Analysis of meniscal degeneration and meniscal gene expression

Osteoarthritis (OA) is a disease characterized by the breakdown of articular cartilage and the formation of osteophytes. However, it has been gradually realized that OA is not merely an articular cartilage disease, but a disease of the whole joint [1, 2]. OA synovial membrane and subchondral bone have drawn considerably attention recently. Aberrant gene expression in OA synovium, OA fibroblast-like synoviocytes (FLS) and OA subchondral bone has been detected [3‐6]. These findings suggest that OA synoviocytes and subchondral bone cells may be involved in the disease process of OA.

Few studies have investigated the potential role of OA meniscal cells in the disease process of OA. The knee menisci are specialized tissues that play a vital role in load transmission, shock absorption and joint stability. Knee menisci may absorb most of the shock generated at the joint because their combined mass is greater than that of the articular cartilage. The current dogma is that menisci protect the articular cartilage, but play a minimal role in the disease process of OA unless they are injured. However, increasing evidence suggests that knee menisci may not be passive bystanders in the disease process of OA. It has been reported that meniscal degeneration is a feature of OA knee joints as revealed by magnetic resonance imaging [7, 8]. There is a strong association between meniscal damage/degenerative tears and cartilage loss [9]. In addition, it has been found that meniscal degeneration contributes to joint space narrowing [10]. Taken together, these findings and observations suggest that pathological changes and extracellular matrix degeneration also occur in OA menisci. OA menisci, similar to OA synovial membrane and OA subchondral bone, may play an active role in the disease process of OA.

In the present study, we examined the prevalence of meniscal degeneration in OA patients who underwent joint replacement surgery, and analyzed the correlation between the degeneration of menisci and the degeneration of articular cartilage. We also examined differential gene expression between OA meniscal cells and normal meniscal cells to test the hypothesis that OA meniscal cells are different from normal meniscal cells and may display a disease-specific gene expression profile. The determination of the differential gene expression between OA and normal meniscal cells may not only provide experimental evident to support our hypothesis, but also may reveal new therapeutic targets for OA therapy and uncover novel disease markers for early diagnosis of OA.

In the present study, we found that more than eighty five percent (85.7%) of the OA patients who underwent joint replacement surgery in our Medical Center had severe degenerative menisci (grades 4 or 3), indicating that meniscal degeneration is common in OA patients. We also found that meniscal degeneration correlated positively with articular cartilage degeneration in OA patients. The Spearman's correlation coefficient (r) was 0.672 (p < 0.0001). Our finding is consistent with previous reports that meniscal degeneration/tears is a feature of OA knee joints [7], that meniscal degeneration contributes to joint space narrowing [10], that there is a strong association between meniscal damage and cartilage loss [9], and that degenerative meniscal tears are positively associated with the severity of articular degeneration compared with other types of meniscal tears [14]. Taken together, our findings suggest that meniscal degeneration in OA, similar to cartilage degeneration, is a major degenerative process regardless of whether it is primary or secondary. Further studies such as examination of the complete patient histories may provide information regarding the cause-result relationship between meniscal degeneration and cartilage degeneration.

If meniscal degeneration is a general feature of OA, one would like to assess whether OA meniscal cells are different from normal meniscal cells and may play a role in the development of OA. To this end, we examined the differential gene expression between OA meniscal cells and normal control meniscal cells. We have recently reported that numerous genes that were classified in the biological process of immune response displayed elevated expression in OA FLS compared to rheumatoid arthritis (RA) FLS. HLA-DPA1 was expressed in OA FLS (hTERT-OA 13A FLS) 16 fold higher than that in RA FLS (hTERT-RA 516 FLS) [4]. Consistently, many genes that were classified in the biological process of immune response including HLA-DPA1 also displayed elevated expression in OA meniscal cells (Table 3). The findings indicate that OA meniscal cells, similar to OA FLS, may be involved in the inflammatory process observed in OA.

Calcium-containing crystals are found in the joint fluid of up to 65% of OA patients, and the presence of these crystals correlates with the radiographic evidence of cartilaginous degeneration [15‐18]. There is evidence indicating that crystals may promote joint degeneration [19‐21]. Most recently, it was demonstrated that the inhibition of meniscal calcification by calcium phosphocitrate, a potent anti-calcification agent, was accompanied by a significant reduction in the degeneration of articular cartilage in Hartley guinea pigs [22]. These data suggest that excessive meniscal calcification may play a role in OA. However, there have been no studies to investigate the alterations in OA meniscal cells or to identify the candidate disease genes that are potentially responsible for the excessive meniscal calcification in OA. In the present study, we found that many genes which are involved in biomineral formation such as ENPP1 [23], in phosphate metabolic process such as ITGB2 and in calcium ion transport such as calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C) are expressed at an elevated levels in OA meniscal cells compared to normal meniscal cells. These findings are consistent with clinical observations that meniscal calcification is more severe in OA menisci [24, 25] and that calcium content in OA menisci is positively correlated with the stage of meniscal degeneration [26]. In addition to the genes listed in Table 3, several other genes that have been previously implicated in pathological calcification were also detected (Table 4). They were ankylosis progressive homolog (ANKH) [27, 28], matrix Gla protein (MGP) [29, 30] and tuftelin (TUFT1) [31‐33]. Taken together, our findings suggest that OA meniscal cells may be actively involved in the meniscal calcification process in OA.

Many genes that have been previously found to be expressed at elevated levels in other types of OA cells or tissues such as in OA articular cartilage and OA bone were also detected in this study. They were integrin, beta 8 (ITGB8) [34], insulin-like growth factor binding protein 7 (IGFBP7) [34], fibromodul (FMOD) [28], cathepsin S (CTSS) [35], secreted frizzled-related protein 4 (SFRP4) [34], bone marrow stromal cell antigen 1 (BST1) [35], collagen, type XI, alpha 1 (COL11A1) [34], collagen, type V, alpha 1 (COL5A1) [34], PTGES [36] and ADAMTS5 [37]. These genes were expressed at significantly elevated levels in OA meniscal cells compared to normal meniscal cells (Tables 3 and 4). ADAMTS5 is a major cartilage matrix degrading enzyme and has been implicated in articular cartilage degeneration previously [38, 39]. The elevated expression of ADAMTS5 in OA meniscal cells suggests that ADAMTS5 may play a role in meniscal degeneration.

Furthermore, many genes that have been previously found to be expressed at decreased levels in other types of OA cells or tissues were also detected in this study. They are MMP10 [34], MMP12 [40], tenascin XB (TNXB) [34], fusion (FUS) [28], melanoma cell adhesion molecule (MCAM) [35], and T-box 5 (TBX5) [5]. These genes were expressed at decreased levels in OA meniscal cells compared to normal meniscal cells (Tables 3 and 4). The consistency between our findings and the previous findings provides strong support for our hypothesis that OA meniscal cells are different from normal meniscal cells and may play an active role in the development of OA.

Our study has some limitations which should be considered. The control meniscal cells we used were derived from the menisci of osteosarcoma patients and were not optimal normal control meniscal cells. To minimize this limitation, we only collected overtly normal-appearing meniscal and cartilage specimens (grade 0) from osteosarcoma patients whose tumors were located far away from the knee joints. In addition, we analyzed the differential gene expression using meniscal cells rather than using meniscal tissue specimens directly to eliminate the effect of different drugs that might be taken by OA and osteosarcoma patients at the time of surgery.

Most meniscal cells synthesize type I collagen as their major collagen product. The meniscal cells in the inner, central nonvascularized region of meniscus synthesize type II collagen. It is an open question at this time as to whether a single cell type exists in the meniscus that displays a fibroblast-like phenotype or a chondrocyte-like phenotype depending on its environment, or whether two or more distinct cell types exist. Therefore, it is not absolutely certain at this time whether the differences in the gene expressions detected in this study reflect the overall differences between OA meniscal cells and the control meniscal cells or only reflect the differences between a subpopulation of OA meniscal cells and a subpopulation of the control meniscal cells. Another limitation is that the age of osteosarcoma patients was younger than the age of OA patients. Therefore, certain genes we detected may be age-related rather disease-specific. It is difficult to obtain age-matched control meniscal specimens because osteosarcoma occurs often in younger patients while OA occurs mostly in older patients. We will continue this line of study when more age matched normal control meniscal specimens become available in the future. In spite of these limitations, the consistency between our findings and the previous findings that many of the genes we detected are also abnormally expressed in other OA cell types/tissues [4, 5, 23, 28, 34‐36, 40] suggests that many of the differential gene expressions detected in this study are disease-specific.

Our findings suggest that OA is not merely a cartilage disease, but also a disease of the menisci. OA meniscal cells may play an active role in the disease process of OA. Investigation of the gene expression profile of OA meniscal cells compared to normal meniscal cells may reveal new therapeutic targets for OA therapy and uncover novel disease markers for early diagnosis of OA.