Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats.

Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.

Bone matrix, cellularity, and structural changes in a rat model with high-turnover osteoporosis induced by combined ovariectomy and a multiple-deficient diet.

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro-computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro-computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants.

Implications of combined ovariectomy and glucocorticoid (dexamethasone) treatment on mineral, microarchitectural, biomechanical and matrix properties of rat bone.

Osteoporosis is one of the deleterious side effects of long-term glucocorticoid therapy. Since the condition is particularly aggressive in postmenopausal women who are on steroid therapy, in this study we have attempted to analyse the combined effect of glucocorticoid (dexamethasone) treatment and cessation of oestrogen on rat bone. The dual aim was to generate osteoporotic bone status in a short time scale and to characterise the combination of glucocorticoid-postmenopausal osteoporotic conditions. Sprague Dawley rats (N = 42) were grouped randomly into three groups: untreated control, sham-operated and ovariectomized-steroid (OVX-Steroid) rats. Control animals were euthanized with no treatment [Month 0 (M0)], while sham and OVX-Steroid rats were monitored up to 1 month (M1) and 3 months (M3) post laparotomy/post OVX-Steroid treatment. Histology, dual-energy X-ray absorptiometry (DXA), micro-computed tomography (micro-CT), and biomechanical and mRNA expression analysis of collagenous, non-collagenous matrix proteins and osteoclast markers were examined. The study indicated enhanced osteoclastogenesis and significantly lower bone mineral density (BMD) in the OVX-Steroid rats with Z-scores below -2.5, reduced torsional strength, reduced bone volume (BV/TV%), significantly enhanced trabecular separation (Tb.S), and less trabecular number (Tb.N) compared with sham rats. Osteoclast markers, cathepsin K and MMP 9 were upregulated along with Col1α1 and biglycan with no significant expression variation in fibronectin, MMP 14, LRP-5, Car II and TNC. These results show higher bone turnover with enhanced bone resorption accompanied with reduced torsional strength in OVX-Steroid rats; and these changes were attained within a short timeframe. This could be a useful model which mimics human postmenopausal osteoporosis that is associated with steroid therapy and could prove of value both in disease diagnosis and for testing generating and testing biological agents which could be used in treatment.

Quantitative assessment of microcirculation and diffusion in the bone marrow of osteoporotic rats using VCT, DCE-MRI, DW-MRI, and histology.

BACKGROUND: Etiologic and pathophysiologic role of functional bone marrow processes is not fully understood especially in the case of osteoporosis. PURPOSE: To investigate the role of vascularization and diffusion in rat models of osteoporosis through a cross-correlation between non-invasive in-vivo imaging and invasive ex-vivo imaging of bone, bone marrow, and in particular of microcirculation. MATERIAL AND METHODS: Osteoporosis was induced in rats by combining ovariectomy (OVX) with calcium and Vitamin D3 deficiency, or with glucocorticoid (dexamethasone). For comparison, controls underwent a sham surgery. In in-vivo investigations, animals (n = 36) were examined by volumetric CT (VCT) and MRI at 1, 3, or 12 months post surgery. Using VCT, bone morphology was monitored and relative bone density r within pelvis was extracted. With DCE-MRI and DW-MRI, parameters A (amplitude), Kep (exchange rate constant), and ADC (apparent diffusion coefficient) were acquired for regions of lumbar vertebrae, pelvis, and femur. In ex-vivo investigations, selective histological sections of pelvis were either stained with hematoxylin and eosin (HE stain) for quantifying vessel size and density or immunostained for collagen IV and α-smooth muscle actin to assess vessel maturity (SMA/collagen IV ratio). RESULTS: After 12 months, decrease in DCE-MRI parameter Kep was found in all locations of osteoporotic rats (strongest in femur and lumbar vertebrae) while no significant differences were seen for parameter A and DW-MRI parameter ADC. Furthermore, vessel rarefication and maturation were observed on the histological level in animals with osteoporotic phenotype. In particular in the pelvis, the osteoporotic individuals (irrespective of the osteoporosis inducers applied) exhibited decreased Kep, significantly reduced vessel density, significantly increased vessel maturity, as well as statistically unaltered A, ADC, and vessel diameter. CONCLUSION: Changes in microcirculation but not diffusion in the bone marrow of osteoporotic rats are detected by DCE-MRI and DW-MRI due to vessel rarefication and maturation.

Induction of osteoporosis with its influence on osteoporotic determinants and their interrelationships in rats by DEXA.

BACKGROUND: As women are the population most affected by multifactorial osteoporosis, research is focused on unraveling the underlying mechanism of osteoporosis induction in rats by combining ovariectomy (OVX) either with calcium, phosphorus, vitamin C and vitamin D2/D3 deficiency, or by administration of glucocorticoid (dexamethasone). MATERIAL/METHODS: Different skeletal sites of sham, OVX-Diet and OVX-Steroid rats were analyzed by Dual Energy X-ray Absorptiometry (DEXA) at varied time points of 0, 4 and 12 weeks to determine and compare the osteoporotic factors such as bone mineral density (BMD), bone mineral content (BMC), area, body weight and percent fat among different groups and time points. Comparative analysis and interrelationships among osteoporotic determinants by regression analysis were also determined. RESULTS: T scores were below-2.5 in OVX-Diet rats at 4 and 12 weeks post-OVX. OVX-diet rats revealed pronounced osteoporotic status with reduced BMD and BMC than the steroid counterparts, with the spine and pelvis as the most affected skeletal sites. Increase in percent fat was observed irrespective of the osteoporosis inducers applied. Comparative analysis and interrelationships between osteoporotic determinants that are rarely studied in animals indicate the necessity to analyze BMC and area along with BMD in obtaining meaningful information leading to proper prediction of probability of osteoporotic fractures. CONCLUSIONS: Enhanced osteoporotic effect observed in OVX-Diet rats indicates that estrogen dysregulation combined with diet treatment induces and enhances osteoporosis with time when compared to the steroid group. Comparative and regression analysis indicates the need to determine BMC along with BMD and area in osteoporotic determination.

Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition.

BACKGROUND: The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies. METHODS: Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro. RESULTS: The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone. CONCLUSIONS: Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

Characterization of bone turnover and energy metabolism in a rat model of primary and secondary osteoporosis.

BACKGROUND: An experimental rat model served for evaluation of bone- and energy metabolism in early and late stages of osteoporosis. For the early stage, we hypothesized that bilateral ovariectomy (OVX)+multi-deficiency diet (OVXD; depletion of vitamin D, calcium, vitamin K, phosphorus) would induce increased bone turnover while the late stage would be characterized by enhanced bone catabolism. Obesity, insulin resistance and hyperleptinemia would be seen during the whole course of disease. Healthy female Sprague Dawley rats (n=41) aged 10 weeks were randomly assigned to sham and treatment groups and sacrificed at 3, 12, and 14 months after the study began. RESULTS: In the early phase, OVXD was associated with an increase in body weight, but not, however, in later stages. There was a decrease in bone mineral density and relative bone volume (BV/TV) as assessed by Dual Energy X-ray Absorptiometry and micro computed tomography that was most severe in the later stages of disease, indicating bone catabolism. Osteocalcin limiting bone formation was increased initially, whereas later stages (14 months) were characterized by elevated osteopontin, suggesting bone remodeling. Severe hyperparathyroidism was present during all stages of disease. Only the early phases of disease were characterized by hyperinsulinemia and increased adrenocorticotrophic stimulating hormone, whereas in the late stage hypoleptinemia rather than hyperleptinemia was seen. CONCLUSION: Markers of bone and energy metabolism reflected both an increased bone turn over and ongoing bone remodeling associated with initial hyperinsulinemia. Osteopontin and osteocalcin can be used to differentiate early and late stages of osteoporosis.

Applicability of ToF-SIMS for monitoring compositional changes in bone in a long-term animal model.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a well-established technique in material sciences but has not yet been widely explored for implementation in life sciences. Here, we demonstrate the applicability and advantages of ToF-SIMS analysis for the study of minerals and biomolecules in osseous tissue. The locally resolved analysis of fragment ions deriving from the sample surface enables imaging and differentiation of bone tissue and facilitates histology on non-stained cross sections. In a rat model, bilateral ovariectomy combined with either a multi-deficiency diet or steroid treatment was carried out to create osteoporotic conditions. We focused our study on the Ca content of the mineralized tissue and monitored its decline. Calcium mass images of cross sections show the progressive degenerative changes in the bone. We observed a decreased Ca concentration in the edge region of the trabeculae and a decline in the Ca/P ratio. Additionally, we focused on the non-mineralized matrix and identified fragment ions that are characteristic for the collagen matrix. We observed trabeculae with wide ranges of non-mineralized collagen for the diet group owing to an impaired mineralization process. Here, the advantage of coeval monitoring of collagen and minerals indicated an osteomalacic model rather than an osteoporotic one.

Implications of combined ovariectomy/multi-deficiency diet on rat bone with age-related variation in bone parameters and bone loss at multiple skeletal sites by DEXA.

BACKGROUND: Osteoporosis is a multi-factorial, chronic, skeletal disease highly prevalent in post-menopausal women and is influenced by hormonal and dietary factors. Because animal models are imperative for disease diagnostics, the present study establishes and evaluates enhanced osteoporosis obtained through combined ovariectomy and deficient diet by DEXA (dual-energy X-ray absorptiometry) for a prolonged time period. MATERIAL/METHODS: Sprague-Dawley rats were randomly divided into sham (laparotomized) and OVX-diet (ovariectomized and fed with deficient diet) groups. Different skeletal sites were scanned by DEXA at the following time points: M0 (baseline), M12 (12 months post-surgery), and M14 (14 months post-surgery). Parameters analyzed included BMD (bone mineral density), BMC (bone mineral content), bone area, and fat (%). Regression analysis was performed to determine the interrelationships between BMC, BMD, and bone area from M0 to M14. RESULTS: BMD and BMC were significantly lower in OVX-diet rats at M12 and M14 compared to sham rats. The Z-scores were below -5 in OVX-diet rats at M12, but still decreased at M14 in OVX-diet rats. Bone area and percent fat were significantly lower in OVX-diet rats at M14 compared to sham rats. The regression coefficients for BMD vs. bone area, BMC vs. bone area, and BMC vs. BMD of OVX-diet rats increased with time. This is explained by differential percent change in BMD, BMC, and bone area with respect to time and disease progression. CONCLUSIONS: Combined ovariectomy and deficient diet in rats caused significant reduction of BMD, BMC, and bone area, with nearly 40% bone loss after 14 months, indicating the development of severe osteoporosis. An increasing regression coefficient of BMD vs. bone area with disease progression emphasizes bone area as an important parameter, along with BMD and BMC, for prediction of fracture risk.

Effects of multi-deficiencies-diet on bone parameters of peripheral bone in ovariectomized mature rat.

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.