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.

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.

Association of clinical parameters of operatively treated thoracolumbar fractures with quality of life parameters.

PURPOSE: The intention of the current work was to assess the association between clinical parameters and seven different quality of life (QoL) instruments after surgical treatment of thoracolumbar spinal fractures after an average follow-up of 4.2 years. METHODS: The following human-related quality of life and PRO measures of 66 patients were correlated to clinical parameters such as fingertip-to-floor distance (FFD), Schober measurement, pressure and percussion pain in the lumbopelvine area (PPP), and paravertebral muscle tension: reALOS, SF-36, VAS, VAS spine score, BDI, the GBB-24, and the IES-R. RESULTS: Overall, there was a significant association between the clinical parameters of the thoracolumbar spine such as PPP, paravertebral muscle tension, FFD and Schober's sign on one side, and the seven tested instruments on the other side. CONCLUSIONS: PPP and FFD as well as a small Schober measurement are clinical parameters which significantly influence QoL after surgical treatment of thoracolumbar fractures.

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.

Percutaneous lateral ankle stabilization: an anatomical investigation.

BACKGROUND: The current study investigated the topographic anatomy of the percutaneous anatomical lateral ankle stabilization in relation to the neurovascular hindfoot structures. The study should serve as an aid for performance of this new minimally invasive technique. MATERIALS AND METHODS: Eleven cadaver specimens were dissected exposing the nerves, vessels, ligaments and tendons. The portals and transosseous tunnels were performed with Kirschner wires. All distances of the Kirschner wires and the neurovascular structures were measured with reference to clearly identifiable bony landmarks. RESULTS: On the medial side the average distance of the Kirschner wire to the medial calcaneal branch of the tibial nerve was found to be 7 mm (SD±4). The medial calcaneal branch was hit twice by the transosseous Kirschner wire. On the lateral side the mean distance of the fibular exit point of the Kirschner wire to the sural nerve was 13 mm (SD±4). The closest distance of the superficial peroneal nerve to the footprint of the ATFL averaged 11.5 mm (SD±3) and its anterior location in relation to the tip of the fibula was 28 mm (SD±8). The posterior tibial artery was found at a mean distance of 41 mm from the perforating Kirschner wire (SD±6). CONCLUSION: The current study introduced a novel percutaneous approach to treat chronic ankle instability that had minimal risk to neurovascular structures in a cadaver model. CLINICAL RELEVANCE: Further clinical studies must be undertaken to identify whether this technique would also be superior when compared with open surgery.

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.

Change of mechanical vertebrae properties due to progressive osteoporosis: combined biomechanical and finite-element analysis within a rat model.

For assessing mechanical properties of osteoporotic bone, biomechanical testing combined with in silico modeling plays a key role. The present study focuses on microscopic mechanical bone properties in a rat model of postmenopausal osteoporosis. Female Sprague-Dawley rats were (1) euthanized without prior interventions, (2) sham-operated, and (3) subjected to ovariectomy combined with a multi-deficiencies diet. Rat vertebrae (corpora vertebrae) were imaged by micro-CT, their stiffness was determined by compression tests, and load-induced stress states as well as property changes due to the treatment were analyzed by finite-element modeling. By comparing vertebra stiffness measurements with finite-element calculations of stiffness, an overall microscopic Young's modulus of the bone was determined. Macroscopic vertebra stiffness as well as the microscopic modulus diminish with progression of osteoporosis by about 70 %. After strong initial changes of bone morphology, further decrease in macroscopic stiffness is largely due to decreasing microscopic Young's modulus. The micromechanical stress calculations reveal particularly loaded vertebra regions prone to failure. Osteoporosis-induced changes of the microscopic Young's modulus alter the fracture behavior of bone, may influence bone remodeling, and should be considered in the design of implant materials.

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.

Bone formation induced by strontium modified calcium phosphate cement in critical-size metaphyseal fracture defects in ovariectomized rats.

The first objective was to investigate new bone formation in a critical-size metaphyseal defect in the femur of ovariectomized rats filled with a strontium modified calcium phosphate cement (SrCPC) compared to calcium phosphate cement (CPC) and empty defects. Second, detection of strontium release from the materials as well as calcium and collagen mass distribution in the fracture defect should be targeted by time of flight secondary ion mass spectrometry (TOF-SIMS). 45 female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) SrCPC (n = 15), (2) CPC (n = 15), and (3) empty defect (n = 15). Bilateral ovariectomy was performed and three months after multi-deficient diet, the left femur of all animals underwent a 4 mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with SrCPC or CPC or was left empty. After 6 weeks, histomorphometric analysis showed a statistically significant increase in bone formation of SrCPC compared to CPC (p = 0.005) and the empty defect (p = 0.002) in the former fracture defect zone. Furthermore, there was a statistically significant higher bone formation at the tissue-implant interface in the SrCPC group compared to the CPC group (p < 0.0001). These data were confirmed by immunohistochemistry revealing an increase in bone-morphogenic protein 2, osteocalcin and osteoprotegerin expression and a statistically significant higher gene expression of alkaline phosphatase, collagen10a1 and osteocalcin in the SrCPC group compared to CPC. TOF-SIMS analysis showed a high release of Sr from the SrCPC into the interface region in this area compared to CPC suggesting that improved bone formation is attributable to the released Sr from the SrCPC.

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures.

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.