2. Analysis of the human scapholunate ligament ECM
First, our analysis of elastic fibers as determined by VHF histochemistry showed very low amount of these ECM molecules all samples, with no differences among samples (Figure 3). Elastic fibers were thin and parallel oriented to the longitudinal axis of the abundant collagen network. Quantitative analyses showed significant differences only between the P2 region and FT and RT control tissues and between P1 and FT (Table 1). In contrast, the quantitative analysis of fibrillar collagen fibers identified by PSR histochemical staining revealed that all control tissues and the six regions of the SLIL analyzed here contained high amounts of collagen (Figure 3). Differences were statistically significant for most zones, especially for P1, which showed significantly lower amounts of collagen than the six control tissues (Table 1). Interestingly, comparisons between the two zones distinguished within each region in this study (D1 vs. D2, M1 vs. M2 and P1 vs. P2) were statistically significant for the amount of fibrillar collagen fibers identified by PSR (Table 1).
Then, we quantified three types of collagens by immunohistochemistry, and we found that, in general, control tissues tended to show higher expression of Col-I, Col-III and Col-IV than the SLIL, except for AC (Figure 4 and Table 1). For Col-I, we found that most SLIL regions significantly differed from all CTR tissues, with regions D2, P1 and P2 showing the lowest amounts of this type of collagen. Within the regions, we found that D1 was significantly higher than D2. Regarding Col-III, most SLIL regions expressed low amounts of these fibrillar components, except M2, which was highly positive for this ECM component. Most control tissues tended to express high amounts of type-III collagen, with the only exception of AC. Differences were significant for most comparisons, including the comparisons within each region, but were non-significant for the comparison of AC vs. D2, M1 and P1. For Col-IV, we found that most control tissues and SLIL zones contained very few amounts of this protein, whilst M2 was highly positive. Differences between M2 and all types of control tissues were statistically significant, as well as the differences between M1 and M2 (Table 1).
When the ECM proteoglycans were identified by AB histochemistry (Figure 5 and Table 1), we found that most regions of the SLIL were enriched in these non-fibrillar components of the tissue ECM, especially in the case of the zones M1, M2 and P2. However, the presence of these components was low in most control tissues, with the exception of AC, which was statistically higher than all the other samples (SLIL regions and controls). M1, M2 and P2 were statistically higher than all control tissues, whereas D2 was higher than D1 and P2, than P1. When versican was analyzed, we found that this protein playing an important role in ECM homeostasis was highly positive in D2 and P1, with significant differences with AC, CL and RT in both cases, whereas very low amounts of VRS were found in M1, which statistically differed from all control tissues except RT. Comparisons within the SLIL regions revealed statistical differences between M1 and M2 and between P1 and P2 (Figure 5 and Table 1).
In addition, the analysis of glycoproteins using PAS histochemistry (Figure 5 and Table 1) revealed that the highest expression corresponded again to AC control tissues. For the SLIL zones, M2, P1 and P2 showed the highest glycosaminoglycans content, with statistical differences with all the control tissues. Differences were also statistically significant for the comparison within each SLIL region (P1 vs. P2, M1 vs. M2, and D1 vs. D2).