Background. Resveratrol is a polyphenolic compound commonly found in the skins of red grapes. Sirtuin 1 (SIRT1) is a human gene that is activated by resveratrol and has been shown to promote longevity and boost mitochondrial metabolism. We examined the effect of resveratrol on normal and osteoarthritic (OA) human chondrocytes. Methods. Normal and OA chondrocytes were incubated with various concentrations of resveratrol (1 µM, 10 µM, 25 µM and 50 µM) and cultured for 24, 48 or 72 hours or for six weeks. Cell proliferation, gene expression, and senescence were evaluated. Results. SIRT1 was significantly upregulated in normal chondrocytes with resveratrol concentrations of 25 µM and 50 µM on both two- (2D) (both p = 0.001) and three-dimensional (3D) cultures (p = 0.008 and 0.001, respectively). It was significantly upregulated in OA chondrocytes treated with 10 µM, 25 µM and 50 µM resveratrol on 2D cultures (p = 0.036, 0.002 and 0.001, respectively) and at 50 µM concentration on 3D cultures (p = 0.001). At 72 hours, the expression of collagen (COL)-10, aggrecan (AGG), and runt-related transcription factor 2 (RUNX2) was significantly greater in both 25 µM (p = 0.011, 0.006 and 0.015, respectively) and 50 µM (p = 0.019, 0.004 and 0.002, respectively) resveratrol-treated normal chondrocyte cultures. In OA chondrocytes, expression of COL10 and RUNX2 was significantly greater in 25 µM (p = 0.004 and 0.024) and 50 µM (p = 0.004 and 0.019) cultures at 72 hours on 3D cultures. Conclusions. At concentrations of 25 µM and/or 50 µM, resveratrol treatment significantly upregulates SIRT1 gene expression in normal and osteoarthritic chondrocytes. Resveratrol induces chondrocytes into a hypertrophic state through upregulation of COL1, COL10, and RUNX2. Cite this article: Bone Joint Res 2014;3:51–9

The ability of the body to constantly maintain metabolism homeostasis while fulling the heightened energy and macromolecule demand is crucial to ensure successful tissue healing outcomes. Studies investigating the local metabolic environment during healing are scarce to date. Here, using Type 2 Diabetes (T2D) as a study model, we investigate the impact of metabolism dysregulation on scaffold-guided large-volume bone regeneration. Our study treated wild-type or T2D rats with 5 mm critical-sized femoral defects with 3D-printed polycaprolactone (PCL) scaffolds with 70% porosity. Metabolomics was leveraged for a holistic view of metabolism alteration as healing progress and correlated to regenerated bone tissue volume and quality assessed using micro-computed tomography (µ-CT), histology, and immunohistology. Semi-targeted metabolomics analysis indicated dysregulation in the glycolysis and TCA cycle – the main energy production pathways, in T2D compared to healthy animals. The abundance of metabolites substrates, i.e., amino acids – for protein/ extracellular matrix synthesis was also affected in T2D. Tissue-level metabolites observations aligned with morphological observation with less newly formed bone observed in T2D than wild-type rats. This study enlightens the metabolism landscape during scaffold-guided large-volume bone regeneration in wild-type vs. T2D to further guide the personalization of the scaffold to drive successful regeneration

Healthy bone metabolism is a tightly coupled dynamic process that relies on a balance between bone resorption (catabolism) by osteoclasts and bone formation (anabolism) by osteoblasts. Traditionally, tissue-engineering approaches for non-union fracture repair employ local anabolic therapeutic delivery strategies that target mesenchymal stem cells (MSCs) and osteoblasts to induce bone formation, however, the challenge of healing non-union defects depends on the cause of defect e.g. trauma or disease, and targeting bone formation alone is often not sufficient. Our research focuses on utilising both anabolic therapeutics, including recombinant human bone morphogenic protein (rhBMP) −2 and parathyroid hormone (PTH). (1–34). , and anti-catabolic bisphosphonates (BPs) to target bone metabolism. A major challenge with harnessing a combined dosing regimen is controlling the release of the individual therapeutics to target cells. We have developed a number of polymer-ceramic based biomaterial delivery systems, including injectable and implantable scaffolds, for the controlled release of rhBMP-2 and the BP zoledronic acid (ZA) and demonstrated their efficacy in vivo. A dual therapeutic load provided a synergistic enhancement of bone regeneration, demonstrating significantly increased bone formation and remodelling compared to anabolic therapies alone. Utilising hydroxyapatite as the ceramic phase in our scaffolds further increased bone formation, demonstrating the polymer-ceramic scaffolds to be osteoconductive in the absence of therapeutics. In addition, we have demonstrated the manipulation of bone metabolism through a specific dosing regimen of PTH. (1–34). , a therapeutic traditionally used as an anabolic, to induce bone remodelling and drive healing in BP loaded fractures. Our research to date has shown that optimising the delivery and regimen of anabolic and anti-catabolic therapeutics to control bone metabolism, augments the bone regenerative potential of these therapeutics in orthopaedic applications

Previously, we have demonstrated reduced biomechanical bone strength and matrix quality in Tachykinin (Tac)1-deficient mice lacking the sensory neuropeptide substance P (SP). A similar distortion of bone microarchitecture was described for α-calcitonin gene-related pepide (α-CGRP)-deficient mice. In previous studies we observed alterations in cell survival and differentiation capacity of bone cells isolated from wildtype mice when stimulated with SP and α-CGRP. We assume that changes in sensory neurotransmitter balance modulate bone cell metabolism thereby possibly contributing to inferior bone quality. In order to explore this hypothesis, we investigated and compared metabolic parameters in osteoblasts and osteoclasts isolated from SP- and α-CGRP-deficient mice and wildtype (WT) controls. Bone marrow-derived macrophages (BMMs) and osteoblast-like cells from female C57Bl/6J (WT-control), Tac1-deficient (Tac1-/−) and α-CGRP-deficient (α-CGRP-/−) mice were isolated and differentiated according to established protocols (Niedermair et al., 2014). Cell metabolism studies were performed for enzyme activity and cell survival. We observed reduced numbers of BMM from Tac1-/− and α-CGRP-/− mice after initial seeding compared to WT but no changes in viability. Osteoblast-like cells from Tac1-/− mice tend to migrate out faster from bone chips compared to WT-controls whereas migration of osteoblast-like cells from α-CGRP-/− mice was not affected. Osteoblasts and osteoclast/BMM cultures from WT mice endogenously synthesize and secrete SP as well as α-CGRP at a picomolar range. We found no changes regarding BMM or osteoblast proliferation from both, Tac1-/− and α-CGRP-/− mice when compared to WT-controls. Caspase 3/7-activity was reduced by trend in osteoclast/BMM cultures of α-CGRP-/− mice and significantly reduced in osteoclast/BMM cultures of Tac1-/− mice compared to WT-controls. We found significantly higher Caspase 3/7-activity in osteoblasts of Tac1-/− mice after 14 days of osteogenic culture conditions when compared to WT-controls whereas osteoblasts of α-CGRP-/− mice were unaffected. Cathepsin K enzyme activity was significantly reduced in osteoclast/BMM cultures of Tac1-/− and α-CGRP-/− mice compared to WT-controls. ALP activity of Tac1-/− osteoblasts was higher after 7 days and reduced after 21 days of osteogenic culture compared to WT-controls whereas ALP activity of osteoblasts of α-CGRP-/− mice was unchanged. Acccording to our in vitro observations, we suggest some reduction in bone resorption rate but concomitantly a reduction in bone formation rate in Tac1-/− mice compared to WT-controls resulting in a net bone loss in these mice as bone resorption is faster than bone formation. Furthermore, we assume that bone resorption rate is slightly reduced in α-CGRP-/− mice but bone formation rate seems to be unchanged. Therefore we hypothesize that additional conditions present in vivo might contribute to the inferior bone properties of α-CGRP-/− mice

We describe a model which can be used for in vitro biocompatibility assays of biomaterials. We studied the in vitro response of human osteoarthritis or rheumatoid arthritis fibroblast-like synoviocytes to Al. 2. O. 3. or ZrO. 2. particles by analyzing the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) and the metabolism of arachidonic acid via lipoxygenase and cyclo-oxygenase pathways. Our results show that, in these cells and under our experimental conditions, Al. 2. O. 3. and ZrO. 2. did not significantly modify the synthesis of IL-1 and IL-6 or the metabolism of arachidonic acid

A defining characteristic of the Nucleus Pulposus (NP) and the inner AF is the very limited vascular supply and low pH that imposes metabolic constraints on the disc cells. Interference with the normal physiology of the NP niche, by activities linked to changes in oxygen diffusion across the endplate leads to dysregulated niche function. Hypoxia Inducible Factor-1 (HIF-1) and HIF-2 are robustly and constitutively expressed by cells of the NP. Our recent work has shown that expression of HIF-1 is indispensable for NP cell survival in vivo and suggests an important role of HIF-1 in NP cell metabolic program. This talk will discuss central role of HIF-1 as metabolic and pH homeostatic regulator of NP cells and possible implication for a therapeutic strategy to treat disc degeneration

Breast and other cancers commonly metastasize to bone to cause bone destruction, pain, fractures hypercalcemia and muscle weakness. Recently, we described a specific molecular mechanism by which bone-derived transforming growth factor (TGF)-beta, released as a consequence of tumor-induced bone destruction causes muscle dysfunction, before the loss of muscle mass. Circulating TGF-beta induces oxidation of the ryanodine receptor (RYR1) on the sarcoplasmic reticulum of skeletal muscle to induce calcium leak and muscle weakness. Blocking TGF-beta, or its release from bone (with bisphosphonates), preventing oxidation of or stabilizing RyR1 all prevented muscle weakness in mouse models of breast cancer bone metastases. In addition to these effects on skeletal muscle, circulating TGF-beta may act on beta cells of the pancreas to impair insulin secretion and result in glucose intolerance. These and other potential systemic effects of TGF-beta released from the tumor-bone microenvironment or from cancer treatment-induced bone destruction implicate bone as a major source of systemic effects of cancer and cancer treatment. Therapy to block the systemic effects of the bone microenvironment will improve morbidity associated with bone metastases and cancer treatment.

Introduction

Tendinopathies are debilitating and painful conditions. They are believed to result from repetitive overuse, which can create micro-damage that accumulates over time, and initiates a catabolic cell response. The aetiology of tendinopathy remains poorly understood, therefore the ideal treatment remains unclear. However, current data support the use of eccentric exercise as an effective treatment. In a previous study, we have shown that eccentric loading generates perturbations in the tendon at 10Hz, which is not present during other less effective loading regimes. Consequently, we hypothesis that 10Hz loading initiates an increased anabolic response in tenocytes, that can promote tendon repair.

Intervertebral disc (IVD) degeneration presents a harsh microenvironment characterised by low glucose, low oxygen and matrix acidity posing a significant challenge for cell-based therapies. The objective of this work was to assess the effect of primed bone marrow derived stem cells (BMSC) and articular chondrocytes (AC) in different pH (7.1, 6.8 and 6.5) conditions and assess metabolic activity in terms of oxygen (O₂) and glucose consumption as well as lactate production. Secondly, we investigated pH effects on cell viability and matrix accumulation capacity. Primary cells were encapsulated in alginate beads and cultured in disc-like conditions (5% O₂, 5mM glucose, pH 7.1, 6.8 and 6.5). For growth factor priming, cells were cultured with 10ng/ml TGF-β3 at a pH of 7.4 for 14 days prior to being subjected to acidic pH conditions. AC exhibited superior cell viability and sGAG deposition compared to BMSC at all pH levels which was further enhanced after priming. Priming also reduced O₂ consumption of AC for all pH conditions while lactate production profiles of both cell types were altered with decreasing extracellular pH. This work demonstrates the importance of cell type selection to sustain disc-like microenvironmental conditions. Results show that BMSCs that have not been primed may need additional factors to sustain the harsh acidic microenvironment. In contrast, AC were capable of sustaining the low pH conditions better than BMSC and accumulated more similar disc-like matrix in all conditions. Overall this study highlights that AC may be advantageous for disc regeneration and warrant further investigation for disc repair.

Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model were constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. We demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α–impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α–induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Our results revealed that melatonin can reverse TNF-α–impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD

Osteoporosis (OP) and osteoarthritis (OA) are leading causes of musculoskeletal dysfunction in elderly, with chondrocyte senescence, inflammation, oxidative stress, subcellular organelle dysfunction, and genomic instability as prominent features. Age-related intestinal disorders and gut dysbiosis contribute to host tissue inflammation and oxidative stress by affecting host immune responses and cell metabolism. Not surprisingly, the development of OP and OA correlate with dysregulations of the gut microflora in rodents and humans. Intestinal microorganisms produce metabolites, including short-chain fatty acids, bile acids, trimethylamine N-oxide, and liposaccharides, affecting mitochondrial function, metabolism, biogenesis, autophagy, and redox reactions in chondrocytes to regulate joint homeostasis. Modulating the abundance of specific gut bacteria, like Lactobacillus and Bifidobacterium, by probiotics or fecal microbiota transplantation appears to suppress age-induced chronic inflammation and oxidative damage in musculoskeletal tissue and holds potential to slow down OP development. The talk will highlight treatment options with probiotics or metabolites for modulating the progression of OA and OP

Aims. This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation. Methods. In this study, 60 rats were included in a titanium rod implantation model and underwent subsequent guanylate cyclase treatment. Imaging, histology, and biomechanics were used to evaluate the osseointegration of rats in each group. First, the impact of VIT on bone integration in aged rats with iron overload was investigated. Subsequently, VIT was employed to modulate the differentiation of MC3T3-E1 cells and RAW264.7 cells under conditions of iron overload. Results. Utilizing an OVX rat model, we observed significant alterations in bone mass and osseointegration due to VIT administration in aged rats with iron overload. The observed effects were concomitant with reductions in bone metabolism, oxidative stress, and inflammation. To elucidate whether these effects are associated with osteoclast and osteoblast activity, we conducted in vitro experiments using MC3T3-E1 cells and RAW264.7 cells. Our findings indicate that iron accumulation suppressed the activity of MC3T3-E1 while enhancing RAW264.7 function. Furthermore, iron overload significantly decreased oxidative stress levels; however, these detrimental effects can be mitigated by VIT treatment. Conclusion. Collectively, our data provide compelling evidence that VIT has the potential to reverse the deleterious consequences of iron overload on osseointegration and bone mass during ageing. Cite this article: Bone Joint Res 2024;13(9):427–440

As high incidences of tendinopathies are observed particularly in those who intensively use their tendons, we assume that pathological changes are caused, at least partially, by mechanical overload. This has led to the so-called overload hypothesis, explaining the development of tendinopathies by structural failure resulting from excessive load. At the same time, tendon loading is an important part in tendon rehabilitation. Currently, exercise treatment approaches such as eccentric training or heavy load resistance training are widely applied in tendinopathy rehabilitation, with good clinical results such as an improvement in function and a reduction in pain. Particularly those rehabilitative approaches which impose high strains on the tendon may induce an adaptation of the tendon's mechanical properties such as increased tendon stiffness. An increased tendon stiffness is often interpreted as desirable, as it may protect the tendon from overloading and thus prevent future strain injuries. However, the tendinopathic tendon is not necessarily less stiff than the tendon in the contralateral leg and an improvement in tendon stiffness is not necessarily accompanied by an improvement in tendon pain or function. In addition, metabolic factors, resulting e.g. in low-level systemic inflammation, may contribute to pathological tendon tissue changes and are not necessarily affected by an exercise program, while nutritional interventions or dietary supplements may potentially affect tendon cell metabolism. Indeed, dietary supplements have been introduced as an additional therapeutic approach in the treatment of tendinopathies in recent years, and their positive curative effects have been reported for both the general population and athletes. In the management of tendinopathies, it may thus be advisable if therapeutic approaches aim to address both tendon mechanics and tendon metabolism for better treatment effectiveness and a sustainable improvement in pain and function

Nitric oxide is a free radical which in vivo is solely produced during the conversion of the amino acid arginine into citrulline by nitric oxide synthase enzymes. Recently, the importance of nitric oxide on inflammation and bone metabolism has been investigated. However, the knowledge regarding possible in vitro effects of arginine supplementation on chondrogenic differentiation is limited. ATDC5, a cell line which is derived from mouse teratocarcinoma cells and which is characterized as chondrogenic cell line, were proliferated in Dulbecco's Modified Eagle Medium (DMEM)/F12 and subsequently differentiated in proliferation medium supplemented with insulin, transferrin and sodium-selenite and where arginine was added in four different concentrations (0, 7.5, 15 and 30 mM). Samples were harvested after 7 or 10 days and were stored at −80 °C for subsequent RNA isolation for qPCR analysis. To determine chondrogenic differentiation, Alcian Blue staining was performed to stain the proteoglycan aggrecan, which is secreted by differentiated ATDC5 cells. All measurements were performed in triplo. Alcian Blue staining showed a qualitative increase of proteoglycan aggrecan secretion in differentiated ATDC5 cells after treatment with 7 and 15 mM arginine, with additional increased expression of ColII, ColX, Bmp4 and Bmp6. Treatment with 30 mM arginine inhibited chondrogenic differentiation and expression of aforementioned genes, however, Cox-2 and Vegfa gene expression were increased in these samples. Bmp7 was not significantly expressed in any experimental condition. The obtained results are suggestive for a dose-dependent effect of arginine supplementation on chondrogenic differentiation and associated gene expression, with 7.5 and 15 mM as most optimal concentrations and implications for apoptosis after incubation with 30 mM arginine. A future recommendation would be to investigate the effects of citrulline in a similar experiment, as this shows even more promising results to enhance the nitric oxide metabolism in sepsis and bone healing

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods. MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging. The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription. Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic. Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024

Platelet Rich Plasma (PRP), either rich (L-PRP) or poor (P-PRP) of leukocytes, is frequently used as an anti-inflammatory and regenerative tool in osteoarthritis (OA). PRP contains proteins but not genes as it is derived from megakaryocytes. Proteomics but not metabolomics of PRP was recently studied. Metabolomics is a field of ‘omics’ research involved in comprehensive portrayal of the small molecules, metabolites, in the metabolome. These small molecules can be endogenous metabolites or exogenous compounds found in an organism (1). Our aim was to determine the difference between L-PRP and P-PRP. A cross-sectional clinical study was designed in six recreational male athletes between the ages of 18 and 35 years. 3 mL P-PRP and 3 mL -LPRP was prepared from 60 mL of venous blood after treating with 9 mL of sodium citrate and centrifugation at 2.700 rpm for 10 min. Half of the prepared PRP's were frozen at −20°C for a week. Fresh and frozen samples were analyzed at the Q-TOF LC/MS device after thawing to room temperature. Untargeted metabolomic results revealed that the metabolomic profile of the L-PRP and P-PRP were significantly different from each other. A total of 33.438 peaks were found. Statistically significant (p<0.05) peaks were uploaded to the MetaboAnalyst 5.0 platform. Exogenous out of 2.308 metabolites were eliminated and metabolites found significant for our study were subjected to pathway analysis. Steroid biosynthesis, sphingolipid metabolism and metabolism of lipid pathways were affected. In the L-PRP samples, Nicotinamide riboside (FC: 2.2), MHPG (FC: 3.0), estrone sulfate (FC: 7.5), thiamine diphosphate (FC: 2.0), leukotriene E4 (FC: 7.5), PC(18:1 (9Z)e/2:0) (FC: 9.8) and Ap4A (FC: 2.1) were higher compared to P-PRP. C24 sulfatide (FC: −11.8), 3-hexaprenyl-4,5-dihydroxybenzoic acid (FC: −2.8) metabolites were furthermore lower in P-PRP. Clinical outcomes of PRP application should consider these metabolic pathways in future studies (2)

Abstract. Objectives. Osteoarthritis (OA) is a painful and debilitating disorder of diarthroidal joints. Progressive degeneration of the cartilage extracellular matrix (ECM) together with abnormal chondrocyte characteristics occur leading to a switch to a fibroblast-like phenotype and production of mechanically-weak cartilage. Early changes to chondrocytes within human cartilage have been observed including chondrocyte swelling. [1]. together with the development of thin cytoplasmic processes which increase in number and length with degeneration. [2]. Changes to chondrocyte phenotype in degenerate cartilage are associated with F-actin redistribution and stress fibres (SF) formation, leading to morphologically-dedifferentiated (fibroblast-like) chondrocytes. [3,4]. It is unclear if these processes are a consequence of ‘passive’ cell swelling into a defective ECM or an ‘active’ event driven by changes in cell metabolism resulting in alterations to cell shape. To address this, we have quantified and compared the distribution and levels of F-actin, a key cytoskeletal protein involved in the formation of cytoplasmic processes, within in situ chondrocytes in non-degenerate and mildly degenerate human cartilage. Methods. Human femoral head cartilage was obtained from 21 patients [15 females, 6 males, average age 69.6yrs, (range 47–90yrs)] following femoral neck fracture, with Ethical Approval and patient's permission. Cartilage explants were removed from areas graded non-degenerate grade 0 (G0) or mildly degenerate grade 1 (G1) and cultured for up to 3wks in Dulbecco's Modified Eagle's Medium (DMEM) +/− 25% human serum (HS). In situ chondrocytes were stained with CMFDA (5-chloromethylfluoresceindiacetate, Cell-Tracker Green®) and phalloidin (F-actin labelling) and imaged by confocal microscopy and analysed quantitatively using ImageJ and Imaris® software. Results. There were significant increases in the total amount (TA) of F-actin and its distribution [intense punctuate (IP) and intense areas (IA)] between the whole chondrocyte populations of G0 and G1 cartilage (P=0.0356; 0.0112; 0.016, respectively). Where the volume of chondrocytes was divided into normal (<1000 µm³) and swollen (≥1000 µm³) cells, F-actin TA increased in swollen cells (P=0.036 within G0 and G1, and P=0.0009 between grades) compared to chondrocytes of normal volume in each grade. Moreover, IP and IA within and between G0 and G1 were higher compared to normal chondrocytes (with P<0.0001 for IP and P<0.001 for IA). In addition, tissue culture experiments demonstrated that 90% of chondrocytes with cytoplasmic processes had strong F-actin intensity (either IP or IA with P<0.0001). Furthermore, 83% of this F-actin was associated with cytoplasmic processes, with >65% situated at the base of the process (P<0.0001). Conclusions. The increases in chondrocyte F-actin levels (TA) and its localisation (IP, IA) appear to be associated with cell swelling and development of cytoplasmic processes, which are both characteristics of early OA cartilage. [1]. This suggests the formation of chondrocyte cytoplasmic processes is an ‘active’ event potentially involving changes to matrix metabolism rather than a ‘passive’ cell swelling into a defective extracellular matrix. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction:. Exercise has showed to reduce pain and improve function in patients with discogenic low back pain (LBP). Although there is currently no biologic evidence that the intervertebral disc (IVD) can respond to physical exercise in humans, a recent study has shown that chronic running exercise is associated with increased IVD hydration and hypertrophy1. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types, including chondrocytes2. This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs). Our hypothesis is that irisin may improve hNPCs metabolism and proliferation. METHODS:. The hNPCs, isolated from discectomy surgical waste material (n = 5), were expanded and encapsulated in alginate beads. The hNPCs were treated with: i) only growth medium (control); ii) medium with recombinant irisin (r-IR) at different concentrations (5, 10 and 25 ng / mL); iii) medium with Interleukin-1β (IL1β); iv) medium with IL1β for 24 h and then with IL1β and r-IR; v) medium with r-IR for 24 h and then with r-IR and IL1 β. We evaluated proliferation (trypan blue and PicoGreen), metabolic activity (MTT), nitrite concentration (Griess), and expression levels of catabolic and anabolic genes via real-time polymerase chain reaction (qPCR). Each analysis was performed in triplicate for each donor and each experiment was performed three times. Data were expressed as mean ± S.D. One-way ANOVA was used for the groups under exam. RESULTS:. Irisin increased hNPCs proliferation (p < 0.001), metabolic activity at 10 ng/mL (p < 0.05), and GAG content at concentration of 10 ng/mL and 25 ng/mL (p < 0.01; p < 0.001, respectively). The production of nitrites, used as an indicator of cellular oxidative stress, was significantly decreased (p < 0.01). Gene expression levels compared to the control group increased for COL2A1 (p < 0.01), ACAN (p < 0.05), TIMP-1 and −3 (p < 0.01), while a decrease in mRNA levels of MMP-13 (p < 0.05) and IL1β (p < 0.001) was noticed. r-IR pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p < 0.001), as well as a decrease of IL-1β (p < 0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-IR led to an increment of hNPC metabolic activity (p < 0.001), COL2A1 gene expression (p < 0.05) and a reduction of IL-1β (p < 0.05) and ADAMTS-5 gene levels (p < 0.01). CONCLUSIONS:. The present study suggested that irisin may stimulate hNPCs proliferation, metabolic activity, and anabolism by reducing the expression of IL-1β and catabolic enzymes while promoting the synthesis of extracellular matrix components. Furthermore, this myokine was able to blunt the catabolic effect of in vitro inflammation. Our results indicate that irisin may be one of the mediators by which physical exercise and muscle tissues modulate IVD metabolism, thus suggesting the existence of a biological cross-talk mechanism between the muscle and the IVD

Varus malalignment increases the susceptibility of cartilage to mechanical overloading, which stimulates catabolic metabolism to break down the extracellular matrix and lead to osteoarthritis (OA). The altered mechanical axis from the hip, knee to ankle leads to knee joint pain and ensuing cartilage wear and deterioration, which impact millions of the aged population. Stabilization of the remaining damaged cartilage, and prevention of further deterioration, could provide immense clinical utility and prolong joint function. Our previous work showed that high tibial osteotomy (HTO) could shift the mechanical stress from an imbalanced status to a neutral alignment. However, the underlying mechanisms of endogenous cartilage stabilization after HTO remain unclear. We hypothesize that cartilage-resident mesenchymal stem cells (MSCs) dampen damaged cartilage injury and promote endogenous repair in a varus malaligned knee. The goal of this study is to further examine whether HTO-mediated off-loading would affect human cartilage-resident MSCs' anabolic and catabolic metabolism. This study was approved by IACUC at Xi'an Jiaotong University. Patients with medial compartment OA (52.75±6.85 yrs, left knee 18, right knee 20) underwent open-wedge HTO by the same surgeons at one single academic sports medicine center. Clinical data was documented by the Epic HIS between the dates of April 2019 and April 2022 and radiographic images were collected with a minimum of 12 months of follow-up. Medial compartment OA with/without medial meniscus injury patients with unilateral Kellgren /Lawrence grade 3–4 was confirmed by X-ray. All incisions of the lower extremity healed well after the HTO operation without incision infection. Joint space width (JSW) was measured by uploading to ImageJ software. The Knee injury and Osteoarthritis Outcome Score (KOOS) toolkit was applied to assess the pain level. Outerbridge scores were obtained from a second-look arthroscopic examination. RNA was extracted to quantify catabolic targets and pro-inflammatory genes (QiaGen). Student's t test for two group comparisons and ANOVA analysis for differences between more than 2 groups were utilized. To understand the role of mechanical loading-induced cartilage repair, we measured the serial changes of joint space width (JSW) after HTO for assessing the state of the cartilage stabilization. Our data showed that HTO increased the JSW, decreased the VAS score and improved the KOOS score significantly. We further scored cartilage lesion severity using the Outerbridge classification under a second-look arthroscopic examination while removing the HTO plate. It showed the cartilage lesion area decreased significantly, the full thickness of cartilage increased and mechanical strength was better compared to the pre-HTO baseline. HTO dampened medial tibiofemoral cartilage degeneration and accelerate cartilage repair from Outerbridge grade 2 to 3 to Outerbridge 0 to 1 compared to untreated varus OA. It suggested that physical loading was involved in HTO-induced cartilage regeneration. Given that HTO surgery increases joint space width and creates a physical loading environment, we hypothesize that HTO could increase cartilage composition and collagen accumulation. Consistent with our observation, a group of cartilage-resident MSCs was identified. Our data further showed decreased expression of RUNX2, COL10 and increased SOX9 in MSCs at the RNA level, indicating that catabolic activities were halted during mechanical off-loading. To understand the role of cartilage-resident MSCs in cartilage repair in a biophysical environment, we investigated the differentiation potential of MSCs under 3-dimensional mechanical loading conditions. The physical loading inhibited catabolic markers (IL-1 and IL-6) and increased anabolic markers (SOX9, COL2). Knee-preserved HTO intervention alleviates varus malalignment-related knee joint pain, improves daily and recreation function, and repairs degenerated cartilage of medial compartment OA. The off-loading effect of HTO may allow the mechanoregulation of cartilage repair through the differentiation of endogenous cartilage-derived MSCs

The use of mesenchymal stem cell (MSCs) for intervertebral disc (IVD) regeneration has been extensively explored in the last two decades. MSCs are potent cell types that can be easily and safely harvested due to their abundancy and availability. Moreover, they are characterized by the capacity to differentiate towards IVD cells as well as release growth factors to support resident cell metabolism and recruit local progenitor cells to induce endogenous repair of degenerated IVDs. This talk will outline the characteristics of the main MSC sources and their effect towards IVD regeneration based on available preclinical and clinical evidence. In addition, innovative aspects of MSC-derived cell-free therapies will also be discussed