The Establishment of Intervertebral Endplate Degeneration: A Novel in-vivo Diurnal-Axial Controllable Loading Model in Rat Tail


 Background. Cartilage endplate (CEP) acts as an important mechanical barrier and nutrient channel for the intervertebral disc (IVD). Its vulnerability to excessive loading and subsequent degeneration might be an initial trigger for IVD degeneration. This study aims to build a new in-vivo animal model of rat-tail CEP degeneration through implementing a controllable axial compressive device.Methods. An axial controllable compressive load was conducted on the 8–9 th coccygeal IVD of adult rat tail through puncturing 4 Kirschner wires into its superior and inferior vertebra. The exact compression loads were controlled and adjusted by a remote controller. The compression and decompression periods were set as 8 h/16 h per day. The control group was set as no device installation, while the Kirschner wires were punctured without compression in the sham group. After each 4, 8 and 12 weeks, 3 rats in each group were sacrificed and compressed disc were analyzed.Results. After compression, the targeted CEP degenerated gradually with time. Through 12 weeks' loading, the CEP structure collapsed completely and the Schmorl's nodes formed and penetrated from the cartilage layer into the vertebra. When compared with the control group, the number of micro porosities in the CEP becomes extremely scarce with loading, while the content of aggrecan and collagen II decreased significantly. However, these significant changes did not show up when sham and control groups were compared.Conclusions. A new excessive force induced CEP degeneration animal model was developed with its contribution as a practical alternative for further studying mechanisms of CEP degeneration.


Background
The cartilage endplate (CEP) is an important component of the intervertebral disc tissue. It is a cartilagelike tissue that connects the upper and lower vertebral bodies with the annulus brosus (AF) and nucleus pulposus. As one of the biomechanical barriers, the superior and inferior CEP can absorb and disperse various forms of mechanical forces. The special micro-porous structure of the CEP tissue serves as one of the most important channels for supplying nutrition from vertebrae to the gel-like nucleus pulposus (NP) [1]. Once the endplate is degenerated, the abnormal changes such as disrupted nutrition supply and atypical intramedullary pressure to the cellular microenvironment within the structures of the disc cells would nally lead to tissue breakdown and functional impairments [2,3].
Although human intervertebral disc (IVD) is susceptible to various forms of forces, axial load plays an important role in the pathogenesis of its degeneration [4,5]. Some studies believed that the cartilaginous endplate is the weakest point under the compression of axial force and the excessive damage caused by it acting as the initiating factor of IVD degeneration [6]. Therefore, to establish an axial force-induced animal model of CEP degeneration and observe its morphological changes during the process of degeneration is critically important.
In the past, many helpful efforts have been made for building an effective animal models of CEP degeneration, such as osteoporosis modeling, autoimmune response and chemicals injection [7][8][9], yet in vivo biomechanical approach have not been considered in these studies. Despite these, in animal models of IVD (mainly on NP), biomechanical apparatus as ilizarov-type external xator was applied by many studies [10][11][12]. Moreover, it is widely modi ed for use in the eld of orthopedic surgery in order to correct bone deformities. However, the disadvantages of this apparatus are also prominent such as unchanged loading module and imprecise compressive forces during long-term experiment. Most importantly, according to our knowledge, its function as inducing CEP degeneration has not been studied. Therefore, in the current study we designed a novel and reliable model for causing CEP degeneration on rat tail IVD.
By means of penetrating four Kirschner wires into two adjacent vertebrae, this custom-build compressive device with controllable loading module was applied to the rat tail. Our aim of this study was to use this device for providing stable axial compressive forces in long term and to observe the effects of these forces to the pathological changes of CEP during different phases of degeneration.

Ethical Approval and Animal Grouping
The experimental protocols were reviewed and approved by the Ethics Committee on Animal Studies of Shanghai East Hospital, Tongji University School of Medicine. Animal welfare was assessed and guided strictly following the guideline in Research Animal welfare of Shanghai East Hospital. Thirty threemonths-old male Sprague-Dawley rats (SD) were used in this study (SLAC Laboratory Animal Co., Ltd, Shanghai, China). All rats were group-housed in standard plastic cages by 3 per cage and raised in the speci c pathogen-free (SPF) laboratory of Shanghai east hospital. A total of twenty seven rats were randomly assigned into three groups: the operational Group A applied with compressive device (n = 9), the Sham Group B carrying devices without compression (n = 9), and the control Group C (n = 9), respectively.
Three rats were randomly chosen for sacri ce in each group at time frames (4, 8 and 12 weeks).

Establishment Of The Compression Device
The custom-made device consists of three main parts (Fig. 1). 1) For the compressive unit, one geared motor equipped with screw rod were penetrated through three aluminum rings (with four drilled holes); one pressure sensor was inserted in between two aluminum rings (Fig. 1a, b). 2) The control box that connects the compressive unit through wire provides one screen which shows the compressive force, one signal receiving unit and one power bank that connects to the above two units for electrical energy (inside of the box), as shown in Fig. 1c. 3) An infrared remote controller was provided for increasing or decreasing compressive force, as shown in Fig. 1d.

Surgical Procedures
As shown in Fig. 2, the model was both installed in the tail of SD rats in operation group A and the sham group B. Animals were anesthetized with the injection of 4% chloral hydrate (0.1 ml/kg body wt, Sangon Biotech, Shanghai, China) by means of intra-peritoneal local anesthesia (Fig. 2a). ). The prone position was taken for the operation. First, the compressive unit was penetrated through the tail. Four Kirschner wires (0.9 mm) were respectively inserted into the center of 8th and 9th coccygeal vertebrae (Co 8/9) through two aluminum rings. Then an instruction of increasing force was taken by pressing the button on the infrared remote controller in order to press the disc between Co 8 and 9. As previous studies described [10,13], 1.3 MPa compressive stress was installed to the targeted disc. The diameters and area of Co 8/9 disc was measured and calculated using a Vernier caliper tightened to the skin of Co 8/9 disc (Fig. 2b). Then the force value that needed to be conducted was calculated according to pressure-force-area formula. The force value in Newton (N) can be displayed in real time on the screen of the control box, therefore an increased compression command was continuously given to the control box until the predetermined force value was reached (Fig. 2c). The loading duration was set as 8-hours loading and 16hours free loads in one single day [14]. A protection of shield was then installed around the compressive unit in case of rat-biting damage to the connecting wires. For rats in sham operation group B, force compression command was not performed after the puncture of Kirschner wires. Amoxicillin trihydrate subcutaneous injection (7 mg/kg body wt, Yuanye Bio-Technology Co., Ltd, Shanghai, China) was used to prevent infection within one week after the surgery. Pain killer analgesic ibuprofen granules (5 mg/kg body wt, HPGC, Harbin, China) for one week were used by intragastric administration.

Mri Examinations
After 4, 8 and 12 weeks of the operation, the Co 8/9 disc of each 3 rats in Group A, B and C were respectively sacri ced for magnetic resonance imaging (MRI) examination. A MRI scanner (Achieva 3.0T; Philips Medical Systems, Best, the Netherlands) was used with the following parameters: T1-weighted imaging: TR = 300 ms, TE = 20 ms, FOV = 60mm × 60 mm, data matrix = 148 × 150, and slice thickness = 1 mm. T2-weighted imaging: TR = 5000 ms, TE = 50 ms, FOV = 60 mm × 60 mm, data matrix = 200 × 200, and slice thickness = 1 mm. Two senior radiologists independently read and assessed the MRI image results. The degree of IVD was assessed using the P rrmann grading classi cation system [15].

Histological Evaluations
The disc sections were stained with hematoxylin and eosin (HE) and Safranin-O Fast Green (SOFG) and analyzed qualitatively to observe the morphological changes of degenerated cartilage endplates using a microscope (Leica DM6000B, Microsystems, Wetzlar, Germany). A histological classi cation system was used to assess the characteristics of degeneration [17].

Immunohistochemistrical Staining
Matrix collagen II and collagen II were performed as markers of endplate and IVD degeneration. The immunohistochemical staining was performed using the following primary antibodies: mouse monoclonal collagen II antibody (1:150, ab185430, Abcam, Cambridge, UK); mouse monoclonal collagen II antibody (1:150 dilution, ab3773, Abcam, Cambridge, UK). For detection, 3, 30-diaminobenzidine was used; positive cells were stained brown. Nuclei were counter-stained blue with hematoxylin. All stained preparations were photographed under a microscope (Leica DM6000B, Microsystems, Wetzlar, Germany).
Then the stained sections were semi-quantitatively analyzed by means of Image-Pro Plus 7.0 software (Media Cybernetics, Inc., Rockville, USA). The average optical density was measured on the images at 400 × magni cation.

Statistical analysis
Two-tailed Student t-test was performed using SPSS 20.0 version (IBM Inc., Chicago, IL, USA). The experiment data were presented as mean ± standard deviation (SD). The level of statistical signi cance was set at P < 0.05.

Animals
There are in total 27 rats were included in this study. However, after the loading operation, there is one rat in Group A failed to carry the loading device due to the ischemic necrosis of tail. In Group B, one rat died of the anesthesia intolerance during the device installing operation.

Mri Results
In the operational group, after continuous loading, the signal of Co8/9 disc decreased signi cantly (Fig. 3A). In the sham operation group, the Co8/9 vertebral Kirschner wire penetration marks were observed, showing a low signal; no signi cant signal changes of the loaded discs at 4, 8, and 12 weeks after loading were observed (Fig. 3B). In the control group, however, the T2 images of the Co 8/9 intervertebral disc showed high signal during the whole experimental process; The boundary between CEP and NP was clear and the intervertebral space height was normal (Fig. 3C). Table 1 shows the grading scores of loaded disc in three different groups. Table 1 The grading scores of Co8/9 in three different groups, showing with ve levels and equivalent number. He & Sofg Staining And Scoring Results In the control group, the gross structure and thickness of CEP was complete and uniform; The distribution of micro-porosity with regular size can be observed. The NP cells were rounded like and there was no sign of no cell cluster or brosis. The AF ring has a well-organized and lamellar architecture. The boundaries between CEP, NP and AF were well-de ned (shown in Fig. 4, 5 control group a1-a4).
In the sham surgery group, the structure of CEP was also normal and complete. However, the appearance of a curved AF structure was observed. The SOFG histological scores after 4-12weeks' loading were 0.4 ± 0.8, 0.7 ± 0.6, and 1.1 ± 0.5, respectively (Table 2). In the experimental group: 1) after 4weeks' loading, in the degenerated CEP, there were more in ammatory cells accumulating inside of the micro-porosity region. At the same time, the diameter of the microporosity increased (c1/c3 in Figs. 4 and 5); the size and the water content of the NP decreased (c1/c4 in Figs. 4 and 5). The AF was compressed into an S-type cycle under loading (c2 in Figs. 4 and 5). The SOFG histological score was 5.4 ± 2.1 on average (Table 2); 2) After 8 weeks' loading, a signi cant thinned CEP was shown. The number of micro-porosities in CEP decreased and its structure became irregular. The calci ed NP ruptured through the superior and inferior endplates (Schmorl's node) as shown in d1/d4 of Fig. 4, 5; The AF was severely ruptured and disordered. The SOFG histological score averaged 7.6 ± 2.5 ( Table 2). 3) After 12weeks' loading, the micro-porosities in the CEP almost disappeared, and the structure of CEP was severely broken. The notochordal cells almost disappeared, and a large amount of cartilage brosis and calci cation occurred in the central region. More NP tissues penetrated into the vertebral area, and AF was severely ruptured and disordered (e1-e4 in Fig. 4, 5). The SOFG histological score averaged as 11.3 ± 1.2 ( Table 2).

Immunohistochemistrical results
For the aggrecan expressions in CEP and NP, they dropped dramatically after the abnormal mechanical loading was applied to. After 4-and 8-weeks' compression, the complete tissues structure collapsed and enlarged NP and CEP cells expressed very limited aggrecan (Fig. 6c-e and Fig. 8). However, there are no signi cant differences of aggrecan expressions in both NP and CEP when the control and the sham groups were compared, respectively (Fig. 6a, b and Fig. 8).
In the control group and the sham-operated group, the collagen II contents in the CEP and NP were rich and concentrated (as shown in Fig. 7a, b), and there were no statistical expression differences between the two groups. After 4-8 weeks' loading, this matrix gradually fragmented and dispersed, and its content decreased signi cantly in NP but not obviously in CEP. Fibroblast-like cells shown in the NP area (Fig. 7c, d). After 12 weeks' loading, both in the structure of NP and CEP, the positive stainings were severely reduced (Fig. 7e). As shown in Figs. 7 and 8, when comparing the expression of matrix collagen II in CEP and NP between the control and the loading group in each time point, the differences were statistically signi cant. (P < 0.05).

Discussion
Although the CEP tissue plays an important role in IVD, it has not been extensively studied compared with NP, possibly because the tissue layer is so thin rendering it di cult to harvest [18]. However, the structural failure always started in the endplate, indicating that these thin cartilage layers are the weak parts susceptible to excessive mechanical loading [19,20]. In this study, with the aim of inducing CEP degeneration, we established a novel diurnal-axial loading device based on Ilizarov-type apparatus and applied it to rat tail in vivo.
This device was developed in order to provide a stable and precise axial loading force. The loading force created by the compressive unit would drive the motion of two aluminum rings on the coccygeal of rat tail. The Kirschner wires that penetrated the two adjacent vertebrae through aluminum rings were driven close to each other. The targeted disc was then compressed with pre-set force that can be precisely adjusted following the monitor. Meanwhile, in order to test the penetrating effects of wires to the vertebra and the following potential effect to CEP, the sham operational group was set and examined. No signi cant signs of CEP degeneration were found in the sham group, suggesting the little effect of wire punctuation to the cause of CEP degeneration. There are many loading devices built for inducing cartilage degeneration. One rat tail bending model was established by Lindblom et al. [21]. They found that disc on the concave curved side degenerated signi cantly with direct damage to the tissue structure and cell number. Further, the degree of degeneration heavily depends on time and the extent of bending force applied to the rat tail. Another classic model, Ilizarov-type compressive model, was widely used by many researchers due to its reliable installation and satisfactory modeling results [11,[22][23][24]. However, a set of springs were mainly used in these studies as the provider of compression loads. A stable and reliable compressive effect cannot last long due to the spring fatigue. Meanwhile, the static loading mode is not comparable to the physiological mechanical loading conditions of CEP during daily activities.
In our study, after loading, the targeted Co8/9 disc degenerated with time. The histological staining scores were 5.4 ± 2.1, 7.6 ± 2.5 and 11.3 ± 1.2 after 4, 8 and 12 weeks, respectively. The loaded disc showed the sign of severe and signi cant degeneration: the NP was severely dehydrated and brotic, while the number of intervertebral cells were largely decreased. The micro-porosities in CEP disappeared during the phase of degeneration. Under loading, the degenerated discs ruptured through the cracked CEP with the formation of Schmorl's nodules. Kuga et al. [25] found in a fatigue stress test of spinal functional units investigate that the AF is prone to rupture; and the CEP is a weak area that susceptible to repetitive motion stress. Zehra et al. [26] found that abnormal pressure loading can cause the failure of CEP showing that the CEP is thinning with degeneration. They also found that the degree of IVD is strongly associated with multi and large CEP defects, which might contribute to the volume and compression change inside the NP [27]. Maclean JJ et al. [28] observed the effects of short-term high-force compressive loading to the rat intervertebral disc by means of Ilizarov-type device. Their ndings show that abnormal compression can cause a decreased expression of collagen I and II and an increase d expression of catabolic genes such as collagen IIase-1 etc., which may lead to the breakdown of collagen II, a signi cant key extracellular matrix in the intervertebral disc.
With the progression of degeneration, the extracellular matrix collagen II and collagen II gradually disappear. Immunohistochemical stainings showed when comparing with the control group, the expression of collagen II in NP of the experimental group was reduced by 39.8%, 65.4% and 80.7% at 4W, 8W, 12 weeks after the operation; and were 22.5%. 56.2% and 71.3% in CEP; For collagen II, the expression in the experimental group were reduced by 42.6%, 57.9% and 73.4%, after the operation; were 48.6%, 74.8% and 86.5% in CEP, respectively. At the same time, there was no signi cant difference in the expression of the aggrecan and collagen II in the Co8/9 disc of sham operation group compared with the control group (P = 0.36). Since the modeling procedure acquired the penetration of vertebral body, whether the nutritional disorder would be a confounding factor which affecting the results is also the focus of this experiment. Studies found that high-force loading could cause intracellular environmental disorders, the activation of in ammatory factors and the production of matrix metalloproteinases, in the end, leading to the continuous decomposition and destruction of extracellular matrix. Yamazaki et al. [29] proposed that abnormal pressure load would up-regulate the catabolism related factors and induce the expression increase of matrix degrading and proteolytic enzyme. Ariga et al. [30] conducted an ex vivo study and observed increased number of apoptotic CEP cells with increased static mechanical loading force.
There are several limitations in the current study. First of all, from the perspective of physiological characteristics. This model simply takes the axial compression load into consideration, while the realistic biomechanics of the human intervertebral disc are more complicated. Based on our knowledge there is no in-vivo study which simulates the effects of comprehensive mechanical effects such as torsion and shear force to the CEP [31]. Second, the applied loading force and loading frequency of the model are manually controlled in a diurnally setting mode yet cannot mimic the physiological loading condition of the human spine. Thirdly, rat was selected in our experiment as the model. Compared with large animals, the extracellular matrix of the IVD in small animal like rat contains higher water content and the existence of notochordal cells in its disc made the results hard to be comparable with human discs. In the future, a compressive device with multi-frequencies and loading settings controlled by a wireless terminal should be developed. Further, large animal models such as goat, cattle and even ape with similar spine biomechanical backgrounds are to be considered. JT, interpreted data and revised the paper.

Conclusion
MJY, interpreted data, made the gures and nalized the paper.
LJL, designed and funded the study, revised and nalized the paper.