DISCUSSION
Despite its crucial role as a major stabilizer of the wrist bone, the exact structure of the human SLIL is not properly understood. For this reason, in the present study, we evaluated the human SLIL using an array of histological, histochemical and immunohistochemical methods, trying to shed light on the specific composition of each zone of the SLIL. In the first place, we analyzed each SLIL region after establishing two subregions in each of the classically defined regions (dorsal, membranous and palmar) (Berger, 1996; Berger et al., 1991). This subdivision of each region was done to define the histological characteristics of each zone of the ligament with higher precision. In fact, the original classification was made based on the gross aspect of the SLIL, what may not coincide with the histological features of each zone of the SLIL.
The results of our histological analysis revealed that the SLIL was heterogeneous, and each subregion had specific histological characteristics. First, our histological analysis using HE staining confirmed that the dorsal region of the SLIL consisted of a dense ECM with dispersed elongated cells that partially resembles a human ligament, as other authors already demonstrated (Berger, 1996; Berger et al., 1991; Berger & Blair, 1984; Sokolow & Saffar, 2001). However, the three-dimensional disposition of the fibers and the cell distribution were different in both subregions of this area (D1 and D2), which supports the idea that the dorsal area consisted of two distinct zones. Then, the analysis of the membranous region revealed the presence of a dense tissue containing cells resembling human chondrocytes that were surrounded by a well-defined pericellular matrix and a capsule, as it is the case of the human cartilage. Although the similitude of the membranous region with human fibrocartilage was previously preconized (Berger, 1996; Berger et al., 1991; Berger & Blair, 1984), we found that the ECM was more dense in M2 than in M1, and both the cells and the ECM in M1 were more similar to hyaline chondrocytes than to fibrochondrocytes, supporting again the possibility that both subregions could be histologically different. Finally, our characterization of the palmar region revealed a structure containing abundant fibers and scattered spindle-shaped cells, as previously described (Berger, 1996; Berger et al., 1991; Berger & Blair, 1984). Again, differences in fiber orientation and cell content were detected between P1 and P2, suggesting again that this region could be heterogeneous.
To confirm our hypothesis that each region could be heterogeneous, we analyzed the ECM composition at each subregion. The ECM plays a key role in controlling the physical properties of human tissues, supporting compressive and extension forces and providing resilience and shock absorption capacity, especially during continuous biomechanical stress (Hoffmann et al., 2019). For this reason, understanding of the ECM configuration at each subregion of the SLIL could contribute to understand the pathomechanics of the human carpus (Wolff & Wolfe, 2016). In this milieu, we first analyzed the presence of ECM fibers in the SLIL and compared the results with control tissues. Regarding elastic fibers, we found that these components were very scarcely present in the SLIL and in control tissues. Although elastic fibers have been found in high amounts at certain ligaments such as the nuchal ligament or the ligamentum flavum, its presence is location-specific, and many ligaments have very low amounts of these fibers (Hill et al., 2020). Despite the low amount of elastic fiber found within the SLIL, it is probably that these fibers, which run parallel to the collagen networks, contribute to the resistance of this complex structure to tensile or shear stress (Henninger et al., 2019).
Then, we analyzed the presence and distribution of collagen fibers. Collagen is the main fibrillar component of the ECM and plays a crucial role in controlling tissue stiffness and resistance to tensile forces, as these fibers can store elastic energy by stretching the flexible regions of the fibrils in their triple-helix tridimensional structure (Silver et al., 2003, 2021). Collagen fibers are typically very abundant in mature tendons and ligaments (Barros et al., 2002) and in other mechanically demanding areas, where they are the main responsible for mechanotransduction of incoming forces. As expected, we found that all SLIL regions and control tissues contained high amounts of collagen fibers, with some differences among samples. Among controls, the higher concentration of collagen was found in AC, which is known to contain high amounts of these fibers (Bloebaum et al., 2021). Regarding the SLIL, the highest contents of collagen fibers were found in D1 and M1, whereas the lowest amounts of collagen corresponded to both subregions of the palmar region (P1 and P2). These results are in agreement with previous biomechanical studies suggesting that the palmar region could be mechanically weaker than the dorsal region (Kakar et al., 2019), and could contribute to understand why most SLIL lesions begin with a disruption of the palmar region of this structure (Andersson & Garcia-Elias, 2013). Interestingly, differences between both subregions of each region (D1 vs. D2, M1 vs. M2 and P1 vs. P2) were statistically significant, suggesting again that each region could be heterogeneous. Strikingly, our analysis of specific types of collagens also revealed differences among areas, with the highest contents of collagens types I, III and IV corresponding to the M2 subregion, with significant differences between both subregions of each region for some collagen types. Specifically, M1 was significantly different to M2 for collagens III and IV. The fact that M2 showed the highest contents of the three types of collagens is consistent with the possibility that M2 could be more similar to a hyaline cartilage than a fibrous cartilage, since it has been demonstrated that hyaline cartilage contains high amounts of collagens I, III and IV (Alcaide-Ruggiero et al., 2021).
In addition, we assessed the presence of relevant non-fibrillar components of the ECM in the tissues analyzed in this work. Non-fibrillar ECM components are fundamental molecules able to control the biomechanical properties of human tissues by regulating the tissue response to external mechanical forces (Ghadie et al., 2021). Specifically, proteoglycans and glycosaminoglycans mediate collagen fibrils alignment and regulate water content of the ECM, which in turn, is able to control tissue stiffness (Müller et al., 2004; Smith & Melrose, 2015). Our results showed that the human SLIL was enriched in these components, and proteoglycans and glycoproteins tended to be more abundant in SLIL than in control tissues, with the exception of AC, which showed the highest contents of all tissues. Interestingly, the membranous and palmar regions displayed the most intense signal for both types of components, although the levels of AC were not reached. When versican was analyzed, we found that the lowest expression of this proteoglycan corresponded to AC, M1, M2 and P2. Again, significant differences were found between both subregions of the dorsal, membranous and palmar regions for all non-fibrillar components, except for some specific comparisons.
In order to furtherly characterize each zone, we analyzed the phenotype of cells found at each area using S100 immunohistochemistry, a typical cartilage-linked marker that is especially expressed by hyaline chondrocytes (Yammani, 2012). The finding that cells were positive at the M1 area followed by M2, along with the ECM structure showing the typical capsule and ECM arrangement of the human cartilage, confirms the cartilaginous nature of these zones. Furthermore, the analysis of blood vessels showed very few vessels at M1 and M2, as it is the case of cartilage. In general, these results, support the idea that the membranous region could indeed be composed by a variety of cartilage tissue, and that M1 could be more similar to hyaline cartilage than M2.
Finally, we aimed to assess the presence of stem cells at each zone of the SLIL in order to identify those areas that could have higher self-regeneration potential. Our results suggest that the SLIL in general contains low amounts of stem cells, what is in agreement with previous reports questioning the intrinsic healing capability of the human SLIL (Minami et al., 2003). However, the fact that subregion D2 could be more enriched in stem cells than other areas may imply that the dorsal area has more intrinsic regenerative potential, what could be related to the presence of stronger mechanical forces affecting the dorsal region of the SLIL, as compared with other regions (Kakar et al., 2019).
Altogether, these results support our hypothesis that each region of the human SLIL could be structurally heterogeneous. In fact, a global analysis of the ECM evaluation results using hierarchical clustering confirmed the existence of differences within the SLIL. Remarkably, the D1 zone was structurally more similar to ligaments and tendons used as controls than to other areas of the SLIL, although the control tissue showing the lowest distance with D1 was a fibrocartilage (TF). Thus, our results suggest that the rest of SLIL zones shared ECM composition similarities with hyaline cartilage (AC), with M1 showing the closest distance with AC. Although the SLIL is generally considered to be histologically related to a human fibrocartilage (Berger, 1996; Sokolow & Saffar, 2001), our comprehensive analysis suggests that this structure could be more close to an articular cartilage, with high heterogeneity among zones.
The present study has several limitations. First, results should be confirmed in a larger cohort of human hand donors. Second, biomechanical studies should be carried out in the future to correlate our histological results with biomechanical data of stiffness and elasticity of each specific zone of the SLIL. Finally, the study could be complemented with the implant of different biomaterials or bioartificial tissues generated by tissue engineering to determine the potential usefulness of this approach to repair the human SLIL, as previously reported for other wrist ligaments (Lui et al., 2021).
To our knowledge, this is the first study that characterized the SLIL after stablishing a subclassification of the classical SLIL regions. Overall, our results support the idea that each subregion has a definite structure and composition not only at a morphological level, but also when the expression of relevant tissue components were analyzed. These results could be clinically relevant, as they could contribute to explain why most disruptions of the human SLIL typically commence at the palmar region, and support the surgical approaches based on a reinforcement of this region. In addition, the higher similitude of the SLIL with a cartilage-like tissue contributes to understand the poor regenerative properties of the human SLIL, especially in the areas devoid of blood vessels. However, the heterogeneity of this structure supports the idea that certain zones, such as D2, could have more intrinsic regeneration potential derived from the presence of stem cells. In consequence, lesions affecting the zones with lower regeneration potential, especially M1 and M2, should be treated with the use of tissue grafts containing mesenchymal stem cells or other approaches based on tissue engineering and regenerative medicine, as suggested for the human knee meniscus (Kwon et al., 2019). In addition, our results suggest that surgical repair of injuries affecting the membranous region should preferentially be repaired using cartilage grafts that can be obtained from articular surfaces, meniscus grafts or other similar anatomical regions, as described for the knee joint repair (Ashton Tan et al., 2022). However, surgical treatment of the dorsal region of the SLIL could be favored by the use of grafts whose histological structure is more similar to this region, such as the human ligaments and tendons. Future works carried out in vivo should confirm or not this statement.