Effects of vibration treatment on tibial bone of ovariectomized rats analyzed by in vivo micro‐CT

Daily low‐amplitude, high‐frequency whole‐body vibration (WBV) treatment can increase bone formation rates and bone volume in rodents. Its effects vary, however, with vibration characteristics and study design, and effects on 3D bone microstructure of ovariectomized animals over time have not been documented. Our goal was to determine the effects of WBV on tibial bone of ovariectomized, mature rats over time using an in vivo micro‐CT scanner. Adult rats were divided into: ovariectomy (OVX) (n = 8), SHAM‐OVX (n = 8), OVX and WBV treatment (n = 7). Eight weeks after OVX, rats in the vibration group were placed on a vibrating platform for 20 min at 0.3 g and 90 Hertz. This was done 5 days a week for six weeks, twice a day. Zero, 8, 10, 12 and 14 weeks after OVX, in vivo micro‐CT scans were made (vivaCT 40, Scanco Medical AG) of the proximal and diaphyseal tibia. After sacrifice, all tibiae were dissected and tested in three‐point bending. In the metaphysis between 8 to 12 weeks after OVX, WBV treatment did not alter structural parameters compared to the OVX group and both groups continued to show deterioration of bone structure. In the epiphysis, structural parameters were not altered. WBV also did not affect cortical bone and its bending properties. To summarize, no substantial effects of 6 weeks of low‐magnitude, high‐frequency vibration treatment on tibial bone microstructure and strength in ovariectomized rats were found. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:62–69, 2010     

Biological reaction to polyethylene particles in a murine calvarial model is highly influenced by age

Particle‐induced osteolysis is driven by multiple factors including bone metabolism, inflammation, and age. The objective of this study was to determine the influence of age on polyethylene (PE) particle‐induced osteolysis in a murine calvarial model comparing 2‐month‐old (young) versus 24‐month‐old (old) mice. After PE particle implantation, calvaria were assessed at days (D) 3, D7, D14, and D21 via chemoluminescent imaging for inflammation (L‐012 probe). In addition micro‐computed tomography (micro‐CT) and histomorphometry end points addressed the bone reaction. Inflammation peaked at D7 in young mice and D14 in old mice. Using micro‐CT, a nadir of mature bone was recorded at D7 for young mice, versus D21 for old mice. Besides, regenerating bone peaked at distinct timepoints: D7 for young mice versus D21 for old mice. In the young mice group, the histomorphometric findings correlated with micro‐CT regenerating bone findings at D7, associated with ample osteoïd deposition. No osteoïd could be histologically quantified in the old mice group at D7. This study demonstrated that the biological reaction to polyethylene particles is highly influenced by age. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:574–580, 2016.     

Strontium ranelate treatment enhances hydroxyapatite‐coated titanium screws fixation in osteoporotic rats

Increased bone turnover with excessive bone resorption and decreased bone formation is known to impair implant fixation. Strontium ranelate is well known as an effective antiosteoporotic agent by its dual effect of antiresorbing and bone‐forming activity. This study was designed to evaluate the effect of systemic strontium ranelate (SR) treatment on fixation of hydroxyapatite (HA)‐coated titanium screws in ovariectomized (OVX) rats. Twelve weeks after being OVX (n = 30) or sham (n = 10) operated, 40 female Sprague–Dawley rats received unilateral implants in the proximal tibiae. The OVX rats were randomly divided into the following groups: OVX, OVX + SR_L (“_L” refers to low SR dose of 500 mg/kg/day), OVX + SR_H (“_H” refers to high SR dose of 1000 mg/kg/day).Twelve weeks after treatment, bone blocks with implants were evaluated with micro‐CT and biomechanical push‐out tests. Compared to OVX animals, SR treatment increased the bone volume ratio by 51.5% and 1.1‐fold, the percentage osteointegration by 1.0‐fold and 1.9‐fold in micro‐CT evaluation, and the maximal force by 1.9‐fold and 3.3‐fold in biomechanical push‐out test, for the low and high dose of SR, respectively. Significant correlation between micro‐CT and biomechanical properties demonstrated that trabecular parameters played an important role in predicting the biomechanical properties of implant fixation. Our findings suggest that SR treatment can dose‐dependently improve HA‐coated screw fixation in OVX rats and facilitate the stability of the implant in the osteoporotic bone. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:578–582, 2010     

Antioxidant impregnated ultra‐high molecular weight polyethylene wear debris particles display increased bone remodeling and a superior osteogenic:osteolytic profile vs. conventional UHMWPE particles in a murine calvaria model

Periprosthetic osteolysis remains a major limitation of long‐term successful total hip replacements with ultra‐high molecular weight polyethylene (UHMWPE) bearings. As intra and extracellular reactive oxygen species are know to contribute to wear debris‐induced osteoclastic bone resorption and decreased osteoblastic bone formation, antioxidant doped UHMWPE has emerged as an approach to reduce the osteolytic potential of wear debris and maintain coupled bone remodeling. To test this hypothesis in vivo, we evaluated the effects of crosslinked UHMWPE wear debris particles (AltrX^?), versus similar wear particles made from COVERNOX^? containing UHMWPE (AOX^?), in an established murine calvaria model. Eight‐week‐old female C57B/6 mice (n = 10/Group) received a pre‐op micro‐CT scan prior to surgical implantation of the UHMWPE particles (2mg), or surgery without particles (sham). Dynamic labeling was performed by intraperitoneal injection of calcein on day 7 and alizarin on day 9, and the calvaria were harvested for micro‐CT and histology on day 10. Surprisingly, we found that AOX particles induced significantly more bone resorption (1.72‐fold) and osteoclast numbers (1.99‐fold) vs. AltrX (p < 0.001). However, AOX also significantly induced 1.64‐fold more new bone formation vs. AltrX (p < 0.01). Moreover, while the osteolytic:osteogenic ratio of both particles was very close to 1.0, which is indicative of coupled remodeling, AOX was more osteogenic (Slope = 1.13 ± 0.10 vs. 0.97 ± 0.10). Histomorphometry of the metabolically labeled undecalcified calvaria revealed a consistent trend of greater MAR in AOX vs. AltrX. Collectively, these results demonstrate that anti‐oxidant impregnated UHMWPE particles have decreased osteolytic potential due to their increased osteogenic properties that support coupled bone remodeling. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:845–851, 2016.

     

Effect of zoledronic acid on spinal fusion outcomes in an ovariectomized rat model of osteoporosis

To evaluate the effect of zoledronic acid (ZA) on spinal fusion in ovariectomized (OVX) rats. Female SD rats (n = 50) were OVX or sham‐operated and randomized into five groups: Sham, OVX control, ZOL‐20 (20 µg/kg), ZOL‐100 (100 µg/kg), and ZOL‐500 (500 µg/kg). Eight weeks after OVX, bilateral lumbar spinal fusion was performed using autologous iliac bone with ZA or saline according to the grouping. The lumbar spines were harvested at 8 weeks and subjected to radiographic, manual palpation, micro‐computed tomographic (micro‐CT), and histological analysis. The manual palpation result differed significantly only between the ZOL‐500 (fused: partially fused: not fused, 9:0:0) and OVX control (4:2:3) (p < 0.05). The radiographic scales were also differed significantly only between these two groups. According to the micro‐CT results, the bone volume fraction (BV/TV) were significantly higher in all ZA‐treated groups (54.2%, 65.9%, and 73.6%) than OVX control (43.7%) (p < 0.01). At clinical dose or lower, ZA didn't alter the spinal fusion, but a higher dose increased the spinal fusion rate significantly. This study suggests ZA may have a positive effect on spinal fusion in the presence of osteoporosis, and spinal fusion surgery outcome is not likely to be altered by ZA at clinical dose. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1297–1304, 2015.     

Differential effects of biologic versus bisphosphonate inhibition of wear debris‐induced osteolysis assessed by longitudinal micro‐CT

Aseptic loosening of total joint replacements is caused by wear debris‐induced osteoclastic bone resorption, for which bisphosphonates (BPs) and RANK antagonists have been developed. Although BPs are effective in preventing metabolic bone loss, they are less effective for inflammatory bone loss. Because this difference has been attributed to the antiapoptotic inflammatory signals that protect osteoclasts from BP‐induced apoptosis, but not RANK antagonists, we tested the hypothesis that osteoprotegerin (OPG) is more effective in preventing wear debris‐induced osteolysis than zoledronic acid (ZA) or alendronate (Aln) in the murine calvaria model using in vivo micro‐CT and traditional histology. Although micro‐CT proved to be incompatible with titanium (Ti) particles, we were able to demonstrate a 3.2‐fold increase in osteolytic volume over 10 days induced by polyethylene (PE) particles versus sham controls (0.49 ± 0.23mm³ versus 0.15 ± 0.067mm³; p < 0.01). Although OPG and high‐dose ZA completely inhibited this PE‐induced osteolysis (p < 0.001), pharmacological doses of ZA and Aln were less effective but still reached statistical significance (p < 0.05). Traditional histomorphometry of the sagital suture area of calvaria from both Ti and PE‐treated mice confirmed the remarkable suppression of resorption by OPG (p < 0.001) versus the lack of effect by physiological BPs. The differences in drug effects on osteolysis were largely explained by the significant difference in osteoclast numbers observed between OPG versus BPs in both Ti‐ and PE‐treated calvaria; and linear regression analyses that demonstrated a highly significant correlation between osteolysis volume and sagittal suture area versus osteoclast numbers (p < 0.001). © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1340–1346, 2008     

Effects of LLLT in combination with bisphosphonate on bone healing in critical size defects: a histological and histometric study in rat calvaria

The purpose of this study was to analyze histologically the effect of low-level laser therapy (LLLT) in combination with bisphosphonate on bone healing in surgically created critical size defects (CSD) in rat calvaria. One hundred Wistar female rats sham operated (sham) and ovariectomized (Ovx) were maintained untreated for 1 month to allow for the development of osteopenia in the Ovx animals. A CSD was made in the calvarium of each rat, and the animals were divided into five groups according to following treatments: (1) sham rats (control), (2) Ovx rats, (3) Ovx rats treated with LLLT, (4) Ovx rats treated with bisphosphonate, and (5) Ovx rats treated with bisphosphonate and LLLT. Groups 4 and 5 were irrigated with 1 ml of bisphosphonate, and groups 3 and 5 were submitted to LLLT (GaAlAs), 660 nm, 24 J, and 0.4285 W/cm² on the CSD. Ten animals of each treatment were killed at 30 and 60 days. Histomorphometric assessments, using image analysis software, and histological analyses were performed. No defect was completely regenerated with the bone. Histometrically, it can be observed that groups 3 (37.49?±?1.94%, 43.11?±?2.39%) and 5 (35.05?±?1.57%, 41.07?±?1.89%) showed a significant bone neoformation when compared to groups 1 (16.81?±?1.57%, 27.54?±?1.49%), 2 (11.68?±?0.98%, 22.51?±?1.05%), and 4 (14.62?±?1.70%, 25.67?±?1.41%) in all experimental periods (P?<?0.05). It was possible to conclude that the LLLT associated or not with bisphosphonate treatment was effective for stimulating bone formation in CSD in the calvaria of rats submitted to ovariectomy.     

应用microCT技术对中药干预去卵巢小鼠骨组织微结构的分析

目的应用micro CT技术观察健骨颗粒对C57小鼠去卵巢骨质疏松模型骨组织骨量、骨微结构及生物力学的影响。方法将4周龄清洁级C57小鼠30只随机分为三组(假手术组10只、卵巢切除小鼠两组各10只),两组卵巢切除小鼠术后1周开始,分别用健骨颗粒和生理盐水进行灌胃,假手术组用生理盐水灌胃。2个月后,小鼠左胫骨行micro CT检测及图像分析、右胫骨行生物力学三点抗压最大载荷检测。结果与假手术组比较,去卵巢组BMC、BMD、Mean、BV、BS显著下降(P0.01),TMC下降(0.01P0.05);与去卵巢组比较,去卵巢中药组BMC、BMD、Mean指标显著提高(P0.01);图像分析结果显示去卵巢组较假手术组骨皮质薄,骨小梁数量少,形态细小、不连续呈扭曲或断裂状等明显骨质疏松病理特征,去卵巢中药组介于两者之间。生物力学结果显示三组胫骨三点抗压最大载荷均具有明显差异(P0.01)。结论去卵巢2月成功建立小鼠绝经后骨质疏松症模型;健骨颗粒抗骨质疏松作用明显,主要通过增加骨量和改善骨小梁微结构来最终提高骨强度;micro CT对骨质疏松参数分析简洁、高效,图像多维、全面,与传统的检测方法相比,具有一定的优势。     

Antisense oligonucleotide targeting TNF‐α can suppress co‐cr‐mo particle‐induced osteolysis

The most common cause of implant failure in joint replacement is aseptic loosening due to particle‐induced osteolysis. TNF‐α has been shown to be one of the key factors in the process of osteoclastogenesis. Anti‐TNF agents are useful in the treatment of joint inflammation related to osteolysis. This study investigated the effect of a single subcutaneous dose of an antisense oligonucleotide (ASO) on particle‐induced osteolysis. We utilized the murine calvaria osteolysis model in C57BL/J6 mice. Bone resorption was measured by the toluidine blue staining. Osteoclasts were detected by tartrate resistant acid phosphatase (TRAP) staining assay and were quantified by a TRAP quantification kit. Results show that bone resorption is 0.347 ± 0.09 mm² in mice with particle implantation, and decreased to 0.123 ± 0.05 mm² and 0.052 ± 0.02 mm² after ASO treatment with low and high doses, respectively. The number of osteoclasts in animal calvaria treated with ASO is reduced compared with that of untreated animals, and the quantification results indicate that about 90% of osteoclastogenesis is suppressed by the ASO. In addition, the osteoclastogenesis can be reestablished by the addition of TNF‐α. In conclusion, we demonstrate that the antisense oligonucleotide targeting to TNF‐α can suppress osteolysis induced by metal particles in a murine calvaria model. This new finding may be of value in the search for novel therapeutic methods for implant loosening. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1114–1120, 2008     

Comparison of bone loss induced by ovariectomy and neurectomy in rats analyzed by in vivo micro‐CT

We hypothesized that osteoporosis due to estrogen deficiency progresses faster than due to disuse and that at the same amount of bone loss, disuse leads to less favorable bone structure and mechanical properties than estrogen deficiency. Adult rats were either ovariectomized (OVX) (n = 9) or neurectomized (NX) (n = 8). At week 0, 1, 2, 3, and 4, in vivo micro‐CT scans were made of the proximal tibia. Segmented CT‐scans at weeks 0 and 4 were used to build a 3D voxel‐based micro finite element model (FEM). Displacement in the longitudinal direction was prescribed at the proximal end leading to a compression step of 1%. The severe reduction in metaphyseal bone volume fraction was not significantly different between OVX and NX. Epiphyseal bone loss was less severe in both groups, and BV/TV was significantly lower after NX. Trabecular separation and degree of anisotropy in the metaphysis and connectivity and trabecular number in the epiphysis were significantly more deteriorated after NX. FEM‐derived stiffness decreased in both groups, but more after NX. Osteoporosis due to estrogen‐deficiency progressed overall at a rate similar to osteoporosis due to disuse. At the same amount of induced bone loss, disuse led to more deteriorated bone structure and mechanical properties than estrogen deficiency. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1521–1527, 2009     

Promotion of normal healing of bone defects under estrogen deficiency by implantation of beta‐tricalcium phosphate composed of rod‐shaped particles

We compared the healing of bone defects in ovariectomized rats implanted with beta‐tricalcium phosphate (β‐TCP) composed of rod‐shaped particles, which were prepared using the applied hydrothermal method (HTCP), and that of bone defects implanted with conventional β‐TCP composed of globular‐shaped particles (CTCP), which were prepared by normal sintering. Eight‐week‐old female Wistar rats were ovariectomized, and 2 weeks after the operation, 0.5‐ to 0.6‐mm diameter spherical granules of each ceramic were implanted in a bone defect created in the distal end of the femur. Four, 8, and 12 weeks after implantation, the amount of newly formed bone implanted with HTCP was significantly larger than that implanted with CTCP and was equivalent to that in non‐ovariectomized sham‐operated rats. Without implantation, spontaneous repair of the trabecular bone was barely observed. The physiological structure of the trabecular network was maintained in the region implanted with HTCP, but that in the region implanted with CTCP was severely destroyed. Gene expression microarray analysis revealed that the expression of genes involved in interferon signaling pathways was upregulated in osteoclasts cultured on HTCP compared with that cultured on CTCP. Our results suggest that the microstructure of β‐TCP affected the biological behavior of osteoclasts and regulated local bone metabolism. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:189–196, 2014.     

Osteoinductivity of demineralized bone matrix in immunocompromised mice and rats is decreased by ovariectomy and restored by estrogen replacement

The osteoinduction potential of human demineralized bone matrix (DBM) in females with low estrogen (E2) is unknown. Moreover, the osteoinductivity of commercial human DBM is tested in male athymic rats and mice, but DBM performance in these animals may not reflect performance in female animals or provide information on E2's role in the process. To gain insight, human DBM was implanted bilaterally in the gastrocnemius of twenty-four athymic female mice (10 mg/implant) and twenty-four athymic female rats (15 mg/implant). Eight animals in each group were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized with E2-replacement (OVX+E2) via subcutaneous slow release capsules of 17beta-estradiol. OVX and OVX+E2 animals were pair-fed to SHAM animals. Four animals from each group were euthanized at 35 days and four at 56 days. Animal weight, uterine weight, and blood estrogen levels confirmed that pair feeding, ovariectomy, and E2 replacement were successful. Histological sections of implanted tissues were evaluated qualitatively for absence or presence of DBM, ossicle formation, and new bone or cartilage using a previously developed qualitative scoring system (QS) and by histomorphometry to obtain a quantitative assessment of osteoinduction. OVX mice had a small but significant QS decrease at 35 days compared to SHAM mice, confirmed by quantitative measurement of ossicle, marrow space, and new bone areas. The QS in rats was not affected by OVX but histomorphometry showed decreased new bone in OVX rats, which was restored by E2. The QS indicated that the number of new bone sites was not reduced by OVX in rats or mice at 56 days, but the relative amount of new bone v. marrow space was affected and differed with animal species. Residual DBM was less in OVX animals, indicating that DBM resorption was affected. Cartilage was present in rats but not in mice, suggesting that endochondral ossification was slower and indicating that bone graft studies in these species are not necessarily comparable. These results show the importance of E2 in human DBM-induced bone formation and suggest that E2 may be needed for clinical effectiveness in post-menopausal women.     

Changes in bone sclerostin levels in mice after ovariectomy vary independently of changes in serum sclerostin levels

We examined the effects that ovariectomy had on sclerostin mRNA and protein levels in the bones of 8‐week‐old mice that were either sham‐operated (SHAM) or ovariectomized (OVX) and then euthanized 3 or 6 weeks later. In this model, bone loss occurred between 3 and 5 weeks postsurgery. In calvaria, ovariectomy significantly decreased sclerostin mRNA levels at 6 weeks postsurgery (by 52%) but had no significant effect at 3 weeks. In contrast, sclerostin mRNA levels were significantly lower in OVX femurs at 3 weeks postsurgery (by 53%) but equal to that of SHAM at 6 weeks. The effects of ovariectomy on sclerostin were not a global response of osteocytes because they were not mimicked by changes in the mRNA levels for two other relatively osteocyte‐specific genes: DMP‐1 and FGF‐23. Sclerostin protein decreased by 83% and 60%, at 3 and 6 weeks postsurgery in calvaria, respectively, and by 38% in lumbar vertebrae at 6 weeks. We also detected decreases in sclerostin by immunohistochemistry in cortical osteocytes of the humerus at 3 weeks postsurgery. However, there were no significant effects of ovariectomy on sclerostin protein in femurs or on serum sclerostin at 3 and 6 weeks postsurgery. These results demonstrate that ovariectomy has variable effects on sclerostin mRNA and protein in mice, which are dependent on the bones examined and the time after surgery. Given the discrepancy between the effects of ovariectomy on serum sclerostin levels and sclerostin mRNA and protein levels in various bones, these results argue that, at least in mice, serum sclerostin levels may not accurately reflect changes in the local production of sclerostin in bones. Additional studies are needed to evaluate whether this is also the case in humans. © 2013 American Society for Bone and Mineral Research.     

局部单次注射辛伐他汀增强大鼠疏松骨骼内固定强度的实验研究

 目的 探讨疏松骨骼局部注射辛伐他汀对内固定强度的影响及其作用机制。方法 取3月龄雌性SD大鼠24只,切除双侧卵巢制备大鼠骨质疏松模型。3个月后将动物随机分为三组。右侧股骨髁植入钛合金螺钉后于髓腔内分别注射5 mg、10　mg辛伐他汀或空白PBS缓冲液。术后1个月行骨密度测定、螺钉周围骨微结构定量分析,测定螺钉最大载荷,通过免疫组织化学染色观察BMP-2的表达。结果 辛伐他汀5 mg组、10　mg组骨密度分别为(201±23.3)、(207.9±23.5) mg/cm²,与空白PBS缓冲液组(170.8±13.8) mg/cm²比较差异有统计学意义;螺钉骨整合率分别为51.4%±3.0%、52.6%±4.1%,较空白PBS缓冲液组(27.3%±4.9%)增加,螺钉周边各项骨微结构指标均改善,差异有统计学意义;螺钉的最大载荷分别为(161.5±9.4)、(161.9±11.4) N,高于空白PBS缓冲液组(145.7±9.6) N,差异有统计学意义;BMP-2的表达较空白PBS缓冲液组增强。结论 疏松骨骼局部单次注射小剂量辛伐他汀可促进内植物周边的骨整合,增强内固定的稳定性;其作用机制与局部注射辛伐他汀促进BMP-2的高表达有关。     

Combined treatment with PTH (1-34) and OPG increases bone volume and uniformity of mineralization in aged ovariectomized rats

The combination of PTH with OPG has been proposed as a potential therapy in patients with severe osteoporosis. In the present study, we examined the bone material of aged ovariectomized (OVX) rats treated either with PTH (1-34) or OPG alone or in combination of both. The micro- and nanostructural characteristics of the mineralized bone were evaluated using quantitative backscattered electron imaging (qBEI) and small-angle X-ray scattering (SAXS). Rats (n=68) were either sham-operated or ovariectomized (OVX) at the age of 3 months, and 15 months later, OVX animals were treated either with vehicle, OPG (10 mg/kg), PTH (80 microg/kg) or a combination of both during 5.5 months. All treatments were by subcutaneous injection, 3 days per week. Secondary metaphyseal spongiosa from distal femora was assessed for mineralized bone volume (BV/TV), for the mean Ca-concentration (Camean), the width of the bone mineralization density distribution (Cawidth), as well as the average mineral particle thickness parameter (T) and the degree of alignment of the mineral particles (rho). A remarkable increase of BV/TV up to 139% (P<0.001) was observed in the PTH-treated groups independently of OPG. Camean was slightly increased (+1.7%, P<0.05) in the OPG-treated group. Cawidth was reduced (-6.4%, P<0.01, and -8.9%, P<0.001) in animals treated with OPG and PTH+OPG, respectively. In contrast, Cawidth in sham-operated rats was 16.0% (P<0.001) higher than in OVX. The T parameter was not altered in the trabecular bone within the group of treated and untreated OVX rats. However, the non-ovariectomized animals exhibited a significantly lower T value (-7.1%, P<0.01) with respect to OVX. In conclusion, qBEI and SAXS data of OVX rats suggest that PTH alone was responsible for increase of bone volume, whereas OPG positively influenced the homogeneity and density of mineralization without affecting the nanostructure of the bone material.     

Genetic and hormonal control of bone volume,architecture, and remodeling in XXY mice

Klinefelter syndrome is the most common chromosomal aneuploidy in men (XXY karyotype, 1 in 600 live births) and results in testicular (infertility and androgen deficiency) and nontesticular (cognitive impairment and osteoporosis) deficits. The extent to which skeletal changes are due to testosterone deficiency or arise directly from gene overdosage cannot be determined easily in humans. To answer this, we generated XXY mice through a four‐generation breeding scheme. Eight intact XXY and 9 XY littermate controls and 8 castrated XXY mice and 8 castrated XY littermate controls were euthanized at 1 year of age. Castration occurred 6 months prior to killing. A third group of 9 XXY and 11 XY littermates were castrated and simultaneously implanted with a 1‐cm Silastic testosterone capsule 8 weeks prior to sacrifice. Tibias were harvested from all three groups and examined by micro–computed tomography and histomorphometry. Blood testosterone concentration was assayed by radioimmunoassay. Compared with intact XY controls, intact androgen‐deficient XXY mice had lower bone volume (6.8% ± 1.2% versus8.8% ± 1.7%, mean ± SD, p = .01) and thinner trabeculae (50 ± 4 µm versus 57 ± 5 µm, p = .007). Trabecular separation (270 ± 20 µm versus 270 ± 20 µm) or osteoclast number relative to bone surface (2.4 ± 1.0/mm² versus 2.7 ± 1.5/mm²) did not differ significantly. Testosterone‐replaced XXY mice continued to show lower bone volume (5.5% ± 2.4% versus 8.1% ± 3.5%, p = .026). They also exhibited greater trabecular separation (380 ± 69 µm versus 324 ± 62 µm, p = .040) but equivalent blood testosterone concentrations (6.3 ± 1.8 ng/mL versus 8.2 ± 4.2 ng/mL, p = .28) compared with testosterone‐replaced XY littermates. In contrast, castration alone drastically decreased bone volume (p < .001), trabecular thickness (p = .05), and trabecular separation (p < .01) to such a great extent that differences between XXY and XY mice were undetectable. In conclusion, XXY mice replicate many features of human Klinefelter syndrome and therefore are a useful model for studying bone. Testosterone deficiency does not explain the bone phenotype because testosterone‐replaced XXY mice show reduced bone volume despite similar blood testosterone levels. © 2010 American Society for Bone and Mineral Research.     

Comparison of Skeletal Effects of Ovariectomy Versus Chemically Induced Ovarian Failure in Mice

Bone loss associated with menopause leads to an increase in skeletal fragility and fracture risk. Relevant animal models can be useful for evaluating the impact of ovarian failure on bone loss. A chemically induced model of menopause in which mice gradually undergo ovarian failure yet retain residual ovarian tissue has been developed using the chemical 4‐vinylcyclohexene diepoxide (VCD). This study was designed to compare skeletal effects of VCD‐induced ovarian failure to those associated with ovariectomy (OVX). Young (28 day) C57Bl/6Hsd female mice were dosed daily with vehicle or VCD (160 mg/kg/d, IP) for 15 days (n = 6–7/group) and monitored by vaginal cytology for ovarian failure. At the mean age of VCD‐induced ovarian failure (～6 wk after onset of dosing), a different group of mice was ovariectomized (OVX, n = 8). Spine BMD (SpBMD) was measured by DXA for 3 mo after ovarian failure and OVX. Mice were killed ～5 mo after ovarian failure or OVX, and bone architecture was evaluated by μCT ex vivo. In OVX mice, SpBMD was lower than controls 1 mo after OVX, whereas in VCD‐treated mice, SpBMD was not lower than controls until 2.9 mo after ovarian failure (p < 0.05). Both VCD‐induced ovarian failure and OVX led to pronounced deterioration of trabecular bone architecture, with slightly greater effects in OVX mice. At the femoral diaphysis, cortical bone area and thickness did not differ between VCD mice and controls but were decreased in OVX compared with both groups (p < 0.05). Circulating androstenedione levels were preserved in VCD‐treated mice but reduced in OVX mice relative to controls (p < 0.001). These findings support that (1) VCD‐induced ovarian failure leads to trabecular bone deterioration, (2) bone loss is attenuated by residual ovarian tissue, particularly in diaphyseal cortical bone, and (3) the VCD mouse model can be a relevant model for natural menopause in the study of associated bone disorders.     

Effect of body weight on osteopenia in ovariectomized rats

Summary Bilateral ovariectomies or sham surgeries were performed in female Sprague Dawley rats that were 78 days of age and weighed an average of 210 g. Food was available ad libitum to the control rats and to a group of ovariectomized rats (obese OVX). The food consumption of a second group of ovariectomized rats (weight-matched OVX) was restricted to match their body weights to those of the control rats. All rats were sacrificed at 14 weeks postovariectomy. Radioimmunoassay of terminal serum estradiol confirmed the success of ovariectomy. The estradiol concentration in control rats was 24.9±20.2 pg/ml, whereas the hormone was undetectable (<10 pg/ml) in both groups of OVX rats. The final body weights of control and weight-matched OVX rats were nearly identical (∼260 g). In contrast, obese OVX rats weighed significantly more than both of the above groups (∼320 g,P<0.001). The proximal tibia and lumbar vertebra were processed undecalcified for quantitative bone histomorphometry. Tibial trabecular bone volume (TBV) was determined to be 17.6±4.5%, 7.9±5.3%, and 3.6±3.1% for the control, obese OVX, and weight-matched OVX groups, respectively. Tibial TBV for both OVX groups was significantly less than the control value (P<0.001). The difference in tibial TBV between obese OVX and weight-matched OVX rats was also statistically significant (P<0.02). Histologic indices of bone resorption and formation were indicative of increased bone turnover in the proximal tibia of both OVX groups. In comparison to control rats, both groups of OVX rats exhibited a strong trend for a reduction in vertebral TBV, but no significant differences were observed among the three groups. Our results suggest that increased body weight provides partial protection against osteopenia in the long bones of OVX rats. However, it is important to note that this protective effect is only partial and that marked osteopenia develops in the long bones of OVX rats regardless of body weight.     

Comparison of ex vivo and in vivo micro‐computed tomography of rat tibia at different scanning settings

The parameters of a micro‐computed tomography (μCT) scan, including whether a bone is imaged in vivo or ex vivo, determine the quality of the resulting image. In turn, this impacts the accuracy of the trabecular and cortical outcomes. The absolute impact of μCT scanning at different voxel sizes and whether the sample is imaged in vivo or ex vivo on the morphological outcomes of the proximal tibia in the rat is unknown. The right proximal tibia of 6‐month‐old Sham‐control and ovariectomized (OVX) rats (n = 8/group) was scanned using μCT (SkyScan 1176, Bruker, Kontich, Belgium) using three sets of parameters (9 μm ex vivo, 18 μm ex vivo, 18 μm in vivo) to compare the trabecular and cortical outcomes. Regardless of scan protocols, differences between Sham and OVX groups were observed as expected. At a voxel size of 18 μm, scanning in vivo or ex vivo had no effect on any of the outcomes measured. However, compared to a 9 μm voxel size scan, imaging at 18 μm resulted in significant underestimation of the connectivity density (p < 0.05) of the trabecular bone and a significant overestimation (p < 0.05) of the trabecular indices (trabecular thickness, degree of anisotropy) and of the cortical indices (cortical bone area, cortical area fraction, cortical thickness) in both Sham and OVX rats. These results suggest the benefit to scanning the proximal tibia of rats at a voxel size as low as 9 μm, although considerations must be made for the increased acquisition time, anesthesia, animal welfare, and radiation exposure associated with lower voxel size in vivo scanning. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1690–1698, 2017.

     

Unfocused extracorporeal shock wave therapy as potential treatment for osteoporosis

Extracorporeal shock wave therapy (ESWT) influences the differentiation of bone marrow stroma cells towards osteoprogenitors and increases the expression of several growth factors. To assess whether unfocused ESWT might serve as a treatment for osteoporosis, we examined the bone architecture dynamics of ESWT‐treated and untreated rat tibiae using in vivo micro‐computed tomography (CT) scanning. In addition, the effects of ESWT on fracture healing, using a bilateral fibula osteotomy, were examined. Unilateral unfocused ESWT with 2,000 pulses and an energy flux density of 0.16 mJ/mm² was applied to the hind leg of ovariectomized and sham‐ovariectomized rats. A single treatment with unfocused ESWT resulted in a higher trabecular bone volume fraction (BV/TV) in the proximal tibia of the sham‐ovariectomized animals. Three weeks after ESWT, BV/TV was 110% of baseline BV/TV in treated legs versus 101% in untreated contralateral control legs (p = 0.001) and 105% of baseline BV/TV versus 95% at 7 weeks after ESWT (p = 0.0004). In ovariectomized rats, shock wave treatment resulted in a diminished bone loss. At 7 weeks, the BV/TV of the treated legs was 50% of baseline BV/TV, whereas in untreated control legs this was 35% (p = 0.0004). ESWT did not influence acute fracture healing. This study shows that bone microarchitecture can be affected by unfocused shock waves, and indicates that unfocused ESWT might be useful for the treatment of osteopenia and osteoporosis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1528–1533, 2009