular bone loss and cortical thickening in mice. Transplanted cells were engrafted in bone marrow, along trabeculae, and in periosteal and endosteal envelopes. None of the osteocytes were of donor origin. Bone microarchitecture of transplanted mice changed to tend toward the donor phenotype. INTRODUCTION: Osteopenia and osteoporosis are complications of bone marrow transplants (BMT) attributed to related chemotherapy. However, the specific influence of total body irradiation (TBI) is unknown. METHODS: We investigated the effects of TBI and BMT on bone mass and microarchitecture by micro-CT. Eighteen C57Bl/6 (B6) mice receiving lethal TBI had a BMT with marrow cells from green fluorescent protein--transgenic-C57Bl/6 (GFP) mice. Transplanted (T(GFP)B6), B6, and GFP mice were euthanized 1, 3, and 6 months after BMT or at a related age. RESULTS: T(GFP)B6 presented a dramatic bone loss compared with B6 and did not restore their trabecular bone mass over time, despite a cortical thickening 6 months after BMT. Serum testosterone levels were not significantly reduced after BMT. During aging, GFP mice have less trabeculae, thicker cortices, but a narrower femoral shaft than B6 mice. From 3 months after BMT, cortical characteristics of T(GFP)B6 mice differed statistically from B6 mice and were identical to those of GFP mice. GFP(+) cells were located along trabecular surfaces and in periosteal and endosteal envelopes, but none of the osteocytes expressed GFP. CONCLUSION: Our findings suggest that engrafted cells did not restore the irradiation-induced trabecular bone loss, but reconstituted a marrow microenvironment and bone remodeling similar to those of the donor. The effects of irradiation and graft on bone remodeling differed between cortical and trabecular bone.

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells.

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b(-) cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b(-) cells increased the number of AlkP(+) cells from the plastic adherent fraction from 6.3% +/- 0.8 to 56% +/- 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b(-) fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.

Disuse and orchidectomy have additional effects on bone loss in the aged male rat.

INTRODUCTION: A severely osteopenic rat model was obtained by combining orchidectomy (ORX) and disuse (due to local paralysis induced by botulinum toxin [BTX] in the quadriceps muscle). METHODS: Forty-two aged male rats (5-6 months old) were randomized into three groups: 18 were SHAM operated; 6 were ORX; and 18 were ORX and BTX injected in the right hindlimb. One, two, and three months after surgery, bone mass (BV/TV) and microarchitectural parameters (Tb.Th, Tb.N, Tb.Sp, Tb.Pf, and structure model index [SMI]) were measured by microcomputed tomography (microCT) on the primary and secondary spongiosa of the femur. Osteoid parameters (OS/BS, O.Th), the number of osteoclasts (Nb.Oc), and the mineral apposition rate (Ct.MAR, Cn.MAR) were measured by histology. The serum tartrate-resistant acid phosphatase (TRAcP) 5b activity was measured by immunoassay. RESULTS: ORX induced a decrease of BV/TV, Tb.N and an increase of Tb.Sp, Tb.Pf, and SMI on both primary and secondary spongiosa. ORX and BTX had cumulative effects on bone loss, since differences were maximized on the right femur. The decrease in BV/TV reached -65%. Osteoid parameters and mineral apposition rate increased during the time course of the study. A peak of serum TRAcP was found at 7 days post-ORX. TRAcP levels reached the highest values in the ORX-BTX groups and the effect lasted longer than in the group with ORX alone. The association of ORX-BTX induced a greater bone resorption, due to the removal of complete trabeculae, compared to ORX alone. CONCLUSION: This model induced a severe and rapid bone loss and can be used to explore pharmacological- and biomaterial-based countermeasures.

Comparative effects of five bisphosphonates on apoptosis of macrophage cells in vitro.

Bisphosphonates (BPs) inhibits bone resorption by reducing osteoclastic activity; they induce osteoclast apoptosis. Pathophysiology of prostheses loosening is complex and implies an inflammatory reaction secondary to the phagocytosis of wear debris by macrophages with a secondary increased bone resorption by osteoclasts. BPs inhibit proliferation and cause cell death in macrophages by induction of apoptosis. We have used mouse macrophage-like J774.1 cells to evaluate the effects of five BPs. J774A.1 cells were cultured in a standard culture medium for 2-days. BPs (alendronate, pamidronate, etidronate, risedronate, zoledronic acid) were added in the medium at concentration of 10(-6) to 10(-4)M during 3 days. Cells were studied by fluorescence microscopy after staining with the fluorescent dye Hoescht H33342 and the percentage of apoptotic cells was determined on 300 nuclei. Cells were analyzed by flow cytofluorometry after staining with annexin V-FITC (for counting apoptotic cells) and propidium iodide (for necrotic/late-apoptotic cells) on 2000 cells. Etidronate did not cause significant apoptosis or necrosis, at any concentration. Alendronate and pamidronate caused apoptosis and death only at very high concentration [10(-4)M]. On the contrary, apoptotic and necrotic cells were evidenced with risedronate or zoledronic acid at lower concentrations. These effects were dose-dependant and occurred when concentration reached [10(-5)M]. The number of apoptotic cells was higher with zoledronic acid and then with risedronate. Cytofluorometry appeared superior to cytologic analysis in the investigation of macrophage apoptosis, since necrotic cells loose contact with the glass slides and are not identifiable in cytological counts. Some amino-BPs appear to induce apoptosis in macrophages.

Relations between radiograph texture analysis and microcomputed tomography in two rat models of bone metastases.

AIMS: Osteolytic (Walker 256, W256) and osteoblastic (MatLyLu, MLL) metastases were induced to investigate their effect on bone architecture by microcomputed tomography (microCT) and texture analysis of radiographs. METHODS: Fischer and Copenhagen rats received an intracardiac injection with W256/MLL cells, respectively. Femur and tibia radiographs were analyzed by texture analysis with run lengths and fractal algorithms. Microarchitecture was analyzed on primary and secondary spongiosa by microCT. RESULTS: W256 and MLL induced a decrease of trabecular bone mass, a disconnection of trabeculae and an increased conversion of plates into pillars. On radiographs and 3-dimensional models of W256 rats, a disappearance of the primary spongiosa was observed. On radiographs and 3-dimensional models of MLL rats, osteolytic lesions were observed as disseminated dark areas. Run length and fractal analyses were altered in both metastases. CONCLUSION: W256 and MLL cells induced two different patterns of osteolysis. Texture analysis of radiographs is a useful technique to explore trabecular bone changes.

Evaluation of an injectable bone substitute (betaTCP/hydroxyapatite/hydroxy-propyl-methyl-cellulose) in severely osteopenic and aged rats.

The use of injectable biomaterials is of interest in osteoporotic patients to locally restore bone mass in sites at risk of fracture. An injectable bone substitute (IBS1 made of betaTCP/hydroxyapatite as a calcium phosphate substitute and hydroxy-propyl-methyl-cellulose as a polymer carrier) was used in a severely osteopenic rat model obtained by combining orchidectomy (ORX) and disuse (paralysis induced by botulinum toxin - BTX). Fifty-six aged male rats were randomized into three groups: 18 were SHAM operated; 38 were ORX and BTX injected in the right hindlimb; they constituted the OP (osteoporotic) group. One month after ORX-BTX surgery, 20 of these OP rats received a IBS1 injection in the right femur (OP-IBS1 rats). Animals were studied at the time of IBS1 injection 1 month post ORX-BTX (M1), 1 month (M2) and 2 months (M3) after IBS1 injection. Bone mass (BV/TV) and microarchitectural parameters were measured by microCT. BV/TV was decreased after ORX-BTX; ORX and BTX had cumulative effects on bone loss (differences maximized on the right femur). BV/TV (combining the volume of both bone and material in OP-IBS1 rats) was elevated at M1 but decreased at M2. Marked bone formation was found onto the biomaterial granules but bone had a woven texture. A marked increase in the number of nonosteoclastic TRAcP+ cells was found in the implanted area. IBS1 induced new bone formation shortly after implantation but both IBS1 and woven bone were resorbed without inducing lamellar bone. Biomaterial trials must be conducted with long-term implantation periods, in aged osteoporotic animals.

Notch is constitutively active in Theileria-transformed B cells and can be further stimulated by the filarial nematode-secreted product, ES-62.

Theileria parva-infected B cells express Jagged-1 and activate Notch signalling in a parasite-dependent manner. ES-62, a filarial nematode-secreted phosphorylcholine-containing glycoprotein, is able to further stimulate Notch-mediated signalling in parasitized cells. Notch is also activated to a similar extent by addition of exogenous IL-10, and this occurs prior to any increase in proliferation in T. parva-infected B cells.

Biodegradability of poly (2-hydroxyethyl methacrylate) in the presence of the J774.2 macrophage cell line.

The degradation of cross-linked and linear poly(2-hydroxyethyl methacrylate) (pHEMA), was examined in vitro with J774.2 cells. pHEMA microbeads were prepared with both types of polymers. Only cells in contact with the microbeads increased their production of lysosomal enzymes (TRAcP and ANAE) and released large amounts of reactive oxygen species with both types of pHEMA microbeads. Electron microscopy showed that macrophages were able to erode the surface of linear pHEMA but unable to erode the surface of the cross-linked polymer. Cells appeared wrapped by the linear pHEMA surface, but those cultured on the cross-linked polymer were only laying at the surface. After cell culture, the surface roughness of pHEMA slices was observed by atomic force microscopy (AFM). There was a significant increase in roughness (R(a)) of the surface of linear pHEMA slices cultured with J774.2 cells whereas no difference in R(a) between the surface of cross-linked pHEMA slices could be measured. AFM image of the hydrated materials were done: the surface of linear pHEMA swelled considerably in saline whereas the hydrated cross-linked polymer did not differ from the air-dried appearance. In conclusion, linear pHEMA swells in biological fluids, activates macrophages in close contact with the polymer and can be progressively eroded.

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.

Melanin affinity of N-(2-diethylaminoethyl)-4-iodobenzamide, an effective melanoma imaging agent.

The cellular uptake and incorporation in macromolecules of iodine-125 labelled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), a melanoma imaging agent, was studied using human melanoma cells M3Dau (amelanotic) and M4Beu (melanotic). The interaction between [125I]BZA and synthetic melanin was examined in various conditions of incubation. The results showed that uptake was high only for M4Beu, whereas the incorporation in trichloroacetic acid-precipitable proteins was very low for both model cell lines, with no correlation with melanin content. Experiments with synthetic melanin showed that BZA binding to melanin was saturable and reversible, and involved several types of interaction. The influence of the ionic environment indicated that electrostatic forces play a role in the affinity, and the decrease in binding produced by the presence of an alcohol in the medium suggested that hydrophobic interactions may be involved in the binding mechanism. This was supported by the Scatchard analysis, which revealed two classes of binding sites, and the determination of two association constants (K1 = 3.9 +/- 1.9 x 106/M and K2 = 2.9 +/- 0.9 x 104/M). The affinity of BZA for melanin might explain the good results obtained in a phase II clinical trial for the diagnosis of malignant melanoma metastases, in which the specificity was 100%.

Synthesis, characterization and comparative biodistribution study of a new series of p-iodine-125 benzamides as potential melanoma imaging agents.

Iodobenzamides are reported to possess some affinity for melanoma. In order to identify the compound having the most appropriate pharmacokinetic properties as a potential melanoma imaging agent, thirteen new [125I]radioiodobenzamides with a butylene amide-amine spacer and various substituents on the terminal amino group were investigated. Their synthesis, radioiodination and biodistribution in B16 melanoma bearing C57BL6 mice are described and compared to [125I] labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), our reference compound. Changes in the terminal amino constituents induced modifications of lipophilicity, tumor uptake and organ distribution. The dimethylaminobutyl iodobenzamide appeared to be the most promising radiopharmaceutical imaging agent for the detection of melanoma and its metastases.

Distribution of I-BZA (N-2-diethylaminoethyl-4-iodobenzamide) in grafted melanoma and normal skin: a study by secondary ion mass spectroscopy.

Iodobenzamides labelled with radioactive iodine are undergoing clinical evaluation as imaging and potential therapeutic agents in malignant melanomas. However, the uptake mechanism in melanic tissues remains controversial. Using secondary ion mass spectrometry (SIMS), we studied the microscopic distribution of N-(2 diethylaminoethyl)-4 iodobenzamide (I-BZA) in B16 murine melanoma inoculated to C57BL/6J1 Co mice as well as in normal pigmented skin. SIMS provides specific detection of iodine-127 atoms entering 127I-BZA composition. In B16 melanoma, 127I-BZA distribution was found to be heterogeneous, with focal areas of high concentration corresponding to cells rich in melanin pigments. In skin, SIMS analysis showed 127I-BZA distribution appearing as multiple small selective concentration areas within the epidermis. The number of these foci decreased from the stratum basale towards the stratum corneum. In both tissues, the intracellular location appeared specifically intracytoplasmic, with no apparent nuclear uptake. Distribution of this molecule mirrored that of melanin pigments. There was no enhancement of uptake at the membrane site. These results suggest that, in melanic tumors as well as in normal pigmented tissue, specific uptake of 127I-BZA occurs in pigment cells, with a possible link to melanin pigments.

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.

Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblast-like cells.

The uncertainties about the transmission of prion proteins from xenogenic grafts prepared from bovine bone has led to the reconsideration of allogenic bone as a grafting material. Allografting is a complementary technique to autografting nowadays when large bone volumes are necessary. Several preparation techniques have been proposed. Fresh-frozen, freeze-dried and gamma irradiation are the most common. However, a large amount of lipids is present in the medullary spaces (near 70% in weight for a human femoral head). They are known to strongly influence the biocompatibility of the bone graft. The exact changes of lipids upon the sterilization and storage processes are poorly known. The aims of the present study were to appreciate the effects of gamma irradiation on medullary lipids and to identify the cytotoxicity of gamma-irradiated bank bone with/without lipid on cultures of osteoblast-like cells. Bone cores from 8 femoral heads retrieved during prosthesis surgery for arthritis were prepared with a drilling trephine. Cores were either sterilized by gamma radiations (25000 gray) or kept frozen until lipid extraction and lipofuschine-like dosage by Folch's method and fluorometric study. Peroxidated lipids appeared 2 to 3-fold higher in the gamma-irradiated cores than in frozen ones. Slices were prepared from bone cores and were transferred on confluent osteoblast-like cell layers (Saos-2). The raw slices (containing lipids) did not induce cell death. On the other hand, cell death was dramatically increased around the gamma-irradiated slices. Defatted slices which had been sterilized by gamma radiations or UV did not induce cell death. Defatting procedures should be added when preparing bone allografts in human bone banks.

Constitutive PI3-K activity is essential for proliferation, but not survival, of Theileria parva-transformed B cells.

Theileria is an intracellular parasite that causes lymphoproliferative disorders in cattle, and infection of leucocytes induces a transformed phenotype similar to tumour cells, but the mechanisms by which the parasite induces this phenotype are not understood. Here, we show that infected B lymphocytes display constitutive phosphoinositide 3-kinase (PI3-K) activity, which appears to be necessary for proliferation, but not survival. Importantly, we demonstrate that one mechanism by which PI3-K mediates the proliferation of infected B lymphocytes is through the induction of a granulocyte-monocyte colony-stimulating factor (GM-CSF)-dependent autocrine loop. PI3-K induction of GM-CSF appears to be at the transcriptional level and, consistently, we demonstrate that PI3-K is also involved in the constitutive induction of AP-1 and NF-kappaB, which characterizes Theileria-infected leucocytes. Taken together, our results highlight a novel strategy exploited by the intracellular parasite Theileria to induce continued proliferation of its host leucocyte.

Theileria annulata in CD5(+) macrophages and B1 B cells.

Theileria parasites infect and transform bovine leukocytes. We have analyzed laboratory-established Theileria sp.-infected leukocyte lines and observed that transformed macrophages express CD5. Low-level expression of CD5 by macrophages was further confirmed on three independent Theileria annulata clinical isolates from Tunisia. Interestingly, the fourth CD5(+) clinical isolate (MB2) was morphologically different, expressed surface immunoglobulin M (IgM) and BoLA class II, and had rearranged Ig light-chain genes. To demonstrate that MB2 did indeed contain CD5(+) B cells, individual clonal lines were obtained by limiting dilution, and CD5 expression and Ig gene rearrangement were confirmed. This suggests that in natural infections T. annulata can invade and transform CD5(+) B cells.

Evaluation in mice of some iodinated melanoma imaging agents using cryosectioning and multi-wire proportional counting.

AMBIS 4000, a multi-wire proportional counter, was calibrated for iodine-125 measurements. The detector displayed a linear response over a wide dynamic range. Using whole-body mice cryosections, a linear relationship could be established between count rate per area (cpm/mm2) measured with the AMBIS 4000 detector and the count rate per gram (dpm/g) determined with an NaI(Tl) scintillation detector. A calibration curve could, thus be constructed. This new method allowed direct visual and quantitative evaluation of the biodistribution of a short series of 125I-labelled benzamides in melanoma-bearing mice. All the compounds studied showed good tumoral targeting ability. For one of them, ortho-N-(2-diethylaminoethyl)-4-iodobenzamide, liver and lung uptake decreased rapidly after dosing, making it a suitable tracer for scintigraphic detection of malignant melanoma.

Upregulation of Jun and Fos family members and permanent JNK activity lead to constitutive AP-1 activation in Theileria-transformed leukocytes.

Theileria parasitises bovine leukocytes and transforms them into proliferating, metastatic tumours, where the infection resembles a leukaemia-like disease. We have studied the signal transduction pathways leading to activation of the transcription factor AP-1 in different transformed leukocytes. Parasite infection leads to an up-regulation of all members of the Jun/Fos family of proteins and surprisingly, this occurs in the absence of any detectable ERK, or p38 MAP kinase activity. In the parasitised B-sarcoma TBL3, AP-1 induction occurs in the absence of any JNK activity. In contrast, in infected macrophage and B-cell lines, AP-1 transcriptional activity is strictly associated with the parasite-induced constitutive activation of JNK and subsequent c-Jun N-terminal phosphorylation. Thus, constant AP-1 transcriptional activity involves both an upregulation in the levels of Jun and Fos proteins and constitutive JNK activation.

A new quantitative method to evaluate the biodistribution of a radiolabelled tracer for melanoma using whole-body cryosectioning and a gaseous detector: comparison with conventional tissue combustion technology.

Whole-body autoradiography (WBA) and multi-wire proportional counting allow for spatial imaging of the radioactive material present in the tissues and organs of dehydrated animal sections. AMBIS 4000 counting of whole-body cryosections offers a sensitive, accurate and reproducible novel method for the quantitative measurement of the tissue distribution of a [14C] radiopharmaceutical. Intensity of AMBIS 4000 counting (net cpm/mm2) and concentration of radioactivity (nCi/g) were linearly related, yielding a standard curve. Evaluating biodistribution (a) provides pharmacokinetic data for predicting the potential tissue deposition of an absorbed dose of radioactivity in man, and (b) allows visual and quantitative evaluation of radioactivity in small anatomical structures that otherwise could not be detected by conventional tissue combustion technology. This new method of WBA, coupled with AMBIS 4000 counting, should prove a valuable method for pharmacodynamic studies, and afford a predictive tool for nuclear medicine by assessing specific targeting of selected tissues.