In vitro response of osteoarthritic chondrocytes and fibroblast-like synoviocytes to a 500–730 kDa hyaluronan amide derivative Paola Brun, 1 Barbara Zavan, 1 Vincenzo Vindigni, 2 Antonella Schiavinato, 3 Assunta Pozzuoli, 4 Claudio Iacobellis, 4 Giovanni Abatangelo 1 1 Department of Biomedical Sciences, University of Padova, Italy 2 Clinic of Plastic Surgery, University of Padova, Italy 3 Fidia Farmaceutici, Quality and Development, Abano Terme, Italy 4 Dipartimento di Scienze Chirurgiche, Oncologiche e Gastroenterologiche, University of Padova, Italy Received 14 September 2011; revised 7 June 2012; accepted 12 June 2012 Published online 1 August 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32771 Abstract: The aim of this study was to compare the effects of native hyaluronan (HA) with that of its hexadecylamide deriv- ative (HYADD) on proliferation of fibroblast-like synoviocytes (FLS) and chondrocytes. The production of inflammatory and anti-inflammatory cytokines was also analyzed in FLS cul- tures. The proliferation of osteoarthritis (OA) chondrocytes was enhanced when cells were treated with 0.5–1.5 mg mL 1 of HA or HYADD V R 4-G. This effect was completely suppressed by the anti-CD44 antibody. At 0.5 to 1 mg mL 1 , HA and HYADD V R 4-G did not influence the proliferation of normal or pathological FLS; however, at the higher concentration (1.5 mg mL 1 ), HYADD V R 4-G did significantly inhibit cell prolifera- tion. As to effects on inflammation, a significant increase in the expression of the IL-10 gene was observed when FLS were pretreated with tumor necrosis factor alpha and then cul- tured in the presence of 0.5 mg mL 1 HYADD V R 4-G or HA. The effects of HA derivatives on FLS proliferation and production of anti-inflammatory cytokines indicate that they may be of therapeutic benefit in OA. The longer residence time in the joint cavity, the increased viscoelasticity, and the anti-inflam- matory potential of HYADD V R 4-G make it a better candidate than native HA for OA therapy. V C 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 100B: 2073–2081, 2012. Key Words: hyaluronan, fibroblast-like synoviocytes, chon- drocytes, CD44, IL-10 How to cite this article: Brun P, Zavan B, Vindigni V, Schiavinato A, Pozzuoli A, Iacobellis C, Abatangelo G. 2012. In vitro response of osteoarthritic chondrocytes and fibroblast-like synoviocytes to a 500–730 kDa hyaluronan amide derivative. J Biomed Mater Res Part B 2012:100B:2073–2081. INTRODUCTION The mechanical and biological functions of articular joints are regulated by synovial fluid and a critical component of this fluid is hyaluronan (HA), a molecule produced by fibro- blast-like synoviocytes (FLS) in the joint cavity. 1–3 In rheuma- toid arthritis (RA) and osteoarthritis (OA), the most common chronic joint disorders worldwide, synoviocytes proliferate at an accelerated rate, giving rise to synovial membrane hyper- plasia and progressive cartilage disruption. 4–6 The pathogene- sis of these disabling diseases is not fully understood, but many authors correlate this hyperplasia to pathological changes in the joint environment, such as increased proin- flammatory cytokines (IL-1, IL 2, IL 6, IL 7, and IL 8), 7–9 decreased concentration and molecular weight (MW) of HA in synovial fluid 10–12 and defective apoptosis. 13,14 Recent studies have shown that chondrocyte apoptosis and cartilage degradation might be prevented by neutralizing inflamma- tion, 15,16 and in particular, anti-inflammatory and chondro- protective properties of interleukin-10 in the synovial fluid of patients with knee OA have been described. 17 The beneficial effects of intra-articular HA therapy on both reducing pain and promoting function demonstrate the key role of hyaluronic acid in OA. The underlying mecha- nisms may be due to its chondroprotective and anti-inflam- matory effects. The efficacy of articular HA 500–730 kDa therapy in OA has been demonstrated in animal models of experimental OA 18,19 and in clinical trials. 20,21 It is possible that receptor-mediated effects of HA and derivatives lead to changes in chondrocyte, 22–25 synoviocyte, 11,26 and subchon- dral bone osteoblast 27 metabolism. These effects are de- pendent on both the MW and concentration of the molecule. In particular, low- to mid-MW preparations seem to affect chondrocyte proliferation more than high MW molecules. 28– 30 HA at 500–730 kDa was shown to induce proliferation of normal chondrocytes in culture at concentrations ranging from 0.25 to 0.75 mg mL 1 . 31 This effect was mediated by CD44, the principle HA receptor that has a crucial role in many physiological and pathological processes. HA is also used to restore natural viscoelasticity, resulting in improved joint functionality and decreased symptoms. 13–32 As the Correspondence to: P. Brun; e-mail: paola.brun@unipd.it Contract grant sponsor: Italian Institute of Health; contract grant number: MIUR 2009-2010 V C 2012 WILEY PERIODICALS, INC. 2073