Multi-organ phenotypes in mice lacking latent TGFß binding protein 2 (LTBP2).

BACKGROUND: Latent TGFß 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 TGFß, 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.

Cortical bone relationships are maintained regardless of sex and diet in a large population of LGXSM advanced intercross mice.

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 (r2: 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.

Fracture healing is delayed in the absence of gasdermin-interleukin-1 signaling.

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1ß (IL-1ß) 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-1ß 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.

Osteoblast-Specific Wnt Secretion Is Required for Skeletal Homeostasis and Loading-Induced Bone Formation in Adult Mice.

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; WlsF/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 (WlsF/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).

Estimation of load conditions and strain distribution for in vivo murine tibia compression loading using experimentally informed finite element models.

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.

MicroCT for Scanning and Analysis of Mouse Bones.

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).

Proliferating osteoblasts are necessary for maximal bone anabolic response to loading in mice.

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.

Old Mice Have Less Transcriptional Activation But Similar Periosteal Cell Proliferation Compared to Young-Adult Mice in Response to in vivo Mechanical Loading.

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.

Congenital lipodystrophy induces severe osteosclerosis.

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.

Effects of High-Fat Diet and Body Mass on Bone Morphology and Mechanical Properties in 1100 Advanced Intercross Mice.

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 (F34 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.

Evaluation of loading parameters for murine axial tibial loading: Stimulating cortical bone formation while reducing loading duration.

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 µÏµ and a transition from lamellar-woven bone near -2,000 µÏµ. 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 µÏµ 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.

Mechanical comparison of iliosacral reconstruction techniques after sarcoma resection.

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.

Activation of Wnt Signaling by Mechanical Loading Is Impaired in the Bone of Old Mice.

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.

Aging diminishes lamellar and woven bone formation induced by tibial compression in adult C57BL/6.

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 µÎµ and -3000 µÎµ (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 µÎµ did not alter cortical bone volume but loading to a peak strain of -3000 µÎµ 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 µÎµ 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.

Experimental and finite element analysis of strains induced by axial tibial compression in young-adult and old female C57Bl/6 mice.

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 µÎµ and a tensile strain on the antero-medial face of +1450 µÎµ. 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.

Adaptation of tibial structure and strength to axial compression depends on loading history in both C57BL/6 and BALB/c mice.

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.

Enhanced periosteal and endocortical responses to axial tibial compression loading in conditional connexin43 deficient mice.

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.

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged 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 µÎµ endocortical; -2350 µÎµ 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.

The effects of screw length on stability of simulated osteoporotic distal radius fractures fixed with volar locking plates.

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.

Connexin43 deficiency reduces the sensitivity of cortical bone to the effects of muscle paralysis.

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.