The effects of early-stage OA on FA composition of the IFP were examined in the rabbit ACLT model, 2 and 8 weeks after surgical intervention. The main findings of this study were that i) ACLT changed the infrapatellar n-3/n-6 PUFA ratio towards a pro-inflammatory phenotype, ii) the FA profile of the IFP was altered as early as 2 weeks post-ACLT, iii) the ACLT-induced alterations in the proportions of particular FA became more pronounced at 8 weeks, and iv) the FA signature of the contralateral knee resembled that of the unoperated control in most aspects, but with some noteworthy exceptions.
The observed reduction in the n-3/n-6 PUFA ratio of the IFP may contribute to the inflammation and cartilage degradation in early OA. It is also in concordance with our data showing increased mRNA expression of inflammatory and cartilage-degrading factors interleukin IL-6, matrix metalloproteinase MMP-3, and MMP-13 in articular cartilage of similarly-treated rabbits at 2 weeks post-ACLT [24]. As an extrasynovial organ, the IFP does not directly interact with cartilage, but it has been proposed to be a source of adipokines, cytokines, FA, and LM that could contribute to the pathophysiological processes in OA [3, 15]. Other health issues that are associated with OA include obesity and low-grade systemic inflammation [2, 25]. Obesity is also linked to altered dietary and, consequently, body n-3/n-6 PUFA balance that has potential implications in OA [6, 7]. The n-3/n-6 PUFA ratio is of importance, as n-6 PUFA increase COX-2 protein levels and PGE2 production in chondrocytes [11, 26]. The increased proportion of 20:4n-6 in the IFP of the ACLT group is in line with earlier findings of Gierman et al., who observed that the secretion of 20:4n-6 was higher from the IFP of OA patients compared to post-mortem donors without OA [15].
In contrast, n-3 PUFA are metabolized into less inflammatory or pro-resolving LM [12, 27], and have anti-destructive effects on cartilage [16, 17]. In chondrocytes, n-3 PUFA, with 20:5n-3 being the most effective, reduce expression of COX-2, IL-1α and IL-1β, tumor necrosis factor-α, aggrecanases ADAMTS4–5, MMP-3, and MMP-13 [16]. In addition, n-3 PUFA may participate in bone remodeling by favoring osteoblastogenesis [28]. The elevated 22:6n-3 percentage in the ACLT knees of the rabbits is similar to earlier findings from OA patients [15]. As the IFP has been shown to induce both protective and disease-aggravating activities in OA [4], pro-resolving LM, such as resolvins, protectins, and maresins, derived from 22:6n-3 could potentially contribute to the resolution pathways that are activated in OA [27, 29]. In addition to joint health, long-chain n-3 PUFA can have beneficial effects on dyslipidaemia and the cardiovascular system [14].
The FA profile of the IFP was already altered 2 weeks post-ACLT. The affected FA were generally of minor proportions, and the most interesting change was the reduction in the n-3/n-6 PUFA ratio. According to previous findings in the rabbit model, the first signs of OA have typically been documented 4 weeks post-ACLT [21, 30,31,32]. These include reduced proteoglycan (PG) content, fibrillation, and lowered biomechanical stiffness [20, 33]. Bone mineral density has been shown to be decreased and blood flow increased in periarticular bone at 2 weeks post-ACLT [34]. Ojanen et al. reported a loss of fixed charge density from articular cartilage 2 weeks post-ACLT [22]. The FA signature in adipose tissue/plasma is known to change rapidly in response to various stimuli, for instance, fasting and dietary changes [35, 36]. Against this background, the altered FA profiles after 2 weeks of intervention were expected and could represent early responses to inflammation and tissue damage. This could also be connected to the reduced fixed charge density of PG reported previously [22].
As shown in Fig. 3, ACLT reduced the relative proportions of 16:1n-7, 18:3n-3, and C18 MUFA at 8 weeks post-ACLT, while percentages of longer-chain SFA and MUFA tended to increase simultaneously with several C20–22 PUFA. Many of these changes emerged at 2 weeks already and would be consistent with a situation where cellular membranes, and FA abundant in their PL pool, had increased in proportion at the expense of TAG residing in storage lipid droplets. For example, adipocyte sphingomyelin is rich in long-chain SFA and MUFA [37]. The overall pattern of FA modifications observed in the rabbits is not unique to this model, as similar changes have been documented in adipose tissue during food deprivation of rodents [36], supporting the present FA results in the rabbits with a slightly negative energy balance compared to the initial body mass. The IFP has been assumed to be resistant to starvation [4], but recent data have shown that its volume can decrease due to weight loss [38]. The effects of OA on the composition of the IFP have not been studied systematically. However, it is known that OA can increase vascularization, inflammatory infiltration, and thickness of the interlobular septa, i.e., fibrosis [39]. Moreover, OA patients with IFP lymphocytic infiltration have thicker lobuli septa and smaller adipose lobuli than OA patients without lymphocytic infiltration. These adaptations may contribute to the observed changes in the infrapatellar FA composition after ACLT.
There were some notable differences in the FA profiles between the contralateral and control knees 8 weeks post-ACLT. The contralateral knees are often used as within-animal controls in ACLT studies [30, 31, 33]. However, altered post-operative loading patterns may affect cartilage and bone in the contralateral joint [34, 40]. Such changes have been documented, for instance, in dynamic elastic modulus, PG concentration, collagen content and orientation angle, permeability, and fibril network modulus of articular cartilage between the control and contralateral knees in this animal model [21, 32]. When compared to the unoperated control knee, the present study revealed an increase in the n-3 PUFA sum and a decrease in the MUFA sum, findings that are not trivial to explain. One possibility is that the systemic redistribution of circulation following the operation, and the consequent uneven flow of metabolites to the limbs, may have affected the tissues of both the operated and the contralateral knee. This might involve the neural regulation of circulation, but inflammatory signaling and edema in the operated knee could also have metabolic reflections in other parts of the body that would not be present in the unoperated controls. In addition, the distribution of n-3 PUFA to the peripheral circulation could be used to induce the resolution phase of inflammation, which employs n-3 PUFA-derived LM, such as resolvins. The previously described changes in gait and muscle use in the contralateral knee of OA patients emphasize the importance of treating the contralateral knee as an anatomical site affected by the diseased joint [41]. Even though the FA signature of the contralateral knee remained similar to that of the unoperated control in most aspects, its use as an unaffected control for studying the FA profile of the IFP cannot be recommended.
The proportion of total MUFA was also reduced in the ACLT knees 8 weeks post-ACLT. The possible role of MUFA in OA is not clear, but 18:1n-9 has exerted anti-destructive and anti-inflammatory effects on chondrocytes and cartilage in vitro [11]. The present findings are in line with this notion, as the proportion of total MUFA was reduced post-ACLT even though the decreasing trend in 18:1n-9, the most abundant individual MUFA, did not reach significance. Previously, 18:1n-9 has been identified as a critical metabolite for discriminating between early and late-stage OA, with increased levels in synovial fluid during disease progression [18]. Regarding SFA, 16:0 has been documented to inhibit cartilage destruction in vitro [11], but the present study did not find any effects of ACLT on the proportions of major SFA.
There are some limitations in the present study. The control group numbers were relatively small, but similar numbers of rabbits/joints have been used successfully in previous publications [21]. Based on earlier studies, OA changes in this model are systematic, and a relatively small number of samples has yielded statistical differences in primary outcome variables. For statistical comparison, the minimum number of joints in each group would be 6, which was attained. To reduce the number of experimental animals, both knee joints were used from the control group animals, as has been done previously [21, 32]. The contralateral knees were not sham-operated to avoid infection and to make the present study comparable to earlier ones. Unfortunately, the food intake of the animals was not recorded during the study. For this reason, it is not known, whether there were differences in the energy balance or in the intake of essential PUFA precursors between the study groups. However, since all groups received the same diet, dietary effects on the observed differences in FA profiles can be assumed to be minor. Another limitation was that the FA composition was not determined from separate neutral and PL fractions but from total lipids and, thus, it mostly reflected the IFP storage fats rather than the membrane lipids.
The progression of OA in the rabbit ACLT model is rapid [8]. Early stages of secondary OA can develop within weeks and, thus, the selected model served well to address the aims of this study. Smaller species, such as rats and mice, have knee joint surfaces that are too small to allow for cell deformation experiments that were conducted in these animals. Our choice of rabbits allows for comparisons with earlier studies with the same surgical protocol. The present study provides novel information about the role of the IFP in inflammation, which has relevance in the progression of OA [4]. Studying the normal and abnormal loading of the knee joint, and the patellofemoral and tibiofemoral joints, is not possible in cell cultures or explants. The rabbit knee differs in anatomy and size of intra-articular structures, as well as in the range of motion from the human knee [42]. These differences have to be considered when translating the present results to the context of early OA in humans. Nevertheless, the basic bony, ligamentous, and muscular structures are similar [42, 43], and OA development, albeit much faster in rabbits than in humans, includes a similar array of pathological events [8]. Thus, the FA phenomena described here could have relevance in the development of human OA.
