This prospective cohort of 12 patients with acute intra-articular proximal tibia fractures is, to the best of our knowledge, the largest study to report on cytokine levels using the contralateral knee joint for comparison and the association of intra-articular cytokine levels with 3- and 12-month postoperative clinical outcomes. Acute inflammatory cytokines have been implicated as one of many factors contributing to joint degeneration in intra-articular joint injury leading to PTOA [1,2,3, 5, 6].
This study found a significant increase of nearly all the pro-inflammatory cytokines investigated, such as IL-1β, IL-2, IL-6, IL-8, IL-12p70, TNF-α, IFN-y, MMP-1, MMP-3, and MMP-9. Moreover, all anti-inflammatory cytokines IL-1RA, IL-4, IL-10, and IL-13 were elevated significantly compared to the healthy contralateral knee. Only the metabolic mediator cytokine TGF-β3 had a significant decrease in its level. No cartilage degradation products ACG and CTX-2 proved to be significantly elevated. Furthermore, several pro- and anti-inflammatory cytokines and metabolic mediators were correlated to 12-month postoperatively clinical outcomes. The results of this study supports the current understanding of the inflammatory response behind the development of PTOA and could supplement the surgical treatment of intra-articular fractures [2, 6, 16,17,18,19].
Cytokines were significantly increased in the fractured knee compared to the contralateral knee
The production of IL-6 in joints usually occurs as a response to increased cytokine levels of IL-1β and TNF-α [19, 20]. Previous studies have confirmed the synergy of IL-1β and TNF-α to decrease the synthesis of the building blocks of the extracellular matrix (ECM) and increase the synthesis of proteolytic enzymes such as MMPs [19, 21]. Moreover, the pro-inflammatory cytokines have been shown to stimulate cells to synthesis other inflammatory cytokines, thus promoting their production in a self-propelling way [6, 16, 19, 20]. The cytokines observed to act this way are IL-1β, IL-6, IL-8, and TNF-α [16, 19]. In this study, IL-6 had the largest fracture/contralateral knee ratio (2618) in our data (Table 3). IL-6 has been shown to promote osteoclast differentiation and bone resorption [6, 19]. Haller et al. demonstrated a significantly elevated level of IL-6 in the injured knee, which persisted through to the second time point of aspiration . A large upregulation of IL-6 might have an impact over time and, therefore, play an important role in the development of PTOA. We also found that IL-8, IL-1β, TNF-α, and MMP-1 had a higher ratio than the other cytokines measured (212, 40, 20, and 74) compared to levels in the healthy knee (Table 3). Lieberthal et al. speculate that the lack of adequate, post-injury control of the pro-inflammatory cytokines in some patients maintains the chronic inflammation and tissue damage that leads to PTOA . The possible synergy and autocrine behavior of these cytokines might have an important role in the development of the acute phase of chronic inflammation post-injury due to their large upregulation .
Another important finding was that the anti-inflammatory cytokines IL-1RA and IL-10 had a more than 40-fold increase (334 and 46) in their ratio (Table 3). IL-10 has been shown to stimulate the production of IL-1RA, type 2 collagen and ACG production as well as inhibiting MMPs, chondrocyte apoptosis and downregulating IL-1β and TNF-α [19, 22]. The presence of increased anti-inflammatory cytokines might suggest that a chondroprotective milieu is stimulated as a response to the pro-inflammatory cytokines. Furthermore, the lack of increased concentration of the metabolic mediators, indicate that the cartilage was not influenced by the inflammation, at the time of aspiration (Table 3). However, further research would be needed to investigate the balance between pro- and anti-inflammatory cytokines in the acute milieu in intra-articular knee fractures to understand the timeline and mechanism where the inflammatory process tends towards a chronic state of inflammation leading to PTOA.
Clinical outcomes after 12 months correlate with several cytokines
Interestingly, we found that several pro-inflammatory cytokines were correlated with 12-month postoperative clinical outcomes, indicating that upregulation of acute pro-inflammatory cytokines might correlate with knee pain and the overall quality of life (Table 4). This, in contrast to the fact that all clinical parameters recorded in this study were improved between the 3- and 12-month follow-up (Table 2). Surprisingly, anti-inflammatory cytokines IL-1RA, IL-10, and IL-13 were positively correlated with 12-month postoperative outcomes (Table 4). These findings suggest that a response to the pro-inflammatory cytokines might have an impact on the clinical outcome as well. A similar study conducted on tibial plateau fractures by Haller et al. found that the anti-inflammatory cytokines IL-1RA and IL-10 remained elevated at a second time point of aspiration, and that the concentration of IL-1RA increased .
The clinical outcome VAS in activity at 12 months, correlated with numerous cytokines, such as IL-1β, IL-2, IL-8, TNF-α, IL-1RA, IL-10, and TGF-β1 (Table 4). All the cytokines except TGF-β1 were significantly elevated in the fractured knee (Table 3). Surprisingly, the levels of cytokines IL-1RA and IL-10 were positively correlated with VAS in activity at 12 months. This finding was unexpected and suggests that IL-1RA and IL-10 may have a role in developing early symptoms of PTOA. Further research would be needed to identify the potential negative role of anti-inflammatory cytokines in the joint milieu, before drawing conclusions. These findings and the findings of Haller et al. raise the question of the role of transient versus prolonged cytokine elevation, whether it is pro- or anti-inflammatory and the role of these cytokines on the chondrocyte physiology . Lastly, in this study, the cytokine level of cartilage degradation products ACG and CTX-2 had no significant correlation with outcomes and was slightly but not significantly elevated in the fractured knee (Tables 3,4). This result may be explained be the early timing of aspiration. Another possible explanation for this is that the cartilage is slightly resistant against the initial inflammation caused by the fracture. This only becomes a problem when the inflammation persists, and the concentration of cartilage degradation products increase. Further research should investigate the possible correlation of inflammatory cytokines with clinical outcomes in a larger population and serial sampling during follow-ups to provide more information about the joint milieu in intra-articular knee fractures.
Monitoring cartilage health with serum levels of cytokines
We found that an elevation of IL-6 was negatively correlated to plasma IL-6 level (Supplement 1). This finding suggests that an upregulation of IL-6 may be detected in the fractured joint due to the timing of sampling collection. Measurement of the levels of IL-6 in serum at a second time point could show an increase in its level as it has been shown to have a persistently elevated level in the SF collected from fractured knees . However, the concentration of cytokines in serum is not only dependent of the inflammatory process in the fractured knee, but the overall regulation of cytokines in the body. In this study, TNF-α and TNF-β where both positively correlated with serum levels, while TNF-α was significantly elevated in the fractured knee (Table 3, Supplement 1). Furthermore, TNF-α has been shown to stimulate the production of other cytokines . The positive correlation with VAS at 12 months in this study could open the possibility of investigating it as a potential biomarker of early-onset PTOA during follow-up (Table 4).
Lastly, we found that the levels of the metabolic mediators TGF-β2 and -β3 were correlated with SF of the fractured knee, with only TGF-β3 showing a significant decrease in the injured knee (Table 3, Supplement 1). In vitro studies have shown that the TGF-β signaling pathway switches its protective role in normal cartilage to a more damaging role in advanced OA. Both overactivity and lack of activity could suggest that only a narrow range of TGF-β can maintain cartilage health [23, 24]. Therefore, TGF-β mediators could perhaps be used to monitor the level of cartilage health through serum sampling. However, it might take some time before an elevation of cytokines in the fractured knee joint causes an increase (or decrease) of serum cytokines. Therefore, further investigation where sampling serum is a part of the follow-up, could reveal suitable cytokines that could serve as a diagnostic biomarker of early-onset PTOA.
A discussion of the last exploratory outcome would go beyond the scope of this paper. However, it is worth mentioning that future studies should, if possible, include the measurement of these variables presented in this paper during follow-up, as they may perhaps influence or help predict clinical outcomes.
The first limitation of this study is the small sample size (n = 12). It is possible that with a larger study, these cytokines might contribute additional information regarding clinical outcomes. However, we have demonstrated that some cytokines correlate with clinical outcome after 12 months. Second, the follow-up period might not be long enough to see any significant changes in outcome. Therefore, future studies with follow-up beyond 12 months are suggested. Third, we can’t be sure that we didn’t alter the concentration of proteins in some knees more than others, since the volume of SF in each knee before injecting the saline was unknown. Fourth, due to the relatively small number of patients included, a multiple comparison cannot statistically be justified. However, our results may generate additional hypotheses for future research. Lastly, 1 patient was lost to follow-up after 12-months.