Introduction. Analogous to articular cartilage, changes in spatial chondrocyte organisation have been proposed to be a strong indicator for local tissue degeneration and destruction in the intervertebral disc (IVD). While a progressive structural and functional degradation of the extracellular (ECM) and pericellular (PCM) matrix occurs in osteoarthritic cartilage, these processes have not yet been biomechanically elucidated in the IVD. We aimed to evaluate the local stiffness of the ECM and PCM in the anulus fibrosus of the IVD on the basis of local cellular spatial organisation. Method. Using atomic force microscopy, we measured the elastic modulus of the local ECM and PCM in human disc samples using the spatial chondrocyte patterns as an image-based biomarker. Result. By measuring tissue from 30 patients, we found a significant difference in the elastic moduli of the PCM in clusters when compared to the healthy patterns single cells (p=0.029), pairs (p=0.016), and string formations (p=0.010) whereas the values of the elastic moduli of the ECM only reached statistical significance when clusters were compared with string formations. The ECM/PCM ratio ranged from 0.62 to 0.89. Overall, the reduced elastic moduli in clusters demonstrates that cluster formation is not only a morphological phenomenon describing disc degeneration, but it marks a compromised biomechanical functioning. Conclusion. This study is the first to describe and quantify the differences in the elastic moduli of the ECM in relation to the PCM in the anulus fibrosus of the IVD by means of atomic force microscopy on the basis of spatial chondrocyte organisation. Advanced disc degeneration is accompanied by a biomechanically compromised tissue functioning

It is known that the gait dynamics of elderly substantially differs from that of young people. However, it has not been well studied how this age-related gait dynamics affects the knee biomechanics, e.g., cartilage mechanical response. In this study, we investigated how aging affects knee biomechanics in a female population using subject-specific computational models. Two female subjects (ages of 23 and 69) with no musculoskeletal disorders were recruited. Korea National Institute for Bioethics Policy Review Board approved the study. Participants walked at a self-selected speed (SWS), 110% of SWS, and 120% of SWS on 10 m flat ground. Three-dimensional marker trajectories and ground reaction forces (Motion Analysis, USA), and lower limbs’ muscle activities were measured (EMG, Noraxon USA). Knee cartilage and menisci geometries were obtained from subjects’ magnetic resonance images (3T, GE Health Care). An EMG-assisted musculoskeletal finite element modeling workflow was used to estimate knee cartilage tissue mechanics in walking trials. Knee cartilage and menisci were modeled using a transversely isotropic poroviscoelastic material model. Walking speed in SWS, 110%, and 120% of SWS were 1.38 m/s, 1.51 m/s, and 1.65 m/s for the young, and 1.21 m/s, 1.34 m/s and 1.46 m/s for the elderly, respectively. The maximum tensile stress in the elderly tibial cartilage was ~25%, ~33%, and ~32% lower than the young at SWS, 110%, and 120% of SWS, respectively. These preliminary results suggest that the cartilage in the elderly may not have enough stimulation even at 20% increases in walking speed, which may be one reason for tissue degeneration. To enhance these findings, further study with more subjects and different genders will investigate how age-related gait dynamics affects knee biomechanics. Acknowledgments: Australian NHMRC Ideas Grant (APP2001734), KITECH (JE220006)

Introduction. Promoting bone mass homeostasis keeps skeleton away from osteoporosis. a-Ketoglutarate (a-KG) is an indispensable intermediate of tricarboxylic acid cycle (TCA) process for cellular energy production. a-KG mitigates cellular senescence, tissue degeneration, and oxidative stress. We investigated whether a-KG affected osteoblast activity or osteoporosis development. Method. Serum and bone specimens were biopsied from 26 patients with osteoporosis or 24 patients without osteoporosis who required spinal surgery. Ovariectomized or aged mice were fed 0.25% or 0.75% a-KG in drinking water for 8 – 12 weeks ad libitum. Bone mineral density, trabecular/cortical bone microarchitecture, mechanical strength, bone formation, and osteoclastic erosion were investigated using mCT, material testing device, in vivo calcein labelling, and TRAP histochemical staining. Serum a-KG, osteocalcin, and TRAP5b levels were quantified using ELISA kits. Bone-marrow mesenchymal cells and macrophages were incubated osteogenic and osteoclastogenic media. Histone H3K27me3 levels and enrichment were investigated using immunoblotting and chromatin precipitation-PCR. Result. Serum a-KG levels in patients with osteoporosis were less than controls; and were correlated with T-scores of hips (R2 = 0.6471, P < 0.0001) and lumbar spine (R2 = 0.7235, P < 0.001) in osteoporosis (AUC = 0.9941, P < 0.001). a-KG supplement compromised a plethora of osteoporosis signs in ovariectomized or aged mice, including bone mass loss, trabecular bone microarchitecture deterioration, and mechanical strength loss. It elevated serum osteocalcin levels and decreased serum TRAP5b. a-KG preserved caclein-labelling bone formation and repressed osteoclast resorption. It reversed osteogenic differentiation of bone-marrow stromal cells and reduced osteoclast formation in ovariectomized mice. Mechanically, a-KG attenuated H3K27 hypermethylation and Runx2 transcription repression, improving mineralized matrix production in osteogenic cells. Conclusion. Decreased serum a-KG is correlated with human and murine osteoporosis. a-KG reverses bone loss by repressing histone methylation in osteoblasts. This study highlighted a-KG supplement as a new biochemical option for protecting osteoporosis

Chronic Achilles tendinopathy is characterised by sub-acute inflammation with pro-inflammatory type 1 macrophages (M1), tissue degeneration and consequent partial or total tendon injury. Control of the inflammatory response and M1-to-M2 macrophage polarisation can favour tendon healing both directly and indirectly, by allowing for the regenerative process driven by local mesenchymal stem cells. Ten patients (3 females and 7 males aged between 32 and 71 years old) with partial Achilles tendon injury were treated with injections of autologous peripheral blood mononuclear cells (PB-MNCs). The cell concentrate was obtained from 100-120 cc of each patient's blood with a selective point-of-care filtration system. PB-MNCs remained trapped in the filter and were injected immediately after sampling. Around 60% of the PB-MNC concentrate was injected directly into the injured area, while the remaining 40% was injected in smaller amounts into the surrounding parts of the Achilles tendon affected by tendinosis. All patients were evaluated both clinically with the help of the American Orthopaedic Foot & Ankle Society (AOFAS) scale, and radiologically (MRI examination) at baseline and 2 months after the PB-MNC injection. A clinical reassessment with the AOFAS scale was also performed 6 months after the intervention. The rehabilitation protocol implied full weight-bearing walking immediately after the procedure, light physical activity 3-4 days after the injection, and physiotherapist-assisted stretching exercises and eccentric training. In all patients, functional and radiological signs of tendon healing processes were detected as early as 2 months after a single treatment and the AOFAS scale rose from the initial mean value of 37.5 (baseline) to 85.4 (6 months). Our preliminary results indicate that regenerative therapies with PB-MNCs can prove useful for partial Achilles tendon injuries as a valid alternative to surgical options, especially when other conservative approaches have failed. Advantages of this therapy include rapid execution, no need for an operating theatre, easy reproducibility, quick recovery and good tolerability regardless of the patient's age (the procedure is not to be performed in subjects who are below 18 years old). Further studies on the topic are recommended to confirm these observations

Tendon tissue equilibrium very heavily depends on appropriate mechanical loading within a narrow, and still poorly defined, physiological range. We will present an overview of our recent work on the tendon cell-matrix interactions that drive tissue homeostasis, matrix remodelling and eventual tissue degeneration, and discuss a roadmap for unravelling these mechanically regulated signalling pathways for the development of effective treatment strategies. Our data suggest that tissue damage accumulates in the tendon until “intrinsic repair mechanisms” are overwhelmed. At this point, the metabolic cost of extracellular matrix remodeling exceeds the locally available nutrient supply. We hypothesize that upon reach S43.1 ing this “Metabolic Tipping Point”, the vascular system is recruited along with accompanying nerve supply (and pain) and the tissue enters into a chronic disease state characterized by high matrix turnover and increasingly poor tissue quality. In this paradigm, a delicate mechanically regulated balance exists between recruitment and suppression of the extrinsic vascular system by the resident tendon core cells. Upon injury or damage, this regulation in turn steers the tissue towards either functional remodeling or chronic tendon disease

Objective. Full-thickness cartilage defects are commonly found in symptomatic knee patients, and are associated with progressive cartilage degeneration. Although the risk of defect progression to degenerative osteoarthritis is multifactorial, articular cartilage defects change contact mechanics and the mechanical response of tissue adjacent to the defect. The objective of this study was to quantify changes in intra-tissue strain patterns occurring at the defect rim and opposing tissue in an experimental model mimicking in vivo cartilage-on-cartilage contact conditions. Methods. Macroscopically intact osteochondral explants with smooth surfaces were harvested form the femoral condyles of 9 months old bovine knees. Two groups were tested; reference group with intact cartilage (n=8) and defect group with a full thickness cylindrical defect (diameter 8 mm) in one cartilage surface from each pair (n=8). The explants with defect articular surface and the opposing intact cartilage were compressed at ∼0.33 times body weight (350N) during cycles of 2s loading followed by 1.4s unloading. In plane tissue deformations were measured using displacement encoded imaging with stimulated echoes (DENSE) on a 9.4T MRI scanner. A two-sample t-test was used to assess statistical significance (p<0.05) of differences in maximal Green-Lagrange strains between the defect, opposing surface and intact reference cartilage. Results. Strain levels were elevated in the cartilage neighbouring the defect rim and in the opposing articulating surface. Similar to intact cartilage, compressive and tensile strains presented a depth dependent variation. The maximal strains profiles were highest in the superficial zone and decreased with depth for all explants, except for the shear strains in the cartilage opposing the defect which were constant. The maximal tensile strain in the middle and superficial zone were significantly higher for the defect cartilage (3.97±1.99% and 4.52±2.04%) compared to the intact reference (1.91±1.13% and 2.53±1.27%), indicating that the defect edges are bulging towards the defect. The shear strains were significantly higher (∼1.5x) throughout cartilage depth of the defect rim compared to the intact reference cartilage. However, in the cartilage opposing the defect, shear strains were significantly lower (∼0.5x) compared to the intact cartilage representing less matrix distortion. No significant difference in maximal compressive strains were observed between the opposing intact and defect at all cartilage depths. Conclusions. Presence of isolated full thickness cartilage defects will affect the cartilage deformations. Even under pure compressive loading alone, the altered contact mechanics resulted in excessive strains at tissue adjacent to the defect potentially damaging the cartilage and inducing tissue degeneration

Introduction. The exact mechanisms leading to tendinopathies and tendon ruptures remain poorly understood while their occurrence is clearly associated with exercise. Overloading is thought to be a major factor contributing to the development of tendon pathologies. However, as animal studies have shown, heavy loading alone won't cause tendinopathies. It has been speculated, that malfunctioning adaptation or healing processes might be involved, triggering tendon tissue degeneration. By analysing the expression of the entirety of degrading enzymes (degradome) in pathological and non-pathological, strained and non-strained tendon tissue, the aim of this study was to identify common or opposite patterns in gene regulation. This approach may generate new targets for future studies. Materials and Methods. RNA was extracted from different tendon tissues: normal (n=7), tendinopathic (n=4) and ruptured (n=4) Achilles tendon; normal (n=4) and tendinopathic (n=4) posterior tibialis tendon; normal hamstrings tendon with or without subjection to static strain (n=4). The RNA was reverse transcribed, then pooled per group The expression of 538 protease genes was analysed using Taqman low-density array quantitative RT-PCR. To be considered relevant, changes had to be at least 4fold and measurable at a level below 36 Cts. Results. In general, there was little common regulation when exercised was compared with pathological tissue. The expression of PAMR1 and TNFαIP3 was upregulated with exercise (169-fold and 78-fold), Achilles tendinopathy (9724-fold and 7-fold) and Achilles tendon rupture (1809-fold and 10-fold), while DDI1, PSMB11 and PSH2 which were down-regulated with exercise were upregulated with Achilles pathology. Discussion. The newly found targets may deliver insights into the initiation and progression of tendon pathologies: PAMR1, a regeneration associated muscle protease which has been shown to be downregulated in Duchenne muscular dystrophy and upregulated in regenerating muscle fibers, might also be involved in tendon regeneration; TNFαIP3, which negatively regulates the NF-κB/pro-inflammatory pathway, could have anti-inflammatory function in tendon regeneration. PSMB11 and PSH2 are for the first time shown to be expressed in tendon and regulated in tendon pathology. Using this approach we were able to generate new targets and to add information on function, regulation and expression sites of recently identified proteins

Introduction. The ability of tendons to withstand stress generally decreases with age, often resulting in increased tissue degeneration and decreased regeneration capacity. However, the underlying molecular and cellular mechanisms of tendon senescence remain poorly characterized. Therefore, the aim of the current study was to identify genes showing an age-dependent altered expression profile in tendons. Materials and Methods. A suppression-subtractive-hybridization (SSH) screen comparing cDNA libraries generated from Achilles tendons of mature-adult (3 months) and old (18 months) female C57BL/6 mice was conducted. Subsequently, the differential expression of the identified genes was validated by RT-qPCR and selected genes were then further analysed by immunohistochemistry and Western blot. To investigate age-related structural alterations in the collagenous extracellular matrix we applied SHG-microscopy and TEM. In vitro experiments with young and old tendon derived stem/progenitor cells (TDSCs) involved wounding assays, tendon-like constructs as well as collagen gel contraction assays. Results. Among 168 identified genes, several ECM genes showed a differential expression, including Col1a1, Col3a1, fibronectin, fibromodulin, thrombospondin-1, decorin, biglycan, lysyl oxidase, and Sparc. As evidenced by RT-qPCR the mRNA levels of these genes were down-regulated in old tendons and in old TDSCs. Additionally, protein content of SPARC and Lysyl oxidase was diminished in vitro in cellular extracts from old TDSCs. The impact of Sparc on tendon ageing was further analysed in young and old Sparc−/− C57BL/6 as well as in age-matched wildtype mice. Tendons of Sparc−/− mice are generally thinner and TEM revealed thinner collagen fibrils and a larger interfibrillar area. Further, TDSCs of old and Sparc−/− tendons formed thinner in vitro tendon constructs, showed a higher collagen gel contraction capacity, and display altered cell-ECM adhesion and cell migration properties when compared to young wildtype cells. Employing SHG-microscopy we further observed age-related changes in the collagenous structure of Achilles tendons. Discussion. The decreased expression of ECM proteins and modulators thereof in old tendons in combination with structural changes is potentially associated with an increased risk of tendon injury in the elderly, since structure and composition of the tendon are directly related to its function

Introduction. Tendon healing begins with inflammation and results in an incomplete repair with fibrosis, culminating in tendon pathology along with tissue degeneration. Inflammatory mediators regulate the expression of growth factors, and members of the TGFβ superfamily including BMPs have been suggested to play a key role in the development of fibrosis. In established tendon diseases where inflammation and reparative processes persists, the cellular phenotype of tendon cells has been implied to undergo a transformation from that of normal tissue. This study investigates the inflammation-driven mechanisms of tendon pathology using an in vitro tendon cell model. We hypothesized that cells from diseased tendons will exhibit dysregulation of TGFβ superfamily members in response to inflammatory mediators when compared to cells derived from healthy tendons. Materials and Methods. Diseased human tendon cells were isolated from patients with large to massive rotator cuff tears (n=4). Cells isolated from healthy human hamstring tendons served as control tissue (n=5). Cells were treated with human recombinant IL-1β (5ng/ml), oncostatin M (10ng/ml), IL-6 (10ng/ml), IL-10 (10ng/ml) in serum-free medium, or serum-free medium alone (control) for 24 hours. Cell viability was monitored by Alamar Blue assay, and expression of TGFB1, TGFBR1, TGFBR2, CTGF, BMP2 and BMP7 were quantified by quantitative reverse transcription polymerase chain reaction (RT-QPCR). Results. Cytokine stimulation did not significantly influence cell viability in either group. In diseased cells, IL-1β induced a 4.9-fold increase in BMP2 compared to control cells (p=0.032). There were no significant changes in the expression of other TGFβ superfamily genes after stimulation with other cytokines. CTGF was significantly increased in diseased compared to healthy cells following IL-1β stimulation (p=0.0295). No other genes showed differential regulation by inflammatory cytokines between diseased and healthy cells. Discussion. This work suggests that BMP-2, a growth factor related to cell differentiation, is dysregulated with IL-1β stimulation and plays a key role in the development of tendon diseases. Differences in IL-1β-induced CTGF expression suggests increased responsiveness of diseased cells to this cytokine. BMP-2 could be an important growth factor in the development of tendon diseases and further investigation of its role in chronic inflamed tissue is warranted

Background. Osteoarthritis (OA), is characterised with a loss of cartilage and pain in affected joints. It is this pain which most patients associate with their condition. Intra-articular (IA) hyaluronan (HA) has been shown to reduce the pain associated with OA both in animal models and in clinical trials. There are purified HA available and in recent years hyaluronan hydrogels, where the material has been cross-linked into networks, have become available. One of these cross-linked HA hydrogels is Durolane¯. This study has sought to evaluate the effect of Durolane in an in vivo model of osteoarthritis. Methods. Mice (C57BL/6, 12 weeks) were obtained from Jackson Labs and all protocols were approved by Rush IACUC. Joint injury was initiated by TGFb1 injection as described [1]. Mice were given IA injections of 200 ng TGFb1, at days 1 and 3 delivered in a 6 ul volume into the rear right knee joint only. Twenty four hours after the second injection of TGFb1 10 ul of Durolane was injected into the same knee joint. All animals were exercised daily on a treadmill to induce tissue degeneration. Three groups of animals were evaluated: Naïve (n = 4), TGFb1 + saline (n = 5) and TGFb1 + Durolane (n = 5). Running performance was monitored daily and 15 days post injections, gait was assessed quantitatively using the TreadScan gait analysis system (CleverSys). Results. Combined treatment of IA TGFb1 and treadmill running results in rapid and reproducible OA-like joint tissue remodelling in injected knee joints, including cartilage erosion, synovial and joint capsule fibrosis and chondrophyte accumulation along joint margins [2]. It was clear that the injections of TGFb1 + saline into the rear right knee joint caused impairment in gait, such as limping and difficulty to maintain treadmill running. In comparison the TGFb1 + Durolane treated animals showed running behaviours similar to that seen in untreated naïve mice. Quantitative assessment of gait using the TreadScan system, for a number of gait parameters, confirmed that Durolane returned the gait in these animals with induced OA closer to the gait of naïve animals. For example the stance time, described as time elapsed while the foot is in contact with the tread in its stance phase, being 185.81 ms (SD 34.85) for naïve, 249.67 ms (SD 37.58) for TGFb1 + saline and 214.86 ms (SD 28.1) for TGFb1 + Durolane treated animals. Single factor ANOVA for primary comparison between TGFb1 + Durolane and TGFb1 + saline provided a significant improvement for the Durolane group (p < 0.05). Conclusions. This study has demonstrated that a single IA injection of Durolane can improve gait in this non-surgical model of OA confirming earlier data that Durolane provides anti-nociceptive effects in a model of joint pain [3]

Objectives

The effects of disease progression and common tendinopathy treatments on the tissue characteristics of human rotator cuff tendons have not previously been evaluated in detail owing to a lack of suitable sampling techniques. This study evaluated the structural characteristics of torn human supraspinatus tendons across the full disease spectrum, and the short-term effects of subacromial corticosteroid injections (SCIs) and subacromial decompression (SAD) surgery on these structural characteristics.

We assessed the predictive value of the macroscopic and detailed microscopic appearance of the coracoacromial ligament, subacromial bursa and rotator-cuff tendon in 20 patients undergoing subacromial decompression for impingement in the absence of full-thickness tears of the rotator cuff. Histologically, all specimens had features of degenerative change and oedema in the extracellular matrix. Inflammatory cells were seen, but there was no evidence of chronic inflammation. However, the outcome was not related to cell counts.

At three months the mean Oxford shoulder score had improved from 29.2 (20 to 40) to 39.4 (28 to 48) (p < 0.0001) and at six months to 45.5 (36 to 48) (p < 0.0001). At six months, although all patients had improved, the seven patients with a hooked acromion had done so to a less extent than those with a flat or curved acromion judged by their mean Oxford shoulder scores of 43.5 and 46.5 respectively (p = 0.046). All five patients with partial-thickness tears were within this group and demonstrated less improvement than the patients with no tear (mean Oxford shoulder scores 43.2 and 46.4, respectively, p = 0.04). These findings imply that in the presence of a partial-thickness tear subacromial decompression may require additional specific treatment to the rotator cuff if the outcome is to be improved further.