Effect of patient specificity on predicting knee cartilage degeneration in obese adults: Musculoskeletal finite-element modeling of data from the CAROT trial.

Obesity is a known risk factor for development of osteoarthritis (OA). Numerical tools like finite-element (FE) models combined with degenerative algorithms have been developed to understand the interplay between OA and obesity. In this study, we aimed to predict knee cartilage degeneration in a cohort of obese adults to investigate the importance of patient-specific information on degeneration predictions. We used a validated FE modeling approach and three different age-dependent functions (step-wise, exponential, and linear) to simulate cartilage degradation under overloading in the knee joint. Gait motion analysis and magnetic resonance imaging data from 115 obese individuals with knee OA were used for musculoskeletal and FE modeling. Cartilage degeneration predictions were contrasted with Kellgren-Lawrence (KL) and Boston-Leeds Osteoarthritis Knee Score (BLOKS) grades. The findings show that overall, the similarities between numerical predictions and clinical measures were better for the medial (average area under the curve (AUC) = 0.62) compared to the lateral compartment (average AUC = 0.52) of the knee. Classification results for KL grades, full patient-specific models and patient-specific geometry with generic gait data showed higher AUC values (AUC = 0.71 and AUC = 0.68, respectively) compared to generic geometry and patient-specific gait (AUC = 0.48). For BLOKS grades, AUC values for both full patient-specific models and for patient-specific geometry with generic gait locomotion were higher (AUC = 0.66 and AUC = 0.64, respectively) compared to when the generic geometry and patient-specific gait were used (AUC = 0.53). In summary, our study highlights the importance of considering individual information in knee OA prediction. Nevertheless, our findings suggest that personalized gait play a smaller role in the OA prediction and classification capacity than personalized joint geometry.

Multi-Modal in Vitro Experiments Mimicking the Flow Through a Mitral Heart Valve Phantom.

PURPOSE: Fluid-structure interaction (FSI) models are more commonly applied in medical research as computational power is increasing. However, understanding the accuracy of FSI models is crucial, especially in the context of heart valve disease in patient-specific models. Therefore, this study aimed to create a multi-modal benchmarking data set for cardiac-inspired FSI models, based on clinically important parameters, such as the pressure, velocity, and valve opening, with an in vitro phantom setup. METHOD: An in vitro setup was developed with a 3D-printed phantom mimicking the left heart, including a deforming mitral valve. A range of pulsatile flows were created with a computer-controlled motor-and-pump setup. Catheter pressure measurements, magnetic resonance imaging (MRI), and echocardiography (Echo) imaging were used to measure pressure and velocity in the domain. Furthermore, the valve opening was quantified based on cine MRI and Echo images. RESULT: The experimental setup, with 0.5% cycle-to-cycle variation, was successfully built and six different flow cases were investigated. Higher velocity through the mitral valve was observed for increased cardiac output. The pressure difference across the valve also followed this trend. The flow in the phantom was qualitatively assessed by the velocity profile in the ventricle and by streamlines obtained from 4D phase-contrast MRI. CONCLUSION: A multi-modal set of data for validation of FSI models has been created, based on parameters relevant for diagnosis of heart valve disease. All data is publicly available for future development of computational heart valve models.

Overexpression of the plastidial pseudo-protease AtFtsHi3 enhances drought tolerance while sustaining plant growth.

With climate change, droughts are expected to be more frequent and severe, severely impacting plant biomass and quality. Here, we show that overexpressing the Arabidopsis gene AtFtsHi3 (FtsHi3OE) enhances drought-tolerant phenotypes without compromising plant growth. AtFtsHi3 encodes a chloroplast envelope pseudo-protease; knock-down mutants (ftshi3-1) are found to be drought tolerant but exhibit stunted growth. Altered AtFtsHi3 expression therefore leads to drought tolerance, while only diminished expression of this gene leads to growth retardation. To understand the underlying mechanisms of the enhanced drought tolerance, we compared the proteomes of ftshi3-1 and pFtsHi3-FtsHi3OE (pFtsHi3-OE) to wild-type plants under well-watered and drought conditions. Drought-related processes like osmotic stress, water transport, and abscisic acid response were enriched in pFtsHi3-OE and ftshi3-1 mutants following their enhanced drought response compared to wild-type. The knock-down mutant ftshi3-1 showed an increased abundance of HSP90, HSP93, and TIC110 proteins, hinting at a potential downstream role of AtFtsHi3 in chloroplast pre-protein import. Mathematical modeling was performed to understand how variation in the transcript abundance of AtFtsHi3 can, on the one hand, lead to drought tolerance in both overexpression and knock-down lines, yet, on the other hand, affect plant growth so differently. The results led us to hypothesize that AtFtsHi3 may form complexes with at least two other protease subunits, either as homo- or heteromeric structures. Enriched amounts of AtFtsH7/9, AtFtsH11, AtFtsH12, and AtFtsHi4 in ftshi3-1 suggest a possible compensation mechanism for these proteases in the hexamer.

Systemically administered zoledronic acid activates locally implanted synthetic hydroxyapatite particles enhancing peri-implant bone formation: A regenerative medicine approach to improve fracture fixation.

Fracture fixation in an ageing population is challenging and fixation failure increases mortality and societal costs. We report a novel fracture fixation treatment by applying a hydroxyapatite (HA) based biomaterial at the bone-implant interface and biologically activating the biomaterial by systemic administration of a bisphosphonate (zoledronic acid, ZA). We first used an animal model of implant integration and applied a calcium sulphate (CaS)/HA biomaterial around a metallic screw in the tibia of osteoporotic rats. Using systemic ZA administration at 2-weeks post-surgery, we demonstrated that the implant surrounded by HA particles showed significantly higher peri­implant bone formation compared to the unaugmented implants at 6-weeks. We then evaluated the optimal timing (day 1, 3, 7 and 14) of ZA administration to achieve a robust effect on peri­implant bone formation. Using fluorescent ZA, we demonstrated that the uptake of ZA in the CaS/HA material was the highest at 3- and 7-days post-implantation and the uptake kinetics had a profound effect on the eventual peri­implant bone formation. We furthered our concept in a feasibility study on trochanteric fracture patients randomized to either CaS/HA augmentation or no augmentation followed by systemic ZA treatment. Radiographically, the CaS/HA group showed signs of increased peri­implant bone formation compared with the controls. Finally, apart from HA, we demonstrated that the concept of biologically activating a ceramic material by ZA could also be applied to ß-tricalcium phosphate. This novel approach for fracture treatment that enhances immediate and long-term fracture fixation in osteoporotic bone could potentially reduce reoperations, morbidity and mortality. STATEMENT OF SIGNIFICANCE: • Fracture fixation in an ageing population is challenging. Biomaterial-based augmentation of fracture fixation devices has been attempted but lack of satisfactory biological response limits their widespread use. • We report the biological activation of locally implanted microparticulate hydroxyapatite (HA) particles placed around an implant by systemic administration of the bisphosphonate zoledronic acid (ZA). The biological activation of HA by ZA enhances peri­implant bone formation. •Timing of ZA administration after HA implantation is critical for optimal ZA uptake and consequently determines the extent of peri­implant bone formation. • We translate the developed concept from small animal models of implant integration to a proof-of-concept clinical study on osteoporotic trochanteric fracture patients. • ZA based biological activation can also be applied to other calcium phosphate biomaterials.

In Situ Loading and Time-Resolved Synchrotron-Based Phase Contrast Tomography for the Mechanical Investigation of Connective Knee Tissues: A Proof-of-Concept Study.

Articular cartilage and meniscus transfer and distribute mechanical loads in the knee joint. Degeneration of these connective tissues occurs during the progression of knee osteoarthritis, which affects their composition, microstructure, and mechanical properties. A deeper understanding of disease progression can be obtained by studying them simultaneously. Time-resolved synchrotron-based X-ray phase-contrast tomography (SR-PhC-µCT) allows to capture the tissue dynamics. This proof-of-concept study presents a rheometer setup for simultaneous in situ unconfined compression and SR-PhC-µCT of connective knee tissues. The microstructural response of bovine cartilage (n = 16) and meniscus (n = 4) samples under axial continuously increased strain, or two steps of 15% strain (stress-relaxation) is studied. The chondrocyte distribution in cartilage and the collagen fiber orientation in the meniscus are assessed. Variations in chondrocyte density reveal an increase in the top 40% of the sample during loading, compared to the lower half. Meniscus collagen fibers reorient perpendicular to the loading direction during compression and partially redisperse during relaxation. Radiation damage, image repeatability, and image quality assessments show little to no effects on the results. In conclusion, this approach is highly promising for future studies of human knee tissues to understand their microstructure, mechanical response, and progression in degenerative diseases.

Exploratory neutron tomography of articular cartilage.

OBJECTIVE: To investigate the feasibility of using neutron tomography to gain new knowledge of human articular cartilage degeneration in osteoarthritis (OA). Different sample preparation techniques were evaluated to identify maximum intra-tissue contrast. DESIGN: Human articular cartilage samples from 14 deceased donors (18-75 years, 9 males, 5 females) and 4 patients undergoing total knee replacement due to known OA (all female, 61-75 years) were prepared using different techniques: control in saline, treated with heavy water saline, fixed and treated in heavy water saline, and fixed and dehydrated with ethanol. Neutron tomographic imaging (isotropic voxel sizes from 7.5 to 13.5 µm) was performed at two large scale facilities. The 3D images were evaluated for gradients in hydrogen attenuation as well as compared to images from absorption X-ray tomography, magnetic resonance imaging, and histology. RESULTS: Cartilage was distinguishable from background and other tissues in neutron tomographs. Intra-tissue contrast was highest in heavy water-treated samples, which showed a clear gradient from the cartilage surface to the bone interface. Increased neutron flux or exposure time improved image quality but did not affect the ability to detect gradients. Samples from older donors showed high variation in gradient profile, especially from donors with known OA. CONCLUSIONS: Neutron tomography is a viable technique for specialized studies of cartilage, particularly for quantifying properties relating to the hydrogen density of the tissue matrix or water movement in the tissue.

Validation of fluid-structure interaction simulations of the opening phase of phantom mitral heart valves under physiologically inspired conditions.

BACKGROUND AND OBJECTIVE: Atrioventricular valve disease is a common cause of heart failure, and successful surgical or interventional outcomes are crucial. Patient-specific fluid-structure interaction (FSI) modeling may provide valuable insights into valve dynamics and guidance of valve repair strategies. However, lack of validation has kept FSI modeling from clinical implementation. Therefore, this study aims to validate FSI simulations against in vitro benchmarking data, based on clinically relevant parameters for evaluating heart valve disease. METHODS: An FSI model that mimics the left heart was developed. The domain included a deformable mitral valve of different stiffnesses run with different inlet velocities. Five different cases were simulated and compared to in vitro data based on the pressure difference across the valve, the valve opening, and the velocity in the flow domain. RESULTS: The simulations underestimate the pressure difference across the valve by 6.8-14 % compared to catheter measurements. Evaluation of the valve opening showed an underprediction of 5.4-7.3 % when compared to cine MRI, 2D Echo, and 3D Echo data. Additionally, the simulated velocity through the valve showed a 7.9-8.4 % underprediction in relation to Doppler Echo measurements. Qualitative assessment of the velocity profile in the ventricle and the streamlines of the flow in the domain showed good agreement of the flow behavior. CONCLUSIONS: Parameters relevant to the diagnosis of heart valve disease estimated by FSI simulations showed good agreement when compared to in vitro benchmarking data, with differences small enough not to affect the grading of heart valve disease. The FSI model is thus deemed good enough for further development toward patient-specific cases.

Impact of storage time prior to cryopreservation on mechanical properties of aortic homografts.

Optimal time spans in homograft procurement are still debatable among tissue banks and needs to be further investigated. Cell viability decreases at longer preparation intervals, but the effect on collagen and elastic fibers has not been investigated to the same extent. These fibers are of importance to the homograft elasticity and strength. The objective of this study was to analyze the mechanical properties of homograft tissue at different time spans in the procurement process. Ten aortic homografts were collected at the Tissue Bank in Lund. Twelve samples were obtained from each homograft, cryopreserved in groups of three after 2-4 days, 7-9 days, 28-30 days, and 60-62 days in antibiotic decontamination. Mechanical testing was performed with uniaxial tensile tests, calculating elastic modulus, yield stress and energy at yield stress. Two randomly selected samples were assessed with light microscopy. Procurement generated a total of 120 samples, with 30 samples in each time group. Elastic modulus and yield stress was significantly higher in samples cryopreserved after 2-4 days (2.7 MPa (2.5-5.0) and 0.78 MPa (0.68-1.0)) compared to 7-9 days (2.2 MPa (2.0-2.6) and 0.53 MPa (0.46-0.69)), p = 0.008 and 0.011 respectively. Light microscopy did not show any difference in collagen and elastin at different time spans. There was a significant decrease in elastic modulus and yield stress after 7 days of decontamination at 4 °C compared to 2-4 days. This could indicate some deterioration of elastin and collagen at longer decontamination intervals. Clinical significance of these findings remains to be clarified.

Micro- and nanostructure specific X-ray tomography reveals less matrix formation and altered collagen organization following reduced loading during Achilles tendon healing.

Recovery of the collagen structure following Achilles tendon rupture is poor, resulting in a high risk for re-ruptures. The loading environment during healing affects the mechanical properties of the tendon, but the relation between loading regime and healing outcome remains unclear. This is partially due to our limited understanding regarding the effects of loading on the micro- and nanostructure of the healing tissue. We addressed this through a combination of synchrotron phase-contrast X-ray microtomography and small-angle X-ray scattering tensor tomography (SASTT) to visualize the 3D organization of microscale fibers and nanoscale fibrils, respectively. The effect of in vivo loading on these structures was characterized in early healing of rat Achilles tendons by comparing full activity with immobilization. Unloading resulted in structural changes that can explain the reported impaired mechanical performance. In particular, unloading led to slower tissue regeneration and maturation, with less and more disorganized collagen, as well as an increased presence of adipose tissue. This study provides the first application of SASTT on soft musculoskeletal tissues and clearly demonstrates its potential to investigate a variety of other collagenous tissues. STATEMENT OF SIGNIFICANCE: Currently our understanding of the mechanobiological effects on the recovery of the structural hierarchical organization of injured Achilles tendons is limited. We provide insight into how loading affects the healing process by using a cutting-edge approach to for the first time characterize the 3D micro- and nanostructure of the regenerating collagen. We uncovered that, during early healing, unloading results in a delayed and more disorganized regeneration of both fibers (microscale) and fibrils (nanoscale), as well as increased presence of adipose tissue. The results set the ground for the development of further specialized protocols for tendon recovery.

Multimodal and multiscale characterization reveals how tendon structure and mechanical response are altered by reduced loading.

Tendons are collagen-based connective tissues where the composition, structure and mechanics respond and adapt to the local mechanical environment. Adaptation to prolonged inactivity can result in stiffer tendons that are more prone to injury. However, the complex relation between reduced loading, structure, and mechanical performance is still not fully understood. This study combines mechanical testing with high-resolution synchrotron X-ray imaging, scattering techniques and histology to elucidate how reduced loading affects the structural properties and mechanical response of rat Achilles tendons on multiple length scales. The results show that reduced in vivo loading leads to more crimped and less organized fibers and this structural inhomogeneity could be the reason for the altered mechanical response. Unloading also seems to change the fibril response, possibly by altering the strain partitioning between hierarchical levels, and to reduce cell density. This study elucidates the relation between in vivo loading, the Achilles tendon nano-, meso­structure and mechanical response. The results provide fundamental insights into the mechanoregulatory mechanisms guiding the intricate biomechanics, tissue structural organization, and performance of complex collagen-based tissues. STATEMENT OF SIGNIFICANCE: Achilles tendon properties allow a dynamic interaction between muscles and tendon and influence force transmission during locomotion. Lack of physiological loading can have dramatic effects on tendon structure and mechanical properties. We have combined the use of cutting-edge high-resolution synchrotron techniques with mechanical testing to show how reduced loading affects the tendon on multiple hierarchical levels (from nanoscale up to whole organ) clarifying the relation between structural changes and mechanical performance. Our findings set the first step to address a significant healthcare challenge, such as the design of tailored rehabilitations that take into consideration structural changes after tendon immobilization.

3D Finite Element Models Reconstructed From 2D Dual-Energy X-Ray Absorptiometry (DXA) Images Improve Hip Fracture Prediction Compared to Areal BMD in Osteoporotic Fractures in Men (MrOS) Sweden Cohort.

Bone strength is an important contributor to fracture risk. Areal bone mineral density (aBMD) derived from dual-energy X-ray absorptiometry (DXA) is used as a surrogate for bone strength in fracture risk prediction tools. 3D finite element (FE) models predict bone strength better than aBMD, but their clinical use is limited by the need for 3D computed tomography and lack of automation. We have earlier developed a method to reconstruct the 3D hip anatomy from a 2D DXA image, followed by subject-specific FE-based prediction of proximal femoral strength. In the current study, we aim to evaluate the method's ability to predict incident hip fractures in a population-based cohort (Osteoporotic Fractures in Men [MrOS] Sweden). We defined two subcohorts: (i) hip fracture cases and controls cohort: 120 men with a hip fracture (<10 years from baseline) and two controls to each hip fracture case, matched by age, height, and body mass index; and (ii) fallers cohort: 86 men who had fallen the year before their hip DXA scan was acquired, 15 of which sustained a hip fracture during the following 10 years. For each participant, we reconstructed the 3D hip anatomy and predicted proximal femoral strength in 10 sideways fall configurations using FE analysis. The FE-predicted proximal femoral strength was a better predictor of incident hip fractures than aBMD for both hip fracture cases and controls (difference in area under the receiver operating characteristics curve, ΔAUROC = 0.06) and fallers (ΔAUROC = 0.22) cohorts. This is the first time that FE models outperformed aBMD in predicting incident hip fractures in a population-based prospectively followed cohort based on 3D FE models obtained from a 2D DXA scan. Our approach has potential to notably improve the accuracy of fracture risk predictions in a clinically feasible manner (only one single DXA image is needed) and without additional costs compared to the current clinical approach. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Multi-scale characterization of the spatio-temporal interplay between elemental composition, mineral deposition and remodelling in bone fracture healing.

Bone mineralization involves a complex orchestration of physico-chemical responses from the organism. Despite extensive studies, the detailed mechanisms of mineralization remain to be elucidated. This study aims to characterize bone mineralization using an in-vivo long bone fracture healing model in the rat. The spatio-temporal distribution of relevant elements was correlated to the deposition and maturation of hydroxyapatite and the presence of matrix remodeling compounds (MMP-13). Multi-scale measurements indicated that (i) zinc is required for both the initial mineral deposition and resorption processes during mature mineral remodeling; (ii) Zinc and MMP-13 show similar spatio-temporal trends during early mineralization; (iii) Iron acts locally and in coordination with zinc during mineralization, thus indicating novel evidence of the time-events and inter-play between the elements. These findings improve the understanding of bone mineralization by explaining the link between the different constituents of this process throughout the healing time. STATEMENT OF SIGNIFICANCE: Bone mineralization involves a complex orchestration of physico-chemical responses from the organism, the detailed mechanisms of which remain to be elucidated. This study presents a highly novel multi-scale multi-modal investigation of bone mineralization using bone fracture healing as a model system. We present original characterization of tissue mineralization, where we relate the spatio-temporal distribution of important trace elements to a key matrix remodeling compound (MMP-13), the initial deposition and maturation of hydroxyapatite and further remodeling processes. This is the first time that mineralization has been probed down to the nanometric level, and where key mineralization components have been investigated to achieve a comprehensive and mechanistic understanding of the underlying mineralization processes during bone healing.

Spatiotemporal and microstructural characterization of heterotopic ossification in healing rat Achilles tendons.

Achilles tendon rupture is a common debilitating medical condition. The healing process is slow and can be affected by heterotopic ossification (HO), which occurs when pathologic bone-like tissue is deposited instead of the soft collagenous tendon tissue. Little is known about the temporal and spatial progression of HO during Achilles tendon healing. In this study we characterize HO deposition, microstructure, and location at different stages of healing in a rat model. We use phase contrast-enhanced synchrotron microtomography, a state-of-the-art technique that allows 3D imaging at high-resolution of soft biological tissues without invasive or time-consuming sample preparation. The results increase our understanding of HO deposition, from the early inflammatory phase of tendon healing, by showing that the deposition is initiated as early as one week after injury in the distal stump and mostly growing on preinjury HO deposits. Later, more deposits form first in the stumps and then all over the tendon callus, merging into large, calcified structures, which occupy up to 10% of the tendon volume. The HOs were characterized by a looser connective trabecular-like structure and a proteoglycan-rich matrix containing chondrocyte-like cells with lacunae. The study shows the potential of 3D imaging at high-resolution by phase-contrast tomography to better understand ossification in healing tendons.

Minor variations in multicellular life cycles have major effects on adaptation.

Multicellularity has evolved several independent times over the past hundreds of millions of years and given rise to a wide diversity of complex life. Recent studies have found that large differences in the fundamental structure of early multicellular life cycles can affect fitness and influence multicellular adaptation. Yet, there is an underlying assumption that at some scale or categorization multicellular life cycles are similar in terms of their adaptive potential. Here, we consider this possibility by exploring adaptation in a class of simple multicellular life cycles of filamentous organisms that only differ in one respect, how many daughter filaments are produced. We use mathematical models and evolutionary simulations to show that despite the similarities, qualitatively different mutations fix. In particular, we find that mutations with a tradeoff between cell growth and group survival, i.e. "selfish" or "altruistic" traits, spread differently. Specifically, altruistic mutations more readily spread in life cycles that produce few daughters while in life cycles producing many daughters either type of mutation can spread depending on the environment. Our results show that subtle changes in multicellular life cycles can fundamentally alter adaptation.

Heterotopic mineral deposits in intact rat Achilles tendons are characterized by a unique fiber-like structure.

Heterotopic mineralization entails pathological mineral formation inside soft tissues. In human tendons mineralization is often associated with tendinopathies, tendon weakness and pain. In Achilles tendons, mineralization is considered to occur through heterotopic ossification (HO) primarily in response to tendon pathologies. However, refined details regarding HO deposition and microstructure are unknown. In this study, we characterize HO in intact rat Achilles tendons through high-resolution phase contrast enhanced synchrotron X-ray tomography. Furthermore, we test the potential of studying local tissue injury by needling intact Achilles tendons and the relation between tissue microdamage and HO. The results show that HO occurs in all intact Achilles tendons at 16 weeks of age. HO deposits are characterized by an elongated ellipsoidal shape and by a fiber-like internal structure which suggests that some collagen fibers have mineralized. The data indicates that deposition along fibers initiates in the pericellular area, and propagates into the intercellular area. Within HO deposits cells are larger and more rounded compared to tenocytes between unmineralized fibers, which are fewer and elongated. The results also indicate that multiple HO deposits may merge into bigger structures with time by accession along unmineralized fibers. Furthermore, the presence of unmineralized regions within the deposits may indicate that HOs are not only growing, but mineral resorption may also occur. Additionally, phase contrast synchrotron X-ray tomography allowed to distinguish microdamage at the fiber level in response to needling. The needle injury protocol could in the future enable to elucidate the relation between local inflammation, microdamage, and HO deposition.

Correction: A musculoskeletal finite element model of rat knee joint for evaluating cartilage biomechanics during gait.

[This corrects the article DOI: 10.1371/journal.pcbi.1009398.].

Predicting the formation of different tissue types during Achilles tendon healing using mechanoregulated and oxygen-regulated frameworks.

During Achilles tendon healing in rodents, besides the expected tendon tissue, also cartilage-, bone- and fat-like tissue features have been observed during the first twenty weeks of healing. Several studies have hypothesized that mechanical loading may play a key role in the formation of different tissue types during healing. We recently developed a computational mechanobiological framework to predict tendon tissue production, organization and mechanical properties during tendon healing. In the current study, we aimed to explore possible mechanobiological related mechanisms underlying formation of other tissue types than tendon tissue during tendon healing. To achieve this, we further developed our recent framework to predict formation of different tissue types, based on mechanobiological models established in other fields, which have earlier not been applied to study tendon healing. We explored a wide range of biophysical stimuli, i.e., principal strain, hydrostatic stress, pore pressure, octahedral shear strain, fluid flow, angiogenesis and oxygen concentration, that may promote the formation of different tissue types. The numerical framework predicted spatiotemporal formation of tendon-, cartilage-, bone- and to a lesser degree fat-like tissue throughout the first twenty weeks of healing, similar to recent experimental reports. Specific features of experimental data were captured by different biophysical stimuli. Our modeling approach showed that mechanobiology may play a role in governing the formation of different tissue types that have been experimentally observed during tendon healing. This study provides a numerical tool that can contribute to a better understanding of tendon mechanobiology during healing. Developing these tools can ultimately lead to development of better rehabilitation regimens that stimulate tendon healing and prevent unwanted formation of cartilage-, fat- and bone-like tissues.

Nanoscale characterization of collagen structural responses to in situ loading in rat Achilles tendons.

The specific viscoelastic mechanical properties of Achilles tendons are highly dependent on the structural characteristics of collagen at and between all hierarchical levels. Research has been conducted on the deformation mechanisms of positional tendons and single fibrils, but knowledge about the coupling between the whole tendon and nanoscale deformation mechanisms of more commonly injured energy-storing tendons, such as Achilles tendons, remains sparse. By exploiting the highly periodic arrangement of tendons at the nanoscale, in situ loading of rat Achilles tendons during small-angle X-ray scattering acquisition was used to investigate the collagen structural response during load to rupture, cyclic loading and stress relaxation. The fibril strain was substantially lower than the applied tissue strain. The fibrils strained linearly in the elastic region of the tissue, but also exhibited viscoelastic properties, such as an increased stretchability and recovery during cyclic loading and fibril strain relaxation during tissue stress relaxation. We demonstrate that the changes in the width of the collagen reflections could be attributed to strain heterogeneity and not changes in size of the coherently diffracting domains. Fibril strain heterogeneity increased with applied loads and after the toe region, fibrils also became increasingly disordered. Additionally, a thorough evaluation of radiation damage was performed. In conclusion, this study clearly displays the simultaneous structural response and adaption of the collagen fibrils to the applied tissue loads and provide novel information about the transition of loads between length scales in the Achilles tendon.