Addition of mannitol to hyaluronic acid enhances the inhibition of matrix metalloprotease expression in IL-1β stimulated human C-20/A4 chondrocytes

Osteoarthritis (OA) of the knee and hip is ranked as the 11th leading raison of disability and its prevalence keeps going up over the last past decades...


Introduction
Osteoarthritis (OA) of the knee and hip is ranked as the 11 th leading raison of disability and its prevalence keeps going up over the last past decades [1]. In the United States, over 9 million people suffer from symptomatic knee OA [2]. One third of adults over the age of 60 have radiographic evidence of this condition and 12.1% experience pain and/or disability [3]. Despite the high prevalence of knee OA and the high level of disability related to the disease progression, only few patients undergo joint replacement with a mean time from symptoms onset to Total Knee Replacement (TKR) of about 19 years [3,4]. Such a long evolution explains the huge need of new non-surgical therapies, which can alleviate pain and improve the quality of life with a better effectiveness than the current therapeutic modalities. The therapeutic approach of knee OA is based on the combination of non-pharmacological and pharmacological modalities, widely described in multiple guidelines [5][6][7]. Among the pharmacological modalities, Intra-Articular (IA) injections of hyaluronic acid (HA), also named Viscosupplementation (VS), have been ranked as the most effective treatment for alleviating knee OA pain [8], despite some controversies remain regarding the interpretation of the meta-analyses [9][10][11][12]. Hyaluronic acid is a non sulfated High Molecular Weight (HMW) glycosaminoglycan naturally present in ECM and synovial fluid that plays a major role in cartilage lubrication [13,14]. The HA lubricating properties explain that HA injections for treatment of OA are classified by the majority of health agencies worldwide, as a medical device but not as a drug. However, it has been now well evidenced that IA HA has also multiple pharmacological effects on molecular signaling pathways and contributes to the joint homeostasis. The multiple mechanisms by which HA may act as an anti-inflammatory and a structure-modifying drug for OA have been developed in a recent review [15]. and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) [16] synthesis and activity. Furthermore IL-1β stimulates Nitric Oxide (NO) synthesis and oxygen free radicals production that increases the damaging effects on cartilage [17]. The ECM degradation and particularly that of its two major components, aggrecan and type 2 collagen, is mainly due to MMP-1,

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Oxygen Species (ROS) degrade HA macromolecules, leading to increased levels of HA fragments of low molecular weight with pro-inflammatory properties that worsen again the inflammatory cascade [15]. The important role of oxidative stress resulting from chronic overproduction of ROS is often underestimated. Oxygen free radicals damage DNA, increase cell apoptosis, contribute to degradation of the ECM and inhibit proteoglycans synthesis [18].
The NADPH oxidase enzymes constitute a large enzyme family which function is dedicated to the production of reactive oxygen species (ROS). One of our recent work showed that the NADPPH oxidase 4 (Nox4) is the major source of ROS production in human chondrocytes and in the C-20/A4 human chondrocyte cell line [19,20] in which Nox4-derived ROS regulate collagenase 1 (MMP-1) expression and chondrocyte death consecutive to IL-1β stimulation.
Interestingly a number of hydroxyl rich polysaccharides, such as HA, have also antioxidant properties and are themselves inhibitors of oxygen free radical −O2* , underlining the importance of HA concentration [21]. However HA is also very sensitive to oxidative stress [22]. HA macromolecules, containing many OH groups, react with ROS, resulting in the rupture of the macromolecular chains and accelerated degradation of the highly viscous solution. The rapid depolymerisation of HA is a major reason for the short intraarticular half-life of viscosupplements made of non-cross-linked HA, cross-linking being another way to protect HA from degradation by ROS [21]. Several studies have shown that the addition of a polyol with ROS scavenging properties, such as mannitol or sorbitol, protect HA from ROS degradation [21][22][23][24]. Safety and efficacy of a mannitol-modified HA viscosupplement, HANOX-M, has been assessed in a large scale multicenter, prospective doubleblind controlled trial [25] in patients with knee OA. The study demonstrated that HANOX-M was as safe and effective than a high molecular weight HA viscosupplément not containing mannitol, but with a more rapid onset of action than that of the latter [26]. Due to the antioxidant effect of mannitol we hypothesized that, besides its better resistance to degradation, the mannitol-modified HA might have a more pronounced effect than HA alone on inflammation and MMPs related ECM degradation. The aim of the study was to compare in vitro the effect of the addition of mannitol to HA versus HA alone on the level of ROS production and the NOX-4 and MMPs expression upon IL-1β stimulation.

Statistical Analysis
Data are presented as means +/-SD, significance levels were assessed using Student's paired t test. A p-value of 0.05 or less between groups was considered to indicate a statistically significant difference.
All quantitative data were generated using biological replicates in triplicate unless stated otherwise and are expressed as the mean plus 95% confidence interval. Data normality was tested by a Shapiro-Wilk test. For each experiment, p-values were determined using a paired Student test. All analyses were conducted using StatView (SAS institute, US).

Discussion
It has become increasingly apparent that HA has the capacity not only to alleviate pain but also to protect and restore the chondral matrix via multiple molecular pathways [15].
Therefore it is legitimate to consider HA not only as a pain-killer but as a background therapy for OA with structure-modifying OA, thanks to its potential structure-modifying effect.