Low-Input RNA-Sequencing in Patients with Cartilage Lesions, Osteoarthritis, and Healthy Cartilage

Objective To analyze and compare cartilage samples from 3 groups of patients utilizing low-input RNA-sequencing. Design Cartilage biopsies were collected from patients in 3 groups ( n = 48): Cartilage lesion (CL) patients had at least ICRS grade 2, osteoarthritis (OA) samples were taken from patients undergoing knee replacement, and healthy cartilage (HC) was taken from ACL-reconstruction patients without CLs. RNA was isolated using an optimized protocol. RNA samples were assessed for quality and sequenced with a low-input SmartSeq2 protocol. Results RNA isolation yielded 48 samples with sufficient quality for sequencing. After quality control, 13 samples in the OA group, 9 in the HC group, and 9 in the CL group were included in the analysis. There was a high degree of co-clustering between the HC and CL groups with only 6 genes significantly up- or downregulated. OA and the combined HC/CL group clustered significantly separate from each other, yielding 659 significantly upregulated and 1,369 downregulated genes. GO-term analysis revealed that genes matched to cartilage and connective tissue development terms. Conclusion The gene expression profiles from the 3 groups suggest that there are no major differences in gene expression between cartilage from knees with a cartilage injury and knees without an apparent cartilage injury. OA cartilage, as expected, showed markedly different gene expression from the other 2 groups. The gene expression profiles resulting from this low-input RNA-sequencing study offer opportunities to discover new pathways not previously recognized that may be explored in future studies.

Concomitant full-thickness cartilage lesions do not affect patient-reported outcomes at minimum 10-year follow-up after ACL reconstruction

Purpose To compare patients with a concomitant full-thickness cartilage lesion and anterior cruciate ligament (ACL) injury to patients with an isolated ACL injury at 10–15 years post ACL reconstruction. Methods This is a longitudinal follow-up of a cohort of 89 patients that were identified in the Norwegian National Knee Ligament Registry and included in the index study in 2007. The study group consisted of 30 patients that underwent ACL reconstruction and had a concomitant, isolated full-thickness cartilage lesion (International Cartilage Repair Society [ICRS] grade 3–4). Each study patient was matched with two control patients who underwent ACL reconstruction but had no cartilage lesions (ICRS grade 1–4) (n = 59). At a median follow-up of 10.2 years (range 9.9–15.6), 65 patients (74%) completed the Knee Injury and Osteoarthritis Outcome Score (KOOS), which was the main outcome measure, resulting in 23 pairs after matching. Results At a follow-up of 10–15 years after ACL reconstruction, no significant differences in KOOS were found between patients with a concomitant full-thickness cartilage lesion and patients without cartilage lesions. There was also no significant difference between the two groups when comparing the change over time in KOOS scores from preoperative to follow-up. Both groups showed significant improvement in all KOOS subscales from preoperative to follow-up, except for in the Symptoms subscale for the control group. The greatest improvement was in the QoL subscale for the study group. Conclusion ACL-reconstructed patients with a full-thickness cartilage lesion did not report worse outcomes at 10–15 years after surgery compared with patients with an isolated ACL injury. Our findings support that there is no long-term negative effect of a concomitant cartilage lesion in an ACL-reconstructed knee. These findings should be considered when discussing treatment and informing about the expected long-term outcome after ACL reconstruction to patients with such combined injuries. Level of evidence II.

Factors affecting Baker cyst volume, with emphasis on cartilage lesion degree and effusion in the young and middle-aged population

Background The principal aim of this study was to investigate the presence of factors affecting Baker’s cyst volume in young and middle-aged populations. Methods Open cyst excision with valve and capsule repair, as well as knee arthroscopy, were used to treat eighty-five patients. The cases were categorized in terms of age, effusion, chondral lesion degree, meniscal tear degree, and Lindgren scores. An ultrasonography (USG) device was used to calculate the cyst volume. The IBM-SPSS 22 program was used for statistical analysis and to assess the relationships between variables using Spearman’s correlation tests. Results The degree of chondral lesion was moderately and positively correlated with cyst volume in the total population (correlation coefficient: 0.469; p < 0.05). The degree of the chondral lesion was moderately and positively correlated with the degree of effusion (correlation coefficient: 0.492; p < 0.005). The cyst volume was weakly and positively correlated with the degree of effusion (correlation coefficient: 0.20; the correlation was at the limits of statistical significance p = 0.07 < 0.08). Conclusions This study revealed that an increase in chondral lesion severity increases the amount of effusion and cyst volume.

Etiology of Cartilage Lesions Does Not Affect Clinical Outcomes of Patellofemoral Autologous Chondrocyte Implantation

Objective To determine the relationship between cartilage lesion etiology and clinical outcomes after second-generation autologous chondrocyte implantation (ACI) in the patellofemoral joint (PFJ) with a minimum of 2 years’ follow-up. Methods A retrospective review of all patients that underwent ACI in the PFJ by a single surgeon was performed. Seventy-two patients with a mean follow-up of 4.2 ± 2.0 years were enrolled in this study and were stratified into 3 groups based on the etiology of PFJ cartilage lesions: patellar dislocation (group 1; n = 23); nontraumatic lesions, including chondromalacia, osteochondritis dissecans, and degenerative defects (group 2; n = 28); and other posttraumatic lesions besides patellar dislocations (group 3; n = 21). Patient’s mean age was 29.6 ± 8.7 years. Patients in group 1 were significantly younger (25.4 ± 7.9 years) than group 2 (31.7 ± 9.6 years; P = 0.025) and group 3 (31.5 ± 6.6 years; P = 0.05). Body mass index averaged 26.2 ± 4.3 kg/m2, with a significant difference between group 1 (24.4 ± 3.2 kg/m2) and group 3 (28.7 ± 4.5 kg/m2; P = 0.005). A clinical comparison was established between groups based on patient-reported outcome measures (PROMs) and failure rates. Results Neither pre- nor postoperative PROMs differed between groups ( P > 0.05). No difference was seen in survivorship between groups (95.7% vs. 82.2% vs. 90.5%, P > 0.05). Conclusion Cartilage lesion etiology did not influence clinical outcome in this retrospective study after second generation ACI in the PFJ. Level of Evidence Level III, retrospective comparative study.

POS0277 ANABOLIC EFFECT OF LNA043, A NOVEL DISEASE-MODIFYING OSTEOARTHRITIS DRUG CANDIDATE: RESULTS FROM AN IMAGING-BASED PROOF-OF-CONCEPT TRIAL IN PATIENTS WITH FOCAL ARTICULAR CARTILAGE LESIONS

Background:LNA043 is a modified, recombinant version of the human angiopoietin-like 3 (ANGPTL3) protein acting directly on cartilage-resident cells to transmit its cartilage anabolic effect. A first-in-human study previously demonstrated the favourable safety profile and the modulation of several pathways involved in cartilage homeostasis and osteoarthritis (OA)1. A previous proof-of-mechanism imaging study used high field (7 Tesla) magnetic resonance imaging (MRI) to show formation of hyaline-like tissue after a single injection of 20 mg LNA043 (unpublished data).Objectives:To evaluate non-invasively the chondro-regenerative capacity of multiple intra-articular (i.a.) injections of LNA043 in patients with articular cartilage lesions in the knee (NCT03275064).Methods:This was a randomised, double-blind, placebo (PBO)-controlled, proof-of-concept study in patients with a partial thickness cartilage lesion. In total, 58 patients (43 [20 mg LNA043]; 15 [PBO]), stratified by lesion type (condylar or patellar) were treated with 4 weekly i.a. injections. The primary endpoint was T2 relaxation time measurement as a marker of collagen fiber network, and cartilage lesion-volume was a secondary endpoint, both using 3-Tesla MRI. Assessments were performed at baseline, weeks (wks) 8, 16, 28 and 52 (the latter in 23/58 patients). While lesion-volume for the secondary endpoint was determined from manually segmented images, the cartilage volume of 21 sub-regions spanning the entire knee was also measured from 3D isotropic MR images employing an automated segmentation prototype software (MR Chondral Health 2.1 [MRCH], Siemens Healthcare)2. An exploratory analysis evaluated the treatment effect for the additive volume of the 3 subregions in the weight-bearing area of the medial femur.Results:No change in T2 relaxation time was detected between treatment and PBO groups. Manual segmentation showed continuous filling of the cartilage lesions up to wk 28 in LNA043-treated patients with femoral lesions (p=0.08, vs PBO) while no effect was detected for patients with patellar lesions. Given the limitations of measuring small, irregularly shaped lesions with manual image-analysis, the MRCH approach was used (Figure 1). In the medial femoral weight-bearing region, refilling was detected over time (Δ=123 mm3 at wk 28, N= 37, p= 0.05). No overgrowth was detected in the lateral femoral condyles without cartilage damage. The overall safety profile was favourable; only mild/moderate local reactions were reported, including a higher incidence of joint swelling (9.3% vs 0%) and arthralgia (11.6% vs 6.7%) for LNA043 vs PBO resolving spontaneously or with paracetamol/NSAIDs. No anti-drug antibodies were detected.Conclusion:Treatment with 4 weekly i.a. injections of 20 mg LNA043 resulted in regeneration of damaged cartilage in patients with femoral articular cartilage lesions. Automated measurement of cartilage volume in the femoral index region was able to detect a relevant treatment effect and was found to be more sensitive than the manual segmentation method. No sign of cartilage overgrowth was observed in healthy femoral regions. A Phase 2b study in patients with mild to moderate knee OA is in preparation.References:[1]Scotti et al. ACR Convergence 2020; Abstract #1483[2]Juras et al. Cartilage 2020; Sep 29:1-12Disclosure of Interests:Siegfried Trattnig: None declared, Celeste Scotti Shareholder of: Novartis, Employee of: Novartis, Didier Laurent Shareholder of: Novartis, Employee of: Novartis, Vladimir Juras: None declared, Scott Hacker Grant/research support from: Novartis, Brian Cole: None declared, Libor Pasa: None declared, Roman Lehovec: None declared, Pavol Szomolanyi: None declared, Esther Raithel Employee of: Siemens Healthcare GmbH, Franziska Saxer Shareholder of: Novartis, Employee of: Novartis, Jens Praestgaard Shareholder of: Novartis, Employee of: Novartis, Fabiola La Gamba Shareholder of: Novartis, Employee of: Novartis, José L. Jiménez Employee of: Novartis, David Sanchez Ramos Shareholder of: Novartis, Employee of: Novartis, Ronenn Roubenoff Shareholder of: Novartis, Employee of: Novartis, Matthias Schieker Shareholder of: Novartis, Employee of: Novartis

AB0788 MARKER OF CARTILAGE TISSUE LESION IMMUNOPATHOGENESIS OF RHEUMATOID ARTHRITIS

Background:Collagen type 2 is the basic protein of cartilaginous tissue composing over 80% of its mass. Finding excessive antibodies to collagen type 2 and immune complexes is a diagnostic and prognostic criterion of immune lesion of articular cartilage.Objectives:To study production of antibodies to collagen type 2 in patients with rheumatoid arthritis using immobilized magnetically controlled forms.Methods:The antigen was represented by commercial formulation of collagen type 2 produced by Serva (Sweden). Antibodies to collagen type 2 were determined in the patients’ blood serum by way of a technique of immunoenzyme assay (ELISA test) using immobilized magnetic sorbents. Magnetic sorbents were polyacrylamide granules from 10 to 100 micron in size containing magnetic material and collagen type 2. We studied 30 apparently healthy donor individuals and 92 patients with a confirmed diagnosis of rheumatoid arthritis.Results:A study of sera from rheumatoid arthritis patients revealed antibodies to collagen type 2 in 63 patients (68.48%). The correlation coefficient between anti-collagen type 2 antibodies, and IgA / IgM amounted to 0.28 and 0.36, correspondingly. At stage 1 and 2 of disease activity the level of antibodies was higher than in donors (р<0.001). The highest level of antibodies to collagen type 2 was seen in patients with stage 3 of disease activity (0.55±0.02). The amount of antibodies to collagen type 2 in patients with visceral manifestations of rheumatoid arthritis was no different from that in patients without any additional organ involvement, which is hardly surprising since collagen type 2 is mostly localized in articular cartilage and is practically absent from the connective tissue of other organs and systems.Conclusion:Thus the presence of antibodies to collagen type 2 correlates with the disease activity and is an important marker of articular cartilage lesion in patients with rheumatoid arthritis.Disclosure of Interests:None declared

The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability

Osteoarthritis is the most prevalent systemic musculoskeletal disorder characterised by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here we sought to examine the contribution of accelerated growth to osteoarthritis development using a murine model of excessive longitudinal growth. Suppressor of cytokine signalling 2 (SOCS2) is a negative regulator of growth hormone (GH) signalling, thus mice deficient in SOCS2 (Socs2-/-) display accelerated bone growth. We examined vulnerability of Socs2-/- mice to osteoarthritis following surgical induction of disease (destabilisation of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. We observed significant increase in number (WT DMM: 532± 56; WT sham: 495± 45; KO DMM: 169± 49; KO sham: 187± 56; P<0.01) and density (WT DMM: 2.2± 0.9; WT sham: 1.2± 0.5; KO DMM: 13.0± 0.5; KO sham: 14.4± 0.7) of growth plate bridges in Socs2-/- in comparison to wild-type (WT). Histological examination of WT and Socs2-/- knees revealed articular cartilage damage with DMM in comparison to sham (WT DMM: 3.4± 0.4; WT sham: 0.3± 0.05 (P<0.05); KO DMM: 3.2± 0.8; KO sham: 0.8± 0.3). Articular cartilage lesion severity scores (mean and maximum) were similar in WT and Socs2-/- mice with either DMM, or with ageing. Micro-CT analysis revealed significant decreases in SCB thickness, epiphyseal trabecular number and thickness in the medial compartment of Socs2-/-, in comparison to WT (P<0.001). DMM had no effect on the SCB thickness in comparison to sham in either genotype. Together these data suggest that enhanced GH signalling through SOCS2 deletion accelerates growth plate fusion, however this has no effect on osteoarthritis vulnerability in this model.

MiR-30b-5p Influences Chronic Exercise Arthritic Injury by Targeting Hoxa1

We identified the role of miR-30b-5p in chronic exercise arthritic injury. Rats with chronic exercise arthritic injury received treatment with miR-30b-5p antagomiR. H&E and Safranin O-fast green staining were performed. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected. The binding relationship between homeobox A1 (Hoxa1) and miR-30b-5p was revealed. After manipulating the expressions of miR-30b-5p and/or Hoxa1 in chondrocytes, the viability, apoptosis and migration of chondrocytes were assessed. The levels of molecules were determined by qRT-PCR or Western blot. MiR-30b-5p antagomiR ameliorated articular cartilage lesion and destruction, reduced Mankin’s score and the levels of TNF-α, IL-1β, miR-30b-5p, matrix metallopeptidase 13 (MMP-13), and cleaved caspase-3, and increased relative thickness and the levels of Hoxa1, Aggrecan and type II collagen (COLII) in model rats. MiR-30b-5p up-regulation decreased Hoxa1 level, viability, migration and induced apoptosis, whereas miR-30b-5p down-regulation produced the opposite effects. MiR-30b-5p up-regulation increased the levels of MMP-13 and cleaved caspase-3, but decreased those of Aggrecan and COLII in chondrocytes. However, the action of miR-30b-5p up-regulation on chondrocytes was reversed by Hoxa1 overexpression. In conclusion, miR-30b-5p is involved in cartilage degradation in rats with chronic exercise arthritic injury and regulates chondrocyte apoptosis and migration by targeting Hoxa1.