Immature articular cartilage and subchondral bone covered by menisci are potentially susceptive to mechanical load

In intact knee joints, tibial cartilage is divided into 2 regions: the cartilage covered by the menisci and that uncovered by menisci. Cartilage uncovered by menisci bears the body weight directly, whereas cartilage covered by menisci does so indirectly through the menisci. The pattern of loading during locomotion has a substantial influence on the general characteristic of the regional variation of cartilage [1]. Therefore, cartilage covered by menisci is thinner than cartilage uncovered by menisci [2]. However, it has been suggested that post-meniscectomy structural changes in the knee lead to alterations in the tibial contact area and cartilage thickness [3]. Kinematic changes have been implicated as a cause of osteoarthritis (OA) [4], and broaden areas of cartilage degeneration [5]. Recently, tibial cartilage covered by menisci may be damaged after meniscectomy exclusively [6]. Currently, these were thought to be because of increase contact stress following reduced contact area [3]. However, recent studies indicated that cartilage covered by menisci have different histological character compare to cartlilage uncovered by menisci [1, 7, 8]. Therefore, investigating the regional differences between cartilage covered by menisci and uncovered by menisci is important for understanding the mechanism of cartilage degradation after meniscectomy.

Meniscectomy have been performed not only to adult but also to children in such a case of discoid meniscus [9]. Caution is required when treating children because premature arthritis is common in patients who undergo total meniscectomy during childhood [9]. Previous study showed that injury to skeletally immature cartilage are vulnerable to load-induced injury than mature cartilage [10]. Therefore, understanding whether cartilage covered by menisci is susceptive to mechanical loading in immature knee is important for prevention of OA changes.

Evaluation of mechanical properties is important for investigating the cartilage degeneration process [11], and it is believed that changes in mechanical properties are superior to alterations in the composition of the cartilage matrix [12]. The mechanical properties of cartilage may vary according to whether it is covered or uncovered by menisci by the reason why mechanical environment differ in these regions [1, 7, 8]. Several studies showed that articular cartilage covered by menisci was stiffer [1, 7] and thinner [1] compared to cartilage uncovered by menisci. These studies focused on only the mechanical properties and histological findings of the main components of the cartilage matrix, such as glycosaminoglycan and collagen, in regions covered and uncovered by menisci. However, the mechanical properties of cartilage are affected by not only proteoglycan and collagen content [13‐15], but also water content [15] and collagen integrity [15, 16]. Therefore, the reason why mechanical properties differ in regions covered by menisci and uncovered by menisci is still unknown.

Of each of the cartilage layers, the superficial layer of articular cartilage is thought to be particularly important in determining its mechanical properties [17]. Recent studies have reported that superficial collagen have a role for countervail to compression stress and shear stress [17, 18]. Moreover, it is thought to have different collagen subtype, I, II, and III compare to middle layer, deep layer which have only type II collagen [19]. However, potential differences in collagen fiber structure between these two regions, especially in the superficial layer of cartilage, which affect cartilage mechanical properties, are yet to be determined.

Subchondral bone may have a substantial role in the OA process. In previous studies, knee radiographs have shown property changes and scleroses in the subchondral bone [20, 21]. In recent studies, these subchondral bone changes have been evaluated using micro-computed tomography (micro-CT) in animal OA models [22‐25] and human OA [26, 27]. These studies have shown that the components of subchondral bone architecture, including bone mineral density (BV/TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), were altered in OA and that these bone changes precede cartilage damage [28, 29]. These subchondral bone architecture thought to be associated with local load magnitude during locomotion, a spatial shift in the contact area could place loads on a region of subchondral bone that may not adapt to the rapidly increased load after meniscectomy [30]. Understanding site-specific subchondral bone architecture is important because extreme mechanical load transferred to the osteoporotic subchondral bone may cause microfracture, which may lead to scleroses of the subchondral bone [20]. Although mechanical load transferred to the subchondral bone may differ between regions covered and uncovered by menisci, to our knowledge, no study has focused on the differences in subchondral bone architecture between these regions.

Thus, the main aim of the present study was to investigate the mechanical and histological properties, particularly the collagen fiber structure of the superficial cartilage layer and aspects of subchondral bone architecture that may differ between cartilage covered and uncovered by menisci using immature knee. Our hypothesis is 1) the mechanical properties of the cartilage differ between in regions covered and uncovered by menisci, 2) collagen fiber of the superficial zone shows low density in cartilage covered by menisci, 3) subchondral trabecular bone architecture shows low density in cartilage covered by menisci.

In this study, we compared the mechanical and histological properties, water content, and SEM findings of cartilage and micro-CT findings of subchondral bone architecture between the regions covered and uncovered by menisci. Our results revealed differences in the mechanical properties and structures of articular cartilage and subchondral bone between the 2 regions.

We found that the cartilage was thinner in the regions covered by menisci (Figure 2). This result is consistent with that of previous studies [16, 33]. A report, which showed that the thickness of the cartilage covered by menisci changed after meniscectomy [33], indicated that menisci protect the cartilage covered by menisci from extreme direct mechanical loading.

The present study showed that the mechanical properties of the tibial articular cartilage differed between the regions covered and uncovered by menisci (Figure 3). Previous studies [8] have reported that the tibial plateau cartilage covered by menisci showed higher deformity to compression load than the cartilage uncovered by menisci and the result of the present study is consistent with previous finding. Although it is unclear what these differences in mechanical properties between the 2 regions may mean, our findings, showing low density of collagen in the superficial layer (Figure 6) may contribute to mechanical properties. Superficial layer of cartilage is component 10-20% of cartilage thickness [34] and it have ability to maintain fluid load support to compression loading [15]. In our study, creep normalized to cartilage thickness from indentation method is almost 20% in cartilage covered by menisci, nearly twice compare to cartilage uncovered by menisci. Therefore, our indentation methods may mainly reflects superficial structure and partly middle and deep layer structure. Water content is related to permeability [35, 36], which may indicate matrix resistance to pressure-induced fluid flow [37], fixed charge density [38], and superficial collagen integrity [18, 39]. Previous studies have determined high water content in degenerated cartilage and have reported that this cartilage shows greater deformation of the solid matrix [36, 37]. Our findings showing low proteoglycan content (Figures 4 and 5), low collagen density in the superficial layer (Figure 6), and high water content in covered regions (Figure 7) suggest that covered regions have lower matrix resistance to mechanical load, and that the cartilage in covered regions show high deformity to compression load.

In our study, attention should be paid for interpretation of the results. Because, previous studies showed growth effects on the collagen structure [40] and collagen content can double during the maturation process [41]. In addition, the organization of the collagen fibers progressed from a somewhat random collagen meshwork in immature cartilage to a more organized arrangement with maturation [42]. Previous studies showed that immature cartilage have a vulnerability of soft superficial layer to compressive injury [43]. Therefore, the histology and ultrastructure of collagen fibers by SEM observations from this study might not be translated to mature sample, additionally to human due to of anatomical differences [44]. However, our findings, which immature cartilage covered by menisci have low cartilage matrix, might partly support early degenerative changes in cartilage after total meniscectomy in child knee [9].

In OA, subchondral scleroses are one of the earliest radiographic changes detected. This change often occurs before joint space narrowing of knee OA [20, 21]. Once scleroses occur, bone volume and bone density increase, thus causing thickening of the subchondral bone [20, 45]. This change may be due to bone remodeling in response to repetitive microfracture by overloading [46, 47]. We observed that bone density was low in the covered regions (Figure 9A, B); this implies that subchondral bone in covered regions is weak with regards to excessive mechanical load, and that excessive repetitive mechanical load is the cause of microfractures and scleroses in the covered regions. Our results showing thinner cartilage, low collagen density, and low proteoglycan content in the superficial layer of cartilage as well as high water content in covered regions suggest that the cartilage in covered regions is susceptible to excessive mechanical load, and that extreme mechanical load is thereby transmitted to the subchondral bone more easily. Although this study was not performed in humans, the weakness seen in the subchondral bone together with the weakness of the superficial layer of cartilage in covered regions suggests that covered regions are more susceptible to subchondral microfracture and subchondral bone scleroses. Although the weakness to excessive mechanical load seen in the superficial layer of cartilage and subchondral bone in the covered regions is compensated for by the menisci, i.e., the meniscus–cartilage–subchondral bone unit receives mechanical load, this compensation does not occur after meniscectomy. Therefore, it is not the meniscus–cartilage–subchondral bone unit but the cartilage–subchondral bone unit in the covered regions that receives the mechanical load in this situation. After meniscectomy, the contact area between the femur and tibia changes [3] and contact mechanical load between the femur and tibia increases [3]. Therefore, meniscectomy leads to subchondral bone microfracture [48, 49], subchondral bone scleroses in covered regions [30], and cartilage degeneration [30, 33, 50]. Our findings strongly support the reports that suggest that subchondral bone scleroses and cartilage degeneration often occur after meniscectomy.

This study has several potential limitations. First, we used immature, 6-month-old porcine samples. There may have been some differences in collagen architecture and proteoglycan content as compared to mature porcine [51] or other species [43, 52], therefore our finding might not be translated directly to mature samples or to humans. Second, we obtained from tibial cartilage covered by menisci and uncovered by menisci, however the menisci are known to shift slightly during joint movement. Some of the region could receive mechanical loading both meniscus-uncovered and meniscus-covered, therefore the results cannot still be trusted to depend purely on the menisci, but instead they may reflect mechanical adaptation [53]. Third, we evaluated collagen density in superficial layer of cartilage using SEM system. Although SEM observation indicate lower collagen density in cartilage covered by menisci, which partly support immunohistochemical finding of type II collagen, this is a limitation preventing of results with statistical support because SEM observation is a qualitative evaluation. Fourth limitation is the technique used to measure mean cartilage thickness. In this study, we evaluated mean cartilage thickness of samples obtained from the osteochondral plugs by using a stereoscopic microscope. Thus, the mean cartilage thicknesses of the regions covered and uncovered by menisci may have differed a little from the actual cartilage thickness. Fifth, we evaluated bone architecture by determining BV/TV, Tb.Th, and Tb.Sp on the basis of the findings of micro-CT analysis; however, these measurements do not always provide proper reflections of the mechanical properties of subchondral bone [54].

In conclusion, our study confirmed that the cartilage covered by menisci differs from that uncovered with respect to its cartilage thickness, mechanical properties, proteoglycan content, water content, and collagen architecture. For the first time, we found that the collagen architecture of the superficial zone in covered regions showed a low density upon SEM analysis, and that the architecture of the subchondral bone covered by menisci showed a low bone density and thinner trabecular bone upon micro-CT analysis. These findings indicate that immature cartilage in covered regions is potentially susceptive to excessive mechanical load. This weakness may be compensated for by the menisci, which overcome mechanical loads in normal conditions. Therefore, our findings support the view that immature cartilage degeneration and subchondral microfracture occur easily after meniscectomy, which exposes the previously covered regions of the cartilage to direct mechanical load. Further study is needed to assess how structural changes and degeneration occur more easily in cartilage covered by menisci after meniscectomy in vivo.