Geroprotectors and Skeletal Health: Beyond the Headlines.

Osteoporosis and osteoarthritis are the most common age-related diseases of the musculoskeletal system. They are responsible for high level of healthcare use and are often associated with comorbidities. Mechanisms of ageing such as senescence, inflammation and autophagy are common drivers for both diseases and molecules targeting those mechanisms (geroprotectors) have potential to prevent both diseases and their co-morbidities. However, studies to test the efficacy of geroprotectors on bone and joints are scant. The limited studies available show promising results to prevent and reverse Osteoporosis-like disease. In contrast, the effects on the development of Osteoarthritis-like disease in ageing mice has been disappointing thus far. Here we review the literature and report novel data on the effect of geroprotectors for Osteoporosis and Osteoarthritis, we challenge the notion that extension of lifespan correlates with extension of healthspan in all tissues and we highlight the need for more thorough studies to test the effects of geroprotectors on skeletal health in ageing organisms.

PTH(1-34) treatment and/or mechanical loading have different osteogenic effects on the trabecular and cortical bone in the ovariectomized C57BL/6 mouse.

In preclinical mouse models, a synergistic anabolic response to PTH(1-34) and tibia loading was shown. Whether combined treatment improves bone properties with oestrogen deficiency, a cardinal feature of osteoporosis, remains unknown. This study quantified the individual and combined longitudinal effects of PTH(1-34) and loading on the bone morphometric and densitometric properties in ovariectomised mice. C57BL/6 mice were ovariectomised at 14-weeks-old and treated either with injections of PTH(1-34); compressive loading of the right tibia; both interventions concurrently; or both interventions on alternating weeks. Right tibiae were microCT-scanned from 14 until 24-weeks-old. Trabecular metaphyseal and cortical midshaft morphometric properties, and bone mineral content (BMC) in 40 different regions of the tibia were measured. Mice treated only with loading showed the highest trabecular bone volume fraction at week 22. Cortical thickness was higher with co-treatment than in the mice treated with PTH alone. In the mid-diaphysis, increases in BMC were significantly higher with loading than PTH. In ovariectomised mice, the osteogenic benefits of co-treatment on the trabecular bone were lower than loading alone. However, combined interventions had increased, albeit regionally-dependent, benefits to cortical bone. Increased benefits were largest in the mid-diaphysis and postero-laterally, regions subjected to higher strains under compressive loads.

The longitudinal effects of ovariectomy on the morphometric, densitometric and mechanical properties in the murine tibia: A comparison between two mouse strains.

Oestrogen deficiency-related bone loss in the ovariectomized (OVX) mouse is a common model for osteoporosis. However, a comprehensive in vivo assessment of intervention-related changes in multiple bone properties, and in multiple mouse strains, is required in order to identify an appropriate model for future evaluation of novel anti-osteoporotic therapies. The aim of this study was to evaluate the effect of OVX on the morphometric and densitometric properties measured in the microCT images and the mechanical properties estimated with finite element models of the tibia in two mouse strains, C57BL/6 and BALB/c. 14-weeks-old female C57BL/6 and BALB/c mice were divided into two groups per strain: (1) ovariectomized, (2) non-operated control. The right tibia was scanned at baseline (14 weeks) and then every two weeks thereafter, until 24-weeks-old, using in vivo microCT. Changes in trabecular and cortical bone morphometry, spatiotemporal changes in densitometric properties and in mechanical properties (from micro-finite element (µFE) analysis) were computed. Differences between OVX and non-operated controls were evaluated by ANCOVA, adjusted for 14-weeks baseline. In morphometry, trabecular bone mass was significantly reduced in both C57BL/6 and BALB/c from four weeks following surgery. Though the OVX-effect was transient in BALB/c as bone mass reached skeletal homeostasis. OVX inhibited the age-related thickening of cortical bone only in C57BL/6. In both strains, increments in bone mineral content were significantly lower with OVX only in the proximal tibia, with intervention-related differences increasing with time. OVX had no effect on µFE estimates of stiffness nor failure load in either strain. The results of this study show strain-, time- and region-(trabecular or cortical) dependent changes in morphometric and densitometric properties. These findings highlight the importance of choosing an appropriate mouse model and time points for research of treatments against accelerated bone resorption.

Longitudinal effects of Parathyroid Hormone treatment on morphological, densitometric and mechanical properties of mouse tibia.

The use of Parathyroid Hormone (PTH) as bone anabolic is limited due to cost-benefit assessments. Preclinical studies evaluating the effects of PTH on bone have reported variable and often contradictory results. Here, we have applied a new approach using a combination of in-vivo longitudinal µCT, image processing techniques and finite element models to monitor early local changes in the whole tibia (divided in 40 compartments) and mechanical properties of female C57BL/6J mice treated with PTH 1-34, compared to controls. Compared with standard 3D bone morphometric analysis, our new approach allowed detection of much smaller and localised changes in bone mineral content (BMC) at very early time points (1 week vs 3 weeks with standard methods) and showed that changes do not occur uniformly over time and across the anatomical space. Indeed, in the PTH treated mice, significant changes in BMC were observed in the medial and posterior sectors of the proximal tibia, a week after treatment, and in the medial sector of the tibia midshaft region a week later (p < 0.05). By the third week, two thirds of the regions showed significantly higher values of BMC (p < 0.05). The effect of PTH on bone regional volume is similar to that on BMC, but there is almost no effect of PTH on bone tissue mineral density. The differences in estimated mechanical properties became significant after three weeks of treatment (p < 0.05). These results provide the first evidence of an early and localised PTH effect on murine bone, and show that our novel partitioning approach, compared to the standard evaluation protocol, allows a more precise quantification of bone changes following treatment, which would facilitate preclinical testing of novel mono- and/or combination therapies throughout the bone.

Development of a protocol to quantify local bone adaptation over space and time: Quantification of reproducibility.

In vivo micro-computed tomography (µCT) scanning of small rodents is a powerful method for longitudinal monitoring of bone adaptation. However, the life-time bone growth in small rodents makes it a challenge to quantify local bone adaptation. Therefore, the aim of this study was to develop a protocol, which can take into account large bone growth, to quantify local bone adaptations over space and time. The entire right tibiae of eight 14-week-old C57BL/6J female mice were consecutively scanned four times in an in vivo µCT scanner using a nominal isotropic image voxel size of 10.4µm. The repeated scan image datasets were aligned to the corresponding baseline (first) scan image dataset using rigid registration. 80% of tibia length (starting from the endpoint of the proximal growth plate) was selected as the volume of interest and partitioned into 40 regions along the tibial long axis (10 divisions) and in the cross-section (4 sectors). The bone mineral content (BMC) was used to quantify bone adaptation and was calculated in each region. All local BMCs have precision errors (PE%CV) of less than 3.5% (24 out of 40 regions have PE%CV of less than 2%), least significant changes (LSCs) of less than 3.8%, and 38 out of 40 regions have intraclass correlation coefficients (ICCs) of over 0.8. The proposed protocol allows to quantify local bone adaptations over an entire tibia in longitudinal studies, with a high reproducibility, an essential requirement to reduce the number of animals to achieve the necessary statistical power.

The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity.

Osteoarthritis (OA) is the most prevalent and debilitating joint disease, and there are currently no effective disease-modifying treatments available. Multiple risk factors for OA, such as aging, result in progressive damage and loss of articular cartilage. Autonomous circadian clocks have been identified in mouse cartilage, and environmental disruption of circadian rhythms in mice predisposes animals to OA-like damage. However, the contribution of the cartilage clock mechanisms to the maintenance of tissue homeostasis is still unclear. Here, we have shown that expression of the core clock transcription factor BMAL1 is disrupted in human OA cartilage and in aged mouse cartilage. Furthermore, targeted Bmal1 ablation in mouse chondrocytes abolished their circadian rhythm and caused progressive degeneration of articular cartilage. We determined that BMAL1 directs the circadian expression of many genes implicated in cartilage homeostasis, including those involved in catabolic, anabolic, and apoptotic pathways. Loss of BMAL1 reduced the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and NFATC2 and decreased expression of the major matrix-related genes Sox9, Acan, and Col2a1, but increased p-SMAD1/5 levels. Together, these results define a regulatory mechanism that links chondrocyte BMAL1 to the maintenance and repair of cartilage and suggest that circadian rhythm disruption is a risk factor for joint diseases such as OA.

Evaluation of in-vivo measurement errors associated with micro-computed tomography scans by means of the bone surface distance approach.

In vivo micro-computed tomography (µCT) scanning is an important tool for longitudinal monitoring of the bone adaptation process in animal models. However, the errors associated with the usage of in vivo µCT measurements for the evaluation of bone adaptations remain unclear. The aim of this study was to evaluate the measurement errors using the bone surface distance approach. The right tibiae of eight 14-week-old C57BL/6 J female mice were consecutively scanned four times in an in vivo µCT scanner using a nominal isotropic image voxel size (10.4 µm) and the tibiae were repositioned between each scan. The repeated scan image datasets were aligned to the corresponding baseline (first) scan image dataset using rigid registration and a region of interest was selected in the proximal tibia metaphysis for analysis. The bone surface distances between the repeated and the baseline scan datasets were evaluated. It was found that the average (±standard deviation) median and 95th percentile bone surface distances were 3.10 ± 0.76 µm and 9.58 ± 1.70 µm, respectively. This study indicated that there were inevitable errors associated with the in vivo µCT measurements of bone microarchitecture and these errors should be taken into account for a better interpretation of bone adaptations measured with in vivo µCT.

Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and progressive establishment of high trabecular bone mass.

Adult Ibsp-knockout mice (BSP-/-) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice, while impairing primary mineralization.

Absence of bone sialoprotein (BSP) impairs primary bone formation and resorption: the marrow ablation model under PTH challenge.

Bone sialoprotein (BSP) is highly expressed in early bone deposition and may play a part in primary bone mineralization. We previously showed that while BSP−/− mice have a mild secondary bone phenotype and are responsive to mechanical (unloading) and hormonal (ovariectomy, parathyroid hormone (PTH)) challenges, repair of a cortical bone defect, which involves primary bone deposition is significantly delayed in these mice. In the present study, we investigated the role of BSP in a pure model of primary bone modeling. Bone marrow was ablated by trans-epiphysis aspiration in the femora of BSP+/+ and BSP−/− mice, and 7 days post surgery µCT analysis showed vigorous new bone formation in the shaft of BSP+/+ animals but much less in BSP−/− mice. After 14 days, the volume of medullary bone was significantly decreased as expected in BSP+/+ mice, while it remained stable in the BSP−/−. Osteoid thickness and surface were higher in BSP−/− at day 7, suggesting delayed mineralization, while osteoclast surface and number were significantly lower at day 14, a stage of high medullary bone resorption. At day 7, mRNA expression of early osteoblast marker genes (RUNX2, osterix, alkaline phosphatase, osteopontin) did not differ between the two genotypes, while markers of terminal differentiation (MEPE, DMP1, osteocalcin) as well as receptor activator of NF-kappaB ligand (RANKL) and tartrate-resistant acid phosphatase (TRAP) were significantly lower in BSP−/− than in BSP+/+ mice. PTH treatment maintained the volume of medullary bone up to 12 days after ablation in BSP+/+ mice, but failed to do so in BSP−/− mice. PTH significantly increased bone formation rate in both genotype, while it reduced osteoclast number and surface in BSP+/+, but not in BSP−/− medullary bone. In summary, medullary bone formation after marrow ablation is blunted in BSP−/− mice, with delayed resorption and impaired response to PTH. These findings confirm the hypothesis of a crucial role for BSP in primary ossification, which has long been suspected for mineralization, but here extends to bone deposition and turnover.

Inositol polyphosphate 4-phosphatase B as a regulator of bone mass in mice and humans.

Osteoporosis is a multifactorial genetic disease characterized by reduction of bone mass due to dysregulation of osteoclast differentiation or maturation. Herein, we identified a regulator of osteoclastogenesis, the murine homolog of inositol polyphosphate 4-phosphatase type IIα (Inpp4bα). Expression of Inpp4bα is detected from early osteoclast differentiation to activation stage. Targeted expression of native Inpp4bα ex vivo repressed whereas phosphatase-inactive Inpp4bα stimulated osteoclast differentiation. Inpp4bα acts on intracellular calcium level that modulates NFATc1 nuclear translocation and activation. In vivo mice deficient in Inpp4b displayed increased osteoclast differentiation rate and potential resulting in decreased bone mass and osteoporosis. Importantly, INPP4B in human was identified as a susceptibility locus for osteoporosis. This study defined Inpp4b as a major modulator of the osteoclast differentiation and as a gene linked to variability of bone mineral density in mice and humans.

Mice lacking bone sialoprotein (BSP) lose bone after ovariectomy and display skeletal site-specific response to intermittent PTH treatment.

Bone sialoprotein (BSP) belongs to the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, whose members play multiple and distinct roles in the development, turnover, and mineralization of bone and dentin. The functions of BSP in bone remodeling are not yet well established. We previously showed that BSP knockout (BSP(-/-)) mice exhibit a higher trabecular bone volume, concomitant with lower bone remodeling, than wild-type (BSP(+/+)) mice. To determine whether bone turnover can be stimulated in the absence of BSP, we subjected BSP(+/+) and BSP(-/-) mice to catabolic [ovariectomy (OVX)] or anabolic (intermittent PTH administration) hormonal challenges. BSP(-/-) mice progressively develop hypocalcemia and high serum PTH between 2 and 4 months of age. Fifteen and 30 d after OVX, microtomography analysis showed a significant decrease of trabecular bone volume in tibiae of both genotypes. Histomorphometric parameters of bone formation and resorption were significantly increased by OVX. PTH treatment resulted in an increase of trabecular thickness and both bone formation and resorption parameters at all skeletal sites in both genotypes and a decrease of trabecular bone volume in tibiae of BSP(+/+) but not BSP(-/-) mice. PTH increased cortical thickness and bone area in BSP(+/+) but not BSP(-/-) mice and stimulated the bone formation rate specifically in the endosteum of BSP(+/+) mice and the periosteum of BSP(-/-) mice. PTH enhanced the expression of RANKL, MEPE, and DMP1 in both genotypes but increased OPG and OPN expression only in BSP(-/-) mice. In conclusion, despite the low basal turnover, both catabolic and anabolic challenges increase bone formation and resorption in BSP(-/-) mice, suggesting that compensatory pathways are operative in the skeleton of BSP-deficient mice. Although up-regulation of one or several other SIBLINGs is a possible mechanism, further studies are needed to analyze the interplay and cross-regulation involved in compensating for the absence of BSP.

Bone sialoprotein deficiency impairs osteoclastogenesis and mineral resorption in vitro.

Bone sialoprotein (BSP) and osteopontin (OPN) belong to the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members interact with bone cells and bone mineral. Previously, we showed that BSP knockout (BSP(-/-) ) mice have a higher bone mass than wild type (BSP(+/+) ) littermates, with very low bone-formation activity and reduced osteoclast surfaces and numbers. Here we report that approximately twofold fewer tartrate-resistant acid phosphatase (TRACP)-positive cells and approximately fourfold fewer osteoclasts form in BSP(-/-) compared with BSP(+/+) spleen cell cultures. BSP(-/-) preosteoclast cultures display impaired proliferation and enhanced apoptosis. Addition of RGD-containing proteins restores osteoclast number in BSP(-/-) cultures to BSP(+/+) levels. The expression of osteoclast-associated genes is markedly altered in BSP(-/-) osteoclasts, with reduced expression of cell adhesion and migration genes (αV integrin chain and OPN) and increased expression of resorptive enzymes (TRACP and cathepsin K). The migration of preosteoclasts and mature osteoclasts is impaired in the absence of BSP, but resorption pit assays on dentine slices show no significant difference in pit numbers between BSP(+/+) and BSP(-/-) osteoclasts. However, resorption of mineral-coated slides by BSP(-/-) osteoclasts is markedly impaired but is fully restored by coating the mineral substrate with hrBSP and partly restored by hrOPN coating. In conclusion, lack of BSP affects both osteoclast formation and activity, which is in accordance with in vivo findings. Our results also suggest at least some functional redundancy between BSP and OPN that remains to be clarified.

Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis.

Bone sialoprotein (BSP) and osteopontin (OPN) are both highly expressed in bone, but their functional specificities are unknown. OPN knockout (-/-) mice do not lose bone in a model of hindlimb disuse (tail suspension), showing the importance of OPN in bone remodeling. We report that BSP(-/-) mice are viable and breed normally, but their weight and size are lower than wild-type (WT) mice. Bone is undermineralized in fetuses and young adults, but not in older (> or =12 mo) BSP(-/-) mice. At 4 mo, BSP(-/-) mice display thinner cortical bones than WT, but greater trabecular bone volume with very low bone formation rate, which indicates reduced resorption, as confirmed by lower osteoclast surfaces. Although the frequency of total colonies and committed osteoblast colonies is the same, fewer mineralized colonies expressing decreased levels of osteoblast markers form in BSP(-/-) versus WT bone marrow stromal cultures. BSP(-/-) hematopoietic progenitors form fewer osteoclasts, but their resorptive activity on dentin is normal. Tail-suspended BSP(-/-) mice lose bone in hindlimbs, as expected. In conclusion, BSP deficiency impairs bone growth and mineralization, concomitant with dramatically reduced bone formation. It does not, however, prevent the bone loss resulting from loss of mechanical stimulation, a phenotype that is clearly different from OPN(-/-) mice.