Correlation of radiographic and histopathological changes with IL-17 and advanced oxidation protein products in knee osteoarthritic individuals with metabolic syndrome

Patient selection

Total 78 patients with advanced knee osteoarthritis, meeting the standards established by the American College of Rheumatology (ACR) [16] and undergoing total knee replacement surgery, were enrolled in the study at the Orthopedic Surgery Department of Medicare Cardiac and General Hospital, Karachi, from December 2022 to October 2023. The sample size was determined using the software “Select Statistical Services,” with the study power set at 80 % and a confidence interval of 95 %. The patients were divided into two groups: 40 in the MetS group and 38 in the non-MetS group. The study was granted approval by Institutional Review & Ethics Board (IREB) and Board of Advance Studies & Research (BASR) of Baqai Medical University, Karachi. Written agreement was acquired from the participants in compliance with the Declaration of Helsinki. The inclusion criteria comprised diagnosed cases of advanced knee OA patients with metabolic syndrome undergoing knee replacement surgery. The exclusion criteria ruled out patients who had a history of previous joint surgery or trauma, and those with preexisting conditions like various forms of arthritis, cancer or chronic inflammatory diseases, and patients currently taking steroids or anticancer medicines.

The diagnosis of metabolic syndrome followed the criteria outlined by the National Cholesterol Education Program Adult Treatment Panel III (ATPIII) for Asians, which defines metabolic syndrome based on the presence of three or more specified risk factors (components): higher waist circumference (102 cm (≥40 in) for men, 88 cm (≥35 in) for women), increased triglycerides (TG) (≥150 mg/dl), low high-density lipoproteins (HDL) cholesterol (<40 mg/dl in men, < 50 mg/dl in women), high BP (≥130/85 mmHg), hyperglycemia (≥100 mg/dl) [17].

Demographic and clinical parameters

A detailed demographic and clinical history of the patient along with various anthropometric measurements were carefully recorded (Table 1) maintaining the patient confidentiality. Patients were classified into three groups as per WHO classification of obesity according to their body mass index (BMI): normal weight (BMI 18.5–24.9 kg/m²), overweight (BMI 25–29.9 kg/m²), and obese (BMI≥30 kg/m²) [18].

Sample collection

Tissue processing and histological analyses

Histopathological evaluation

Articular cartilage was histologically graded according to the Osteoarthritis Research Society International (OARSI) score system [22].

The scoring of microscopic features of the IFP was graded as under:

1. Grade 0=no presence of lymphocytic infiltration;

2. Grade 1=presence of perivascular mononuclear cell infiltration;

3. Grade 2=both perivascular and interstitial mononuclear cell infiltration.

For analyzing vascularization in the IFP, a number of vessels were counted in four randomly selected fields from a single tissue slice at a magnification of × 200. Subsequently, the mean number of vessels was calculated [23].

Morphometric analysis

Images of H&E-stained sections of IFP captured at × 100 magnification were converted into binary images (black and white) for analysis using a microscope (Optika, Italy) equipped with specialized imaging and measuring software, Optika ProView software. Adipocytes were represented by white regions, while the black regions represented the boundaries, facilitating the calculation of adipocyte number and area [24].

Immunoassays measurements

The enzyme-linked immunosorbent assay kit from Human AOPPs Abbexa, Cambridge, and the Human IL-17 ELISA kit (R&D System, Minneapolis, MN, USA) were employed to measure the concentrations of AOPPs and IL-17 in serum and synovial fluid, following the instructions provided by the manufacturer.

Statistical analysis

SPSS statistical software, IBM-SPSS version 23.0 was used to perform the data analyses. Independent sample t-test was utilized for normally distributed values, while the Pearson chi-squared test was employed to compare observed frequencies of categorical data. Spearman correlation coefficients were used for ordinal data, and Pearson correlation coefficients were used for continuous numerical data to evaluate correlations after adjusting for age and gender, with statistical significance at a p-value <0.05.

Demographic and clinical characteristics of patients

In our study, we observed a higher frequency of OA in females, with mean ages of 63.9 ± 7.7 in the non-MetS group and 63.9 ± 6.8 in the MetS group (p>0.05) (Table 1). This finding is supported by various studies suggesting that beyond the age of 50, the incidence of OA in women increases more rapidly than in men, indicating a potential influence of menopause on OA progression [25]. Furthermore, OA patients in the MetS group exhibited higher mean BMI and triglyceride levels (p<0.04, p<0.001, respectively), whereas mean HDL levels were significantly less in the OA with MetS patients compared to the non-MetS group patients (p<0.001), as determined by the Independent sample t-test (Table 1).

Radiological and histopathological grading

In this study, the OARSI scoring system for cartilage degradation and the Kellgren–Lawrence (KL) grading system for radiological changes were used, as various studies have demonstrated their complementary roles in assessing different aspects of joint degeneration [15], 26]. The Pearson Chi-Square test revealed positive association of histological cartilage grade with IFP grade (p<0.05) between the studied groups. Conversely, KL grading demonstrated consistent results across groups, indicating an insignificant association between them (p<0.37) (Table 2). These findings align with those of Yasuda et al. who observed no significant association between radiographic changes in knee OA and individual or cumulative components of MetS [27]. However, contrary to these results, another study has reported significantly advanced radiological changes in the MetS group [28] (Figure 1).

Association of number of MetS components with histological and radiological severity of OA

The radiological (p=0.667) and histological grading of cartilage (p=0.061) in OA patients did not show a significant association with the number of components present in metabolic syndrome (MetS), likely due to the small sample size. In contrast, histological grading of the IFP significantly increased in OA patients with an increasing number of MetS components (p=0.004) using Pearson’s Chi-Square test. These results are in line with a study that demonstrates significant association of metabolic disorders with OA severity [1].

Figure 1:

Anterior-posterior (AP) radiographs of the knee from OA patients. (a) MetS group has marked osteoarthritic changes exhibiting, loss of medial joint space (white arrow head), moderate osteophyte formation (black arrow head), and discrete sclerosis with cysts formation (blue arrow head). (b) Non-MetS group shows advanced narrowing of joint space (white arrow head), osteophyte formation (black arrow head), and discrete subchondral sclerosis (blue arrow head).

Histological features of IFP and articular cartilage

Histological analysis of IFP in the MetS group revealed higher grades of inflammation, extensive fibrosis, and morphometric changes, such as increased vascularization (p=0.00) (Figure 3c) and adipocyte size (p=0.00) (Figure 3e), along with a diminished adipocyte cell count (p=0.00) (Figure 3d) compared to the OA non-MetS patients, as determined by the independent sample t-test (Table 2). These findings are supported by an earlier study reporting similar changes in the IFP of OA patients compared to patients undergoing anterior cruciate ligament (ACL) reconstruction [24].

Figure 2:

Histological examination of articular cartilage in OA patients. (2a) Non-MetS group indicates (i,ii) increased cartilage thickness (indicated on the scale), decreased surface erosion (blue arrow), intact tidemark (black arrow), organized chondrocytes (c), and a thinner subchondral plate (green arrow); (2 b) MetS group demonstrates (i) decreased cartilage thickness, more surface erosion (blue arrow), preserved tidemark (black arrow), and subchondral plate thinning (green arrow). (Ii) shows surface fissuring (blue arrow), tide mark duplication in early stages and presence of cell clusters near surface (c) (H & E stain, magnification×40). (2c) Comparison of articular cartilage thickness in OA patients between the MetS and non-MetS groups, (p=0.07).

Figure 3:

Histological analysis of infrapatellar fat pad (IFP) between studied groups in OA patients. (a) Histological characterization of IFP; (i) MetS group has increased collagen fibers density (blue arrow) and vascularization (black arrow). In contrast, (ii) non-MetS group shows lower collagen fibers density (blue arrow) distributed among clusters of adipocytes and fewer blood vessels (black arrow) (H & E stain, magnification×40). (b) Adipocyte morphology evaluation in (i) non-MetS group and (ii) MetS group shows an enlargement in adipocyte size and a reduction in the number of adipocytes in MetS group (p<0.05), (3c) comparison of number of vessels in the IFP of OA patients between studied groups, (p<0.05). (3d and 3e) Comparison of adipocyte cell count and adipocyte size in the IFP of OA patients between studied groups (p=0.00).

In MetS group, elevated grades of cartilage degeneration (p<0.05) were noted (Figure 2b, Table 2). However, mean difference in cartilage thickness was not significant among the groups (p=0.070) (Table 2, Figure 2). Pragasam and Venkatesan observed comparable changes in the articular cartilage in an animal-based study, associating them to MetS [29]. We also observed thickening of the subchondral plate, along with the infiltration of vascular elements from the subchondral bone and adjacent marrow space into the calcified cartilage. This process led to the duplication of the tidemark and localized thinning of the cartilage (Figure 2b). These findings are in accordance to earlier studies that have reported similar changes in the cartilage structure [14], 30].

Spearman’s rank correlation between histological data of the IFP and articular cartilage with molecular markers

BMI (p=0.000) and histological cartilage grade (p=0.007) showed positive correlations with the histological grade of the IFP of overall cohort. Moreover, IFP histological grading exhibited positive correlations with vascularization (p=0.001), and adipocyte size (p=0.013) while demonstrating a negative correlation with adipocyte cell count (p=0.007) and cartilage thickness (p=0.003) of overall cohort (Table 3).

Histological cartilage grade demonstrated a positive correlation with AOPPs levels in both serum and SF (p=0.011 & p=0.035 respectively), while IFP histological grading showed positive correlation with AOPPs levels in both SF (p=0.001) and serum (p=0.005) and IL-17 levels in both SF (p=0.005) and serum (p=0.033) in overall cohort (Table 3). There was no significant correlation of radiological grades with histological grades of cartilage (p=0.184) and IFP (p=0.935).

Correlation between molecular markers in serum and SF using Pearson correlation

A significantly positive correlation was found between serum IL-17 and SF IL-17 (p=0.00) (Figure 4a) and, similarly, a significantly positive correlation was observed between serum AOPPs and SF AOPPs (p=0.00) (Figure 4b).

Figure 4:

Graphical representation of correlation analysis of various markers among the investigated groups. (a) IL-17 levels in SF and serum (p<0.05). (b) AOPPs levels in serum and SF (p<0.05). (c) Levels of IL-17 and AOPPs in the serum (p<0.05. (4d) Levels of IL-17 and AOPPs in SF (p<0.05) using Pearson correlation analysis.

Furthermore, serum IL-17 exhibited a positive correlation with AOPPS in both serum (p=0.00) (Figure 4c) and SF (r=0.682. p=0.00). Likewise, SF IL-17 showed a positive correlation with the AOPPs in both serum (r=0.693. p=0.00) and SF (p=0.00) (Figure 4d).

Logistic regression analysis of samples from OA patients with MetS

In univariate model, cases of MetS group are more likely associated with obesity, high TG levels, low HDL, diabetes, high IFP grade, increase in vascularization, decrease in adipocyte cell count, increase in adipocyte size (area), high SF AOPPs levels, high SF IL-17 levels, and high serum IL-17 levels (Table 4).

Whereas in multivariate model after adjusted with age and gender, it was found that MetS group samples were more likely associated with high TG, low HDL, diabetes, high IFP grade, increased IFP vascularization, adipocyte cell count were less likely associated with, increased adipocyte size (area), high SF levels of AOPPs, and high serum levels of IL-17. Above findings showed statistically significant p-value (p<0.05) (Table 4).