Inbred Strain‐Specific Response to Biglycan Deficiency in the Cortical Bone of C57BL6/129 and C3H/He Mice

Inbred strain‐specific differences in mice exist in bone cross‐sectional geometry, mechanical properties, and indices of bone formation. Inbred strain‐specific responses to external stimuli also exist, but the role of background strain in response to genetic deletion is not fully understood. Biglycan (bgn) deficiency impacts bone through negative regulation of osteoblasts, resulting in extracellular matrix alterations and decreased mechanical properties. Because osteoblasts from C3H/He (C3H) mice are inherently more active versus osteoblasts from other inbred strains, and the bones of C3H mice are less responsive to other insults, it was hypothesized that C3H mice would be relatively more resistant to changes associated with bgn deficiency compared with C57BL6/129 (B6;129) mice. Changes in mRNA expression, tissue composition, mineral density, bone formation rate, cross‐sectional geometry, and mechanical properties were studied at 8 and 11 wk of age in the tibias of male wildtype and bgn‐deficient mice bred on B6;129 and C3H background strains. Bgn deficiency altered collagen cross‐linking and gene expression and the amount and composition of mineral in vivo. In bgn's absence, changes in collagen were independent of mouse strain. Bgn‐deficiency increased the amount of mineral in both strains, but changes in mineral composition, cross‐sectional geometry, and mechanical properties were dependent on genetic background. Bgn deficiency influenced the amount and composition of bone in mice from both strains at 8 wk, but C3H mice were better able to maintain properties close to wildtype (WT) levels. By 11 wk, most properties from C3H knockout (KO) bones were equal to or greater than WT levels, whereas phenotypic differences persisted in B6;129 KO mice. This is the first study into mouse strain‐specific changes in a small leucine‐rich proteoglycan gene disruption model in properties across the bone hierarchy and is also one of the first to relate these changes to mechanical competence. This study supports the importance of genetic factors in determining the response to a gene deletion and defines biglycan's importance to collagen and mineral composition in vivo.     

Exercise-induced changes in the cortical bone of growing mice are bone- and gender-specific

Fracture risk and mechanical competence of bone are functions of bone mass and tissue quality, which in turn are dependent on the bone's mechanical environment. Male mice have a greater response to non-weight-bearing exercise than females, resulting in larger, stronger bones compared with control animals. The aim of this study was to test the hypothesis that short-term weight-bearing running during growth (21 days starting at 8 weeks of age; 30 min/day; 12 m/min; 5 degrees incline; 7 days/week) would similarly have a greater impact on cross-sectional geometry and mechanical competence in the femora and tibiae of male mice versus females. Based on the orientation of the legs during running and the proximity of the tibia to the point of impact, this response was hypothesized to be greatest in the tibia. Exercise-related changes relative to controls were assayed by four-point bending tests, while volumetric bone mineral density and cross-sectional geometry were also assessed. The response to running was bone- and gender-specific, with male tibiae demonstrating the greatest effects. In male tibiae, periosteal perimeter, endocortical perimeter, cortical area, medial-lateral width and bending moment of inertia increased versus control mice suggesting that while growth is occurring in these mice between 8 and 11 weeks of age, exercise accelerated this growth resulting in a greater increase in bone tissue over the 3 weeks of the study. Exercise increased tissue-level strain-to-failure and structural post-yield deformation in the male tibiae, but these post-yield benefits came at the expense of decreased yield deformation, structural and tissue-level yield strength and tissue-level ultimate strength. These results suggest that exercise superimposed upon growth accelerated growth-related increases in tibial cross-sectional dimensions. Exercise also influenced the quality of this forming bone, significantly impacting structural and tissue-level mechanical properties.     

Short-Term Exercise in Mice Increases Tibial Post-Yield Mechanical Properties While Two Weeks of Latency Following Exercise Increases Tissue-Level Strength

We have previously shown that exercise during growth increases post-yield deformation in C57BL6/129 (B6;129) male tibiae at the expense of reduced pre-yield deformation and structural and tissue strength. Other research in the literature indicates that increased mineral content, cross-sectional geometry and structural strength due to exercise can be maintained or increased after exercise ends for as long as 14 weeks. It was therefore hypothesized that after our exercise protocol ended, effects of exercise on mechanical properties would persist, resulting in increased post-yield behavior and rescued strength versus age-matched control mice. Beginning at 8 weeks of age, exercise consisted of running on a treadmill (30 min/day, 12 m/min, 5° incline) for 21 consecutive days. At the end of running and 2 weeks later, in the cortical bone of the tibial mid-diaphyses of B6;129 male mice, changes due to exercise and latency following exercise were assayed by mechanical tests and analyses of cross-sectional geometry. Exercise increased structural post-yield deformation compared with weight-matched control mice, without changes in bone size or shape, suggesting that exercised-induced changes in pre-existing bone quality were responsible. Over the 2-week latency period, no growth-related changes were noted in control mice, but exercise-induced changes resulted in increased tissue stiffness and strength versus mice sacrificed immediately after exercise ended. Our data indicate that periods of exercise followed by latency can alter strength, stiffness, and ductility of bone independent of changes in size or shape, suggesting that exercise may be a practical way to increase the quality of the bone extracellular matrix.     

Biglycan deficiency interferes with ovariectomy-induced bone loss.

Biglycan is a matrix proteoglycan with a possible role in bone turnover. In a 4-week study with sham-operated or OVX biglycan-deficient or wildtype mice, we show that biglycan-deficient mice are resistant to OVX-induced trabecular bone loss and that there is a gender difference in the response to biglycan deficiency. INTRODUCTION: Biglycan (bgn) is a small extracellular matrix proteoglycan enriched in skeletal tissues, and biglycan-deficient male mice have decreased trabecular bone mass and bone strength. The purpose of this study was to investigate the bone phenotype of the biglycan-deficient female mice and to investigate the effect of estrogen depletion by ovariectomy (OVX). MATERIALS AND METHODS: OVX or sham operations were performed on 21-week-old mice that were divided into four groups: wt sham (n = 7), wt OVX (n = 9), bgn-deficient sham (n = 10) and bgn-deficient OVX (n = 10). The mice were killed 4 weeks after surgery. Bone mass and bone turnover were analyzed by peripheral quantitative computed tomography (pQCT), biochemical markers, and histomorphometry. RESULTS AND CONCLUSIONS: In contrast to the male mice, there were only few effects of bgn deficiency on bone metabolism in female mice, showing a clear gender difference. However, when stressed by OVX, the female bgn knockout (KO) mice were resistant to the OVX-induced trabecular bone loss. The wt mice showed a decrease in trabecular bone mineral density by pQCT measurements, a decrease in trabecular bone volume (BV/TV), and an increase in mineral apposition rate. In contrast, no significant changes were detected in bgn KO mice after OVX. In addition, analysis of the bone resorption marker deoxypyridinoline showed no significant increase in the bgn KO OVX mice compared with bgn KO sham mice. Measurements of serum osteoprotegerin (OPG) and RANKL revealed increased levels of OPG and decreased levels of RANKL in the bgn KO mice compared with wt mice. In conclusion, the bgn deficiency protects against increased trabecular bone turnover and bone loss in response to estrogen depletion, supporting the concept that bgn has dual roles in bone, where it may modulate both formation and resorption ultimately influencing the bone turnover process.     

Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues.

Decorin (dcn) and biglycan (bgn), two members of the family of small leucine-rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a "subclinical" cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn- and dcn-deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double-knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double-KO animals in which progeroid changes are observed also in the skin. Ultrastructural analysis of bone collagen fibrils in bone of double-KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked "serrated fibril" morphology. The phenotype of the double-KO animal mimics directly the rare progeroid variant of human Ehlers-Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and den deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.     

Age-related osteoporosis in biglycan-deficient mice is related to defects in bone marrow stromal cells.

Biglycan (bgn) is an extracellular matrix proteoglycan that is enriched in bone and other skeletal connective tissues. Previously, we generated bgn-deficient mice and showed that they developed age-dependent osteopenia. To identify the cellular events that might contribute to this progressive osteoporosis, we measured the number of osteogenic precursors in the bone marrow of normal and mutant mice. The number of colonies, indicative of the colony-forming unit potential of fibroblasts (CFU-F), gradually decreased with age. By 24 weeks of age, colony formation in the bgn knockout (KO) mice was significantly more reduced than that in the wild type (wt) mice. This age-related reduction was consistent with the extensive osteopenia previously shown by X-ray analysis and histological examination of 24-week-old bgn KO mice. Because bgn has been shown previously to bind and regulate transforming growth factor beta (TGF-beta) activity, we also asked whether this growth factor would affect colony formation. TGF-beta treatment significantly increased the size of the wt colonies. In contrast, TGF-beta did not significantly influence the size of the bgn colonies. An increase in apoptosis in bgn-deficient bone marrow stromal cells (BMSCs) was observed also. The combination of decreased proliferation and increased apoptosis, if it occurred in vivo, would lead to a deficiency in the generation of mature osteoblasts and would be sufficient to account for the osteopenia developed in the bgn KO mice. The bgn KO mice also were defective in the synthesis of type I collagen messenger RNA (mRNA) and protein. This result supports the suggestion that the composition of the extracellular matrix may be regulated by specific matrix components including bgn.     

Tibial changes in experimental disuse osteoporosis in the monkey

We studied the mechanical properties and structural changes in the monkey tibia with disuse osteoporosis and during subsequent recovery. Bone bending stiffness was evaluated in relationship to microscopic changes in cortical bone and Norland bone mineral analysis. Restraint in the semireclined position produced regional losses of bone most obviously in the anterior-proximal tibiae. Following 6 months of restraint, the greatest losses of bone mineral in the proximal tibiae ranged from 23% to 31%; the largest changes in bone stiffness ranged from 36% to 40%. Approximately 8 1/2 months of recovery were required for restoration of normal bending properties. However, even after 15 months of recovery, bone mineral content did not necessarily return to normal levels. Histologically, resorption cavities in cortical bone were seen within 1 month of restraint; by 2 1/2 months of restraint there were large resorption cavities subperiosteally, endosteally, and intracortically. After 15 months of recovery, the cortex consisted mainly of first-generation haversian systems. After 40 months, the cortex appeared normal with numerous secondary and tertiary generations of haversian systems.     

Genetic Variations in Bone Density, Histomorphometry, and Strength in Mice

The purpose of this study was to assess breed-related differences in bone histomorphometry, bone biomechanics, and serum biochemistry in three mouse breeds shown to differ in bone mineral density (BMD) (as measured by DXA) and bone mineral content (BMC). Femurs, tibiae, and sera were collected from 16-week-old C3H/HeJ {C3H}, C57BL/6J {BL6}, and DBA/2J {DBA}mice (n = 12/breed). Data collected included BMC and BMD (femora), histomorphometry of cancellous (distal femur) and cortical bone (diaphyseal tibiae and femora), bone strength (femora), and serum alkaline phosphatase (ALP). Consistent with previous reports, BMC and BMD were higher in C3H than in BL6 or DBA mice. The higher BMD in the C3H breed was associated with greater cancellous bone volume, cortical bone area, periosteal bone formation rate, biomechanical strength, and serum ALP. However, mid-diaphyseal total femoral and tibial cross-sectional area and moment of inertia were greatest in BL6, intermediate in C3H, and lowest in DBA mice. The specific distribution of cortical bone in C3H, BL6, DBA mice represents a difference in adaptive response to similar mechanical loads in these breeds. This difference in adaptive response may be intrinsic to the adaptive mechanism, or may be intrinsic to the bone tissue material properties. In either case, the bone-adaptive response to ordinary mechanical loads in the BL6 mice yields bones of lower mechanical efficiency (less stiffness per unit mass of bone tissue) and does not adapt as well as that of the C3H mice where the final product is a bone with greater resistance to bending under load. We suggest that the size, shape, and BMD of the bone are a result of breed-specific genetically regulated cellular mechanisms. Compared with the C3H mice, the lower BMD in BL6 mice is associated with long bones that are weaker because the larger cross-sectional area fails to compensate completely for their lower BMD and BMC. Received: 16 November 1999 / Accepted: 19 April 2000 / Online publication: 27 July 2000     

Tibial changes in experimental disuse osteoporosis in the monkey

Summary We studied the mechanical properties and structural changes in the monkey tibia with disuse osteoporosis and during subsequent recovery. Bone bending stiffness was evaluated in relationship to microscopic changes in cortical bone and Norland bone mineral analysis. Restraint in the semireclined position produced regional losses of bone most obviously in the anterior-proximal tibiae. Following 6 months of restraint, the greatest losses of bone mineral in the proximal tibiae ranged from 23% to 31%; the largest changes in bone stiffness ranged from 36% to 40%. Approximately 8 ? months of recovery were required for restoration of normal bending properties. However, even after 15 months of recovery, bone mineral content did not necessarily return to normal levels. Histologically, resorption cavities in cortical bone were seen within 1 month of restraint; by 2 ? months of restraint there were large resorption cavities subperiosteally, endosteally, and intracortically. After 15 months of recovery, the cortex consisted mainly of first-generation haversian systems. After 40 months, the cortex appeared normal with numerous secondary and tertiary generations of haversian systems.     

Quantitative trait loci for femoral size and shape in a genetically heterogeneous mouse population.

The aim of this study was to examine the genetic effects on cortical bone geometry. Genotypes from 487 mice were compared with geometric traits obtained from microCT. We found 14 genetic markers that associate with geometric traits, showing the complexity of genetic control over bone geometry. INTRODUCTION: Previous studies have shown that genetic background affects bone characteristics, particularly bone mineral density, in both mouse and human populations. Much less is known, however, about the effects of polymorphic genes on bone size, shape, and mechanical integrity. In this study, we investigated the genetic determinants of geometric properties of cortical bone in mice. MATERIALS AND METHODS: This study used a genetically heterogeneous mouse population, which is denoted UM-HET3 stock and is derived as the progeny of (BALB/cJ X C57BL/6J) F1 females and (C3H/HeJ X DBA/2J) F1 males. The experimental group consisted of 487 female UM-HET3 mice. Genotypic data from 99 polymorphic genetic loci was obtained from the mice at 4 weeks of age. At 18 months of age, the mice were humanely killed, and the right femurs were scanned with microcomputed tomography to assess geometric properties of cortical bone. A permutation-based test was used to detect significant associations between genetic markers and geometric traits. This test generates experiment-wise p values, which account for the effect of testing multiple hypotheses. An experiment-wise p < or = 0.05 was considered statistically significant. RESULTS: Fourteen genetic markers were found to significantly associate with one or more geometric traits. Two markers (D3Mit62 and D4Mit155) were associated with traits describing bone size; 2 (D12Mit167 and D14Mit170) were linked with traits describing bone shape; and 10 (D1Nds2, D5Mit95, D6Mit216, D7Mit91, D8Mit51, D9Mit110, D11Mit83, D15Mit100, D15Mit171, and D17Mit46) were associated with both size and shape. CONCLUSIONS: Our results indicate that the genetic control of cortical bone geometry is complex and that femoral size and shape may be influenced by different, although overlapping, groups of polymorphic loci.     

Bone formation is impaired in a model of type 1 diabetes

The effects of type 1 diabetes on de novo bone formation during tibial distraction osteogenesis (DO) and on intact trabecular and cortical bone were studied using nonobese diabetic (NOD) mice and comparably aged nondiabetic NOD mice. Diabetic mice received treatment with insulin, vehicle, or no treatment during a 14-day DO procedure. Distracted tibiae were analyzed radiographically, histologically, and by microcomputed tomography (microCT). Contralateral tibiae were analyzed using microCT. Serum levels of insulin, osteocalcin, and cross-linked C-telopeptide of type I collagen were measured. Total new bone in the DO gap was reduced histologically (P < or = 0.001) and radiographically (P < or = 0.05) in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice (P < or = 0.001) and normalized with insulin treatment. Evaluation of the contralateral tibiae by microCT and mechanical testing demonstrated reductions in trabecular bone volume and thickness, cortical thickness, cortical strength, and an increase in endosteal perimeter in diabetic animals, which were prevented by insulin treatment. These studies demonstrate that bone formation during DO is impaired in a model of type 1 diabetes and preserved by systemic insulin administration.     

Mechanical Load Increases in Bone Formation via a Sclerostin‐Independent Pathway

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long‐term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost?/? and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (?9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost?/? tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost?/? mice over their normal load controls. The increased BV was associated with significantly increased mid‐shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost?/? mice than in wild type controls. Thus, long‐term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load. © 2014 American Society for Bone and Mineral Research.     

Transgenic over-expression of plasminogen activator inhibitor-1 results in age-dependent and gender-specific increases in bone strength and mineralization

The plasminogen activation system (PAS) and its principal inhibitor, plasminogen activator inhibitor-1 (PAI-1), are recognized modulators of matrix. In addition, the PAS has previously been implicated in the regulation of bone homeostasis. Our objective was to study the influence of active PAI-1 on geometric, biomechanical, and mineral characteristics of bone using transgenic mice that over-express a variant of human PAI-1 that exhibits enhanced functional stability.

Femora were isolated from male and female, wildtype (WT) and transgenic (PAI-1.stab) mice at 16 and 32 weeks of age (n = 10). Femora were imaged via DEXA for BMD and μCT for cortical mid-slice geometry. Torsional testing was employed for biomechanical properties. Mineral composition was analyzed via instrumental neutron activation analysis. Female femora were further analyzed for trabecular bone histomorphometry (n = 11). Whole animal DEXA scans were performed on PAI-1.stab females and additional transgenic lines in which the functional domains of the PAI-1 protein were specifically disrupted.

Thirty-two week female PAI-1.stab femora exhibited decreased mid-slice diameters and reduced polar moment of area compared to WT, while maintaining similar cortical bone width. Greater biomechanical strength and stiffness were demonstrated by 32 week PAI-1.stab female femora in addition to a 52% increase in BMD. PAI-1.stab trabecular bone architecture was comparable to WT. Osteoid area was decreased in PAI-1.stab mice while mineral apposition rate increased by 78% over WT. Transgenic mice expressing a reactive-site mutant form of PAI-1 showed an increase in BMD similar to PAI-1.stab, whereas transgenic mice expressing a PAI-1 with reduced affinity for vitronectin were comparable to WT.

Over-expression of PAI-1 resulted in increased mineralization and biomechanical properties of mouse femora in an age-dependent and gender-specific manner. Changes in mineral preceded increases in strength/stiffness and deterred normal cross-sectional expansion of cortical bone in females. Trabecular bone was not altered in PAI-1.stab mice whereas MAR increased significantly, further supporting mineral changes as the underlying factor in strength differences. The primary influence of PAI-1 occurred during a period of basal bone remodeling, attributing a role for this system in remodeling as opposed to development. Comparison of transgenic lines indicates that PAI-1's influence on bone is dependent on its ability to bind vitronectin, and not on its proteolytic activity. The impact of PAI-1 on mouse femora supports a regulatory role of the plasminogen activation system in bone homeostasis, potentially elucidating novel targets for the treatment of bone disease.     

Risedronate does not reduce mechanical loading-related increases in cortical and trabecular bone mass in mice

To establish whether the combination of anti-resorptive therapy with mechanical loading has a negative, additive or synergistic effect on bone structure, we assessed the separate and combined effects of risedronate and non-invasive dynamic loading on trabecular and cortical bone. Seventeen-week-old female C57BL/6 mice were given daily subcutaneous injections of vehicle (n=20) or risedronate at a dose of 0.15, 1.5, 15 or 150 μg/kg/day (n=10 in each) for 17 days. From the fourth day of treatment, the right tibiae were subjected to a single period of axial loading (40 cycles/day) for three alternate days per week for two weeks. The left tibiae were used as internal controls. Trabecular and cortical sites in the tibiae were analyzed by high-resolution micro-computed tomography and imaging of fluorochrome labels. In the non-loaded tibiae, treatment with the higher doses of risedronate at 15 or 150 μg/kg/day resulted in higher trabecular bone volume and trabecular number than in vehicle-treated controls, whereas such treatment was associated with no differences in cortical bone volume at any dose. In the loaded tibiae, loading induced increases in trabecular and cortical bone volume compared with contra-lateral controls primarily through increased trabecular thickness and periosteal expansion, respectively, independently of risedronate treatment. In conclusion, the response to mechanical loading in both trabecular and cortical bone in mice is therefore not impaired by short-term treatment with risedronate, even over a 1000-fold dose range. In considering the optimization of treatments for osteoporosis, it is reassuring that anti-resorptive therapy and mechanical loading can exert independent beneficial effects.     

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. © 2011 American Society for Bone and Mineral Research     

Connexin 43 deficiency attenuates loss of trabecular bone and prevents suppression of cortical bone formation during unloading

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and has been demonstrated as an integral component of skeletal homeostasis. In the present study, we sought to further refine the role of Cx43 in the response to mechanical unloading by subjecting skeletally mature mice with a bone‐specific deletion of Cx43 (cKO) to 3 weeks of mechanical unloading via hindlimb suspension (HLS). The HLS model was selected to recapitulate the effects of skeletal unloading due to prolonged bed rest, reduced activity associated with aging, and spaceflight microgravity. At baseline, the cortical bone of cKO mice displayed an osteopenic phenotype, with expanded cortices, decreased cortical thickness, decreased bone mineral density, and increased porosity. There was no baseline trabecular phenotype. After 3 weeks of HLS, wild‐type (WT) mice experienced a substantial decline in trabecular bone volume fraction, connectivity density, trabecular thickness, and trabecular tissue mineral density. These deleterious effects were attenuated in cKO mice. Conversely, there was a similar and significant amount of cortical bone loss in both WT and cKO. Interestingly, mechanical testing revealed a greater loss of strength and rigidity for cKO during HLS. Analysis of double‐label quantitative histomorphometry data demonstrated a substantial decrease in bone formation rate, mineralizing surface, and mineral apposition rate at both the periosteal and endocortical surfaces of the femur after unloading of WT mice. This suppression of bone formation was not observed in cKO mice, in which parameters were maintained at baseline levels. Taken together, the results of the present study indicate that Cx43 deficiency desensitizes bone to the effects of mechanical unloading, and that this may be due to an inability of mechanosensing osteocytes to effectively communicate the unloading state to osteoblasts to suppress bone formation. Cx43 may represent a novel therapeutic target for investigation as a countermeasure for age‐related and unloading‐induced bone loss. © 2012 American Society for Bone and Mineral Research.     

Mechanotransduction in bone: genetic effects on mechanosensitivity in mice

Bone formation is enhanced by mechanical loading, but human exercise intervention studies have shown that the response to mechanical loading is variable, with some individuals exhibiting robust osteogenic responses while others respond modestly. Thus, mechanosensitivity - the ability of bone tissue to detect mechanical loads - could be under genetic control. We applied controlled mechanical loading to the ulnae of 20-week-old (adult) female mice derived from three different inbred strains (C3H/He, C57BL/6, and DBA/2), and measured the bone formation response with fluorochrome labels. Mechanical properties, including mechanical strain, second moments of area, and cortical bone material properties, were measured in a group of calibration animals not subjected to in vivo loading. The C3H/He mice were significantly less responsive to mechanical loading than the other two biological strains. Material properties (flexural elastic modulus, ultimate stress) were greatest in the C3H/He cortical tissue. Geometric and areal properties at the midshaft ulna were also greatest in the C3H/He mice. Based on the presumed role of osteocytes in strain detection, we measured osteocyte lacuna population densities in decalcified midshaft ulna sections. Osteocyte lacuna density was not related to mechanosensitivity. Our data suggest that bone mechanosensitivity has a significant genetic component. Identification of the genes that exert their influence on mechanosensitivity could ultimately lead to therapies that enhance bone mass and reduce fracture susceptibility.     

Microfluidic enhancement of intramedullary pressure increases interstitial fluid flow and inhibits bone loss in hindlimb suspended mice

Interstitial fluid flow (IFF) has been widely hypothesized to mediate skeletal adaptation to mechanical loading. Although a large body of in vitro evidence has demonstrated that fluid flow stimulates osteogenic and antiresorptive responses in bone cells, there is much less in vivo evidence that IFF mediates loading‐induced skeletal adaptation. This is due in large part to the challenges associated with decoupling IFF from matrix strain. In this study we describe a novel microfluidic system for generating dynamic intramedullary pressure (ImP) and IFF within the femurs of alert mice. By quantifying fluorescence recovery after photobleaching (FRAP) within individual lacunae, we show that microfluidic generation of dynamic ImP significantly increases IFF within the lacunocanalicular system. In addition, we demonstrate that dynamic pressure loading of the intramedullary compartment for 3 minutes per day significantly eliminates losses in trabecular and cortical bone mineral density in hindlimb suspended mice, enhances trabecular and cortical structural integrity, and increases endosteal bone formation rate. Unlike previously developed modalities for enhancing IFF in vivo, this is the first model that allows direct and dynamic modulation of ImP and skeletal IFF within mice. Given the large number of genetic tools for manipulating the mouse genome, this model is expected to serve as a powerful investigative tool in elucidating the role of IFF in skeletal adaptation to mechanical loading and molecular mechanisms mediating this process. © 2010 American Society for Bone and Mineral Research     

FTIR Microspectroscopic Analysis of Human Iliac Crest Biopsies from Untreated Osteoporotic Bone

Historically, osteoporosis has been defined as a disease in which there is ``too little bone, but what there is, is normal.' As a result of research design and sample selection limitations, published data contradict and confirm the historical definition. Because of these limitations, it has been hard to assess the contribution of mineral quality to mechanical properties, and to select therapeutic protocols that optimize bone mineral properties. The coupling of an optical microscope to an infrared spectrometer enables the acquisition of spectral data at known sites in a histologic section of mineralized tissue without loss of topography and/or orientation. The use of second-derivative spectroscopy coupled with curve-fitting techniques allows the qualitative and quantitative assessment of mineral quality (crystallite size and perfection, mineral:matrix ratio) at well-defined morphologic locations. We have previously applied these techniques to the study of normal human osteonal, cortical, and trabecular bone. The results indicated that the newly deposited bone mineral is less ``crystalline/mature' than the older one. In the present study, Fourier transform infrared microspectroscopy (FTIRM) was applied to the study of human osteonal and cortical bone from iliac crest biopsies of untreated osteoporotic patients. The hypothesis tested was that osteoporotic bone mineral is monotonically different in its properties expressed as ``crystallinity/maturity' than the normal. The results indicate significant differences in the mineral properties as expressed by crystal size and perfection, with the mineral from osteoporotic bone being more crystalline/mature than the normal.     

Alendronate treatment promotes bone formation with a less anisotropic microstructure during intramembranous ossification in rats

There are safety concerns regarding administration of bisphosphonates to children. Little is known about the effects of bisphosphonates on bone matrix organization during bone modeling. The present study examined the effects of alendronate (ALN) on bone matrices formed by intramembranous ossification in the appendicular growing skeleton. ALN was administered to 1-week-old Sprague–Dawley rats at a dose of 0, 35, or 350 μg/kg/week for 4 or 8 weeks. The position of femoral diaphysis formed exclusively by intramembranous ossification was identified, and cross sections of cortical bone at this position were analyzed. Bone mineral density (BMD) and geometric parameters were evaluated using peripheral quantitative computed tomography. The preferential orientation degree of biological apatite (BAp) crystals in the bone longitudinal direction, which shows the degree of bone matrix anisotropy, was evaluated using microbeam X-ray diffraction analysis. We analyzed bone histomorphometrical parameters and performed bone nanomechanical tests to examine the material properties of newly developing cortical bone. The preferential orientation degree of BAp crystals significantly decreased in 35 μg/kg/week ALN-treated groups compared with vehicle-treated groups, although there were no significant differences in BMD between the two groups. The periosteal mineral apposition rate significantly increased in the 35 μg/kg/week ALN-treated group. We found a high negative correlation between bone matrix anisotropy and the regional periosteal mineral apposition rate (r = −0.862, P < 0.001). Nanomechanical tests revealed that 35 μg/kg/week ALN administration caused deterioration of the material properties of the bone microstructure. These new findings suggest that alendronate affects bone matrix organization and promotes bone formation with a less anisotropic microstructure during intramembranous ossification.