Modulation of the mechanical responses of synovial fibroblasts by osteoarthritis-associated inflammatory stressors

Highlights

• SFb from OA patients differ by size and SA-β-gal expression from scope SFb.

• Mechanical stress modifies inflammatory and ECM gene expression of SFb.

• Soluble factors in OA-pre-obese condition media drive SFb inflammatory response.

• Lubricin gene expression is susceptible to both loading and inflammation stressors.

Abstract

Objectives

To compare mechanobiological response of synovial fibroblasts (SFb) from OA patient cohorts under mechanical load and inflammatory stressors for better understanding of SFb homeostatic functions.

Methods

Primary SFb isolated from knee synovium of OA obese (OA-ob:SFb), OA-pre-obese (OA-Pob:SFb), non-OA arthroscopic (scope:SFb), and non-OA arthroscopic with cartilage damage (scope-CD:SFb) were exposed to OA-conditioned media (OACM), derived from OA obese (OA-ob:CM), OA-pre-obese (OA-Pob:CM), and mechanical stretch at either 0 %, 6 % or 10 % for 24 h. Differences in the mRNA levels of genes involved in extracellular matrix production, inflammation and secretory activity were measured.

Results

Despite the significant BMI differences between the OA-ob and OA-Pob groups, OA-Pob has more patients with underlying dyslipidaemia, and low-grade synovitis with higher levels of secreted proteins, CXCL8, COL4A1, CCL4, SPARC and FGF2 in OA-Pob:CM. All primary SFb exhibited anti-proliferative activity with both OA-CM. Mechanical stretch stimulated lubricin production in scope:SFb, higher TGFβ1 and COL1A1 expressions in scope-CD:SFb. OA-Pob:CM stimulated greater detrimental effects than the OA-ob:CM, with higher pro-inflammatory cytokines, IL1β, IL6, COX2 and proteases such as aggrecanases, ADAMTS4 and ADAMTS5, and lower ECM matrix, COL1A1 expressions in all SFb. OA-ob:SFb were unresponsive but expressed higher pro-inflammatory cytokines under OA-Pob:CM treatment.

Conclusion

Both mechanical and inflammatory stressors regulate SFb molecular functions with heterogeneity in responses that are dependent on their pathological tissue of origins. While mechanical stretch promotes a favorable effect with enhanced lubricin production in scope:SFb and TGFβ1 and COL1A1 in scope-CD:SFb, the presence of excessively high OA-associated inflammatory mediators in OA-Pob:CM, predominantly SPARC, CXCL8 and FGF2 drive all SFb regardless of pathology, towards greater pro-inflammatory activities.

Introduction

OA is the most common form of arthritis. Older age, female above 60 years, being overweight or obese, joint trauma, overuse and mechanical overload or abnormal stresses on the joint, can increase the risk of developing OA (Heidari, 2011; Loeser, 2010). The complex pathophysiology of OA involves degeneration and remodelling of the whole joint tissues driven by a combination of cellular changes, inflammatory mediators, matrix degrading enzymes and biomechanical stresses (Loeser et al., 2012; Man and Mologhianu, 2014). The characteristics of endstage-OA include depletion of articular cartilage, thickening of subchondral bone, formation of osteophytes, development of bone marrow lesions, degeneration of ligaments, hypertrophy of joint capsule and synovial inflammation (Loeser et al., 2012).

Chronic inflammation, which is increased by ageing and obesity, underlies OA development through increased oxidative stress, ‘wear-and-tear’ of the joints and abnormal biomechanical loads to the joints (Heidari, 2011). The synovium faces multiple insults, local and systemic inflammatory insults throughout the joint movement stresses themselves often exacerbated by obesity (Smith, 2011). However, research into the synovium or synovial fibroblasts (SFb) within the synovial inflammation apparent during OA progression (Scanzello and Goldring, 2012) is relatively limited.

The knee is a uniaxial hinge synovial joint supporting extension and flexion during the gait cycle. An ideal in vitro replicate of longitudinal direction subjected on muscle tissue or tendon during exercise was reported using the uniaxial cyclic stretch system on muscle cells or tenocytes (Passey et al., 2011). The uniaxial cyclic stretch system, however, partially recapitulates the mechanical stresses experienced by the synovium which has a wider range of movement as it is loosely attached to the inner surface of the articular capsule (Schett et al., 2001; Simkin, 1991). Nevertheless, relative to other muscle tissues, the synovium is continually exposed to a dynamic mechanical stress due to the load of body weight and shear forces of the synovial fluid motion during exercise under both normal and pathological conditions (Schett et al., 2001), which the present study aims to recapitulate using both mechanical loading and inflammatory stressors.

SFb, resident cells in the synovium, play an important role in nourishing chondrocytes, lubricating and maintaining cartilage by contributing to synovial fluid composition as synovial fluid flows during joint movement (Ospelt, 2017). Multiple cellular physiological processes within SFb functions are regulated by mechanical loading including changes in cell orientation, proliferation, cell division synthesis and breakdown of extracellular matrix proteins (Guilak, 2011; Yanagida-Suekawa et al., 2013). Studies in rabbits demonstrated enhanced secretion of hyaluronan by SFb following cyclic stretch (Ingram et al., 2008). Obesity is associated with OA progression via increased joint loading by excess weight and through inflammatory adipocytokines secretion (Richter et al., 2015). Mechanical stress also attenuates the production of inflammatory mediators of prostaglandin E₂ (PGE₂) by SFb (Sambajon et al., 2003). However, how mechanical stress, obesity and inflammatory mediators are integrated or how these factors affect SFb function remains unclear.

This study evaluated the responses of OA-SFb to mechanical loading, inflammatory stressors and combinations of both. Changes in cell morphology, expression of selected genes linked to functions such as production of extracellular matrix and inflammatory mediators have been analysed. Functional and molecular responses of SFb derived from endstage-OA were compared with SFb responses from non-inflammatory knee pathologies without osteoarthritis. Our findings suggest that metabolic and inflammatory insults impact the morphology and molecular functions of synovial fibroblasts, including effects induced by mechanical stretch. In particular, the OA-Pob:CM-associated inflammatory mediators promote cartilage destructive pathways.