Single impact injury of Vertebral Endplates, without Structural Disruption Initiates Disc Degeneration In Vitro


 Background : Intervertebral disc degeneration is usually attributed to ageing, genetic, mechanical, and nutritional factors et al. It has been acknowledged that the degenerative process is associated with an aberrant cell-medicated response to structural failures, such as vertebral burst fracture, radial fissures, and endplate fracture. Vertebral endplate trauma, due to, Kirschner wire use or drill holes, can induce degenerative changes of the intervertebral disc (IVD). However, whether a single impact injury of the endplates without structural disruption, which is common seen in the clinic, is sufficiently to initiate disc degeneration is still controversial. This study is to further evolve an in vitro impact injury model of IVD and to investigate if a single impact injury of the endplates without structural disruption can initiate intervertebral disc degeneration(IVDD). Methods. Rats spinal segments (from L1/2 to L5/6, n=54) were harvested and randomly assigned into three groups: Control (n=18), Low Impact (12 J/cm 3 , n=18) and High Impact (25 J/cm 3 , n=18). Samples in both of the impact groups were subjected pure axial impact loading using a custom-made apparatus, and cultured for 14 days. The degenerative process was investigated by using histomorphology and real-time PCR. Results: The discs in both of the impact groups showed significant degenerative changes at 14 days, both of which showed much higher histological scores and up-regulation of the catabolic (MMP-9, MMP-13) genes transcription than that of the control group ( P ＜0.05). The discs with endplate fracture compared to that with intact endplate also showed strongly up-regulated catabolic (MMP-9, MMP-13) genes transcription, and more significant degenerative changes based on the histological scoring ( P ＜0.05). No significant difference of anabolic (TGF-β, Col1α1, Col3α1) genes transcription was found between different groups( P ＞0.05). Conclusion: This study demonstrated that a single impact loading (12 J/cm 3 ) on the spinal segments of the rats could initiate IVDD at 14 days after injury and not only endplate impairment but also a single impact loading without structural disruption could also promote IVDD.

and nutritional factors, which is characterized by a decrease in proteoglycans, cell population, water content, and loss of disc height [4]. The environment and behavioral factors are also believed to influence this degenerative process [5].
It has been acknowledged that the degenerative process is associated with an aberrant cellmedicated response to structural failures, such as vertebral burst fracture, radial fissures, herniation, and endplate fracture [6]. Vertebral endplate trauma, due to, Kirschner wire use or drill holes, can induce degenerative changes of the endplate and the intervertebral disc (IVD) [7]. Cinotti et al., for instance, demonstrated degenerative changes in the annulus and nucleus, such as decreases in amounts of water, proteoglycans and cells as well as significant morphological alterations [7].
However, whether a single impact injury of the endplate without structural disruption, which is common seen in the clinic [8], is sufficiently to initiate disc degeneration is still controversial. This can be ascribed to the lack of an appropriate model.
Since a genuine in vivo impact injury model is ethically challenging, the availability of an experimental animal model that consistently reproduces the disease after impact injury would facilitate the investigations of post-traumatic degenerative process. Although several animal trauma models have been established [9][10][11][12], the different nature of injury causes different emphasis on matrix remodeling, apoptosis, and inflammation and hence poses the question of clinical relevance, in particular as none of them mimic the clinical situation. Therefore, the goal of this study was to further evolve an in vitro impact injury model and to investigate if a single impact injury of the endplate without structural disruption can initiate intervertebral disc degeneration. We hypothesize that not only structural disruption of the endplate but also a single impact injury without structural impairment can initiate post-traumatic disc degeneration.

Methods
Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) if not stated otherwise.
Full-Organ IVD Culture Model and Trauma Induction. The animal study was also approved by the institutional review board and animal care committee of the Sun Yat-sen university (2013A-204).

Animal Groups And Impact Loading
Thirty Sprague-Dawley rats (0.4-0.5 kg, male, 6-months-old) obtained from Sun Yat-sen university animal facility were euthanatized with 10% chloral hydrate at a dose of 5 ml/kg via intra-peritoneal administration. Subsequently, fifty-four Spinal segments (IVD/endplate with approximately 3 mm of the adjacent vertebral bodies) from L1/2 to L5/6 (3/animal) were isolated less than 4 fours, and flushed with 0.9% NaCI containing 50 µl/ml penicillin. The segments were trimmed transversely by bone saw (IsoMet, Buehler, Lake Bluff, IL) with the cranial and caudal cutting planes parallel to each other at a height of 7.08 ± 0.21 mm, and perpendicular with respect to the cranial/caudal axis of the segment. The segments were then randomly assigned into three groups: Control (n = 18), Low Impact (12 J/cm 3 , n = 18) and High Impact (25 J/cm 3 , n = 18). The specimens were then subjected to single impact load using a custom-made apparatus, which guarantees axial load. The impact force was recorded using a piezoelectric loadcell (Kistler) (Fig. 1A). Pilot-experiment revealed 25 J/cm 3 as the threshold energy for endplate failure, at which the endplate was expected to fracture in half of the specimens. The height of each specimen was calculated as the vertical distance between the two vertebral cross sections, and was recorded before and after impact loading. Based on the pilotexperiment, the specimens with a height decrease more than 10% indicates endplate fracture, whereas less than 10% decrease indicates endplate intact.
After impact, samples were washed with 0.9% NaCI containing 50 µl/ml penicillin for three times, and then cultured at 37℃, 5% CO 2 in DMEM (Dulbecco's Modified Eagle Medium, DMEM) with 2% fetal calf serum, 1% Pen/Strep, 50 mg/ml ascorbate-2-phosphate and 0.1% Primocin. Half of the samples were collected at day 7, and the other half were collected at day 14 for histology and mRNA analysis. The assignment of the specimens in each group was shown in Table 1. Table 1 The assignment of the specimens in each group.

Control
Low The changes of the intervertebral disc degeneration were investigated. The disc degeneration assessment scoring system that we developed based on our prior work [13,14] was used to assess the anulus fibrosus, the cellularity of the nucleus pulposus, and the matrix of the nucleus pulposus through sections (Table 2). Histomorphometric assessment was performed by an orthopedic researcher (L.S), who was blinded to the different treatments between groups. All histologic sections were reviewed one month after the first examination to determine the intraobserver reliability. The average score of the two measurements for each specimen was used for the statistical analysis. Table 2 The scoring system of lumbar intervertebral disc degeneration.
Each gene expression was quantified by real-time PCR using CFX96 Real-Time System (Bio-Rad, Herculus, CA, USA). Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold change in comparison with the control group. Table 3 Sequences of primers used in the real-time PCR.
GACTGGCCAGCCTTAGTT TG GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Col1α1, collagenⅠα1; Col3α1, collagen Ⅲα1; MMP-9, Matrix metalloproteinases-9; MMP-13, Matrix metalloproteinases-13; IL-6, Interleukin-6 Statistical Analysis SPSS 25.0 software (SPSS Inc., Chicago, IL) was used for univariate analysis of variance. The data between groups were compared by using t test. The results of histological scores were analyzed using the Wilcoxon signed ranks test, with a confidence interval of 95%. To assess intraobserver reliability, we used the intraclass correlation coefficient for average and single measurement. The agreement of intraclass correlation coefficient was rated as follows: 0 to 0.4, fair agreement; 0.41 to 0.60, moderate agreement; 0.61 to 0.8, substantial agreement, and 0.81 to 1.00, excellent agreement [17]. Statistical significance was indicated at P 0.05.

Results
The impact time was less than 0.008Sec for both Low Impact and High Impact groups (Fig. 1B, D).
With the applied trauma-protocol, there was no specimen with endplate fracture in the Low Impact group. In the High Impact group, endplate fracture occurred in 9 specimens, which has been confirmed by CT scan and histological evaluation (Fig. 2). After impact injury, the average height of specimens in the Low and High Impact groups decreased 3.49 ± 0.62% and 7.93 ± 1.34% respectively, both of which showed significant differences in comparison with the control group (P 0.05, Fig. 3).

Histological Findings
The intervertebral discs in the control group appeared normal. The nucleus pulposus contained abundant cells and sounded by large zones of acellular matrix, and the annulus fibrosus showed normal organization of fibrocartilage lamellae (Fig. 4A, F, K). At day 7, the discs in the Low Impact and High Impact groups showed no significant degenerative changes ( Fig. 4B,C,G,H,I,M), although both of the histological scores were higher than that of the control group (Fig. 5A). There was also no significant difference in the histological score between the discs with endplate fractures and that with intact endplates in the High Impact groups (P 0.05, Fig. 5B).
At day 14, the discs in both of the Impact groups showed significant degenerative changes, where the nucleus pulposus comprised relatively few cells (Fig. 4D,E,I,J), with less deeply stained proteoglycans ( Fig. 4D,E) relative to the control group. The annulus fibrosus showed less organized fibrocartilage lamellae, as compared with the control group, and the collagen fibers formed a wavy arrangement (Fig. 4N,O). Both of the histological scores in the Low and High Impact groups were significantly higher than that of the control group (P 0.05, Fig. 5A). There was no significant difference in the histological score between the Low and High Impact groups (P 0.05, Fig. 5A). However, the average histological score of discs with endplate fracture was much higher than that with intact endplate in the High Impact group (P 0.05, Fig. 5B). The intralclass correlation coefficient was 0.923 for a single measurement, which showed strong agreement.

Real-time Pcr
Both of the Low and High impact injury caused an up-regulation of the catabolic (MMP-9, MMP-13) genes transcription in comparison with the control group after injury, especially in the Low Impact group at day 14 (P 0.05, Fig. 6A,B). The pro-inflammatory (IL-6) gene transcription was also strongly up-regulated in the Low Impact group at day 7 (P 0.05, Fig. 6C); however, a subsequent decrease of gene transcription was noticed at day 14. There were no significant differences of the IL-6 gene transcription in the High Impact group at day 7 and day 14 (P 0.05, Fig. 6C). For the anabolic (TGF-β, Col1α1, Col3α1) genes, col1α1 and col3α1 were strongly up-regulated at day 7 in the High Impact group but reverse to down-regulation at day 14 (Fig. 6D,E). Although col3α1 was also up-regulated at day 7 and day 14 in the Low Impact group, there was no significant difference in comparison with the control level. Gene expression of TGF-β was not changed in both of the Low and High Impact groups (Fig. 6F).
In comparison with the discs with intact endplate, the catabolic (MMP-9, MMP-13) genes transcription were strongly up-regulated at day 7 in the discs with endplate fracture(P 0.05, Fig. 7). Although the up-regulations of anabolic (TGF-β, Col1α1, Col3α1) genes transcription were also much higher but did not reach significance. Due to the limited number of samples at day 14, we could not find any significant difference in each gene expression between the disc with intact endplate and that with endplate fracture (data were not shown).

Discussion
Our aims were to develop an in vitro impact injury model that can mimic the clinical situation, and to investigate if a single impact injury without structural disruption can initiate intervertebral disc degeneration. Therefore, histomorphometric and biological analysis after in vitro trauma were performed on spinal segments with different level of impact loading. The results showed that, not only structural disruption of the endplate but also a single impact injury without structural impairment can initiate post-traumatic disc degeneration.
Although intervertebral disc degeneration(IVDD) related to impact injury in the clinic is not uncommon, whether and how it influence the onset of the IVDD changes is still not well documented.
In order to investigate the post-traumatic degenerative process, in vivo and in vitro models have been developed. Animal trauma models are usually divided into three main classes: stab incision models, overload models, and endplate perforation models [11]; however, none of them mimic the clinical situation. In this study, we desigened a dropped-weight apparatus for the sterile induction of impact injury and an accompanying injury of the adjacent intervertebral disc, using different level of energy.
The histomorphological findings showed that the proteoglycans in nucleus pulposus decreased significantly and the fibrocartilage lamellae showed less organized in the low-and high-impact injury groups, compared with the control groups. These changes were consistent with the previous studies showing loss of proteoglycans and less organized collagens during the initial phase of disc degeneration [18][19][20].
The degenerative changes in the IVDs, such as composition of extracellular matrix (ECM), loss of disc cells, proteoglycans and water content, have been suggested to be the consequence of an upregulation of catabolic matrix metalloproteinases (MMPs) and pro-inflammatory (IL-1β, TNF-α, IL-6) gene transcriptions [21][22][23][24][25]. Consistent with the previous reports, we found that mRNA expressions of catabolic (MMP-9, MMP-13) genes were up-regulated in the impact injury groups, especially at day 14 in the low-impact group. The pro-inflammatory (IL-6) gene were also up-regulated at day 7 in both the low and high-impact groups. The correlation between inflammatory gene regulation and catabolic gene transcription may indicate interactions between their transcription regulations. It has been reported that, the activity of MMPs is not limited to matrix cleavage; they also modulate the inflammatory response [26], which may explain why degenerative discs were also sensitive to proinflammatory stimuli [27]. It has been reported that, TGF-β was a major up-stream regulator of collagens and proteoglycans [28]. However, TGF-β gene transcription was not affected in this study.
The remodeling response of TGF-β may be compensated by the inhibiting effect of the proinflammatory cytokines [29].This need to be verified by further study.
It is well known that, the nutritional pathways that into the nucleus pulposus of human intervertebral discs are mainly by diffusion through the central portion of the end-plate from these marrow space cartilage contacts [9]. Thus, a functional intact endplate is crucial to transport nutrients and waste products by diffusion [30,31].Dudli et al. [15] characterized the process of disc degeneration using an in vitro full-organ model, and elucidated that burst endplates, but not equienergetic loading promotes disc degeneration. However, Chan et al [32]. reported that, impact loads which was generally subtraumatic, could also cause cumulative damage and injury to the lumbar spine. In the current study, we also found that discs with intact endplate in the impact groups also showed degenerative signs based on the histomorphological findings and the catabolic (MMP-9, MMP-13) genes transcription, although it was not so significant as that with endplate fracture. This indicates that not only endplate fracture but also a single impact injury can initiate degenerative changes of intervertebral disc.
Taken together, an in vitro intervertebral disc degeneration model with impact injury has been developed and applied to demonstrate that not only structural disruption of the endplate but also a single impact injury without structural impairment can initiate post-traumatic disc degenerative changes, which are confirmed by histological changes such as less deeply stained proteoglycans and less organized fibrocartilage lamellae. This is also accompanied by the induction of collagenases (MMP-9, MMP-13) and up-regulation of pro-inflammatory (IL-6) gene transcription, which are recognized to be involved in disc degeneration.
However, there are several limitations to the study. Firstly, there are some biomechanical and anatomic differences between the spine of the rats and that of the human; however, the objective of this study was to evolve an in vitro impact injury model of IVD and to investigate the roles of a single impact injury of the endplates without structural disruption on the the process of intervertebral disc degeneration(IVDD). This presented model established by axial impact loading could accurately mimics the loading conditions in patients suffered endplate trauma and the results may provide some clues for us to investigate the initiation of disc degeneration after axial impact injury of endplates.
In addition, drawing conclusions from animal models as well as from in vitro study is always problematic. The increased gene transcription that demonstrated with quantitative PCR, may not essentially induce a functional gen product, as mechanisms like post-transcriptional modifications or gene silencing may interfere. With matrix metalloproteinases, just a small aspect of the characteristics of disc degeneration was studied. Finally, due to the limited number of samples in this study, we did not focus on the difference in the gene expression between the discs with intact endplate and that with endplate fracture. So, further studies are warranted to verify the results of the current study.

Conclusions
This current study demonstrates that a single impact loading (12 J/cm3) on the spinal segments of rats could initiate intervertebral disc degeneration (IVDD) at 14 days after injury and not only endplate impairment but also a single impact loading without structural disruption could also promote IVDD.

Ethics approval and consent to participate
The animal study was also approved by the institutional review board and animal care committee of the Sun Yat-sen university (2013A-204).

Consent for publication
All the authors consent to publish this manuscript   This graph shows the mean percentage of decrease of sample height between groups.

Availability of data and materials
There was a significant decrease in the height changes of High-impact group in comparison with the Control and the Low-impact groups (*P 0.05 vs Control and Low-impact groups).  The histological scores of the IVDs between different groups at day 7 and day 14 after injury. A: This graph showed that both of the histological scores in the Low and High Impact groups were significantly higher than that of the control group (* P 0.05 vs. Control group).
B: This graph showed that the average histological score of discs with endplate fracture was much higher than that with intact endplate in the High Impact group at 14 days after injury(* P 0.05 vs. Control group; † P 0.05 vs. High Impact group).

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