hAF matrix structural analysis with herniation progression
The hAF was obtained from herniated IVD biopsies collected from patients undergoing microdiscectomy. A macroscopic dissection of IVD samples to isolate the AF tissue was first performed (Fig. 1A). hAF from herniated samples was separated into three categories, according to the information provided by the neurosurgeon: protused, contained and uncontained (Fig. 1B). As controls, hAF was obtained from non-herniated IVD of AIS patients [n = 6, median age 16 (15–19.5 years old), 3 males/3 females]. Age, gender and Pfirrmann grade distribution per hernia type can be observed in Fig. 1 C1, 1C2 and 1C3, respectively. Gender distribution is similar in the scoliosis and uncontained groups. In contained hernias, almost two thirds of the samples were from females, and in protused samples, the reverse was observed. Pfirrmann grade distribution among herniated samples shows a low presence of samples with grade V in all categories. In addition, the samples with Pfirrmann grade III and IV are evenly distributed, except in contained hernias, in which samples with Pfirrmann grade III are more numerous (Fig. 1 C3).
ECM ultra-structural alterations in hAF were first analysed, regarding matrix density, collagen fibres maturation and distribution/orientation. For that, TEM analysis was performed in representative samples from hernias with distinct containment levels (Fig. 2A). Highly organized collagen fibre bundles (white arrows) together with a few disorganized fibres were observed (red arrows), both in scoliosis (Fig. 2A, a, e, i) and in protused hernias (Fig. 2A, b, f, j). In the contained hernias, a disorganized fibre arrangement was more prominent (red arrows), although some organization (white arrows) was still present (Fig. 2A, c, g, k). On the other hand, uncontained hernias exhibit loss of lamellar organization of the fibres, being these randomly dispersed within the tissue (red arrows) (Fig. 2A, d, h, l).
In addition, the analysis of the thickness of collagen fibres was performed by the analysis of PSR staining under polarized light, as previously described [32]. This evaluation allows the identification of collagen fibres thickness, depending on their birefringence under polarized light. The visualization of red and yellow collagen fibres correspond to thicker fibres, while green correspond to thinner fibres, related with more youthful collagens [33]. This analysis is usually associated with Col I and collagen III (Col III) fibres, although, in this study, the fibres are most probably associated with Col I, since Col III is not abundant in AF and is described only as a pericellular protein within the tissue [34, 35]. Nevertheless, this analysis should be performed using second harmonic microscopy [36]. Figure 2B shows representative images of hAF samples stained with PSR and imaged under polarized light, while Fig. 2C shows the proportion of the collagen fibres (in red, yellow and green) for the different conditions analysed. Results clearly show a lower frequency (< 7%) of the red birefringence (thicker fibres) for all the samples, in comparison with the more mature fibres (yellow/green birefringence). Moreover, fibres with red birefringence were reduced in the herniated samples, compared with AIS. On the other hand, the thinner fibres (green) were present in higher proportion (38–86%) in all the conditions analysed, while the frequency of collagen fibres with intermediate thickness (yellow) range from 12 to 60%. Together with thicker fibres, the intermediate size collagen fibres are also reduced in all herniated samples compared with AIS control, more specifically in protused hernias (12%, **p < 0.01). These fibres slightly increase in contained hernias (30%, p = 0.0718) compared with protused hernias.
In what concerns thinner collagen fibres, an opposite trend is observed. In this case, the AF from all herniated discs present a higher proportion of green fibres, compared with AIS discs, being this significantly higher in the case of protused hernias (87.85%, **p < 0.01). With herniation progression, the presence of these fibres is slightly reduced (69.35%, p = 0.0656 contained hernias). These results suggest an increased synthesis of new collagen fibres in the AF from herniated discs, compared with AIS controls, particularly in protused hernias.
hAF matrix biochemical analysis with herniation progression
A biochemical analysis of the ECM of hAF was performed by histological/IHC analysis of Col I, Col II, FN and sGAG, generally lost during fibrosis (Fig. 3A) (low magnification images are presented in Supplementary Figure S2). From the images, the percentage of staining area was quantified and the results are presented as the median percentage of area or positive cells and respective interquartile range for each marker (Fig. 3B). Figure 3A (a–d) shows representative images for sGAG staining (blue) in the hAF of different samples. By AB/PSR staining, it can be observed a clear higher stained area of sGAG (blue) in hAF from most AIS samples [77.1% (43.8–90.6)] compared to all herniated samples, particularly in contained hernias [7.1% (2.1–21.3), p = 0.0515] and uncontained hernias [1.78% (0.2–81.9), p = 0.0555].
Regarding the expression of Col I (Fig. 3A (e–h) and 3B), it is highly present in AF tissue from AIS samples (Fig. 3A, e) [84.0% (65.4–98.1)] and significantly decreased in the AF of all herniated samples [8.0% (3.1–16.0), **p < 0.01, in protused hernias; 11.9% (2.2–49.7), **p < 0.01, in contained hernias; and 13.5% (1.4–47.4) *p < 0.05, in uncontained hernias] (Fig. 3A, f–h). Col II stained area was also assessed (Fig. 3A (i–l) and 3B), showing an opposite trend of Col I. The stained area of Col II is lower in hAF from AIS samples [20.5% (3.5–35.8)] (Fig. 3A, i) increasing in hAF from herniated samples (Fig. 3A, j–l). This increase is close to significant in hAF of contained hernias [65.4% (32.8–82.5), p = 0.0717] and uncontained hernias [68.9% (26.8–88.8), p = 0.0976].
Relatively to FN expression (Fig. 3A (m–p) and 3B), the results show low and heterogeneous FN expression in protused hernias [0.5% (0.04–15.8)], with 2 (out of 9) samples showing higher FN stained area (about 25.8 and 30.3%). Nevertheless, FN expression tends to increase in hAF from herniated IVD, being significantly higher in contained hernias samples [23.5% (3.2–59.0), *p < 0.05], and in uncontained samples [14.8% (6.6–42.8), p = 0.0516].
In addition, to discard that the differences observed might be due to age differences, the interaction between the variables “age” and “hernia containment” for all the markers analysed was addressed using a multivariate analysis. The p values obtained (Fig. 3C) demonstrate an absence of interaction between the variables “age” and “herniation progression” (p > 0.05) for sGAG, Col I, Col II and FN presence in hAF, reinforcing that the differences in the biochemical composition of hAF ECM with herniation progression are not due to age differences between the different donors.
Moreover, for each herniation stage (protused, contained and uncontained), a linear regression between hAF ECM stained area and donor age was performed (Fig. 4). With this analysis, it is possible to verify a positive correlation for sGAG and Col I with ageing only in the group of hernias contained by PLL (r2 = 0.2346, p = 0.0488 for sGAGs and r2 = 0.259, p = 0.037 for Col I, respectively), suggesting that, within this herniation containment level, Col I and sGAG increase with ageing.
hAF fibrotic analysis with herniation progression at the cell level
Additionally, relevant cellular markers to tissue/IVD fibrosis (α-SMA+ cells, MMP12+ cells and CD68) were also evaluated in hAF samples by IHC analysis.
The α-SMA+ cells were absent from hAF of AIS samples (Fig. 5A (a–d) and 5B), as well as in a high percentage of herniated samples (in 33.3% of the protused hernias, 47% of the contained hernias and 38.5% of the uncontained hernias), suggesting a high heterogeneity of the α-SMA expression in hAF. Nevertheless, in the samples presenting α-SMA+ cells, an increased expression of α-SMA in the AF from herniated tissues was observed, ranging from 2.27 to 94.71%, when compared to AIS samples.
In what concerns MMP12 (Fig. 5A (e–h) and 5B), another marker associated with IVD fibrosis, heterogeneity was also observed, since MMP12+ cells were absent in 33.3% of AIS samples, in 11.1% protused hernias, in 5.9% of contained hernias and in 9.1% of uncontained samples. No differences were observed regarding the presence of MMP12 in the different herniated conditions, with most samples exhibiting MMP12+ cells below 50%.
Furthermore, macrophage presence in hAF samples was also assessed by the expression of CD68+ cells (a common marker used for macrophages) (Fig. 5A, i–l, and Fig. 5B). CD68+ cells were observed in 33% of AIS samples, in 24% of contained hernias and in 11.7% of uncontained samples, but not in protused hernias (Fig. 5A,i–l). Macrophages were present in AF tissue both, dispersed (Fig. 5A, k) and agglomerated (Fig. 5A, l), the last predominantly in hAF tissue borders (Fig. 5A, l). Due to this observation, macrophage infiltration area was quantified (Fig. 5B), instead of percentage of positive cells. Nevertheless, the quantification of CD68+ area revealed no significant differences between hernia conditions.
As previously described for ECM markers, the interaction between the variables “hernia containment” and “age” was addressed for α-SMA expression using a multivariate analysis (Fig. 5C), but no interaction was found between the two variables (p value = 0.291). Moreover, no linear correlations were found between the increase of α-SMA expression with ageing for each hernia containment group.