Osteocyte calcium signals encode strain magnitude and loading frequency in vivo.

Osteocytes are considered to be the major mechanosensory cells of bone, but how osteocytes in vivo process, perceive, and respond to mechanical loading remains poorly understood. Intracellular calcium (Ca2+) signaling resulting from mechanical stimulation has been widely studied in osteocytes in vitro and in bone explants, but has yet to be examined in vivo. This is achieved herein by using a three-point bending device which is capable of delivering well-defined mechanical loads to metatarsal bones of living mice while simultaneously monitoring the intracellular Ca2+ responses of individual osteocytes by using a genetically encoded fluorescent Ca2+ indicator. Osteocyte responses are imaged by using multiphoton fluorescence microscopy. We investigated the in vivo responses of osteocytes to strains ranging from 250 to 3,000 [Formula: see text] and frequencies from 0.5 to 2 Hz, which are characteristic of physiological conditions reported for bone. At all loading frequencies examined, the number of responding osteocytes increased strongly with applied strain magnitude. However, Ca2+ intensity within responding osteocytes did not change significantly with physiological loading magnitudes. Our studies offer a glimpse into how these critical bone cells respond to mechanical load in vivo, as well as provide a technique to determine how the cells encode magnitude and frequency of loading.

Increased in vivo patellofemoral loading after total knee arthroplasty in resurfaced patellae.

PURPOSE: The primary purpose of the study was to investigate if and how patellar bone tracer uptake (BTU) distribution in SPECT/CT is influenced by patellar resurfacing and the position of femoral and tibial TKA component position. METHODS: A total of 104 knees of 103 consecutive patients who underwent primary TKA were prospectively investigated. Primary patellar resurfacing was done in 40 knees while 64 had a TKA without patellar resurfacing. All patients underwent clinical assessment using the knee society score (KSS) and standardized radiographs and Tc-99m-HDP-SPECT/CT before and 12 and 24 months after TKA. Measurements of BTU including intensity and anatomical distribution pattern in eight different patellar regions were performed. Tibial and femoral TKA component position was assessed from 3D reconstructed CT data. Patellar height, thickness and tilt were measured and the distance between the tibial tuberosity and the trochlear groove (TT-TG) was measured. Univariate analysis was performed to identify differences between the two groups (p < 0.05). RESULTS: Significantly higher BTU was found in the anterior, non-articular, areas of the patella in patients who underwent patellar resurfacing (p < 0.05). The BTU pattern was similar between the groups, as the maximal uptake in both groups was seen in the superior posterior parts and the minimal uptake was seen in the inferior anterior parts. The mean postoperative KSS was significantly higher in the unresurfaced group after 12 months (p < 0.05), but with no significant difference after 24 months. CONCLUSIONS: Based on the findings of the present study, patellar resurfacing is related to significantly higher BTU in the anterior parts of the patella and lower clinical outcomes. In light of these results, routine patellar resurfacing as part of a primary TKA might be reevaluated. SPECT/CT enables a precise localization of the BTU and might be considered as the ideal imaging modality for evaluation and investigate of patellofemoral disorders after TKA.

Adaptive enhancement of apatite crystal orientation and Young's modulus under elevated load in rat ulnar cortical bone.

Functional adaptation refers to the active modification of bone structure according to the mechanical loads applied daily to maintain its mechanical integrity and adapt to the environment. Functional adaptation relates to bone mass, bone mineral density (BMD), and bone morphology (e.g., trabecular bone architecture). In this study, we discovered for the first time that another form of bone functional adaptation of a cortical bone involves a change in bone quality determined by the preferential orientation of apatite nano-crystallite, a key component of the bone. An in vivo rat ulnar axial loading model was adopted, to which a 3-15 N compressive load was applied, resulting in approximately 440-3200 µÉ› of compression in the bone surface. In the loaded ulnae, the degree of preferential apatite c-axis orientation along the ulnar long axis increased in a dose-dependent manner up to 13 N, whereas the increase in BMD was not dose-dependent. The Young's modulus along the same direction was enhanced as a function of the degree of apatite orientation. This finding indicates that bone has a mechanism that modifies the directionality (anisotropy) of its microstructure, strengthening itself specifically in the loaded direction. BMD, a scalar quantity, does not allow for load-direction-specific strengthening. Functional adaptation through changes in apatite orientation is an excellent strategy for bones to efficiently change their strength in response to external loading, which is mostly anisotropic.

In vivo load on knee, hip and spine during manual materials handling with two lifting techniques.

It is generally accepted that the lifting technique strongly influences physical loads within the human body and, thus, the risk of musculoskeletal disorders. However, there is a lack of knowledge regarding whether particular lifting techniques are effective in reducing loads. Hence, this retrospective study quantified (partly published) in vivo loads at joints within the human body during two typical lifting techniques, stoop lifting and squat lifting. Patients who had received instrumented implants underwent in vivo load measurements at either the knee (two patients), the hip (eight patients), or the upper lumbar spine (four patients) while lifting a 10 kg weight frontally with either straight (stoop) or bent (squat) knees. Contact forces and moments and the orientation of the contact force vector were determined and examined using the paired t test of Statistical Parametric Mapping. The two lifting techniques did not differ in terms of load magnitudes but did differ in terms of directions: (i) at the hip joint, the load vector varied significantly (p < 0.05) in the frontal and sagittal planes, (ii) at the knee joint, the load vector differed significantly (p < 0.05) in the sagittal plane (iii) while the load vector and magnitude did not differ at the upper lumbar spine (p > 0.05). Our findings indicate that the lifting technique causes changes in the orientation rather than the magnitude of lower extremity joint contact loads. Even though this quantification could only be performed in a small group of patients, the quantification of the relevance of such lifting technique recommendations will hopefully guide future recommendations towards a more scientific interpretation.

Computationally derived endosteal strain and strain gradients correlate with increased bone formation in an axially loaded murine tibia model.

Osteoporosis is a common metabolic bone disorder characterized by low bone mass and microstructural degradation of bone tissue due to a derailed bone remodeling process. A deeper understanding of the mechanobiological phenomena that modulate the bone remodeling response to mechanical loading in a healthy bone is crucial to develop treatments. Rodent models have provided invaluable insight into the mechanobiological mechanisms regulating bone adaptation in response to dynamic mechanic stimuli. This study sheds light on these aspects by means of assessing the mechanical environment of the cortical and cancellous tissue to in vivo dynamic compressive loading within the mouse tibia using microCT-based finite element model in combination with diaphyseal strain gauge measures. Additionally, this work describes the relation between the mid-diaphyseal strains and strain gradients from the finite element analysis and bone formation measures from time-lapse in vivo tibial loading with a fluorochrome-derived histomorphometry analysis. The mouse tibial loading model demonstrated that cancellous strains were lower than those in the midshaft cortical bone. Sensitivity analyses demonstrated that the material property of cortical bone was the most significant model parameter. The computationally-modeled strains and strain gradients correlated significantly to the histologically-measured bone formation thickness at the mid-diaphyseal cross-section of the mouse tibia.

Elucidating the mechano-molecular dynamics of TRAP activity using CRISPR/Cas9 mediated fluorescent reporter mice.

Osteoclasts are essential for bone remodeling by adapting their resorptive activity in response to their mechanical in vivo environment. However, the molecular mechanisms underlying this process remain unclear. Here, we demonstrated the role of tartrate-resistant acid phosphatase (TRAP, Acp5), a key enzyme secreted by osteoclasts, in bone remodeling and mechanosensitivity. Using CRISPR/Cas9 reporter mice, we demonstrated bone cell reporter (BCRIbsp/Acp5) mice feature fluorescent TRAP-deficient osteoclasts and examined their activity during mechanically driven trabecular bone remodeling. Although BCRIbsp/Acp5 mice exhibited trabecular bone impairments and reduced resorption capacity in vitro, RNA sequencing revealed unchanged levels of key osteoclast-associated genes such as Ctsk, Mmp9, and Calcr. These findings, in conjunction with serum carboxy-terminal collagen crosslinks (CTX) and in vivo mechanical loading outcomes collectively indicated an unaltered bone resorption capacity of osteoclasts in vivo. Furthermore, we demonstrated similar mechanoregulation during trabecular bone remodeling in BCRIbsp/Acp5 and wild-type (WT) mice. Hence, this study provides valuable insights into the dynamics of TRAP activity in the context of bone remodeling and mechanosensation.

Lower-limb internal loading and potential consequences for fracture healing.

Introduction: Mechanical loading is known to determine the course of bone fracture healing. We hypothesise that lower limb long bone loading differs with knee flexion angle during walking and frontal knee alignment, which affects fracture healing success. Materials and methods: Using our musculoskeletal in silico modelling constrained against in vivo data from patients with instrumented knee implants allowed us to assess internal loads in femur and tibia. These internal forces were associated with the clinical outcome of fracture healing in a relevant cohort of 178 extra-articular femur and tibia fractures in patients using a retrospective approach. Results: Mean peak forces differed with femoral compression (1,330-1,936 N at mid-shaft) amounting to about half of tibial compression (2,299-5,224 N). Mean peak bending moments in the frontal plane were greater in the femur (71-130 Nm) than in the tibia (from 26 to 43 Nm), each increasing proximally. Bending in the sagittal plane showed smaller mean peak bending moments in the femur (-38 to 43 Nm) reaching substantially higher values in the tibia (-63 to -175 Nm) with a peak proximally. Peak torsional moments had opposite directions for the femur (-13 to -40 Nm) versus tibia (15-48 Nm) with an increase towards the proximal end in both. Femoral fractures showed significantly lower scores in the modified Radiological Union Scale for Tibia (mRUST) at last follow-up (p < 0.001) compared to tibial fractures. Specifically, compression (r = 0.304), sagittal bending (r = 0.259), and frontal bending (r = -0.318) showed strong associations (p < 0.001) to mRUST at last follow-up. This was not the case for age, body weight, or localisation alone. Discussion: This study showed that moments in femur and tibia tend to decrease towards their distal ends. Tibial load components were influenced by knee flexion angle, especially at push-off, while static frontal alignment played a smaller role. Our results indicate that femur and tibia are loaded differently and thus require adapted fracture fixation considering load components rather than just overall load level.

Patellar tendon elastic properties derived from in vivo loading and kinematics.

Patellar complications frequently limit the success of total knee arthroplasty. In addition to the musculoskeletal forces themselves, patellar tendon elastic properties are essential for driving patellar loading. Elastic properties reported in the literature exhibit high variability and appear to differ according to the methodologies used. Specifically in total knee arthroplasty patients, only limited knowledge exists on in vivo elastic properties and their corresponding loads. For the first time, we report stiffness, Young's modulus, and forces of the patellar tendon, derived from four patients with telemetric total knee arthroplasties using a combined imaging and measurement approach. To achieve this, synchronous in vivo telemetric assessment of tibio-femoral contact forces and fluoroscopic assessment of knee kinematics, along with full body motion capture and ground reaction forces, fed musculoskeletal multi-body models to quantify patellar tendon loading and elongation. Mechanical patellar tendon properties were calculated during a squat and a sit-stand-sit activity, with resulting tendon stiffness and Young's modulus ranging from 511 to 1166 N/mm and 259 to 504 MPa, respectively. During these activities, the patellar tendon force reached peak values between 1.31 and 2.79 bodyweight, reaching levels of just âˆ¼0.5 bodyweight below the tibio-femoral forces. The results of this study provide valuable input data for mechanical simulations of the patellar tendon and the whole resurfaced knee.

Positive interactions of mechanical loading and PTH treatments on spatio-temporal bone remodelling.

Osteoporosis is one of the most common skeletal diseases, but current therapies are limited to generalized antiresorptive or anabolic interventions, which do not target regions that would benefit from improvements to skeletal health. To improve the evaluation of treatment plans, we used a spatio-temporal multiscale approach that combines longitudinal in vivo micro-computed tomography (micro-CT) and in silico subject-specific finite element modeling to quantitatively map bone adaptation changes due to disease and treatment at high resolution. Our findings show time and region-dependent modifications in bone remodelling following one and two sets of mechanical loading and/or pharmacological interventions. The multiscale results highlighted that the distal section was unaffected by mechanical loading alone but the proximal tibia had the greatest gain from positive interactions of combined therapies. Mechanical loading abated the catabolic effect of PTH, but the main benefit of combined treatments occurred from the additive interactions of the two therapies in periosteal apposition. These results provide detailed insight into the efficacy of combined treatments, facilitating the optimisation of dosage and treatment duration in preclinical mouse studies, and the development of novel interventions for skeletal diseases. STATEMENT OF SIGNIFICANCE: Combined mechanical loading and pharmacotherapy have the potential to slow osteoporosis-induced bone loss but current therapies do not target the regions in need of strengthening. We show for the first time spatial region-dependant interactions between PTH and mechanical loading treatment in OVX mouse tibiae, highlighting local regions in the tibia that benefitted from separate and combined treatments. Combined experimental-computational analysis also detailed the lasting period of each treatment per location in the tibia, the extent of positive (or negative) interactions of the combined therapies, and the impact of each treatment on the regulation of bone adaptation spatio-temporally. This approach can be used to create hypothesis about the interactions of different treatments to optimise the design of biomaterials and medical interventions.

Dynamic Knee Joint Line Orientation Is Not Predictive of Tibio-Femoral Load Distribution During Walking.

Some approaches in total knee arthroplasty aim for an oblique joint line to achieve an even medio-lateral load distribution across the condyles during the stance phase of gait. While there is much focus on the angulation of the joint line in static frontal radiographs, precise knowledge of the associated dynamic joint line orientation and the internal joint loading is limited. The aim of this study was to analyze how static alignment in frontal radiographs relates to dynamic alignment and load distribution, based on direct measurements of the internal joint loading and kinematics. A unique and novel combination of telemetrically measured in vivo knee joint loading and simultaneous internal joint kinematics derived from mobile fluoroscopy ("CAMS-Knee dataset") was employed to access the dynamic alignment and internal joint loading in 6 TKA patients during level walking. Static alignment was measured in standard frontal postoperative radiographs while external adduction moments were computed based on ground reaction forces. Both static and dynamic parameters were analyzed to identify correlations using linear and non-linear regression. At peak loading during gait, the joint line was tilted laterally by 4°-7° compared to the static joint line in most patients. This dynamic joint line tilt did not show a strong correlation with the medial force (R 2: 0.17) or with the mediolateral force distribution (pseudo R 2: 0.19). However, the external adduction moment showed a strong correlation with the medial force (R 2: 0.85) and with the mediolateral force distribution (pseudo R 2: 0.78). Alignment measured in static radiographs has only limited predictive power for dynamic kinematics and loading, and even the dynamic orientation of the joint line is not an important factor for the medio-lateral knee load distribution. Preventive and rehabilitative measures should focus on the external knee adduction moment based on the vertical and horizontal components of the ground reaction forces.

Bone remodelling in the mouse tibia is spatio-temporally modulated by oestrogen deficiency and external mechanical loading: A combined in vivo/in silico study.

Osteoporosis disrupts the healthy remodelling process in bone and affects its mechanical properties. Mechanical loading has been shown to be effective in stimulating bone formation to mitigate initial bone loss. However, no study has investigated the effects of repeated mechanical loading, with a pause of one week in between, in the mouse tibia with oestrogen deficiency. This study uses a combined experimental and computational approach, through longitudinal monitoring with micro-computed tomography, to evaluate the effects of loading on bone adaptation in the tibiae of ovariectomised (OVX) C57BL/6 mice from 14 to 22 weeks of age. Micro-FE models coupled with bone adaptation algorithms were used to estimate changes in local tissue strains due to OVX and mechanical loading, and to quantify the relationship between local strain and remodelling. The first in vivo mechanical loading increased apposition, by 50-150%, while resorption decreased by 50-60%. Both endosteal and periosteal resorption increased despite the second mechanical loading, and periosteal resorption was up to 70% higher than that after the first loading. This was found to correlate with an initial decrease in average strain energy density after the first loading, which was lower and more localised after the second loading. Predictions of bone adaptation showed that between 50 and 90% of the load-induced bone apposition is linearly strain driven at the organ-level, but resorption is more biologically driven at the local level. The results imply that a systematic increase in peak load or loading rate may be required to achieve a similar bone adaptation rate in specific regions of interests.

Load-sharing in the lumbosacral spine in neutral standing & flexed postures - A combined finite element and inverse static study.

Understanding load-sharing in the spine during in-vivo conditions is critical for better spinal implant design and testing. Previous studies of load-sharing that considered actual spinal geometry applied compressive follower load, with or without moment, to simulate muscle forces. Other studies used musculoskeletal models, which include muscle forces, but model the discs by simple beams or spherical joints and ignore the articular facet joints. This study investigated load-sharing in neutral standing and flexed postures using a detailed Finite Element (FE) model of the ligamentous lumbosacral spine, where muscle forces, gravity loads and intra-abdominal pressure, as predicted by a musculoskeletal model of the upper body, are input into the FE model. Flexion was simulated by applying vertebral rotations following spine rhythm measured in a previous in-vivo study, to the musculoskeletal model. The FE model predicted intradiscal pressure (IDP), strains in the annular fibers, contact forces in the facet joints, and forces in the ligaments. The disc forces and moments were determined using equilibrium equations, which considered the applied loads, including muscle forces and IDP, as well as forces in the ligaments and facet joints predicted by the FE model. Load-sharing was calculated as the portion of the total spinal load carried along the spine by each individual spinal structure. The results revealed that spinal loads which increased substantially from the upright to the flexed posture were mainly supported by the discs in the upright posture, whereas the ligaments' contribution in resisting shear, compression, and moment was more significant in the flexed posture.

Spatial relationships between bone formation and mechanical stress within cancellous bone.

Bone adapts to mechanical stimuli. While in vivo mechanical loading has been shown to increase the density of cancellous bone, theory suggests that the relationship between tissue stress/strain and subsequent bone formation occurs at the scale of individual trabeculae. Here we examine bone formation one week following mechanical stimulus. Three bouts of cyclic loading (300 cycles/day on 3 consecutive days) were applied to caudal vertebrae of female rats (n=7). Bone formation was determined using three-dimensional images of fluorescent markers of bone formation (0.7×0.7×5.0µm(3)) and local tissue stress/strain was determined using high-resolution finite element models. Three days of mechanical stimuli resulted in an increase in mineralizing surface (loaded: 17.68±2.17%; control: 9.05±3.20%; mean±SD) and an increase in the volume of bone formed (loaded: 7.09±1.97%; control: 1.44±0.50%). The number of bone formation sites was greater in loaded animals (650.71±118.54) than pinned not loaded controls (310.71±91.55), a difference that was explained by the number of formation sites at regions with large local tissue strain energy density (SED). In addition, the probability of observing bone formation was greater at locations of the microstructure experiencing greater SED, but did not exceed 32%, consistent with prior work. Our findings demonstrate that bone formation in the week following a short term mechanical stimulus occurs near regions of bone tissue experiencing high tissue SED, although the ability of finite element models to predict the locations of bone formation remains modest and further improvements may require accounting for additional factors such as osteocyte distribution or fluid flow.

Development of an in vivo rabbit ulnar loading model.

Ulnar and tibial cyclic compression in rats and mice have become the preferred animal models for investigating the effects of mechanical loading on bone modeling/remodeling. Unlike rodents, rabbits provide a larger bone volume and normally exhibit intracortical Haversian remodeling, which may be advantageous for investigating mechanobiology and pharmaceutical interventions in cortical bone. Therefore, the objective of this study was to develop and validate an in vivo rabbit ulnar loading model. Ulnar tissue strains during loading of intact forelimbs were characterized and calibrated to applied loads using strain gauge measurements and specimen-specific finite element models. Periosteal bone formation in response to varying strain levels was measured by dynamic histomorphometry at the location of maximum strain in the ulnar diaphysis. Ulnae loaded at 3000 microstrain did not exhibit periosteal bone formation greater than the contralateral controls. Ulnae loaded at 3500, 4000, and 4500 microstrain exhibited a dose-dependent increase in periosteal mineralizing surface (MS/BS) compared with contralateral controls during the second week of loading. Ulnae loaded at 4500 microstrain exhibited the most robust response with significantly increased MS/BS at multiple time points extending at least 2weeks after loading was ceased. Ulnae loaded at 5250 microstrain exhibited significant woven bone formation. Rabbits required greater strain levels to produce lamellar and woven bone on periosteal surfaces compared with rats and mice, perhaps due to lower basal levels of MS/BS. In summary, bone adaptation during rabbit ulnar loading was tightly controlled and may provide a translatable model for human bone biology in preclinical investigations of metabolic bone disease and pharmacological treatments.

Experimental and finite element analysis of strains induced by axial tibial compression in young-adult and old female C57Bl/6 mice.

Axial compression of the mouse tibia is used to study strain-adaptive bone (re)modeling. In some studies, comparisons between mice of different ages are of interest. We characterized the tibial deformation and force-strain relationships in female C57Bl/6 mice at 5-, 12- and 22-months age. A three-gauge experimental method was used to determine the strain distribution at the mid-diaphysis, while specimen-specific finite element analysis was used to examine strain distribution along the tibial length. The peak strains in the tibial mid-diaphyseal cross-section are compressive and occur at the postero-lateral apex. The magnitudes of these peak compressive strains are 1.5 to 2 times those on the opposite, antero-medial face (a site often used for strain gauge placement). For example, -10 N force applied to a 5-months old mouse engenders a peak compressive strain of -2800 µÎµ and a tensile strain on the antero-medial face of +1450 µÎµ. The orientation of the neutral axis at the mid-diaphysis did not differ with age (p=0.46), indicating a similar deformation mode in young and old tibiae. On the other hand, from 5- to 22-months there is a 25% reduction in cortical thickness and moment of inertia (p<0.05), resulting in significantly greater tibial strain magnitudes in older mice for equivalent applied force (p<0.05). We conclude that comparisons of tibial loading responses in young-adult and old C57Bl/6 tibiae are facilitated by similar deformation pattern across ages, but that modest adjustment of force levels is required to engender matching peak strains.

The effects of misalignment during in vivo loading of bone: techniques to detect the proximity of objects in three-dimensional models.

Theories of mechanical adaptation of bone suggest that mechanical loading causes bone formation at discrete locations within bone microstructure experiencing the greatest mechanical stress/strain. Experimental testing of such theories requires in vivo loading experiments and high-resolution finite element models to determine the distribution of mechanical stresses. Finite element models of in vivo loading experiments typically assume idealized boundary conditions with applied load perfectly oriented on the bone, however small misalignments in load orientation during an in vivo experiment are unavoidable, and potentially confound the ability of finite element models to predict locations of bone formation at the scale of micrometers. Here we demonstrate two different three-dimensional spatial correlation methods to determine the effects of misalignment in load orientation on the locations of high mechanical stress/strain in the rodent tail loading model. We find that, in cancellous bone, the locations of tissue with high stress are maintained under reasonable misalignments in load orientation (p<0.01). In cortical bone, however, angular misalignments in the dorsal direction can alter the locations of high mechanical stress, but the locations of tissue with high stress are maintained under other misalignments (p<0.01). We conclude that, when using finite element models of the rodent tail loading model, small misalignments in loading orientation do not affect the predicted locations of high mechanical stress within cancellous bone.

Effects of knee flexion on the femoropopliteal artery: a computational study.

During knee flexion, the muscles of the upper leg impose various loads on the underlying femoropopliteal artery resulting in radial compression, bending, torsion, axial extension and axial compression. Measuring the dynamic force environment of the femoropopliteal artery and quantifying its resulting deformation characteristics is an essential input to peripheral device design. The goal of this study was to create an anatomically accurate, three dimensional finite element model capable of capturing the loading conditions and deformation characteristics of the femoropopliteal artery during knee flexion. Three dimensional geometries of the muscle, bone, arterial and soft tissues of the leg were constructed from CT scan data and meshed for finite element analysis. Knee flexion was simulated and deformation characteristics of length change (axial compression), curvature, radial compression and axial twist were quantified and compared to previous experimental studies. The model predicts 8.23% shortening and an average curvature of 0.294±0.26 cm(-1) in the vessel after knee flexion, with maximum stresses of 61.17 kPa and maximum strains of 0.16%. The model created replicates known in vivo deformation characteristics seen previously in angiographic images and for the first time associates femoropopliteal artery deformation characteristics with stress and strain levels within the arterial tissue.