A Novel Primary Cilium-Mediated Mechanism Through which Osteocytes Regulate Metastatic Behavior of Both Breast and Prostate Cancer Cells.

Bone metastases are a common cause of suffering in breast and prostate cancer patients, however, the interaction between bone cells and cancer cells is poorly understood. Using a series of co-culture, conditioned media, human cancer spheroid, and organ-on-a-chip experiments, this study reveals that osteocytes suppress cancer cell proliferation and increase migration via tumor necrosis factor alpha (TNF-α) secretion. This action is regulated by osteocyte primary cilia and associated intraflagellar transport protein 88 (IFT88). Furthermore, it shows that cancer cells block this mechanism by secreting transforming growth factor beta (TGF-ß), which disrupts osteocyte cilia and IFT88 gene expression. This bi-directional crosstalk signaling between osteocytes and cancer cells is common to both breast and prostate cancer. This study also proposes that osteocyte inhibition of cancer cell proliferation decreases as cancer cells increase, producing more TGF-ß. Hence, a positive feedback loop develops accelerating metastatic tumor growth. These findings demonstrate the importance of cancer cell-osteocyte signaling in regulating breast and prostate bone metastases and support the development of therapies targeting this pathway.

Human vascularised synovium-on-a-chip: a mechanically stimulated, microfluidic model to investigate synovial inflammation and monocyte recruitment.

Healthy synovium is critical for joint homeostasis. Synovial inflammation (synovitis) is implicated in the onset, progression and symptomatic presentation of arthritic joint diseases such as rheumatoid arthritis and osteoarthritis. Thus, the synovium is a promising target for the development of novel, disease-modifying therapeutics. However, target exploration is hampered by a lack of good pre-clinical models that accurately replicate human physiology and that are developed in a way that allows for widespread uptake. The current study presents a multi-channel, microfluidic, organ-on-a-chip (OOAC) model, comprising a 3D configuration of the human synovium and its associated vasculature, with biomechanical and inflammatory stimulation, built upon a commercially available OOAC platform. Healthy human fibroblast-like synoviocytes (hFLS) were co-cultured with human umbilical vein endothelial cells (HUVECs) with appropriate matrix proteins, separated by a flexible, porous membrane. The model was developed within the Emulate organ-chip platform enabling the application of physiological biomechanical stimulation in the form of fluid shear and cyclic tensile strain. The hFLS exhibited characteristic morphology, cytoskeletal architecture and matrix protein deposition. Synovial inflammation was initiated through the addition of interleukin-1ß(IL-1ß) into the synovium channel resulting in the increased secretion of inflammatory and catabolic mediators, interleukin-6 (IL-6), prostaglandin E2 (PGE2), matrix metalloproteinase 1 (MMP-1), as well as the synovial fluid constituent protein, hyaluronan. Enhanced expression of the inflammatory marker, intercellular adhesion molecule-1 (ICAM-1), was observed in HUVECs in the vascular channel, accompanied by increased attachment of circulating monocytes. This vascularised human synovium-on-a-chip model recapitulates a number of the functional characteristics of both healthy and inflamed human synovium. Thus, this model offers the first human synovium organ-chip suitable for widespread adoption to understand synovial joint disease mechanisms, permit the identification of novel therapeutic targets and support pre-clinical testing of therapies.

Organ-on-a-Chip and Microfluidic Platforms for Oncology in the UK.

Organ-on-chip systems are capable of replicating complex tissue structures and physiological phenomena. The fine control of biochemical and biomechanical cues within these microphysiological systems provides opportunities for cancer researchers to build complex models of the tumour microenvironment. Interest in applying organ chips to investigate mechanisms such as metastatsis and to test therapeutics has grown rapidly, and this review draws together the published research using these microfluidic platforms to study cancer. We focus on both in-house systems and commercial platforms being used in the UK for fundamental discovery science and therapeutics testing. We cover the wide variety of cancers being investigated, ranging from common carcinomas to rare sarcomas, as well as secondary cancers. We also cover the broad sweep of different matrix microenvironments, physiological mechanical stimuli and immunological effects being replicated in these models. We examine microfluidic models specifically, rather than organoids or complex tissue or cell co-cultures, which have been reviewed elsewhere. However, there is increasing interest in incorporating organoids, spheroids and other tissue cultures into microfluidic organ chips and this overlap is included. Our review includes a commentary on cancer organ-chip models being developed and used in the UK, including work conducted by members of the UK Organ-on-a-Chip Technologies Network. We conclude with a reflection on the likely future of this rapidly expanding field of oncological research.

Techniques for Visualization and Quantification of Primary Cilia in Chondrocytes.

Primary cilia regulate and coordinate a variety of cell signaling pathways important in chondrocyte physiology and cartilage development, health, and disease. Despite this, the chondrocyte primary cilium and its associated role in cartilage biology remains poorly understood. Key to elucidating primary cilia structure and function in chondrocytes is the ability to visualize this unique structure. Here we describe materials and methods for immunofluorescence labeling, microscopy, and measurement of chondrocyte primary cilia.

Oscillations of the circadian clock protein, BMAL-1, align to daily cycles of mechanical stimuli: a novel means to integrate biological time within predictive in vitro model systems.

Purpose: In vivo, the circadian clock drives 24-h rhythms in human physiology. Isolated cells in vitro retain a functional clockwork but lack necessary timing cues resulting in the rapid loss of tissue-level circadian rhythms. This study tests the hypothesis that repeated daily mechanical stimulation acts as a timing cue for the circadian clockwork. The delineation and integration of circadian timing cues into predictive in vitro model systems, including organ-on-a-chip (OOAC) devices, represent a novel concept that introduces a key component of in vivo physiology into predictive in vitro model systems. Methods: Quiescent bovine chondrocytes were entrained for 3 days by daily 12-h bouts of cyclic biaxial tensile strain (10%, 0.33 Hz, Flexcell) before sampling during free-running conditions. The core clock protein, BMAL-1, was quantified from normalised Western Blot signal intensity and the temporal oscillations characterised by Cosinor linear fit with 24-h period. Results: Following entrainment, the cell-autonomous oscillations of the molecular clock protein, BMAL-1, exhibited circadian (24 h) periodicity (p < 0.001) which aligned to the diurnal mechanical stimuli. A 6-h phase shift in the mechanical entrainment protocol resulted in an equivalent shift of the circadian clockwork. Thus, repeated daily mechanical stimuli synchronised circadian rhythmicity of chondrocytes in vitro. Conclusion: This work demonstrates that daily mechanical stimulation can act as a timing cue that is sufficient to entrain the peripheral circadian clock in vitro. This discovery may be exploited to induce and sustain circadian physiology within into predictive in vitro model systems, including OOAC systems. Integration of the circadian clock within these systems will enhance their potential to accurately recapitulate human diurnal physiology and hence augment their predictive value as drug testing platforms and as realistic models of human (patho)physiology. Supplementary Information: The online version contains supplementary material available at 10.1007/s44164-022-00032-x.

Collagen pre-strain discontinuity at the bone-Cartilage interface.

The bone-cartilage unit (BCU) is a universal feature in diarthrodial joints, which is mechanically-graded and subjected to shear and compressive strains. Changes in the BCU have been linked to osteoarthritis (OA) progression. Here we report existence of a physiological internal strain gradient (pre-strain) across the BCU at the ultrastructural scale of the extracellular matrix (ECM) constituents, specifically the collagen fibril. We use X-ray scattering that probes changes in the axial periodicity of fibril-level D-stagger of tropocollagen molecules in the matrix fibrils, as a measure of microscopic pre-strain. We find that mineralized collagen nanofibrils in the calcified plate are in tensile pre-strain relative to the underlying trabecular bone. This behaviour contrasts with the previously accepted notion that fibrillar pre-strain (or D-stagger) in collagenous tissues always reduces with mineralization, via reduced hydration and associated swelling pressure. Within the calcified part of the BCU, a finer-scale gradient in pre-strain (0.6% increase over ~50µm) is observed. The increased fibrillar pre-strain is linked to prior research reporting large tissue-level residual strains under compression. The findings may have biomechanical adaptative significance: higher in-built molecular level resilience/damage resistance to physiological compression, and disruption of the molecular-level pre-strains during remodelling of the bone-cartilage interface may be potential factors in osteoarthritis-based degeneration.

Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion.

Women with ductal carcinoma in situ (DCIS) have an increased risk of progression to invasive breast cancer. Although not all women with DCIS will progress to invasion, all are treated as such, emphasising the need to identify prognostic biomarkers. We have previously shown that altered myoepithelial cells in DCIS predict disease progression and recurrence. By analysing DCIS duct size in sections of human breast tumour samples, we identified an associated upregulation of integrin ß6 and an increase in periductal fibronectin deposition with increased DCIS duct size that associated with the progression of DCIS to invasion. Our modelling of the mechanical stretching myoepithelial cells undergo during DCIS progression confirmed the upregulation of integrin ß6 and fibronectin expression in isolated primary and cell line models of normal myoepithelial cells. Our studies reveal that this mechanostimulated DCIS myoepithelial cell phenotype enhances invasion in a TGFß-mediated upregulation of MMP13. Immunohistochemical analysis identified that MMP13 was specifically upregulated in DCIS, and it was associated with progression to invasion. These findings implicate tissue mechanics in altering the myoepithelial cell phenotype in DCIS, and that these alterations may be used to stratify DCIS patients into low and high risk for invasive progression.

Reversible changes in the 3D collagen fibril architecture during cyclic loading of healthy and degraded cartilage.

Biomechanical changes to the collagen fibrillar architecture in articular cartilage are believed to play a crucial role in enabling normal joint function. However, experimentally there is little quantitative knowledge about the structural response of the Type II collagen fibrils in cartilage to cyclic loading in situ, and the mechanisms that drive the ability of cartilage to withstand long-term repetitive loading. Here we utilize synchrotron small-angle X-ray scattering (SAXS) combined with in-situ cyclic loading of bovine articular cartilage explants to measure the fibrillar response in deep zone articular cartilage, in terms of orientation, fibrillar strain and inter-fibrillar variability in healthy cartilage and cartilage degraded by exposure to the pro-inflammatory cytokine IL-1ß. We demonstrate that under repeated cyclic loading the fibrils reversibly change the width of the fibrillar orientation distribution whilst maintaining a largely consistent average direction of orientation. Specifically, the effect on the fibrillar network is a 3-dimensional conical orientation broadening around the normal to the joint surface, inferred by 3D reconstruction of X-ray scattering peak intensity distributions from the 2D pattern. Further, at the intrafibrillar level, this effect is coupled with reversible reduction in fibrillar pre-strain under compression, alongside increase in the variability of fibrillar pre-strain. In IL-1ß degraded cartilage, the collagen rearrangement under cyclic loading is disrupted and associated with reduced tissue stiffness. These finding have implications as to how changes in local collagen nanomechanics might drive disease progression or vice versa in conditions such as osteoarthritis and provides a pathway to a mechanistic understanding of such diseases. STATEMENT OF SIGNIFICANCE: Structural deterioration in biomechanically loaded musculoskeletal organs, e.g., joint osteoarthritis and back pain, are linked to breakdown and changes in their collagen-rich cartilaginous tissue matrix. A critical component enabling cartilage biomechanics is the ultrastructural collagen fibrillar network in cartilage. However, experimental probes of the dynamic structural response of cartilage collagen to biomechanical loads are limited. Here, we use X-ray scattering during cyclic loading (as during walking) on joint tissue to show that cartilage fibrils resist loading by a reversible, three-dimensional orientation broadening and disordering mechanism at the molecular level, and that inflammation reduces this functionality. Our results will help understand how changes to small-scale tissue mechanisms are linked to ageing and osteoarthritic progression, and development of biomaterials for joint replacements.

Mechanical Stimulation Modulates Osteocyte Regulation of Cancer Cell Phenotype.

Breast and prostate cancers preferentially metastasise to bone tissue, with metastatic lesions forming in the skeletons of most patients. On arriving in bone tissue, disseminated tumour cells enter a mechanical microenvironment that is substantially different to that of the primary tumour and is largely regulated by bone cells. Osteocytes, the most ubiquitous bone cell type, orchestrate healthy bone remodelling in response to physical exercise. However, the effects of mechanical loading of osteocytes on cancer cell behaviour is still poorly understood. The aim of this study was to characterise the effects of osteocyte mechanical stimulation on the behaviour of breast and prostate cancer cells. To replicate an osteocyte-controlled environment, this study treated breast (MDA-MB-231 and MCF-7) and prostate (PC-3 and LNCaP) cancer cell lines with conditioned media from MLO-Y4 osteocyte-like cells exposed to mechanical stimulation in the form of fluid shear stress. We found that osteocyte paracrine signalling acted to inhibit metastatic breast and prostate tumour growth, characterised by reduced proliferation and invasion and increased migration. In breast cancer cells, these effects were largely reversed by mechanical stimulation of osteocytes. In contrast, conditioned media from mechanically stimulated osteocytes had no effect on prostate cancer cells. To further investigate these interactions, we developed a microfluidic organ-chip model using the Emulate platform. This new organ-chip model enabled analysis of cancer cell migration, proliferation and invasion in the presence of mechanical stimulation of osteocytes by fluid shear stress, resulting in increased invasion of breast and prostate cancer cells. These findings demonstrate the importance of osteocytes and mechanical loading in regulating cancer cell behaviour and the need to incorporate these factors into predictive in vitro models of bone metastasis.

Modelling TGFßR and Hh pathway regulation of prognostic matrisome molecules in ovarian cancer.

In a multi-level "deconstruction" of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix (ECM) molecules, COL11A1, cartilage oligomeric matrix protein, FN1, versican, cathepsin B, and COL1A1, are upregulated in cancer. Using biopsies, we identified significant associations between TGFßR activity, Hedgehog (Hh) signaling, and these ECM molecules and studied the associations in mono-, co-, and tri-culture. Activated omental fibroblasts (OFs) produced more matrix than malignant cells, directed by TGFßR and Hh signaling cross talk. We "reconstructed" omental metastases in tri-cultures of HGSOC cells, OFs, and adipocytes. This combination was sufficient to generate all six ECM proteins and the matrisome expression signature. TGFßR and Hh inhibitor combinations attenuated fibroblast activation and gel and ECM remodeling in these models. The tri-culture model reproduces key features of omental metastases and allows study of diseased-associated ECM.

A human multi-cellular model shows how platelets drive production of diseased extracellular matrix and tissue invasion.

Guided by a multi-level "deconstruction" of omental metastases, we developed a tetra (four cell)-culture model of primary human mesothelial cells, fibroblasts, adipocytes, and high-grade serous ovarian cancer (HGSOC) cell lines. This multi-cellular model replicated key elements of human metastases and allowed malignant cell invasion into the artificial omental structure. Prompted by findings in patient biopsies, we used the model to investigate the role of platelets in malignant cell invasion and extracellular matrix, ECM, production. RNA (sequencing and quantitative polymerase-chain reaction), protein (proteomics and immunohistochemistry) and image analysis revealed that platelets stimulated malignant cell invasion and production of ECM molecules associated with poor prognosis. Moreover, we found that platelet activation of mesothelial cells was critical in stimulating malignant cell invasion. Whilst platelets likely activate both malignant cells and mesothelial cells, the tetra-culture model allowed us to dissect the role of both cell types and model the early stages of HGSOC metastases.

Polycystin-2 Is Required for Chondrocyte Mechanotransduction and Traffics to the Primary Cilium in Response to Mechanical Stimulation.

Primary cilia and associated intraflagellar transport are essential for skeletal development, joint homeostasis, and the response to mechanical stimuli, although the mechanisms remain unclear. Polycystin-2 (PC2) is a member of the transient receptor potential polycystic (TRPP) family of cation channels, and together with Polycystin-1 (PC1), it has been implicated in cilia-mediated mechanotransduction in epithelial cells. The current study investigates the effect of mechanical stimulation on the localization of ciliary polycystins in chondrocytes and tests the hypothesis that they are required in chondrocyte mechanosignaling. Isolated chondrocytes were subjected to mechanical stimulation in the form of uniaxial cyclic tensile strain (CTS) in order to examine the effects on PC2 ciliary localization and matrix gene expression. In the absence of strain, PC2 localizes to the chondrocyte ciliary membrane and neither PC1 nor PC2 are required for ciliogenesis. Cartilage matrix gene expression (Acan, Col2a) is increased in response to 10% CTS. This response is inhibited by siRNA-mediated loss of PC1 or PC2 expression. PC2 ciliary localization requires PC1 and is increased in response to CTS. Increased PC2 cilia trafficking is dependent on the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4) activation. Together, these findings demonstrate for the first time that polycystins are required for chondrocyte mechanotransduction and highlight the mechanosensitive cilia trafficking of PC2 as an important component of cilia-mediated mechanotransduction.

Corrigendum: Mechanical Stimulation: A Crucial Element of Organ-on-Chip Models.

[This corrects the article DOI: 10.3389/fbioe.2020.602646.].

Mechanical Stimulation: A Crucial Element of Organ-on-Chip Models.

Organ-on-chip (OOC) systems recapitulate key biological processes and responses in vitro exhibited by cells, tissues, and organs in vivo. Accordingly, these models of both health and disease hold great promise for improving fundamental research, drug development, personalized medicine, and testing of pharmaceuticals, food substances, pollutants etc. Cells within the body are exposed to biomechanical stimuli, the nature of which is tissue specific and may change with disease or injury. These biomechanical stimuli regulate cell behavior and can amplify, annul, or even reverse the response to a given biochemical cue or drug candidate. As such, the application of an appropriate physiological or pathological biomechanical environment is essential for the successful recapitulation of in vivo behavior in OOC models. Here we review the current range of commercially available OOC platforms which incorporate active biomechanical stimulation. We highlight recent findings demonstrating the importance of including mechanical stimuli in models used for drug development and outline emerging factors which regulate the cellular response to the biomechanical environment. We explore the incorporation of mechanical stimuli in different organ models and identify areas where further research and development is required. Challenges associated with the integration of mechanics alongside other OOC requirements including scaling to increase throughput and diagnostic imaging are discussed. In summary, compelling evidence demonstrates that the incorporation of biomechanical stimuli in these OOC or microphysiological systems is key to fully replicating in vivo physiology in health and disease.

Solution-Processed Epitaxial Growth of Arbitrary Surface Nanopatterns on Hybrid Perovskite Monocrystalline Thin Films.

Semiconductor surface patterning at the nanometer scale is crucial for high-performance optical, electronic, and photovoltaic devices. To date, surface nanostructures on organic-inorganic single-crystal perovskites have been achieved mainly through destructive methods such as electron-beam lithography and focused ion beam milling. Here, we present a solution-based epitaxial growth method for creating nanopatterns on the surface of perovskite monocrystalline thin films. We show that high-quality monocrystalline arbitrary nanopatterns can form in solution with a low-cost simple setup. We also demonstrate controllable photoluminescence from nanopatterned perovskite surfaces by adjusting the nanopattern parameters. A seven-fold enhancement in photoluminescence intensity and a three-time reduction of the surface radiative recombination lifetime are observed at room temperature for nanopatterned MAPbBr3 monocrystalline thin films. Our findings are promising for the cost-effective fabrication of monocrystalline perovskite on-chip electronic and photonic circuits down to the nanometer scale with finely tunable optoelectronic properties.

Sub-toxic levels of Co2+ are anti-inflammatory and protect cartilage from degradation caused by IL-1ß.

BACKGROUND: Cobalt ions from some orthopaedic implants induce a dose-dependent cytotoxic and pro-inflammatory response. Recent studies show that sub-toxic levels of cobalt influence actin organisation regulating fibroblasts and macrophages behaviour. However little is known about the influence of sub-toxic levels of cobalt on articular cartilage biology and biomechanics. Previously, we have reported that IL-1ß signalling in chondrocytes, is regulated by primary cilia and associated intraflagellar transport. Since primary cilia expression is modulated by actin organisation, we set out to test the hypothesis that sub-toxic levels of cobalt regulate cilia expression and IL-1ß signalling thereby influencing articular cartilage degradation. METHODS: Isolated chondrocytes and bovine cartilage explants were subjected to Co2+ in the presence and absence of IL-1ß. Primary cilia were monitored by confocal immunofluorescence. Nitric oxide and PGE2 release were used to monitor IL-1ß signalling. Degradation of cartilage matrix was assessed by the release of sGAG and the biomechanical properties of the tissue in uniaxial unconfined compression. FINDINGS: Sub-toxic levels of Co2+ (50 µM) blocked IL-1ß-induced primary cilia elongation in isolated chondrocytes. This was associated with disruption of pro-inflammatory signalling in both isolated chondrocytes and cartilage explants, and inhibition of cartilage matrix degradation and loss of biomechanical properties. INTERPRETATION: This study reveals that low levels of cobalt ions are anti-inflammatory, preventing cartilage degradation in response to IL-1ß. This mechanism is associated with regulation of primary cilia elongation. These observations provide new insight into the potential beneficial role of cobalt and may lead to novel mechanisms for controlling cartilage inflammation.

Nanoscale Mapping Reveals Functional Differences in Ion Channels Populating the Membrane of Primary Cilia.

BACKGROUND/AIMS: The primary cilium is a nanoscale membrane protrusion believed to act as a mechano-chemical sensor in a range of different cell types. Disruptions in its structure and signalling have been linked to a number of medical conditions, referred to as ciliopathies, but remain poorly understood due to lack of techniques capable of investigating signal transduction in cilia at nanoscale. Here we set out to use latest advances in nanopipette technology to address the question of ion channel distribution along the structure of primary cilium. METHODS: We used glass nanopipettes and Scanning Ion Conductance Microscopy (SICM) to image 3D topography of intact primary cilia in inner medullary collecting duct (IMCD) cells with nanoscale resolution. The high-resolution topographical images were then used to navigate the nanopipette along the structure of each cilium and perform spatially resolved single-channel recordings under precisely controlled mechanical and chemical stimulation. RESULTS: We have successfully obtained first single-channel recordings at specific locations of intact primary cilia. Our experiments revealed significant differences between the populations of channels present at the ciliary base, tip and within extra-ciliary regions in terms of mean conductance and sensitivity to membrane displacement as small as 100 nm. Ion channels at the base of cilium, where mechanical strain is expected to be the highest, appeared particularly sensitive to the mechanical displacement. CONCLUSION: Our results suggest the distribution of ion channels in the membrane of primary cilia is non-homogeneous. The relationship between the location and function of ciliary ion channels could be key to understanding signal transduction in primary cilia.

Ciliary exclusion of Polycystin-2 promotes kidney cystogenesis in an autosomal dominant polycystic kidney disease model.

The human PKD2 locus encodes Polycystin-2 (PC2), a TRPP channel that localises to several distinct cellular compartments, including the cilium. PKD2 mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD) and affect many cellular pathways. Data underlining the importance of ciliary PC2 localisation in preventing PKD are limited because PC2 function is ablated throughout the cell in existing model systems. Here, we dissect the ciliary role of PC2 by analysing mice carrying a non-ciliary localising, yet channel-functional, PC2 mutation. Mutants develop embryonic renal cysts that appear indistinguishable from mice completely lacking PC2. Despite not entering the cilium in mutant cells, mutant PC2 accumulates at the ciliary base, forming a ring pattern consistent with distal appendage localisation. This suggests a two-step model of ciliary entry; PC2 first traffics to the cilium base before TOP domain dependent entry. Our results suggest that PC2 localisation to the cilium is necessary to prevent PKD.

Proteoglycan degradation mimics static compression by altering the natural gradients in fibrillar organisation in cartilage.

Structural and associated biomechanical gradients within biological tissues are important for tissue functionality and preventing damaging interfacial stress concentrations. Articular cartilage possesses an inhomogeneous structure throughout its thickness, driving the associated variation in the biomechanical strain profile within the tissue under physiological compressive loading. However, little is known experimentally about the nanostructural mechanical role of the collagen fibrils and how this varies with depth. Utilising a high-brilliance synchrotron X-ray source, we have measured the depth-wise nanostructural parameters of the collagen network in terms of the periodic fibrillar banding (D-period) and associated parameters. We show that there is a depth dependent variation in D-period reflecting the pre-strain and concurrent with changes in the level of intrafibrillar order. Further, prolonged static compression leads to fibrillar changes mirroring those caused by removal of extrafibrillar proteoglycans (as may occur in aging or disease). We suggest that fibrillar D-period is a sensitive indicator of localised changes to the mechanical environment at the nanoscale in soft connective tissues. STATEMENT OF SIGNIFICANCE: Collagen plays a significant role in both the structural and mechanical integrity of articular cartilage, allowing the tissue to withstand highly repetitive loading. However, the fibrillar mechanics of the collagen network in cartilage are not clear. Here we find that cartilage has a spatial gradient in the nanostructural collagen fibril pre-strain, with an increase in the fibrillar pre-strain with depth. Further, the fibrillar gradient changes similarly under compression when compared to an enzymatically degraded tissue which mimics age-related changes. Given that the fibrils potentially have a finite capacity to mechanically respond and alter their configuration, these findings are significant in understanding how collagen may alter in structure and gradient in diseased cartilage, and in informing the design of cartilage replacements.