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Bone & Joint Research
Vol. 12, Issue 4 | Pages 259 - 273
6 Apr 2023
Lu R Wang Y Qu Y Wang S Peng C You H Zhu W Chen A

Aims. Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant (gynura bicolor), has demonstrated anti-inflammatory properties in various diseases. We aimed to explore the chondroprotective effect of DHCA on OA and its potential mechanism. Methods. In vitro, interleukin-1 beta (IL-1β) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of collagen II, aggrecan, SOX9, inducible nitric oxide synthase (iNOS), IL-6, matrix metalloproteinases (MMPs: MMP1, MMP3, and MMP13), and signalling molecules associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Immunofluorescence analysis assessed the expression of aggrecan, collagen II, MMP13, and p-P65. In vivo, a destabilized medial meniscus (DMM) surgery was used to induce mice OA knee joints. After injection of DHCA or a vehicle into the injured joints, histological staining gauged the severity of cartilage damage. Results. DHCA prevented iNOS and IL-6 from being upregulated by IL-1β. Moreover, the IL-1β-induced upregulation of MMPs could be inhibited by DHCA. Additionally, the administration of DHCA counteracted IL-1β-induced downregulation of aggrecan, collagen II, and SOX9. DHCA protected articular cartilage by blocking the NF-κB and MAPK pathways. Furthermore, DHCA mitigated the destruction of articular cartilage in vivo. Conclusion. We present evidence that DHCA alleviates inflammation and cartilage degradation in OA chondrocytes via suppressing the NF-κB and MAPK pathways, indicating that DHCA may be a potential agent for OA treatment. Cite this article: Bone Joint Res 2023;12(4):259–273


Bone & Joint Research
Vol. 10, Issue 9 | Pages 558 - 570
1 Sep 2021
Li C Peng Z Zhou Y Su Y Bu P Meng X Li B Xu Y

Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of transforming growth factor beta 1 (TGF-β1) was downregulated, while that of TGF-β2, Mothers against decapentaplegic homolog 3 (SMAD3), and WNT11 were upregulated in DDH, and alpha smooth muscle actin (αSMA), a key myofibroblast marker, showed marginal increase. In vitro studies showed that fibroblast proliferation was suppressed in DDH, which was associated with cell cycle arrest in G0/G1 and G2/M phases. Cell cycle regulators including Cyclin B1 (CCNB1), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), Cyclin-dependent kinase 1 (CDK1), E2F1, cell division cycle 6 (CDC6), and CDC7 were downregulated in DDH. Conclusion. DDH is associated with the loss of collagen fibres and fibroblasts, which may cause loose joint capsule formation. However, the degree of differentiation of fibroblasts to myofibroblasts needs further study. Cite this article: Bone Joint Res 2021;10(9):558–570


Bone & Joint Research
Vol. 7, Issue 2 | Pages 157 - 165
1 Feb 2018
Sun Y Kiraly AJ Sun AR Cox M Mauerhan DR Hanley EN

Objectives. The objectives of this study were: 1) to examine osteophyte formation, subchondral bone advance, and bone marrow lesions (BMLs) in osteoarthritis (OA)-prone Hartley guinea pigs; and 2) to assess the disease-modifying activity of an orally administered phosphocitrate ‘analogue’, Carolinas Molecule-01 (CM-01). Methods. Young Hartley guinea pigs were divided into two groups. The first group (n = 12) had drinking water and the second group (n = 9) had drinking water containing CM-01. Three guinea pigs in each group were euthanized at age six, 12, and 18 months, respectively. Three guinea pigs in the first group were euthanized aged three months as baseline control. Radiological, histological, and immunochemical examinations were performed to assess cartilage degeneration, osteophyte formation, subchondral bone advance, BMLs, and the levels of matrix metalloproteinse-13 (MMP13) protein expression in the knee joints of hind limbs. Results. In addition to cartilage degeneration, osteophytes, subchondral bone advance, and BMLs increased with age. Subchondral bone advance was observed as early as six months, whereas BMLs and osteophytes were both observed mainly at 12 and 18 months. Fibrotic BMLs were found mostly underneath the degenerated cartilage on the medial side. In contrast, necrotic BMLs were found almost exclusively in the interspinous region. Orally administered CM-01 decreased all of these pathological changes and reduced the levels of MMP13 expression. Conclusion. Subchondral bone may play a role in cartilage degeneration. Subchondral bone changes are early events; formation of osteophytes and BMLs are later events in the OA disease process. Carolinas Molecule-01 is a promising small molecule candidate to be tested as an oral disease-modifying drug for human OA therapy. Cite this article: Y. Sun, A. J. Kiraly, A. R. Sun, M. Cox, D. R. Mauerhan, E. N. Hanley Jr. Effects of a phosphocitrate analogue on osteophyte, subchondral bone advance, and bone marrow lesions in Hartley guinea pigs. Bone Joint Res 2018;7:157–165. DOI:10.1302/2046-3758.72.BJR-2017-0253


Bone & Joint Research
Vol. 4, Issue 5 | Pages 84 - 92
1 May 2015
Hamamura K Nishimura A Iino T Takigawa S Sudo A Yokota H

Objectives. Salubrinal is a synthetic agent that elevates phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) and alleviates stress to the endoplasmic reticulum. Previously, we reported that in chondrocytes, Salubrinal attenuates expression and activity of matrix metalloproteinase 13 (MMP13) through downregulating nuclear factor kappa B (NFκB) signalling. We herein examine whether Salubrinal prevents the degradation of articular cartilage in a mouse model of osteoarthritis (OA). Methods. OA was surgically induced in the left knee of female mice. Animal groups included age-matched sham control, OA placebo, and OA treated with Salubrinal or Guanabenz. Three weeks after the induction of OA, immunoblotting was performed for NFκB p65 and p-NFκB p65. At three and six weeks, the femora and tibiae were isolated and the sagittal sections were stained with Safranin O. Results. Salubrinal suppressed the progression of OA by downregulating p-NFκB p65 and MMP13. Although Guanabenz elevates the phosphorylation level of eIF2α, it did not suppress the progression of OA. Conclusions. Administration of Salubrinal has chondroprotective effects in arthritic joints. Salubrinal can be considered as a potential therapeutic agent for alleviating symptoms of OA. Cite this article: Bone Joint Res 2015;4:84–92


Bone & Joint Research
Vol. 11, Issue 1 | Pages 40 - 48
27 Jan 2022
Liao W Sun J Wang Y He Y Su K Lu Y Liao G Sun Y

Aims. In the repair of condylar cartilage injury, synovium-derived mesenchymal stem cells (SMSCs) migrate to an injured site and differentiate into cartilage. This study aimed to confirm that histone deacetylase (HDAC) inhibitors, which alleviate arthritis, can improve chondrogenesis inhibited by IL-1β, and to explore its mechanism. Methods. SMSCs were isolated from synovium specimens of patients undergoing temporomandibular joint (TMJ) surgery. Chondrogenic differentiation potential of SMSCs was evaluated in vitro in the control, IL-1β stimulation, and IL-1β stimulation with HDAC inhibitors groups. The effect of HDAC inhibitors on the synovium and condylar cartilage in a rat TMJ arthritis model was evaluated. Results. Interleukin (IL)-1β inhibited the chondrogenic differentiation potential of SMSCs, while the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and panobinostat (LBH589), attenuated inhibition of IL-1β-induced SMSC chondrogenesis. Additionally, SAHA attenuated the destruction of condylar cartilage in rat TMJ arthritis model. IL-6 (p < 0.001) and matrix metalloproteinase 13 (MMP13) (p = 0.006) were significantly upregulated after IL-1β stimulation, while SAHA and LBH589 attenuated IL-6 and MMP13 expression, which was upregulated by IL-1β in vitro. Silencing of IL-6 significantly downregulated MMP13 expression and attenuated IL-1β-induced chondrogenesis inhibition of SMSCs. Conclusion. HDAC inhibitors SAHA and LBH589 attenuated chondrogenesis inhibition of SMSC induced by IL-1β in TMJ, and inhibition of IL-6/MMP13 pathway activation contributes to this biological progress. This study provides a theoretical basis for the application of HDAC inhibitors in the treatment of TMJ arthritis. Cite this article: Bone Joint Res 2022;11(1):40–48


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_1 | Pages 6 - 6
2 Jan 2024
Liu W Feng M Xu P
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More and more evidences showed that cartilage harbored local progenitor cells that could differentiate toward osteoblast, chondrocyte, and adipocyte. However, our previous results showed that osteoarthritis derived chondroprogenitor cells (OA-CPC) exhibited strong osteogenic potential even in chondrogenic condition. How to promote their chondrogenic potential is the key for cartilage repair and regeneration in osteoarthritis. Recently, lipid availability was proved to determine skeletal progenitor fate. Therefore, we aim to determine whether lipid inhibition under 3D culture condition could enhance OA-CPC chondrogenesis. Moreover, glucose concentration was also evaluated for chondrogenic capacity. Although there are many researches showed that lower glucose promotes chondrogenesis, in our results, we found that OA-CPC in high concentration of glucose (4.5g/L) with lipid inhibitor (GW1100) showed strongest chondrogenic potential, which could form largest cell pellet with strong proteoglycan staining, COL II expression and no COL I expression. Besides, COL2A1 was increased and COL10A1 was decreased significantly by GW1100 under high glucose condition in 2D culture. Interestingly, although the expression level of MMP13 was not changed by GW1100 at RNA and protein level, less MMP13 protein secreted out of cell nuclear. In summary, we estimated that higher glucose and lower lipid supplies benefit OA-CPC chondrogenesis and cartilage repair


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_8 | Pages 109 - 109
11 Apr 2023
Amado I Hodgkinson T Mathavan N Murphy C Kennedy O
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Post-traumatic osteoarthritis (PTOA) is a subset of osteoarthritis, which occurs secondary to traumatic joint injury which is known to cause pathological changes to the osteochondral unit. Articular cartilage degradation is a primary hallmark of OA, and is normally associated with end-stage disease. However, subchondral bone marrow lesions are associated with joint injury, and may represent localized bone microdamage. Changes in the osteochondral unit have been traditionally studied using explant models, of which the femoral-head model is the most common. However, the bone damage caused during harvest can confound studies of microdamage. Thus, we used a novel patellar explant model to study osteochondral tissue dynamics and mechanistic changes in bone-cartilage crosstalk. Firstly, we characterized explants by comparing patella with femoral head models. Then, the patellar explants (n=269) were subjected to either mechanical or inflammatory stimulus. For mechanical stimulus 10% strain was applied at 0.5 and 1 Hz for 10 cycles. We also studied the responses of osteochondral tissues to 10ng/ml of TNF-α or IL-1β for 24hrs. In general the findings showed that patellar explant viability compared extremely well to the femoral head explant. Following IL-1β or TNF-α treatment, MMP13, significantly increased three days post exposure, furthermore we observed a decrease in sulfate glycoaminoglycan (sGAG) content. Bone morphometric analysis showed no significant changes. Contrastingly, mechanical stimulation resulted in a significant decrease sGAG particularly at 0.5Hz, where an increase in MMP13 release 24hrs post stimulation and an upregulation of bone and cartilage matrix degradation markers was observed. Furthermore, mechanical stimulus caused increases in TNF-α, MMP-8, VEGF expression. In summary, this study demonstrates that our novel patella explant model is an excellent system for studying bone-cartilage crosstalk, which responds well to both mechanical and inflammatory stimulus and is thus of great utility in the study of PTOA


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_9 | Pages 64 - 64
17 Apr 2023
Bermudez-Lekerika P Croft A Crump K Wuertz-Kozak K Le Maitre C Gantenbein B
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Previous research has shown catabolic cell signalling induced by TNF-α and IL-1β within intervertebral (IVD) cells. However, these studies have investigated this in 2D monolayer cultures, and under hyper-physiological doses. Thus, we aim to revisit the catabolic responses of bovine IVD cells in vitro in 3D culture under increasing doses of TNF-α or IL-1β stimulation at three different timepoints. Primary bovine nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated and expanded for two weeks. Subsequently, NP and AF cells were encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks for phenotype recovery. Re-differentiated cells were stimulated with 0.1, 1 and 10 ng/ml TNF-α or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation. A dose-dependent upregulation of catabolic markers was observed in both cell types after one day of TNF-α or IL-1β stimulation. 10 ng/ml TNF-α stimulation induced a significant upregulation (p<0.05) of ADAMTS4, MMP3 and MMP13 in AF cells after one day of stimulation. Similarly, MMP3 upregulation showed a strong trend (p=0.0643) in NP cells. However, no effects on expression were seen after seven days. In addition, no significant difference between treatments in COL2, COL1 and ACAN expression was observed, and cell viability was not reduced at any time point, regardless of the treatment. We demonstrate a dose-dependent upregulation of catabolic markers in NP and AF cells under TNF-α or IL-1β stimulation, with a significant upregulation of ADAMTS4, MMP3 and MMP13 genes in AF cells after one day of treatment. Notably, after seven days of treatment, the dose-dependent effects were no longer observed possibly due to an adaptation mechanism of IVD cells to counter the metabolic shift


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_8 | Pages 54 - 54
1 Aug 2020
Bisson D Haglund L Kocabas S Ouellet J Saran N
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Adolescent idiopathic scoliosis (AIS) is a poorly understood progressive curvature of the spine. The 3-dimmensionnal spinal deformation brings abnormal biomechanical stresses on the load-bearing organs. We have recently reported for the first time the presence of facet joint cartilage degeneration comparable to age-related osteoarthritis in scoliotic adolescents. To better understand the degenerative mechanisms and explore new therapeutic possibilities, we focused on Toll-like receptors (TLRs) which are germline-encoded pattern recognition receptors that recognize pathogens and endogenous proteins such as fragmented extracellular matrix components (alarmins) present in intervertebral discs (IVD) and articular cartilage. Once activated, they regulate the production pro-inflammatory cytokines, proteases and neurotrophins which can lead to matrix catabolism, inflammation and potentially pain. These mechanisms have however not been studied in the context of AIS or facet joints. Facet joints of AIS patients undergoing corrective surgery and of cadaveric donors (non-scoliotic) were collected from consenting patients or organ donors with ethical approval. Cartilage biopsies and chondrocytes were isolated using 3mm biopsy punches and collagenase type 2 digestion respectively. qPCR was used to assess gene expression of the degenerative factors (MMP3, MMP13, IL-1ß, IL-6, IL-8) The biopsies were cut into two equal halves, one was treated for 4 days with a TLR2 agonist (Pam2CSK4, Invivogen) in serum-free chondrocyte media while the other one was cultured in media alone. MMP3, MMP13, IL-6 and IL-8 ELISAs and DMMB assays were performed on the biopsy cultured media. The ex vivo cartilage was then fixed, cryosectionned and also stained with SafraninO-Fast Green dyes. Baseline gene expression levels of TLR1,−2,−4,−6 were all upregulated in scoliotic chondodryctes compared to non-scoliotic. Pearson correlation analysis revealed that all TLR1,−2,−4,−6 gene expression correlated strongly and significantly with degenerative markers (MMP3, MMP13, IL-6, IL-8) in scoliotic chondrocytes but not in non-scoliotic. (Figure 1) When monolayer facet joint chondrocytes were activated with Pam2CSk4, there was a significant upregulation in previously described degenerative markers, TLR2 and NGF, a potent neurotrophin. These findings were strengthened by protein secretion analysis of select markers such as MMP-3, −13, IL-6 and IL-8 which were all upregulated after TLR2 activation. The scoliotic biopsies which were treated with Pam2CSK4 had a significant loss of proteoglycan content as shown by histology, was reflected in the proteoglycan content found in the media by DMMB. TLR gene expression levels were upregulated and correlated with proteases and pro-inflammatory cytokines in degenerating scoliotic cartilage, suggesting they promote cartilage degradation, especially considering the lack of correlations in non-scoliotic healthy cartilage. Furthermore, when TLRs are activated by Pam2CSK4 it triggers the release of the same proteases and pro-inflammatory cytokines in our ex vivo experiment. All this exacerbates the loss of proteoglycan in the cartilage ex vivo model after four days of insult with a TLR2 specific agonist. These results suggest that TLRs are an important pathway partaking in the cartilage degeneration of scoliotic facet joints and potentially all cartilage beyond our scope. Future studies aim at blocking TLRs to alleviate proteolysis and inflammation. For any figures or tables, please contact the authors directly


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_13 | Pages 26 - 26
1 Nov 2021
Amado I Hodgkinson T Murphy C Kennedy O
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Introduction and Objective. Traditionally, osteoarthritis (OA) has been associated mostly with degradation of cartilage only. More recently, it has been established that other joint tissues, in particular bone, are also centrally involved. However, the link between these two tissues remains unclear. This relationship is particularly evident in post-traumatic OA (PTOA), where bone marrow lesions (BMLs), as well as fluctuating levels of inflammation, are present long before cartilage degradation begins. The process of bone-cartilage crosstalk has been challenging to study due to its multi-tissue complexity. Thus, the use of explant model systems have been crucial in advancing our knowledge. Thus, we developed a novel patellar explant model, to study bone cartilage crosstalk, in particular related to subchondral bone damage, as an alternative to traditional femoral head explants or cylindrical core specimens. The commonly used osteochondral explant models are limited, for our application, since they involve bone damage during harvest. The specifics aim of this study was to validate this novel patellar explant model by using IL-1B to stimulate the inflammatory response and mechanical stimulation to determine the subsequent developments of PTOA. Materials and Methods. Lewis rats (n=48) were used to obtain patellar and femoral head explants which were harvested under an institutional ethical approval license. Explants were maintained in high glucose media (containing supplements), under sterile culture conditions. Initially, we characterised undamaged patellar explants and compared them with the commonly used femoral head. First, tissue viability was assessed using an assay of metabolic activity and cell damage. Second, we created chemical and mechanical damage in the form of IL-1B treatment, and mechanical stimulation, to replicate damage. Standard biochemical assays, histological assays and microstructural assays were used to evaluate responses. For chemical damage, explants were exposed to 10ng/ml of IL-1B for 24 hours at 0, 1, 3 and 7 days after harvesting. For mechanical damage, tissues were exposed to mechanical compression at 0.5 Hz, 10 % strain for 10 cycles, for 7 days. Contralateral patellae served as controls. In both groups, sGAG, ADAMTS4, and MMP-13 were measured as an assessment of representative cartilage responses while ALP, TRAP and CTSK were assessed as a representative of bone responses. In addition to this, histomorphometric, and immunohistochemical, evaluations of each explant system were also carried out. Results. Our results confirm that the patellar explant system is an excellent ex vivo model system to study bone-cartilage crosstalk, and one which does not induce any bone damage at the time of tissue harvest. We successfully established culture conditions to maintain viability in these explants for up to 28 days. Rat IL-1B treatment resulted in increased both proteoglycan content and bone metabolism markers after 7 days when compared with the controls. To confirm this finding, qualitative immunohistochemical staining showed chondrocytes increased expression of MMP13 after treatment with IL-1B. Furthermore, we observed that the levels of ADAMTS4 decreased in 48 hours after IL-1B exposure. Contrastingly IL-1B treatment had the opposite effect on CTSK markers when compared with the control. Mechanically compressed patellae showed a decrease in compressive moduli from day 3 to day 7, suggesting that tissue remodelling may have taken place as a compensatory mechanism in response to damage. In addition, MMP13 release decreased over 48 hours after mechanical compression, while TRAP levels were increased compared with the control. Conclusions. Thus, we successfully demonstrated that IL-1B and mechanical stimulation affects both bone and cartilage tissues independently in this system, which may have relevance in the understanding of bone-cartilage crosstalk after injury and how this is involved in PTOA development


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 100 - 100
1 Jul 2014
Smith M Schiavinato A Little C
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Introduction. Osteoarthritis (OA) involves pathological change in all joint tissues, including cartilage degradation and synovitis. Synovial inflammation is significantly associated with pain severity and incidence in knee OA. It is becoming evident that synovitis also plays an active role in the initiation and progression of cartilage erosion in OA, through direct secretion of catabolic enzymes as well as factors that stimulate chondrocyte catabolic activity. Therapeutic agents that target both synovitis and cartilage pathology are likely to be maximally beneficial in treating pain and slowing cartilage breakdown in OA. We have previously shown that an amide-derivative of HA (HYMOVIS™) was superior to native HA of the same MW in improving gait, and reducing synovial hyperplasia in a sheep OA model. In the present study the mechanisms whereby the chemically modified HA may be beneficial were examined using chondrocytes and synovial fibroblasts from knees of OA patients. Patients & Methods. Chondrocytes (HAC, n=6) and synovial fibroblasts (HSF, n=6) were isolated from OA patients at the time of knee replacement. HYMOVIS™ (0, 0.5, 1.0 or 1.5mg/mL) was added to simultaneously or 1 hour before interleukin-1β (IL1, 2ng/mL). Cultures were terminated 30 minutes later for Bioplex. ®. quantitation of p-JNK, p-NFκB and p-p38; or 24 hours later for RNA isolation and analysis of gene expression by real time RT-PCR, and measurement of MMP13 activity in the media. Only statistically significant results are reported. Results. In HAC in the absence of IL1, HYMOVIS™ decreased MMP13, ADAMTS5, PTGS2 and IL6 and increased COL2A1 mRNA (2–10fold). In HSF in absence of IL1, HYMOVIS™ decreased TIMP1, TIMP3, CD44, IL6 and increased PTGS2 (2–3fold). In HAC and HSF, IL1 increased expression of MMP1, MMP13, PTGS2, IL6 (>100fold), ADAMTS4 (∼10 fold), all phosphoproteins (3–10fold), and APMA-activated MMP13 activity in media. IL1 increased expression of ADAMTS5 (∼10fold) only in HSF. As expected, IL1 reduced expression of the key matrix proteins in HAC (2–3 fold decrease in COL2A1 and ACAN) and HSF (2 fold decrease in COL1A1). When added simultaneously with IL1, HYMOVIS™ decreased expression of MMP13, ADAMTS5, PTGS2, IL6 expression, and normalised matrix protein expression in both HAC and HAS. Pre-incubation with HYMOVIS™ for 1 hour inhibited IL1-stimulated p-JNK, p-NFκB and p-p38 in both cell types (excluding p-JNK in HSF). In HAC, HYMOVIS™ pre-incubation was superior to simultaneous addition in reducing expression of MMP1, MMP13, ADAMTS4, PTGS2, and IL6 expression. There was a less dramatic effect of HYMOVIS™ pre-incubation on gene expression in HSF compared with HAC. The inhibitory effects of HYMOVIS™ on IL1 stimulated gene expression in HAC and HSF was partially ameliorated by pre-incubation with a CD-44 blocking antibody. Discussion/Conclusions. The present studies have demonstrated several potential key mechanisms whereby the intra-articular injection of a hexadecylamide-derivative of HA (HYMOVIS™) may have both symptom and disease-modifying effects in OA. The previously described increased joint retention of the hexadecylamide-derivative, might act in a similar manner to the pre-incubation studies in our cell culture studies, to reduce the initiation of degradative events with recurrent/cyclic inflammatory episodes that typify OA


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_8 | Pages 127 - 127
11 Apr 2023
Nau T Cutts S Naidoo N
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There is an evolving body of evidence that demonstrates the role of epigenetic mechanisms, such as DNA-methylation in the pathogenesis of OA. This systematic review aims to summarize the current evidence of DNA methylation and its influence on the pathogenesis of OA. A pre-defined protocol in alignment with the PRISMA guidelines was employed to systematically review eight bibliographic databases, to identify associations between DNA-methylation of articular chondrocytes and osteoarthritis. A search of Medline (Ovid), Embase, Web-of-Science, Scopus, PubMed, Cinahl (EBSCOhost), Cochrane Central and Google Scholar was performed between 1st January 2015 to 31st January 2021. Data extraction was performed by two independent reviewers. During the observation period, we identified 15 gene specific studies and 24 genome wide methylation analyses. The gene specific studies mostly focused on the expression of pro-inflammatory markers, such as IL8 and MMP13 which are overexpressed in OA chondrocytes. DNA hypomethylation in the promoter region resulted in overexpression, whereas hypermethylation was seen in non-OA chondrocytes. Others reported on the association between OA risk genes and the DNA methylation pattern close to RUNX2, which is an important OA signal. The genome wide methylation studies reported mostly on differentially methylated regions comparing OA chondrocytes and non-OA chondrocytes. Clustering of the regions identified genes that are involved in skeletal morphogenesis and development. Differentially methylated regions were seen in hip OA and knee OA chondrocytes, and even within different regions of an OA affected knee joint, differentially methylated regions were identified depending on the disease stage. This systematic review demonstrates the growing evidence of epigenetic mechanisms, such as DNA methylation, in the pathogenesis of OA. In recent years, there has been a focus on the interplay between OA risk genes and DNA methylation changes which revealed a reactivation of genes responsible for endochondral ossification during development. These are important findings and may help to identify eventual future therapeutic targets. However, the current body of literature is mostly showing the differences in DNA methylation of OA chondrocytes and non-OA chondrocytes, but a true longitudinal analysis demonstrating the DNA methylation changes actually happening is still not available


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_9 | Pages 81 - 81
17 Apr 2023
Rambacher K Gennrich J Schewior R Lang S Pattappa G Zihlmann C Stiefel N Zellner J Docheva D Angele P
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Meniscus tears have been treated using partial meniscectomy to relieve pain in patients, although this leads to the onset of early osteoarthritis (OA). Cell-based therapies can help preserve the meniscus, although the presence of inflammatory cytokines compromises clinical outcomes. Anti-inflammatory drugs (e.g. celecoxib), can help to reduce pain in patients and in vitro studies suggest a beneficial effect on cytokine inhibited matrix content. Previously, we have demonstrated that the inhibitory effects of IL-1β can be countered by culture under low oxygen tension or physioxia. The present study sought to understand whether physioxia, celecoxib or combined application can counter the inhibitory effects IL-1β inhibited meniscus cells. Human avascular and vascular meniscus cells (n =3) were isolated and expanded under 20% (hyperoxia) or 2% (physioxia) oxygen. Cells were seeded into collagen scaffolds (Geistlich, Wolhusen) and cultured for 28 days either in the presence of 0.1ng/mL IL-1β, 5µg/mL celecoxib or both under their expansion oxygen conditions. Histological (DMMB, collagen I and collagen II immunostaining), GAG content and gene expression analysis was evaluated for the scaffolds. Under hyperoxia, meniscus cells showed a significant reduction in GAG content in the presence of IL-1β (*p < 0.05). Celecoxib alone did not significantly increase GAG content in IL-1β treated cultures. In contrast, physioxic culture showed a donor dependent increase in GAG content in control, IL-1β and celecoxib treated cultures with corresponding histological staining correlating with these results. Additionally, gene expression showed an upregulation in COL1A1, COL2A1 and ACAN and a downregulation in MMP13 and ADAMTS5 under physioxia for all experimental groups. Physioxia alone had a stronger effect in countering the inhibitory effects of IL-1β treated meniscus cells than celecoxib under hyperoxia. Preconditioning meniscus cells under physioxia prior to implantation has the potential to improve clinical outcomes for cell-based therapies of the meniscus


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_2 | Pages 116 - 116
2 Jan 2024
Šećerović A Ristaniemi A Crivelli F Heub S Weder G Ferguson S Ledroit D Grad S
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Intervertebral disc (IVD) degeneration is inadequately understood due to the lack of in vitro systems that fully mimic the mechanical and biological complexity of this organ. We have recently made an advancement by developing a bioreactor able to simulate physiological, multiaxial IVD loading and maintain the biological environment in ex vivo IVD models [1]. To validate this new bioreactor system, we simulated natural spine movement by loading 12 bovine IVDs under a combination of static compression (0.1 MPa), cyclic flexion/extension (±3˚, ±6˚ or 0-6˚) and cyclic torsion (±2˚, ±4˚ or 0-4˚) for more than 10’000 (0.2 Hz) or 100’000 (1 Hz) cycles over 14 days. A higher number of cycles increased the release of glycosaminoglycans and nitric oxide, as an inflammation marker, whereas fewer cycles maintained these two factors at physiological levels. All applied protocols upregulated the expression of MMP13 in the outermost annulus fibrosus (AF), indicating a collagen degradation response. This was supported by fissures observed in the AF after a longer loading duration. Increasing loading cycles induced high cell death in the nucleus pulposus and inner AF, while with fewer cycles, high cell viability was maintained in all IVD regions, irrespective of the magnitude of rotation. Less frequent multiaxial loading maintains IVD homeostasis while more frequent loading initiates an IVD degenerative profile. Specifically, the morphological and molecular changes were localized in the AF, which can be associated with combined flexion/extension and torsion. More loading cycles induced region-specific cell death and a higher release of extracellular matrix molecules from the innermost IVD regions, likely associated with longer exposure to static compression. Altogether, we demonstrated the advantages of the multiaxial bioreactor to study region-specific response in the IVD, which will allow a more profound investigation of IVD degeneration under different combinations of motions


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_2 | Pages 135 - 135
2 Jan 2024
Füllemann P Jörimann T Bella E Stoddart M Matthys R Verrier S
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Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies. Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used. Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control. Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain. Acknowledgments: Funded by the AO Foundation, StrainBot sponsored by RISystemAG & PERRENS 101 GmbH


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_2 | Pages 96 - 96
2 Jan 2024
Bauer C Moser L Otahal A Kern D Dammerer D Zantop T Nehrer S
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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


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_3 | Pages 33 - 33
1 Apr 2018
Timur UT Emans P van Rhijn L Welting T
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Introduction. Cartilage homeoprotein 1 (CART-1) is a homeoprotein which has been suggested to play a role in chondrocyte differentiation and in skeletal development. It is expressed mainly in prechondrocytic mesenchymal condensations. Patients with mutations in the CART-1 gene display several craniofacial abnormalities, suggesting that CART-1 has a functional role in craniofacial skeletal development. However, its target genes and position in the established chondrogenic pathways is poorly documented. Given the fact that CART-1 is expressed predominantly in the chondrocyte lineage and its role in skeletal development, we hypothesized that CART-1 regulates expression of several pivotal genes involved in chondrogenic differentiation. Methods. The coding sequence of human CART-1 was custom synthesized with optimized codon usage and cloned into a p3XFLAG-CMV-7.1 expression vector. FLAG-CART-1 was transiently overexpressed in SW1353 cells by polyethyleneimine-mediated transfection (1,000 ng of plasmid/well in 12-well plates). FLAG-Empty vector was used as a negative control. FLAG-CART-1 overexpression was confirmed by means of anti-FLAG immunoblotting. To investigate a potential connection between CART-1 and established key chondrogenic pathways, TGFβ3 (10 ng/mL) was added to SW1353 cells in CART-1 overexpression cultures or their appropriate controls. Cells were harvested 48 hours after transfection and mRNA expression of several genes involved in chondrogenic differentiation was determined by qRT-PCR. Data represent three separate experiments performed in technical triplicate. Results. Overexpression of CART-1 was confirmed on protein level. CART-1 significantly upregulated the expression of hypertrophic markers MMP13 and COLX, while the expression of RUNX2, ALP and COL1 was significantly downregulated. The expression of COL2A1 and SOX9 was not altered in the presence of CART1. TGFβ3 significantly decreased MMP13 expression in SW1353 cultures, but induced the expression of COLX, RUNX2 and COL1. This TGFβ3 dependent behaviour was reversed when CART-1 was overexpressed in these cultures. Conclusion. Our results implicate a functional role for the homeodomain protein CART-1 in controlling the expression of several markers involved in chondrocyte differentiation and show important interactions with other signaling pathways involved in chondrogenic differentiation. Current efforts focus on further elucidating the connection between CART-1 and other chondrogenic pathways


Bone & Joint Research
Vol. 5, Issue 9 | Pages 412 - 418
1 Sep 2016
Ye S Ju B Wang H Lee K

Objectives. Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration. Methods. Levels of IL-18 were measured in the blood of patients with intervertebral disc degenerative disease and in control patients. Human NP and AF cells were cultured in a NP cell medium and treated with IL-18 or IL-18 plus BMP-2. mRNA levels of target genes were measured by real-time polymerase chain reaction, and protein levels of aggrecan, type II collagen, SOX6, and matrix metalloproteinase 13 (MMP13) were assessed by western blot analysis. Results. The serum level of patients (IL-18) increased significantly with the grade of IVD degeneration. There was a dramatic alteration in IL-18 level between the advanced degeneration (Grade III to V) group and the normal group (p = 0.008) Furthermore, IL-18 induced upregulation of the catabolic regulator MMP13 and downregulation of the anabolic regulators aggrecan, type II collagen, and SOX6 at 24 hours, contributing to degradation of disc matrix enzymes. However, BMP-2 antagonised the IL-18 induced upregulation of aggrecan, type II collagen, and SOX6, resulting in reversal of IL-18 mediated disc degeneration. Conclusions. BMP-2 is anti-catabolic in human NP and AF cells, and its effects are partially mediated through provocation of the catabolic effect of IL-18. These findings indicate that BMP-2 may be a unique therapeutic option for prevention and reversal of disc degeneration. Cite this article: S. Ye, B. Ju, H. Wang, K-B. Lee. Bone morphogenetic protein-2 provokes interleukin-18-induced human intervertebral disc degeneration. Bone Joint Res 2016;5:412–418. DOI: 10.1302/2046-3758.59.BJR-2016-0032.R1


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_10 | Pages 25 - 25
1 May 2017
Vickers L Thorpe A Sammon C Le Maitre C
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Introduction. Current strategies to treat back pain address the symptoms but not the underlying cause. Here we are investigating a novel hydrogel material (NPgel) which can promote MSC differentiation to Nucleus pulposus cells. Current in vitro studies have only explored conditions that mimic the native disc microenvironment. Here, we aim to determine the stem cells regenerative capacity under conditions that mimic the degenerate environment seen during disc degeneration. Methods. hMSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5%) with ± calcium (2.5mM and 5.0mM CaCl. 2. ), IL-1β and TNFα either individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by Alamar blue assay. Histological and immunohistochemical analysis investigated altered matrix and matrix degrading enzyme expression. Results. Viability of hMSCs was maintained under all culture conditions. Matrix deposition of glycosaminoglycans were observed under all conditions, MMP13 expression was upregulated by calcium but not by pro-inflammatory cytokines IL-1β and TNFα. Conclusions. We are developing an in vitro modelling system which can be used to test novel therapies within a degenerate microenvironment. Interestingly, our preliminary findings suggest calcium is a major contributor to regulating MMP13 in this model system. Investigating the degenerate niche will identify targets for inhibition to provide the correct niche to promote regeneration of the IVD. No conflict of interest. Funding: BMRC, MERI Sheffield Hallam University, for joint funding the Daphne Jackson Trust fellowship


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_3 | Pages 68 - 68
1 Apr 2018
Riedl M Koch M Freimoser F Pattappa G Zellner J Docheva D Angele P Pfeifer C
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Introduction. Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA. Methods. Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2μM after 14 days of chondrogenic pre-differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14. The pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13) and histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry. Results. Hypertrophy was reduced by addition of BMS at day 14 and further reduced by addition from the beginning. BMS treatment resulted in smaller cells under hypertrophic conditions, higher collagen II content in chondrogenic groups and reduction in collagen X production and ALP activity in every condition. Gene expression data for hypertrophic markers, collagen X and MMP13, were upregulated under the influence of BMP4 but a distinct downregulation in MMP13 expression was shown upon addition of BMS during the late stage differentiation and further reduced upon addition during early stage chondrogenesis. Furthermore, Collagen X expression was reduced by early BMS treatment. Discussion. The treatment with the RAR IA, BMS, attenuated hypertrophic changes in chondrogenically differentiated hMSCs as demonstrated by histology, immunohistochemistry and PCR. These findings suggest an additional approach to attenuate hypertrophy in chondrogenically differentiated hMSCs. Current studies are exploring the timing and dose of BMS to most efficaciously prevent hypertrophy