Three-Dimensional Quantitative Morphometric Analysis (QMA) for In Situ Joint and Tissue Assessment of Osteoarthritis in a Preclinical Rabbit Disease Model

This work utilises advances in multi-tissue imaging, and incorporates new metrics which define in situ joint changes and individual tissue changes in osteoarthritis (OA). The aims are to (1) demonstrate a protocol for processing intact animal joints for microCT to visualise relevant joint, bone and cartilage structures for understanding OA in a preclinical rabbit model, and (2) introduce a comprehensive three-dimensional (3D) quantitative morphometric analysis (QMA), including an assessment of reproducibility. Sixteen rabbit joints with and without transection of the anterior cruciate ligament were scanned with microCT and contrast agents, and processed for histology. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Subsequently, 3D QMA was performed; including measures of cartilage, subchondral cortical and epiphyseal bone, and novel tibio-femoral joint metrics. Reproducibility of the QMA was tested on seven additional joints. A significant correlation was observed in cartilage thickness from matching histology-microCT pairs. The lateral compartment of operated joints had larger joint space width, thicker femoral cartilage and reduced bone volume, while osteophytes could be detected quantitatively. Measures between the in situ tibia and femur indicated an altered loading scenario. High measurement reproducibility was observed for all new parameters; with ICC ranging from 0.754 to 0.998. In conclusion, this study provides a novel 3D QMA to quantify macro and micro tissue measures in the joint of a rabbit OA model. New metrics were established consisting of: an angle to quantitatively measure osteophytes (σ), an angle to indicate erosion between the lateral and medial femoral condyles (ρ), a vector defining altered angulation (λ, α, β, γ) and a twist angle (τ) measuring instability and tissue degeneration between the femur and tibia, a length measure of joint space width (JSW), and a slope and intercept (m, Χ) of joint contact to demonstrate altered loading with disease progression, as well as traditional bone and cartilage and histo-morphometry measures. We demonstrate correlation of microCT and histology, sensitive discrimination of OA change and robust reproducibility.     

Imaging of experimental osteoarthritis in small animal models

Normally, tissue alterations in small animal models for osteoarthritis (OA) are assessed by time-consuming and destructive histology or biochemical assays. Some high resolution imaging modalities are used for longitudinal monitoring of the OA disease process in vivo. microCT is one of these imaging modalities, which is known for superb high-resolution imaging of bone architecture alterations. A major drawback of microCT is that it has low soft-tissue contrast, which makes direct imaging of cartilage impossible. The use of microCT in combination with negatively charged radiopaque contrast agents enables imaging of cartilage degeneration. We demonstrate the possibility of microCT to image cartilage degeneration as a consequence of experimental OA, by the use contrast enhanced microCT in vivo in a rat model for OA. Furthermore, for the assessment of alterations in molecular processes involved in OA we used the recently developed technique of multi pinhole SPECT. This enables us to assess molecular processes involved in experimental OA in a rat at sub-millimeter level. Here we show quantification of subchondral bone turnover in an OA rat knee. These new techniques demonstrate the possibilities of quantitative experimental OA assessment in small animal models such as mice and rats and might enable substitution of the conventional destructive methods.     

Role of recombinant plasmid pEGFP-N1-IGF-1 transfection in alleviating osteoporosis in ovariectomized rats

Decreased levels of serum insulin-like growth factor-1 (IGF-1) have been proven to cause osteoporosis. Gene transfer of IGF-1 offers an attractive technology to treat skeletal metabolic disorders including osteoporosis, but the viral vectors are limited by their high antigenicity and immune response. Our purpose was to investigate the expression of a non-invasive vector, recombinant plasmid enhanced green fluorescent protein-N1 (pEGFP-N1) that transferred IGF-1 gene into ovariectomized (OVX) rats in vivo and evaluate the effect of this therapy on osteoporosis. OVX or sham operations were performed in 60 female, 7-month-old unmated SD rats. 12 weeks after OVX operation, the vectors were transfected to the 10-month-old rats and experimental data were detected from 48 h to 7 week after transfection. Our results showed that remarkable expression of fluorescence and serum IGF-1 was observed in the rats transfected by recombinant plasmids, indicating that IGF-1 gene was successfully transferred to OVX rats by injecting the vector through hydrodynamic method via the tail vein. The bone metabolism index including serum alkaline phosphatase, the histomorphometric parameters of lumbar vertebra including trabecular area percentage, trabecular thickness, trabecular number and trabecular separation, and the bone mineral density (BMD) and biomechanical parameters of lumbar vertebra including BMD, maximum condensing force, crushing strength in OVX rats transfected by pEGFP-N1-IGF-1 were improved remarkably compared with OVX+pEGFP-N1 rats, indicating that the transfection of recombinant plasmid pEGFP-N1-IGF-1 played a significant role in alleviating osteoporosis in rats induced by OVX. This encouraged a potential approach of IGF-1 gene therapy to the treatment of osteoporosis.     

A polysaccharide from the dried rhizome of Drynaria fortunei (Kunze) J. Sm. prevents ovariectomized (OVX)-induced osteoporosis in rats

In the present study, a homogenous polysaccharide (DFPW) was isolated and purified from the dried rhizome of Drynaria fortunei, and its protective effect against osteoporosis was investigated in ovariectomized (OVX) rats. Histological analysis indicated that oral administration of DFPW (100 and 400 mg/kg) for 12 weeks significantly improved trabecular bone mass, as demonstrated by the increase in trabecular area, trabecular thickness and its number in OVX rats. Furthermore, the decline of bone mineral density and bone mineral content including Ca, P and Mg induced by OVX was reversed by the DFPW administration. This function was achieved by the decreased levels of the bone turnover markers, such as serum ALP, urinary deoxypyridinoline (DPD), Ca and P excretions. Besides, DFPW improved biomechanical parameters (maximum load, energy, Young's, modulus and maximum stress) to strengthen the hardness and strength femoral diaphysis in OVX rats. These results strongly suggested that DFPW might be a hopeful alternative therapeutics to treat postmenopausal osteoporosis.     

European Society of Biomechanics S.M. Perren Award 2008: using temporal trends of 3D bone micro-architecture to predict bone quality

In longitudinal studies, three-dimensional (3D) bone images are acquired at sequential time points essentially resulting in four-dimensional (4D) data for an individual. Based on the 4D data, we propose to calculate temporal trends and project these trends to estimate future bone architecture. Multiple consecutive deformation fields, calculated with Demons deformable image registration algorithm, were extrapolated on a voxel-by-voxel basis. Test data were from in vivo micro-computed tomography (microCT) scans of the proximal tibia of Wistar rats that were either ovariectomized (OVX; N=5) or sham operated (SHAM; N=6). Measurements performed at baseline, 4 and 8 weeks were the basis to predict the 12 week data. Predicted and actual 12 week data were compared using qualitative (3D rendering) and quantitative (geometry, morphology and micro-finite element, microFE) methods. The results indicated a voxel-based linear extrapolation scheme yielded mean geometric errors that were smaller than the voxel size of 15 microm. Key morphological parameters that were estimated included bone volume ratio (BV/TV; mean error 0.4%, maximum error 9%), trabecular thickness (Tb.Th; -1.1%, 11%), connectivity density (Conn.D; 9.0%, 18.5%) and the apparent Young's modulus (E(1); 6.0%, 32%). These data demonstrated a promising and novel approach for quantitatively capturing in vivo bone dynamics at the local trabecular level. The method does not require an a priori understanding of the diseases state, and can provide information about the trends of the bone remodeling process that may be used for better monitoring and treatment of diseases such as osteoporosis.     

Pulsed electromagnetic fields prevent osteoporosis in an ovariectomized female rat model: a prostaglandin E2-associated process

With the use of Helmholtz coils and pulsed electromagnetic field (PEMF) stimulators to generate uniform time varying electromagnetic fields, the effects of extremely low frequency electromagnetic fields on osteoporosis and serum prostaglandin E(2) (PGE(2)) concentration were investigated in bilaterally ovariectomized rats. Thirty-five 3 month old female Sprague-Dawley rats were randomly divided into five different groups: intact (INT), ovariectomy (OVX), aspirin treated (ASP), PEMF stimulation (PEMF + OVX), and PEMF stimulation with aspirin (PEMF + ASP) groups. All rats were subjected to bilateral ovariectomy except those in INT group. Histomorphometric analyses showed that PEMF stimulation augmented and restored proximal tibial metaphyseal trabecular bone mass (increased hard tissue percentage, bone volume percentage, and trabecular number) and architecture (increased trabecular perimeter, trabecular thickness, and decreased trabecular separation) in both PEMF + OVX and PEMF + ASP. Trabecular bone mass of PEMF + OVX rats after PEMF stimulation for 30 days was restored to levels of age matched INT rats. PEMF exposure also attenuated the higher serum PGE(2) concentrations of OVX rats and restored it to levels of INT rats. These experiments demonstrated that extremely low intensity, low frequency, single pulse electromagnetic fields significantly suppressed the trabecular bone loss and restored the trabecular bone structure in bilateral ovariectomized rats. We, therefore, conclude that PEMF may be useful in the prevention of osteoporosis resulting from ovariectomy and that PGE(2) might relate to these preventive effects.     

动态观察去卵巢骨质疏松大鼠股骨骨微结构的变化

目的:绝经后骨质疏松是好发于中老年女性人群中的骨代谢疾病,去卵巢骨质疏松大鼠模型是国内外通用的模拟绝经后骨质疏松发生的经典动物模型,本研究通过观察去卵巢骨质疏松大鼠股骨骨微结构的动态变化,为骨质疏松大鼠模型的临床应用提供理论参考依据。方法:将90只3月龄雌性SD大鼠按体重分层后随机分为基础组(10只)、假手术组(40只)和去卵巢组(40只)。分别在手术前(基础组)和后的3、6、12、24周,腹主动脉取血处死基础组以及假手术组和去卵巢组大鼠,每组各8-10只。每组中随机取6只大鼠,对其左股骨行micro-CT扫描及三维结构重建。选择股骨远端距生长板远端1 mm处,2.0 mm×3.5 mm,厚0.9 mm的骨组织为感兴趣区域,对感兴趣区域进行骨形态计量学分析。结果:与0周组比较,从去卵巢3周开始一直持续到24周,去卵巢组大鼠股骨vBMD、BV/TV和Tb.N显著降低,Tb.Sp和SMI显著升高,而Tb.Th无显著变化;与0周组比较,从假手术后3周开始一直到24周,假手术组所有检测指标均无显著变化。与同周龄假手术组比较,从去卵巢3周开始一直持续到24周,去卵巢组大鼠股骨Tb.N、BV/TV和vBMD显著降低,Tb.Sp显著升高,而Tb.Th没有显著变化。从去卵巢6周开始一直到24周,去卵巢组大鼠SMI显著增加。结论:3月龄大鼠股骨远端的骨微结构在去卵巢3周时就出现显著变化。提示,采用3月龄大鼠进行抗骨质疏松药物筛选时,去卵巢3周后就可以进行药物处理。     

降钙素对骨质疏松大鼠骨密度形态计量学与骨代谢的影响

目的探讨降钙素(密盖息)对骨质疏松大鼠骨密度、骨形态计量学影响以及与血钙、磷、维生素D代谢和生长因子的关系。方法用摘除大鼠双侧卵巢的方式制备骨质疏松模型(OVX),实验动物分为4个组:模型对照组、密盖息治疗组,盐酸雷洛昔芬治疗组,假手术组。应用HOLOGIC第4代双能X线4500W骨密度仪测定大鼠腰椎、股骨上段骨密度值(BMD);以骨形态计量学测股骨骨小梁面积、矿化沉积率;用ELISA法测定血清IGF-1水平和血清25OHVitD浓度以及血淋巴细胞维生素D受体(VDR)含量。结果密盖息治疗组、盐酸雷洛昔芬治疗组均较OVX组腰椎、股骨上段骨密度增高,组间比较差异有显著性(P<0.01)。密盖息治疗组较盐酸雷洛昔芬治疗组股骨上段骨密度增高,两组之间差异有显著性(P<0.01)。密盖息治疗组骨小梁面积明显增加、矿化沉积率增高。密盖息治疗组、盐酸雷洛昔芬治疗组血清IGF-1浓度值、血清25-OHVitD浓度值升高,与OVX组比较差异有显著性(P<0.01)。各组血淋巴细胞VDR含量无明显变化,与OVX组比较差异无显著性(P>0.05)。结论密盖息能够预防腰椎、股骨上段骨密度丢失,使骨小梁面积明显增加、矿化沉积率增高并且血清IGF-1及血清25-OHVitD浓度值升高,但对VDR含量无明显作用。     

Comparison of the Therapeutic Effects of Yeast-incorporated Gallium with those of Inorganic Gallium on Ovariectomized Osteopenic Rats

The purpose of this study was to verify the effect of organic gallium on ovariectomized osteopenic rats. Thirty Wistar female rats used were divided into three groups: (1) sham-operation rats (control), (2) ovariectomized (OVX) rats with osteopenia, and (3) OVX rats with osteopenia treated with organic gallium. Treatments were performed over an 8-week period. At sacrifice, the fifth lumbar vertebral body, one tibia, one femur, and the fourth lumbar vertebrae were removed, subjected to micro-CT for determination of trabecular bone structure, and then processed for histomorphometry to assess bone turnover. The femoral neck was used for mechanical compression testing. Treatment with organic gallium increased bone volume in OVX animals. Organic gallium-treated animals had significant increases in trabecular and cortical thickness and bone strength. The plasma total calcium and inorganic phosphate concentrations in OVX rats decreased and bone mineral content in the lumbar vertebrae and femur increased after treatment with organic gallium. These data provide an important proof of concept that organic gallium may represent a powerful approach to treating or reversing severe osteoporosis in humans.     

Doxycycline effects on mechanical and morphometrical properties of early- and late-stage osteoarthritic bone following anterior cruciate ligament injury.

As posttraumatic osteoarthritis (OA) progresses, the mechanical and morphometrical properties of the subchondral bone change and may be linked to damage of the articular cartilage. Potentially to slow that progression, doxycycline was administered orally twice daily (4 mg.kg(-1).day(-1)) in skeletally mature canines after anterior cruciate ligament transection (ACLX). To test if doxycycline significantly altered the structure and function of OA bone, we tested cancellous bone mechanical properties, measured bone mineral content, and analyzed bone structure by microcomputed tomography. Our investigation focused on subchondral trabecular bone changes in the medial femoral condyle at 36 and 72 wk after ACLX. Significant mechanical changes discovered at 36 wk post-ACLX were less obvious at 72 wk in both treated and ACLX groups. Doxycycline treatment conserved bone strain energy density at 72 wk. Doxycycline had little effect on the degradation of superficial osseous tissue at 36 wk post-ACLX; by 72 wk, doxycycline in an ACLX model limited subchondral bone loss within the first 3 mm of periarticular bone with established OA. Significant bone loss occurred in the deeper trabecular bone for all groups. Substantial architectural adaptation within deeper trabecular bone accompanied changes in mechanics in early and established OA.     

Analysis of Bacteria,Parasites, and Heavy Metals in Lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis>) and Rocket Salad (<Emphasis Type="Italic">Eruca sativa</Emphasis> L.) Irrigated with Treated Effluent from a Biological Wastewater Treatment Plant

The purpose of this study was to verify the effect of organic gallium on ovariectomized osteopenic rats. Thirty Wistar female rats used were divided into three groups: (1) sham-operation rats (control), (2) ovariectomized (OVX) rats with osteopenia, and (3) OVX rats with osteopenia treated with organic gallium. Treatments were performed over an 8-week period. At sacrifice, the fifth lumbar vertebral body, one tibia, one femur, and the fourth lumbar vertebrae were removed, subjected to micro-CT for determination of trabecular bone structure, and then processed for histomorphometry to assess bone turnover. The femoral neck was used for mechanical compression testing. Treatment with organic gallium increased bone volume in OVX animals. Organic gallium-treated animals had significant increases in trabecular and cortical thickness and bone strength. The plasma total calcium and inorganic phosphate concentrations in OVX rats decreased and bone mineral content in the lumbar vertebrae and femur increased after treatment with organic gallium. These data provide an important proof of concept that organic gallium may represent a powerful approach to treating or reversing severe osteoporosis in humans.     

Combined Oral Administration of Bovine Collagen Peptides with Calcium Citrate Inhibits Bone Loss in Ovariectomized Rats

Purpose

Collagen peptides (CPs) and calcium citrate are commonly used as bone health supplements for treating osteoporosis. However, it remains unknown whether the combination of oral bovine CPs with calcium citrate is more effective than administration of either agent alone.

Methods

Forty 12-week-old Sprague-Dawley rats were randomly divided into five groups (n = 8) for once-daily intragastric administration of different treatments for 3 months at 3 months after ovariectomy (OVX) as follows: sham + vehicle; OVX + vehicle; OVX + 750 mg/kg CP; OVX + CP-calcium citrate (75 mg/kg); OVX + calcium citrate (75 mg/kg). After euthanasia, the femurs were removed and analyzed by dual energy X-ray absorptiometry and micro-computed tomography, and serum samples were analyzed for bone metabolic markers.

Results

OVX rats supplemented with CPs or CP-calcium citrate showed osteoprotective effects, with reductions in the OVX-induced decreases in their femoral bone mineral density. Moreover, CP-calcium citrate prevented trabecular bone loss, improved the microarchitecture of the distal femur, and significantly inhibited bone loss with increased bone volume, connectivity density, and trabecular number compared with OVX control rats. CP or CP-calcium citrate administration significantly increased serum procollagen type I N-terminal propeptide levels and reduced serum bone-specific alkaline phosphatase, osteocalcin, and C-telopeptide of type I collagen levels.

Conclusions

Our data indicate that combined oral administration of bovine CPs with calcium citrate inhibits bone loss in OVX rats. The present findings suggest that combined oral administration of bovine CPs with calcium citrate is a promising alternative for reducing bone loss in osteopenic postmenopausal women.     

Beneficial effects of losartan on vascular injury induced by advanced glycosylation end products and their receptors in spontaneous hypertension rats

The purpose of this study was to examine the effects of ENA Actimineral Resource A (ENA-A), seaweed origin alkaline water, on postmenopausal osteoporosis in ovariectomized (OVX) rats. The 12-week old Wistar rats were divided randomly into 4 groups: ovariectomized (OVX), OVX plus 0.5% ENA-A, OVX plus 5% ENA-A and OVX plus 10% ENA-A. A histopathological analysis indicated that ENA-A could prevent OVX-induced bone loss by increasing femur trabecular bone area in a dose-dependent manner. ENA-A significantly (p < 0.05) increased serum estradiol levels, decreased serum osteocalcin activity and suppressed serum pyridinoline (PYD) levels. The in vitro effects of ENA-A were also studied using MC3T3-E1 cells. ENA-A significantly stimulated cell proliferation and increased both ALP activity and calcium deposition in a dose-dependent manner. These results suggest that the treatment of ovariectomized rats with ENA-A not only prevents bone resorption but also appears to maintain the cancellous bone structure of postmeopausal osteoporosis.     

Quantification of subchondral bone changes in a murine osteoarthritis model using micro-CT

In the past few years there has been a considerable interest in the role of bone in osteoarthritis. Despite the increasing evidence of the involvement of bone in osteoarthritis, it remains very difficult to attribute the cause or effect of changes in subchondral bone to the process of osteoarthritis. Although osteoarthritis in mice provides a useful model to study changes in the subchondral bone, detailed quantification of these changes is lacking. Therefore, the goal of this study was to quantify subchondral bone changes in a murine osteoarthritis model by use of micro-computed tomography (micro-CT). We induced osteoarthritis-like characteristics in the knee joints of mice using collagenase injections, and after four weeks we calculated various 3D morphometric parameters in the epiphysis of the proximal tibia. The collagenase injections caused cartilage damage, visible in histological sections, particularly on the medial tibial plateau. Micro-CT analysis revealed that the thickness of the subchondral bone plate was decreased both at the lateral and the medial side. The trabecular compartment demonstrated a small but significant reduction in bone volume fraction compared to the contralateral control joints. Trabeculae in the collagenase-injected joints were thinner but their shape remained rod-like. Furthermore, the connectivity between trabeculae was reduced and the trabecular spacing was increased. In conclusion, four weeks after induction of osteoarthritis in the murine knee subtle but significant changes in subchondral bone architecture could be detected and quantified in 3D with micro-CT analysis.     

A micro-computed tomography study of the trabecular bone structure in the femoral head

The goal of this study was to characterize the trabecular microarchitecture of the femoral head using micro-computed tomography (ICT). Femoral head specimens were obtained from subjects following total hip replacement. Cylindrical cores from the specimens were scanned to obtain 3-D images with an isotropic resolution of 26 Im. Bone structural parameters were evaluated on a per millimeter basis: relative bone volume (BV/TV), trabecular number (Tb.N), thickness (Tb.Th) and separation (Tb.Sp), structure model index (SMI), and connectivity (Conn.D). The ICT data show that the first two millimeters, starting at the joint surface, are characterized by more plate-like trabeculae, and are significantly denser than the underlying trabecular bone. Regional differences in the trabecular architecture reveal that the superior pole has significantly higher BV/TV, Tb.N and Tb.Th values, with lower Tb.Sp compared to the inferior and side poles. Because subchondral bone is essential in the load attenuation of joints, the difference in bone structure between the subchondral and trabecular bone might arise from the different functions each have within joint-forming bones. The denser trabecular structure of the superior pole as compared to the inferior pole can be interpreted as a functional adaptation to higher loading in this area.     

两种骨丢失动物模型骨质,甲状旁腺激素和降钙素的变化比较

本文观察了模拟失重１４天、２１天大鼠和卵巢切除（ＯＶＸ）３０天、６０天大鼠第６胸椎（Ｔ６）、第３腰椎（Ｌ３）和股骨干骨量、骨生物力学特性,血ＰＴＨ和ＣＴ变化特点。实验结果显示,悬吊１４天大鼠Ｔ６和Ｌ３矿盐密度显著增加,２１天Ｔ６矿盐密度显著增加,但Ｌ３矿盐密度显著降低。ＯＶＸ大鼠Ｔ６矿盐密度无变化,但Ｌ３矿盐密度显著降低。悬吊１４天、２１天大鼠股骨干近侧１／３段矿盐含量显著降低,ＯＶＸ６０天大鼠股骨干近侧１／３段矿盐含量显著降低。悬吊大鼠骨力学特性降低较ＯＶＸ大鼠严重。悬吊大鼠血ＰＴＨ无变化,但ＣＴ显著增加,ＯＶＸ大鼠ＰＴＨ、ＣＴ显著降低。实验结果提示,模拟失重大鼠有骨矿盐再分布现象发生,ＯＶＸ大鼠无此现象。悬吊模拟失重大鼠骨质量的降低较ＯＶＸ大鼠严重。在两个动物模型中,皮质骨和松质骨均受影响。     

Therapeutic effects of bone marrow mesenchymal stem cells-derived exosomes on osteoarthritis

Mesenchymal stem cells (MSCs) have shown chondroprotective effects in clinical models of osteoarthritis (OA). However, effects of MSC-derived exosomes on OA remain unclear. The study aimed to investigate the therapeutic potential of exosomes from human bone marrow MSCs (BM-MSCs) in alleviating OA. The anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgery were performed on the knee joints of a rat OA model, followed by intra-articular injection of BM-MSCs or their exosomes. In addition, BM-MSC-derived exosomes were administrated to primary human chondrocytes to observe the functional and molecular alterations. Both of BM-MSCs and BM-MSC-derived exosomes alleviated cartilage destruction and subchondral bone remodelling in OA rat model. Administration of BM-MSCs and exosomes could reduce joint damage and restore the trabecular bone volume fraction, trabecular number and connectivity density of OA rats. In addition, in vitro assays showed that BM-MSCs-exosomes could maintain the chondrocyte phenotype by increasing collagen type II synthesis and inhibiting IL-1β–induced senescence and apoptosis. Furthermore, exosomal lncRNA MEG-3 also reduced the senescence and apoptosis of chondrocytes induced by IL-1β, indicating that lncRNA MEG-3 might partially account the anti-OA effects of BM-MSC exosomes. The exosomes from BM-MSCs exerted beneficial therapeutic effects on OA by reducing the senescence and apoptosis of chondrocytes, suggesting that MSC-derived exosomes might provide a candidate therapy for OA treatment.     

Comparison between quantitative X-ray imaging,dual energy X-ray absorptiometry and microCT in the assessment of bone mineral density in disuse-induced bone loss

Objectives:

We recently introduced a new methodology called quantitative X-ray imaging (qXRI) to investigate bone mineral density in isolated rodent bones. The aims of the present study were to compare DXA and microCT with qXRI in a rat model of disuse osteoporosis.

Methods:

Fourteen Copenhagen rats were injected with a single dose of botulinum toxin (BTX - 2 UI) in the right Mus quadriceps femoris. The left hindlimb serves as control. Areal BMD and vBMD were determined with a Hologic Discovery-W device and a Skyscan 1172 microcomputed tomograph (microCT). Absorbing material density (AMD) was determined on digitized X-ray images obtained with a Faxitron M020 device.

Results:

All three methods highlighted significant lower values for aBMD, vBMD and AMD in trabecular and cortical bone in the BTX-injected side. In trabecular bone, aBMD, vBMD and AMD were significantly correlated with BV/TV. In cortical bone, only aBMD and vBMD were significantly correlated with cortical bone mass On the other hand, only AMD was significantly correlated with the mechanical parameters bending strength and bending modulus.

Conclusions:

qXRI is a rapid and cheap method to assess trabecular bone mass in isolated rodent bones and can be used as a surrogate for the densitometry of small animals.     

Biomechanical properties of bones from rats treated with sevelamer

Sevelamer hydrochloride is used for ten years in patients on dialysis as a phosphate binder. We have previously shown that oral application of sevelamer prevents the bone loss and increases the bone volume in ovariectomized rats. In this study we further analysed the biomechanical properties of bones from rats treated with sevelamer utilizing a threepoint bending test to determine the mechanical properties of the cortical bone of the mid-shaft femur, while the indentation test was used to determine the mechanical properties of cancellous bone in the marrow cavity of the distal femoral metaphysis. Parameters analyzed included: maximum load (F(u)), stiffness (S), energy absorbed (W), toughness (T) and ultimate strength (sigma). The intrinsic properties, stress, elastic modulus and toughness were determined from measured maximum load, strains, stiffness, energy absorbed, outer and inner diameters, and calculated bone cross-sectional moment of inertia. Sevelamer was given to rats for 25 weeks with a content of 3% of sevelamer in a standard diet, starting immediately following ovariectomy (OVX). Animals were divided to the following groups: (1) Sham; (2) Sham + sevelamer 3%; (3) OVX; (4) OVX + sevelamer 3%. Our results showed that sevelamer particularly influenced the rat trabecular bone by increasing the maximum load for 26.2%, energy absorbed for 24.2% and the ultimate strength for 26.2% in sham animals treated with sevelamer 3%, as compared to sham rats. Sevelamer 3% in OVX rats also increased the maximum load for 71.4%, stiffness for 70.7%, energy absorbed for 55.9% and the ultimate strength for 71.3% as compared to OVX controls. In the three bending test sevelamer had a very little effect on preventing loss of bone strenght in the cortical bone. These results collectively suggest that sevelamer improves bone biomechanical properties, mainly affecting trabecular bone quality in both normal and ovariectomized rats.