Pauli C et al. used Pauli’s microscopic grading system to validate the changes observed in three separate areas (femoral side, tibial side, and inner border) of ageing and osteoarthritic (OA) menisci . In our study, the meniscal biopsies were taken only from the inner border and all the sample sections were cut from the site of the tear. Additionally, we focused on torn meniscus in this study, which is different from ageing and OA menisci based on in Pauli’s research. Therefore, we modified the Pauli’s grading system to make it more suitable for our research (Table 3). In our results, meniscal biopsies collected from the patients with meniscal tear (group B and group C) showed evidence of hyaline degeneration and loss of collagen fibre organisation. Although the features from the meniscal samples in both the groups were very different from those obtained from the amputee controls (group A), they were similar to those in the OA patients, as described by Pauli et al. . Vacuoloid changes and cell cluster formation in the meniscus may occur as a consequence of alterations in the tissue structure during OA development [28,29,30,31]. We found that the torn menisci in groups B and C exhibited vacuoloid changes or/and abnormal cell clusters. Consistent with our findings, Battistelli M et al. found vacuoloid changes in the menisci from traumatic and end-stage OA patients . However, contrary to Battistelli’s observation, we evaluated the score for the surface of the inner border in groups B and C to be 3 (Table 4), as we found that all torn menisci showed severe fraying and disruption on the surface. Microscopically, we found that there were varying degrees of degenerative pathological changes in the torn menisci versus normal menisci, thus providing new insights into the pathological changes in the meniscus after injury, especially when combined with a torn ACL.
In our research, a 34-year-old female patient in group C with a certain type of complex meniscal tear (flap and horizontal tear) in the posterior horn, who had undergone arthroscopic surgery 16 months after injury, showed vacuoloid changes, cell clusters, mostly unorganised collagen fibres and some hyaline degeneration. She was the only patient with severe meniscal degeneration (score of 8). Several studies demonstrated that age, gender, BMI, sports activities and time interval from injury to surgery influenced the risk of meniscal degeneration [8, 33]. Consequently, the combined injury pattern (meniscal tear and ACL tear) might not be the only factor contributing to meniscus degeneration in this case and other factors, such as higher age, feminine gender, longer interval between injury and arthroscopy, might also increase the risk of meniscal degradation as well. We will attempt to address these assumptions in our future studies.
It is considered that meniscus degeneration, similar to cartilage degradation, is due to metabolic imbalances and is characterised by increased synthesis and activity of matrix metalloproteinases (MMPs) and aggrecanases [11, 34]. Several studies have demonstrated that both MMP13 and ADAMTS5 are master ECM degenerative enzymes that have been previously considered to be major contributors to the development of joint degeneration [35, 36]. Brophy’ findings suggested that the expression level of MMP13 was higher in patients with a combined meniscal and ACL tear compared with the patients with only meniscal tear. However, there was no significant difference in the ADAMTS5 expression in both groups . Similarly, our results demonstrated that in the presence of meniscal tear, the expression of MMP13 and ADAMTS5 is elevated, especially when it is associated with ACL injury, but without any significant difference in the ADAMTS5 levels between the groups B and C. Moreover, these results were consistent with the immunohistochemical staining results. The overexpression of matrix-degrading genes is likely to be responsible for the degradation of both aggrecan and collagen. Besides, it is implied that the catabolic processes might have occurred in the meniscus after it was torn, and when combined with ACL injury, these substantial changes appear to become significant. Furthermore, the ECM degenerative enzymes that are produced in the meniscus probably not only act on the meniscus itself but can also be released into the joint cavity to degrade the cartilage, which could play a critical role in the subsequent articular degradation.
Conversely, when compared with patients with a normal meniscus, the expression of ACAN in patients with meniscal tear was decreased. Additionally, ACAN was expressed at dramatically lower levels in patients with a combined meniscal and ACL rupture. These findings indicate that the anabolic ability decreases in the lacerated meniscus, which could explain why the torn meniscus has less potential for repair, particularly when combined with ACL injury. In addition, the metabolism of the meniscal tissues resected during surgery may provide vital insights into the condition of the integral articular health, which is probably an important indicator for predicting future degradation and the latent risk for subsequent development of OA . However, contrary to the “significantly lower” values shown in Brophy’ results , we found the levels of ACAN were not significantly different between groups B and C. We speculate that this discrepancy might be due to the differences in the patients’ age as all the patients we recruited were under 40, which is in contrast with some of the patients in Brophy’ study who were 60 years old. Age might be a factor in influencing the gene expression in meniscal tissue.
Type X collagen (COL10A1) is a short, network-forming collagen specifically expressed by hypertrophic chondrocytes and is regarded as an important hypertrophic marker of chondrocytes [38, 39]. Behrendt reported that TNF-α increased the expression of COL10A1 in the meniscus of explants [38, 39]. Brophy suggested that COL10A1 was the most prominent mRNA to be elevated in OA meniscus compared to that in the injured meniscus . Similarly, our findings showed that meniscal tears result in elevated expression of the COL10A1 in the meniscal tissues compared with the normal meniscus. In addition, combined meniscal and ACL damage has a higher tendency to get exacerbated than insular meniscal damage. Furthermore, vacuoloid changes and cell clusters of fibrochondrocytes with reduced lacuna in the torn meniscus can be clearly seen via microscopic observation, suggesting that the phenotype of some fibrochondrocytes in the torn meniscus switch from normal to hypertrophic. It is well known that chondrocyte hypertrophy contributes to cartilage degeneration during the progression of OA . Therefore, this may be another piece of evidence to support that in the long-run, the meniscus tends to degenerate after meniscal tear, especially when accompanied by ACL rupture.
Recent research showed that CEBPβ, a transcription factor, is essential for the degeneration of cartilage in OA, which mediates the promotion of catabolic activities involving the up-regulation of MMP3, MMP13, and ADAMTS5 [16, 17]. CEBPβ is also an important regulator in facilitating the transition of chondrocytes from proliferative to hypertrophic form, which expresses type X collagen . Consequently, higher levels of CEBPβ in patients with a meniscal tear, particularly in those with meniscal and ACL tears, might be especially correlated to higher expression of the downstream genes relative to cartilage degeneration noted earlier, which ultimately lead to elevated degradation of ECM. In other words, our observations support the possibility that the up-regulation of CEBPβ can explain the overexpression of MMP13, ADAMTS5, and COL10A1. Yet, surprisingly, there is still a lack of compelling evidence in support of this hypothesis, and this warrants further investigation.
MicroRNAs (miRNAs), small non-coding single-stranded RNAs, play an increasingly crucial role in OA progression [18, 42]. Some authors have suggested that miR-193b-3p affects chondrocyte ageing by regulating aggrecan, type-II collagen, and SOX9 . Likewise, Tracey suggested that miR-455-3p exacerbates the process of OA by regulating TGF signalling and suppressing the Smad2/3 pathway . Taking the aforementioned points into account, we initially inferred that the four miRNAs were probably involved in the inflammatory responses of meniscus destruction. One attractive possibility is that they regulate the target upstream signalling pathways or specific transcription factors, such as CEBPβ, NFKBIA, Runx2, SOX5, SOX9, MAPK1, SMAD3, and BMPR2 , respectively or synergistically, which gives rise to aberrant expression of multiple catabolic and anabolic genes. However, the exact processes used by the four miRNAs were unclear. Therefore, further investigations, such as cell culture, animal experiment and luciferase assay, are required.
It is known that both the IKDC and Lysholm scales are perceived as valid, reliable, and responsive self-reported outcome measures . Our short-term results showed that the postoperative scores were significantly higher than the preoperative ones, suggesting that the clinical efficacy of arthroscopic surgery ranges from good to excellent. To minimise the effects caused by differences in the follow-up duration, we selected a minimum postoperative period of 15 months as the follow-up time, as we believe that after arthroscopy, normal knee function recovers to a relatively stable level in the patients within this duration. Nevertheless, regardless of Lysholm or IKDC scores, individuals with combined meniscal and ACL injuries showed less favourable postoperative outcomes than those with isolated meniscal tears, indicating that the knee with the associated rupture has lower functional rehabilitation and athletic ability. It was perplexing that there were significant differences in the Lysholm score while the IKDC score did not change significantly between the two groups after surgery. However, this inconsistency can be explained by the design of both the scores. The Lysholm score focuses on the symptoms and daily activities, while the IKDC score highlights the sports-related functions. Moreover, patients need more time to regain their exercise capacity after surgery.
Patients with combined injuries appear to have suffered more severe trauma and undergone a more complicated arthroscopic operation involving ACL reconstruction, which might be the most rational explanation for the lower scores. However, the discrepancy in postoperative outcomes also suggests that the joints in these patients might be more seriously damaged, which could be due to the torn meniscus not only at the biomechanics level but also at the molecular level. There was a moderately negative correlation between the MMP3:TIMP2/3 ratio in the knee synovial fluid and preoperative Lysholm score (the greater the ratio, the worse the Lysholm score) . Similarly, Scanzello et al. reported that CCR7 and CCL19 expression in the biopsies of knee synovium showed strong negative associations with preoperative Lysholm scores in meniscectomy patients. Additionally, IL-8 and CCL5 were moderately but not significantly associated with Lysholm scores . In the follow-up, the relative expression levels of CCL19 and CCR7 in the synovium were associated with greater postoperative improvements in the Lysholm score . Consequently, there might be a potential relationship between the preoperative or postoperative Lysholm score and the molecular markers in the meniscus that were shown in our results. This provides a good starting point for discussion and further research.
This study has several limitations. For instance, the number of patients is too small. Moreover, the general health conditions including sex and smoking status should be taken into account as well. Furthermore, our clinical follow-up results lack direct evidence, such as postoperative X-ray and magnetic resonance imaging results to demonstrate that patients with combined injury patterns have more serious degeneration of meniscus and articular cartilage compared with patients with isolated meniscal tears. Here, we have compiled most of the work, which has so far only scratched the surface of this prolific field of research, and further studies need to be performed to address these issues.