Mouse models of accelerated aging in musculoskeletal research for assessing frailty, sarcopenia, and osteoporosis - A review.

Musculoskeletal aging encompasses the decline in bone and muscle function, leading to conditions such as frailty, osteoporosis, and sarcopenia. Unraveling the underlying molecular mechanisms and developing effective treatments are crucial for improving the quality of life for those affected. In this context, accelerated aging models offer valuable insights into these conditions by displaying the hallmarks of human aging. Herein, this review focuses on relevant mouse models of musculoskeletal aging with particular emphasis on frailty, osteoporosis, and sarcopenia. Among the discussed models, PolgA mice in particular exhibit hallmarks of musculoskeletal aging, presenting early-onset frailty, as well as reduced bone and muscle mass that closely resemble human musculoskeletal aging. Ultimately, findings from these models hold promise for advancing interventions targeted at age-related musculoskeletal disorders, effectively addressing the challenges posed by musculoskeletal aging and associated conditions in humans.

Microvascular Disease Associates with Larger Osteocyte Lacunae in Cortical Bone in Type 2 Diabetes Mellitus.

Clinical studies indicate that microvascular disease (MVD) affects bone microstructure and decreases bone strength in type 2 diabetes mellitus (T2D). Osteocytes are housed in small voids within the bone matrix and lacunae and act as sensors of mechanical forces in bone. These cells regulate osteoclastic bone resorption and osteoblastic bone formation as well as osteocytic perilacunar remodeling. We hypothesized that MVD changes morphometric osteocyte lacunar parameters in individuals with T2D. We collected iliac crest bone biopsies from 35 individuals (10 female, 25 male) with T2D with MVD (15%) or without MVD (21%) with a median age of 67 years (interquartile range [IQR] 62-72 years). The participants were included based on c-peptide levels >700 pmol L-1, absence of anti-GAD65 antibodies, and glycated hemoglobin (HbA1c) levels between 40 and 82 mmol mol-1 or 5.8% and 9.7%, respectively. We assessed osteocyte lacunar morphometric parameters in trabecular and cortical bone regions using micro-computed tomography (micro-CT) at a nominal resolution of 1.2 µm voxel size. The cortical osteocyte lacunar volume (Lc.V) was 7.7% larger (p = 0.05) and more spherical (Lc.Sr, p < 0.01) in the T2D + MVD group. Using linear regression, we found that lacunar density (Lc.N/BV) in trabecular but not cortical bone was associated with HbA1c (p < 0.05, R 2 = 0.067) independently of MVD. Furthermore, Lc.V was larger and Lc.Sr higher in the center than in the periphery of the trabecular and cortical bone regions (p < 0.05). In conclusion, these data imply that MVD may impair skeletal integrity, possibly contributing to increased skeletal fragility in T2D complicated by MVD. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A 3D-Printed Assemblable Bespoke Scaffold as a Versatile Microcryogel Carrier for Site-Specific Regenerative Medicine.

Advances in additive manufacturing have led to diverse patient-specific implant designs utilizing computed tomography, but this requires intensive work and financial implications. Here, Digital Light Processing is used to fabricate a hive-structured assemblable bespoke scaffold (HIVE). HIVE can be manually assembled in any shape/size with ease, so a surgeon can create a scaffold that will best fit a defect before implantation. Simultaneously, it can have site-specific treatments by working as a carrier filled with microcryogels (MC) incorporating different biological factors in different pockets of HIVE. After characterization, possible site-specific applications are investigated by utilizing HIVE as a versatile carrier with incorporated treatments such as growth factors (GF), bioceramic, or cells. HIVE as a GF-carrier shows a controlled release of bone morphogenetic protein/vascular endothelial growth factor (BMP/VEGF) and induced osteogenesis/angiogenesis from human mesenchymal stem cells (hMSC)/human umbilical vein endothelial cells (HUVECs). Furthermore, as a bioceramic-carrier, HIVE demonstrates enhanced mineralization and osteogenesis, and as a HUVEC carrier, it upregulates both osteogenic and angiogenic gene expression of hMSCs. HIVE with different combinations of MCs yields a distinct local effect and successful cell migration is confirmed within assembled HIVEs. Finally, an in vivo rat subcutaneous implantation demonstrates site-specific osteogenesis and angiogenesis.

Trabecular bone remodeling in the aging mouse: A micro-multiphysics agent-based in silico model using single-cell mechanomics.

Bone remodeling is regulated by the interaction between different cells and tissues across many spatial and temporal scales. Notably, in silico models are regarded as powerful tools to further understand the signaling pathways that regulate this intricate spatial cellular interplay. To this end, we have established a 3D multiscale micro-multiphysics agent-based (micro-MPA) in silico model of trabecular bone remodeling using longitudinal in vivo data from the sixth caudal vertebra (CV6) of PolgA(D257A/D257A) mice, a mouse model of premature aging. Our in silico model includes a variety of cells as single agents and receptor-ligand kinetics, mechanomics, diffusion and decay of cytokines which regulate the cells' behavior. We highlighted its capabilities by simulating trabecular bone remodeling in the CV6 of five mice over 4 weeks and we evaluated the static and dynamic morphometry of the trabecular bone microarchitecture. Based on the progression of the average trabecular bone volume fraction (BV/TV), we identified a configuration of the model parameters to simulate homeostatic trabecular bone remodeling, here named basal. Crucially, we also produced anabolic, anti-anabolic, catabolic and anti-catabolic responses with an increase or decrease by one standard deviation in the levels of osteoprotegerin (OPG), receptor activator of nuclear factor kB ligand (RANKL), and sclerostin (Scl) produced by the osteocytes. Our results showed that changes in the levels of OPG and RANKL were positively and negatively correlated with the BV/TV values after 4 weeks in comparison to basal levels, respectively. Conversely, changes in Scl levels produced small fluctuations in BV/TV in comparison to the basal state. From these results, Scl was deemed to be the main driver of equilibrium while RANKL and OPG were shown to be involved in changes in bone volume fraction with potential relevance for age-related bone features. Ultimately, this micro-MPA model provides valuable insights into how cells respond to their local mechanical environment and can help to identify critical pathways affected by degenerative conditions and ageing.

Mesenchyme-derived vertebrate lonesome kinase controls lung organogenesis by altering the matrisome.

Vertebrate lonesome kinase (VLK) is the only known secreted tyrosine kinase and responsible for the phosphorylation of a broad range of secretory pathway-resident and extracellular matrix proteins. However, its cell-type specific functions in vivo are still largely unknown. Therefore, we generated mice lacking the VLK gene (protein kinase domain containing, cytoplasmic (Pkdcc)) in mesenchymal cells. Most of the homozygous mice died shortly after birth, most likely as a consequence of their lung abnormalities and consequent respiratory failure. E18.5 embryonic lungs showed a reduction of alveolar type II cells, smaller bronchi, and an increased lung tissue density. Global mass spectrometry-based quantitative proteomics identified 97 proteins with significantly and at least 1.5-fold differential abundance between genotypes. Twenty-five of these had been assigned to the extracellular region and 15 to the mouse matrisome. Specifically, fibromodulin and matrilin-4, which are involved in extracellular matrix organization, were significantly more abundant in lungs from Pkdcc knockout embryos. These results support a role for mesenchyme-derived VLK in lung development through regulation of matrix dynamics and the resulting modulation of alveolar epithelial cell differentiation.

Hierarchical Porous Monoliths of Steel with Self-Reinforcing Adaptive Properties.

Porous structures offer an attractive approach to reduce the amount of natural resources used while maintaining relatively high mechanical efficiency. However, for some applications the drop in mechanical properties resulting from the introduction of porosity is too high, which has limited the broader utilization of porous materials in industry. Here, it is shown that steel monoliths can be designed to display high mechanical efficiency and reversible self-reinforcing properties when made with porous architectures with up to three hierarchical levels. Ultralight steel structures that can float on water and autonomously adapt their stiffness are manufactured by the thermal reduction and sintering of 3D printed foam templates. Using distinct mechanical testing techniques, image analysis, and finite element simulations, the mechanisms leading to the high mechanical efficiency and self-stiffening ability of the hierarchical porous monoliths are studied. The design and fabrication of mechanically stable porous monoliths using iron as a widely available natural resource is expected to contribute to the future development of functional materials with a more sustainable footprint.

Abnormal morphological features of osteocyte lacunae in adolescent idiopathic scoliosis: A large-scale assessment by ultra-high-resolution micro-computed tomography.

AIM: Abnormal osteocyte lacunar morphology in adolescent idiopathic scoliosis (AIS) has been reported while the results were limited by the number of osteocyte lacunae being quantified. The present study aimed to validate previous findings through (a) comparing morphological features of osteocyte lacunae between AIS patients and controls in spine and ilium using a large-scale assessment, and (b) investigating whether there is an association between the acquired morphological features of osteocyte lacunae and disease severity in AIS. METHOD: Trabecular bone tissue of the facet joint of human vertebrae on both concave and convex sides at the apex of the scoliotic curve were collected from 4 AIS and 5 congenital scoliosis (CS) patients, and also at the same anatomic site from 3 non-scoliosis (NS) subjects intraoperatively. Trabecular bone tissue from ilium was obtained from 12 AIS vs 9 NS subjects during surgery. Osteocyte lacunae were assessed using ultra-high-resolution micro-computed tomography. Clinical information such as age, body mass index (BMI) and radiological Cobb angle of the major curve were collected. RESULTS: There was no significant difference between density of osteocyte lacuna and bone volume fraction (BV/TV) between groups. A total of 230,076 and 78,758 osteocyte lacunae from facet joints of apical vertebra of scoliotic curve and iliac bone were included in the analysis, respectively. In facet joint bone biopsies, lacunar stretch (Lc.St) was higher, and lacunar equancy (Lc.Eq), lacunar oblateness (Lc.Ob), and lacunar sphericity (Lc.Sr) were lower in AIS and CS groups when compared with NS group. CA side was associated with higher Lc.St when compared with CX side. In iliac bone biopsies, Lc.Ob was higher and lacunar surface area (Lc.S) was lower in AIS group than NS group. Median values of Lc.St, Lc.Eq and Lc.Sr were significantly associated with radiological Cobb angle with adjustment for age and BMI (R-squared: 0.576, 0.558 and 0.543, respectively). CONCLUSIONS: This large-scale assessment of osteocyte lacunae confirms that AIS osteocyte lacunae are more oblate in iliac bone that is less influenced by asymmetric loading of the deformed spine than the vertebrae. Shape of osteocyte lacunae in iliac bone is associated with radiological Cobb angle of the major curve in AIS patients, suggesting the likelihood of systemic abnormal osteocyte morphology in AIS. Osteocyte lacunae from concave side of scoliotic curves were more stretched in both AIS and CS groups, which is likely secondary to asymmetric mechanical loading.

The major urinary protein gene cluster knockout mouse as a novel model for translational metabolism research.

Scientific evidence suggests that not only murine scent communication is regulated by major urinary proteins, but that their expression may also vary in response to metabolism via a yet unknown mechanism. Major urinary proteins are expressed mainly in the liver, showing a sexually dimorphic pattern with substantially higher expression in males. Here, we investigate the metabolic implications of a major urinary protein knockout in twelve-week-old male and female C57BL/6N mice during ad libitum feeding. Despite both sexes of major urinary protein knockout mice displayed numerically increased body weight and visceral adipose tissue proportions compared to sex-matched wildtype mice, the main genotype-specific metabolic differences were observed exclusively in males. Male major urinary protein knockout mice exhibited plasma and hepatic lipid accumulation accompanied by a hepatic transcriptome indicating an activation of lipogenesis. These findings match the higher major urinary protein expression in male compared to female wildtype mice, suggesting a more distinct reduction in energy requirements in male compared to female major urinary protein knockout mice. The observed sex-specific anabolic phenotype confirms a role of major urinary protein in metabolism and, since major urinary proteins are not expressed in humans, suggests the major urinary protein knockout mouse as a potential alternative model for translational metabolism research which needs to be further elucidated.

Tissue-Level Regeneration and Remodeling Dynamics are Driven by Mechanical Stimuli in the Microenvironment in a Post-Bridging Loaded Femur Defect Healing Model in Mice.

Bone healing and remodeling are mechanically driven processes. While the generalized response to mechanical stimulation in bone is well-understood, much less is known about the mechanobiology-regulating tissue-scale bone formation and resorption during the reparative and remodeling phases of fracture healing. In this study, we combined computational approaches in the form of finite element analysis and experimental approaches by using a loaded femoral defect model in mice to investigate the role of mechanical stimulation in the microenvironment of bone. Specifically, we used longitudinal micro-computed tomography to observe temporal changes in bone at different densities and micro-finite element analysis to map the mechanics of the microenvironment to tissue-scale formation, quiescence (no change in bone presence between time points), and resorption dynamics in the late reparative and remodeling phases (post bridging). Increasing levels of effective strain led to increasing conditional probability of bone formation, while decreasing levels of effective strain led to increasing probability of bone resorption. In addition, the analysis of mineralization dynamics showed both a temporal and effective strain level-dependent behavior. A logarithmic-like response was displayed, where the conditional probability of bone formation or resorption increased rapidly and plateaued or fell rapidly and plateaued as mechanical strain increased.

Large-scale osteocyte lacunar morphological analysis of transiliac bone in normal and osteoporotic premenopausal women.

Bone's ability to adapt is governed by the network of embedded osteocytes, which inhabit individual pores called lacunae. The morphology of these lacunae and their resident osteocytes are known to change with age and diseases such as postmenopausal osteoporosis. However, it is unclear whether alterations in lacunar morphology are present in younger populations with osteoporosis. To investigate this, we implemented a previously validated methodology to image and quantify the three-dimensional morphometries of lacunae on a large scale with ultra-high-resolution micro-computed tomography (microCT) in transiliac bone biopsies from three groups of premenopausal women: control n = 39; idiopathic osteoporosis (IOP) n = 45; idiopathic low BMD (ILBMD) n = 19. Lacunar morphometric parameters were measured in both trabecular and cortical bone such as lacunar density (Lc.N/BV), lacunar volume (Lc.V), and lacunar sphericity (Lc.Sr). These were then compared against each other and also with previously measured tissue morphometries such as bone volume density (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and others. We detected no differences in lacunar morphology between the IOP, ILBMD and healthy premenopausal women. In contrast, we did find significant differences between lacunar morphologies including Lc.N/BV, Lc. V, and Lc. Sr in cortical and trabecular regions within all three groups (p < 0.001), which was consistent with our previous findings on a subgroup of the healthy group. Furthermore, we discovered strong correlations between Lc. Sr from trabecular regions with the measured BV/TV (R = -0.90, p < 0.05). The findings and comprehensive lacunar dataset we present here will be a crucial foundation for future investigations of the relationship between osteocyte lacunar morphology and disease.

Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging.

Fracture healing is regulated by mechanical loading. Understanding the underlying mechanisms during the different healing phases is required for targeted mechanical intervention therapies. Here, the influence of individualized cyclic mechanical loading on the remodelling phase of fracture healing was assessed in a non-critical-sized mouse femur defect model. After bridging of the defect, a loading group (n = 10) received individualized cyclic mechanical loading (8-16 N, 10 Hz, 5 min, 3 × /week) based on computed strain distribution in the mineralized callus using animal-specific real-time micro-finite element analysis with 2D/3D visualizations and strain histograms. Controls (n = 10) received 0 N treatment at the same post-operative time-points. By registration of consecutive scans, structural and dynamic callus morphometric parameters were followed in three callus sub-volumes and the adjacent cortex showing that the remodelling phase of fracture healing is highly responsive to cyclic mechanical loading with changes in dynamic parameters leading to significantly larger formation of mineralized callus and higher degree of mineralization. Loading-mediated maintenance of callus remodelling was associated with distinct effects on Wnt-signalling-associated molecular targets Sclerostin and RANKL in callus sub-regions and the adjacent cortex (n = 1/group). Given these distinct local protein expression patterns induced by cyclic mechanical loading during callus remodelling, the femur defect loading model with individualized load application seems suitable to further understand the local spatio-temporal mechano-molecular regulation of the different fracture healing phases.

Large-scale quantification of human osteocyte lacunar morphological biomarkers as assessed by ultra-high-resolution desktop micro-computed tomography.

Ultra-high-resolution imaging of the osteocyte lacuno-canalicular network (LCN) three-dimensionally (3D) in a high-throughput fashion has greatly improved the morphological knowledge about the constituent structures - positioning them as potential biomarkers. Technologies such as serial focused ion beam/scanning electron microscopy (FIB/SEM) and confocal scanning laser microscopy (CLSM) can image in extremely high resolution, yet only capture a small number of lacunae. Synchrotron radiation computed tomography (SR-CT) can image with both high resolution and high throughput but has a limited availability. Desktop micro-computed tomography (micro-CT) provides an attractive balance: high-throughput imaging on the micron level without the restrictions of SR-CT availability. In this study, accuracy, reproducibility, and sensitivity of large-scale quantification of human osteocyte lacunar morphometries were assessed by ultra-high-resolution desktop micro-computed tomography. For this purpose, thirty-one transiliac human bone biopsies containing trabecular and cortical regions were imaged using ultra-high-resolution desktop micro-CT at a nominal isotropic voxel resolution of 1.2 µm. The resulting 3D images were segmented, component labeled, and the following morphometric parameters of 7.71 million lacunae were measured: Lacunar number (Lc.N), density (Lc.N/BV), porosity (Lc.TV/BV), volume (Lc.V), surface area (Lc.S), surface area to volume ratio (Lc.S/Lc.V), stretch (Lc.St), oblateness (Lc.Ob), sphericity (Lc.Sr), equancy (Lc.Eq), and angle (Lc.θ). Accuracy was quantified by comparing automated lacunar identification to manual identification. Mean true positive rate (TPR), false positive rate (FPR), and false negative rate (FNR) were 89.0%, 3.4%, and 11.0%, respectively. Regarding the reproducibility of lacunar morphometry from repeated measurements, precision errors were low (0.2-3.0%) and intraclass correlation coefficients were high (0.960-0.999). Significant differences between cortical and trabecular regions (p<0.001) existed for Lc.N/BV, Lc.TV/BV, local lacunar surface area (), and local lacunar volume (), all of which demonstrate the sensitivity of the method and are possible biomarker candidates. This study provides the foundation required for future large-scale morphometric studies using ultra-high-resolution desktop micro-CT and high-throughput analysis of millions of osteocyte lacunae in human bone samples.

Real-time finite element analysis allows homogenization of tissue scale strains and reduces variance in a mouse defect healing model.

Mechanical loading allows both investigation into the mechano-regulation of fracture healing as well as interventions to improve fracture-healing outcomes such as delayed healing or non-unions. However, loading is seldom individualised or even targeted to an effective mechanical stimulus level within the bone tissue. In this study, we use micro-finite element analysis to demonstrate the result of using a constant loading assumption for all mouse femurs in a given group. We then contrast this with the application of an adaptive loading approach, denoted real time Finite Element adaptation, in which micro-computed tomography images provide the basis for micro-FE based simulations and the resulting strains are manipulated and targeted to a reference distribution. Using this approach, we demonstrate that individualised femoral loading leads to a better-specified strain distribution and lower variance in tissue mechanical stimulus across all mice, both longitudinally and cross-sectionally, while making sure that no overloading is occurring leading to refracture of the femur bones.

Spatio-temporal characterization of fracture healing patterns and assessment of biomaterials by time-lapsed in vivo micro-computed tomography.

Thorough preclinical evaluation of functionalized biomaterials for treatment of large bone defects is essential prior to clinical application. Using in vivo micro-computed tomography (micro-CT) and mouse femoral defect models with different defect sizes, we were able to detect spatio-temporal healing patterns indicative of physiological and impaired healing in three defect sub-volumes and the adjacent cortex. The time-lapsed in vivo micro-CT-based approach was then applied to evaluate the bone regeneration potential of functionalized biomaterials using collagen and bone morphogenetic protein (BMP-2). Both collagen and BMP-2 treatment led to distinct changes in bone turnover in the different healing phases. Despite increased periosteal bone formation, 87.5% of the defects treated with collagen scaffolds resulted in non-unions. Additional BMP-2 application significantly accelerated the healing process and increased the union rate to 100%. This study further shows potential of time-lapsed in vivo micro-CT for capturing spatio-temporal deviations preceding non-union formation and how this can be prevented by application of functionalized biomaterials. This study therefore supports the application of longitudinal in vivo micro-CT for discrimination of normal and disturbed healing patterns and for the spatio-temporal characterization of the bone regeneration capacity of functionalized biomaterials.

Application of subject-specific adaptive mechanical loading for bone healing in a mouse tail vertebral defect.

Methods to repair bone defects arising from trauma, resection, or disease, continue to be sought after. Cyclic mechanical loading is well established to influence bone (re)modelling activity, in which bone formation and resorption are correlated to micro-scale strain. Based on this, the application of mechanical stimulation across a bone defect could improve healing. However, if ignoring the mechanical integrity of defected bone, loading regimes have a high potential to either cause damage or be ineffective. This study explores real-time finite element (rtFE) methods that use three-dimensional structural analyses from micro-computed tomography images to estimate effective peak cyclic loads in a subject-specific and time-dependent manner. It demonstrates the concept in a cyclically loaded mouse caudal vertebral bone defect model. Using rtFE analysis combined with adaptive mechanical loading, mouse bone healing was significantly improved over non-loaded controls, with no incidence of vertebral fractures. Such rtFE-driven adaptive loading regimes demonstrated here could be relevant to clinical bone defect healing scenarios, where mechanical loading can become patient-specific and more efficacious. This is achieved by accounting for initial bone defect conditions and spatio-temporal healing, both being factors that are always unique to the patient.

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 (R 2 = 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.

Effects of Early Life Stress on Bone Homeostasis in Mice and Humans.

Bone pathology is frequent in stressed individuals. A comprehensive examination of mechanisms linking life stress, depression and disturbed bone homeostasis is missing. In this translational study, mice exposed to early life stress (MSUS) were examined for bone microarchitecture (µCT), metabolism (qPCR/ELISA), and neuronal stress mediator expression (qPCR) and compared with a sample of depressive patients with or without early life stress by analyzing bone mineral density (BMD) (DXA) and metabolic changes in serum (osteocalcin, PINP, CTX-I). MSUS mice showed a significant decrease in NGF, NPYR1, VIPR1 and TACR1 expression, higher innervation density in bone, and increased serum levels of CTX-I, suggesting a milieu in favor of catabolic bone turnover. MSUS mice had a significantly lower body weight compared to control mice, and this caused minor effects on bone microarchitecture. Depressive patients with experiences of childhood neglect also showed a catabolic pattern. A significant reduction in BMD was observed in depressive patients with childhood abuse and stressful life events during childhood. Therefore, future studies on prevention and treatment strategies for both mental and bone disease should consider early life stress as a risk factor for bone pathologies.

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

In vivo micro-CT has already been used to monitor microstructural changes of bone in mice of different ages and in models of age-related diseases such as osteoporosis. However, as aging is accompanied by frailty and subsequent increased sensitivity to external stimuli such as handling and anesthesia, the extent to which longitudinal imaging can be applied in aging studies remains unclear. Consequently, the potential of monitoring individual mice during the entire aging process-from healthy to frail status-has not yet been exploited. In this study, we assessed the effects of long-term in vivo micro-CT imaging-consisting of 11 imaging sessions over 20 weeks-on hallmarks of aging both on a local (i.e., static and dynamic bone morphometry) and systemic (i.e., frailty index (FI) and body weight) level at various stages of the aging process. Furthermore, using a premature aging model (PolgA(D257A/D257A)), we assessed whether these effects differ between genotypes. The 6th caudal vertebrae of 4 groups of mice (PolgA(D257A/D257A) and PolgA(+/+)) were monitored by in vivo micro-CT every 2 weeks. One group was subjected to 11 scans between weeks 20 and 40 of age, whereas the other groups were subjected to 5 scans between weeks 26-34, 32-40 and 40-46, respectively. The long-term monitoring approach showed small but significant changes in the static bone morphometric parameters compared to the other groups. However, no interaction effect between groups and genotype was found, suggesting that PolgA mutation does not render bone more or less susceptible to long-term micro-CT imaging. The differences between groups observed in the static morphometric parameters were less pronounced in the dynamic morphometric parameters. Moreover, the body weight and FI were not affected by more frequent imaging sessions. Finally, we observed that longitudinal designs including baseline measurements at young adult age are more powerful at detecting effects of in vivo micro-CT imaging on hallmarks of aging than cross-sectional comparisons between multiple groups of aged mice subjected to fewer imaging sessions.

Enhancing the regenerative effectiveness of growth factors by local inhibition of interleukin-1 receptor signaling.

Although growth factors (GFs) are key molecules for regenerative medicine, their use has been limited by issues associated with suboptimal delivery systems and incomplete understanding of their signaling dynamics. Here, we explored how proinflammatory signals affect GF regenerative potential. Using bone regeneration in mouse, we found that the regenerative capacity of two clinically relevant GFs (BMP-2 and PDGF-BB) is impaired by interleukin-1 receptor (IL-1R1). Mechanistically, IL-1R1 activation in bone-forming cells desensitizes them to GFs and accelerates senescence. Moreover, administration of the GFs triggers IL-1 release by macrophages. To provide localized and sustained IL-1R1 inhibition, we engineered IL-1R antagonist (IL-1Ra) to bind the extracellular matrix (ECM) very strongly and demonstrate that codelivering GFs with ECM-binding IL-1Ra induces superior regeneration. Thus, we highlight that GF regenerative activity is hindered by proinflammatory signals, and GF-based therapies should integrate immunomodulation. Particularly, ECM-binding IL-1Ra holds clinical translational potential by enhancing efficacy of GF therapies.

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.