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.