**1. Introduction**

Osteoarthritis (OA) is a prevalent degenerative arthritic disease, a chronic condition that causes pain and disability among elderly patients [1,2]. An estimated 10% to 15% of all adults aged over 60 have some degree of OA [3], with prevalence doubling since the mid-20th century [4]. OA has been defined as a "whole joint" and multifactorial disease, characterized by synovial inflammation, progressive loss of articular cartilage and remodeling of the underlying bone [5,6].

Although OA physiopathology is still not completely understood, chronic inflammation is known to play a critical role in disease development and progression, with accumulating evidence supporting the association between OA pathology and di fferent markers of inflammation [7]. OA cartilage and synovium overexpress cytokines and pro-inflammatory mediators that stimulate the accumulation of proteolytic enzymes, aggrecanases and matrix metalloproteinases (MMPs) responsible for the extracellular matrix (ECM) degradation, and for mediating detrimental e ffects through innate immunity signals [8,9]. In particular, MMP3 is known to mediate the integrity of various constituents of the ECM, such as collagens (types II, III, IV, V, VII, IX, X), fibronectin, elastin, proteoglycans, directly or through the activation of other pro-MMPs and pro-TNF α, in OA [10]. This molecular condition together with chondrocyte di fferentiation into a hypertrophic phenotype, result in loss of the ability to restore the ECM with consequent cartilage degradation. Basic calcium phosphate (BCP) deposition in the cartilage and synovial membrane is closely associated with OA inflammation, and contributes to local tissue damage and failed tissue repair, further intensifying hyaline articular cartilage loss and progressive joint deterioration [11–13].

Current osteoarthritis prevention and treatment are still very limited and unsatisfactory, with therapeutics focused mainly in drugs which improve pain or symptoms, such as topic and oral nonsteroidal anti-inflammatory drugs, acetaminophen, and opioids [14]. Although there are some advances in the design of new molecules to target cartilage repair and bone, or to treat inflammation and pain, at present, no e ffective OA drugs have ye<sup>t</sup> been approved [15], making the search for new potential molecules a priority to overcome the growing burden of OA.

In addition, the lack of reliable models able to simulate the physiologic OA scenario, contributes to slow down the discovery of novel preventive or therapeutic agents. Monolayer (2D) cell culture approaches are limited by the lack of a physiologic context, while three-dimensional human tissue systems, although taking into consideration cell–cell and cell–extracellular matrix interactions, are still not demonstrative of the OA heterogeneity [16,17]. Preclinical animal models represent a more complex system, but are not totally representative of the human physiopathology, frequently leading to the failure of therapeutic responses in a later stage of the drug validation process.

In the search of e ffective drugs that might prevent or slow down the development of the disease, natural products derived from plants and marine organisms, remain a source of new molecular entities for the treatment of chronic inflammatory related diseases, including osteoarthritis [18,19]. Dietary supplements, of natural and synthetic origin, representing a nutritional and health benefit, were already associated with OA in human clinical trials. Although most were associated with OA pain relief, some were shown to modify the inflammatory OA process, by balancing anabolic and catabolic joint events, and promoting the synthesis of structural articulation precursors [20–22].

Among natural products, those containing phenolic rings, such as the flavonoids and some meroterpenoids, are usually provided of interesting biological activities, and have been shown to modulate cytokines such as tumor necrosis factorα (TNF α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), with a crucial role in chronic inflammatory and autoimmune diseases [23]. Some terpenoids based drugs are already available in the pharmaceutical market such as artemisinin and paclitaxel (Taxol ®), acting as antimalarial and anticancer drugs, respectively [24].

In recent years, a series of meroterpenoids isolated from the brown alga *Cystoseira usneoides* have been shown to exhibit anti-inflammatory and antioxidant activities, by reducing the secretion of pro-inflammatory cytokines and downregulating the expressions of COX-2 and iNOS enzymes in THP-1 activated macrophages [25–28]. Among those, amentadione (YP) (Figure S1) showed

radical-scavenging activity and demonstrated a significant role in reducing the production of TNF α in LPS-stimulated THP-1 human macrophages [26]. These results led us to further investigate the anti-inflammatory action of this pure marine compound and its potential as a novel cartilage protective agen<sup>t</sup> in an OA context. For this purpose, we designed an OA preclinical pipeline consisting of an in vitro 2D-cell based system followed by an ex vivo explant-based and co-culture OA models. Our aim is to evaluate the potential protective e ffect of YP in the interplay between mineralization and inflammatory processes involved in OA development and progression.
