TRPV4 mediates cell damage induced by hyperphysiological compression and regulates COX2/PGE2 in intervertebral discs

Background Aberrant mechanical loading of the spine causes intervertebral disc (IVD) degeneration and low back pain. Current therapies do not target the mediators of the underlying mechanosensing and mechanotransduction pathways, as these are poorly understood. This study investigated the role of the mechanosensitive transient receptor potential vanilloid 4 (TRPV4) ion channel in dynamic compression of bovine nucleus pulposus (NP) cells in vitro and mouse IVDs in vivo. Methods Degenerative changes and the expression of the inflammatory mediator cyclooxygenase 2 (COX2) were examined histologically in the IVDs of mouse tails that were dynamically compressed at a short repetitive hyperphysiological regime (vs sham). Bovine NP cells embedded in an agarose-collagen hydrogel were dynamically compressed at a hyperphysiological regime in the presence or absence of the selective TRPV4 antagonist GSK2193874. Lactate dehydrogenase (LDH) and prostaglandin E2 (PGE2) release, as well as phosphorylation of mitogen-activated protein kinases (MAPKs), were analyzed. Degenerative changes and COX2 expression were further evaluated in the IVDs of trpv4-deficient mice (vs wild-type; WT). Results Dynamic compression caused IVD degeneration in vivo as previously shown but did not affect COX2 expression. Dynamic compression significantly augmented LDH and PGE2 releases in vitro, which were significantly reduced by TRPV4 inhibition. Moreover, TRPV4 inhibition during dynamic compression increased the activation of the extracellular signal-regulated kinases 1/2 (ERK) MAPK pathway by 3.13-fold compared to non-compressed samples. Trpv4-deficient mice displayed mild IVD degeneration and decreased COX2 expression compared to WT mice. Conclusions TRPV4 therefore regulates COX2/PGE2 and mediates cell damage induced by hyperphysiological dynamic compression, possibly via ERK. Targeted TRPV4 inhibition or knockdown might thus constitute promising therapeutic approaches to treat patients suffering from IVD pathologies caused by aberrant mechanical stress

Longitudinal assessment of frailty and osteosarcopenia in an in vivo model of premature aging

Frailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. Although anabolic interventions such as mechanical stimuli are known to promote bone and muscle mass, it remains unclear whether the ability of the musculoskeletal system to sense mechanical signals is maintained with age. A better understanding of the pathophysiology of osteosarcopenia will help to identify interventions to strengthen the musculoskeletal system, which ultimately will be beneficial for the prevention and/or treatment of frailty. With the advent of longitudinal in vivo phenotyping techniques, animal models are of increasing interest in aging studies as disease progression can be monitored over time in multiple tissues of individual animals. This not only provides a more comprehensive analysis of multi-system dysfunctions but also reduces the number of required animals. However, a suitable animal model mimicking frailty and osteosarcopenia is still lacking. Therefore, the presented thesis has been divided into three aims: (i) To develop an approach to study bone mechanobiology in vivo across multiple scales (ii) To develop an approach to study frailty and osteopenia in vivo in a model of premature aging (iii) To longitudinally assess frailty and osteosarcopenia in an in vivo model of premature aging. To address the first aim, a previously developed methodological platform known as “Local in vivo Environment (LivE) histochemistry” was optimized to investigate the relationship between loading frequency and bone adaptation across multiple scales. Specifically, the combination of longitudinal micro-CT imaging with micro-finite element (micro-FE) analysis of mouse caudal vertebrae subjected to either static or cyclic loading at varying frequencies showed that bone adaptation to load is controlled by local mechanical signals with net bone changes logarithmically dependent on loading frequency. In order to assess whether the link between bone remodeling and the local mechanical environments can also be observed at the cellular level, a rigid 2D-3D registration was used to map osteocytes, the orchestrators of bone remodeling, identified on 2D histology sections to the 3D micro-CT data. Following this, the expression of the anti-anabolic and anti-catabolic signaling factors Sclerostin and RANKL were evaluated both globally and locally in relation to the mechanical and remodeling environments. Consistent with the anabolic responses observed at the tissue level, cyclic loading resulted in a down-regulation of osteocytic Sclerostin and RANKL expression on a global level. In line with this, the RANKL expression was lower in regions close to bone formation than in regions close to bone resorption, thus suggesting that alterations in protein levels are locally linked to the in vivo microenvironment. In a second part of this thesis, a long-term in vivo micro-CT imaging approach was coupled with longitudinal assessments of the clinical mouse frailty index (FI), a tool to quantify the accumulation of health deficits, in order to evaluate the suitability of a mouse model of premature aging as a model for frailty and senile osteoporosis. Furthermore, as the proposed method requires repeated scanning over a long period, potential biasing effects of radiation, anesthesia and handling associated with in vivo micro-CT imaging were investigated. Although the long-term imaging approach can lead to small but significant changes in bone morphometric parameters, the comparison between genotypes was not impaired, and the overall health status of the animal (i.e., FI and body-weight) was not affected. Moreover, this study demonstrated that longitudinal designs including baseline measurements already at young age are more powerful at detecting age-related phenotypic changes than those including multiple groups with fewer imaging sessions. Finally, in order to evaluate the suitability of the PolgA model as a model for frailty and osteosarcopenia, the thus established long-term FI and in vivo micro-CT approach was combined with extensive musculoskeletal phenotyping. Concomitant to a higher rate of deficit accumulation, PolgA mice displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as the functionality thereof. In addition to lower muscle weights and fiber area, PolgA showed impairments in grip-strength and concentric muscle forces. Longitudinal micro-CT imaging of the 6th caudal vertebrae showed that PolgA had reduced bone micro-architectural integrity as well as lower bone turnover, thus mimicking senile osteoporosis as observed in humans. Lastly, this study showed that PolgA mutation altered the response to various anabolic stimuli in skeletal muscle and bone, indicating that the mechanoregulation of the musculoskeletal system may indeed change with age. In summary, the application of the multiscale bone mechanobiology approach was successful to improve our understanding of the relationship between loading frequency and trabecular bone adaptation in vivo. Secondly, the application of long-term in vivo micro-CT imaging combined with longitudinal FI assessments was shown to be pivotal for monitoring the development of frailty and senile osteoporosis in PolgA mice. Lastly, the integration of comprehensive musculoskeletal phenotyping showed that prematurely aged PolgA mice mimic multiple signs of frailty and of osteosarcopenia and thus provide a powerful model to improve our understanding of frailty and the aging musculoskeletal system. Taken together, the development of a multiscale mechanobiology approach as well as the identification of a model of frailty and osteosarcopenia provide the groundwork to elucidate the pathophysiology of osteosarcopenia and to test potential interventions, which ultimately will be constructive towards the prevention and/or treatment of frailty

Effects of long-term in vivo micro-CT imaging on hallmarks of osteopenia and frailty in aging mice

ISSN:1932-620

3D Local in vivo Environment (LivE) imaging for single cell protein analysis of bone tissue

The molecular processes behind pathological bone remodelling seen in diseases such as osteoporosis are unclear. However, a recently developed methodological platform known as Local in vivo Environment (LivE) imaging has been used to link cellular expression data to the local remodelling and mechanical environment in 2D sections of bone tissue. The method therefore can be used to give insight into which proteins are important for pathological bone remodelling. However, the cells within bone tissue exist as a 3D network. Therefore extension of LivE to accommodate 3D data may provide additional physiologically relevant information that is not possible to determine using 2D analysis alone. This will have implications for the further understanding of the cellular basis that underlies bone diseases such as osteoporosis. Here the LivE imaging technique is expanded to incorporate data from cells in a three dimensional manner via a serial sectioning technique. The methodological steps involved in the LivE imaging approach are defined and the optimisation steps performed are explained in detail

Geroprotectors: A role in the treatment of frailty

The proportion of the population over the age of 65 is growing the most rapidly due to the longevity revolution. Frailty is prevalent in this age group and strongly associated with disability and hospitalization, having a significant impact on the costs of health and social care. New effective interventions to delay or reverse frailty are urgently required. Geroprotectors are a new class of drugs, which target fundamental mechanisms of ageing and show promise in delaying the onset of or boosting resilience in frail older people. However, there are challenges to their clinical translation. Here we review the literature for evidence that frailty can be delayed or reversed and geroprotectors can improve frailty in murine models and in patients. We will then discuss the challenges, which make their clinical testing complex and propose potential options for moving forward

Hallmarks of Frailty and Osteosarcopenia in Prematurely Aged PolgA (D257A/D257A) Mice

Background Frailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty, suitable animal models are needed. Methods To evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild‐type littermates (PolgA(+/+), WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors, and bone was assessed. Results PolgA(D257A/D257A) accumulated health deficits at a higher rate compared with WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, P < 0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (−14%, P < 0.05, −23%, P < 0.0001) and fibre area (−20%, P < 0.05, −22%, P < 0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip strength and concentric muscle forces (P < 0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signalling response of the mechanistic target of rapamycin complex 1, myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro‐computed tomography analysis of the sixth caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular, and cortical thickness, P < 0.05) as well as reduced remodelling activities (e.g. bone formation and resorption rate, P < 0.05) compared with WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load‐induced bone adaptation, suggesting reduced mechanosensitivity with age. Conclusions PolgA(D257A/D257A) mutation leads to hallmarks of age‐related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.ISSN:2190-5991ISSN:2190-600

Mechanostat parameters estimated from time-lapsed in vivo micro-computed tomography data of mechanically driven bone adaptation are logarithmically dependent on loading frequency

Mechanical loading is a key factor governing bone adaptation. Both preclinical and clinical studies have demonstrated its effects on bone tissue, which were also notably predicted in the mechanostat theory. Indeed, existing methods to quantify bone mechanoregulation have successfully associated the frequency of (re)modeling events with local mechanical signals, combining time-lapsed in vivo micro-computed tomography (micro-CT) imaging and micro-finite element (micro-FE) analysis. However, a correlation between the local surface velocity of (re)modeling events and mechanical signals has not been shown. As many degenerative bone diseases have also been linked to impaired bone (re)modeling, this relationship could provide an advantage in detecting the effects of such conditions and advance our understanding of the underlying mechanisms. Therefore, in this study, we introduce a novel method to estimate (re)modeling velocity curves from time-lapsed in vivo mouse caudal vertebrae data under static and cyclic mechanical loading. These curves can be fitted with piecewise linear functions as proposed in the mechanostat theory. Accordingly, new (re)modeling parameters can be derived from such data, including formation saturation levels, resorption velocity moduli, and (re)modeling thresholds. Our results revealed that the norm of the gradient of strain energy density yielded the highest accuracy in quantifying mechanoregulation data using micro-finite element analysis with homogeneous material properties, while effective strain was the best predictor for micro-finite element analysis with heterogeneous material properties. Furthermore, (re)modeling velocity curves could be accurately described with piecewise linear and hyperbola functions (root mean square error below 0.2 µm/day for weekly analysis), and several (re)modeling parameters determined from these curves followed a logarithmic relationship with loading frequency. Crucially, (re)modeling velocity curves and derived parameters could detect differences in mechanically driven bone adaptation, which complemented previous results showing a logarithmic relationship between loading frequency and net change in bone volume fraction over 4 weeks. Together, we expect this data to support the calibration of in silico models of bone adaptation and the characterization of the effects of mechanical loading and pharmaceutical treatment interventions in vivo.ISSN:2296-418

Hallmarks of frailty and osteosarcopenia in prematurely aged PolgA (D257A/D257A)

Background Frailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty, suitable animal models are needed. Methods To evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild‐type littermates (PolgA(+/+), WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors, and bone was assessed. Results PolgA(D257A/D257A) accumulated health deficits at a higher rate compared with WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, P < 0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (−14%, P < 0.05, −23%, P < 0.0001) and fibre area (−20%, P < 0.05, −22%, P < 0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip strength and concentric muscle forces (P < 0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signalling response of the mechanistic target of rapamycin complex 1, myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro‐computed tomography analysis of the sixth caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular, and cortical thickness, P < 0.05) as well as reduced remodelling activities (e.g. bone formation and resorption rate, P < 0.05) compared with WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load‐induced bone adaptation, suggesting reduced mechanosensitivity with age. Conclusions PolgA(D257A/D257A) mutation leads to hallmarks of age‐related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.ISSN:2190-5991ISSN:2190-600

Mechano-Regulation of Trabecular Bone Adaptation Is Controlled by the Local in vivo Environment and Logarithmically Dependent on Loading Frequency

It is well-established that cyclic, but not static, mechanical loading has anabolic effects on bone. However, the function describing the relationship between the loading frequency and the amount of bone adaptation remains unclear. Using a combined experimental and computational approach, this study aimed to investigate whether trabecular bone mechano-regulation is controlled by mechanical signals in the local in vivo environment and dependent on loading frequency. Specifically, by combining in vivo micro-computed tomography (micro-CT) imaging with micro-finite element (micro-FE) analysis, we monitored the changes in microstructural as well as the mechanical in vivo environment [strain energy density (SED) and SED gradient] of mouse caudal vertebrae over 4 weeks of either cyclic loading at varying frequencies of 2, 5, or 10 Hz, respectively, or static loading. Higher values of SED and SED gradient on the local tissue level led to an increased probability of trabecular bone formation and a decreased probability of trabecular bone resorption. In all loading groups, the SED gradient was superior in the determination of local bone formation and resorption events as compared to SED. Cyclic loading induced positive net (re)modeling rates when compared to sham and static loading, mainly due to an increase in mineralizing surface and a decrease in eroded surface. Consequently, bone volume fraction increased over time in 2, 5, and 10 Hz (+15%, +21% and +24%, p ≤ 0.0001), while static loading led to a decrease in bone volume fraction (−9%, p ≤ 0.001). Furthermore, regression analysis revealed a logarithmic relationship between loading frequency and the net change in bone volume fraction over the 4 week observation period (R2 = 0.74). In conclusion, these results suggest that trabecular bone adaptation is regulated by mechanical signals in the local in vivo environment and furthermore, that mechano-regulation is logarithmically dependent on loading frequency with frequencies below a certain threshold having catabolic effects, and those above anabolic effects. This study thereby provides valuable insights toward a better understanding of the mechanical signals influencing trabecular bone formation and resorption in the local in vivo environment.ISSN:2296-418

Evaluation of longitudinal time-lapsed in vivo micro-CT for monitoring fracture healing in mouse femur defect models

Longitudinal in vivo micro-computed tomography (micro-CT) is of interest to non-invasively capture the healing process of individual animals in preclinical fracture healing studies. However, it is not known whether longitudinal imaging itself has an impact on callus formation and remodeling. In this study, a scan group received weekly micro-CT measurements (week 0–6), whereas controls were only scanned post-operatively and at week 5 and 6. Registration of consecutive scans using a branching scheme (bridged vs. unbridged defect) combined with a two-threshold approach enabled assessment of localized bone turnover and mineralization kinetics relevant for monitoring callus remodeling. Weekly micro-CT application did not significantly change any of the assessed callus parameters in the defect and periosteal volumes. This was supported by histomorphometry showing only small amounts of cartilage residuals in both groups, indicating progression towards the end of the healing period. Also, immunohistochemical staining of Sclerostin, previously associated with mediating adverse radiation effects on bone, did not reveal differences between groups. The established longitudinal in vivo micro-CT-based approach allows monitoring of healing phases in mouse femur defect models without significant effects of anesthesia, handling and radiation on callus properties. Therefore, this study supports application of longitudinal in vivo micro-CT for healing-phase-specific monitoring of fracture repair in mice.ISSN:2045-232