Evidence for the role of connexin 43-mediated intercellular communication in the process of intracortical bone resorption via osteocytic osteolysis.

BACKGROUND: Connexin 43 (Cx43) is the predominant gap junction protein in bone. Mice with a bone-specific deletion of Cx43 (cKO) have an osteopenic cortical phenotype. In a recent study, we demonstrated that cKO mice are resistant to bone loss induced by hindlimb suspension (HLS), an animal model of skeletal unloading. This protective effect occurred primarily as a result of lower osteoclast-mediated bone resorption. Interestingly, we also documented a significant increase in cortical osteocyte apoptosis and reduced sclerostin production. In the present study, we investigated whether osteocytic osteolysis - bone resorption by osteocytes within lacunae - is induced by HLS and the potential effect of Cx43 deficiency on this process during unloading. METHODS: 6-month-old male Cx43 cKO or wild-type (WT) mice were subjected to three weeks of HLS (Suspended) or normal loading conditions (Control) (n = 5/group). Lacunar morphology and tartrate-resistant acid phosphatase (TRACP) staining were assessed on sections of femur cortical bone. Experimental groups were compared via two-way ANOVA. RESULTS: Empty lacunae were 26% larger in cKO-Control vs. WT-Control (p < 0.05), while there was no difference in the size of empty lacunae between Control and Suspended within WT or cKO (p > 0.05). Similarly, there was a trend (p = 0.06) for 11% larger lacunae containing viable osteocytes for cKO-Control vs. WT-Control, with no apparent effect of loading condition. There was no difference in the proportion of TRACP + cells between WT-Control and cKO-Control (p > 0.05); however, WT-Suspended mice had 246% more TRACP + osteocytes compared WT-Control mice (p < 0.05). There was no difference in TRACP staining between cKO-Control and cKO-Suspended (p > 0.05). CONCLUSIONS: Prior to undergoing apoptosis, osteocytes in cKO mice may be actively resorbing their respective lacunae via the process of osteocytic osteolysis. Interestingly, the proportion of TRACP + osteocytes increased dramatically following unloading of WT mice, an effect that was not observed in cKO mice subjected to HLS. The results of the present study provide initial evidence that osteocytic osteolysis is occurring in cortical bone in response to mechanical unloading. Furthermore, Cx43 deficiency appears to protect against osteocytic osteolysis in a manner similar to the inhibition of unloading-induced osteoclast activation that we have documented previously.

Ionizing Radiation and Bone Loss: Space Exploration and Clinical Therapy Applications.

Damage to normal, nontumor bone tissue following therapeutic irradiation increases the risk of fracture among cancer patients. For example, women treated for various pelvic tumors have been shown to have a greater than 65% increased incidence of hip fracture by 5 years postradiotherapy. Another practical situation in which exposure to ionizing radiation may negatively impact skeletal integrity is during extended spaceflight missions. There is a limited understanding of how spaceflight-relevant doses and types of radiation can influence astronaut bone health, particularly when combined with the significant effects of mechanical unloading experienced in microgravity. Historically, negative effects on osteoblasts have been studied. Radiation exposure has been shown to damage osteoblast precursors. Damage to local vasculature has been observed, ranging from decreased lumen diameter to complete ablation within the irradiated volume, causing a state of hypoxia. These effects result in suppression of bone formation and a general state of low bone turnover. More recently, however, we have demonstrated in pre-clinical mouse models, a very rapid but transient increase in osteoclast activity after exposure to spaceflight and clinically relevant radiation doses. Combined with long-term suppression of bone formation, this skeletal damage may cause long-term deficits. This review will present a broad set of literature outlining our current set knowledge of both clinical therapy and space exploration exposure to ionizing radiation. Additionally, we will discuss prevention of the initial osteoclast-mediated bone loss, the need to promote normal bone turnover and long-term quality of bone tissue, and our hypothesized molecular mechanisms.

Space Radiation and Bone Loss.

Exposure to ionizing radiation may negatively impact skeletal integrity during extended spaceflight missions to the moon, Mars, or near-Earth asteroids. However, our understanding of the effects of radiation on bone is limited when compared to the effects of weightlessness. In addition to microgravity, astronauts will be exposed to space radiation from solar and cosmic sources. Historically, radiation exposure has been shown to damage both osteoblast precursors and local vasculature within the irradiated volume. The resulting suppression of bone formation and a general state of low bone-turnover is thought to be the primary contributor to bone loss and eventual fracture. Recent investigations using mouse models have identified a rapid, but transient, increase in osteoclast activity immediately after irradiation with both spaceflight and clinically-relevant radiation qualities and doses. Together with a chronic suppression of bone formation after radiation exposure, this acute skeletal damage may contribute to long-term deterioration of bone quality, potentially increasing fracture risk. Direct evidence for the damaging effects of radiation on human bone are primarily demonstrated by the increased incidence of fractures at sites that absorb high doses of radiation during cancer therapy: exposures are considerably higher than what could be expected during spaceflight. However, both the rapidity of bone damage and the chronic nature of the changes appear similar between exposure scenarios. This review will outline our current knowledge of space and clinical exploration exposure to ionizing radiation on skeletal health.

Gap Junctions and Biophysical Regulation of Bone Cells.

Communication between osteoblasts, osteoclasts, and osteocytes is integral to their ability to build and maintain the skeletal system and respond to physical signals. Various physiological mechanisms, including nerve communication, hormones, and cytokines, play an important role in this process. More recently, the important role of direct, cell-cell communication via gap junctions has been established. In this review, we demonstrate the integral role of gap junctional intercellular communication (GJIC) in skeletal physiology and bone cell mechanosensing.

Administration of high-dose macrophage colony-stimulating factor increases bone turnover and trabecular volume fraction.

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that plays a critical role in early osteoclastogenesis. To characterize the skeletal effects of M-CSF, we administered soluble M-CSF to mice. It was hypothesized that M-CSF would stimulate bone formation through coupled activity of osteoclasts and osteoblasts. Twenty-four male C57BL/6 J mice (n = 12/group, aged 7 weeks) received subcutaneous injections of human M-CSF [5 mg/(kg day)] or inert vehicle (VEH) for 21 days. M-CSF increased serum bone turnover markers (+57% TRAP-5b and +44% osteocalcin). Microcomputed tomography revealed an anabolic effect on tibial trabecular bone, with higher bone volume fraction (+35%), connectivity density (+79%), and number (+18%), as well as lower trabecular separation (-18%). M-CSF had no significant effect on cortical bone mineral content, geometry, or strength. There was no change in quantitative histomorphometry parameters of femoral cortical bone. These results reveal the complex, site-specific effects of M-CSF. In particular, we have demonstrated an anabolic effect of M-CSF on trabecular bone achieved through coupled activation of osteoblasts. However, in contrast to previous studies, M-CSF was found to have no effect on cortical bone. M-CSF was demonstrated to significantly influence both bone modeling and remodeling in relatively young animals.

Early increase in osteoclast number in mice after whole-body irradiation with 2 Gy X rays.

Bone loss is a consequence of exposure to high-dose radiotherapy. While damage to bone vasculature and reduced proliferation of bone-forming osteoblasts has been implicated in this process, the effect of radiation on the number and activity of bone-resorbing osteoclasts has not been characterized. In this study, we exposed mice to a whole-body dose of 2 Gy of X rays to quantify the early effects of radiation on osteoclasts and bone structural properties. Female C57BL/6 mice (13 weeks old) were divided into two groups: irradiated and nonirradiated controls. Animals were killed humanely 3 days after radiation exposure. Analysis of serum chemistry revealed a 14% increase in the concentration of tartrate resistant acid phosphatase (TRAP)-5b, a marker of osteoclast activity, in irradiated mice (P < 0.05). Osteoclast number (+44%; P < 0.05) and osteoclast surface (+213%; P < 0.001) were elevated in TRAP-stained histological sections of tibial metaphyses. No significant change was observed in osteoblast surface or osteocalcin concentration or in trabecular microarchitecture (i.e. bone volume fraction) as measured through microcomputed tomography (P > 0.05). This study provides definitive, quantitative evidence of an early, radiation-induced increase in osteoclast activity and number. Osteoclastic bone resorption may represent a contributor to bone atrophy observed after therapeutic irradiation.

Development of a low-dose anti-resorptive drug regimen reveals synergistic suppression of bone formation when coupled with disuse.

Safe and effective countermeasures to spaceflight-induced osteoporosis are required to mitigate the potential for mission-critical fractures and ensure long-term bone health in astronauts. Two anti-resorptive drugs, the bisphosphonate zoledronic acid (ZOL) and the anti-receptor activator of NF-kappaB ligand protein osteoprotegerin (OPG), were investigated to find the minimum, comparable doses that yield a maximal increase in bone quality, while minimizing deleterious effects on turnover and mineralization. Through a series of five trials in normally loaded female mice (n = 56/trial), analysis of trabecular volume fraction and connectivity using microcomputed tomography, along with biomechanical testing, quantitative histomorphometry, and compositional analysis, was used to select 45 microg/kg ZOL and 500 microg/kg OPG as doses that satisfy these criteria. These doses were then examined for their ability to mitigate bone loss following short-term unloading through hindlimb suspension (HLS). Seventy-two mice were prophylactically administered ZOL, OPG, or PBS and assigned to loaded control or 2-wk HLS groups (n = 12 for each of 6 groups). Both anti-resorptives were able to preserve trabecular microarchitecture and femoral elastic and maximum force in HLS mice (+30-40% ZOL/OPG vs. PBS). In HLS mice, anti-resorptive dosing reduced resorption perimeter at the femoral endocortical surface by 30% vs. PBS. In loaded control mice, anti-resorptives produced no change in bone formation rate; however, reductions in bone formation rate brought about by HLS were exacerbated by anti-resorptive treatment, suggesting synergistic inhibition of osteoblasts during disuse. Refined anti-resorptive dosing will tend to target countermeasures to the period of disuse, resulting in faster recovery and less adverse effects for astronauts.

Spaceflight-relevant types of ionizing radiation and cortical bone: Potential LET effect?

Extended exposure to microgravity conditions results in significant bone loss. Coupled with radiation exposure, this phenomenon may place astronauts at a greater risk for mission-critical fractures. In a previous study, we identified a profound and prolonged loss of trabecular bone (29-39%) in mice following exposure to an acute, 2 Gy dose of radiation simulating both solar and cosmic sources. However, because skeletal strength depends on trabecular and cortical bone, accurate assessment of strength requires analysis of both bone compartments. The objective of the present study was to examine various properties of cortical bone in mice following exposure to multiple types of spaceflight-relevant radiation. Nine-week old, female C57BL/6 mice were sacrificed 110 days after exposure to a single, whole body, 2 Gy dose of gamma, proton, carbon, or iron radiation. Femora were evaluated with biomechanical testing, microcomputed tomography, quantitative histomorphometry, percent mineral content, and micro-hardness analysis. Compared to non-irradiated controls, there were significant differences compared to carbon or iron radiation for only fracture force, medullary area and mineral content. A greater differential effect based on linear energy transfer (LET) level may be present: high-LET (carbon or iron) particle irradiation was associated with a decline in structural properties (maximum force, fracture force, medullary area, and cortical porosity) and mineral composition compared to low-LET radiation (gamma and proton). Bone loss following irradiation appears to be largely specific to trabecular bone and may indicate unique biological microenvironments and microdosimetry conditions. However, the limited time points examined and non-haversian skeletal structure of the mice employed highlight the need for further investigation.

Histopathologic review of pineal parenchymal tumors identifies novel morphologic subtypes and prognostic factors for outcome.

BACKGROUND: Pineal parenchymal tumors (PPTs) are rare neoplasms of the central nervous system, and data concerning clinical outcomes are limited. The purpose of this study was to define the clinical behavior of PPT according to current histopathologic criteria and identify prognostic factors to guide therapeutic decisions. METHODS: Seventy-five patients treated for PPT at a single institution between 1992 and 2015 were retrospectively identified. Forty-five resection specimens were available and re-reviewed. Freedom from progression (FFP) and overall survival (OS) were estimated using the Kaplan-Meier method and compared using log-rank tests. RESULTS: Median follow-up was 4.1 years. All patients initially underwent surgery; 78% of patients with PPT of intermediate differentiation (PPTID) and all patients with pineoblastoma received adjuvant therapy. Pathologic re-review refined classification in 27% of cases, with the majority of these being adult patients with pineal tumors originally classified as pineoblastomas that more accurately resembled PPTID based on the 2007 WHO classification. CLASSIFICATION: Our histologic review also identified that PPTIDs can be classified into small-cell and large-cell morphologic subtypes, which have distinct clinical outcomes. Tumor grade, extent of resection, and neuraxis spread were prognostic for FFP. PPTID subtype, extent of resection, and neuraxis spread were prognostic for OS. Genetic analysis of a pineoblastoma case identified somatic mutations of DICER1, ARID1A, and KDM5C genes. CONCLUSIONS: PPTIDs can be classified into 1 of 2 novel morphologic subtypes that are associated with distinct clinical outcomes. Tumor grade, neuraxis spread, and extent of resection also influence outcome for patients with PPT.

Phylogeny of marine Gregarines (Apicomplexa)--Pterospora, Lithocystis and Lankesteria--and the origin(s) of coelomic parasitism.

Gregarines constitute a large group of apicomplexans with diverse modes of nutrition and locomotion that are associated with different host compartments (e.g. intestinal lumena and coelomic cavities). A broad molecular phylogenetic framework for gregarines is needed to infer the early evolutionary history of apicomplexans as a whole and the evolutionary relationships between the diverse ultrastructural and behavioral characteristics found in intestinal and coelomic gregarines. To this end, we sequenced the SSU rRNA gene from (1) Lankesteria abbotti from the intestines of two Pacific appendicularians, (2) Pterospora schizosoma from the coelom of a Pacific maldanid polychaete, (3) Pterospora floridiensis from the coelom of a Gulf Atlantic maldanid polychaete and (4) Lithocystis sp. from the coelom of a Pacific heart urchin. Molecular phylogenetic analyses including the new sequences demonstrated that several environmental and misattributed sequences are derived from gregarines. The analyses also demonstrated a clade of environmental sequences that was affiliated with gregarines, but as yet none of the constituent organisms have been described at the ultrastructural level (apicomplexan clade I). Lankesteria spp. (intestinal parasites of appendicularians) grouped closely with other marine intestinal eugregarines, particularly Lecudina tuzetae, from polychaetes. The sequences from all three coelomic gregarines branched within a larger clade of intestinal eugregarines and were similarly highly divergent. A close relationship between Pterospora schizosoma (Pacific) and Pterospora floridiensis (Gulf Atlantic) was strongly supported by the data. Lithocystis sp. was more closely related to a clade of marine intestinal gregarines consisting of Lankesteria spp. and Lecudina spp. than it was to the Pterospora clade. These data suggested that coelomic parasitism evolved more than once from different marine intestinal eugregarines, although a larger taxon sample is needed to further explore this inference.

Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice.

Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20mg/kg) or an inert vehicle (VEH), 24h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P<0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.

Shifting paradigms on the role of connexin43 in the skeletal response to mechanical load.

Gap junctions (GJs) are membrane-spanning channels that allow for the movement of small molecules across cell membranes. Connexin43 (Cx43) is the predominant GJ protein in bone. In vitro studies suggest that gap junctional intercellular communication (GJIC) sensitizes bone cells to mechanical signals. Additionally, mechanical signals detected by osteocytes are communicated to osteoblasts via GJIC, and osteocytic Cx43 hemichannels release anabolic factors, such as PGE2 and ATP, in response to mechanical load. These findings and others have led to near consensus among researchers in the field that GJIC, hemichannels or connexins facilitate the anabolic response of bone to mechanical load and, in their absence, bone would be less sensitive to load. However, recent in vivo evidence suggests the opposite is true. Studies from our laboratory and others demonstrate that Cx43-deficient mice have an increased anabolic response to mechanical load and are protected against the catabolic effects of mechanical unloading. These developments suggest a paradigm shift in our understanding of connexins, GJIC, and mechanotransduction in bone. That is, inhibiting bone cell Cx43 expression or GJIC has a beneficial effect on bone's response to its mechanical environment, preserving bone during unloading and enhancing its formation during loading. Here, we review literature in support of this hypothesis and suggest a mechanism by which Cx43, through interaction with WNT/ß-catenin signaling, moderates both arms of bone remodeling.

Interdependence of muscle atrophy and bone loss induced by mechanical unloading.

Mechanical unloading induces muscle atrophy and bone loss; however, the time course and interdependence of these effects is not well defined. We subjected 4-month-old C57BL/6J mice to hindlimb suspension (HLS) for 3 weeks, euthanizing 12 to 16 mice on day (D) 0, 7, 14, and 21. Lean mass was 7% to 9% lower for HLS versus control from D7-21. Absolute mass of the gastrocnemius (gastroc) decreased 8% by D7, and was maximally decreased 16% by D14 of HLS. mRNA levels of Atrogin-1 in the gastroc and quadriceps (quad) were increased 99% and 122%, respectively, at D7 of HLS. Similar increases in MuRF1 mRNA levels occurred at D7. Both atrogenes returned to baseline by D14. Protein synthesis in gastroc and quad was reduced 30% from D7-14 of HLS, returning to baseline by D21. HLS decreased phosphorylation of SK61, a substrate of mammalian target of rapamycin (mTOR), on D7-21, whereas 4E-BP1 was not lower until D21. Cortical thickness of the femur and tibia did not decrease until D14 of HLS. Cortical bone of controls did not change over time. HLS mice had lower distal femur bone volume fraction (-22%) by D14; however, the effects of HLS were eliminated by D21 because of the decline of trabecular bone mass of controls. Femur strength was decreased approximately 13% by D14 of HLS, with no change in tibia mechanical properties at any time point. This investigation reveals that muscle atrophy precedes bone loss during unloading and may contribute to subsequent skeletal deficits. Countermeasures that preserve muscle may reduce bone loss induced by mechanical unloading or prolonged disuse. Trabecular bone loss with age, similar to that which occurs in mature astronauts, is superimposed on unloading. Preservation of muscle mass, cortical structure, and bone strength during the experiment suggests muscle may have a greater effect on cortical than trabecular bone.

Connexin 43 deficiency desensitizes bone to the effects of mechanical unloading through modulation of both arms of bone remodeling.

Connexin 43 (Cx43) is a gap junction protein that plays an integral role in the skeletal response to mechanical loading and unloading. In a previous study, we demonstrated preservation of trabecular bone mass and cortical bone formation rate in mice with an osteoblast/osteocyte-selective deficiency of Cx43 (cKO) following mechanical unloading via hindlimb suspension (HLS). In the present study, we sought to define the potential mechanisms underlying this response. Following three weeks of HLS, mRNA levels of Sost were significantly greater in wild-type (WT)-Suspended mice vs. WT-Control, while there was no difference between cKO control and cKO-Suspended. Unloading-induced decreases in P1NP, a serum marker of bone formation, were also attenuated in cKO-Suspended. The proportion of sclerostin-positive osteocytes was significantly lower in cKO-Control vs. WT-Control (-72%, p<0.05), a difference accounted for by the presence of numerous empty lacunae in the cortical bone of cKO vs. WT. Abundant TUNEL staining was present throughout the cortical bone of the tibia and femur, suggesting an apoptotic process. There was no difference in empty lacunae in the trabecular bone of the tibia or femur. Trabecular and cortical osteoclast indices were lower in cKO-Suspended vs. WT-Suspended; however, mRNA levels of the gene encoding RANKL increased similarly in both genotypes. Connexin 43 deficient mice experience attenuated sclerostin-mediated suppression of cortical bone formation and lower cortical osteoclast activity during unloading. Preservation of trabecular bone mass and attenuated osteoclast activity during unloading, despite an apparent lack of effect on osteocyte viability at this site, suggests that an additional mechanism independent of osteocyte apoptosis may also be important. These findings indicate that Cx43 is able to modulate both arms of bone remodeling during unloading.

Inhibition of GSK-3ß rescues the impairments in bone formation and mechanical properties associated with fracture healing in osteoblast selective connexin 43 deficient mice.

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated ß-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, ß-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3ß, a key component of the ß-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3ß activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating ß-catenin expression and thus bone formation during fracture repair.

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.

Effect of proton irradiation followed by hindlimb unloading on bone in mature mice: a model of long-duration spaceflight.

Bone loss associated with microgravity unloading is well documented; however, the effects of spaceflight-relevant types and doses of radiation on the skeletal system are not well defined. In addition, the combined effect of unloading and radiation has not received much attention. In the present study, we investigated the effect of proton irradiation followed by mechanical unloading via hindlimb suspension (HLS) in mice. Sixteen-week-old female C57BL/6 mice were either exposed to 1 Gy of protons or a sham irradiation procedure (n=30/group). One day later, half of the mice in each group were subjected to four weeks of HLS or normal loading conditions. Radiation treatment alone (IRR) resulted in approximately 20% loss of trabecular bone volume fraction (BV/TV) in the tibia and femur, with no effect in the cortical bone compartment. Conversely, unloading induced substantially greater loss of both trabecular bone (60-70% loss of BV/TV) and cortical bone (approximately 20% loss of cortical bone volume) in both the tibia and femur, with corresponding decreases in cortical bone strength. Histological analyses and serum chemistry data demonstrated increased levels of osteoclast-mediated bone resorption in unloaded mice, but not IRR. HLS+IRR mice generally experienced greater loss of trabecular bone volume fraction, connectivity density, and trabecular number than either unloading or irradiation alone. Although the duration of unloading may have masked certain effects, the skeletal response to irradiation and unloading appears to be additive for certain parameters. Appropriate modeling of the environmental challenges of long duration spaceflight will allow for a better understanding of the underlying mechanisms mediating spaceflight-associated bone loss and for the development of effective countermeasures.

Effects of combined insulin-like growth factor 1 and macrophage colony-stimulating factor on the skeletal properties of mice.

BACKGROUND/AIM: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. MATERIALS AND METHODS: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). RESULTS: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (-13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. CONCLUSION: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.