Background
Low back pain is a highly disabling condition carrying the potential for high social, economic, and individual effects [
1]. Alterations in the architecture, biochemistry, and biomechanics of the intervertebral disc (IVD) can induce back pain and referred pain, regardless of neurological impairment [
2]. The cartilage endplate (CEP), which plays a role in providing nutrition to the disc, and lactate content of the IVD have been implicated in the process of disc degeneration [
3]. Although the etiological event or agent responsible for primary IVD degeneration (IVDD) has not been clearly identified, one major theory regarding the biomechanical failure of the CEP matrix, wherein structural damage to the collagen network (due to abnormal joint loading) reduces the restraining force capacity of the CEP [
4]. This reduced restraint force of the CEP allows for increased swelling of the IVD by proteoglycans (PG), increased hydration, and, ultimately, the loss of PGs and a corresponding loss of the functional integrity of the disc. A previous study has confirmed that subchondral bone resorption was associated with early development of cartilage, which precedes significant cartilage thinning and subchondral bone sclerosis [
5]. Thus, the ability to detect changes in the biochemical composition of the CEP and subchondral bone could enhance our understanding of cartilage physiology and pathophysiology in IVDD and, potentially, our ability to diagnose, monitor, and treat IVDD diseases in the longer term.
Imaging can be used to identify late stage changes in the IVD, once structural compromise has taken place. Detection of IVDD in its early stage, using modalities able to provide information on biochemical alterations of the structures of the IVD, has the potential to shed light on new biological therapeutic approaches [
6]. Previous studies have evaluated the potential application of quantitative magnetic resonance imaging (MRI) as a diagnostic tool for IVDD in its early stages by attempting to correlate the MRI signal to alterations in the structure of the nucleus pulposus (NP) and annulus fibrosus (AF) [
7‐
9]. A similar approach has been used to identify early degenerative changes of the temporomandibular joint [
10], lumbar facet joints [
11] and knee cartilage [
12]. However, few studies have evaluated the CEP and subchondral bone [
6] owing to the difficulty in visualizing these structures using conventional imaging sequences. Among MRI technologies, T2 relaxation time allows quantification of the content of water and PG, and has been used in previous studies for early detection of cartilage abnormalities, as well as to monitor the response to therapy by previous results [
12]. Some researchers have also demonstrated that cartilage displays a significant magnetization transfer (MT) effect; it has been suggested that collagen is the predominant macromolecular component of cartilage contributing to this effect [
7,
13]. Therefore, T2 relaxation time and the MT Ratio (MTR) could provide superior information on the early molecular and physiological alterations of the IVD for improved identification of IVDD in its early stages, as well as to evaluate outcomes of biological treatments. Research is needed, however, to determine how T2 relaxation time and the MTR vary with the concentration of water and PG or collagen, respectively, and how the measures are affected by pathological changes in IVD tissue.
Therefore, the first aim of this study was to determine the correlations between MRI signals and the biochemical status of IVDs in experimental and normal control dogs, following IVDD induced by annular-puncture. The second aim was to investigate the association between T2 or MTR and IVDD, with particular focus on the sensitivity of MRI-based measures to biochemical changes in the CEP and subchondral bone. The third aim was to define a method with sufficient sensitivity to detect early changes in the signal intensity in the CEP and subchondral bone.
Discussion
This study aimed to evaluate the correlation between biochemical changes in the IVD and T2 relaxation times and MTR. The T2 relaxation times were found to be a more sensitive measure, detecting changes in the CEPZ earlier than the MTR. We demonstrated that two quantitative MR-based measures of the changes in the biochemical content of degenerating IVDs are correlated, indicating a complementary relationship between physiological and biochemical alterations in IVDD. We, therefore, propose that the two complementary strategies are necessary to better reveal subtle molecular alterations and, thus, further our understanding of the progression of IVDD. The use of continuous small rectangle drawings to define the ROIs of the intervertebral area improved our ability to detect differences in the SIs of the CEPZ.
Experimental models are used to study imaging methods in a controlled environment and with known onset of the pathological process. The canine or porcine stab incision model is a well-documented experimental disc degeneration model which alters the biochemistry and matrix composition of the discs within 1 to 3 months [
33‐
35]. Many researches have confirmed that quantitative MRI techniques (such as T2 relaxation time [
36], MTR [
37], T1
p [
38], MR spectroscopy [
39] and diffusion weighted imaging [
20]) have the potential to quantitatively evaluate deterioration in the molecular composition and structural integrity of IVDs. However, most of these studies have focused on the NP or AF [
9,
17,
33,
34,
36], and the very early changes in the CEPZ after stab incisions have not been documented with MRI. It is, therefore, not known how early changes in discs become detectable with MRI, and especially using MTR or T2 relaxation time measurements.
In our study, MTR measurements were not significantly decreased in the CEPZ from baseline through 8 weeks post-surgery. In contrast, there was a slight increase in T2 values of the CEPZ over the first 4 weeks post-operatively, followed by a significant decrease through the 12 weeks of post-operative follow-up.
In vitro studies have reported correlations between T2 relaxation time measurements and the mechanical, histological and biochemical properties of cartilage [
40]. Pathophysiological processes of early cartilage degeneration are characterized by an initial deterioration of the collagen network, followed by loss of PG content, causing increased mobility of water and, consequently, increased water content within the cartilage; this increase in water content within the cartilage can be detected by T2 relaxation time [
41]. Sun et al. [
33] confirmed that degenerative changes in the IVD could be detected as early as 1 week post-operatively, or earlier, using T2 relaxation time Therefore, T2 relaxation time provide a high degree of sensitivity and accuracy to detect IVDD at an earlier stage. The results of our study support this application of T2 relaxation times. We reported a significant at 12 weeks and a consistent increase in the MTR for the NP and AF, and a decrease in T2 values, which are consist with outcomes of previous studies [
8,
9,
15,
17,
32] (Fig.
7). These MR-based findings were supported by biochemical analysis which showed evidence of dehydration and decreased content of PG and increased collagen content [
9,
15,
32,
36] (Table
3).
An obvious increase in T2 values for cartilage [
42] and slight increase in MTR [
4,
15] have been shown to be associated with OA or OA-related morphologic abnormalities. However, in our study, both signal intensities were decreased in the CEPZ at 8 and 12 weeks post-operatively. This result can be explained by the fact that degeneration of the CEPZ is accompanied by a decrease in content of water, collagen type II, and PG [
43]. In a pilot evaluation of the MTR of the IVD, the researchers found an increased level of MT effect between the macromolecular-bound protons and the free-water protons in degenerated discs [
4].
In vitro articular cartilage studies measuring MTR have demonstrated that the concentration and structure of the collagen matrix are the major parameters influencing the MT; however, this effect of the collagen concentration and structure was not evident in the CEPZ in our results. The reason for this may be linked to the varying tissue composition and material properties between the CEPZ and articular cartilage content. Unlike the CEP, the increase in articular cartilage area and thickness that was detected using both MR-based modalities may reflect early osteoarthritic changes, including PG loss accompanied by an increase in water content, due to a loosening of the collagen matrix [
44], and chondrocyte hypertrophy [
45], which are different from endplate changes. MT changes were also correlated with tissue structure and PG content [
4]. Although the baseline cartilage MT parameter was correlated primarily with the collagen content, the correlation to CEPZ was low in our study (
r = 0.392). The changes for the NP and AF were, however, relatively high, in agreement with previous studies quantifying degenerative changes reported in IVDs [
9,
15,
32,
36].
In our study, T2 and MTR showed low to strong correlations with IVDD, indicating that these MR-based parameters are sensitive to disc alterations. These differences in strength of correlation could result from different sensitivity features of the T2 and MTR parameters [
9,
17]. In our results, T2 was sufficiently sensitive to differentiate normal and degenerative states in the CEPZ, AF and NP components of the IVDs (Table
4). In contrast, the MTR differentiated between the control and degenerated discs only at week 12. Moreover, we found obvious degeneration of the CEPZ from pre-operative baseline to post-operative week 12, especially in terms of the cell number and extracellular matrix of CEPZ on histological images (Fig.
3d–l), which are indicative of depletion of the PG and water content. The Picrosirius Red stain also showed the disorder in the continuity and structural integrity of the CEPZ (Fig.
4d1–
i1). The histological endplate score also showed stronger sensitivity to T2 values than MTR. Our results, therefore, show that T2 is more sensitive than MTR to identify changes in the CEPZ under different conditions of degeneration. Indeed, both quantitative MR were sensitive to biochemical changes in the AF and NP. Moreover, the MTR changes in the degenerated discs at 4 weeks (EPZ, 0.15 %; AF, 10.83 %; NP, 12.55 %), 8 weeks (EPZ, 10.79 %; AF, 34.46 %; NP, 67.04 %), and 12 weeks (EPZ, 23.60 %; AF, 42.27 %; NP, 90.54 %), compared to the normal discs, was lower than the corresponding T2 changes at 4 weeks (CEPZ, 6.57 %; AF, 25.19 %; NP, 32.07 %); 8 weeks (18.97 %; 35.53 %; 49.87 %); and 12 weeks (32.01 %; 49.01 %; 65.45 %). Previous researchers have reported that a tear in the AF affects the fluid mechanics of the IVD, causing immediate loss of water and PG aggregates from the disc [
46]. This loss of water is balanced by an increased synthesis of PG by the cells immediately following injury, which results in the rehydration of tissue if the lesion is small enough [
30]. In our study, the SIs fluctuated for the two MR-based quantitative measures over the first 4 weeks post-operatively, due to the relatively larger stab injury used in our study to induce IVDD. The larger dynamic range of T2 sensitivity to disc damage, compared to that of the MTR, indicates that T2 can be used for the detection of early biochemical changes related to IVDD, especially for the CEPZ. Niinimaki et al. [
34] reported a negligible change in the water content of the NP water between healthy and punctured porcine discs (93 %–90 %). These conflicting results may arise from the different measurement methods used the studies. We propose that the definition of ROIs through continuous drawing, without omitting any texture signal information, provides a more accurate method for representing the true change in IVDD compared to a manual location of a large ROI in the targeted area.
An inappropriate choice of ROIs (due to differing anatomical characteristic between the CEPZ and the AF or NP regions) can create substantive partial volume effects. Definition of optimal method to measure ROI by quantitative MR for the CEPZ is still rare. In their study evaluating the effects of stem cell and hydrogel therapies on the CEPZ, Bendtsen et al. [
6] placed the ROI over the subchondral bone and endplate directly. In contrast, in their study correlating lumbar facet joints to IVD using T2 mapping, Stelzeneder et al. [
11] placed the ROIs for the facet joints by first drawing the ROI on the axial echo image of the T2 maps sequence across both articular surfaces at once on each side, and then, transferring the drawn region by “copy and paste” into the T2 maps. A similar image analysis method was used to evaluate articular or hip cartilage [
12,
47]. The canine EP comprises a mean 6 % (3 to 11 %) of the total width (i.e., intervertebral distance) of the IVD, about 0.22 ± 0.06 mm [
18]. The subchondral bone forms a virtual epiphysis that may play the same role as the CEP in the young healthy non-chondrodystrophic dog [
18]. It is always difficult to distinguish the above structures and, therefore, the CEPZ (which includes the subchondral region and the CEP) was used in our study, as referenced in previous research [
20]. This technique allows for measurement of the different tissues based on the number of pixels that compose it. This method of distinguishing different tissues by the difference in neighboring pixels is the gold standard, compared to other image-based measurements. Base on the above anatomical characteristics of canine, the continuous small rectangle drawing of ROIs (1 mm
2, 2.35 mm
2, and 0.70 mm
2) were placed on the anterior, middle, and posterior regions of the IVD, separately from cephalic to caudal vertebrae. All data were transferred into Excel to only select three or four values from the peak or trough of the curves, using a minus 2 SD threshold to filter out surrounding tissue.
Limitations
Our study is limited by characteristics of any pilot study. Firstly, the observation time is relatively short, with a small number of animals included, and potential technical errors in MR measurements. Secondly, partial volume effects were still evident in our results, despite adopting methods deemed to be gold standards for measurement. We did not systematically compare our methods to other which have been reported. Despite these limitations, we were still able to identify subtle differences in the IVDs at different measurement times and to correlate these MR-based quantitative measures to biochemical content. Thirdly, we did not compare our results to advanced imaging techniques, such as T2* mapping [
47], ADC [
34] or Ultrashort Echo Time [
48], or with results of T2 mapping and MTR, such as UTE imaging which can directly visualize and quantify the true cartilaginous EP. However, many previous studies have confirmed the usefulness of T2 mapping and MTR to diagnose in IVDD [
9,
15,
32,
36]. Fourthly, differences in IVDs in dogs and humans also limit the application of findings to humans. In dogs, the disc is smaller than in humans. Consequently, the diffusion distance from the peripheral edge of the annulus to the center of the disc is much shorter in dogs than the equivalent distance in the human disc. In addition, discs in dogs have notochordal cells, whereas in the human, these cells are greatly diminished [
27]. Despite these anatomical differences, it is important to note that the clinical presentation, macroscopic and microscopic appearance, diagnostic, and treatment of IVDD are similar in humans and dogs [
49‐
51]. Experimental models have the advantages of allowing a standardized evaluation of biomechanical, histochemical, and morphologic phenomena of the degenerative and reparative process in the lumbar spine, directly from initiation of the process [
23]. Therefore, dogs have frequently been used in research as a transitional animal model for studying the pathological process of human discs [
18,
27,
50,
52]. On the other hand, the primary criticism of disc injury models is that the rapid advancement of degeneration does not replicate changes seen in human degeneration, which tends to develop over the course of many years. Although this model does not truly reflect the course of human disc degeneration, similar histological and biomechanical changes have been previously reported [
52,
53], and these similarities are confirmed in our study. For this reason, injury-mediated degeneration is a powerful technique to study the basic science of disc degeneration and to develop therapeutic strategies to regenerate tissue according to the comparable endpoints of both injury-initiated degeneration and human degeneration.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CC, ZWJ, and ZHH performed experimental surgery. TG, WL, and YT performed radiological and MRI evaluation. JHW and DLW performed histological evaluation. QH performed ELIAS analysis. DKR and CC conceived of the study and participated in its design. CC, ZWJ and ZHH drafted the manuscript. HL performed the statistical analysis. DKR revised the manuscript. All authors read and approved the final manuscript.