Osteoarthritis (OA), the most common form of arthritis, is a degenerative disorder that disrupts the musculoskeletal system (1). Despite the identification of mechanical, biochemical, and genetic factors, the precise etiology of OA is not entirely elucidated (2, 3). OA is the 11th leading cause of global disability (4), with knee joint involvement being common (2, 5). Knee OA (KOA) affects 3.6% of the total population (nearly 250 million people) worldwide, whereas the prevalence of symptomatic KOA is almost 35% among adults over the age of 60 years (6–9).

In addition to well-known risk factors for KOA, including age, race, sex, prior joint injury, and anatomical joint shape (1, 10), oxidative stress and inflammation also contribute to the pathogenesis of KOA (11–14). Oxidative stress is caused by the overproduction of reactive species, known as pro-oxidants, and the incapability of the antioxidant defense system to scavenge these species, which consequently causes cellular damage and destruction (15, 16). Previous studies have demonstrated the upregulation of reactive species in chondrocytes and cartilage tissue of patients with OA (10, 14, 17, 18). Studies in models of OA have also demonstrated that scavenger enzymes in cartilage are reduced (19, 20).

Matrix metalloproteinases (MMPs), a family of extracellular proteases, are responsible for collagen degradation in the cartilage of OA joints (21). MMPs, especially MMP-1 and MMP-13, cause irreversible destruction of the cartilage matrix through the digestion of type 2 collagen and the subsequent release of matrix proteoglycan from the cartilage (22). The involvement of oxidative stress in MMPs expression has been well documented (23, 24). Accordingly, treatment with antioxidant compounds has been associated with reduced MMP-1 and MMP-9 expression in previous studies (25).

Vitamin D is a well-known main factor for skeletal health and normal bone and cartilage metabolism. It has been proposed to have a protective role against oxidative stress by activating the protective nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-KEAP1) antioxidant pathway. Therefore, vitamin D may exert its antioxidant effects by up-regulating some of the antioxidant enzymes (26, 27). In addition, it may have potential modulating effects on the expression of certain MMPs (28). Low serum levels of 25(OH)D, the biomarker for vitamin D status, have been associated with increased knee cartilage loss (29), cartilage structural changes (30), quadriceps muscle weakness (31), and more incredible knee pain (32) in patients with KOA. Furthermore, Zhang et al. (33) indicated that the risk of progression of OA doubled in participants with low levels of vitamin D. Very little, however, is known about the biological mechanisms via which vitamin D is involved in preventing OA progression (34). To date, findings on the relationship between vitamin D status and oxidative stress biomarkers are mostly related to investigations done on patients with other illnesses, such as multiple sclerosis (35), liver disease (36), and obesity (37). However, the question of whether the conclusions of these patients can also be generalized to patients with KOA remains unanswered. In addition, the available data regarding the relationship between vitamin D status and MMPs in patients with KOA are limited. Therefore, we conducted a case–control study that aimed to evaluate the association between vitamin D status with oxidative stress biomarkers and serum levels of MMPs in patients with KOA.

General characteristics of the study participants according to their vitamin D status are presented in Table 1. In this case–control study, the mean age of 65 healthy controls was 48.1 ± 7.8 years and 47.75% of them were males. Of 124 patients with KOA, 60 patients (48.38%) had sufficient levels of vitamin D (41.8 ± 9.2 ng/mL) and 64 patients (51.62%) had insufficient vitamin D (19.7 ± 5.5 ng/mL) levels. The mean serum levels of vitamin D in healthy controls were not significantly different from KOA patients with sufficient vitamin D levels (42.7 ± 9.6 ng/mL versus 41.8 ± 9.2 ng/mL). In patients with sufficient vitamin D levels, the number of people who took vitamin D and calcium supplements was significantly higher compared to the KOA patients with insufficient vitamin D levels and healthy controls (60% versus 39.1 and 40%, respectively; p = 0.031). There were no significant differences in demographic and body composition measures among the study groups (p > 0.05).

Table 2 indicates the serum concentrations of oxidative stress markers in healthy controls and patients with KOA according to vitamin D status. KOA patients with vitamin D insufficiency had higher levels of MDA, TOS, SOD, OSI, and lower levels of PON-1 and TAC versus KOA patients with sufficient vitamin D status (p < 0.05). The differences in CAT and GPx were not significant among the study groups (p > 0.05).

Figure 1 indicates the serum concentrations of MMP-1, MMP-3, MMP-13, and COMP between healthy controls and patients with KOA according to vitamin D status. Among the three groups, patients with insufficient vitamin D levels had significantly higher MMP-1 and MMP-13 levels compared to the sufficient vitamin D patients (p < 0.0001) and healthy controls (p = 0.001). No significant differences were seen in MMP-3 and COMP among the study groups (p > 0.05).

The results of multiple linear regression analysis investigating the association between serum 25(OH)D and oxidative stress biomarkers and matrix metalloproteinases are shown in Table 3. After controlling for potential confounders including age, sex, BMI, cigarette smoking, physical activity, and use of vitamin D and calcium supplements, serum 25(OH)D levels were inversely correlated with MDA, TOS, SOD, OSI, MMP-1, and MMP-13 and positively associated with TAC (p < 0.0001). The associations of 25(OH)D levels with GPX, CAT, PON-1 MMP-3, and COMP levels were not significant.

According to our results, KOA patients showed lower vitamin D and TAC levels and higher levels of MDA, TOS, and OSI compared to the healthy controls. The levels of SOD in KOA patients who had vitamin D insufficiency were significantly higher, and the levels of PON-1 were significantly lower compared to the KOA patients with sufficient vitamin D. Significant inverse relationships were seen between serum 25(OH)D and MDA, TOS, SOD, OSI, MMP-1, and MMP-13. However, serum 25(OH)D was positively associated with TAC. As far as we know, the current research is the first case–control study on the association between vitamin D status and oxidative stress markers and MMPs in patients with KOA.

According to the clinical practice guidelines of the Endocrine Society Task Force on vitamin D, the sufficient amount of serum 25(OH)D levels for overall health and bone in normal people should be ≥30 ng/mL. Serum vitamin D concentration of <12 ng/mL is considered vitamin D deficiency which leads to osteomalacia in adults and rickets in children (47). Vitamin D is involved in bone mineralization and bone health (48, 49). It has a protective role against KOA development (33). However, the related mechanisms are not well recognized (34). Inflammation and oxidative stress are involved in the pathogenesis of KOA (11–14). Vitamin D has anti-oxidative and anti-inflammatory activities, making it able to upregulate some of the antioxidant enzymes (26, 27) and modulate the expression of immune-related factors and certain MMPs (28). The associations between 25(OH)D and oxidative stress biomarkers (35–37, 50), as well as some MMPs (51), have been demonstrated. However, these associations have not been investigated in patients with OA.

We showed a lower level of vitamin D and a higher level of oxidative stress biomarkers in KOA patients compared to healthy control subjects, as shown by increased MDA levels, TOS, and OSI, as well as decreased TAC levels. Consistent with the present study, in a 2-year observational study, insufficient levels of vitamin D (<30 ng/mL) was significantly higher among young KOA patients (46.57%) compared to normal cases (24%) (52). Furthermore, Zhang et al. indicated that the risk of progression of OA doubled in participants with vitamin D levels of <15 ng/mL (33). Nonetheless, a meta-analysis reported no relationship between serum 25(OH) vitamin D levels and the KOA risk, however, a significant association was detected between lower vitamin D concentrations and KOA progression (53). The discrepancy between the results of different studies could be due to the different cut-off points defined for vitamin D levels. In a survey of 1,478 children aged 7–11 years, serum 25(OH)D concentrations of <20 ng/mL were defined as vitamin D deficiency, between 20 and 30 ng/mL were defined as vitamin D insufficiency, and > 30 ng/mL were defined as vitamin D sufficiency. In this study a negative relationship between serum vitamin D levels and markers of oxidative stress was declared (54).

In another cross-sectional study, insufficient 25(OH)D levels of <20 ng/mL were associated with increased markers of endothelial activation, oxidative stress, and inflammation in obese children (37). These associations were also reported in patients with chronic hepatitis C (55). All of these findings are in agreement with our results. On the other hand, daily supplementation with 5,000 IU vitamin D in patients with diabetes for 12 weeks did not affect biomarkers of oxidative stress (56).

The relationship between vitamin D and MMPs is observed in some in vivo and in vitro studies. 1,25-dihydroxycholecalciferol hindered MMPs activity and expression in leucocytes infected with Mycobacterium tuberculosis (57). In addition, vitamin D inhibited the expression and activity of MMP-9 in human lung fibroblasts (58) and MMP-2 in patients with chronic rhinosinusitis (59).

Insufficient vitamin D levels and their effect on degenerative alterations and articular cartilage increase the risk of OA (49, 60). Various mechanisms might describe the association between vitamin D with oxidative stress markers and MMPs. Vitamin D activates Nrf2 gene expression, which regulates some antioxidant and detoxifying enzymes (61–63) and has an inverse correlation with the accumulation of mitochondrial reactive oxygen species (ROS) (64, 65). Thus, Nrf2 protects cells against oxidative stress (66). On the other hand, it has been shown that 25(OH)D blocks interleukin-1β mediated inhibition of tissue inhibitors of MMPs (58).

It is not known whether oxidative stress markers and MMPs are expressed and produced within the OA joints as well as if the serum levels of these markers could reflect the local levels of them in the synovial fluid of the knee. Further investigation is required to clarify this.

We, for the first time, evaluated the relationship of vitamin D status with oxidative stress biomarkers and MMPs in KOA cases and control cases with sufficient and insufficient vitamin D levels. The subjects were geographically the same, with approximately equal rates of sun exposure. Also, our panel of biomarkers was fairly comprehensive concerning oxidative stress status (MDA, TAC, TOS, SOD, GPX, CAT, PON-1, and OSI) and MMPs (MMP-1, MMP-3, and MMP 13). However, the study also had some limitations that should be considered. Because of the study case–control design, we cannot report cause and effect relationships, and the findings could be biased due to unrecognized confounders, similar to observational studies. We did not use magnetic resonance imaging which would provide more information about the early KOA. Moreover, no radiographic examination of the knees was performed for healthy participants, so asymptomatic KOA may be present in the control group. We could not able to assess the subject’s dietary intakes to study the dietary total antioxidant capacity. Further research is necessary to clarify the mechanisms underlying this association.

We provided further evidence indicating that KOA patients have lower vitamin D levels than control cases without considering taking vitamin D supplements. Also, a reverse association between vitamin D status and serum concentrations of oxidative stress biomarkers such as MDA, TOS, SOD, and OSI, as well as MMP-1 and MMP-13 was detected. TAC was positively correlated with serum vitamin D levels. More studies with prospective designs and large sample sizes are needed to prove these results.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Zabol University of Medical Sciences (Code: IR.ZBMU.REC.1398.208). The patients/participants provided their written informed consent to participate in this study.

FA and SG contributed to study concept, search, data analysis, and drafting of the manuscript. SA and SH-S contributed to data processing, data analysis, and drafting of the manuscript. SA supervised the research. All authors read and approved the final manuscript.

This study was supported by Zabol University of Medical Sciences, Zabol, Iran.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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