In this study, we found that the plasma ccf-mtDNA content was higher in patients with KOA than in HC. In addition, ccf-mtDNA significantly correlated with KOA severity based on the Spearman analysis of the correlation of ccf-mtDNA levels with the X-ray KL classification and WOMAC scores. Logistic regression analysis revealed that ccf-mtDNA may be a risk factor for developing KOA. Therefore, ccf-mtDNA is a marker of mitochondrial dysfunction and cellular stress, and detecting its levels may help prevent and treat KOA.

Consistent with the epidemiological results, we included a high proportion of female participants (80%) in this study, indicating a greater prevalence of KOA among females than males. Additionally, 45% of patients in the KOA group were obese and the age of the patients increased as the K-L grade increased, suggesting that sex, obesity, and age may be risk factors in the pathogenesis of KOA, similar to the findings of a previous study [18].

Notably, the mitochondria do not have the same complex DNA repair mechanisms as the nucleus, and the absence of protective histones in mtDNA is responsible for the damage. Subsequently, the damaged mtDNA fragments are released into the cytoplasm or extracellular space [19, 20]. Following the release into the extracellular space, mtDNA fragments such as ccf-mtDNA can be detected in the blood or other body fluid samples [21, 22]. ccf-mtDNA behaves similarly to DAMPs by activating toll-like receptor 9 (TLR9), inflammatory vesicles, the interferon gene stimulator pathway, and the stimulator pathway to induce inflammation [23]. Increasing evidence suggests that innate response pathways may significantly influence the onset and progression of KOA, particularly TLR [24,25,26]. Reports revealed that TLR-9 activates nuclear transcription factor-κB (NF-κB) transcription factors, leading to the production of pro-inflammatory cytokines, thereby promoting the development of KOA [27]. Mitochondrial dysfunction maintains an active regenerative cycle through oxidative stress, increased ROS, and mtDNA damage, which are regarded as hallmarks of chronic degenerative diseases such as KOA. The accumulation of ROS and mtDNA damage can activate the NF-κB pathway, which majorly regulates inflammation [28]. Significant increases in ccf-mtDNA levels have been observed in diseases with chronic inflammatory states, such as trauma, sepsis, aging, cancer, and immune-mediated diseases [29,30,31,32,33]. In this study, we found that plasma ccf-mtDNA levels were significantly higher in patients with KOA than in HC. However, our findings are not consistent with those of Panagopoulou et al. Panagopoulou et al. did not find a significant difference in plasma ccf-mtDNA levels between patients with KOA and those of HC (P = 0.852) [34]. This inconsistency could be attributed to several factors. In their study, the comparison of ccf-mtDNA between patients with KOA and HC was a secondary analysis that did not strictly control for major confounders. Additionally, the study by Panagopoulou et al. had a small sample size, which may have led to unstable estimates. Finally, this inconsistency could also be due to other factors, such as differences in assay methods (TaqMan method vs. SYBR Green method) or patient ethnicity (Chinese vs. Greek). Hence, further large, well-controlled studies are required to validate these results.

Furthermore, we categorized patients with KOA into four groups following the X-ray KL classification criteria based on the severity of KOA imaging. Additionally, we used WOMAC to assess the severity of symptoms in these patients. Märtens et al. performed a study on the correlation of radiographic changes and pain sensation in shoulder osteoarthritis with patient age. The results show that age was positively correlated with the X-ray K-L classification; the higher the K-L classification, the older the age [35]. Similarly, in this study, we found that as the radiographic K-L grade increased, the age of patients also tended to increase. Furthermore, the WOMAC pain, stiffness, joint function, and total scores were significantly higher as the K-L grade increased, which suggests that the severity of KOA imaging findings is associated with the severity of its symptoms. However, further studies revealed that plasma ccf-mtDNA levels increased sequentially from KOA I–IV groups, suggesting that ccf-mtDNA may be involved in the development of KOA. Notably, joint pain, stiffness, and dysfunction are common symptoms of KOA that affect and quality of life of patients with KOA [36]. Spearman correlation analysis showed that plasma ccf-mtDNA positively correlated with the K-L classification, WOMAC total score, pain score, and joint function score; however, no correlation was found with the stiffness score, suggesting that plasma ccf-mtDNA levels could reflect the severity of KOA. Meanwhile, we analyzed the number of affected joints, and our results showed that ccf-mtDNA content did not significantly differ between patients with bilateral KOA and those with a single KOA. However, ccf-mtDNA content in patients with bilateral KOA tends to be higher than that in patients with single KOA, which may be attributed to the small sample size; hence partially masking the correlation between ccf-mtDNA and the K-L grade. Therefore, future studies with larger sample sizes are needed to investigate the relationship between ccf-mtDNA and the number of affected joints in patients with KOA.

In the early stage of KOA, before irreversible structural alterations occur in the joints, several biomarkers in blood already show alterations. These include inflammatory indicators such as C-reactive protein and interleukin-6 (IL-6), cartilage metabolism indicators, including type II collagen carboxy-terminal telopeptide and cartilage oligomeric matrix protein, lipid metabolism indicators such as cholesterol and fatty acids, and circulating nucleic acids such as microRNAs and cfDNA [37,38,39]. Detection biomarkers in plasma reflecting the severity of the disease can predict the destruction of bone and cartilage at an earlier stage, help to further understand the pathogenesis of KOA, and provide a new research direction for the treatment of the disease. The higher number of mtDNA copies makes it easier to detect mtDNA content than nuclear DNAs in body fluid samples, such as plasma and serum, where the total DNA concentration is deficient [40]. In addition, ccf-mtDNA as a liquid biopsy biomarker has the advantages of noninvasiveness and repeated sampling [41]. Therefore, detection of abnormal changes in ccf-mtDNA has become an increasingly important tool for early disease diagnosis. However, plasma ccf-mtDNA is a non-specific marker, as it is significantly elevated in other inflammatory immune diseases, mitochondrial diseases, and tumors [42,43,44]. Because most KOA patients are older, they are likely to have some associated comorbidities, which may lead to increased ccf-mtDNA levels. Therefore, future studies need larger sample sizes to control for confounding effects of other diseases through multivariate analysis. The irreversible nature of cartilage damage in the knee joints of patients with KOA makes it difficult to treat, and the etiology and pathogenesis of the disease have not yet been clarified. Therefore, it is clinically important to control the risk factors to reduce morbidity. Studies have shown that age, sex, and obesity are risk factors for KOA [45]. In this study, plasma ccf-mtDNA was found to be a pathogenic factor for KOA. In addition to the risk factors reported in previous studies, attention should be paid to patients with elevated plasma ccf-mtDNA levels so that timely measures can be taken to treat KOA.

In stratified analyses, we found that plasma ccf-mtDNA levels were significantly higher in patients with KOA; however, plasma ccf-mtDNA levels greatly differed between patients with KOA and HC, patients with age > 60.1, weight > 60, or BMI > 23.69 compared with patients with age ≤ 60.1, weight ≤ 60, or BMI ≤ 23.69. The close association between age, obesity, and KOA may have masked the relatively modest association conferred by a greater amount of ccf-mtDNA. Furthermore, because of the lack of statistical validity, we cannot exclude the possibility that a smaller number of participants may have contributed to these observations. Therefore, larger independent studies are needed to further explore the interaction between ccf-mtDNA and key demographic variables associated with KOA risk.

Previous findings suggest that ccf-mtDNA may contribute to inflammation and, therefore, may be directly involved in the pathogenesis of KOA. mtDNA activates TLR9 in the endolysosomal membrane when its fragments are released extracellularly and persist in the extracellular fluid, such as ccf-mtDNA. Therefore, activating NF-κB and the transcription of pro-inflammatory genes [46]. Notably, hydroxychloroquine has potential as a disease-modifying osteoarthritis drug (DMOAD) for treating KOA owing to its inhibitory effect on TLR signaling and ability to promote its degradation via a pro-inflammatory pathway [47]. Recent studies have shown that the cGAS-STING pathway may be activated by cytoplasmic mtDNA, and its activation by mtDNA leads to the expression of tumor necrosis factor-alpha (TNF-α) and IL-6, cytokines that are therapeutic targets for KOA [42, 47].

The present study has some limitations. First, only the plasma levels of ccf-mtDNA were measured in this study, whereas the levels of ccf-mtDNA in the joint fluid were not; therefore, the correlation between ccf-mtDNA and KOA in the joint fluid remains unclear. Second, this study was affected by many potential confounding factors. Future studies with large sample sizes, varying patient-level factors, and controlling for confounding factors are needed. Additionally, patients with KOA undergo surgical or exercise therapy shortly after admission, so we cannot determine the progression of their disease and, therefore, cannot detect the progression of these patients through analysis of baseline ccf-mtDNA. Hence, future community studies are needed to investigate the association between plasma ccf-mtDNA and the progression of KOA in patients. Finally, the study’s design was cross-sectional, which did not allow us to conclude a causal relationship between ccf-mtDNA and KOA development. More prospective cohort studies are needed to further confirm this finding.