Coeleveld, K.

Lafeber, F.P.

Mastbergen, S.C.

Meij, B.P.

Popov-Celeketic, J.

Teunissen, M.

Tryfonidou, M.A.

Utrecht University Repository

Abstracts / Osteoarthritis and Cartilage 28 (2020) S20eS85S76Results: By mCT analyses, GP length inwild-typemice reduces from ~260mm to 130 mm between 4 and 10 weeks of age. Deletion of IFT88 at 4, 6 or8 weeks of age resulted in longer GPs in cKO mice (solid bars) at everytimepoint compared with control mice (open bars, Fig 1A). Two weeksafter tamoxifen, cKO GP lengths were not statistically significantly dif-ferent to controls at time of treatment, indicating inhibition of growthplate closure. No changeswere observed in the epiphyseal plate but therewere increases in trabecular bone density immediately below the GP incKO mice. Interestingly, some cKO mice exhibited extremely long GP atthe edges of the tibia, which appeared as large holes by mCT (Fig 1B),whilst the centre of the GP appeared less affected. Histological sections ofGP confirmed longer growth plates with increases in both proliferativebut, most notably, hypertrophic chondrocyte populations. The large,often bi-lateral holes, observed by mCT were largely filled with dis-organised hypertrophic chondrocytes (Fig 1C), which IHC analysis indi-cates were expressing type X collagen (Coll X) (Fig 1D). Intriguingly,double (femoral and sciatic) neurectomy, at 8 weeks of age, also resultedin large, often bilateral, disorganised populations of hypertrophic chon-drocytes, but only in the contralateral (loaded) limb.Conclusions: In contrast to premature closure of the GP observed atearlier timepoints in development, deletion of IFT88 in the adolescentGP results in longer growth plates and inhibition of GP closure due tothe failure of ossification. Histological analyses suggest deletion of IFT88does not impair Hh-PTHrP stimulated proliferation or hypertrophicdifferentiation, but interferes with the ossification process at thechondro-osseous junction. This phenotype is most pronounced in thelateral edges of the GP, possibly supporting a role for IFT88 in GPmechanotransduction. The role of mechanics in influencing GP ossifi-cation is further supported by a similar phenotype seen in the con-tralateral limb of double neurectomisedmice. Quite howmechanics andIFT88 interact to control GP closure is currently unclear.86KNEE JOINT DISTRACTION-INDUCED SHIFT FROM CATABOLIC TOANABOLIC STATE OCCURS AFTER THE DISTRACTION PERIODM. Teunissen 1, J. Popov-Celeketic 2, K. Coeleveld 2, B.P. Meij 1,F.P. Lafeber 2, M.A. Tryfonidou 1, S.C. Mastbergen 2. 1Utrecht Univ.,Utrecht, Netherlands; 2Univ. Med. Ctr. (UMC) Utrecht, Utrecht,NetherlandsPurpose: Knee joint distraction (KJD) is a validated joint-preservingtreatment strategy for severe osteoarthritis (OA) that provides long-term clinical and structural improvement. Data from both humantrials and animal models indicate clear cartilage regeneration from 6months and onwards post-KJD. However, recent work showed thatduring distraction, the balance between catabolic and anabolic indi-cators is directed towards catabolism, as indicated by collagen type 2markers, proteoglycan (PG) turnover and a catabolic transcriptionprofile [unpublished data]. The focus of the present study was toinvestigate cartilage changes directly and 10 weeks after joint dis-traction in order to elucidate the shift from a catabolic to an anaboliccartilage state.Methods: Knee OA was induced bilaterally in 8 dogs according to thegroove model. After 10 weeks of OA induction, all 8 animals weretreated with knee joint distraction on the right side, employing theleft knee as an OA control. After 8 weeks of distraction, 4 dogs wereeuthanized directly (KJDdirect), and after 10 weeks of follow-up the 4remaining dogs (KJDþ10). Macroscopic and microscopic cartilagedegeneration was assessed using the OARSI canine scoring system.RT-qPCR was used to determine relative expression of aggrecan(ACAN)w collagen type II (COL2a1), cartilage oligomeric matrix protein(COMP) and matrix metalloproteinase-3 (MMP3) in the cartilage. PGcontent was determined by the Alcian Blue assay and the synthesis ofPGs was determined using 35SO42- as a tracer.Results: Directly after KJD, macroscopic cartilage damage of the righttibial plateau was higher as compared to the left OA control (OARSIscore KJDdirect : 1.7±0.2 vs 0.6±0.3; p < 0.001). 10 weeks post-KJD thisdifference persisted (OARSI score KJDþ10: 1.4±0.6 vs 0.6±0.3; p ¼0.05). Microscopically, an increase in the total OARSI score was seenafter 10 weeks post-KJD. This was mainly due to an increase ofchondrocyte clusters at 10 weeks of follow-up, resulting in anincrease in the sub score chondrocyte pathology. Remarkedly the subscore intensity of proteoglycan staining decreased directly after KJD(indicating a loss of PGs) but increased after 10 weeks of follow-up(see also figure), suggesting a mixed response depending on the itemscored. Cartilage gene expression analysis showed downregulation ofCOL2a1 (-1.3 ± 0.3), ACAN (-4.4 ± 1.0, p < 0.01) and COMP (-1.7 ± 0.5)directly after knee joint distraction compared to the left OA controlsuggesting enhanced catabolic activity during KJD. In contrast, after10 weeks of follow-up the expression of COL2a1 and COMP wereincreased in the right distracted knee as compared to the left OAcontrol (2.6 ± 1.1 and 2.5 ± 1.2 respectively) as well as compared tothe situation directly after KJD (3.3 ± 1.4 and 4.2 ± 2.0), though not allchanges reached statistical significance. Similar, expression of MMP3was upregulated directly after KJD (4.4 ± 0.8) and downregulated 10weeks post-KJD (-3.3 ± 0.8). Biochemical analysis of the tibia cartilagedirectly after KJD revealed a lower PG content compared to the OAjoint (20.1±10.3 mg/g vs 23.7±11.7 mg/g). At 10 weeks post-KJD thisdifference in PG content was gone (24.8±6.8 mg/g vs 25.4±7.8 mg/g).The PG synthesis rate directly after KJD appeared significantly lowervs. OA (1.4±0.6 nmol/h.g vs 5.9±4.4 nmol/h.g; p < 0.001)). Conversely,10 weeks post-KJD this difference disappeared (3.7±1.2 nmol/h.g vs2.9±0.8 nmol/h.g), and the synthesis rate in the distracted knee wasincreased compared to directly after distraction (p < 0.01) indicating ashift upon follow-up.Conclusions: Further in-depth investigation of the material is ongoingand also includes the other joint tissues such as the bone and thesynovial tissue. Irrespective, these first results on cartilage changessuggest that the shift from a catabolic to an anabolic state occurs withinthe weeks after joint distraction. As such, the post-distraction periodseems to be essential in identifying key-players that support intrinsiccartilage repair.Acknowledgements: Financial support was received from: TTWTechnology Foundation: Perspectief P15-23, Dutch Arthritis Society(ReumaNederland) Long term Research Program LLP9 and LLP12Abstracts / Osteoarthritis and Cartilage 28 (2020) S20eS85 S7787MECHANOBIOLOGICAL RESPONSES IN SYNOVIUM: INSIGHTS INTOTHE BENEFITS OF EXERCISE AND THE ROLE OF INFLAMMATION INKNEE OSTEOARTHRITISH.T. Philpott 1, T. Birmingham 1, S. MacDonald 1, B. Lanting 1, B. Fiset 2,L. Walsh 2, T. Appleton 1. 1Western Univ., London, ON, Canada; 2McGillUniv., Montreal, QC, CanadaPurpose: Exercise improves pain and joint function in patients withknee osteoarthritis (OA), including after total knee arthroplasty. How-ever, many patients limit exercise due to pain, reporting increased painwith activity and relieved with rest. The mechanisms leading to theclinical benefits of exercise over time despite pain during activity inknee OA are poorly understood. Joint pain signals emanate from mul-tiple innervated joint tissues, especially synovium. The synovium is acritical tissue for maintenance of joint health and undergoes markedchanges during OA. Knee synovium is stretched during walking (flex-ion-extension) at predictable frequencies, but the mechanobiology ofsynovium during the gait cycle is unknown. Since synovial inflamma-tion (synovitis) is strongly associated with pain symptoms and painsensitization in OA, responses to exercise may depend on the degree ofinflammation in OA joints. Here, we assessed synovial tissue mecha-nobiological responses to high and low frequency mechanical stretchloading and whether these responses are altered by the burden ofinflammation.Methods: Synovial tissue samples were collected during total kneearthroplasty from 6 patients with end-stage knee OA. Each sample wassplit into 3 equally matched pieces and subjected to cyclic mechanical10% stretch for 30minutes at low (20 Hz, tomodel very slowwalking 40steps/min) or high (60 Hz, to model fast, more painful walking 120steps/min) frequency or static (non-stretch control). Inflammationburden was determined as high (n¼3) or low (n¼3) using the semi-quantitative OMERACT grading system for musculoskeletal ultrasoundand synovial tissue histology (sub-intimal leukocyte infiltration). Fol-lowing mechanical stimulation, tissue samples were incubated in freshculture media for 6 hours prior to whole tissue RNA isolation for RNAsequencing. RNA sequences were aligned and sorted by coordinates, tothe NCBI human genome. Quantification of genes was performed infeatureCounts, DESeq2 was used to normalize feature counts and finddifferentially expressed genes, and pathway enrichment was deter-mined using KEGG analysis.Results: The mean age of patients included in the study was 66.5 (þ/-9.65) and mean BMI was 36.1 (þ/- 5.61), with 4 males and 2 females.All patients had end-stage knee OA with Kellgren-Lawrence grade 3-4. Gene lists were compared between stretch conditions (control vslow frequency; control vs high frequency), and then within stretchconditions and between tissues with high versus low levels ofinflammation. 1885 mechanoresponsive genes were significantlydysregulated by low frequency stretch versus 168 mechanores-ponsive genes by high frequency stretch. Common pathways thatwere highly enriched by both low and high frequency stretch weregenes involved in phagocytosis, Toll-like receptor signaling, NF-kappa-B signaling, and TGF-beta signaling. Pathways uniquelyenriched by low frequency stretch include genes involved in osteo-clast differentiation, necroptosis, NOD-like receptor signaling, andthose suggestive of adaptive immunity (Th1 and Th2 differentiation,B cell receptor signaling). Pathways uniquely enriched by high fre-quency stretch include genes involved in innate immunity includingcytokine receptor interaction, chemokine signaling, and natural killercell-mediated cytotoxicity. In secondary analyses, we explored dif-ferences in mechanically-induced gene expression between patientswith high versus low burden of synovial tissue inflammation. Vennanalyses showed that only 1.8% (17) of genes are mechanoresponsivein both low and high inflammation groups and are therefore purelymechanoresponsive, while 98.2% (945) of significantly mechanores-ponsive genes are influenced by the inflammation status of the tissue.Thus, suggesting that inflammation strongly influences geneexpression in response to stretch.Conclusions: This work explores a mechanobiological basis for fur-ther understanding the clinically-relevant effects of exercise on kneejoints with OA, including mechanisms controlling joint homeostasis,immune cell function and interaction, and pain. We provide evidencethat synovium is mechanoresponsive, with differential transcrip-tional network activation in response to mechanical loadingfrequencies that model less-painful (slow) vs painful (fast) walking.Further, we provide contextual understanding of these effects in thepresence of synovial inflammation, which affects a large proportionof knee OA patients and appears to modulate synovial responses tomechanical loading.88LONG-TERM EFFICACY AND SAFETY OF INTRA-ARTICULARSPRIFERMIN IN PATIENTS WITH KNEE OSTEOARTHRITIS: RESULTSFROM THE 5-YEAR FORWARD STUDYF. Eckstein 1,2, M. Hochberg 3, J. Kraines 4, H. Guehring 5, F. Moreau 4,V. Ona 4, A.R. Bihlet 6, I. Byrjalsen 6, J.R. Andersen 6, B. Daelken 5,O. Guenther 5, C. Ladel 5, M. Michaelis 5, P.G. Conaghan 7. 1 ParacelsusMed. Univ., Salzburg, Austria; 2Chondrometrics GmbH, Ainring,Germany; 3Univ. of Maryland, Baltimore, MD, USA; 4 EMD Serono Res.and Dev. Inst., Inc. (an affiliate of Merck KGaA), Billerica, Darmstadt,MA, Germany; 5Merck KGaA, Darmstadt, Germany; 6Nordic BioSci.,Herlev, Denmark; 7Univ. of Leeds, Leeds, United KingdomPurpose: The 5-year Phase II FORWARD study assessed the effi-cacy and safety of the potential disease-modifying osteoarthritisdrug (DMOAD) sprifermin (recombinant human fibroblast growthfactor 18) in patients with symptomatic, radiographic kneeosteoarthritis (OA). The primary endpoint assessed at Year 2showed dose-dependent modification of total femorotibial joint(TFTJ) cartilage thickness with sprifermin vs placebo (PBO) byquantitative magnetic resonance imaging (qMRI), and this wasfound to be sustained at Year 3 follow-up in a subsequentexploratory analysis. Western Ontario and McMaster UniversitiesOsteoarthritis Index (WOMAC) total scores improved by ~50% inall cohorts, including PBO, at Years 2 and 3. Post-hoc exploratoryanalyses identified a patient “subgroup at risk” of further struc-tural and symptomatic progression, with thinner minimum jointspace width (mJSW) and greater WOMAC pain at baseline, whichdisplayed a clinically relevant differentiation in WOMAC painscore changes between sprifermin (100 mg every 6 months[q6mo]) and PBO at follow-up. Here, we report the long-term 5-year efficacy and safety results in the overall population and the“subgroup at risk”.Methods: Patients were randomised 1:1:1:1:1 to receive intra-articularsprifermin 100 or 30 mg q6mo, 100 or 30 mg q12mo, or PBO, for 18 mo.The treatment period-related analysis for the primary endpoint was 2years, with follow ups over 3 years post treatment (at 3 and 5 years).The intent-to-treat (ITT) population included all randomized patients;the modified (m)ITT population all patients with a baseline and 1qMRI reading up to Year 2. Post-hoc exploratory analysis was conductedin the “subgroup at risk” (minimummedial or lateral JSWof 1.5-3.5 mmand WOMAC pain 40-90 [0-100 scale; 100 ¼ worst pain] at baseline).Treatment differences vs PBO were estimated using a repeated meas-ures model controlling for baseline, treatment, time, pooled countryand treatment by time interaction. Confidence intervals (CIs) wereadjusted for multiplicity of treatments using Dunnett adjustment. Lin-ear dose-effect trend tests were performed exploratively at eachtimepoint.Results: 474 (86.3%) patients completed the primary 2-year obser-vation period (including 18 mo of active treatment); 442 (80.5%)and 378 (69%) patients completed the 3 and 5-year extended fol-low-up periods, respectively. The significant dose-response effect ofsprifermin on TFTJ cartilage thickness change seen at Year 2 waspreserved to Year 5 (trend test, p<0.001), and the 0.05 mm greatermean increase in TFTJ cartilage thickness with sprifermin 100 mgq6mo vs PBO seen at Year 2 was sustained to Year 5 (95% CI 0.004,0.095; p¼0.015; Table 1). WOMAC pain scores were improved by~50% from baseline to Year 5 in all cohorts, including PBO (Fig-ure 1). Post-hoc analysis of the “subgroup at risk” (n¼161) identi-fied a potential differentiation in WOMAC pain scores between thesprifermin 100 mg q6mo (highest dose) and PBO groups at Year 2(-5.82; 95% CI -18.87, 7.23), Year 3 (-8.75; CI -22.42, 4.92) and Year5 (-10.08; 95% CI -25.68, 5.53; Figure 2). At Year 5, there was nonotable difference in the incidence of adverse events (AEs), seriousAEs or study discontinuation due to AEs in any sprifermin group vsPBO in the overall population or the “subgroup at risk”. AEs weremostly moderate in severity. A total of 181 patients (33%) reportedserious AEs, but none were deemed related to treatment by theinvestigators. Withdrawals from the study due to AEs were <10%,

Knee joint distraction-induced shift from catabolic to anabolic state occurs after the distraction period

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