Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect. Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1

Objectives. Adult mice lacking the transcription factor NFAT1 exhibit osteoarthritis (OA). The precise molecular mechanism for NFAT1 deficiency-induced osteoarthritic cartilage degradation remains to be clarified. This study aimed to investigate if NFAT1 protects articular cartilage (AC) against OA by directly regulating the transcription of specific catabolic and anabolic genes in articular chondrocytes. Methods. Through a combined approach of gene expression analysis and web-based searching of NFAT1 binding sequences, 25 candidate target genes that displayed aberrant expression in Nfat1. -/-. AC at the initiation stage of OA, and possessed at least four NFAT1 binding sites in the promoter of each gene, were selected and tested for NFAT1 transcriptional activities by chromatin immunoprecipitation (ChIP) and promoter luciferase reporter assays using chondrocytes isolated from the AC of three- to four-month-old wild-type mice or Nfat1. -/-. mice with early OA phenotype. Results. Chromatin immunoprecipitation assays revealed that NFAT1 bound directly to the promoter of 21 of the 25 tested genes encoding cartilage-matrix proteins, growth factors, inflammatory cytokines, matrix-degrading proteinases, and specific transcription factors. Promoter luciferase reporter assays of representative anabolic and catabolic genes demonstrated that NFAT1-DNA binding functionally regulated the luciferase activity of specific target genes in wild-type chondrocytes, but not in Nfat1. -/-. chondrocytes or in wild-type chondrocytes transfected with plasmids containing mutated NFAT1 binding sequences. Conclusion. NFAT1 protects AC against degradation by directly regulating the transcription of target genes in articular chondrocytes. NFAT1 deficiency causes defective transcription of specific anabolic and catabolic genes in articular chondrocytes, leading to increased matrix catabolism and osteoarthritic cartilage degradation. Cite this article: M. Zhang, Q. Lu, T. Budden, J. Wang. NFAT1 protects articular cartilage against osteoarthritic degradation by directly regulating transcription of specific anabolic and catabolic genes. Bone Joint Res 2019;8:90–100. DOI: 10.1302/2046-3758.82.BJR-2018-0114.R1

Objective. In order to effectively utilize mechanical signals in the clinic as a non-drug-based intervention to improve cartilage defect regeneration after surgical treatment, it is essential to identify crucial components of the cellular response that are typical to the anabolic process. The mechanisms behind the effect of mechanical stimulation are, however, not fully understood and the signaling pathways involved in the anabolic response of chondrocytes to mechano-transduction are not well described. Therefore, a genome-wide identification of mechano-regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode was performed in this study and time evolution and re-inducibility of the response was characterized. Design. Osteochondral constructs consisting of a chondrocyte-seeded collagen-scaffold connected to β-tricalcium-phosphate were pre-cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9×10 minutes over 3h) before microarray-profiling was performed. Proteoglycan synthesis was determined by 35S-sulfate-incorporation over 24 hours. Protein alterations were determined by Western blotting. Results. Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45-fold, p=0.016). Among 115 significantly regulated genes, 114 were up-regulated, 48 of them ≥ two-fold. AP-1-relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2-phosphorylation and rising MAP3K4 expression. Expression of proteoglycan-synthesizing enzymes CHSY1 and GALNT4 was load-responsive as were factors associated with the MAPK-, TGF-β-, calcium-, retinoic-acid-, Wnt- and Notch-signaling pathway which were significantly altered. SOX9, BMP4 and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9-signalling was apparently unaltered. Conclusion. This study associates raising SOX9 protein levels, pERK stimulation and increased CHSY1 expression with anabolic loading of chondrocytes and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback-regulators. Knowledge on time evolution of mechanosensitive indicators responding to anabolic loading is crucial to maximize cartilage matrix-deposition for the generation of high-level cartilage replacement tissue

Summary. Anabolic and catabolic signalling processes within IVDs display overlapping pathways, however some pathways were identified as selective to catabolic signalling and inhibition of one of these pathways inhibited some of the catabolic factors induced by IL-1 although NFkB inhibition also affected anabolic expression. Degeneration of intervertebral discs (IVDs) is implicated in 40% of low back pain cases. In the normal disc the balance between anabolic and catabolic processes are carefully balanced. During degeneration this balance is lost in favour of catabolic processes which lead to degradation of the IVD, infiltration of blood vessels and nerves and release of cytokines which sensitise nerves to pain. Interleukin 1 (IL-1) is known to be important in the pathogenesis of IVD degeneration, here we investigated the intracellular signalling pathways activated by IL-1 and those activated by an anabolic factor (CDMP-1) to investigate differential pathways. Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 or CDMP-1 for 30 minutes. Site-specific phosphorylation of 46 signalling molecules were identified using R&D proteome array. The activation of ERK1/2, p38, c-jun, and IkB were confirmed using cell based ELISAs, in addition pNFκB localisation in stimulated cells was determined using immunohistochemisty. Pre-treatment with inhibitors to p38, and NFkB for 30 minutes, followed by stimulation with IL-1 (10ng/mL) or CDMP-1 (10ng/mL) for 24 hours was investigated to determine effects on anabolic and catabolic factors. In addition localisation of phosphorylated c-jun, p38 and NFkB were investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways in vivo. Twenty intracellular signalling pathways were activated following CDMP-1 treatment and 8 signalling pathways activated by IL-1. Of note key classical IL-1 signalling pathways p38 MAPK, ERK 1/2 and JNK were activated by IL-1, however of these ERK 1/2 particularly was also activated by CDMP-1, whilst p38 and c-jun were only activated by IL-1. IL-1 induced activation of NFkB signalling to a greater extent than CDMP-1, these results were confirmed by the ‘in cell ELISAs’. IVD tissue samples displayed immunopositive staining for phosphorylated c-jun, NFkB and p38. Inhibition of p38 signalling inhibited IL-1 induced MMP 13 expression, but had little effect on the induction of IL-8. However inhibitors of NFkB signalling pathway failed to inhibit the induction of MMP 13 but abrogated the induced IL-6 and IL-8 expression. IL-1 induced a complete aberration of aggrecan expression by NP cells in alginate culture, this effect was partly inhibited by p38 MAPK inhibitor but was completely restored by inhibiting NFkB signalling. However the aggrecan expressed in CDMP-1 treated cells was decreased by inhibiting NFkB but not p38. Here, we have shown that anabolic and catabolic signalling processes within IVDs show a number of overlapping pathways, however a number of differential pathways were identified and inhibition of p38 MAPK and NFkB pathways inhibited a number of catabolic processes investigated which were induced by IL-1. Thus inhibition of signalling pathways could be a novel mechanism of inhibiting catabolic processes which could hold promise to inhibit degeneration at early stages of disease but also create the correct tissue niche to promote regeneration of the disc

Mechanical loading is a potent stimulator of bone formation. A screen for genes associated with mechanically-induced osteogenesis implicated the glutamate transporter GLAST-1 (1), in the mechanoresponse. We are investigating whether modulation of glutamate transporters represents a potential anabolic therapy in bone. Bone cells express functional components from each stage of the glutamate signalling pathway and activation of ionotropic glutamate receptors on osteoblasts can increase bone forming activity (2). Five high affinity Na+-dependant excitatory amino acid transporters (EAATs 1-5) regulate glutamatergic signalling. EAAT1 (GLAST-1) is expressed by osteocytes and bone-forming osteoblasts in vivo. We quantified transcripts for EAATs 1-3 and two splice variants (EAAT1a and EAAT1ex9skip) in human osteoblasts (MG63, SaOS-2 and primary) using real time-PCR. EAAT1a expression was very low whilst levels of the dominant negative EAAT1ex9skip were much higher in all cell types. EAAT1 and EAAT3 proteins were detected by immunofluorescence. We also demonstrated that glutamate transporters function in human osteoblasts. Sodium-dependent 14C-labelled glutamate uptake, sensitive to pharmacological EAAT inhibitors (t-PDC, TBOA) and extracellular glutamate concentration (10-500μM) was detected in MG63 and SaOS-2 cells. To determine whether modulation of EAATs can influence bone formation, we used pharmacological inhibitors of EAATs 1-5 (t-PDC and TBOA) and also over-expressed EAAT1exon9skip using antisense oligonucleotides (AONs) targeted to splice donor sequence of exon 9. Experiments were performed in 0-500μM glutamate. Pharmacological inhibition of EAATs over 5-21 days increased alkaline phosphatase activity and mineralisation of SaOS-2 cells and human primary osteoblasts. Over-expression of EAAT1ex9skip significantly increased cell number and decreased cell death as well as significantly increasing PCNA, Osteonectin and Type I collagen mRNAs in MG63 cells. Furthermore, over-expression of EAAT1ex9skip increased mean alkaline phosphatase activity over 48hrs in SaOS-2 cells. These data show that EAATs are expressed and functional in osteoblasts and that pharmaceutical and genetic inhibition of their activity increases bone formation. These mechanically regulated glutamate transporters are important in regulating bone homeostasis and their manipulation may represent a new anabolic therapy for the treatment of disorders such as osteoporosis or non-union fractures

Introduction. Sclerostin has been implicated in mechanotransduction in bone and recent data show a lack of response to loading in the sclerostin transgenic mouse. Sclerostin, the protein product of the SOST gene, is an attractive therapeutic target for low bone mass conditions, including osteoporosis. It is expressed exclusively by mature osteocytes in bone and we have shown that sclerostin targets pre-osteocytes/osteocytes to regulate bone mineralization and osteoclast activity, as well as inducing catabolic gene expression in osteocytes themselves and promoting osteocyte-mediated bone loss (osteocytic osteolysis). The aim of this study was to examine the direct effects of sclerostin on anabolic responses to loading in bone ex vivo. Methods. 10 × 5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 μstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using a second-generation Zetos™ bone loading system. Daily measurements of bone stiffness (Young's modulus), media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made of resin-embedded, non-decalcified samples at the end of the experiment. Gene expression in the bovine bone was examined by real-time RT-PCR. Results. Bovine bone cores showed a steady increase in Young's modulus with daily application of mechanical loading. This increase in stiffness was blocked by the co-addition of sclerostin. Sclerostin also induced bone acidification and a net release of bone calcium, indicated by the decrease in media pH and the relative increase in ionic calcium concentrations in the presence of sclerostin. Sclerostin also completely abrogated loading-induced calcium/calcein uptake. Sclerostin induced an increase in the expression of the bone resorption genes, tartrate resistant acid phosphatase (TRAP), carbonic anhydrase and cathepsin K and induced the release of β-CTX. Histological examination revealed a significant increase in the size of the osteocyte lacunae in sclerostin-treated bone cores, suggesting a role for osteocytic osteolysis in this effect. Discussion/Conclusion. The observation that sclerostin abrogated the loading-induced increase in bone stiffness constitutes direct evidence for a negative effect of sclerostin on the anabolic response to mechanical loading. Our findings may be explained in part by the observation that sclerostin negatively controls mineralization by late osteoblasts and pre-osteocytes (1). It is also possible that osteocytes themselves are capable of releasing bone mineral in response to sclerostin. This study demonstrates that sclerostin directly antagonises the anabolic effects of mechanical loading in the absence of external (circulating, neural, hormonal) influences. The mechanisms, by which sclerostin exerts these effects, warrant further study

Summary Statement. The stable inhibition of miR-214 in the aged osteoporotic rats induced by OVX could be achieved by periodic administration of AntagomiR-214 at a dosage of 4 mg/kg and at an interval of 7 days, which will provide a potential bone anabolic strategy for treatment of osteoprosis. Introduction. MiR-214 has a crucial role in suppressing bone formation and miR-214 inhibition in osteogenic cells may be a potential anabolic strategy for ameliorating osteoporosis (Wang X, et al. 2013). An aged ovariectomised rat has been regarded as a golden model to test bone anabolic agents for reversing established osteoporosis in aged postmenopausal women (Li X, et al. 2009). However, there is still lack of evidence to demonstrate bone anabolic potential of therapeutic inhibition of miR-214 within osteogenic cells in the golden model. So, it should be necessary to establish RNAi-based administration protocol toward stable inhibition of miR-214 at a low level in the golden model. A targeted delivery system specifically facilitating Antagomir-214 approaching osteogenic cells, i.e. (Asp-Ser-Ser). 6. -liposome (Zhang G, et al 2012), was employed in this study. Objectives. This study was to investigate optimal dosage and duration for therapeutic inhibition of miR-214 within osteogenic cells in the aged osteoporotic rats induced by ovariectomy. Materials and Methods. Six-month-old female Sprague-Dawley rats were ovariectomised (OVX) and left untreated for 12 months to establish aged osteoporosis. To determine the optimal dosage for therapeutic inhibition of miR-214, the OVX rats were injected intravenously with the AntagomiR-214 at a dosage of 0.5mg/kg, 1mg/kg, 2mg/kg, 4mg/kg, 6mg/kg and 8mg/kg (n=6 for each dosage group) delivered by (Asp-Ser-Ser). 6. -liposome, respectively. Thereafter, miR-214 expression level in osteogenic cells from bilateral femur was quantified at day 2 post injection by real-time PCR analysis in combination with laser captured dissection (LCM). To determine the optimal duration of miR-214, the OVX rats were intravenously injected with the AntagomiR-214 (AntagomiR-214 group) or non-sense AntagomiR-214 (NC group) delivered by (Asp-Ser-Ser). 6. -liposome at the optimal dosage or (Asp-Ser-Ser). 6. -liposome alone (Vehicle group). Then, the miR-214 level in osteogenic cells from bilateral femur was quantified at 1, 3, 5, 7, 9, 12, 14, 16, 21 day after the single dosing (n=6 for each time-point) by real-time PCR analysis in combination with LCM, respectively. To examine the long-term effect of the AntagomiR-214 after periodic pulsed dosing, the OVX rats were administrated with the AntagomiR-214 at the optimal dosage and duration for 5 repeated injections and then the miR-214 level in osteogenic cells from bilateral femur was quantified by real-time PCR analysis in combination with LCM. Results. The miR-214 level was efficiently decreased in a dose-dependent manner by the AntogomiR-214 and reached the level lower than 10% of the baseline at a dosage of 4 mg/kg at least in the aged osteoporotic rats. The effective duration for miR-214 at a level lower than 50% of the baseline lasted for 7 days in the osteoporotic rats after the single dosing. The miR-214 level was continuously lowered until 28 days and continuously maintained later at the level lower than 10% of the baseline by the 5 pulsed dosing of the AntagomiR-214 at an interval of 7 days and at a dosage of 4 mg/kg in the osteoporotic rats. Conclusions. The stable inhibition of miR-214 for bone anabolic strategy in the aged osteoporotic rats induced by OVX could be achieved by periodic administration of AntagomiR-214 at a dosage of 4 mg/kg and at an interval of 7 days

Regulation of articular cartilage homeostasis is a complex process in which biologic and mechanical factors are involved. Hyperactivation of Wnt signaling, associated with osteoarthritis (OA), could jeopardize the protective anabolic effect of physiological loading. Here, we investigated the role of excessive Wnt signalling in cartilage molecular responses to loading. Human cartilage explants were harvested from hips of donors without OA. The Wnt agonist CHIR99021 was used to activate Wnt signalling 24 hours before cartilage explants were subjected to a loading protocol consisting of 2 cycles of 1 hour of 10% compression at 1 Hz, followed by 1-hour free swelling. Mechano-responsiveness was evaluated using the expression of type II collagen, aggrecan and MMP-13. Expression of known target genes TCF-1 and c-JUN was evaluated as positive control for Wnt and mechanical stimulation, respectively. In the absence of loading, CHIR99021 decreased the expression of the cartilage anabolic genes type II collagen and aggrecan, and increased the levels of MMP-13, corroborating that Wnt hyperactivation disrupts cartilage homeostasis. In the absence of Wnt hyperactivation, the applied loading protocol, representative for a physiologic stimulation by mechanical loading, led to an increase in type II collagen and aggrecan levels. However, when cartilage explants were subjected to mechanical stimulation in the presence of CHIR99021, the expression of cartilage anabolic genes was decreased, indicating changes to the cells’ mechano-responsiveness. Interestingly, mechanical stimulation was able to reduce the expression levels of MMP-13 compared to the condition of CHIR stimulation without loading. Hyperactivation of Wnt signaling switches the anabolic effect of physiologic compressive loading towards a potential catabolic effect and could contribute to the development and progression of OA

Intervertebral disc (IVD) degeneration (IDD) involves imbalance between the anabolic and the catabolic processes that regulate the extracellular matrix of its tissues. These processes are complex, and improved integration of knowledge is needed. Accordingly, we present a nucleus pulposus cell (NPC) regulatory network model (RNM) that integrates critical biochemical interactions in IVD regulation and can replicate experimental results. The RNM was built from a curated corpus of 130 specialized journal articles. Proteins were represented as nodes that interact through activation and inhibition edges. Semi-quantitative steady states (SS) of node activations were calculated. Then, a full factorial sensitivity analysis (SA) identified which out of the RNM 15 cytokines, and 4 growth factors affected most the structural proteins and degrading enzymes. The RNM was further evaluated against metabolic events measured in non-healthy human NP explant cultures, after 2 days of 1ng/ml IL-1B catabolic induction. The RNM represented successfully an anabolic basal SS, as expected in normal IVD. IL-1B was able to increase catabolic markers and angiogenic factors and decrease matrix proteins. Such activity was confirmed by the explant culture measurements. The SA identified TGF-β and IL1RA as the two most powerful rescue mediators. Accordingly, TGFβ signaling-based IDD treatments have been proposed and IL-1RA gene therapy diminished the expression of proteases. It resulted challenging to simulate rescue strategies by IL-10, but interestingly, IL-1B could not induce IL-10 expression in the explant cultures. Our RNM was confronted to independent in vitro measurements and stands for a unique model, to integrate soluble protein signaling and explore IDD. Acknowledgements: European Commission (Disc4All-ITN-ETN-955735)

Mincing cartilage with commercially available shavers is increasingly used for treating focal cartilage defects. This study aimed to compare the impact of mincing bovine articular cartilage using different shaver blades on chondrocyte viability. Bovine articular cartilage was harvested using a scalpel or three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage obtained with a scalpel was minced into fragments smaller than 1 mm. 3. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1, ACAN) and catabolic genes (MMP1, MMP13), Live/Dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes were measured. The study found that mincing cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p<0.001). Gene expression of anabolic genes was reduced, while catabolic genes were increased after day 7 in all shaver conditions. The MMP13/COL2A1 ratio was also increased in all shaver conditions. Confocal microscopy revealed a thin line of dead cells at the lesion site with viable cells below for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p<0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing. Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing. This indicates that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Further experimental and clinical studies are required to standardize the mincing process with a shaver. Acknowledgements: This study received unrestricted funding from KARL STORZ SE & Co. KG

Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide. 1. During degeneration, progressive structural and biochemical changes occur, leading to blood vessel and nerve ingrowth and promoting discogenic pain. 2. In the last decades, several cytokines have been applied to IVD cells in vitro to investigate the degenerative cascade. Particularly, IL-10 and IL-4 have been predicted as important anabolic factors in the IVD according to a regulatory network model based in silico approach. 3. Thus, we aim to investigate the potential presence and anabolic effect of IL-10 and IL-4 in human NP cells (in vitro) and explants (ex vivo) under hypoxia (5% O2) after a catabolic induction. Primary human NP cells were expanded, encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks in 3D for phenotype recovery while human NP explants were cultured for five days. Afterwards, both alginate and explant cultures were i) cultured for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (single treatments) or ii) stimulated with 0.1 ng/ml IL-1β for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (combined treatments). The presence of IL-4 receptor, IL-4 and IL-10 was confirmed in human intact NP tissue (Fig 1). Additionally, IL-4 single and combined treatments induced a significant increase of proinflammatory protein secretion in vitro (Fig. 2A-C) and ex vivo (Fig. 2D and E). In contrast, no significant differences were observed in the secretome between IL-10 single and combined treatments compared to control group. Overall, IL-4 containing treatments promote human NP cell and explant catabolism in contrast to previously reported IL-4 anti-inflammatory performance. 4. Thus, a possible pleiotropic effect of IL-4 could occur depending on the IVD culture and environmental condition. Acknowledgements: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735. For any figures and tables, please contact the authors directly

The aim of this study was to investigate the regenerative effects of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) derived exosomes (WJ-Exos) on human nucleus pulposus cells (hNPCs) in an in vitro 3D model. WJ-Exos were isolated by tangent flow filtration of WJ-MSCs conditioned media and characterized by TEM, WB for markers expression and quantified with NTA. WJ-Exos PKH26-labeled uptake in hNPCs was detected by confocal microscopy. hNPCs, isolated from surgical specimens (n=4), culture expanded in vitro and encapsulated in alginate beads, were pre-treated with IL1β (10 ng/ml) for 24 hours, then with WJ-Exos at 10, 50 and 100 µg/ml. Cells with growth medium were used as control. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess) iii) glycosaminoglycan (GAG) amount (DMBB), iv) histological staining for extracellular matrix (ECM) analysis and v) gene expression levels of catabolic and anabolic genes (qPCR). The investigations were performed in triplicate for each donor. One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D. A dose dependent increase in hNPCs proliferation was noticed at all exos concentrations under study. Cell death decreased significantly in WJ-Exos 50 µg/ml samples (p ≤ 0,05) compared to IL1β treated hNPCs. Nitrite production was significantly attenuated at 10µg/ml of WJ-Exos (p ≤ 0,01). GAG content and histological analysis showed a difference in ECM synthesis between treated and untreated hNPCs (p ≤ 0,05). Catabolic and inflammatory markers were modulated by WJ-Exos at 100 µg/ml concentration (p ≤ 0,05) whereas 10 µg/ml group increased anabolic gene expression levels (p ≤ 0,05). These findings offer new opportunities for the potential use of exosomes as an attractive alternative cell-free strategy of IDD. WJ-MSC exosomes ameliorate hNPCs growth and viability, attenuate ECM degradation and oxidative stress-related IDD progression after IL1β stimulation. Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects

Mechanical loading regulates the metabolism of chondrocytes in cartilage1. Nowadays, studies exploring the in vitro response of cartilage towards loading often rely on bioreactor experiments applying only compressive loading. This is likely not sufficiently representative for the complex multi-directional loading profile in vivo (i.e. where typical compressive and shear loading are both present). The impact of multi-axial loading is specifically relevant in the context of the onset of osteoarthritis (OA) due to joint destabilization. Here, alterations in the 3D loading profile, and in particular increased shear forces, are suggested to initiate catabolic molecular responses leading to cartilage degeneration3. However, in vitro/ex vivo data confirming this hypothesis are currently lacking. Therefore, we aim to investigate how increased shear loading affects the metabolism and ECM deposition of a healthy chondrogenic cell line and if this response is different in osteoarthritic primary chondrocytes. A murine chondrogenic precursor cell line (ATDC5) and primary human osteoarthritic articular chondrocytes (hOACs) were encapsulated in 2.2% alginate disks and cultured in DMEM medium for three days. Hydrogels seeded with the different cell groups were loaded in the TA ElectroForce BioDynamic Bioreactor and subjected to following loading conditions: (a) 10% compression at 1Hz for 1h, (b) 10% compression and 10° shear loading at 1Hz for 1h. Unloaded constructs were used as control. After loading, hydrogel constructs were stabilized in culture medium for 2 hours, to facilitate adequate gene expression responses, before being dissolved and snap frozen. RNA was isolated and gene expression levels specific for anabolic pathways, characterized by extracellular matrix (ECM) genes (Col2a1, Aggrecan and Perlecan), catabolic processes (MMP-3 and MMP-13) and chondrogenic transcription factor (Sox9) were evaluated using RT-qPCR. The TA ElectroForce BioDynamic Bioreactor was successfully set-up to mimic cartilage loading. In ATDC5 cells, compression elicits an increase in all measured ECM genes (Col2a1, Aggrecan and Perlecan) compared to unloaded controls, suggesting an anabolic response. This upregulation is decreased when adding additional shear strain. In contrast to ATDC5 cells, the anabolic response of proteoglycans Aggrecan and Perlecan to compressive loading was lower in osteoarthritic chondrocytes, and Col2a1 expression appeared decreased. Adding shear strain reversed this effect on Col2a1 expression. Multi-directional loading increased transcription factor Sox9 expression compared to compression in both ATDC5 and OA chondrocytes. In OA chondrocytes, both loading regimens increased MMP-3 and MMP-13 expression. Shear loading reduces the anabolic effect of compressive loading in both cell types. OA cells presented more catabolic response to mechanical loading compared to precursors, given the increase in catabolic enzymes MMP-3 and MMP-13

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

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

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

Knee joint distraction (KJD) is a joint-preserving treatment strategy for severe osteoarthritis (OA) that provides long-term clinical and structural improvement. Data from both human trials and animal models indicate clear cartilage regeneration from 6 months and onwards post-KJD. However, recent work showed that during distraction, the balance between catabolic and anabolic indicators is directed towards catabolism, as indicated by collagen type 2 markers, proteoglycan (PG) turnover and a catabolic transcription profile [unpublished data]. The focus of this study was to investigate the cartilage directly and 10 weeks after joint distraction in order to elucidate the shift from a catabolic to an anabolic cartilage state. Knee OA was induced bilaterally in 8 dogs according to the groove model. After 10 weeks of OA induction, all 8 animals received right knee joint distraction, employing the left knee as an OA control. After 8 weeks of distraction, 4 dogs were euthanized and after 10 weeks of follow-up the 4 other dogs. Macroscopic cartilage degeneration and synovial tissue inflammation was assessed using the OARSI canine scoring system. PG content was determined spectrometrically using Alcian Blue dye solution and the synthesis of newly formed PGs was determined using . 35. SO. 4. 2-. as a tracer, as was described before. Directly after KJD, macroscopic cartilage damage of the right tibial plateau was higher compared to the left OA control (OARSI score: 1.7±0.2 vs 0.6±0.3; p < 0.001). 10 weeks post-KJD this difference persisted (OARSI score: 1.4± 0.6 vs 0.6±0.3; p = 0.05). Directly after KJD, there was no difference in synovial inflammation between KJD and OA control (OARSI score: 1.4±0.5). At 10 weeks synovial inflammation increased significantly in the distracted knee (OARSI score: 2.1±0.3 vs 1.4±0.5; p < 0.05). Biochemical analysis of the tibia cartilage directly after KJD revealed a lower PG content (20.1±10.3 mg/g vs 23.7±11.7 mg/g). At 10 weeks post-KJD this difference in PG content was less (24.8±6.8 mg/g vs 25.4±7.8 mg/g). The PG synthesis rate directly after KJD appeared significantly lower vs. OA (1.4±0.6 nmol/h.g vs 5.9±4.4 nmol/h.g; p < 0.001)). However, 10 weeks post-KJD this difference was not detected (3.7±1.2 nmol/h.g vs 2.9±0.8 nmol/h.g), and the synthesis rate in the distracted knee was increased compared to directly after distraction (p < 0.01). Further in-depth investigation of the material is ongoing; these first results suggest that the shift from a catabolic to an anabolic state occurs within the first weeks after joint distraction, mostly reflected in the biochemical changes. As such, the post-distraction period seems to be essential in identifying key-players that support intrinsic cartilage repair

Intervertebral disc degeneration can lead to physical disability and significant pain, while the present therapeutics still fail to biochemically and biomechanically restore the tissue. Stem cell-based therapy in treating intervertebral disc (IVD) degeneration is promising while transplanting cells alone might not be adequate for effective regeneration. Recently, gene modification and 3D-printing strategies represent promising strategies to enhanced therapeutic efficacy of MSC therapy. In this regard, we hypothesized that the combination of thermosensitive chitosan hydrogel and adipose derived stem cells (ADSCs) engineered with modRNA encoding Interleukin − 4 (IL-4) can inhibit inflammation and promote the regeneration of the degenerative IVD. Rat ADSCs were acquired from adipose tissue and transfected with modRNAs. First, the kinetics and efficacy of modRNA-mediated gene transfer in mouse ADSCs were analyzed in vitro. Next, we applied an indirect co-culture system to analyze the pro-anabolic potential of IL-4 modRNA engineered ADSCs (named as IL-4-ADSCs) on nucleus pulposus cells. ModRNA transfected mouse ADSCs with high efficiency and the IL-4 modRNA-transfected ADSCs facilitated burst-like production of bio-functional IL-4 protein. In vitro, IL-4-ADSCs induced increased anabolic markers expression of nucleus pulposus cells in inflammation environment compared to untreated ADSCs. These findings collectively supported the therapeutic potential of the combination of thermosensitive chitosan hydrogel and IL-4-ADSCs for intervertebral disc degeneration management. Histological and in vivo validation are now being conducted

Multiple biochemical biomarkers have been previously investigated for the diagnosis, prognosis and response to treatment of articular cartilage damage, including osteoarthritis (OA). Synovial fluid (SF) biomarker measurement is a potential method to predict treatment response and effectiveness. However, the significance of different biomarkers and their correlation to clinical outcomes remains unclear. This systematic review evaluated current SF biomarkers used in investigation of cartilage degeneration or regeneration in the knee joint and correlated these biomarkers with clinical outcomes following cartilage repair or regeneration interventions. PubMed, Institute of Science Index, Scopus, Cochrane Central Register of Controlled Trials, and Embase databases were searched. Studies evaluating SF biomarkers and clinical outcomes following cartilage repair intervention were included. Two researchers independently performed data extraction and QUADAS-2 analysis. Biomarker inclusion, change following intervention and correlation with clinical outcome was compared. 9 studies were included. Study heterogeneity precluded meta-analysis. There was significant variation in sampling and analysis. 33 biomarkers were evaluated in addition to microRNA and catabolic/anabolic ratios. Five studies reported on correlation of biomarkers with six biomarkers significantly correlated with clinical outcomes following intervention. However, correlation was only demonstrated in isolated studies. This review demonstrates significant difficulties in drawing conclusions regarding the importance of SF biomarkers based on the available literature. Improved standardisation for collection and analysis of SF samples is required. Future publications should also focus on clinical outcome scores and seek to correlate biomarkers with progression to further understand the significance of identified markers in a clinical context

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