European Journal of Cardiovascular Medicine

Osteoarthritis of knee is one of the most prevalent chronic joint diseases and a leading cause of pain, disability and loss of mobility in adults. Osteoarthritis is a complex disorder that is traditionally thought of as a degenerative “wear and tear” disease, but is now known to involve cartilage degradation, subchondral bone changes, synovial inflammation, and biochemical mediator activity [1]. Radiographic assessment is still the mainstay of disease grading and the Kellgren–Lawrence classification is still widely used for the assessment of structural severity of osteoarthritis [2].

Inflammation has an important role in the pathophysiology of osteoarthritis. Low-grade synovial inflammation can lead to the activation of chondrocytes and synoviocytes to produce cytokines, chemokines, prostaglandins, and matrix-degrading enzymes, which can lead to cartilage breakdown and progression of symptoms [3]. This inflammatory component is clinically relevant because the severity of pain in knee OA does not always match the severity of the radiographic changes. Patients with more advanced radiological disease may have only mild symptoms, while others with less advanced radiographic disease may have significant pain and disability.

Systemic inflammatory biomarkers can offer extra biochemical data to complement clinical examination and radiography. High sensitivity C-reactive protein has been investigated as a marker of low-grade systemic inflammation in osteoarthritis. Stürmer et al. found that hsCRP correlated with the severity and extent of osteoarthritis, lending weight to the notion that even a traditionally “degenerative” joint disease may have measurable systemic inflammatory activity [4]. Pearle et al. also showed that high levels of hsCRP correlated with local inflammatory parameters in OA, indicating that systemic hsCRP may be partly a reflection of synovial inflammation [5].

Biologically relevant cytokines in knee osteoarthritis include interleukin-6 and tumour necrosis factor-alpha. IL-6 can promote inflammatory signalling, cartilage catabolism, and pain sensitization. Livshits et al. assessed circulating IL-6 as a predictor of radiographic knee OA in the Chingford Study, and also measured CRP and TNF-α as systemic inflammatory markers [6]. These results suggest that IL-6 and TNF-α should be included in the biomarker assessment of the severity of knee OA.

Local joint inflammation is also closely related to pain. Hill et al. demonstrated that synovitis detected by MRI was linked to knee pain and cartilage loss in knee OA, suggesting that inflammatory changes in the joint may be related to symptom burden [7]. Likewise, Baker et al. showed a high correlation between synovitis detected by contrast-enhanced MRI and the severity of knee pain, and synovitis was more common in painful knees [8]. These observations offer a biological rationale for assessing inflammatory biomarkers in the context of pain scores

Pain assessment is a critical component of the evaluation of OA as pain is the primary symptom that leads to functional limitation and health care seeking behaviour. In the field of rheumatology and osteoarthritis research, pain severity is often quantified using standardized tools like the visual analogue scale, numeric rating scale, and WOMAC pain score [9]. These clinical pain measures, when used in conjunction with biochemical markers, could be used to identify patients with a higher inflammatory pain phenotype.

In the Biochemistry discipline, inflammatory biomarkers could be a simple and clinically useful adjunct to radiological grading and pain scoring. In a tertiary-care hospital like Jawaharlal Nehru Medical College, Wardha, Maharashtra, it would be interesting to study hsCRP, IL-6, TNF-α, and ESR in patients with knee osteoarthritis to determine if these markers are correlated with the severity of the disease and the pain burden in the local population. The purpose of the present prospective observational study is to assess the value of inflammatory markers in predicting the radiological severity and pain scores in knee OA. The findings may help determine whether routine             biochemical      markers  can         support  clinical assessment, risk stratification, and disease monitoring in knee osteoarthritis.

OBJECTIVES

1. To measure serum inflammatory biomarkers, including hsCRP, IL-6, TNF-α, and ESR, in patients with knee osteoarthritis.
2. To assess the association between inflammatory biomarker levels and radiological severity of knee osteoarthritis using Kellgren–Lawrence
3. To evaluate the correlation between inflammatory biomarker levels and pain severity using standard pain scores such as VAS and/or WOMAC pain score.
4. To identify whether inflammatory biomarkers can serve as biochemical predictors of severe knee osteoarthritis and higher pain burden.

Study design and setting

This prospective observational study was conducted in the Department of Biochemistry in collaboration with the Orthopaedics outpatient department at Jawaharlal Nehru Medical College, Wardha, Maharashtra, during 2011. The study included patients with clinically and radiologically diagnosed knee osteoarthritis.

Study population

A total of 120 patients with knee osteoarthritis were enrolled. Patients aged 40 years and above with knee pain and radiological evidence of osteoarthritis were included. Patients with rheumatoid arthritis, gout, septic arthritis, other inflammatory arthropathies, recent knee trauma, previous                   knee     surgery,     acute     infection,     chronic inflammatory disease, malignancy, chronic liver disease,

or current use of systemic corticosteroids or immunosuppressive therapy were excluded.

Clinical assessment

Baseline demographic and clinical details were recorded, including age, sex, body mass index, symptom duration, bilateral knee involvement, hypertension, diabetes mellitus, and current NSAID use. Pain severity was assessed using the Visual Analogue Scale (VAS). Pain and functional limitation were further evaluated using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), including WOMAC pain, function, and total scores. High pain burden was defined as VAS pain score ≥7.

Radiological assessment

Plain radiographs of the knee were obtained and osteoarthritis severity was graded using the Kellgren–Lawrence grading system. Patients were categorized into mild, moderate, and severe osteoarthritis groups according to radiological severity.

Biochemical assessment

Venous blood samples were collected under aseptic precautions. Serum inflammatory biomarkers measured included high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and erythrocyte sedimentation rate (ESR). hsCRP was measured using a high-sensitivity immunoassay, IL-6 and TNF-alpha by enzyme-linked immunosorbent assay, and ESR by the Westergren method.

A composite inflammatory biomarker score was calculated using standardized values of hsCRP, IL-6, TNF-alpha, and ESR. High biomarker burden was derived from this composite inflammatory biomarker profile.

Outcome measures

The primary outcome was the association of inflammatory biomarker levels with radiological severity of knee osteoarthritis. Secondary outcomes included correlation of biomarkers with VAS pain score, WOMAC pain score, WOMAC total score, and assessment of biomarker performance in predicting severe osteoarthritis and high pain burden.

Statistical analysis

Data were summarized as mean ± standard deviation, median with interquartile range, or frequency and percentage. Comparisons across radiological severity groups were performed using one-way ANOVA, Kruskal–Wallis test, or chi-square test, as appropriate. Associations between inflammatory biomarkers, Kellgren–Lawrence grade, VAS pain, and WOMAC scores were assessed using Spearman correlation. ROC curve analysis was used to evaluate biomarker performance for predicting severe osteoarthritis and high pain burden, with optimal cutoffs selected by the Youden index. A p-value <0.05 was considered statistically significant.

Ethical considerations

The study was conducted after approval from the Institutional Ethics Committee of Jawaharlal Nehru Medical College, Wardha. Written informed consent was obtained from all participants before enrolment

Study cohort and clinical characteristics

A total of 120 patients with clinically and radiologically diagnosed knee osteoarthritis were included. Radiological severity was mild in 44 (36.7%) patients, moderate in 45 (37.5%) patients, and severe in 31 (25.8%) patients.

Baseline clinical characteristics are summarized in Table

1. Increasing radiological severity was accompanied by older age, longer symptom duration, more frequent bilateral involvement, greater analgesic use, and higher pain and functional limitation scores. BMI, sex distribution, hypertension, and diabetes mellitus did not differ significantly across severity groups.
   

   Table 1. Baseline clinical characteristics according to radiological severity

   Characteristic	Overall N=120	Mild n=44	Moderate n=45	Severe n=31	Test statistic	p-value
   Age, years	62.2 ± 8.8	56.0 ± 7.8	63.9 ± 7.4	68.3 ± 6.5	F(2,117)=27.49	<0.001
   Female sex, n (%)	69 (57.5%)	25 (56.8%)	28 (62.2%)	16 (51.6%)	χ²(2)=0.86	0.651
   BMI, kg/m2	28.2 ± 3.0	28.3 ± 2.7	28.1 ± 3.2	28.2 ± 3.1	F(2,117)=0.06	0.941
   Symptom  duration, years	4.8       (3.5- 5.8)	3.3 (2.7-4.5)	4.4 (3.8-5.5)	5.9 (5.5-6.7)	H(2)=44.44	<0.001
   Bilateral                         knee involvement, n (%)	34 (28.3%)	6 (13.6%)	10 (22.2%)	18 (58.1%)	χ²(2)=19.00	<0.001
   Hypertension, n (%)	50 (41.7%)	17 (38.6%)	18 (40.0%)	15 (48.4%)	χ²(2)=0.79	0.672
   Diabetes mellitus, n (%)	28 (23.3%)	9 (20.5%)	10 (22.2%)	9 (29.0%)	χ²(2)=0.80	0.671
   Current NSAID use, n (%)	69 (57.5%)	18 (40.9%)	25 (55.6%)	26 (83.9%)	χ²(2)=13.85	<0.001
   VAS pain score	6.6 ± 1.7	4.9 ± 1.1	6.9 ± 1.1	8.5 ± 0.8	F(2,117)=118.60	<0.001
   WOMAC pain score	13.1 ± 4.1	9.2 ± 2.9	14.1 ± 2.5	17.0 ± 2.1	F(2,117)=90.91	<0.001
   WOMAC                     function score	40.1 ± 12.6	27.7 ± 7.6	42.9 ± 7.2	53.5 ± 6.6	F(2,117)=121.27	<0.001
   WOMAC total score	58.3 ± 17.7	40.6 ± 10.9	62.4 ± 9.4	77.7 ± 8.2	F(2,117)=139.96	<0.001

   Continuous variables are presented as mean ± SD or median (IQR); categorical variables are presented as n (%). Between-group comparisons used one-way ANOVA, Kruskal-Wallis test, or chi-square test, as appropriate.

   Inflammatory biomarkers and radiological severity Serum             inflammatory       biomarker                levels                  increased progressively across radiological severity categories (Table 2). All measured biomarkers showed significant

   
   

   differences between mild, moderate, and severe osteoarthritis, with the most pronounced separation observed for TNF-alpha and ESR. The composite biomarker distribution by severity is shown in Figure 1.
   
   

   Table 2. Inflammatory biomarker levels according to radiological severity

   Biomarker	Overall N=120	Mild n=44	Moderate n=45	Severe n=31	Test statistic	p-value
   hsCRP, mg/L	3.69       (2.52- 5.48)	2.38               (1.92- 3.08)	4.08 (3.12-5.66)	5.38         (4.71- 7.02)	H(2)=48.59	<0.001
   IL-6, pg/mL	6.21       (4.65- 7.69)	4.40               (2.99- 5.51)	6.38 (5.81-7.51)	8.50         (7.50- 9.02)	H(2)=70.40	<0.001
   TNF-alpha, pg/mL	17.34  (14.18- 20.16)	12.32  (10.04- 14.46)	17.71     (16.77- 19.28)	22.26    (20.88- 23.55)	H(2)=93.91	<0.001
   ESR, mm/hr	31.0       (24.0- 39.0)	22.5               (15.0- 26.2)	32.0 (29.0-36.0)	42.0         (39.0- 44.5)	H(2)=82.07	<0.001
   High biomarker burden, n (%)	85 (70.8%)	10 (22.7%)	44 (97.8%)	31 (100.0%)	χ²(2)=77.86	<0.001

   Biomarker concentrations are presented as median (IQR), except high biomarker burden, which is presented as n (%).

   Correlation of biomarkers with pain and functional burden

   Inflammatory biomarkers were positively correlated with Kellgren-Lawrence grade and pain severity (Table 3). Correlations were consistent across VAS pain, WOMAC

    

   pain, and WOMAC total score, indicating that higher inflammatory activity was associated with both radiological severity and patient-reported symptom burden. The relationship between composite biomarker score and VAS pain is illustrated in Figure 2.

    

   Table 3. Correlation of inflammatory biomarkers with radiological severity and pain scores

   Biomarker	KL grade	VAS pain	WOMAC pain	WOMAC total
   hsCRP, mg/L	ρ=0.639; t(118)=9.03; p<0.001	ρ=0.590; t(118)=7.94; p<0.001	ρ=0.543; t(118)=7.02; p<0.001	ρ=0.564; t(118)=7.41; p<0.001
   IL-6, pg/mL	ρ=0.790; t(118)=13.99; p<0.001	ρ=0.680; t(118)=10.07; p<0.001	ρ=0.602; t(118)=8.20; p<0.001	ρ=0.675; t(118)=9.93; p<0.001
   TNF-alpha, pg/mL	ρ=0.900; t(118)=22.46; p<0.001	ρ=0.771; t(118)=13.14; p<0.001	ρ=0.684; t(118)=10.19; p<0.001	ρ=0.768; t(118)=13.02; p<0.001
   ESR, mm/hr	ρ=0.849; t(118)=17.48; p<0.001	ρ=0.707; t(118)=10.86; p<0.001	ρ=0.669; t(118)=9.77; p<0.001	ρ=0.731; t(118)=11.65; p<0.001
   Composite biomarker score	ρ=0.913; t(118)=24.29; p<0.001	ρ=0.789; t(118)=13.97; p<0.001	ρ=0.719; t(118)=11.24; p<0.001	ρ=0.784; t(118)=13.74; p<0.001

   Values represent Spearman correlation coefficients with corresponding test statistic and p-value. KL, Kellgren-Lawrence; VAS, Visual Analogue Scale; WOMAC, Western Ontario and McMaster Universities Osteoarthritis Index.

   Predictive performance for severe osteoarthritis and high pain burden

   ROC-based analyses demonstrated good to excellent discrimination for severe radiological osteoarthritis and high pain burden (Table 4). For severe osteoarthritis, the

    

   composite biomarker score, ESR, and TNF-alpha showed the strongest performance. For high pain burden, the composite biomarker score and TNF-alpha provided the highest discriminatory ability.

   
   

    

   Table 4. ROC-based predictive performance of inflammatory biomarkers

   Outcome	Biomarker	AUC (95% CI)	Optimal threshold	Sensitivity	Specificity	Test statistic	p-value
   Severe OA	hsCRP, mg/L	0.811 (0.713-0.909)	3.76	93.5%	66.3%	z=6.21	<0.001
   Severe OA	IL-6, pg/mL	0.915 (0.845-0.985)	6.72	96.8%	77.5%	z=11.59	<0.001
   Severe OA	TNF-alpha, pg/mL	0.962 (0.914-1.000)	18.38	100.0%	83.1%	z=18.85	<0.001
   Severe OA	ESR, mm/hr	0.966 (0.920-1.000)	38.0	93.5%	92.1%	z=20.00	<0.001
   Severe OA	Composite biomarker score	0.970 (0.928-1.000)	0.66	90.3%	95.5%	z=21.64	<0.001
   High                  pain burden	hsCRP, mg/L	0.817 (0.740-0.894)	3.63	82.8%	79.0%	z=8.05	<0.001
   High                  pain burden	IL-6, pg/mL	0.849 (0.779-0.920)	7.54	56.9%	96.8%	z=9.70	<0.001
   High                  pain burden	TNF-alpha, pg/mL	0.905 (0.849-0.962)	16.47	91.4%	75.8%	z=14.06	<0.001
   High                  pain burden	ESR, mm/hr	0.831 (0.756-0.905)	37.0	62.1%	90.3%	z=8.70	<0.001
   High                  pain burden	Composite biomarker score	0.903 (0.846-0.960)	-0.06	87.9%	79.0%	z=13.80	<0.001

   AUC, area under the receiver operating characteristic curve. Optimal thresholds were selected using the Youden index. High pain burden was defined as VAS pain score >=7.

In this prospective observational study of 120 patients with knee osteoarthritis, inflammatory biomarkers demonstrated a clear step-wise relationship with radiological severity, pain and functional limitation. Older age, longer symptom duration, bilateral knee involvement, higher NSAID use, and higher VAS, WOMAC pain, WOMAC function, and WOMAC total scores were

associated with severe osteoarthritis. The median levels of hsCRP, IL-6, TNF-alpha and ESR rose with disease severity, with 100% of severe cases having high biomarker burden. The composite biomarker score, TNF-alpha and ESR had the highest correlation with Kellgren–Lawrence grade and excellent discrimination for severe osteoarthritis by ROC analysis.

Orita et al. studied 47 consecutive patients with knee

osteoarthritis and measured synovial fluid TNF-alpha, IL-6, and NGF by ELISA, and their findings support these findings. All synovial fluid samples contained detectable levels of TNF-alpha and IL-6, but not NGF. TNF-alpha was significantly positively correlated with WOMAC total score (r=0.69, p<0.01), WOMAC pain (r=0.57, p<0.01),

stiffness (r=0.56, p<0.01), and physical function (r=0.64, p<0.01). In contrast, IL-6 was negatively correlated with KL grade and weakly negatively correlated with WOMAC stiffness [10]. This is consistent with the current study's results that inflammatory mediators, particularly composite biomarker burden, are more strongly associated with pain and functional limitation than radiographic grading alone.

Stannus et al. studied 172 older adults over a mean follow-up of 2.9 years and found that quartiles of IL-6 and TNF-alpha were associated with medial tibiofemoral joint space narrowing with odds ratios of 1.42 and 1.47 per quartile, respectively, further supporting the structural relevance of these cytokines. Baseline IL-6 predicted medial and lateral tibial cartilage volume loss, and change in TNF-alpha was associated with medial cartilage volume loss [11]. In contrast, the current study demonstrated high positive correlation of IL-6 and TNF-alpha with KL grade and good ROC performance for severe OA, suggesting their importance as markers of structural disease burden.

The current results are also supported by longitudinal evidence on pain. Stannus et al. conducted a 5-year prospective cohort study of 149 older adults, measuring hsCRP, TNF-alpha, and IL-6, and found that baseline levels of TNF-alpha and IL-6 were associated with change in knee pain while standing, and that change in inflammatory markers predicted worsening pain over time [12]. We also found high cross-sectional correlation between TNF-alpha, IL-6, hsCRP, ESR and pain scores, with TNF-alpha having the highest discrimination for high pain burden.

The inflammatory phenotype concept is also supported by Daghestani et al., who evaluated soluble macrophage biomarkers CD14 and CD163 in knee osteoarthritis. They found that synovial fluid CD14 and CD163 correlated with the number of activated macrophages, CD14 correlated with joint space narrowing, osteophyte severity and self-reported pain, and both CD14 and CD163 correlated with osteophyte progression [13]. CD14 and CD163 were not assessed in the current study, but our high composite biomarker burden in moderate and severe OA patients is consistent with the notion that a subset of knee OA patients has a measurable inflammatory phenotype that is more severe in terms of structure and symptoms.

Imamura et al. assessed 101 patients (53 with painful knee OA and 48 healthy controls). Patients with OA had Kellgren–Lawrence grade 2–4 disease, VAS pain >4, and a mean VAS score of 7.6 ± 1.59. The serum levels of IL-6 and IL-10 were significantly elevated in the OA patients compared to controls, but there was no significant difference in the levels of IL-8 and TNF-alpha. IL-6 was positively correlated with VAS pain, WOMAC pain and WOMAC rigidity, and WOMAC rigidity was a predictor of serum IL-6 in regression analysis [14]. This is consistent with the current study's results that inflammatory biomarkers, especially IL-6 and composite

biomarker burden, are linked to pain and functional severity in knee OA.

Radojcic et al. added more mechanistic data by examining 104 patients with end-stage knee OA with serum C1M, plasma IL-6, synovial fluid IL-6, contrast-enhanced MRI synovitis scoring, WOMAC pain, and neuropathic pain scores. Synovial fluid IL-6 was correlated with parapatellar synovitis and with WOMAC pain, but plasma IL-6 was not correlated with synovitis [15]. This distinction is clinically relevant: the present study employed serum markers, which may partly account for the superior performance of a composite biomarker score, as systemic markers may reflect a more general inflammatory burden than synovial inflammation alone.

Nees et al. assessed synovial fluid cytokines in 34 patients with knee OA undergoing arthroplasty (14 unicompartmental and 20 bicompartmental). Mean knee pain was high, with NRS 7.12 ± 2.29, and mean OKS-12 was 35.15 ± 7.68. Several synovial mediators were correlated with the radiological severity, including IL-6, IL-8, IFN-gamma, SCGF-beta, VEGF, and CXCL1, with the highest correlation with radiological severity for CXCL1 and IL-8. Pain was significantly correlated with IL-7, IL-10, IL-12, IL-13, IFN-gamma, SCGF-beta, and

VEGF, and poorer knee function was correlated with IL-6, IL-10, IL-12, IL-13, IL-18, beta-NGF, SCGF-beta, VEGF,

and CXCL9. Five mediators (IL-10, IL-12, IL-13, SCGF-beta, and VEGF) were correlated with both pain and function [16]. This is consistent with the current study's results that inflammatory biomarker burden is linked to both radiological severity and pain and functional limitation in knee OA.

But there is also published evidence that biomarker–pain relationships are not always linear. Li et al. characterized synovial fluid inflammatory mediators in knee OA and found that WOMAC-assessed pain and neuropathic pain did not correlate consistently with all inflammatory cytokines measured; some cytokines, such as IL-1beta, IL-6, and TNF-alpha, were reported to be higher in patients with less pain [17]. This is in contrast to the current results and indicates that pain in knee OA is multifactorial, with inflammatory, structural, biomechanical and nociplastic mechanisms.

The predictive performance found in the present study is clinically relevant. The composite biomarker score had an AUC of 0.970 for severe OA and 0.903 for high pain burden, which was superior or equivalent to individual biomarkers. This is in line with the use of combined biomarker interpretation, as knee OA inflammation is biologically heterogeneous. Pan et al. also found that IL-6 was independently associated with cartilage volume loss and worse pain trajectory, with less consistent associations for other markers [18]. These results indicate that single biomarkers can be helpful, but composite or panel-based strategies might be more representative of disease complexity.

A balanced interpretation is required as not all population studies have shown strong systemic inflammatory associations. In the Framingham Offspring cohort, Vlad et al. assessed various systemic inflammatory markers and did not observe a consistent relationship with radiographic hand or knee OA [19]. This discrepancy could be due to differences  in  population  selection,  inclusion  ofcommunity-based patients instead of symptomatic patients, timing of biomarkers, and heterogeneity of OA phenotypes. The present cohort, however, comprised clinically symptomatic and radiologically graded knee OA patients, which may account for the stronger biomarker–severity relationship

At the evidence-synthesis level, Dainese et al. found that inflammatory characteristics such as synovitis, effusion, cytokines and CRP are associated with knee pain in OA, but the magnitude of the association depends on the type of marker and how it is measured [20]. This is consistent with the current results, which found that inflammatory markers were consistently linked to pain burden, but the best results were achieved with a composite biomarker score, not any individual analyte.

The present study has clinical implications for biochemical assessment of knee osteoarthritis. Radiographs are still important for structural grading and VAS/WOMAC scores are still important for symptom assessment. But hsCRP, IL-6, TNF-alpha, ESR and particularly a composite inflammatory biomarker score may be useful to identify patients with severe radiological disease and high pain burden. This could be helpful for risk stratification, clinical monitoring, and identification of an inflammatory OA phenotype.

Limitations

The present study was performed in single center with 120 patients and biomarkers were assessed at one time point, so causal relationships and progression prediction cannot be confirmed. Synovial fluid biomarkers and synovitis assessment by MRI were not done. NSAID use was more common in severe OA and could have affected inflammatory marker levels. Nevertheless, the study offers pertinent biochemical data that inflammatory biomarker burden is strongly linked to radiological severity, pain, and functional limitation in knee OA.

Inflammatory biomarkers, particularly TNF-alpha, ESR, IL-6, hsCRP, and the composite biomarker score, were significantly associated with radiological severity, pain, and functional limitation in knee osteoarthritis. These findings suggest that inflammatory biomarkers may serve as useful adjunctive tools for assessing disease severity and symptom burden.

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