Patient demographics, knee function and radiological assessment
In total, 83 patients, including 21 males and 62 females, were enrolled in the study. The average age was 63.4±10.4 years. The average WOMAC score was 25.5 (21.7-29.3); the average ROM was 112.0° (95% CI 108.0-116.1). The patient demographics, knee function, pain intensity and expression of inflammatory mediators in the different K-L grade groups are presented in Table 1. With increasing age, ROM became more limited (Figure 1A), and the WOMAC score increased (Figure 1B); moreover, worsening K-L grade led to limited ROM (r=-0.499, p<0.001) and an increased WOMAC score (r=0.493, p<0.001) (Figure 2A and Figure 2B), which indicated that knee function was gradually lost.
To explore differences in inflammatory mediators among different K-L groups, expression of inflammatory mediators was compared using one-way ANOVA. We found that IL-1β (p=0.002) and IL-6 (p=0.023) expression differed significantly among the four K-L groups (Figure 3A and Figure 3B). Increased expression of IL-1β and IL-6 was found in early-stage of KOA, which suggested that more severe inflammation may be present in KOA early stages. However, no significant differences were found for other inflammatory mediators among the K-L groups.
Significant correlations among different pain scoring systems
To evaluate the intensity of pain in KOA, nociceptive pain was measured using the NRS and VAS, and neuropathic pain was determined using the PainDETECT questionnaire. The WOMAC pain score was recorded to measure function-related pain. NRS and VAS scores were 3.04 (95% CI 2.63-3.44) and 3.55 (95% CI 3.09-4.03) cm, respectively. The WOMAC pain score was 5.05 (95% CI 4.29-5.81); the PainDETECT pain score was 2.89 (95% CI 2.12-3.67).
To further investigate consistency among the different pain scoring systems, correlation coefficients were determined using the Spearman test. As expected, the four measurements of pain correlated highly with each other and exhibited homogeneity (Figure 4A and Figure 4B).
Pain affected knee function and correlated with age and K-L grade
To determine correlations between pain and patient demographic parameters, knee function or radiological grade, pain was analyzed for correlations with age, ROM, WOMAC score and K-L grade using the Spearman test. With an increase in age or K-L grade, NRS, VAS and WOMAC pain scores also increased, as expected (Figure 5A and Figure 5B). Correspondingly, knee function was gradually lost, as shown by a rise in the WOMAC score (Figure 5C) and limited ROM (Figure 5D). Nonetheless, neuropathic pain scores correlated only with the WOMAC score (r=0.384, p<0.000) and not age (r=0.108, p=0.331), K-L grade (r=0.176, p=0.112) or ROM (r=-0.179, p=0.105). In conclusion, age and K-L grade are important factors correlated with KOA pain, and KOA pain significantly affects knee function.
Correlations among pain, patient characteristics and inflammatory cytokines
Inflammatory cytokines play critical roles in pain initiation and development (5). Among inflammatory cytokines, IL-1β, IL-6 and TNF-α are the most important involved in KOA. Although IL-1β, IL-6 and TNF-α have been widely investigated in both experimental models and clinical samples, expression of these cytokines in KOA pain remains unresolved, and different research studies have reached conflicting conclusions (7, 18, 19). Moreover, as mentioned in the introduction section, M-CSF has been found to be important in KOA (10, 11). However, whether SFM-CSF is involved in KOA pain is still unknown.
To investigate correlations between pain and the above inflammatory cytokines, expression of IL-1β, IL-6, TNF-α and M-CSF was measured by ELISA, and the results were analyzed by the Spearman test. As shown in Table 2, IL-1β and IL-6 very weakly correlated negatively with the VAS score; TNF-α correlated negatively with both VAS and NRS scores. In contrast, M-CSF did not show any correlation with any of the pain scores. Moreover, WOMAC-assessed pain and neuropathic pain did not correlate with any of the inflammatory cytokines. In short, the above evidence demonstrates that inflammatory cytokines correlate significantly with KOA pain. In general, expression of IL-1β, IL-6 and TNF-α may be higher in patients with less pain.
To further determine correlations between inflammatory cytokine expression and patient characteristics, expression of IL-1β, IL-6, TNF-α and M-CSF was analyzed for correlations with age, K-L grade, ROM and WOMAC score using the Spearman test (Table 3). IL-1β and IL-6 correlated negatively with age, K-L grade and WOMAC score, whereas IL-6 correlated positively with ROM. In addition, TNF-α correlated negatively with age. In line with the results illustrated in Figure 3, expression of IL-1 and IL-6 was relatively high in the early stage compared with the late stage of KOA, and expression of these two inflammatory cytokines was associated with knee function.
No correlations were found between pain and measured catabolic proteases
Progression of OA is usually accompanied by high matrix catabolism, we hypothesized that high expression of catabolic proteases in the SF promotes the breakdown of cartilage and causes severe synovitis and inflammation, which may lead to increased pain. Among these catabolic proteases, MMPs and ADAMTS5 play important roles in KOA through involvement in the degradation of ECM type II collagen and aggrecan, respectively (20, 21). However, it remains unknown whether these proteases are involved in KOA pain.
To evaluate correlations between these catabolic proteases and pain, MMP-3, MMP-13 and ADAMTS5 levels were measured by ELISA, and the results were correlated with pain scores using the Spearman test (Supplement Table 1). According to the results, only MMP-13 showed a correlation, albeit weak, with the NRS score (r=0.267, p=0.015). No correlations were found between pain scores and the other measured catabolic enzymes. This result indicates that the catabolic cytokines measured may not be involved in KOA pain.
No correlations were found between pain and measured neuropeptides
It is believed that neuropeptides sensitize knee nociceptors and are related to knee pain (19), and SP, NPY, CGRP and BK are considered important neuropeptides involved in OA pain. First, SP in the SF has been reported in KOA and rheumatoid arthritis (22), and the concentration of SP is significantly higher in painful temporomandibular joints than in painless temporomandibular joints (23). Second, expression of NPY in the dorsal root ganglion was found to correlate significantly with OA pain in animal models (24), and SF NPY expression correlates with OA pain (25). Third, CGRP is reportedly involved in the progression and prognosis of KOA and widely distributed in nociceptive pathways in the peripheral and central nervous systems, correlating with numerous types of pain (26-28). Lastly, BK is an inflammatory mediator that can lead to vasodilation and inflammation induction. Although BK has been reported to correlate with biomarkers for cartilage degradation and inflammation in OA (29), the role of BK in pain needs to be further investigated. Therefore, it is necessary to explore whether these neuropeptides play roles in OA pain.
To address this issue, expression of these neuropeptides was analyzed, and the Spearman test was performed to examine correlations with different pain scoring systems (Supplement Table 2). No correlations were found between any of the measured pain scoring systems and these neuropeptides, which suggests that these neuropeptides may not contribute to KOA pain.