Search

Background: Latent TGFβ binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFβ, although it may interfere with the function of other LTBPs or interact with other signaling pathways., Results: Here, we investigate mice lacking Ltbp2 (Ltbp2 -/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype., Conclusions: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM., (© 2023 American Association for Anatomy.)

Introduction: Knowledge of bone structure-function relationships in mice has been based on relatively small sample sets that limit generalizability. We sought to investigate structure-function relationships of long bones from a large population of genetically diverse mice. Therefore, we analyzed previously published data from the femur and radius of male and female mice from the F34 generation of the Large-by-Small advanced intercross line (LGXSM AI), which have over a two-fold continuous spread of bone and body sizes (Silva et al. 2019 JBMR)., Methods: Morphological traits, mechanical properties, and estimated material properties were collected from the femur and radius from 1113 LGXSM AI adult mice (avg. age 25 wks). Males and females fed a low-fat or high-fat diet were evaluated to increase population variation. The data were analyzed using principal component analysis (PCA), Pearson's correlation, and multivariate linear regression., Results: Using PCA groupings and hierarchical clustering, we identified a reduced set of traits that span the population variation and are relatively independent of each other. These include three morphometry parameters (cortical area, medullary area, and length), two mechanical properties (ultimate force and post-yield displacement), and one material property (ultimate stress). When comparing traits of the femur to the radius, morphological traits are moderately well correlated (r 2 : 0.18-0.44) and independent of sex and diet. However, mechanical and material properties are weakly correlated or uncorrelated between the long bones. Ultimate force can be predicted from morphology with moderate accuracy for both long bones independent of variations due to genetics, sex, or diet; however, predictions miss up to 50 % of the variation in the population. Estimated material properties in the femur are moderately to strongly correlated with bone size parameters, while these correlations are very weak in the radius., Discussion: Our results indicate that variation in cortical bone phenotype in the F34 LGXSM AI mouse population can be adequately described by a reduced set of bone traits. These traits include cortical area, medullary area, bone length, ultimate force, post-yield displacement, and ultimate stress. The weak correlation of mechanical and material properties between the femur and radius indicates that the results from routine three-point bending tests of one long bone (e.g., femur) may not be generalizable to another long bone (e.g., radius). Additionally, these properties could not be fully predicted from bone morphology alone, confirming the importance of mechanical testing. Finally, material properties of the femur estimated based on beam theory equations showed a strong dependence on geometry that was not seen in the radius, suggesting that differences in femur size within a study may confound interpretation of estimated material properties., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nicole Migotsky: None Michael D. Brodt: None James M. Cheverud: None Matthew J. Silva COI: Editorial Board Bone, Calcified Tissue International, Journal of Orthopaedic Research, (© 2022 The Authors. Published by Elsevier Inc.)

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1β (IL-1β) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1β and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture., Competing Interests: KS, CW, JX, MB, LY, TY, YA, HH, DH, MS, JS No competing interests declared, YA Reviewing editor, eLife, GM Consultant for Aclaris Therapeutics, Inc, (© 2022, Sun et al.)

Wnt signaling is critical to many aspects of skeletal regulation, but the importance of Wnt ligands in the bone anabolic response to mechanical loading is not well established. Recent transcriptome profiling studies by our laboratory and others show that mechanical loading potently induces genes encoding Wnt ligands, including Wnt1 and Wnt7b. Based on these findings, we hypothesized that mechanical loading stimulates adult bone formation by inducing Wnt ligand expression. To test this hypothesis, we inhibited Wnt ligand secretion in adult (5 months old) mice using a systemic (drug) and a bone-targeted (conditional gene knockout) approach, and subjected them to axial tibial loading to induce lamellar bone formation. Mice treated with the Wnt secretion inhibitor WNT974 exhibited a decrease in bone formation in non-loaded bones as well as a 54% decline in the periosteal bone formation response to tibial loading. Next, osteoblast-specific Wnt secretion was inhibited by dosing 5-month-old Osx-CreERT2; Wls F/F mice with tamoxifen. Within 1 to 2 weeks of Wls deletion, skeletal homeostasis was altered with decreased bone formation and increased resorption, and the anabolic response to loading was reduced 65% compared to control (Wls F/F ). Together, these findings show that Wnt ligand secretion is required for adult bone homeostasis, and furthermore establish a role for osteoblast-derived Wnts in mediating the bone anabolic response to tibial loading. © 2021 American Society for Bone and Mineral Research (ASBMR)., (© 2021 American Society for Bone and Mineral Research (ASBMR).)

The murine tibia compression model, is the gold standard for studying bone adaptation due to mechanical loading in vivo. Currently, a key limitation of the experimental protocol and associated finite element (FE) models is that the exact load transfer, and consequently the loading conditions on the tibial plateau, is unknown. Often in FE models, load is applied to the tibial plateau based on inferences from micro-computed tomography (μCT). Experimental models often use a single strain gauge to assess the three-dimensional (3D) loading state. However, a single strain gauge is insufficient to validate such FE models. To address this challenge, we develop an experimentally calibrated method for identifying the load application region on the tibial plateau based upon measurements from three strain gauges. To achieve this, axial compression was conducted on mouse tibiae (n=3), with strains gauges on three surfaces. FE simulations were performed to compute the strains at the gauge locations as a function of a variable load location. By minimising the error between experimental and FE strains, the precise load location was identified; this was found to vary between tibia specimens. It was further shown that commonly used FE loading conditions, found in literature, did not replicate the experimental strain distribution, highlighting the importance of load calibration. This work provides critical insights into how load is transferred to the tibial plateau. Importantly, this work develops an experimentally informed technique for loading the tibial plateau in FE models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The purpose of this Chapter is to present a detailed description of methods for performing bone Micro-Computed Tomography (microCT) scanning and analysis. MicroCT is an x-ray imaging method capable of visualizing bone at the micro-structural scale, that is, 1-100 µm resolution. MicroCT is the gold-standard method for assessment of 3D bone morphology in studies of small animals. As applied to the small bones of mice or rats, microCT can efficiently and accurately assess bone structure (e.g., cortical bone area [Ct.Ar]) and micro-structure (e.g., trabecular bone volume fraction [Tb.BV/TV]). The particular application described herein is for post mortem mouse femur specimens. The material presented should be generally applicable to many commercially available laboratory microCT systems, although some details are specific to the system used in our lab (Scanco mCT 40; SCANCO Medical AG, Bruttisellen, Switzerland).

Following mechanical loading, osteoblasts may arise via activation, differentiation, or proliferation to form bone. Our objective was to ablate proliferating osteoblast lineage cells in order to investigate the importance of these cells as a source for loading-induced bone formation. We utilized 3.6Col1a1-tk mice in which replicating osteoblast lineage cells can be ablated in an inducible manner using ganciclovir (GCV). Male and female mice were aged to 5- and 12-months and subjected to 5 days of tibial compression. "Experimental" mice were tk-positive, treated with GCV; "control" mice were either tk-negative treated with GCV, or tk-positive treated with PBS. We confirmed that experimental mice had a decrease in tk-positive cells that arose from proliferation. Next, we assessed bone formation after loading to low (7N) and high (11N) forces and observed that periosteal bone formation rate in experimental mice was reduced by approximately 70% for both forces. Remarkably, woven bone formation induced by high-force loading was blocked in experimental mice. Loading-induced lamellar bone formation was diminished but not prevented in experimental mice. We conclude that osteoblast proliferation induced by mechanical loading is a critical source of bone forming osteoblasts for maximal lamellar formation and is essential for woven bone formation., (© 2020 Federation of American Societies for Experimental Biology.)

Mechanical loading is a potent strategy to induce bone formation, but with aging, the bone formation response to the same mechanical stimulus diminishes. Our main objectives were to (i) discover the potential transcriptional differences and (ii) compare the periosteal cell proliferation between tibias of young-adult and old mice in response to strain-matched mechanical loading. First, to discover potential age-related transcriptional differences, we performed RNA sequencing (RNA-seq) to compare the loading responses between tibias of young-adult (5-month) and old (22-month) C57BL/6N female mice following 1, 3, or 5 days of axial loading (loaded versus non-loaded). Compared to young-adult mice, old mice had less transcriptional activation following loading at each time point, as measured by the number of differentially expressed genes (DEGs) and the fold-changes of the DEGs. Old mice engaged fewer pathways and gene ontology (GO) processes, showing less activation of processes related to proliferation and differentiation. In tibias of young-adult mice, we observed prominent Wnt signaling, extracellular matrix (ECM), and neuronal responses, which were diminished with aging. Additionally, we identified several targets that may be effective in restoring the mechanoresponsiveness of aged bone, including nerve growth factor (NGF), Notum, prostaglandin signaling, Nell-1, and the AP-1 family. Second, to directly test the extent to which periosteal cell proliferation was diminished in old mice, we used bromodeoxyuridine (BrdU) in a separate cohort of mice to label cells that divided during the 5-day loading interval. Young-adult and old mice had an average of 15.5 and 16.7 BrdU+ surface cells/mm, respectively, suggesting that impaired proliferation in the first 5 days of loading does not explain the diminished bone formation response with aging. We conclude that old mice have diminished transcriptional activation following mechanical loading, but periosteal proliferation in the first 5 days of loading does not differ between tibias of young-adult and old mice. © 2020 American Society for Bone and Mineral Research., (© 2020 American Society for Bone and Mineral Research.)

Berardinelli-Seip congenital generalized lipodystrophy is associated with increased bone mass suggesting that fat tissue regulates the skeleton. Because there is little mechanistic information regarding this issue, we generated "fat-free" (FF) mice completely lacking visible visceral, subcutaneous and brown fat. Due to robust osteoblastic activity, trabecular and cortical bone volume is markedly enhanced in these animals. FF mice, like Berardinelli-Seip patients, are diabetic but normalization of glucose tolerance and significant reduction in circulating insulin fails to alter their skeletal phenotype. Importantly, the skeletal phenotype of FF mice is completely rescued by transplantation of adipocyte precursors or white or brown fat depots, indicating that adipocyte derived products regulate bone mass. Confirming such is the case, transplantation of fat derived from adiponectin and leptin double knockout mice, unlike that obtained from their WT counterparts, fails to normalize FF bone. These observations suggest a paucity of leptin and adiponectin may contribute to the increased bone mass of Berardinelli-Seip patients., Competing Interests: The authors have declared that no competing interests exist.

Obesity is generally protective against osteoporosis and bone fracture. However, recent studies indicate that the influence of obesity on the skeleton is complex and can be detrimental. We evaluated the effects of a high-fat, obesogenic diet on the femur and radius of 1100 mice (males and females) from the Large-by-Small advanced intercross line (F 34 generation). At age 5 months, bone morphology was assessed by microCT and mechanical properties by three-point bending. Mice raised on a high-fat diet had modestly greater cortical area, bending stiffness, and strength. Size-independent material properties were unaffected by a high-fat diet, indicating that diet influenced bone quantity but not quality. Bone size and mechanical properties were strongly correlated with body mass. However, the increases in many bone traits per unit increase in body mass were less in high-fat diet mice than low-fat diet mice. Thus, although mice raised on a high-fat diet have, on average, bigger and stronger bones than low-fat-fed mice, a high-fat diet diminished the positive relationship between body mass and bone size and whole-bone strength. The findings support the concept that there are diminishing benefits to skeletal health with increasing obesity. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)

Classic studies in bone mechanobiology have established the importance of loading parameters on the anabolic response. Most of these early studies were done using loading methods not currently in favor, and using non-murine species. Our objective was to re-examine the effects of several loading parameters on the response of cortical bone using the contemporary murine axial tibial compression model. We subjected tibias of 5-month old, female C57Bl/6 mice to cyclic (4 Hz) mechanical loading and examined bone formation responses using dynamic and static histomorphometry. First, using a reference protocol of 1,200 cycles/day, 5 days/week for 2 weeks, we confirmed the significant influence of peak strain magnitude on periosteal mineralizing surface (Ps.MS/BS) and bone formation rate (Ps.BFR/BS) (p < 0.05, ANOVA). There was a significant induction of periosteal lamellar bone at a lower threshold of approx. -1,000 μϵ and a transition from lamellar-woven bone near -2,000 μϵ. In contrast, on the endocortical surface, bone formation indices did not exhibit a load magnitude-dependent response and no incidence of woven bone. Next, we found that reducing daily cycle number from 1,200 to 300 to 60 did not diminish the bone formation response (p > 0.05). On the other hand, reducing the daily frequency of loading from 5 consecutive days/week to 3 alternate days/week significantly diminished the periosteal response, from a loading-induced increase in Ps.MS/BS of 38% (loaded vs. control) for 5 days/week to only 15% for 3 days/week (p < 0.05). Finally, we determined that reducing the study duration from 2 to 1 weeks of loading did not affect bone formation outcomes. In conclusion, cyclic loading to -1,800 μϵ peak strain, at 4 Hz and 60 cycles/day for 5 consecutive days (1 week) induces an increase in periosteal lamellar bone formation with minimal incidence of woven bone in 5-month-old C57Bl/6 female mice. Our results provide a basis for reduction of loading duration (daily cycles and study length) without loss of anabolic effect as measured by dynamic histomorphometry. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:682-691, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Aging diminishes bone formation engendered by mechanical loads, but the mechanism for this impairment remains unclear. Because Wnt signaling is required for optimal loading-induced bone formation, we hypothesized that aging impairs the load-induced activation of Wnt signaling. We analyzed dynamic histomorphometry of 5-month-old, 12-month-old, and 22-month-old C57Bl/6JN mice subjected to multiple days of tibial compression and corroborated an age-related decline in the periosteal loading response on day 5. Similarly, 1 day of loading increased periosteal and endocortical bone formation in young-adult (5-month-old) mice, but old (22-month-old) mice were unresponsive. These findings corroborated mRNA expression of genes related to bone formation and the Wnt pathway in tibias after loading. Multiple bouts (3 to 5 days) of loading upregulated bone formation-related genes, e.g., Osx and Col1a1, but older mice were significantly less responsive. Expression of Wnt negative regulators, Sost and Dkk1, was suppressed with a single day of loading in all mice, but suppression was sustained only in young-adult mice. Moreover, multiple days of loading repeatedly suppressed Sost and Dkk1 in young-adult, but not in old tibias. The age-dependent response to loading was further assessed by osteocyte staining for Sclerostin and LacZ in tibia of TOPGAL mice. After 1 day of loading, fewer osteocytes were Sclerostin-positive and, corroboratively, more osteocytes were LacZ-positive (Wnt active) in both 5-month-old and 12-month-old mice. However, although these changes were sustained after multiple days of loading in 5-month-old mice, they were not sustained in 12-month-old mice. Last, Wnt1 and Wnt7b were the most load-responsive of the 19 Wnt ligands. However, 4 hours after a single bout of loading, although their expression was upregulated threefold to 10-fold in young-adult mice, it was not altered in old mice. In conclusion, the reduced bone formation response of aged mice to loading may be due to failure to sustain Wnt activity with repeated loading. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Background: Reconstruction of iliosacral defects following oncologic resection is a difficult clinical problem associated with a high incidence of failure. Technical approaches to this problem are heterogeneous and evidence supporting specific techniques is sparse. Maximizing construct stability may improve union rates and functional outcomes. The purpose of this study is to compare construct stiffness, load to failure, and mechanism of failure between two methods of iliosacral reconstruction in an ex-vivo model to determine if either is mechanically superior., Methods: Eight third-generation composite pelves reconstructed with a plate-and-screw technique were tested against seven pelves reconstructed with a minimal spinal instrumentation technique using axial loading in a double-leg stance model., Findings: The pelves from the plate group demonstrated higher stiffness in the direction of applied load (102.9 vs. 66.8N/mm; p=0.010) and endured a significantly larger maximum force (1416 vs. 1059N; p=0.015) than the rod group prior to failure. Subjectively, the rod-reconstructed pelves were noted to be rotationally unconstrained while pivoting around their single point-of fixation in each segment leading to earlier failure., Interpretation: Plate-reconstruction was mechanically superior to spinal instrumentation in the manner performed in this study. More than one point of fixation in each segment should be achieved to minimize the risk of rotational deformation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aging purportedly diminishes the ability of the skeleton to respond to mechanical loading, but recent data show that old age did not impair loading-induced accrual of bone in BALB/c mice. Here, we hypothesized that aging limits the response of the tibia to axial compression over a range of adult ages in the commonly used C57BL/6. We subjected the right tibia of old (22 month), middle-aged (12 month) and young-adult (5 month) female C57BL/6 mice to peak periosteal strains (measured near the mid-diaphysis) of -2200 με and -3000 με (n=12-15/age/strain) via axial tibial compression (4 Hz, 1200 cycles/day, 5 days/week, 2 weeks). The left tibia served as a non-loaded, contralateral control. In mice of every age, tibial compression that engendered a peak strain of -2200 με did not alter cortical bone volume but loading to a peak strain of -3000 με increased cortical bone volume due in part to woven bone formation. Both loading magnitudes increased total volume, medullary volume and periosteal bone formation parameters (MS/BS, BFR/BS) near the cortical midshaft. Compared to the increase in total volume and bone formation parameters of 5-month mice, increases were less in 12- and 22-month mice by 45-63%. Moreover, woven bone incidence was greatest in 5-month mice. Similarly, tibial loading at -3000 με increased trabecular BV/TV of 5-month mice by 18% (from 0.085 mm3/mm3), but trabecular BV/TV did not change in 12- or 22-month mice, perhaps due to low initial BV/TV (0.032 and 0.038 mm3/mm3, respectively). In conclusion, these data show that while young-adult C57BL/6 mice had greater periosteal bone formation following loading than middle-aged or old mice, aging did not eliminate the ability of the tibia to accrue cortical bone., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Axial compression of the mouse tibia is used to study strain-adaptive bone (re)modeling. In some studies, comparisons between mice of different ages are of interest. We characterized the tibial deformation and force-strain relationships in female C57Bl/6 mice at 5-, 12- and 22-months age. A three-gauge experimental method was used to determine the strain distribution at the mid-diaphysis, while specimen-specific finite element analysis was used to examine strain distribution along the tibial length. The peak strains in the tibial mid-diaphyseal cross-section are compressive and occur at the postero-lateral apex. The magnitudes of these peak compressive strains are 1.5 to 2 times those on the opposite, antero-medial face (a site often used for strain gauge placement). For example, -10 N force applied to a 5-months old mouse engenders a peak compressive strain of -2800 µε and a tensile strain on the antero-medial face of +1450 µε. The orientation of the neutral axis at the mid-diaphysis did not differ with age (p=0.46), indicating a similar deformation mode in young and old tibiae. On the other hand, from 5- to 22-months there is a 25% reduction in cortical thickness and moment of inertia (p<0.05), resulting in significantly greater tibial strain magnitudes in older mice for equivalent applied force (p<0.05). We conclude that comparisons of tibial loading responses in young-adult and old C57Bl/6 tibiae are facilitated by similar deformation pattern across ages, but that modest adjustment of force levels is required to engender matching peak strains., (© 2013 Elsevier Ltd. All rights reserved.)

Tibial compression can increase murine bone mass. However, loading protocols and mouse strains differ between studies, which may contribute to conflicting results. We hypothesized that bone accrual is influenced more by loading history than by mouse strain or animal handling. The right tibiae of 4-month-old C57BL/6 and BALB/c mice were subjected to axial compression (10 N, 3 days/week, 6 weeks). Left tibiae served as contralateral controls to calculate relative changes: (loaded - control)/control. The WashU protocol applied 60 cycles/day, at 2 Hz, with a 10-s rest-insertion between cycles; the Cornell/HSS protocol applied 1,200 cycles/day, at 6.7 Hz, with a 0.1-s rest-insertion. Because sham loading, sedation, and transportation did not affect tibial morphology, unhandled mice served as age-matched controls (AC). Both loading protocols were anabolic for cortical bone, but Cornell/HSS loading elicited a more rapid response that was greater than WashU loading by 13 %. By 6 weeks, cortical bone volume of each loading group was greater than of AC (average + 16 %) and not different from each other. Ultimate displacement and energy to fracture were greater in tibiae loaded by either protocol, and ultimate force was greater with Cornell/HSS loading. At 6 weeks, independent of mouse strain, the WashU protocol produced minimal trabecular bone and the trabecular bone volume fraction of Cornell/HSS tibiae was greater than that of AC by 65 % and that of WashU by 44 %. We concluded that tibial adaptation to loading was more influenced by waveform than mouse strain or animal handling and therefore may have targeted similar osteogenic mechanisms in C57BL/6 and BALB/c mice.

Purpose: Volar plating for distal radius fractures has caused extensor tendon ruptures resulting from dorsal screw prominence. This study was designed to determine the biomechanical impact of placing unicortical distal locking screws and pegs in an extra-articular fracture model., Methods: We applied volar-locking distal radius plates to 30 osteoporotic distal radius models. We divided radiuses into 5 groups based on distal locking fixation: bicortical locked screws, 3 lengths of unicortical locked screws (abutting the dorsal cortex [full length], 75% length, and 50% length to dorsal cortex), and unicortical locked pegs. Distal radius osteotomy simulated a dorsally comminuted, extra-articular fracture. We determined each construct's stiffness under physiologic loads (axial compression, dorsal bending, and volar bending) before and after 1,000 cycles of axial conditioning and before axial loading to failure (2 mm of displacement) and subsequent catastrophic failure., Results: Cyclic conditioning did not alter the constructs' stiffness. Stiffness to volar bending and dorsal bending forces were similar between groups. Final stiffness under axial load was statistically equivalent for all groups: bicortical screws (230 N/mm), full-length unicortical screws (227 N/mm), 75% length unicortical screws (226 N/mm), 50% length unicortical screws (187 N/mm), and unicortical pegs (226 N/mm). Force at 2-mm displacement was significantly less for 50% length unicortical screws (311 N) compared with bicortical screws (460 N), full-length unicortical screws (464 N), 75% length unicortical screws (400 N), and unicortical pegs (356 N). Force to catastrophic fracture was statistically equivalent between groups, but mean values for pegs (749 N) and 50% length unicortical (702 N) screws were 16% to 21% less than means for bicortical (892 N), full-length unicortical (860 N), and 75% length (894 N) unicortical constructs., Conclusions: Locked unicortical distal screws of at least 75% length produce construct stiffness similar to bicortical fixation. Unicortical distal fixation for extra-articular distal radius fractures should be entertained to avoid extensor tendon injury because this technique does not appear to compromise initial fixation., Clinical Relevance: Using unicortical fixation during volar distal radius plating may protect extensor tendons without compromising fixation., (Copyright © 2012 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

The gap junction protein, connexin43 (Cx43) is involved in mechanotransduction in bone. Recent studies using in vivo models of conditional Cx43 gene (Gja1) deletion in the osteogenic linage have generated inconsistent results, with Gja1 ablation resulting in either attenuated or enhanced response to mechanical load, depending upon the skeletal site examined or the type of load applied. To gain further insights on Cx43 and mechanotransduction, we examined bone formation response at both endocortical and periosteal surfaces in 2-month-old mice with conditional Gja1 ablation driven by the Dermo1 promoter (cKO). Relative to wild type (WT) littermates, it requires a larger amount of compressive force to generate the same periosteal strain in cKO mice. Importantly, cKO mice activate periosteal bone formation at a lower strain level than do WT mice, suggesting an increased sensitivity to mechanical load in Cx43 deficiency. Consistently, trabecular bone mass also increases in mutant mice upon load, while it decreases in WT. On the other hand, bone formation actually decreases on the endocortical surface in WT mice upon application of axial mechanical load, and this response is also accentuated in cKO mice. These changes are associated with increase of Cox-2 in both genotypes and further decrease of Sost mRNA in cKO relative to WT bones. Thus, the response of bone forming cells to mechanical load differs between trabecular and cortical components, and remarkably between endocortical and periosteal envelopes. Cx43 deficiency enhances both the periosteal and endocortical response to mechanical load applied as axial compression in growing mice.

There are conflicting data on whether age reduces the response of the skeleton to mechanical stimuli. We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months (p<0.001). There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months (p<0.001). We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 µε endocortical; -2350 µε periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls (p = 0.001). In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias (p<0.05), due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%; p<0.05 vs. other ages). In summary, non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice. We conclude that mechanical loading of the tibia is anabolic for cortical bone in young and middle-aged female BALB/c mice.

We have shown previously that the effect of mechanical loading on bone depends in part on connexin43 (Cx43). To determine whether Cx43 is also involved in the effect of mechanical unloading, we have used botulinum toxin A (BtxA) to induce reversible muscle paralysis in mice with a conditional deletion of the Cx43 gene in osteoblasts and osteocytes (cKO). BtxA injection in hind limb muscles of wild-type (WT) mice resulted in significant muscle atrophy and rapid loss of trabecular bone. Bone loss reached a nadir of about 40% at 3 weeks after injection, followed by a slow recovery. A similar degree of trabecular bone loss was observed in cKO mice. By contrast, BtxA injection in WT mice significantly increased marrow area and endocortical osteoclast number and decreased cortical thickness and bone strength. These changes did not occur in cKO mice, whose marrow area is larger, osteoclast number higher, and cortical thickness and bone strength lower relative to WT mice in basal conditions. Changes in cortical structure occurring in WT mice had not recovered 19 weeks after BtxA injection despite correction of the early osteoclast activation and a modest increase in periosteal bone formation. Thus BtxA-induced muscle paralysis leads to rapid loss of trabecular bone and to changes in structural and biomechanical properties of cortical bone, neither of which are fully reversed after 19 weeks. Osteoblast/osteocyte Cx43 is involved in the adaptive responses to skeletal unloading selectively in the cortical bone via modulation of osteoclastogenesis on the endocortical surface., (Copyright © 2011 American Society for Bone and Mineral Research.)

Purpose: To evaluate the biocompatibility of heavyweight polypropylene (HWPP), lightweight polypropylene (LWPP), and monofilament knit polytetrafluoroethylene (mkPTFE) mesh by comparing biomechanics and histologic response at 1, 3, and 5 months in a porcine model of incisional hernia repair., Methods: Bilateral full-thickness abdominal wall defects measuring 4 cm in length were created in 27 Yucatan minipigs. Twenty-one days after hernia creation, animals underwent bilateral preperitoneal ventral hernia repair with 8 × 10 cm pieces of mesh. Repairs were randomized to Bard(®)Mesh (HWPP, Bard/Davol, http://www.davol.com), ULTRAPRO(®) (LWPP, Ethicon, http://www.ethicon.com), and GORE(®)INFINIT Mesh (mkPTFE, Gore & Associates, http://www.gore.com). Nine animals were sacrificed at each timepoint (1, 3, and 5 months). At harvest, a 3 × 4 cm sample of mesh and incorporated tissue was taken from the center of the implant site and subjected to uniaxial tensile testing at a rate of 0.42 mm/s. The maximum force (N) and tensile strength (N/cm) were measured with a tensiometer, and stiffness (N/mm) was calculated from the slope of the force-versus-displacement curve. Adjacent sections of tissue were stained with hematoxylin and eosin (H&E) and analyzed for inflammation, fibrosis, and tissue ingrowth. Data are reported as mean ± SEM. Statistical significance (P < 0.05) was determined using a two-way ANOVA and Bonferroni post-test., Results: No significant difference in maximum force was detected between meshes at any of the time points (P > 0.05 for all comparisons). However, for each mesh type, the maximum strength at 5 months was significantly lower than that at 1 month (P < 0.05). No significant difference in stiffness was detected between the mesh types or between timepoints (P > 0.05 for all comparisons). No significant differences with regard to inflammation, fibrosis, or tissue ingrowth were detected between mesh types at any time point (P > 0.09 for all comparisons). However, over time, inflammation decreased significantly for all mesh types (P < 0.001) and tissue ingrowth reached a slight peak between 1 and 3 months (P = 0.001) but did not significantly change thereafter (P > 0.09)., Conclusions: The maximum tensile strength of mesh in the abdominal wall decreased over time for HWPP, LWPP, and mkPTFE mesh materials alike. This trend may actually reflect inability to adequately grip specimens at later time points rather than any mesh-specific trend. Histologically, inflammation decreased with time (P = 0.000), and tissue ingrowth increased (P = 0.019) for all meshes. No specific trends were observed between the polypropylene meshes and the monofilament knit PTFE, suggesting that this novel construction may be a suitable alternative to existing polypropylene meshes.

Purpose: Biologic meshes have unique physical properties as a result of manufacturing techniques such as decellularization, crosslinking, and sterilization. The purpose of this study is to directly compare the biocompatibility profiles of five different biologic meshes, AlloDerm(®) (non-crosslinked human dermal matrix), PeriGuard(®) (crosslinked bovine pericardium), Permacol(®) (crosslinked porcine dermal matrix), Strattice(®) (non-crosslinked porcine dermal matrix), and Veritas(®) (non-crosslinked bovine pericardium), using a porcine model of ventral hernia repair., Methods: Full-thickness fascial defects were created in 20 Yucatan minipigs and repaired with the retromuscular placement of biologic mesh 3 weeks later. Animals were euthanized at 1 month and the repair sites were subjected to tensile testing and histologic analysis. Samples of unimplanted (de novo) meshes and native porcine abdominal wall were also analyzed for their mechanical properties., Results: There were no significant differences in the biomechanical characteristics between any of the mesh-repaired sites at 1 month postimplantation or between the native porcine abdominal wall without implanted mesh and the mesh-repaired sites (P > 0.05 for all comparisons). Histologically, non-crosslinked materials exhibited greater cellular infiltration, extracellular matrix (ECM) deposition, and neovascularization compared to crosslinked meshes., Conclusions: While crosslinking differentiates biologic meshes with regard to cellular infiltration, ECM deposition, scaffold degradation, and neovascularization, the integrity and strength of the repair site at 1 month is not significantly impacted by crosslinking or by the de novo strength/stiffness of the mesh.

Whole-body vibration (WBV) is a low-magnitude mechanical stimulus that may be anabolic for bone, yet we recently found that WBV did not improve bone properties in adult mice. Because intermittent parathyroid hormone (PTH) enhances the anabolic effects of high-magnitude skeletal loading, we sought to determine the skeletal effects of WBV in combination with PTH. Seven-month-old male BALB/c mice were assigned to six groups (n = 13-14/group) based on magnitude of applied acceleration (0 or 0.3 G) and PTH dose (0, 10, or 40 µg/kg/day). Mice were exposed to WBV (0.3 G, 90 Hz, sine wave) or sham loading (0 G) for 15 min/day, 5 days/week for 8 weeks. Vehicle or hPTH (1-34) was administered prior to each WBV session. Whole-body bone mineral content increased by ~ 5% from 0 to 8 weeks in the 40 µg/kg PTH group only, independent of WBV loading. Similarly, PTH treatment increased tibial cortical bone volume by ~5% from 0 to 8 weeks, independent of WBV loading. Neither PTH nor WBV stimulated trabecular bone formation. Consistent with the cortical bone effect, tibias from the 40 µg/kg PTH group had significantly greater ultimate force and energy to failure than tibias in the 0 and 10 µg/kg PTH groups, independent of WBV treatment. In summary, 8 weeks of intermittent PTH treatment increased cortical bone volume and strength in adult male BALB/c mice. Daily exposure to low-magnitude WBV by itself did not improve skeletal properties and did not enhance the PTH effect. No WBV-PTH synergy was found in this preclinical study., (Copyright © 2010 Orthopaedic Research Society.)

Background: The purpose of this comparative study is to evaluate the acute fixation strength of mechanical tacking devices and fibrin sealant against polypropylene suture for laparoscopic ventral hernia repair., Methods: Three metallic mechanical tacking devices (ProTack, Salute, EndoANCHOR), 4 absorbable tacking devices (AbsorbaTack, PermaSorb, I-Clip, and SorbaFix), and 2 types of fibrin sealant (Tisseel, Artiss) were compared with 0-polypropylene suture. Three constructs from each device or an amount of sealant sufficient to cover a 3 × 3 cm(2) area were used to affix a 4 × 3 cm piece of absorbable barrier-coated mesh (Proceed, Ethicon, Inc) to the peritoneal surface of porcine abdominal wall. Ten samples were completed for each fixation modality. Acute fixation strength was measured via a lap shear test on an Instron tensiometer., Results: Acute fixation strength was significantly greater for suture (59.7 7.2 N) compared with all laparoscopic tacking devices and to fibrin sealant (P < .001 for all comparisons). Protack (29.5 ± 2.8 N) was stronger than Absorbatack (13.2 ± 3.7 N; P = .029). Protack, Permasorb, SorbaFix, and I-clip were stronger than fibrin sealant (P < .05 for all comparisons)., Conclusions: The acute fixation strengths of metallic or absorbable tacks as well as fibrin sealant are all significantly less than that achieved with polypropylene suture. These factors should be considered in selecting the type of mechanical fixation for patients undergoing laparoscopic ventral hernia repair.

With aging, the skeleton may lose its ability to respond to positive mechanical stimuli. We hypothesized that aged mice are less responsive to loading than young-adult mice. We subjected aged (22 months) and young-adult (7 months) BALB/c male mice to daily bouts of axial tibial compression for 1 week and evaluated cortical and trabecular responses using micro-computed tomography (µCT) and dynamic histomorphometry. The right legs of 95 mice were loaded for 60 rest-inserted cycles per day to 8, 10, or 12 N peak force (generating mid-diaphyseal strains of 900 to 1900 µε endocortically and 1400 to 3100 µε periosteally). At the mid-diaphysis, mice from both age groups showed a strong anabolic response on the endocortex (Ec) and periosteum (Ps) [Ec.MS/BS and Ps.MS/BS: loaded (right) versus control (left), p < .05]. Generally, bone formation increased with increasing peak force. At the endocortical surface, contrary to our hypothesis, aged mice had a significantly greater response to loading than young-adult mice (Ec.MS/BS and Ec.BFR/BS: 22 months versus 7 months, p < .001). Responses at the periosteal surface did not differ between age groups (p > .05). The loading-induced increase in bone formation resulted in increased cortical area in both age groups (loaded versus control, p < .05). In contrast to the strong cortical response, loading only weakly stimulated trabecular bone formation. Serial (in vivo) µCT examinations at the proximal metaphysis revealed that loading caused a loss of trabecular bone in 7-month-old mice, whereas it appeared to prevent bone loss in 22-month-old mice. In summary, 1 week of daily tibial compression stimulated a robust endocortical and periosteal bone-formation response at the mid-diaphysis in both young-adult and aged male BALB/c mice. We conclude that aging does not limit the short-term anabolic response of cortical bone to mechanical stimulation in our animal model., (© 2010 American Society for Bone and Mineral Research.)

MAGP1 is an extracellular matrix protein that, in vertebrates, is a ubiquitous component of fibrillin-rich microfibrils. We previously reported that aged MAGP1-deficient mice (MAGP1Delta) develop lesions that are the consequence of spontaneous bone fracture. We now present a more defined bone phenotype found in MAGP1Delta mice. A longitudinal DEXA study demonstrated age-associated osteopenia in MAGP1Delta animals and muCT confirmed reduced bone mineral density in the trabecular and cortical bone. Further, MAGP1Delta mice have significantly less trabecular bone, the trabecular microarchitecture is more fragmented, and the diaphyseal cross-sectional area is significantly reduced. The remodeling defect seen in MAGP1Delta mice is likely not due to an osteoblast defect, because MAGP1Delta bone marrow stromal cells undergo osteoblastogenesis and form mineralized nodules. In vivo, MAGP1Delta mice exhibit normal osteoblast number, mineralized bone surface, and bone formation rate. Instead, our findings suggest increased bone resorption is responsible for the osteopenia. The number of osteoclasts derived from MAGP1Delta bone marrow macrophage cells is increased relative to the wild type, and osteoclast differentiation markers are expressed at earlier time points in MAGP1Delta cells. In vivo, MAGP1Delta mice have more osteoclasts lining the bone surface. RANKL (receptor activator of NF-kappaB ligand) expression is significantly higher in MAGP1Delta bone, and likely contributes to enhanced osteoclastogenesis. However, bone marrow macrophage cells from MAGP1Delta mice show a higher propensity than do wild-type cells to differentiate to osteoclasts in response to RANKL, suggesting that they are also primed to respond to osteoclast-promoting signals. Together, our findings suggest that MAGP1 is a regulator of bone remodeling, and its absence results in osteopenia associated with an increase in osteoclast number.

Mice may be useful for studies of skeletal aging, but there are limited data on changes in bone structure and strength over their life span. We obtained bones from female and male BALB/c mice at ages 2, 4, 7, 12, and 20 months and evaluated their structural, densitometric, and mechanical properties. MicroCT of the mid-diaphysis of the femur and radius indicated that during skeletal growth (2-7 months) bone cross-sectional size (area, moment of inertia) increased rapidly; during aging (7-20 months) cortical area was maintained, while moment of inertia continued to increase. Bones from females were smaller than those from males at young ages but not at later ages. Changes in whole-bone stiffness and strength reflected the changes in bone size, with a rapid increase from 2 to 7 months, followed by little or no change. In contrast, energy-to-fracture declined with aging. Cortical tissue mineral density increased during growth and was maintained with aging. MicroCT of trabecular bone revealed age-related changes that were site-dependent. The proximal tibia showed a clear pattern of age-related decline in trabecular BV/TV, with progressive decreases after 4 months in both sexes; lumbar vertebra L5 had more modest age-related declines; in contrast, caudal vertebra Ca7 had increasing BV/TV with aging. Overall, we found no evidence that females had more pronounced age-related deterioration than males. We conclude that bones from aging female and male BALB/c mice exhibit many of the changes seen in humans and are therefore a clinically relevant model for studies of skeletal aging.

Low-amplitude, whole-body vibration (WBV) may be anabolic for bone. Animal studies of WBV have not evaluated skeletal effects in aged animals. We exposed 75 male BALB/c mice (7 month/young-adult; 22 month/aged) to 5 weeks of daily WBV (15 min/day, 5 day/wk; 90 Hz sine wave) at acceleration amplitudes of 0 (sham), 0.3, or 1.0 g. Whole-body bone mineral content (BMC) increased with time in 7 month (p < 0.001) but not 22 month (p = 0.34) mice, independent of WBV (p = 0.60). In 7 month mice, lower-leg BMC increased with time in 0.3 and 1.0 g groups (p < 0.005) but not in the sham group (p = 0.09), indicating a positive WBV effect. In 22 month mice, there were no changes with time in lower-leg BMC (p = 0.11). WBV did not affect tibial trabecular or cortical bone structure (by microCT), dynamic indices of trabecular or cortical bone formation, trabecular osteoclast surface, or the mass of the reproductive fat pad (p > 0.05). Each of these outcomes was diminished in 7 month versus 22 month animals (p < 0.05). In summary, 5 weeks of daily exposure to low-amplitude WBV had no skeletal effects in aged male mice. The potential of WBV to enhance bone mass in age-related osteoporosis is not supported in this preclinical study., ((c) 2009 Orthopaedic Research Society.)

People with diabetes have increased risk of fracture disproportionate to BMD, suggesting reduced material strength (quality). We quantified the skeletal effects of type 1 diabetes in the rat. Fischer 344 and Sprague-Dawley rats (12 wk of age) were injected with either vehicle (Control) or streptozotocin (Diabetic). Forelimbs were scanned at 0, 4, 8, and 12 wk using pQCT. Rats were killed after 12 wk. We observed progressive osteopenia in diabetic rats. Trabecular osteopenia was caused by bone loss: volumetric BMD decreased progressively with time in diabetic rats but was constant in controls. Cortical osteopenia was caused by premature arrest of cortical expansion: cortical area did not increase after 4-8 wk in diabetic rats but continued to increase in controls. Postmortem muCT showed a 60% reduction in proximal tibial trabecular BV/TV in diabetic versus control rats, whereas moments of inertia of the ulnar and femoral diaphysis were reduced approximately 30%. Monotonic bending tests indicated that ulna and femora from diabetic animals were approximately 25% less stiff and strong versus controls. Estimates of material properties indicated no changes in elastic modulus or ultimate stress but modest ( approximately 10%) declines in yield stress for diabetic bone. These changes were associated with a approximately 50% increase in the nonenzymatic collagen cross-link pentosidine. Last, cyclic testing showed diminished fatigue life in diabetic bones at the structural (force) level but not at the material (stress) level. In summary, type 1 diabetes, left untreated, causes trabecular bone loss and a reduction in diaphyseal growth. Diabetic bone has greatly increased nonenzymatic collagen cross-links but only modestly reduced material properties. The loss of whole bone strength under both monotonic and fatigue loading is attributed mainly to reduced bone size.

The purpose of this study was to determine the biomechanical characteristics of 16 arthroscopic knots and to determine if locking knots have superior loop security compared to non-locking knots. Sixteen knot types were tied in arthroscopic fashion and tested on a materials testing system. Knots were cyclically loaded to 30 Newtons (N) for 20 cycles and then loaded to failure at 1.25 mm/s. Ten samples of each knot were tied using both #2 Ethibond and #1 PDS II. Load to ultimate failure, load to clinical failure, post-cyclic stiffness, cyclical elongation, ultimate displacement, loop security, and mode of failure were determined for each knot. Nicky's Knot and the French Knot were most consistently ranked within the top five knot types for each of the biomechanical parameters. Locking knots did not improve loop security over non-locking knots.

Introduction: In vitro data suggest that gap junctional intercellular communication mediated by connexin43 (Cx43) plays an important role in bone cell response to mechanical stimulation. We tested this hypothesis in vivo in a model of genetic deficiency of the Cx43 gene (Gja1)., Materials and Methods: Four-month-old female mice with a conditional Gja1 ablation in osteoblasts (ColCre;Gja1(-/flox)), as well as wildtype (Gja1(+/flox)) and heterozygous equivalent (Gja1(-/flox)) littermates (eight per genotype), were subjected to a three-point bending protocol for 5 d/wk for 2 wk. Microstructural parameters and dynamic indices of bone formation were estimated on sections of loaded and control contralateral tibias., Results: ColCre;Gja1(-/flox) mice had significantly thinner cortices, but larger marrow area and total cross-sectional area in the tibial diaphysis, compared with the other groups. The ColCre;Gja1(-/flox) mice needed approximately 40% more force to generate the required endocortical strain. In Gja1(+/flox) mice, the loading regimen produced abundant double calcein labels at the endocortical surface, whereas predominantly single labels were seen in ColCre;Gja1(-/flox) mice. Accordingly, mineral apposition rate and bone formation rate were significantly lower (54.8% and 50.2%, respectively) in ColCre;Gja1(-/flox) relative to Gja1(+/flox) mice. Intermediate values were found in Gja1(-/flox) mice., Conclusions: Gja deficiency results in thinner but larger tibial diaphyses, resembling changes occurring with aging, and it attenuates the anabolic response to in vivo mechanical loading. Thus, Cx43 plays an instrumental role in this adaptive response to physical stimuli.

With aging, the skeleton may have diminished responsiveness to mechanical stimulation. The senescence-accelerated mouse SAMP6 has many features of senile osteoporosis and is thus a useful model to examine how the osteoporotic skeleton responds to mechanical loading. We performed in vivo tibial bending on 4-month-old SAMP6 (osteoporotic) and SAMR1 (control) mice. Loading was applied daily (60 cycles/day, 5 days/week) for 2 weeks at peak force levels that produced estimated endocortical strains of 1,000 and 2,000 microepsilon In a separate group of mice, sham bending was applied. Comparisons were made between right (loaded) and left (nonloaded) tibiae. Tibial bone marrow cells were cultured under osteogenic conditions and stained for alkaline phosphatase (ALP) and alizarin red (ALIZ) at 14 and 28 days, respectively. Tibiae were then embedded in plastic and sectioned, and endocortical bone formation was assessed based on calcein labels. Tibial bending did not alter the osteogenic potential of the marrow as there were no significant differences in ALP or ALIZ staining between loaded and nonloaded bones. Tibial bending activated the formation of endocortical bone in both SAMP6 and SAMR1 mice, whereas sham bending did not elicit an endocortical response. Both groups of mice exhibited bending strain-dependent increases in bone formation rate. We found little evidence of diminished responsiveness to loading in the SAMP6 skeleton. In conclusion, the ability of the SAMP6 mouse to respond normally to an anabolic mechanical stimulus distinguishes it from chronologically aged animals. This finding highlights a limitation of the SAMP6 mouse as a model of senile osteoporosis.

Medial collateral ligament (MCL) injuries heal by a wound repair scar response controlled by a complex cellular and cytokine environment. Many enzymes participate in wound repair, particularly the matrix metalloproteinases. We hypothesize macrophage metalloelastase (MME/MMP-12) deficiency results in impaired healing of MCL injury. One hundred fifty MME-deficient and 150 WT (MME+/+) mice underwent knee MCL transection with the opposite knee as a sham operated control. Mice were sacrificed at 3, 7, 28, 42, and 56 days. At each of the five time points, 15 mice were utilized for biological and 15 were utilized for biomechanical testing. Outcome measures were the presence of macrophages to represent the inflammatory phase of wound healing, collagen synthesis to assay for matrix repair, and biomechanical testing for repair strength. Immunohistochemistry demonstrated significantly fewer macrophages in cut MCLs from MME-deficient mice versus wild-type (WT) mice at 3, 7, 28, and 42 days (all p

Background: Elbow medial ulnar collateral ligament tears often result in pain and instability that may be career threatening in overhead-throwing athletes. Surgical reconstruction is frequently chosen to treat this injury. Ulnar collateral ligament reconstruction as described by Jobe is the most commonly used technique. Testing of this construct has not demonstrated that the biomechanical parameters of the native ligament are restored. A more recent construct, the docking technique, may more reliably reproduce these factors., Hypothesis: Increasing the number of strands of palmaris longus tendon graft used in ulnar collateral ligament reconstruction and tensioning them using the docking technique result in a construct with improved biomechanical parameters as compared with the Jobe technique., Study Design: Controlled laboratory study., Methods: Thirty-three fresh-frozen human cadaveric elbows were randomized into 3 subgroups: Jobe (11), docking (12), and native (10). The Jobe and docking groups underwent reconstruction using their described palmaris tendon graft constructs. The ulnar collateral ligament was left intact in the native group. Elbows were potted and tested using a servohydraulic materials testing machine to apply a valgus moment at 30 degrees of elbow flexion. Maximal moments to failure, stiffness, and strain at maximal moment and with a 3 N.m force applied were determined using a 2-camera motion analysis system to track reflective markers spanning the site., Results: The docking (14.3 N.m) and native (18.8 N.m) subgroups resulted in higher maximal moment to failure than did the Jobe (8.9 N.m) subgroup (P < .001). There was no significant difference between native and docking groups (P > .05). Native ligaments were stiffer (301.4 N.m) than were Jobe (74.3 N.m) or docking (80.8 N.m; P < .001). Native ligaments demonstrated lower strain at maximal force (0.087 mm/mm) and 3 N.m forces (0.030 mm/mm) than did the Jobe (0.198/0.057 mm/mm) or docking (0.287/0.042 mm/mm) subgroups. There was no difference in stiffness or strain between the Jobe and docking subgroups (P > .05)., Conclusion: Neither technique reproduced the biomechanical profile of the native ulnar collateral ligament; the findings of this study suggest that the docking construct may offer initial biomechanical advantage over the Jobe construct.

Gap junctions are hexameric transmembrane channels formed by connexins, and are responsible for direct cell-to-cell communication. The most abundant gap junction protein in bone is connexin43 (Cx43), although connexin45 (Cx45) is also expressed. In the present study, we tested the hypothesis that bone cell responses to mechanical stimulation are dependent on the type of gap junction communication provided by Cx43 in vitro and in an in vivo model of physical load. Application of cyclic stretch to calvaria osteoblasts results in a modest but detectable increase in PGE2 levels, and the amount of PGE2 produced was lower in cells isolated from Cx43 null mice. Mice with an osteoblast-specific deletion of the Cx43 gene were subjected to an in vivo four-point bending protocol on the tibia. This resulted in fast and exuberant formation of woven bone at the region directly below the loading fulcrum in both osteoblast Cx43-deleted and wild-type mice. However, indirect measurement of endosteal bone apposition suggested a less pronounced effect of physical load in Cx43-deficient than in wild-type mice. Taken together, these results indicate that deficiency of Cx43 in osteoblasts attenuates but does not abolish anabolic responses to mechanical strain.

Unlabelled: To examine the link between bone material properties and skeletal fragility, we analyzed the mechanical, histological, biochemical, and spectroscopic properties of bones from a murine model of skeletal fragility (SAMP6). Intact bones from SAMP6 mice are weak and brittle compared with SAMR1 controls, a defect attributed to reduced strength of the bone matrix. The matrix weakness is attributed primarily to poorer organization of collagen fibers and reduced collagen content., Introduction: The contribution of age-related changes in tissue material properties to skeletal fragility is poorly understood. We previously reported that bones from SAMP6 mice are weak and brittle versus age-matched controls. Our present objectives were to use the SAMP6 mouse to assess bone material properties in a model of skeletal fragility and to relate defects in the mechanical properties of bone to the properties of demineralized bone and to the structure and organization of collagen and mineral., Materials and Methods: Femora from 4- and 12-month-old SAMR1 (control) and SAMP6 mice were analyzed using bending and torsional mechanical testing of intact bones, tensile testing of demineralized bone, quantitative histology (including collagen fiber orientation), collagen cross-links biochemistry, and Raman spectroscopic analysis of mineral and collagen., Results: Intact bones from SAMP6 mice have normal elastic properties but inferior failure properties, with 60% lower fracture energy versus SAMR1 controls. The strength defect in SAMP6 bones was associated with a 23% reduction in demineralized bone strength, which in turn was associated with poorer collagen fiber organization, lower collagen content, and higher hydroxylysine levels. However, SAMP6 have normal levels of collagen cross-links and normal apatite mineral structure., Conclusions: Bones from SAMP6 osteoporotic mice are weak and brittle because of a defect in the strength of the bone matrix. This defect is attributed primarily to poorer organization of collagen fibers and reduced collagen content. These findings highlight the role of the collagen component of the bone matrix in influencing skeletal fragility.

Unlabelled: A new SERM, CHF 4227.01, given to 6-month-old female rats immediately after ovariectomy, preserved bone mass and bone microarchitecture without affecting uterus weight. It also decreased serum cholesterol and fat mass in estrogen-deficient rats., Introduction: We tested the effect of a new benzopyran derivative, CHF 4227.01, with selective estrogen receptor modulator (SERM) activity on bone mass and biomechanics in ovariectomized (OVX) female rats in comparison with 17alpha-ethinylestradiol (EST), raloxifene (RLX), and lasofoxifene (LFX)., Materials and Methods: Four doses of CHF 4227.01 (0.001, 0.01, 0.1, and 1 mg/kg body weight [bw]/day) were administered in OVX animals daily by gavage 5 days/week for 4 months. EST was administered at a dose of 0.1 mg/kg bw/day, whereas RLX and LSX were administered at doses of 1 and 0.1 mg/kg bw/day, respectively, by gavage. In one group (Sham), rats were operated but the ovaries not removed; another OVX group was treated only with placebo., Results and Conclusions: Treatment with CHF 4227.01 (1.0 and 0.1 mg/kg bw), EST (0.1 mg/kg bw), LFX (0.1 mg/kg bw), or RLX (1.0 mg/kg bw) prevented bone loss on the lumbar spine and the proximal femur assessed in vivo by DXA. Volumetric BMD obtained by pQCT ex vivo confirmed protection from bone loss in the spine and proximal femur among rats treated with CHF 4227.01. This effect was associated with strong inhibition of bone resorption both histologically and biochemically. Furthermore, CHF 4227.01 preserved trabecular microarchitecture, analyzed by muCT, and maintained biomechanical indices of bone strength in the spine and proximal femur, effects also observed for RLX, whereas LSX was less protective of microarchitecture. CHF 4227.01 treatment did not affect uterine weight, prevented the increase in body weight and fat mass seen in OVX animals, and decreased serum cholesterol to below the average of intact animals. In conclusion, CHF 4227.01 exhibits a promising therapeutic and safety profile as a new SERM on both skeletal and extraskeletal outcomes.

To support future studies of tibial bending in a murine model of senile osteoporosis (SAMP6), we sought to determine the relationship between applied external bending force and peak endocortical strain in the tibiae of SAMP6 and control SAMR1 mice. The lower hindlimbs of mice were loaded by three-point bending in the lateral-medial plane with a support length of 10 mm. Force-periosteal strain relations were first determined using standard strain gauge methods. Finite-element analysis (FEA) models of the tibia-fibula were generated based on microcomputed tomography images. After choosing appropriate boundary conditions, FEA predictions of periosteal strains were within 15% of measured values. FEA revealed a narrow (3-4 mm) region of the central tibia with well-developed bending strains (tension medially, compression laterally); outside this region, we observed high shear strains. Both the strain gauge data and the finite-element simulations indicated that the tibia of the SAMP6 mouse was 20-25% stiffer than the SAMR1 tibia, consistent with a larger moment of inertia and higher cortical bone modulus. Thus, higher levels of force are required to produce the same target values of strain in the SAMP6 tibia. The ratio of periosteal to endocortical strain in the region of interest was similar for the two mouse strains (1.5-1.6). Based on these ratios, we scaled the strain gauge data to estimate the force-endocortical strain relations for the two mouse strains. In conclusion, FEA, with supporting strain gauge measurements, has provided unique insight regarding the strain environment throughout the tibia during three-point bending in mice.

Unlabelled: We used the SAMP6 osteoporotic mouse to examine the link between marrow osteogenic potential and in vivo cortical bone formation. SAMP6 marrow supported less in vitro osteogenesis than marrow from SAMR1 controls; SAMP6 mice had a corresponding deficit in endocortical mineralizing surface. This marrow/endocortical defect did not affect the periosteum, where SAMP6 mice had normal to enhanced bone formation., Introduction: With aging, there may be a reduction in the number or proliferative capacity of bone marrow osteoprogenitors that may contribute to age-related decreases in bone formation. To examine the link between the ability of the marrow to support osteogenesis and age-related changes in bone formation, we measured in vitro and in vivo indices of osteogenesis in a model of osteoporosis, the senescence-accelerated mouse SAMP6., Materials and Methods: Femora and tibias from SAMP6 and SAMR1 (control) mice were harvested at 2, 4, 6, and 12 months of age (168 bones total). Bone marrow cells were cultured under osteogenic conditions and stained for alkaline phosphatase (ALP) and alizarin red. Dynamic indices of bone formation were assessed histologically from calcein labels., Results: ALP+ and alizarin red-positive areas were significantly less in cultures from SAMP6 bones versus SAMR1 (p < 0.05), indicating less osteogenic potential. For example, SAMP6 tibial cultures had 21% less ALP+ area and 36% less alizarin red-positive area than SAMR1. Marrow from tibias had 2-fold greater osteogenesis than femoral marrow (p < 0.001). SAMP6 mice had a deficit in endocortical mineralizing surface across all age groups (p < 0.05), but no deficit in mineral apposition rate. Last, despite the marrow and endocortical deficits, SAMP6 mice had normal or slightly increased periosteal bone formation, consistent with their larger bone size., Conclusion: SAMP6 bone marrow supports less in vitro osteogenesis than SAMR1, consistent with a lower concentration of marrow osteoprogenitors in SAMP6. SAMP6 mice have less endocortical mineralizing surface than SAMR1 at all ages but no detectable deficit in mineral apposition rate, which suggests a reduction in osteoblast number but normal function. Periosteal bone formation is unimpaired in SAMP6 mice, indicating that the marrow/endocortical defect does not affect the periosteal surface.

The senescence accelerated mouse, strain P6 (SAMP6) has been described as a model of senile osteoporosis. Recent results from whole-bone bending tests indicate that, despite having increased moments of inertia, SAMP6 long bones are weak and brittle compared to SAMR1 controls. In the current study we determined material properties of cortical bone from SAMP6 and SAMR1 femora and tibiae by two methods-nanoindentation and whole-bone bending tests combined with simple beam theory. We hypothesized that: (1) SAMP6 mice have reduced cortical bone material properties compared to SAMR1 controls; and (2) modulus estimated from whole-bone bending tests correlates well with modulus determined by nanoindentation. Results from nanoindentation indicated that modulus and hardness are approximately 10% higher in SAMP6 mice compared to SAMR1 controls (p<0.001), a finding consistent with slightly higher mineralization in SAMP6 bones. Despite their superior elastic and hardness properties, the bending failure properties of SAMP6 bones were markedly inferior--ultimate stress and toughness were reduced by 40% and 60%, respectively (p<0.001). Comparisons between the two testing methods for determining modulus showed poor agreement. Modulus estimated from whole-bone bending tests was not correlated with modulus determined by nanoindentation (p=0.054; r2=0.03) and the absolute values differed by a factor of five between the two methods (bending [wet], 6GPa; nanoindentation [dry], 31GPa). Moreover, relative differences between groups were inconsistent between the two methods. We conclude: (1) cortical bone from the SAMP6 mouse has increased modulus and hardness but poor material strength and toughness, which underscores the relevance of the SAMP6 mouse for studies of skeletal fragility, and (2) values of elastic modulus of bone tissue estimated using simple beam theory and bending tests of mouse femora and tibiae are inaccurate and should be interpreted with caution.

The senescence-accelerated mouse, strain P6 (SAMP6), is a model of senile osteoporosis with relatively low bone mineral density (BMD), low rates of bone formation and reduced long-bone bending strength. Seeking to extend previous descriptions of the SAMP6 skeletal phenotype, we assessed the morphological and mechanical properties of vertebrae from SAM mice at 4 and 12 months of age. We hypothesized that, relative to SAMR1 controls, vertebrae from SAMP6 mice have: (1) less trabecular bone, (2) increased endosteal and periosteal bone size and (3) decreased whole-bone strength. Caudal vertebrae from adult female mice (SAMR1 and SAMP6; 4 and 12 months; n = 10-11 per group) were evaluated by micro-computed tomography and mechanical compression testing. SAMP6 vertebrae had 33% less trabecular bone volume in cephalad and caudal end regions than SAMR1 (P < 0.001) due to significant reductions in both trabecular number (P = 0.002) and thickness (P < 0.001). In contrast to previous findings in SAMP6 long bones, SAMP6 vertebrae showed no evidence of increased overall bone dimensions compared to SAMR1, and in fact had smaller total bone area (TA) and endosteal area (EA) at 12 months of age. Whereas SAMR1 vertebrae showed signs of age-related expansion from 4 to 12 months, SAMP6 vertebral dimensions did not change with age. Mechanical properties of SAMP6 vertebrae were not significantly different than SAMR1 vertebrae (stiffness, yield force, ultimate force, displacement at ultimate force, energy to ultimate force; P > 0.05), another finding in contrast to previous results in SAMP6 long bones. In summary, reduced vertebral trabecular bone volume is another feature of the SAMP6 mice with relevance to senile osteoporosis. However, age-related bone expansion and reduced whole-bone strength were not evident in SAMP6 vertebrae, indicating that while the SAMP6 mouse has many features relevant to senile osteoporosis in humans, not all features are observed or detectable at all skeletal sites.

Peripheral quantitative computed tomography (pQCT) is increasingly used for measurement of cortical bone geometry and density in mice. We evaluated the accuracy of pQCT for area and density measurements of thin-walled aluminum phantoms and mouse femora. Aluminum tubes with varying wall thicknesses and femora from 1- to 6-month-old C3H/HeJ (C3H) and C57B1/6J (B6) mice (average cortical thickness 0.14-0.29 mm) were scanned at 70- or 90-microm resolution. pQCT values of area were compared to optical values determined after sectioning, while pQCT density (vBMD) was compared to solid aluminum density or correlated to bone ash content. For the aluminum phantoms, the error in pQCT area and density depended strongly on wall thickness, and density was consistently underestimated. For mouse femora, threshold values were found that produced zero error in bone area for each strain and age group, although the optimal threshold differed between groups. pQCT vBMD correlated strongly with ash content (r2=0.7), although the regression equations differed between strains and the magnitude of the inter-strain difference in vBMD was fourfold greater than the difference in ash content. This finding suggests that pQCT can overestimate the differences in volumetric mineral density between inbred mouse strains whose bones are of different thickness (e.g., C3H vs. B6). In conclusion, both area and density values obtained by pQCT depend strongly on specimen thickness, consistent with a partial volume averaging artifact. Investigators using pQCT to assess cortical bones in mice should be aware of the potential for cortical thickness-dependent errors.

Background: Medial collateral ligament injuries heal by a scar response., Hypothesis: Increased hemorrhage at the site of medial collateral ligament injury improves healing., Study Design: Controlled laboratory study., Methods: Ninety-six mice were divided into two groups. Group 1 mice underwent knee medial collateral ligament transection with the opposite knee as a sham-operated control and group 2 animals additionally had 0.25 ml of tail cut blood pipetted to the medial collateral ligament transection site and sham-operated opposite knee. Ligament specimens were harvested at 3, 7, 21, and 28 days., Results: Immunohistochemical analysis demonstrated peak macrophage counts at day 7 in all transected specimens. Macrophage counts were higher in group 2 than in group 1 at all time points, with a statistically significant increase of macrophages noted at day 7. In situ hybridization demonstrated increased collagen gene expression, with peaks at 7 and 28 days after transection. Group 2 animals showed increased gene expression at all time points as compared with group 1, with a statistically significant increase noted at 7 and 28 days. Biomechanical testing demonstrated progressive healing at each time point. At 28 days, the load to failure was 67% that of the sham-operated knee., Conclusions: This study suggests there is an increased healing response with bleeding at the ligament injury site., Clinical Relevance: Identification of the factors involved with increased healing may allow manipulation of the healing response in the clinical setting.

The hematopoietic-restricted protein Src homology 2-containing inositol-5-phosphatase (SHIP) blunts phosphatidylinositol-3-kinase-initiated signaling by dephosphorylating its major substrate, phosphatidylinositol-3,4,5-trisphosphate. As SHIP(-/-) mice contain increased numbers of osteoclast precursors, that is, macrophages, we examined bones from these animals and found that osteoclast number is increased two-fold. This increased number is due to the prolonged life span of these cells and to hypersensitivity of precursors to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Similar to pagetic osteoclasts, SHIP(-/-) osteoclasts are enlarged, containing upwards of 100 nuclei, and exhibit enhanced resorptive activity. Moreover, as in Paget disease, serum levels of interleukin-6 are markedly increased in SHIP(-/-) mice. Consistent with accelerated resorptive activity, 3D trabecular volume fraction, trabecular thickness, number and connectivity density of SHIP(-/-) long bones are reduced, resulting in a 22% loss of bone-mineral density and a 49% decrease in fracture energy. Thus, SHIP negatively regulates osteoclast formation and function and the absence of this enzyme results in severe osteoporosis.

The senescence accelerated mouse strain P6 (SAMP6) has emerged as a useful model of senile osteoporosis because it has many features of the disease, including low trabecular bone formation and low areal bone density. We further characterized the SAMP6 model of senile osteoporosis by comparing morphological, mechanical, and densitometric properties of femurs and tibias from SAMP6 mice to those of the control strain (SAMR1) at 4 months and 12 months of age. SAMP6 long bones had increased periosteal width and endosteal area (p < 0.05), resulting in an average increase of 30% in moments of inertia (p < 0.05), but no difference in bone area (p > 0.05) compared with control. Despite their increased moments of inertia, long bones from SAMP6 mice were relatively weak and brittle. Ultimate bending moment was reduced by 25%, and both postyield displacement and energy-to-fracture were reduced by 60% compared with SAMR1 controls (p < 0.001). Average cortical ash fraction was increased slightly from 0.74 in SAMR1 to 0.76 in SAMP6 bones (p < 0.05), indicating that increased mineralization may have contributed to the brittleness of SAMP6 bones. The relative differences we observed--increased endosteal and periosteal dimensions, reduced bending strength, increased brittleness, and increased mineralization--are analogous to changes that occur in the aging human skeleton. Moreover, these features were consistently observed in young (4-month) and old (12-month) animals. These findings extend the previous descriptions of the SAMP6 mouse and identify key mechanical features that further validate its relevance as a unique and functionally relevant model of senile osteoporosis.

In vivo murine models are becoming increasingly important in bone research. To establish baseline data for researchers using these models, we studied the long bones from C57BL/6 female mice, a strain that is widely used in bone research. We determined the femoral structural and material properties in both torsion and four-point bending for mice at ages 4-24 weeks. Measurements of femoral cross-sectional geometry and tibial densitometric properties were also obtained. Results indicated that all structural properties (except ultimate energy), changed significantly with age (p < 0.001). Ultimate torque, ultimate moment, torsional rigidity, and bending rigidity all increased to peak values at 20 weeks, whereas ultimate rotation and ultimate displacement decreased to minimum values at 20 weeks. Our data indicate that increases in the material properties contributed more than increases in cross-sectional geometry to the changes in structural rigidity and ultimate load. For example, from 4-20 weeks torsional rigidity increased 1030%, while shear modulus increased 610% and the polar moment of inertia (a measure of the geometric resistance to rotation) increased by only 85%. Changes in the cross-sectional geometry with age were due to increases in periosteal diameter and decreases in endosteal diameter. In general, both torsion and bending techniques revealed large changes in structural and material properties with age. We conclude that peak bone strength is not achieved before 20 weeks in C57BL/6 female mice, and that torsion and four-point bending tests are equally well suited for evaluating mechanical properties of murine long bones.

Postoperative rehabilitation is an important factor in determining functional outcome following intrasynovial flexor tendon repair. We hypothesized that a rehabilitation protocol that produced increased in vivo excursion would lead to increased digital range of motion and tendon strength and decreased adhesion formation in a canine model. Ninety-six flexor digitorum profundus tendons from 48 dogs were cut transversely and repaired by a multistrand suture technique. Postoperative rehabilitation was performed daily with a low excursion-low force (1.7-mm average excursion; < 10 N force) or a high excursion-low force (3.6 mm excursion; < 10 N force) protocol. After death of the dogs at 10, 21, or 42 days, specimens were evaluated for digital range of motion, tensile mechanical properties, elongation of the repair site, and adhesion formation. Our data indicate that the range of motion of digits whose tendons were at low or high excursion was similar to that of controls. Increased in vivo tendon excursion due to synergistic wrist motion did not significantly affect ex vivo flexion of the distal and proximal interphalangeal joints or tendon displacement (p > 0.05). Similarly, tensile properties (ultimate load, repair site rigidity, and repair site strain at 20 N and at failure) and length of the gap at the repair site were not significantly affected by increased excursion (p > 0.05). Severity of adhesion formation was reduced slightly by increased excursion (p = 0.04). Our findings indicate that 1.7 mm of tendon excursion is sufficient to prevent adhesion formation following sharp transection of the canine flexor tendon and that additional excursion provides little added benefit.

Background: Elongation (gap formation) at the repair site has been associated with the formation of adhesions and a poor functional outcome after repair of flexor tendons. Our objectives were to evaluate the prevalence of gap formation in a clinically relevant canine model and to assess the effect of gap size on the range of motion of the digits and the mechanical properties of the tendons., Methods: We performed operative repairs after sharp transection of sixty-four flexor tendons in thirty-two adult dogs. Rehabilitation with passive motion was performed daily until the dogs were killed at ten, twenty-one, or forty-two days postoperatively. Eight tendons ruptured in vivo. In the fifty-six intact specimens, the change in the angles of the proximal and distal interphalangeal joints and the linear excursion of the flexor tendon were measured as a 1.5-newton force was applied to the tendon. The gap at the repair site was then measured, and the isolated tendons were tested to failure in tension., Results: Twenty-nine tendons had a gap of less than one millimeter, twelve had a gap of one to three millimeters, and fifteen had a gap of more than three millimeters. Neither the time after the repair nor the size of the gap was found to have a significant effect on motion parameters (p > 0.05); however, the ultimate force, repair-site rigidity, and repair-site strain at twenty newtons were significantly affected by these parameters (p < 0.05). Testing of the tendons with a gap of three millimeters or less revealed that, compared with the ten-day specimens, the forty-two-day specimens failed at a significantly (90 percent) higher force (p < 0.01) and had a significantly (320 percent) increased rigidity (p < 0.01) and a significantly (60 percent) decreased strain at twenty newtons (p < 0.05). In contrast, the tensile properties of the tendons that had a gap of more than three millimeters did not change significantly with time., Conclusions: Our data indicate that, in a dog model involving sharp transection followed by repair, a gap at the repair site of more than three millimeters does not increase the prevalence of adhesions or impair the range of motion but does prevent the accrual of strength and stiffness that normally occurs with time.

Avulsions or distal transsections of the flexor digitorum profundus tendon are typically repaired by direct suture of tendon to the distal phalanx. The tensile properties of tendon-tobone repairs performed in cadaver fingers using 3 common suture patterns, the Bunnell, the Kessler, and the Kleinert techniques, were compared; 3-0 Prolene (monofilament) suture was used. Repairs done using the Kessler pattern had an average yield force of 30 N, compared to 39 N for the Bunnell and Kleinert patterns. Although these average yield forces were greater than that required for active digital flexion, considerable elongation (average, 8 mm) was measured at a force of 20 N. Data indicated that the safety factor achieved with these repair methods is lower than that achieved with modern tendon-to-tendon repair methods. The authors conclude that the common tendon-to-bone repair techniques are insufficient to withstand the higher forces associated with controlled passive and active motion rehabilitation methods that are currently advocated.

Despite its widespread use as graft material in orthopaedic surgical procedures, morselized cancellous bone has not yet been well characterized from the standpoint of its mechanical properties. To accommodate the noncohesive nature of this loose particulate form of bone, a triaxial compression test apparatus commonly used in engineering soil mechanics was adapted for the testing of fresh-frozen human morselized cancellous bone specimens. Triaxial compression tests were run to 30% axial strain at five different levels of confining pressure ranging from 0.276 to 0.552 MPa. The measured axial stress versus axial strain behavior was bimodal, characterized initially by relatively stiff linear behavior, then by a rapid transition to a much more compliant (but, again, approximately linear) domain until test cessation. The apparent axial moduli of both response regions were found to be nearly linear functions of the transverse confining pressure. As typically prepared surgically, the distribution of particle size was found to have approximately 80% of the bone graft, by weight, encompassed in particles 0.42-3.2 mm in size. Triaxial tests of samples segregated by size showed that particle size had no appreciable effect on apparent material properties. The nominal Young's modulus and Poisson's ratio of morselized cancellous bone were 100 MPa and 0.2, respectively.