Hyaluronic Acid (HA) is responsible for the viscoelastic properties of synovial fluid, which is greatly diminished in osteoarthritic joints in both concentration and molecular weight [1]. The main objective of intra-articular treatment with HA is to restore the viscoelastic properties of the synovial fluid. In a recent review of existing treatment guidelines, HA is recommended for the treatment of knee OA (level of evidence Ia) [2]. It has been demonstrated that HA reduces osteoarthritic pain owing to a reduction of nociceptive and sensorial response in the articular pain receptors [3]. In the same way, in vitro studies [4, 5] have demonstrated the protective effects of HA on the cartilage in experimental models of osteoarthritis. Other in vitro studies have also demonstrated that HA has beneficial effects on the extra-cellular matrix [6], the immune system cells [7] and inflammation mediators [8]. There are several preparations of HA authorized for the treatment of osteoarthritis from different sources, manufacturing process, molecular weight and final concentration. A recent study [9] made it clear that the rheological properties, the molecular weight of HA and its concentration are factors which could determine the intra-articular viability of the molecule. Mechanical and chemical degradation were both reduced in HAs with low molecular weight, with the final concentration of the product being a critical factor. Considering the differences found, and to elucidate whether the different physico-chemical behaviour had, as a consequence, different in vivo activity, we decided to conduct the present study. The comparison is made using an experimental model of osteoarthritis developed by our team [10].

The administration of HA1 for three weeks in rabbits after ten weeks osteoarthritis-induction, had an inhibiting effect on the apoptosis of the chondrocytes compared to the operated Control group (cytometry study), with statistically significant differences (p = 0.0089) similar to that observed in a five week study [10]. However, this difference was not reached in the group treated with HA2 (p = 0.0696). No differences between either treatment groups was found (p = 0.2176). The study of necrosis did not reveal statistically significant differences between the different study groups. The apoptosis levels determined by TUNEL were clearly higher in the Operated Control group, although the high standard deviation values made the differences not statistically significant. This method has been, up to now, considered the Gold Standard [16, 25, 26]. Nevertheless its variability has been questioned in recent investigations. A new method to replace the TUNEL method for another with better possibilities of quantification (for instance chondropoptosis determined by caspase-2L or caspase-3) must therefore be considered for future studies [27]. Gross morphological study did not revealed differences, the advantage of this method regarding a complete visual histological assessment scale (ICRS) [28], is that in small experimental animals as rabbits, valuable samples are not lost they can be employed in other interesting study methods as flow cytometry. In the near future probably immunohistochemical staining should further displace conventional histological techniques [28].

Levels of nitrites in the knees of both groups treated were significantly lower than those in the Operated Control group. Nevertheless, levels in the animals treated with HA1 were very similar to those obtained in the Control group (p = 0.6551), whereas those treated with HA2 were not (p = 0.0001). This was confirmed by the comparison between HA1 and HA2 that revealed significantly lower levels of nitrites in the HA1 group (p = 0.0001). Levels of nitrates were five to eight times lower than nitrites and follow the same pattern of distribution in the different groups; nevertheless, there were no statistically significant differences, probably due to the high standard deviation values found. The differences found when analysing nitrites are maintained when both nitrites and nitrates are considered together as stable metabolites of nitric oxide. Values of HA in synovial fluid did not show any statistical differences between the different study groups. The molecular weight of the HA in the synovial fluid of the operated Control knees was 17% lower than in the control group. The groups treated showed less reduction of HA molecular weight (10% HA2, 13% HA1) as could be expected after the administration of exogenous HA and in accordance with the molecular weights of HA1 and HA2, respectively. According to a consensus reached at the 7^th International Conference on hyaluronan (South California, 2007) both HAs are included as HAs of medium molecular weight, and both have potentially disease-modifying effects [29] as inhibition IL-1β stimulated production of MMP-1, MMP-3 and MMP-13 [30].

Considering the results shown above, it seems to be clear that in vivo activity is not influenced by molecular weight. Recent studies published by our group [9] stated that considering the elasto-viscous nature of HA, not only molecular weight but stability against degradation processes by mechanical and chemical factors, is an important factor. Rheological properties and final concentration are also critical aspects to be taken into account in the final behaviour of the molecule when injected into the articular space. In the study, differences were also found among the HAs of similar molecular weight, especially with HA2 where the fluctuating concentrations observed led to lower stability results than expected [9]. In this study, the animals treated with HA1 not only showed statistically significant lower NO levels regarding untreated animals, but the values were also similar to the ones in the healthy Control group. The animals treated with HA2 showed lower NO values than the untreated animals, but levels were much higher than the control group (statistically significant). In addition the comparison between treatment groups, HA1 and HA2, revealed significantly lower levels of nitrites in the HA1 group. It is known that NO is able to induce apoptosis in chondrocytes and that, moreover, osteoarthritic cartilage contains a higher percentage of cells undergoing apoptosis than normal cartilage [31]. In conclusion, the protective role of HA1 on the osteoarthritic cartilage is also confirmed with similar results to those obtained in previous studies [10]. HA1 and HA2 showed different physico-chemical characteristics and these differences have resulted in different in vivo behaviour. Consequently, the two products cannot be considered as equivalent. HA1 has shown higher effects on inflammation processes than HA2, which could probably provide better results and a long-term protective effect. Along the same line of research, a long-term clinical trial is under development to confirm this protective action in humans, and a new study focus on extracellular matrix is required to confirm differences founded in this study.

Part of this study has been supported by Tedec-Meiji Farma as a project integrated in the University-Industry collaboration program issued by the University of León (Spain). As an university project no economical benefits in any form have been received or will be received from a commercial party directly or indirectly to the subject of this article by the authors. Our group has been working in Hyaluronic Acid projects with this University-Industry plan with almost all Hyaluronic Acid companies including TRB Chemedica AG.