A BIOPSYCHOSOCIAL APPROACH TO PHENOTYPE KNEE OSTEOARTHRITIS PATIENTS AWAITING TOTAL KNEE ARTHROPLASTY: A CROSS-SECTIONAL STUDY

Abstract

Background: Osteoarthritis (OA) is accompanied by an excessive formation of underlying bone caused by homeostatic alterations. It has been described that in OA joints there is an up-regulation of the WNT/β-catenine pathway driving to the differentiation of Mesenchymal Stromal Cells (MSCs) into osteocytes [1]. Glyp-icans 1-6 act as co-receptors of WNT pathway, improving interactions between molecules [2]. On the other hand, NOTUM plays a role as a negative regulator of the pathway preventing WNT binding to its receptors and favouring the soluble form of Glypicans [3]. All these evidences lead us to think about the importance of Glypicans 1-6 and NOTUM in the osteogenic differentiation of MSCs in OA disease. Objectives: To determine the differences at gene and protein levels of Glypicans 1-6 and NOTUM in MSCs between OA patients and controls at baseline and during induced in vitro osteogenic differentiation. Methods: Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) from both, 8 OA patients and 8 healthy donors were isolated from BM samples. BM samples were obtained by joint replacement and traumatic fractures. BM-MSCs were cultured during 21 days with normal culture medium and with osteogenic inducing medium. Cells and supernatant were recovered at 1, 7, 14 and 21 days. Protein levels were determined by ELISA in cells supernatant. Extraction and purification of RNA from cells and cDNA synthesis were performed. Glypicans 1-6 and NOTUM gene-expression was analysed by qPCR and normalized with housekeeping genes β-actin and RNA18S. The data were calculated with the method of fold change (2-ΔΔCt). Statical analysis were performed with GraphPad Prism 8.0. For the detection of outliers, the ROUT method (Q=1%) was used. Data were analyzed using t-test. Results: The mRNA levels of cells with normal culture medium were up-regulated in GPC6 (t=7d; p=0,016) in OA patients. During osteogenic differentiation, was observed a statistically significant up-regulation in levels of OA patients vs controls of GPC2 (t=14d; p=0,034), GPC4 (t=14d, p=0,025; t=21d, p=0,017), GPC5 (t=14d, p=0,007), GPC6 (t=14d, p=0,013) and NOTUM (t=14d, p=0,009) and down-regulation of GPC3 (t=7d, p=0,034). In addition, protein levels of NOTUM were lower compared to the control group in OA BM-MSCs (t=1d, p=0,049) and in the differentiation to osteocytes (t=1d, p=0,049; t=7d, p=0,043; t=14d, p=0,016). GPC3 levels were higher OA patients in all the times and in the two conditions (p < 0,0001). In the differentiation, GPC2 were lower (t=7d, p< 0,0001). GPC1, GPC4 and GPC6 were not present in any case. Conclusion: Our results evidence a dysregulation in the glypicans in BM-MSCs of OA patients and, specially, during osteogenic differentiation. NOTUM, extracel-lular negative regulator of the WNT/β-catenine signaling pathway, is decreased at protein level in OA patients, despite the fact that gene expression is elevated. Thus, our data confirm the differences in expression in NOTUM and Glypicans between OA patients and healthy controls. Further studies are needed proposing these molecules, specially NOTUM, as an effective treatment of the disease. Background: Knee osteoarthritis (KOA) is a heterogenous disease, meaning individuals can present with various signs and symptoms related to different biopsychosocial (BPS) factors [1,2]. Several phenotypes can as such be expected. Phenotyping KOA patients, specifically those awaiting total knee arthroplasty (TKA), could be relevant, because a substantial part of patients (20%) reports chronic post-TKA pain [3]. Various preoperative phenotypic factors related to domains of the BPS model have been described, but consensus and a classification of patients based on these factors is still lacking [4-7]. Objectives: The aim of this exploratory study is to identify phenotypes based on BPS related factors in KOA patients awaiting TKA. Methods: Participants were included if they were diagnosed with KOA and waiting for TKA surgery in one of the four participating hospitals in Belgium and the Netherlands. A cross-sectional latent profile analysis was conducted in MPlus [8] containing the grade of KOA before TKA surgery (structural variable); body mass index and glycated hemoglobin value (metabolic variables); isometric strength of m. Quadriceps and m. Hamstrings of the affected leg, proprioceptive accuracy of the affected leg, and physical function (functional variables); pain intensity scores and symptoms related to altered somatosensory processing (pain-related variables); pain catastrophizing, depression, anxiety symptoms, expectations and satisfaction (psychological variables); and work and education level (social variables). Data were checked for multicollinearity and multivariate outliers. The ideal model was chosen based on qualitative evaluation, goodness of fit and classification uncertainty. Results: 224 participants were included of which 109 women (65.19 +/-8.18 years old) and 108 men (66.92 +/-7.18 years old). A model with 2 phenotypes was found to be most appropriate. Both phenotypes differed in 13 out of 19 continuous variables. Phenotype 1 (72% of all participants) was characterized by scoring better on at least one of all metabolic, functional, psychological and pain-related continuous variables compared to phenotype 2 (28% of all participants), except for glycated hemoglobin value, proprioception, expectations, and widespread temporal summation and conditioned pain modulation (part of soma-tosensory processing variables). Concerning categorical variables, phenotype 1 (72%) was characterized by having a lower probability to have a Kellgren & Law-rence (K&L) scale 2 compared with patients in phenotype 2 (28%). The probabilities of the other categorical variables did not differ between the two phenotypes. Conclusion: A model with 2 phenotypes in KOA patients awaiting TKA appeared the most appropriate, which confirmed the existence of a group that experiences disturbed somatosensory processing signs in combination with worse results on psychological (pain catastrophizing, fear and depression), functional (strength and physical function), structural (higher possibility to have a K&L grade 2 compared to the other phenotype) and metabolic factors (body mass index), and a group that does not present this disruption in combination with better results on the aforementioned BPS factors. Further research is necessary and should also investigate if different phenotypes react differently regarding treatment outcome after TKA. REFERENCES: [1] Dell'Isola, A. et al. PLoS One 13, e0191045 (2018) [2] Bierma-Zeinstra, S. M. A. et al. Arthritis Res. Ther. 13, 213 (2011) [3] Sayah, S. M. et al. J. Arthroplasty 36, 3993-4002.e37 (2021) [4] Baert, I. A. et al. Osteoarthritis Cartilage 24, 213-23 (2016) [5] Wylde, V. et al.