This 2 year clinical study demonstrated that PAAG significantly alleviated lameness in osteoarthritic joints, as assessed by clinical lameness evaluation. A similar outcome was found in a recent pilot randomized controlled study on an experimental OA model in goats, where 75% (3 out of 4) of goats treated with PAAG were non-lame 4 months after the treatment . No adverse effects were observed during the study period in the treated joints, which is consistent with previous studies using PAAG intra-articularly to treat equine OA [16,17,19,20]. PAAG has also proven to be safe in humans for more than 15 years of use [12-14].
The statistical analysis showed a highly significant (P < 0.0001) reduction of the lameness grade after baseline. The estimated OR showed an increased reduction over time from OR = 20 from baseline to month 1 to OR = 58 from baseline to month 24. The largest reduction in lameness grade appeared from baseline to month 1. After month 1 the lameness grade continued to decrease, although the difference between months 1, 3, 6, 12 and 24 was non-significant (p = 0.18). In particular, the OR was very constant from months 6 to 24. Thus, the clinical improvement in lameness grade was already present one month after PAAG treatment. This suggests that the effect of PAAG on OA might occur mainly during the first month after treatment and lasts and increases progressively until 6 months, with a stabilization between 6 and 24 months.
Worsening of the lameness grade following a previous lameness improvement was observed in only 3 horses with baseline lameness grade 1 (n = 3/11). Since no radiographic follow-up was performed in our clinical trial, it is difficult to speculate on the reason of lameness worsening in these horses. None of the horses with baseline lameness grade 2, 3 or 4 showed deterioration in lameness.
This clinical study has also demonstrated that joint effusion grade decreased significantly over time (P < 0.0001). At baseline, joint effusion was absent in only 7% of the horses, while at 24 months the majority of horses (77.5%) showed no joint effusion of the treated joints. Since lameness grade decreased significantly (P < 0.0001) with lower effusion grade, part of the lameness improvement over time can be seen through lowering of the joint effusion.
Although joint effusion was subjectively assessed in this study, PAAG induced a significant decrease in joint effusion in the osteoarthritic joints. However, the mechanism-of-action of PAAG in reducing joint effusion in osteoarthritic joints needs to be investigated.
The majority of horses (86%) had received a previous unsuccessful anti-osteoarthritic treatment, before receiving PAAG, but there was no correlation between the previous treatment and the outcome lameness variable. In some cases (14%), which were mainly among the last included cases, PAAG was used as a first line treatment based on the encouraging results of the first cases of the study.
At 24 months, 90% of the owners were either satisfied or very satisfied with the outcome of this new OA treatment. This is consistent with the outcome as assessed by the veterinary clinicians (82.5% of non-lame horses at 24 months).
Although conventional concepts of OA emphasize the direct and predominant involvement of cartilage and bone in OA development, it is increasingly recognized that the synovium also contributes to the central pathophysiological event of cartilage matrix depletion. Lack of joint lubrication is postulated to play a significant role in the pathogenesis of OA . This emphasizes the role of viscosupplementation, and hence the improvement of lubrication within the joint, in protecting a joint suffering from OA, and reducing the resulting pain. Recently, a study supported the use of intra-articular lubricin as an adjunct to viscosupplementation for retarding cartilage degeneration and possibly the development of post-traumatic OA [25,26].
Precise characterization of the mechanism-of-action of PAAG on osteoarthritic joints has not yet been established, but histopathological observations on joint tissue from horses [Christensen L, personal communication] and goats  have demonstrated that PAAG, like in other soft tissues, becomes integrated within the synovial membrane.
In the goat study , the synovial membrane of the joints injected with PAAG had a better elastance when compared to the synovial membrane of the control joints. Osteoarthritic joints typically show joint stiffness which is a major source of pain in OA. This is supported by a recent study on human knee joint stiffness, which showed that the stiffness co-efficient was higher in individuals with painful OA compared to those with normal knees .
By integrating the synovial membrane, which may probably decrease the joint capsule stiffness and hence the joint stiffness, PAAG might relieve pain of the osteoarthritic joint. This theory is supported by clinical observations in the study population where osteoarthritic joints that responded well to PAAG were no more painful to passive manipulation of the joints.
The inclusion criteria in the present study were strict in order to maximize the validity of the results. Nevertheless, there were some study limitations including a low number of horses, the fact that it was a prospective non controlled clinical study, and the subjective assessment of joint distension. A quantitative measurement of joint circumference could have been performed. This was a multi-centre study, which represented another study limitation due to several clinicians involved in the study, and the potential for inconsistency in application of the lameness grading scale among the clinicians and within clinicians at different examinations . In addition, radiography was not used for the follow-up of OA because of its association with a series of concerns including the insensitivity of radiographs to detect early and small changes and the slow progression of OA being a common finding in clinical trials . Repeatability of application of the radiographic grading system was not assessed.
The present study has shown that PAAG relieved or completely removed the symptoms of lameness and the joint distention in osteoarthritic joints and can be considered as a disease-modifying OA therapeutic agent. A recent study on an OA model in goats  has shown that PAAG reduces the progression of OA as evaluated by MRI and histopathology, which supports the hypothesis that PAAG contributes to a disease-stabilizing affect. Further work investigating the mechanism of action of PAAG in osteoarthritic joints is required.