Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage

The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans—decorin and biglycan—and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue’s ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease.

Preserved periprosthetic bone stock at 5 years post-operatively with uncemented short hip stem in both collared and collarless version

Introduction Previous bone density studies have generally shown bone resorption around both cemented and uncemented total hip arthroplasty (THA) stems. This is presumed to be due to stress shielding. Short stems have been introduced partly to preserve bone in the proximal femur by a more physiological loading of the bone. The purpose of this study was to evaluate bone remodeling around a short, fully hydroxyapatite-coated titanium stem that comes in a collared and collarless version. Patients and methods A prospective cohort of 50 patients included in a study evaluating the Furlong Evolution stem has been followed for 5 years. Examination was done with dual energy X-ray absorptiometry (DXA) postoperatively, at 1, 2 and 5 years. Clinical outcome was followed with radiography and both general and hip specific outcome measures. Results The two versions of the stem behaved similarly regarding bone remodeling. After an initial decrease up to 1 year, bone mineral density (BMD) increased in all Gruen zones up to 2 years and at 5 years bone stock was still preserved compared with postoperatively (net BMD + 1.2% (95% CI − 0.4 to 2.8)). Increase in BMD occurred mainly in the greater trochanter and distally around the stem with a decrease in the calcar area. Both versions showed excellent clinical outcome up to 5 years. Conclusion This short stem seems to preserve proximal bone stock up to 5 years, exhibiting similar behaviour both with and without a collar. Trial registration number and date of registration ClinicalTrials.gov, (identifier: NCT01894854). July 10, 2013.

TAVI-Mimic for testing of heart valve leaflet materials in physiological loading situations

The loading situation of the aortic valve is complex, complicating the identification of innovative approaches for heart valve leaflet materials, e.g. for transcatheter aortic valve implantation (TAVI). Materials engineering experiments allow for screening of materials but especially for durability testing, the consideration of physiological loads is vital/critical for the suitability-assessment of innovative leaflet materials. For this reason, a framework structure for the testing of leaflet materials in physiological loading (TAVI-Mimic) was developed. The exemplary use case for the TAVI-Mimic was a test for calcification propensity of pericardium during durability testing. The TAVI-Mimic was designed as a fourparted frame, based on previous work of our group. The leaflet material can be attached between inner and outer shells without sewing. In a second step, the TAVI-Mimic was optimized regarding radial load-deformation in comparison to a commercial TAVI by means of finite element analysis (FEA) and hydrodynamic characterization in a pulse duplicator system. Mechanical properties dependent on water uptake of different materials for 3D-printing of the TAVI-Mimic were investigated. After optimization, TAVI-Mimics were equipped with glutaraldehyde-fixated pericardial tissue and prototypes were calcified by using a heart valve durability tester and a metastable calcification-liquid, developed in earlier studies. The development of the TAVI-Mimic using FEA and experiments was successful, leading to a radial load dependent deformation of 0.6 mm which correlates with commercial TAVI. Two methacrylic photopolymers were identified for 3D-printing of the TAVI-Mimic and prototypes attached with pericardial tissue were manufactured. Pericardium TAVI-Mimics were calcified in vitro for one week and an average calciumphosphate precipitate of 0.34- 0.54 mg/cm² was measured. The optimization of the TAVR-Mimic led to an improved load-dependent behaviour compared to a commercial prosthesis while testing. The calcification method, combining the TAVI-Mimic, the metastable calcification solution and the durability tester enabled a successfully calcification of pericardial tissue, approaching the in vivo situation.

Programmed NP Cell Death Induced by Mitochondrial ROS in a One-Strike Loading Disc Degeneration Organ Culture Model

Increasing evidence has indicated that mitochondrial reactive oxygen species (ROS) play critical roles in mechanical stress-induced lumbar degenerative disc disease (DDD). However, the detailed underlying pathological mechanism needs further investigation. In this study, we utilized a one-strike loading disc degeneration organ culture model to explore the responses of intervertebral discs (IVDs) to mechanical stress. IVDs were subjected to a strain of 40% of the disc height for one second and then cultured under physiological loading. Mitoquinone mesylate (MitoQ) or other inhibitors were injected into the IVDs. IVDs subjected to only physiological loading culture were used as controls. Mitochondrial membrane potential was significantly depressed immediately after mechanical stress ( P < 0.01 ). The percentage of ROS-positive cells significantly increased in the first 12 hours after mechanical stress and then declined to a low level by 48 hours. Pretreatment with MitoQ or rotenone significantly decreased the proportion of ROS-positive cells ( P < 0.01 ). Nucleus pulposus (NP) cell viability was sharply reduced at 12 hours after mechanical stress and reached a stable status by 48 hours. While the levels of necroptosis- and apoptosis-related markers were significantly increased at 12 hours after mechanical stress, no significant changes were observed at day 7. Pretreatment with MitoQ increased NP cell viability and alleviated the marker changes by 12 hours after mechanical stress. Elevated mitochondrial ROS levels were also related to extracellular matrix (ECM) degeneration signs, including catabolic marker upregulation, anabolic marker downregulation, increased glycosaminoglycan (GAG) loss, IVD dynamic compressive stiffness reduction, and morphological degradation changes at the early time points after mechanical stress. Pretreatment with MitoQ alleviated some of these degenerative changes by 12 hours after mechanical stress. These changes were eliminated by day 7. Taken together, our findings demonstrate that mitochondrial ROS act as important regulators of programmed NP cell death and ECM degeneration in IVDs at early time points after mechanical stress.

Biomechanical Analysis of Posterior Ligaments of Cervical Spine and Laminoplasty

Cervical laminoplasty is a valuable procedure for myelopathy but it is associated with complications such as increased kyphosis. The effect of ligament damage during cervical laminoplasty on biomechanics is not well understood. We developed the C2–C7 cervical spine finite element model and simulated C3–C6 double-door laminoplasty. Three models were created (a) intact, (b) laminoplasty-pre (model assuming that the ligamentum flavum (LF) between C3–C6 was preserved during surgery), and (c) laminoplasty-res (model assuming that the LF between C3–C6 was resected during surgery). The models were subjected to physiological loading, and the range of motion (ROM), intervertebral nucleus stress, and facet contact forces were analyzed under flexion/extension, lateral bending, and axial rotation. The maximum change in ROM was observed under flexion motion. Under flexion, ROM in the laminoplasty-pre model increased by 100.2%, 111.8%, and 98.6% compared to the intact model at C3–C4, C4–C5, and C5–C6, respectively. The ROM in laminoplasty-res further increased by 105.2%, 116.8%, and 101.8% compared to the intact model at C3–C4, C4–C5, and C5–C6, respectively. The maximum stress in the annulus/nucleus was observed under left bending at the C4–C5 segment where an increase of 139.5% and 229.6% compared to the intact model was observed for laminoplasty-pre and laminoplasty-res model, respectively. The highest facet contact forces were observed at C4–C5 under axial rotation, where an increase of 500.7% and 500.7% was observed compared to the intact model for laminoplasty-pre and laminoplasty-res, respectively. The posterior ligaments of the cervical spine play a vital role in restoring/stabilizing the cervical spine. When laminoplasty is performed, the surgeon needs to be careful not to injure the posterior soft tissue, including ligaments such as LF.

Ciliary IFT88 safeguards coordinated epiphyseal vascularisation, resorption and ossification from disruptive physiological mechanical forces

In the musculoskeletal system, appropriate cell and tissue responses to mechanical force delineate morphogenesis and ensure lifelong health. Despite this, how mechanical cues are integrated into biological programmes remains unclear. Primary cilia are microtubule-based organelles that tune a range of cell activities, including signalling cascades activated or modulated, by extracellular biophysical cues. Here, we demonstrate that the inducible, cartilage-specific deletion of Intraflagellar transport protein 88 (IFT88), which reduces ciliation in the adolescent mouse growth plate (GP), uncouples chondrocyte differentiation from cartilage resorption and mineralisation in a mechano-dependent manner. Targeting IFT88, inhibits hypertrophic chondrocyte VEGF expression, vascular recruitment, osteoclastic activity and the replacement of cartilage with bone. These effects are largely restricted to peripheral tibial regions beneath the load-bearing compartments of the knee. Increases in physiological loading, in control mice, also impairs ossification in the peripheral GP, mimicking the effects of IFT88 deletion. Strikingly, limb immobilisation rescues disrupted VEGF and restores epiphyseal dynamics in Ift88cKO mice. These data indicate, that during this pivotal phase in adolescent skeletal maturation that defines the cessation of growth, ciliary IFT88 protects the coordinated ossification of the growth plate from an otherwise disruptive heterogeneity of physiological mechanical forces.

A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1β exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-β1 pathway. Notably, TGF-β1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1β exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

Bone Mechanoregulation Allows Subject-Specific Load Estimation Based on Time-Lapsed Micro-CT and HR-pQCT in Vivo

Patients at high risk of fracture due to metabolic diseases frequently undergo long-term antiresorptive therapy. However, in some patients, treatment is unsuccessful in preventing fractures or causes severe adverse health outcomes. Understanding load-driven bone remodelling, i.e., mechanoregulation, is critical to understand which patients are at risk for progressive bone degeneration and may enable better patient selection or adaptive therapeutic intervention strategies. Bone microarchitecture assessment using high-resolution peripheral quantitative computed tomography (HR-pQCT) combined with computed mechanical loads has successfully been used to investigate bone mechanoregulation at the trabecular level. To obtain the required mechanical loads that induce local variances in mechanical strain and cause bone remodelling, estimation of physiological loading is essential. Current models homogenise strain patterns throughout the bone to estimate load distribution in vivo, assuming that the bone structure is in biomechanical homoeostasis. Yet, this assumption may be flawed for investigating alterations in bone mechanoregulation. By further utilising available spatiotemporal information of time-lapsed bone imaging studies, we developed a mechanoregulation-based load estimation (MR) algorithm. MR calculates organ-scale loads by scaling and superimposing a set of predefined independent unit loads to optimise measured bone formation in high-, quiescence in medium-, and resorption in low-strain regions. We benchmarked our algorithm against a previously published load history (LH) algorithm using synthetic data, micro-CT images of murine vertebrae under defined experimental in vivo loadings, and HR-pQCT images from seven patients. Our algorithm consistently outperformed LH in all three datasets. In silico-generated time evolutions of distal radius geometries (n = 5) indicated significantly higher sensitivity, specificity, and accuracy for MR than LH (p &lt; 0.01). This increased performance led to substantially better discrimination between physiological and extra-physiological loading in mice (n = 8). Moreover, a significantly (p &lt; 0.01) higher association between remodelling events and computed local mechanical signals was found using MR [correct classification rate (CCR) = 0.42] than LH (CCR = 0.38) to estimate human distal radius loading. Future applications of MR may enable clinicians to link subtle changes in bone strength to changes in day-to-day loading, identifying weak spots in the bone microstructure for local intervention and personalised treatment approaches.