Structural and functional heterogeneity of mineralized fibrocartilage at the Achilles tendon-bone insertion.

A demanding task of the musculoskeletal system is the attachment of tendon to bone at entheses. This region often presents a thin layer of fibrocartilage (FC), mineralized close to the bone and unmineralized close to the tendon. Mineralized FC deserves increased attention, owing to its crucial anchoring task and involvement in enthesis pathologies. Here, we analyzed mineralized FC and subchondral bone at the Achilles tendon-bone insertion of rats. This location features enthesis FC anchoring tendon to bone and sustaining tensile loads, and periosteal FC facilitating bone-tendon sliding with accompanying compressive and shear forces. Using a correlative multimodal investigation, we evaluated potential specificities in mineral content, fiber organization and mechanical properties of enthesis and periosteal FC. Both tissues had a lower degree of mineralization than subchondral bone, yet used the available mineral very efficiently: for the same local mineral content, they had higher stiffness and hardness than bone. We found that enthesis FC was characterized by highly aligned mineralized collagen fibers even far away from the attachment region, whereas periosteal FC had a rich variety of fiber arrangements. Except for an initial steep spatial gradient between unmineralized and mineralized FC, local mechanical properties were surprisingly uniform inside enthesis FC while a modulation in stiffness, independent from mineral content, was observed in periosteal FC. We interpreted these different structure-property relationships as a demonstration of the high versatility of FC, providing high strength at the insertion (to resist tensile loading) and a gradual compliance at the periosteal surface (to resist contact stresses). STATEMENT OF SIGNIFICANCE: Mineralized fibrocartilage (FC) at entheses facilitates the integration of tendon in bone, two strongly dissimilar tissues. We focus on the structure-function relationships of two types of mineralized FC, enthesis and periosteal, which have clearly distinct mechanical demands. By investigating them with multiple high-resolution methods in a correlative manner, we demonstrate differences in fiber architecture and mechanical properties between the two tissues, indicative of their mechanical roles. Our results are relevant both from a medical viewpoint, targeting a clinically relevant location, as well as from a material science perspective, identifying FC as high-performance versatile composite.

A Molecular-Scale Understanding of Misorientation Toughening in Corals and Seashells.

Biominerals are organic-mineral composites formed by living organisms. They are the hardest and toughest tissues in those organisms, are often polycrystalline, and their mesostructure (which includes nano- and microscale crystallite size, shape, arrangement, and orientation) can vary dramatically. Marine biominerals may be aragonite, vaterite, or calcite, all calcium carbonate (CaCO3 ) polymorphs, differing in crystal structure. Unexpectedly, diverse CaCO3 biominerals such as coral skeletons and nacre share a similar characteristic: Adjacent crystals are slightly misoriented. This observation is documented quantitatively at the micro- and nanoscales, using polarization-dependent imaging contrast mapping (PIC mapping), and the slight misorientations are consistently between 1° and 40°. Nanoindentation shows that both polycrystalline biominerals and abiotic synthetic spherulites are tougher than single-crystalline geologic aragonite. Molecular dynamics (MD) simulations of bicrystals at the molecular scale reveal that aragonite, vaterite, and calcite exhibit toughness maxima when the bicrystals are misoriented by 10°, 20°, and 30°, respectively, demonstrating that slight misorientation alone can increase fracture toughness. Slight-misorientation-toughening can be harnessed for synthesis of bioinspired materials that only require one material, are not limited to specific top-down architecture, and are easily achieved by self-assembly of organic molecules (e.g., aspirin, chocolate), polymers, metals, and ceramics well beyond biominerals.

Ultrasonic bandgaps in viscoelastic 1D-periodic media: Mechanical modeling and experimental validation.

Multi-material additive manufacturing is receiving increasing attention in the field of acoustics, in particular towards the design of micro-architectured periodic media used to achieve programmable ultrasonic responses. To unravel the effect of the material properties and spatial arrangement of the printed constituents, there is an unmet need in developing wave propagation models for prediction and optimization purposes. In this study, we propose to investigate the transmission of longitudinal ultrasound waves through 1D-periodic biphasic media, whose constituent materials are viscoelastic. To this end, Bloch-Floquet analysis is applied in the frame of viscoelasticity, with the aim of disentangling the relative contributions of viscoelasticity and periodicity on ultrasound signatures, such as dispersion, attenuation, and bandgaps localization. The impact of the finite size nature of these structures is then assessed by using a modeling approach based on the transfer matrix formalism. Finally, the modeling outcomes, i.e., frequency-dependent phase velocity and attenuation, are confronted with experiments conducted on 3D-printed samples, which exhibit a 1D periodicity at length-scales of a few hundreds of micrometers. Altogether, the obtained results shed light on the modeling characteristics to be considered when predicting the complex acoustic behavior of periodic media in the ultrasonic regime.

Ultrasound characterization of the viscoelastic properties of additively manufactured photopolymer materials.

Photopolymer-based additive manufacturing has received increasing attention in the field of acoustics over the past decade, specifically towards the design of tissue-mimicking phantoms and passive components for ultrasound imaging and therapy. While these applications rely on an accurate characterization of the longitudinal bulk properties of the materials, emerging applications involving periodic micro-architectured media also require the knowledge of the transverse bulk properties to achieve the desired acoustic behavior. However, a robust knowledge of these properties is still lacking for such attenuating materials. Here, we report on the longitudinal and transverse bulk properties, i.e., frequency-dependent phase velocities and attenuations, of photopolymer materials, which were characterized in the MHz regime using a double through-transmission method in oblique incidence. Samples were fabricated using two different printing technologies (stereolithography and polyjet) to assess the impact of two important factors of the manufacturing process: curing and material mixing. Overall, the experimentally observed dispersion and attenuation could be satisfactorily modeled using a power law attenuation to identify a reduced number of intrinsic ultrasound parameters. As a result, these parameters, and especially those reflecting transverse bulk properties, were shown to be very sensitive to slight variations of the manufacturing process.

Ultrasound characterization of bioinspired functionally graded soft-to-hard composites: Experiment and modeling.

Functional grading is a distinctive feature adopted by nature to improve the transition between tissues that present a strong mismatch in mechanical properties, a relevant example being the tendon-to-bone attachment. Recent progress in multi-material additive manufacturing now allows for the design and fabrication of bioinspired functionally graded soft-to-hard composites. Nevertheless, this emerging technology depends on several design variables, including both material and mechanistic ingredients, that are likely to affect the mechanical performance of such composites. In this paper, a model-based approach is developed to describe the interaction of ultrasound waves with homogeneous and heterogeneous additively manufactured samples, which respectively display a variation either of the material ingredients (e.g., ratio of the elementary constituents) or of their spatial arrangement (e.g., functional gradients, damage). Measurements are performed using longitudinal bulk waves, which are launched and detected using a linear transducer array. First, model is calibrated by exploiting the signals measured on the homogeneous samples, which allow identifying relationships between the model parameters and the material composition. Second, the model is validated by comparing the signals measured on the heterogeneous samples with those predicted numerically. Overall, the reported results pave the way for characterizing and optimizing multi-material systems that display complex bioinspired features.

Structure and mineralization of the spearing mantis shrimp (Stomatopoda; Lysiosquillina maculata) body and spike cuticles.

Stomatopoda is a crustacean order including sophisticated predators called spearing and smashing mantis shrimps that are separated from the well-studied Eumalacotraca since the Devonian. The spearing mantis shrimp has developed a spiky dactyl capable of impaling fishes or crustaceans in a fraction of second. In this high velocity hunting technique, the spikes undergo an intense mechanical constraint to which their exoskeleton (or cuticle) has to be adapted. To better understand the spike cuticle internal architecture and composition, electron microscopy, X-ray microanalysis and Raman spectroscopy were used on the spikes of 7 individuals (collected in French Polynesia and Indonesia), but also on parts of the body cuticle that have less mechanical stress to bear. In the body cuticle, several specificities linked to the group were found, allowing to determine the basic structure from which the spike cuticle has evolved. Results also highlighted that the body cuticle of mantis shrimps could be a model close to the ancestral arthropod cuticle by the aspect of its biological layers (epi- and procuticle including exo- and endocuticle) as well as by the Ca-carbonate/phosphate mineral content of these layers. In contrast, the spike cuticle exhibits a deeply modified organization in four functional regions overprinted on the biological layers. Each of them has specific fibre arrangement or mineral content (fluorapatite, ACP or phosphate-rich Ca-carbonate) and is thought to assume specific mechanical roles, conferring appropriate properties on the entire spike. These results agree with an evolution of smashing mantis shrimps from primitive stabbing/spearing shrimps, and thus also allowed a better understanding of the structural modifications described in previous studies on the dactyl club of smashing mantis shrimps.

Local anisotropy in mineralized fibrocartilage and subchondral bone beneath the tendon-bone interface.

The enthesis allows the insertion of tendon into bone thanks to several remarkable strategies. This complex and clinically relevant location often features a thin layer of fibrocartilage sandwiched between tendon and bone to cope with a highly heterogeneous mechanical environment. The main purpose of this study was to investigate whether mineralized fibrocartilage and bone close to the enthesis show distinctive three-dimensional microstructural features, possibly to enable load transfer from tendon to bone. As a model, the Achilles tendon-calcaneus bone system of adult rats was investigated with histology, backscattered electron imaging and micro-computed tomography. The microstructural porosity of bone and mineralized fibrocartilage in different locations including enthesis fibrocartilage, periosteal fibrocartilage and bone away from the enthesis was characterized. We showed that calcaneus bone presents a dedicated protrusion of low porosity where the tendon inserts. A spatially resolved analysis of the trabecular network suggests that such protrusion may promote force flow from the tendon to the plantar ligament, while partially relieving the trabecular bone from such a task. Focusing on the tuberosity, highly specific microstructural aspects were highlighted. Firstly, the interface between mineralized and unmineralized fibrocartilage showed the highest roughness at the tuberosity, possibly to increase failure resistance of a region carrying large stresses. Secondly, fibrochondrocyte lacunae inside mineralized fibrocartilage, in analogy with osteocyte lacunae in bone, had a predominant alignment at the enthesis and a rather random organization away from it. Finally, the network of subchondral channels inside the tuberosity was highly anisotropic when compared to contiguous regions. This dual anisotropy of subchondral channels and cell lacunae at the insertion may reflect the alignment of the underlying collagen network. Our findings suggest that the microstructure of fibrocartilage may be linked with the loading environment. Future studies should characterize those microstructural aspects in aged and or diseased conditions to elucidate the poorly understood role of bone and fibrocartilage in enthesis-related pathologies.

Joining soft tissues to bone: Insights from modeling and simulations.

Entheses are complex multi-tissue regions of the musculoskeletal system serving the challenging task of connecting highly dissimilar materials such as the compliant tendon to the much stiffer bone, over a very small region. The first aim of this review is to highlight mathematical and computational models that have been developed to investigate the many attachment strategies present at entheses at different length scales. Entheses are also relevant in the medical context due to the high prevalence of orthopedic injuries requiring the reattachment of tendons or ligaments to bone, which are associated with a rather poor long-term clinical outcome. The second aim of the review is to report on the computational works analyzing the whole tendon to bone complex as well as targeting orthopedic relevant issues. Modeling approaches have provided important insights on anchoring mechanisms and surgical repair strategies, that would not have been revealed with experiments alone. We intend to demonstrate the necessity of including, in future models, an enriched description of enthesis biomechanical behavior in order to unravel additional mechanical cues underlying the development, the functioning and the maintaining of such a complex biological interface as well as to enhance the development of novel biomimetic adhesive, attachment procedures or tissue engineered implants.

Properties and role of interfaces in multimaterial 3D printed composites.

In polyjet printing photopolymer droplets are deposited on a build tray, leveled off by a roller and cured by UV light. This technique is attractive to fabricate heterogeneous architectures combining compliant and stiff constituents. Considering the layer-by-layer nature, interfaces between different photopolymers can be formed either before or after UV curing. We analyzed the properties of interfaces in 3D printed composites combining experiments with computer simulations. To investigate photopolymer blending, we characterized the mechanical properties of the so-called digital materials, obtained by mixing compliant and stiff voxels according to different volume fractions. We then used nanoindentation to measure the spatial variation in mechanical properties across bimaterial interfaces at the micrometer level. Finally, to characterize the impact of finite-size interfaces, we fabricated and tested composites having compliant and stiff layers alternating along different directions. We found that interfaces formed by deposition after curing were sharp whereas those formed before curing showed blending of the two materials over a length scale bigger than individual droplet size. We found structural and functional differences of the layered composites depending on the printing orientation and corresponding interface characteristics, which influenced deformation mechanisms. With the wide dissemination of 3D printing techniques, our results should be considered in the development of architectured materials with tailored interfaces between building blocks.

Mechanical regulation of bone formation and resorption around implants in a mouse model of osteopenic bone.

Although mechanical stimulation is considered a promising approach to accelerate implant integration, our understanding of load-driven bone formation and resorption around implants is still limited. This lack of knowledge may delay the development of effective loading protocols to prevent implant loosening, especially in osteoporosis. In healthy bone, formation and resorption are mechanoregulated processes. In the intricate context of peri-implant bone regeneration, it is not clear whether bone (re)modelling can still be load-driven. Here, we investigated the mechanical control of peri-implant bone (re)modelling with a well-controlled mechanobiological experiment. We applied cyclic mechanical loading after implant insertion in tail vertebrae of oestrogen depleted mice and we monitored peri-implant bone response by in vivo micro-CT. Experimental data were combined with micro-finite element simulations to estimate local tissue strains in (re)modelling locations. We demonstrated that a substantial increase in bone mass around the implant could be obtained by loading the entire bone. This augmentation could be attributed to a large reduction in bone resorption rather than to an increase in bone formation. We also showed that following implantation, mechanical regulation of bone (re)modelling was transiently lost. Our findings should help to clarify the role of mechanical stimulation on the maintenance of peri-implant bone mass.

Bone remodeling and mechanobiology around implants: Insights from small animal imaging.

Anchorage of orthopedic implants depends on the interfacial bonding between the implant and the host bone as well as on the mass and microstructure of peri-implant bone, with all these factors being continuously regulated by the biological process of bone (re)modeling. In osteoporotic bone, implant integration may be jeopardized not only by lower peri-implant bone quality but also by reduced intrinsic regeneration ability. The first aim of this review is to provide a critical overview of the influence of osteoporosis on bone regeneration post-implantation. Mechanical stimulation can trigger bone formation and inhibit bone resorption; thus, judicious administration of mechanical loading can be used as an effective non-pharmacological treatment to enhance implant anchorage. Our second aim is to report recent achievements on the application of external mechanical stimulation to improve the quantity of peri-implant bone. The review focuses on peri-implant bone changes in osteoporotic conditions and following mechanical loading, prevalently using small animals and in vivo monitoring approaches. We intend to demonstrate the necessity to reveal new biological information on peri-implant bone mechanobiology to better target implant anchorage and fracture fixation in osteoporotic conditions. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:584-593, 2018.

Impaired bone formation in ovariectomized mice reduces implant integration as indicated by longitudinal in vivo micro-computed tomography.

Although osteoporotic bone, with low bone mass and deteriorated bone architecture, provides a less favorable mechanical environment than healthy bone for implant fixation, there is no general agreement on the impact of osteoporosis on peri-implant bone (re)modeling, which is ultimately responsible for the long term stability of the bone-implant system. Here, we inserted an implant in a mouse model mimicking estrogen deficiency-induced bone loss and we monitored with longitudinal in vivo micro-computed tomography the spatio-temporal changes in bone (re)modeling and architecture, considering the separate contributions of trabecular, endocortical and periosteal surfaces. Specifically, 12 week-old C57BL/6J mice underwent OVX/SHM surgery; 9 weeks after we inserted special metal-ceramics implants into the 6th caudal vertebra and we measured bone response with in vivo micro-CT weekly for the following 6 weeks. Our results indicated that ovariectomized mice showed a reduced ability to increase the thickness of the cortical shell close to the implant because of impaired peri-implant bone formation, especially at the periosteal surface. Moreover, we observed that healthy mice had a significantly higher loss of trabecular bone far from the implant than estrogen depleted animals. Such behavior suggests that, in healthy mice, the substantial increase in peri-implant bone formation which rapidly thickened the cortex to secure the implant may raise bone resorption elsewhere and, specifically, in the trabecular network of the same bone but far from the implant. Considering the already deteriorated bone structure of estrogen depleted mice, further bone loss seemed to be hindered. The obtained knowledge on the dynamic response of diseased bone following implant insertion should provide useful guidelines to develop advanced treatments for osteoporotic fracture fixation based on local and selective manipulation of bone turnover in the peri-implant region.

In vivo monitoring of bone architecture and remodeling after implant insertion: The different responses of cortical and trabecular bone.

The mechanical integrity of the bone-implant system is maintained by the process of bone remodeling. Specifically, the interplay between bone resorption and bone formation is of paramount importance to fully understand the net changes in bone structure occurring in the peri-implant bone, which are eventually responsible for the mechanical stability of the bone-implant system. Using time-lapsed in vivo micro-computed tomography combined with new composite material implants, we were able to characterize the spatio-temporal changes of bone architecture and bone remodeling following implantation in living mice. After insertion, implant stability was attained by a quick and substantial thickening of the cortical shell which counteracted the observed loss of trabecular bone, probably due to the disruption of the trabecular network. Within the trabecular compartment, the rate of bone formation close to the implant was transiently higher than far from the implant mainly due to an increased mineral apposition rate which indicated a higher osteoblastic activity. Conversely, in cortical bone, the higher rate of bone formation close to the implant compared to far away was mostly related to the recruitment of new osteoblasts as indicated by a prevailing mineralizing surface. The behavior of bone resorption also showed dissimilarities between trabecular and cortical bone. In the former, the rate of bone resorption was higher in the peri-implant region and remained elevated during the entire monitoring period. In the latter, bone resorption rate had a bigger value away from the implant and decreased with time. Our approach may help to tune the development of smart implants that can attain a better long-term stability by a local and targeted manipulation of the remodeling process within the cortical and the trabecular compartments and, particularly, in bone of poor health.

Does mechanical stimulation really protect the architecture of trabecular bone? A simulation study.

Although it is beyond doubt that mechanical stimulation is crucial to maintain bone mass, its role in preserving bone architecture is much less clear. Commonly, it is assumed that mechanics helps to conserve the trabecular network since an "accidental" thinning of a trabecula due to a resorption event would result in a local increase of load, thereby activating bone deposition there. However, considering that the thin trabecula is part of a network, it is not evident that load concentration happens locally on the weakened trabecula. The aim of this work was to clarify whether mechanical load has a protective role for preserving the trabecular network during remodeling. Trabecular bone is made dynamic by a remodeling algorithm, which results in a thickening/thinning of trabeculae with high/low strain energy density. Our simulations show that larger deviations from a regular cubic lattice result in a greater loss of trabeculae. Around lost trabeculae, the remaining trabeculae are on average thinner. More generally, thin trabeculae are more likely to have thin trabeculae in their neighborhood. The plausible consideration that a thin trabecula concentrates a higher amount of strain energy within itself is therefore only true when considering a single isolated trabecula. Mechano-regulated remodeling within a network-like architecture leads to local concentrations of thin trabeculae.

Inverse finite element modeling for characterization of local elastic properties in image-guided failure assessment of human trabecular bone.

The local interpretation of microfinite element (µFE) simulations plays a pivotal role for studying bone structure­function relationships such as failure processes and bone remodeling.In the past µFE simulations have been successfully validated on the apparent level,however, at the tissue level validations are sparse and less promising. Furthermore,intra trabecular heterogeneity of the material properties has been shown by experimental studies. We proposed an inverse µFE algorithm that iteratively changes the tissue level Young's moduli such that the µFE simulation matches the experimental strain measurements.The algorithm is setup as a feedback loop where the modulus is iteratively adapted until the simulated strain matches the experimental strain. The experimental strain of human trabecular bone specimens was calculated from time-lapsed images that were gained by combining mechanical testing and synchrotron radiation microcomputed tomography(SRlCT). The inverse µFE algorithm was able to iterate the heterogeneous distribution of moduli such that the resulting µFE simulations matched artificially generated and experimentally measured strains.

Modeling microdamage behavior of cortical bone.

Bone is a complex material which exhibits several hierarchical levels of structural organization. At the submicron-scale, the local tissue porosity gives rise to discontinuities in the bone matrix which have been shown to influence damage behavior. Computational tools to model the damage behavior of bone at different length scales are mostly based on finite element (FE) analysis, with a range of algorithms developed for this purpose. Although the local mechanical behavior of bone tissue is influenced by microstructural features such as bone canals and osteocyte lacunae, they are often not considered in FE damage models due to the high computational cost required to simulate across several length scales, i.e., from the loads applied at the organ level down to the stresses and strains around bone canals and osteocyte lacunae. Hence, the aim of the current study was twofold: First, a multilevel FE framework was developed to compute, starting from the loads applied at the whole bone scale, the local mechanical forces acting at the micrometer and submicrometer level. Second, three simple microdamage simulation procedures based on element removal were developed and applied to bone samples at the submicrometer-scale, where cortical microporosity is included. The present microdamage algorithm produced a qualitatively analogous behavior to previous experimental tests based on stepwise mechanical compression combined with in situ synchrotron radiation computed tomography. Our results demonstrate the feasibility of simulating microdamage at a physiologically relevant scale using an image-based meshing technique and multilevel FE analysis; this allows relating microdamage behavior to intracortical bone microstructure.

The role of the renal ammonia transporter Rhcg in metabolic responses to dietary protein.

High dietary protein imposes a metabolic acid load requiring excretion and buffering by the kidney. Impaired acid excretion in CKD, with potential metabolic acidosis, may contribute to the progression of CKD. Here, we investigated the renal adaptive response of acid excretory pathways in mice to high-protein diets containing normal or low amounts of acid-producing sulfur amino acids (SAA) and examined how this adaption requires the RhCG ammonia transporter. Diets rich in SAA stimulated expression of enzymes and transporters involved in mediating NH4 (+) reabsorption in the thick ascending limb of the loop of Henle. The SAA-rich diet increased diuresis paralleled by downregulation of aquaporin-2 (AQP2) water channels. The absence of Rhcg transiently reduced NH4 (+) excretion, stimulated the ammoniagenic pathway more strongly, and further enhanced diuresis by exacerbating the downregulation of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC2) and AQP2, with less phosphorylation of AQP2 at serine 256. The high protein acid load affected bone turnover, as indicated by higher Ca(2+) and deoxypyridinoline excretion, phenomena exaggerated in the absence of Rhcg. In animals receiving a high-protein diet with low SAA content, the kidney excreted alkaline urine, with low levels of NH4 (+) and no change in bone metabolism. Thus, the acid load associated with high-protein diets causes a concerted response of various nephron segments to excrete acid, mostly in the form of NH4 (+), that requires Rhcg. Furthermore, bone metabolism is altered by a high-protein acidogenic diet, presumably to buffer the acid load.

Micro-computed tomography based computational fluid dynamics for the determination of shear stresses in scaffolds within a perfusion bioreactor.

Perfusion bioreactors are known to exert shear stresses on cultured cells, leading to cell differentiation and enhanced extracellular matrix deposition on scaffolds. The influence of the scaffold's porous microstructure is investigated for a polycaprolactone (PCL) scaffold with a regular microarchitecture and a silk fibroin (SF) scaffold with an irregular network of interconnected pores. Their complex 3D geometries are imaged by micro-computed tomography and used in direct pore-level simulations of the entire scaffold-bioreactor system to numerically solve the governing mass and momentum conservation equations for fluid flow through porous media. The velocity field and wall shear stress distribution are determined for both scaffolds. The PCL scaffold exhibited an asymmetric distribution with peak and plateau, while the SF scaffold exhibited a homogenous distribution and conditioned the flow more efficiently than the PCL scaffold. The methodology guides the design and optimization of the scaffold geometry.

Local mechanical stimuli regulate bone formation and resorption in mice at the tissue level.

Bone is able to react to changing mechanical demands by adapting its internal microstructure through bone forming and resorbing cells. This process is called bone modeling and remodeling. It is evident that changes in mechanical demands at the organ level must be interpreted at the tissue level where bone (re)modeling takes place. Although assumed for a long time, the relationship between the locations of bone formation and resorption and the local mechanical environment is still under debate. The lack of suitable imaging modalities for measuring bone formation and resorption in vivo has made it difficult to assess the mechanoregulation of bone three-dimensionally by experiment. Using in vivo micro-computed tomography and high resolution finite element analysis in living mice, we show that bone formation most likely occurs at sites of high local mechanical strain (p<0.0001) and resorption at sites of low local mechanical strain (p<0.0001). Furthermore, the probability of bone resorption decreases exponentially with increasing mechanical stimulus (R(2) = 0.99) whereas the probability of bone formation follows an exponential growth function to a maximum value (R(2) = 0.99). Moreover, resorption is more strictly controlled than formation in loaded animals, and ovariectomy increases the amount of non-targeted resorption. Our experimental assessment of mechanoregulation at the tissue level does not show any evidence of a lazy zone and suggests that around 80% of all (re)modeling can be linked to the mechanical micro-environment. These findings disclose how mechanical stimuli at the tissue level contribute to the regulation of bone adaptation at the organ level.