Effect of Coflex interspinous stabilization on the prevention of progression of adjacent segment degeneration after single level and skipped level spinal fusion in a canine model

Effect of Coflex interspinous stabilization on the prevention of progression of adjacent segment degeneration after single level and skipped level spinal fusion in a canine model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Effect of Coflex interspinous stabilization on the prevention of progression of adjacent segment degeneration after single level and skipped level spinal fusion in a canine model Jia-ming Liang, Fang-qi Lin, Jia-ning Ding, Jian-guang Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.12549/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Interspinous spacer (ISPs) was a promising treatment method for adjacent segment degeneration (ASD) after spinal fusion. Coflex, one of ISPs, has been deceived to prevent or decelerate ASD after spinal fusion, while the proof of the effectiveness of such device is still very limited. The purpose of this study was further to investigate the protection role of Coflex in vivo after spinal fusion, when implanted in adjacent segment and middle segment. Methods Three groups of beagles were allocated as follows (n=6): (1)L4-5 lumbar interbody fusion(IF). (2) L4-5 lumbar interbody fusion +L5-6 interspinous Coflex implantation(Cof1).(3) L4-5 and L6-7 interbody fusion+ L5-6 interspinous Coflex implantation (Cof2). In all animals, L5-6 discs were punctured to generate degeneration, and the intact L2–3 disc served as a noninjuries control (Con group). The effectiveness of Coflex on the prevention or deceleration of the progression of ASD was determined by magnetic resonance imaging, gross anatomical observation, histological and immunohistochemically analysis, and Real-time PCR analysis of gene expression. Results The objective disc in every group showed degeneration, however, the degeneration was more significant in IF group than Cof1 and Cof2 groups. MRI and histologic assay demonstrated that the discs of Cof1 and Cof2 groups maintained a relatively well-preserved structure as compared to the discs of IF group. Furthermore, immunohistochemistry analysis and real-time PCR demonstrated that the indicators of disc degeneration, TIMP1, BMP2, Col I, were up-regulated and disc matrix gene, Col II was down-regulated in IF group significantly Conclusions Coflex could decelerate the progression of ASD after spinal fusion, and it holds the same value not only at adjacent segment after single level spinal fusion, but also dose at the middle segment after “skipped” level (nonconsecutive) fusion Orthopedics Coflex interspinous spacer adjacent segmental degeneration spinal fusion canine model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background From now on, the rapidly aging population has become a most common situation in global society, and the increasingly prevalence of lumbar spine disorders have paralleled this trend, unfortunately, most of these clinical conditions affecting lumbar spine cannot exempt from surgery treatment [1 , 2] . Spinal fusion has been employed to treat a variety of spinal disorders, including degenerative disc disease, segmental instability, spondylolisthesis, trauma, and scoliosis for decades [3] . Due to spinal fusion surgery has significantly facilitated patient comfort and mobility, fusion is regarded as a gold standard treatment for degenerative lumbar spine disease. However, we cannot ignore the fact that many researchers have demonstrated that fusion can accelerate the degeneration of adjacent lumbar segment, due to it can lead to excessive stress at unfused adjacent levels [4 , 5] . Therefore, society of spine surgery holds the promise to develop an effective method to prevent or slow down the progress of adjacent segmental degeneration (ASD), after surgical treatment of lumbar degenerative disorders [6] . Many risk factors for ASD have been reported, such as pre-existing degeneration of adjacent discs, facet tropism, post-operative sagittal alignment and so on . Among those risk factors, the elimination of fixed segment motion are thought to play the most important role, as it can increase unfused adjacent segment motion, and then accelerates the degeneration of adjacent segments [7] . Implantation of an interspinous spacer(ISPs) device in adjacent segment is a known feasible technique to tackle with ASD, because it can facilitate to decelerate the degenerative process by providing intervertebral dynamic stability for adjacent segment [6] . Coflex is a dynamic ISP, it is a U-shaped, compressible device which can be implanted between the spinous processes after decompression [8] . As far as we know, there are few studies have focused on the effectiveness of such device to prevent or decelerate the progress of adjacent segment degeneration after spinal fusion. In this study, we postulated that the implantation of Coflex between the spinous processes in adjacent segment after spinal fusion could prevent or decelerate ASD . And it can also play the same important role in the middle segment as a “transition” area in the treatment of “skipped” level (nonconsecutive) disc degeneration ( SLDD ) after fusion were delivered at two “skipped” level, when used in a canine spinal fusion model. Methods Animals and Groups Eighteen healthy and skeletal mature beagles (19 months old on average), weight 16 to 21 kg and average weight 18kg, were used in this study. The experimental animals were provided by the animal experimental center of the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. And experiments of animals were approved by the Animal Research Committee of Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine. All animals were healthy and free of infection, and were magnetic resonance imaging (MRI) scanned of the spine to assure the absence of Intervertebral disc (IVD) degeneration related diseases before the study. 18 beagles were bred and randomly allocated into the following study groups:(1)L4-5 lumbar interbody fusion （ IF ） . (2) L4-5 lumbar interbody fusion +L5-6 interspinous Coflex implantation （ Cof1 ） .(3) L4-5 and L6-7 interbody fusion+ L5-6 interspinous Coflex implantation （ Cof2 ） . In all animals, L5-6 discs were punctured to generate degeneration ， and the intact L2–3 disc served as a noninjuries control (Con group). The animals were followed up for 6 months after the initial operation. The changes in the L5/L6 lumbar discs were assessed using gross anatomical observation, MRI, histology, and gene expression analysis at 3 and 6 months respectively. At each point （ 3 months and 6 months postoperatively ） , 3 beagles were randomly selected from each group to be killed with an excess dose of ketamine hydrochloride and xylazine hydrochloride injection after MRI scan. Preparation of Autologous Iliac Bone Graft 24 hours abstinence for food and 12 hours abstinence for water was maintained before surgery, and Surgical procedures were performed under general anesthesia by intramuscular administration of ketamine hydrochloride injection (0.1 ml/kg) and xylazine hydrochloride (0.08 ml/kg). After anesthetization, the dogs were placed in the lateral position. The incision was 4 cm along the lateral border of the spina iliac anterior superior. After exposure of spina iliac anterior superior, an approximately 3 × 2-cm autologous iliac bone graft was obtained by bone drill. The autologous iliac bone graft was prepared for anterior lumbar interbody fusion. Anterior lumbar interbody fusion and implantation of Coflex When the harvesting of iliac bone graft finished, beagles for experiment were placed in a supine position, median abdominal incision were delivered, then lateral incision approach of the rectus abdominis was achieved, once an opening through the muscle was obtained, carefully protect the peritoneum and reflect it anteriorly by blunt dissection. After retraction of the peritoneum and its contents using a wide retractor, the appropriate involved vertebras were identified. Separate the intervertebral disc and annulus from the cartilaginous endplates of the vertebrae with a thin osteotome, and then the disc was removed by pituitary rongeurs, Kerrison rangers and curets. When interbody space was cleaned and prepared for fusion, predisposed grafts were used to complete interbody fusion. After completion of the fusion, close all layers with absorbable sutures. When the interbody fusion was delivered, the Coflex groups underwent additional implantation of the Coflex (Paradigm Spine, LCC, New York) at the involved level. The beagles were placed in prone position, surgery was performed through a standard posterior midline approach, and then the interspinous ligament was dissected and excised. The device was inserted between the adjacent spinous processes and the flanges were crimped follow the manufacturer’s instructions, so that it was seated fitly. Appropriate placement of the implant and adequate segmental sagittal alignment were identified under C-arm machine. Generation of L5-6 disc degeneration After all of surgical procedures described above were done, the beagles were placed in prone position, L5–6 discs were then punctured with 18-gauge needles （ HuaYi Bio-technology, Shanghai, China ） along the outside of the facet joints under C-arm fluoroscopic guidance until the needle tips reached contralateral side of the discs both in anterior-posterior and lateral radiographs. Following the operation, antibiotic was intravenously delivered in five consecutive days. Beagles were fed in cages, and food and water were placed at a relatively high place, which would force them in erect position more than 8 hours per day. The animals were monitored daily for potential complications or abnormal behavior. Important surgical procedures were shown in Fig. 1. Magnetic resonance imaging MRI (Achieva 3.0T. Philips, Holland) scans were administered to evaluate signal changes in T2-weighted (T2-W; TR 2270 ms, TE 126 ms) images at baseline, 3 and 6 months after the operation in all groups. The subjects were imaged at the time of follow-up using the same scanner and examination protocol, and the slice thickness and interslice gap were 4 mm and 0.4 mm for sagittal. Lumbar IVD degeneration was graded on T2-weighted MR images according to the Pfirrmann classification system on a scale of I–V, where grade V denotes the most degenerate category [9] . All radiological assessments were made independently by two observers, including one radiologist and one orthopedic spine surgeon. when disagreement occurred with respect to the radiological grade, a consensus opinion with involvement of a third observer was sought. Gross anatomical observation At 3 and 6 months, after MRI scan, 3 beagles of each groups were killed with an excess dose of ketamine hydrochloride and xylazine hydrochloride injection, and the spines were harvested. Then the L2-3 and L5–6 discs were isolated intact, bilateral cartilage endplate and some vertebral body were preserved. The discs from each dog were cut coronally at the center of the disc for Gross anatomical observation. Histological and immunohistochemically analysis All L5-6 discs of 18 dogs were cut transversally at the center of the nucleus pulposus (NP). One half of every disc was used for histological studies, and the other half was used for Realtime PCR analysis of gene expression. The NP tissues were isolated immediately, and fixation were done in 10 % neutral-buffered formalin for 72 hours and then processed for paraffin embedding and cut into transversal sections (6 μm thick) using a microtome. The sections were stained with hematoxylin and eosin for evaluation. Immunohistochemical detection of Col I, Col II performed using formalin fixed sections obtained as described above. Briefly, NP tissue sections were maintained at room temperature for 60 minutes and dewaxed by xylene twice (10 minutes each time). The tissues were then rehydrated by a series of 5-minute washed in 100 %, 95 %, 80 %, and 70 % ethanol, followed by 5-minute washed in distilled water and three consecutive 3-minute washed with PBS (PBS; Gibco Grand Island, New York, USA) . Incubation in 3 % hydrogen peroxide for 10 minutes were delivered in purpose of inactivating the endogenous peroxidase, after that, antigen retrieval was performed by heating the samples at 95 °C for 20 minutes in 10 mM sodium citrate (pH 6.0). Nonspecific binding was blocked by incubating with 10 % normal goat serum (Gibco Grand Island, New York, USA) for 20 minutes, then the NP tissue sections were labeled overnight at 4 °C with primary antibody: anti-Col I (1:200dilution), anti-Col II (1:200 dilution), polyclonal antibodies (immunoglobulin G) (Hua An Biotech, Hangzhou , China) . The NP sections were then incubated for 60 minutes each with a horseradish peroxidase-labeled secondary antibody and then streptavidin–peroxidase (Hua An Biotech). After washing with PBS, the sections were incubated with 3,30-diaminobenzidine substrate until a brown color generated. Finally, the sections were counterstained with hematoxylin. Dehydration was performed by using a series of 2-minute washes in 50 %, 70 %, 95 %, 95 %, and 100 % ethanol. After two consequent 2-minute washes with xylene, the slides were sealed with coverslips. To quantify the immunohistochemical results, staining intensity was analyzed using the Image-Pro Plus 6.0 (Media Cybernetics, Rockville, Maryland, USA) . The area of interest in all sections was analyzed, and the mean density was calculated by integrated optical density divided by the area. Real-time PCR analysis of gene expression Total RNA was extracted from the NP using the Trizol reagent (Life Technologies) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using AMV reverse transcriptase (Life Technologies) . After the cDNA had been obtained by reverse transcription, relative gene expressions of COL1, COL2, TIMP-1 and BMP2 were determined by real-time PCR and normalized to the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. These primers were designed using Primer Premier 6.0 software (PREMIER Biosoft, palo alto, California, USA) (Table 1). The Mini Opticon™ Detector System (Life Technologies) and the SYBR Green PCR kit (Life Technologies) were used for Realtime PCR analysis. The real-time PCR consisted of an initial enzyme activation step at 95 °C for 20 seconds, followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 20 seconds. A cycle threshold (Ct) value was obtained for each sample, and triplicate sample values were averaged. The 2–ΔΔCt value was then used to calculate relative expression of each target gene [10] . The data presented (mean) were from three independent experiments in which both sample sets were analyzed in triplicate. Statistical analysis All data were statistically analyzed with GraphPad Prism (v6.0). Error bars in graphical data represent mean ±s.d. Statistical significance was determined using a Mann–Whitney U test, in which p values of p ＜ 0.05 were considered statistically significant. Variance was similar between the groups that were statistically compared. Results MRI assessment The Pfirrmann classification results at the 3 time points are shown in Fig 2 . At 3 and 6 months after surgery, the signal intensities on T2-weighted images of punctured discs in IF group were significantly degenerated as compared to before surgery ( Fig. 2b.c ). At 3 months after surgery, the degree of degeneration in the punctured discs was mainly grade III according to the Pfirrmann classification and grade IV to V at 6 months (Fig 3 ). The MRI results showed a gradual increase in Pfirrmann grade after surgery. In Cof1 and Cof2 groups at 3 months, the signal intensities on T2-weighted images of punctured discs were similar as before surgery （ Fig. 2e.h ） , and at 6 months there are only a slight decrease, no prominent degeneration or disc herniation were observed ( Fig. 2f.i ). For these two groups, Pfirrmann grade were mainly grade I to II at 3 months after surgery, and the degree of degeneration at 6 months were mainly grade II to III ( Fig 3 ). Gross anatomical findings The control discs (L2-3) in all groups demonstrated no degenerative changes at 6 months, as anatomical morphology showed Gel-like nucleus pulposus, discrete fibrous lamellas and there were no cleft or anular disorganization can be identified. The L5-6 discs in IF group showed that tissue defects happened in the nucleus, and fibrous tissue are indistinguishable from annulus. Besides, anular disorganization, cleft extended throughout NP and anulus fibrosus (AF) were also can be seen (Fig. 4a). The Cof1 and Cof2 groups showed slight degeneration happened in NP with NP appeared viscous and suffered peripheral fibrous infiltration. Nucleus and anulus were distinguishable, anulus were well-organized and half ring-shaped and no identified cleft in NP and AF, no prominent degeneration were seen, except for some tiny osteophytes generated at the margin of anterior region of the vertebrae ( Fig. 4b.c ). Histological and immunohistochemical analysis To verify the occurrence of disc degeneration, H & E staining were performed to examine the morphology of the NP and AF. As shown in Fig 5 , the control discs had a normal boundary between the AF and the NP, the cells of the nucleus pulposus were normal, the NP were mixed of large, vacuolated (notochordal) cells and smaller, chondrocyte-like cells, and the AF was well organized parallel. When Compared with controls, noteworthy degenerative morphological changes in the IVDs in IF group were observed after surgery, cracks and ruptures of collagen fibers could be seen in the annulus fibrosus, most contents of normal nucleus pulposus were lost, and there were very few chondrocyte-like cells, In addition, the boundary between the NP and AF was not exist ( Fig. 5b ). Though histologic results showed degeneration in all objective discs, the degenerative changes of the NP in the IF groups were more apparent than those in the Cof1 and Cof2 groups, as shown in (Fig. 5c.d) To further demonstrate the protection effect of Coflex on the objective disc , the NP sections were stained by Col I ， Col II antibodies at 6 months. As shown in Fig 6, the immunohistochemical staining indicated that the NP in the IF discs was significant strongly positive for Col I, compared to the control and Cof1 and Cof2 groups. When compared to the control and other two groups, the staining intensity of Col II decreased significantly in IF group. Immunohistochemical staining intensity analysis were performed by Image-Pro Plus 6.0, the results showed that the staining intensity of Col I was significantly higher and Col II was significantly lower in the IF group than that in the control and other two groups (p <0.05). Gene expression analysis To confirm the protection effect of Coflex on the objective disc, real-time PCR was used to measure the levels of TIMP1, BMP2, Col I, Col II, in the control, IF, Cof1 and Cof2 groups at 3 and 6 months. As shown in (Fig 7) , the levels of a TIMP1, BMP2, Col I was increased in the IF, Cof1, Cof2 groups at two observe time point, and the Col II expression was decreased. At 3 month, TIMP1 mRNA expression markedly increased and Col 2 decreased in discs from the IF group compared with the Cof1 and Cof2 groups （ p ＜ 0.05 ） , while the increase of BMP2 and COL I were not significant. Noteworthily, at 6month, remarkably increase of Col I, TIMP1, BMP2 and decrease of Col 2 were recorded in IF group (p <0.01). In IF group, when comparison was delivered chronologically, the result showed that the TIMP1, BMP2 and Col I increased and Col II decreased more prominently at 6 months compared to those at 3 month (p ＜ 0.01), however, such significant difference were not seen in Cof1 and Cof2 groups. Moreover, as we assumed, no conspicuous difference between Cof1 and Cof2 groups were detected. Together, the results show that adjacent segment inter-body fusion increased the expression of TIMP1, BMP2, Col I and decreased Col II, and such tendency has become more significant with the pass of time. Moreover, the implantation of Coflex can attenuate this effect. Discussion Rigid spinal fusion is the most popular surgical procedure in the management of lumbar instability or lumbar disk herniation (LDH) [11] . Though rigid fusion has been proved to be an effective way to restore the disc space height, reconstruct the stability and alignment of spine, a variety of complications had emerged such as adjacent segment disease with the change of spinal mechanical activities [12] . The rigid fusion decreased the flexibility and mobility of the entire lumbar spine, leading to the center of rotation of vertebral body shifts over the disc, consequently, the stress on the facets and/or disc of the adjacent mobile segment significantly increased [4] . The increased stress will lead to a significant effect on intersegmental mobility and the increase in intradiscal pressure, overall, it accelerates degenerative changes at adjacent unfused segment, especially at the cranial level [13] . Moreover, several researchers have demonstrated that rigid fusion not only play an important role in the initiation of adjacent segment degeneration, but would also make pre-existed degenerative changes in the adjacent level deteriorate [14] . Recently, a long time follow up remarked that adjacent segment disease is a severe problem that causes refractory low back pain, which would cause patients’ as well as surgeons’ dissatisfactions, and revision laminectomy and extension of fusion may become unavoidable in some cases [15] . long segment lumbar arthrodesis would become a mandatory procedure in the treatment of such refractory pain, which leads to spinal deformities and deliver a heavy burden both on individual and nations [16] . For the purpose of avoiding or ameliorating these adverse effects, the appearance of dynamic stabilization devices such as Coflex has brought new hope to spine society, such device is considered to reduce the stress on adjacent segments to achieve relatively ideal mobility, thereby avoid the harmful effects of rigid fusion [17] . Hybrid surgery such as Interspinous device+ interbody fusion has become a promising method to delay the adjacent segment degeneration, and some researchers have investigated its efficacy in clinical use [18] . Though many preliminary evidences have showed its protection role in prevention or deceleration of ASD, there are still some controversy yet to be explored [19] . In our study, we established an L5-6 disc degeneration model in beagles by annular puncture from the posterior approach using 18-gauge needles under C-arm fluoroscopic guidance and observed the role of Coflex in the objective segment after rigid fusion was performed at adjacent segment. we firstly showed that rigid fusion could accelerate ASD, and further demonstrated that Coflex could not completely prevent the deterioration of ASD but can significantly decelerate the progressive of ASD. Moreover, Coflex can demonstrate its protection role both in adjacent segment after one level spinal fusion and in the middle segment as a “transition” area after "skipped" level (nonconsecutive) interbody fusion. Beagles exhibited gradually increasing degenerative changes in IF group as demonstrated by both Pfirrmann classification and histological evaluation after surgery. Moreover, gene expression analysis showed an increase in the mRNA expression levels of Col I, TIMP1 and BMP2, three major indicators of disc degeneration, and a decrease of Col II, a major component of normal disc [20] . These results were consistent with the matrix breakdown observed in human degenerative disc associated with decrease of Col II expression, fibrosis associated with up-regulation of Col 1, and matrix degradation and disc remodeling associated increase of TIMP1 and BMP2 [21] . Interestingly, our data showed that the progress of degeneration was more rapidly in later-3 months than it in pre-3 months, and among the genes expression we investigated, only the change of TIMP1 and Col 2 were significant at 3 months. One explanation to this divergence is that animals were lack of activity due to the pain and feebleness after surgery at first several months, consequently, motion and load bearing of spine were very limited in these periods. When they have rehabilitated and normal activities were restored, significant degeneration were exhibited in IF group at 6 months compared to other groups. Though progressive degeneration can be found in all experimental groups, a significant prevention or deceleration effect was exhibited by Coflex. A variety of animal models have been established for the exploration of intervertebral disc degeneration and treatment [22] . IVD could successfully developed in many models, however, the pathological and molecular changes are different, according to the varied anatomical and physiological structures of different animals. Rodents model is the most popular one among those models, nevertheless, the different structure and components of disc between rodents and humans has limit its accuracy in the study of IVD and treatment [23] . In this study, we chose beagles as animal model because the beagle’s disc is similar with human both in physiology and morphology , in addition, though beagles are quadrupeds animals, they like squatting for most time, such mechanical property was of the same status in human [22, 24] . We force beagles stay in erect position more than 8 hours per day after surgery by place food and water in a high place to reach, which would facilitate the progression of degeneration. A variety of methods have been conceived to achieve the establishment of the model of IVD degeneration, scalpel blade and gauge needle were used in those classic models [21] . According to these previous study , the velocity of the progression of disc is determined by the extent of damage to the annulus pulposus, using scalpel blade can cause an acute disc degeneration within 2 weeks, while a 16 or 18-gauge needle would make it happen in a gradually, relatively slow pattern, which is similar to how its work in human IVD degeneration [25 , 26] . As the purpose of this study is to clarify the ability of Coflex to decelerate or prevent ASD, which is considered as a slow progressive pathological change, a 16-gauge needle was used to develop the IVD degeneration model. In this study, we arranged control and experimental segments in the same dog, which can eliminate individual differences such as age, weight, pre degeneration status. The present study has some limitations. It has been reported that the IVD of beagles have a tendency of degeneration at an earlier age, which might cause difference of existed among objective IVDs to some extent. Therefore, this might bring potential bias to the present study. In addition, Pfirrmann classification is a non-quantitative analysis, quantitative evaluation of MRI was not performed, and the number of animals for histological and immunohistochemically analysis was small. Nevertheless, the study demonstrated the interspinous spacer device Coflex can protect adjacent segment after spinal fusion and decelerate the progression of adjacent IVD degeneration. The protect role can both exhibited well in adjacent segment and the middle segment in the treatment of "skipped" level (nonconsecutive) disc degeneration (SLDD) after fusion were delivered at two “skipped” level. Conclusions The present study demonstrated that the implantation of Coflex could decelerate the progression of adjacent IVD degeneration after spinal fusion. The results support the premises that Coflex is an effective interspinous device in the prevention or deceleration of ASD after spinal fusion, both in adjacent segment and middle segment. Abbreviations ISPs: Interspinous spacer; ASD: adjacent segment degeneration; IF: interbody fusion; Cof1: L4-5 lumbar interbody fusion +L5-6 interspinous Coflex implantation Cof2: L4-5 and L6-7 interbody fusion+ L5-6 interspinous Coflex implantation Con group: noninjuries control SLDD: "skipped" level (nonconsecutive) disc degeneration MRI: magnetic resonance imaging IVD: Intervertebral disc NP: nucleus pulposus AF: anulus fibrosus LDH: lumbar disk herniation Declarations Ethics approval and consent to participate Experiments of animals were approved by the Animal Research Committee of Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine. Acknowledgements This study was supported by the Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital . The authors would like to thank the personnel from the spine surgery clinical team leading by prof. XJG for participating in the surgery and data collection, and the discussion of this project. Funding This study was financially supported by Science and Technology Commission of Shanghai Municipality (NO. SHDC2014102). The funding plays important roles in the design of the study; collection, analysis, and interpretation of data. It also helps in supplying the experimental animals; purchasing the experimental materials etc. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors' contributions XJG conceived the initial idea and the conceptualization, chose the animal model and assisted in the surgery. 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Clin Orthop Relat Res. 2015;473(6):1903-12. Singh K, Masuda K, An HS. Animal models for human disc degeneration. Spine J. 2005;5(6 Suppl):267S-79S. Chen CH, Chiang CJ, Wu LC, Yang CH, Kuo YJ, Tsuang YH, et al. Time course investigation of intervertebral disc degeneration in a rat-tail puncture model. Life Sci. 2016;156:15-20. Zhang Y, Tao H, Gu T, Zhou M, Jia Z, Jiang G, et al. The effects of human Wharton's jelly cell transplantation on the intervertebral disc in a canine disc degeneration model. Stem Cell Res Ther. 2015;6:154. Lei T, Zhang Y, Zhou Q, Luo X, Tang K, Chen R, et al. A novel approach for the annulus needle puncture model of intervertebral disc degeneration in rabbits. Am J Transl Res. 2017;9(3):900-9. Mwale F, Masuda K, Grant MP, Epure LM, Kato K, Miyazaki S, et al. Short Link N promotes disc repair in a rabbit model of disc degeneration. Arthritis Res Ther. 2018;20(1):201. Tables Table 1 Primers designed using Primer Premier 6.0 software Gene Gene bank Sequence TIMP1 NM_001003182.1 5’-ACCTATGCTGCTGGCTGTG-3’ 5’-GGTTTCCAGAGCCGCCAC-3’ BMP2 XM_534351.5 5’-AACTCCACTAACCACGCCATTG-3’ 5’-GGTTGTGGAGGGTTGTGGGT-3’ Col 1 NM_001003090.1 5’- GTAGACACCACCCTCAAG-3’ 5’-GGAAGAGCGGAGAATACT-3’ Col 2 NM_001006951.1 5’-AGCAGCAAGAGCAAGGACAAG-3’ 5’-CCTTCCTCCGCCTGCTGT-3’ GAPDH NM_001003142.2 5’- ATTCCACGGCACAGTCAAG-3’ 5’- GGTGATGCTGGTGCTGAG-3’ Primer Premier 6.0 software from PREMIER Biosoft (palo alto, California, USA) TIMP1 inhibitors of matrix metalloproteinases 1 , BMP2 Bone morphogenetic protein 2 Col 1 type I collagen , Col 2 type II collagen, GAPDH glyceraldehyde-3-phosphate dehydrogenase Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3305","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":124769,"identity":"55382fda-2521-44f2-8193-dc8f76556e79","order_by":1,"name":"Jia-ming Liang","email":"","orcid":"https://orcid.org/0000-0003-3485-0233","institution":"Shanghai Jiao Tong University Affiliated Sixth People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia-ming","middleName":"","lastName":"Liang","suffix":""},{"id":124770,"identity":"ad43424e-8204-48da-a6d1-92eb5599788b","order_by":2,"name":"Fang-qi Lin","email":"","orcid":"","institution":"Shanghai Jiao Tong University Affiliated Sixth People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fang-qi","middleName":"","lastName":"Lin","suffix":""},{"id":124771,"identity":"6bd3d98d-7865-488c-b30d-f6b21f584a69","order_by":3,"name":"Jia-ning Ding","email":"","orcid":"","institution":"Shanghai Jiao Tong University Affiliated Sixth People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia-ning","middleName":"","lastName":"Ding","suffix":""},{"id":124772,"identity":"d1a34d6b-2984-4c34-a45c-3d9613416d48","order_by":4,"name":"Jian-guang Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYFCCAwwMHxgYeBgYmA8c+FBBpBbGGWAtbIkHZ5wh0h5mDjDFY3yYt4UI5QYHj197zPDnjow5e8+HA7wNDPL8Ygfwa5FsOFNuXNj2jMey5+yGA5I7GAxnzk7Ar4Wf4Uya9MyGwzwGN3I3HDA8w5BgcJuAFjaQFp4/QC333zw4kNhGhBZ+huPHpHnYQLbwMBw4SIwWoF/YJGcC/WJwJs3gYMMZCcJ+Mbhx/JnEhz937A2OH378+U+FjTy/NAEtDBJnDBjASQDKJaAcBPjbHyBrGQWjYBSMglGACQD/alANV1j6+gAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jian-guang","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2019-08-02 14:01:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.12549/v1","doiUrl":"https://doi.org/10.21203/rs.2.12549/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":442524,"identity":"4a39bec8-82fb-481d-aa83-8dcf96598b68","added_by":"auto","created_at":"2020-02-04 11:17:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":560114,"visible":true,"origin":"","legend":"Surgical procedure. a. Anterior lumber interbody fusion use prepared iliac bone graft. b. Posterior implantation of Coflex. c.d. Development of the disc degeneration model. Needle tips reached contralateral side of the discs both in anterior-posterior and lateral radiographs.","description":"","filename":"Fig1.surgicalprocedure.jpg","url":"https://assets-eu.researchsquare.com/files/8fde6a2f-588d-4223-ae75-94e419982bf6/v1/Fig1.surgical procedure.jpg"},{"id":442525,"identity":"89f3cc86-4ad3-4b19-b53e-5b065b74c164","added_by":"auto","created_at":"2020-02-04 11:17:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":687710,"visible":true,"origin":"","legend":"Sagittal T2-weighted images by MRI at 3 observed time points. Pre-operative images(a.d.g.) at 3 groups showed hyperintense signal and the homogeneous structures of the L5-6 discs. At 3 months post-operation(b.e.h.), MRI T2-weighted image showed inhomogeneous structure of the disc with an intermediate gray signal intensity in the L5-6 disc in IF-group(b.), while in Cof1 and Cof2 groups(e.h.), hyperintense signal were still seen. At 6 months post-operation(c.f.i.),in IF group(c.), the structure of the disc is inhomogeneous, with a hypointense black signal intensity. The distinction between nucleus and anulus is lost, and the disc collapse even herniation could be seen, however, Cof1 and Cof2 groups(f.i.) only showed high intensity slightly decreased, any further degeneration were not seen.","description":"","filename":"Fig2.MRIscanning.jpg","url":"https://assets-eu.researchsquare.com/files/8fde6a2f-588d-4223-ae75-94e419982bf6/v1/Fig 2. MRI scanning.jpg"},{"id":442526,"identity":"5de9e33f-0997-4cbe-b3b7-fffdd9b65f30","added_by":"auto","created_at":"2020-02-04 11:17:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":383601,"visible":true,"origin":"","legend":"Analysis of signal changes in MRI images with Pfirrmann classification. a. T2-weighted images showed no degeneration in all groups before surgery. b. There was significantly decreased grading of MRI scans in the IF group compared with Cof1 and Cof2 groups at 3 months after surgery(p\u003c0.5). c. MRI grading in the IF was more significantly decreased at 6 months compared with Cof1 and Cof2 after surgery(p\u003c0.001). MRI grading were compared between 3 months and 6 months(b.c.), significant decrease was seen in IF group at 6 month(p\u003c0.001), however, such decrease were not seen in Cof1 and Cof2 groups(p\u003e0.05).","description":"","filename":"Fig3.PfirrmannClassification.jpg","url":"https://assets-eu.researchsquare.com/files/8fde6a2f-588d-4223-ae75-94e419982bf6/v1/Fig 3.Pfirrmann Classification.jpg"},{"id":442527,"identity":"84ad5ea0-d61c-4ab8-90c1-19da13f99ea5","added_by":"auto","created_at":"2020-02-04 11:17:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":287506,"visible":true,"origin":"","legend":"Gross anatomical views of L5/6 disc at 6 months after surgery. a. Disc in the IF group showed nucleus defected, fibrous tissues are indistinguishable from annulus. Anular disorganization, cleft extended throughout NP and AF were also can be seen. b. Disc in the Cof1 and Cof2 groups showed nucleus and anulus were distinguishable, annuls were well-organized, and some tiny osteophytes generated at the margin of anterior region of the vertebrae.","description":"","filename":"Fig4.Grossanatomicalviews.jpg","url":"https://assets-eu.researchsquare.com/files/8fde6a2f-588d-4223-ae75-94e419982bf6/v1/Fig 4. Gross anatomical views .jpg"},{"id":442528,"identity":"10b17f77-5666-4462-80e2-474240a27fd5","added_by":"auto","created_at":"2020-02-04 11:17:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":292919,"visible":true,"origin":"","legend":"Histological images of the NP in 3 groups and the control at 6 months. The paraffin sections of the NP tissues were stained by hematoxylin and eosin. a. Showed intact annulus fibrosus and cell-enriched nucleus pulposus. b. Disc in IF group showed chondrocyte-like cells in nucleus pulposus were approximately disappeared, and cracks among the layers of collagen fibers of the annulus fibrosus emerged. c.d. Discs in Cof1 and Cof2 groups showed intact annulus fibrosus, while the number of chondrocyte-like cells in nucleus pulposus were relatively reduced.","description":"","filename":"Fig5.Histologicalimages.jpg","url":"https://assets-eu.researchsquare.com/files/8fde6a2f-588d-4223-ae75-94e419982bf6/v1/Fig 5.Histological images .jpg"},{"id":442529,"identity":"8b7d3428-9ac5-413d-877b-bbbccdd501c1","added_by":"auto","created_at":"2020-02-04 11:17:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":376178,"visible":true,"origin":"","legend":"Typical immunohistochemical images at 6 months. The immunohistochemical micrographs showed the images of the NP sections which were stained by an antibody against type I collagen (Col I), and type II collagen (Col II). Staining of the Col I for the IF group was stronger than that for the Cof1 and Cof2 groups and the control, while the staining of the Col2 was much weaker.","description":"","filename":"Fig6.immunohistochemicalimages.jpg","url":"https://assets-eu.researchsquare.com/files/8fde6a2f-588d-4223-ae75-94e419982bf6/v1/Fig 6.immunohistochemical images.jpg"},{"id":442530,"identity":"2a86c2cc-a2f0-4cf6-9783-81434bb8bfd1","added_by":"auto","created_at":"2020-02-04 11:17:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":287005,"visible":true,"origin":"","legend":"Real-time PCR analysis. Real-time PCR was used to analyze the levels of the disc matrix components Col I, Col II, and anticatabolic factors such as TIMP1 and growth factors such as BMP2 from the Control, IF, and Cof1, and Cof2 groups. Expression was normalized to the average of the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase). The results showed that TIMP1 mRNA expression markedly increased and Col 2 decreased in discs from the IF group compared with the Cof1 and Cof2 groups at 3 months. *statistical significance(p\u003c0.05). Excepted for the existed differences became more significant, BMP2, and Col I significantly increased at 6 months, **statistical significance(p\u003c0.01).","description":"","filename":"Fig7.geneanalysis.jpg","url":"https://assets-eu.researchsquare.com/files/8fde6a2f-588d-4223-ae75-94e419982bf6/v1/Fig 7.gene analysis.jpg"},{"id":13469469,"identity":"3edde992-8d4f-4a26-98ad-ba906bacc89d","added_by":"auto","created_at":"2021-09-16 21:01:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":814679,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3305/v1/1a2f7bec-2d16-4615-a74d-d92c4daa7bcf.pdf"}],"financialInterests":"","formattedTitle":"Effect of Coflex interspinous stabilization on the prevention of progression of adjacent segment degeneration after single level and skipped level spinal fusion in a canine model","fulltext":[{"header":"Background","content":"\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eFrom now on, the rapidly aging population has become a most common situation in global society, and the increasingly prevalence of lumbar spine disorders have paralleled this trend, unfortunately, most of these clinical conditions affecting lumbar spine cannot exempt from surgery treatment \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[1\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e,\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e2]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e.\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eSpinal fusion has been employed to treat a variety of spinal disorders, including degenerative disc disease, segmental instability, spondylolisthesis, trauma, and scoliosis for decades\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[3]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. Due to spinal fusion surgery has significantly facilitated patient comfort and mobility, fusion is regarded as a gold standard treatment for degenerative lumbar spine disease. However, we cannot ignore the fact that many researchers have demonstrated that fusion can accelerate the degeneration of adjacent lumbar segment, due to it can lead to excessive stress at unfused adjacent levels\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e[4\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e,\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e5]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. Therefore, society of spine surgery holds the promise to develop an effective method to prevent or slow down the progress of adjacent segmental degeneration (ASD), after surgical treatment of lumbar degenerative disorders \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[6]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eMany risk factors for ASD have been reported, such as pre-existing degeneration of adjacent discs, facet tropism, post-operative sagittal alignment and so on\u003cspan style=\"color: black;\"\u003e.\u003c/span\u003e Among those risk factors, the elimination of fixed segment motion are thought to play the most important role, as it can increase unfused adjacent segment motion, and then accelerates the degeneration of adjacent segments\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e[7]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u003cspan style=\"color: black;\"\u003e.\u003c/span\u003e Implantation of an interspinous spacer(ISPs) device in adjacent segment is a known feasible technique to tackle with ASD, because it can facilitate to decelerate the degenerative process by providing intervertebral dynamic stability for adjacent segment \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e[6]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. Coflex is a dynamic ISP, it is a U-shaped, compressible device which can be implanted between the spinous processes after decompression\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[8]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. As far as we know, there are few studies have focused on the effectiveness of such device to prevent or decelerate the progress of adjacent segment degeneration after spinal fusion.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eIn this study, we postulated that the implantation of Coflex between the spinous processes in adjacent segment after spinal fusion could prevent or decelerate \u003cspan style=\"color: black;\"\u003eASD\u003c/span\u003e. And it can also play the same important role in the middle segment as a \u0026ldquo;transition\u0026rdquo; area in the treatment of \u0026ldquo;skipped\u0026rdquo; level (nonconsecutive) disc degeneration (\u003cspan style=\"color: black;\"\u003eSLDD\u003c/span\u003e) after fusion were delivered at two \u0026ldquo;skipped\u0026rdquo; level, when used in a canine spinal fusion model.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAnimals and Groups \u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eEighteen healthy and skeletal mature beagles (19 months old on average), weight 16 to 21 kg and average weight 18kg, were used in this study. The experimental animals were provided by the animal experimental center of the Shanghai Jiao Tong University Affiliated Sixth People\u0026rsquo;s Hospital. And experiments of animals were approved by the Animal Research Committee of Sixth People\u0026rsquo;s Hospital, Shanghai Jiao Tong University School of Medicine. All animals were healthy and free of infection, and were magnetic resonance imaging (MRI) scanned of the spine to assure the absence of Intervertebral disc (IVD) degeneration related diseases before the study. 18 beagles were bred and randomly allocated into the following study groups:(1)L4-5 lumbar interbody fusion\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eIF\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e）\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. (2) L4-5 lumbar interbody fusion +L5-6 interspinous Coflex implantation\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eCof1\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e）\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e.(3) L4-5 and L6-7 interbody fusion+ L5-6 interspinous Coflex implantation \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eCof2\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e）\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. In all animals, L5-6 discs were punctured to generate degeneration \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e，\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eand the intact L2\u0026ndash;3 disc served as a noninjuries control (Con group). The animals were followed up for 6 months after the initial operation. The changes in the L5/L6 lumbar discs were assessed using gross anatomical observation, MRI, histology, and gene expression analysis at 3 and 6 months respectively. At each point\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e3 months and 6 months postoperatively\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e）\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e, 3 beagles were randomly selected from each group to be killed with an excess dose of ketamine hydrochloride and xylazine hydrochloride injection after MRI scan. \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ePreparation of Autologous Iliac Bone Graft\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e24 hours abstinence for food and 12 hours abstinence for water was maintained before surgery, and Surgical procedures were performed under general anesthesia by intramuscular administration of ketamine hydrochloride injection (0.1 ml/kg) and xylazine hydrochloride (0.08 ml/kg). After anesthetization, the dogs were placed in the lateral position. The incision was 4 cm along the lateral border of the spina iliac anterior superior. After exposure of spina iliac anterior superior, an approximately 3 \u0026times; 2-cm autologous iliac bone graft was obtained by bone drill. The autologous iliac bone graft was prepared for anterior lumbar interbody fusion.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAnterior lumbar interbody fusion and implantation of Coflex\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eWhen the harvesting of iliac bone graft finished, beagles for experiment were placed in a supine position, median abdominal incision were delivered, then lateral incision approach of the rectus abdominis was achieved, once an opening through the muscle was obtained, carefully protect the peritoneum and reflect it anteriorly by blunt dissection. After retraction of the peritoneum and its contents using a wide retractor, the appropriate involved vertebras were identified. Separate the intervertebral disc and annulus from the cartilaginous endplates of the vertebrae with a thin osteotome, and then the disc was removed by pituitary rongeurs, Kerrison rangers and curets. When interbody space was cleaned and prepared for fusion, predisposed grafts were used to complete interbody fusion. After completion of the fusion, close all layers with absorbable sutures. When the interbody fusion was delivered, the Coflex groups underwent additional implantation of the Coflex\u003cspan style=\"color: black;\"\u003e (Paradigm Spine, LCC, New York)\u003c/span\u003e at the involved level. The beagles were placed in prone position, surgery was performed through a standard posterior midline approach, and then the interspinous ligament was dissected and excised. The device was inserted between the adjacent spinous processes and the flanges were crimped follow the manufacturer\u0026rsquo;s instructions, so that it was seated fitly. Appropriate placement of the implant and adequate segmental sagittal alignment were identified under C-arm machine. \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eGeneration of L5-6 disc degeneration\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAfter all of surgical procedures described above were done, the beagles were placed in prone position, L5\u0026ndash;6 discs were then punctured with 18-gauge needles\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; color: black;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eHuaYi Bio-technology, Shanghai, China\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; color: black;\"\u003e）\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ealong the outside of the facet joints under C-arm fluoroscopic guidance until the needle tips reached contralateral side of the discs both in anterior-posterior and lateral radiographs. Following the operation, antibiotic was intravenously delivered in five consecutive days. Beagles were fed in cages, and food and water were placed at a relatively high place, which would force them in erect position more than 8 hours per day. The animals were monitored daily for potential complications or abnormal behavior. Important surgical procedures were shown in Fig. 1.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eMagnetic resonance imaging\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eMRI \u003cspan style=\"color: black;\"\u003e(Achieva 3.0T. Philips, Holland) \u003c/span\u003escans were administered to evaluate signal changes in T2-weighted (T2-W; TR 2270 ms, TE 126 ms) images at baseline, 3 and 6 months after the operation in all groups. The subjects were imaged at the time of follow-up using the same scanner and examination protocol, and the slice thickness and interslice gap were 4 mm and 0.4 mm for sagittal. Lumbar IVD degeneration was graded on T2-weighted MR images according to the Pfirrmann classification system on a scale of I\u0026ndash;V, where grade V denotes the most degenerate category \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[9]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e.\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e All radiological assessments were made independently by two observers, including one radiologist and one orthopedic spine surgeon. when disagreement occurred with respect to the radiological grade, a consensus opinion with involvement of a third observer was sought.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eGross anatomical observation\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAt 3 and 6 months, after MRI scan, 3 beagles of each groups were killed with an excess dose of ketamine hydrochloride and xylazine hydrochloride injection, and the spines were harvested. Then the L2-3 and L5\u0026ndash;6 discs were isolated intact, bilateral cartilage endplate and some vertebral body were preserved. The discs from each dog were cut coronally at the center of the disc for Gross anatomical observation.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eHistological and immunohistochemically analysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-indent: 24.0pt; line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAll L5-6 discs of 18 dogs were cut transversally at the center of the nucleus pulposus (NP). One half of every disc was used for histological studies, and the other half was used for Realtime PCR analysis of gene expression. \u003cspan style=\"color: black;\"\u003eThe NP tissues were isolated immediately, and fixation were done in 10 % neutral-buffered formalin for 72 hours and then processed for paraffin embedding and cut into transversal sections (6 \u0026mu;m thick) using a microtome.\u003c/span\u003e The sections were stained with hematoxylin and eosin for evaluation. Immunohistochemical detection of Col I, Col II performed using formalin fixed sections obtained as described above. Briefly, NP tissue sections were maintained at room temperature for 60 minutes and dewaxed by xylene twice (10 minutes each time). The tissues were then rehydrated by a series of 5-minute washed in 100 %, 95 %, 80 %, and 70 % ethanol, followed by 5-minute washed in distilled water and three consecutive 3-minute washed with PBS \u003cspan style=\"color: black;\"\u003e(PBS; Gibco Grand Island, New York, USA)\u003c/span\u003e. \u0026nbsp;Incubation in 3 % hydrogen peroxide for 10 minutes were delivered in purpose of inactivating the endogenous peroxidase, after that, antigen retrieval was performed by heating the samples at 95 \u0026deg;C for 20 minutes in 10 mM sodium citrate (pH 6.0). Nonspecific binding was blocked by incubating with 10 % normal goat serum \u003cspan style=\"color: black;\"\u003e(Gibco Grand Island, New York, USA)\u003c/span\u003e for 20 minutes, then the NP tissue sections were labeled overnight at 4 \u0026deg;C with primary antibody: anti-Col I (1:200dilution), anti-Col II (1:200 dilution), polyclonal antibodies (immunoglobulin G) \u003cspan style=\"color: black;\"\u003e(Hua An Biotech, Hangzhou , China)\u003c/span\u003e. The NP sections were then incubated for 60 minutes each with a horseradish peroxidase-labeled secondary antibody and then streptavidin\u0026ndash;peroxidase \u003cspan style=\"color: black;\"\u003e(Hua An Biotech). \u003c/span\u003eAfter washing with PBS, the sections were incubated with 3,30-diaminobenzidine substrate until a brown color generated. Finally, the sections were counterstained with hematoxylin. Dehydration was performed by using a series of 2-minute washes in 50 %, 70 %, 95 %, 95 %, and 100 % ethanol. After two consequent 2-minute washes with xylene, the slides were sealed with coverslips. To quantify the \u003cspan style=\"color: black;\"\u003eimmunohistochemical results, staining intensity was analyzed using the Image-Pro Plus 6.0 (Media Cybernetics, Rockville, Maryland, USA)\u003c/span\u003e. The area of interest in all sections was analyzed, and the mean density was calculated by integrated optical density divided by the area.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eReal-time PCR analysis of gene expression\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eTotal RNA was extracted from the NP using the Trizol reagent \u003cspan style=\"color: black;\"\u003e(Life Technologies)\u003c/span\u003e according to the manufacturer\u0026rsquo;s instructions. RNA was reverse transcribed into cDNA using AMV reverse transcriptase \u003cspan style=\"color: black;\"\u003e(Life Technologies)\u003c/span\u003e. After the cDNA had been obtained by reverse transcription, relative gene expressions of COL1, COL2, TIMP-1 and BMP2 were determined by real-time PCR and normalized to the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. These primers were designed using Primer Premier 6.0 software \u003cspan style=\"color: black;\"\u003e(PREMIER Biosoft, palo alto, California, USA)\u003c/span\u003e (Table 1). The Mini Opticon\u0026trade; Detector System \u003cspan style=\"color: black;\"\u003e(Life Technologies)\u003c/span\u003e and the SYBR Green PCR kit \u003cspan style=\"color: black;\"\u003e(Life Technologies)\u003c/span\u003e were used for Realtime PCR analysis. The real-time PCR consisted of an initial enzyme activation step at 95 \u0026deg;C for 20 seconds, followed by 40 cycles of 95 \u0026deg;C for 5 seconds and 60 \u0026deg;C for 20 seconds. A cycle threshold (Ct) value was obtained for each sample, and triplicate sample values were averaged. The 2\u0026ndash;\u0026Delta;\u0026Delta;Ct value was then used to calculate relative expression of each target gene\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[10]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. The data presented (mean) were from three independent experiments in which both sample sets were analyzed in triplicate.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eStatistical analysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAll data were statistically analyzed with GraphPad Prism (v6.0). Error bars in graphical data represent mean \u0026plusmn;s.d. Statistical significance was determined using a Mann\u0026ndash;Whitney U test, in which p values of p \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e＜\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e0.05 were considered statistically significant. Variance was similar between the groups that were statistically compared.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eMRI assessment\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eThe Pfirrmann classification results at the 3 time points are shown in \u003cspan style=\"color: black;\"\u003eFig 2\u003c/span\u003e. At 3 and 6 months after surgery, the signal intensities on T2-weighted images of punctured discs in IF group were significantly degenerated as compared to before surgery (\u003cspan style=\"color: black;\"\u003eFig. 2b.c\u003c/span\u003e). At 3 months after surgery, the degree of degeneration in the punctured discs was mainly grade III according to the Pfirrmann classification and grade IV to V at 6 months \u003cspan style=\"color: black;\"\u003e(Fig 3\u003c/span\u003e). The MRI results showed a gradual increase in Pfirrmann grade after surgery. In Cof1 and Cof2 groups at 3 months, the signal intensities on T2-weighted images of punctured discs were similar as before surgery\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eFig. 2e.h\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e）\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e, and at 6 months there are only a slight decrease, no prominent degeneration or disc herniation were observed (\u003cspan style=\"color: black;\"\u003eFig. 2f.i\u003c/span\u003e). For these two groups, Pfirrmann grade were mainly grade I to II at 3 months after surgery, and the degree of degeneration at 6 months were mainly grade II to III (\u003cspan style=\"color: black;\"\u003eFig 3\u003c/span\u003e). \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eGross anatomical findings\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eThe control discs (L2-3) in all groups demonstrated no degenerative changes at 6 months, as anatomical morphology showed Gel-like nucleus pulposus, discrete fibrous lamellas and there were no cleft or anular disorganization can be identified. The L5-6 discs in IF group showed that tissue defects happened in the nucleus, and fibrous tissue are indistinguishable from annulus. Besides, anular disorganization, cleft extended throughout NP and anulus fibrosus (AF) were also can be seen \u003cspan style=\"color: black;\"\u003e(Fig. 4a). \u003c/span\u003eThe Cof1 and Cof2 groups showed slight degeneration happened in NP with NP appeared viscous and suffered peripheral fibrous infiltration. Nucleus and anulus were distinguishable, anulus were well-organized and half ring-shaped and no identified cleft in NP and AF, no prominent degeneration were seen, except for some tiny osteophytes generated at the margin of anterior region of the vertebrae (\u003cspan style=\"color: black;\"\u003eFig. 4b.c\u003c/span\u003e). \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eHistological and immunohistochemical analysis \u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eTo verify the occurrence of disc degeneration, H \u0026amp; E staining were performed to examine the morphology of the NP and AF. As shown in \u003cspan style=\"color: black;\"\u003eFig 5\u003c/span\u003e, the control discs had a normal boundary between the AF and the NP, the cells of the nucleus pulposus were normal, the NP were mixed of large, vacuolated (notochordal) cells and smaller, chondrocyte-like cells, and the AF was well organized parallel. \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eWhen Compared with controls, noteworthy degenerative morphological changes in the IVDs in IF group were observed after surgery, cracks and ruptures of collagen fibers could be seen in the annulus fibrosus, most contents of normal nucleus pulposus were lost, and there were very few chondrocyte-like cells, In addition, the boundary between the NP and AF was not exist (\u003cspan style=\"color: black;\"\u003eFig. 5b\u003c/span\u003e). Though histologic results showed degeneration in all objective discs, the degenerative changes of the NP in the IF groups were more apparent than those in the Cof1 and Cof2 groups,\u003cspan style=\"color: black;\"\u003e as shown in (Fig. 5c.d) To further demonstrate the protection effect of Coflex on the objective disc , the NP sections were stained by Col I\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; color: black;\"\u003e，\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eCol II antibodies at 6 months. As shown in Fig 6, the immunohistochemical staining indicated that the NP in the IF discs was significant strongly positive for Col I, compared to the control and Cof1 and Cof2 groups. When compared to the control and other two groups, the staining intensity of Col II decreased significantly in IF group. Immunohistochemical staining intensity analysis were performed by Image-Pro Plus 6.0, the results showed that the staining intensity of Col I was significantly higher and Col II was significantly lower in the IF group than that in the control and other two groups (p \u0026lt;0.05).\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eGene expression analysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eTo confirm the protection effect of Coflex on the objective disc, real-time PCR was used to measure the levels of TIMP1, BMP2, Col I, Col II, in the control, IF, Cof1 and Cof2 groups at 3 and 6 months. \u003cspan style=\"color: black;\"\u003eAs shown in (Fig 7)\u003c/span\u003e, the levels of a TIMP1, BMP2, Col I was increased in the IF, Cof1, Cof2 groups at two observe time point, and the Col II expression was decreased. \u0026nbsp;At 3 month, TIMP1 mRNA expression markedly increased and Col 2 decreased in discs from the IF group compared with the Cof1 and Cof2 groups\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ep\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e＜\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e0.05\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e）\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e, while the increase of BMP2 and COL I were not significant. Noteworthily, at 6month, remarkably increase of Col I, TIMP1, BMP2 and decrease of Col 2 were recorded in IF group (p \u0026lt;0.01). In IF group, when comparison was delivered chronologically, the result showed that the TIMP1, BMP2 and Col I increased and Col II decreased more prominently at 6 months compared to those at 3 month (p\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%;\"\u003e＜\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e0.01), however, such significant difference were not seen in Cof1 and Cof2 groups. Moreover, as we assumed, no conspicuous difference between Cof1 and Cof2 groups were detected. Together, the results show that adjacent segment inter-body fusion increased the expression of TIMP1, BMP2, Col I and decreased Col II, and such tendency has become more significant with the pass of time. Moreover, the implantation of Coflex can attenuate this effect.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion ","content":"\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eRigid spinal fusion is the most popular surgical procedure in the management of lumbar instability or lumbar disk herniation (LDH) \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[11]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. Though rigid fusion has been proved to be an effective way to restore the disc space height, reconstruct the stability \u003cspan style=\"color: black;\"\u003eand alignment of spine, a variety of complications had emerged such as adjacent segment disease with the change of spinal mechanical activities \u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[12]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. The rigid fusion decreased the flexibility and mobility of the entire lumbar spine, leading to the center of rotation of vertebral body shifts over the disc, consequently, the stress on the facets and/or disc of the adjacent mobile segment significantly increased \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[4]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. The increased stress will lead to a\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e \u003cspan style=\"color: black;\"\u003esignificant effect on intersegmental mobility and the increase in intradiscal pressure, overall, it accelerates degenerative changes at adjacent unfused segment, especially at the cranial level \u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[13]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. Moreover, several researchers have demonstrated that rigid fusion not only play an important role in the initiation of adjacent segment degeneration, but would also make pre-existed degenerative changes in the adjacent level deteriorate \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[14]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eRecently, a long time follow up remarked that adjacent segment disease is a severe problem that causes refractory low back pain, which would cause patients\u0026rsquo; as well as surgeons\u0026rsquo; dissatisfactions, and revision laminectomy and extension of fusion may become unavoidable in some cases \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[15]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. long segment lumbar arthrodesis would become a mandatory procedure in the treatment of such refractory pain, which leads to spinal deformities and deliver a heavy burden both on individual and nations \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[16]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eFor the purpose of avoiding or ameliorating these adverse effects, the appearance of dynamic stabilization devices such as Coflex has brought new hope to spine society, such device is considered to reduce the stress on adjacent segments to achieve relatively ideal mobility, thereby avoid the harmful effects of rigid fusion \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[17]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. Hybrid surgery such as Interspinous device+\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e \u003cspan style=\"color: black;\"\u003einterbody fusion has become a promising method\u003c/span\u003e \u003cspan style=\"color: black;\"\u003eto delay the adjacent segment degeneration, and\u003c/span\u003e \u003cspan style=\"color: black;\"\u003esome researchers have investigated its efficacy in clinical use \u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[18]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. Though many preliminary evidences have showed its protection role in prevention or deceleration of ASD, there are still some controversy yet to be explored \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[19]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eIn our study, we established an L5-6 disc degeneration model in beagles by annular puncture from the posterior approach using 18-gauge needles under C-arm fluoroscopic guidance and observed the role of Coflex in the objective segment after rigid fusion was performed at adjacent segment. we firstly showed that rigid fusion could accelerate ASD, and further demonstrated that Coflex could not completely prevent the deterioration of ASD but can significantly decelerate the progressive of ASD. Moreover, Coflex can demonstrate its protection role both in adjacent segment after one level spinal fusion and in the middle segment as a \u0026ldquo;transition\u0026rdquo; area after \"skipped\" level (nonconsecutive) interbody fusion.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eBeagles exhibited gradually increasing degenerative changes in IF group as demonstrated by both Pfirrmann classification and histological evaluation after surgery. Moreover, gene expression analysis showed an increase in the mRNA expression levels of Col I, TIMP1 and BMP2, three major indicators of disc degeneration, and a decrease of Col II, a major component of normal disc \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[20]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. These results were consistent with the matrix breakdown observed in human degenerative disc associated with decrease of Col II expression, fibrosis associated with up-regulation of Col 1, and matrix degradation and disc remodeling associated increase of TIMP1 and BMP2 \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[21]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. Interestingly, our data showed that the progress of degeneration was more rapidly in later-3 months than it in pre-3 months, and among the genes expression we investigated, only the change of TIMP1 and Col 2 were significant at 3 months. One explanation to this divergence is that animals were lack of activity due to the pain and feebleness after surgery at first several months, consequently, motion and load bearing of spine were very limited in these periods. When they have rehabilitated and normal activities were restored, significant degeneration were exhibited in IF group at 6 months compared to other groups.\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e \u003cspan style=\"color: black;\"\u003eThough progressive degeneration can be found in all experimental groups, a significant prevention or deceleration effect was exhibited by Coflex. \u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eA variety of animal models have been established for the exploration of intervertebral disc degeneration and treatment \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[22]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. IVD could successfully developed in many models, however, the pathological and molecular changes are different, according to the varied anatomical and physiological structures of different animals. Rodents model is the most popular one among those models, nevertheless, the different structure and components of disc between rodents and humans has limit its accuracy in the study of IVD and treatment \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[23]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e.\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e \u003cspan style=\"color: black;\"\u003eIn this study, we chose beagles as animal model because the beagle\u0026rsquo;s disc is similar with human both in physiology and morphology\u003c/span\u003e, \u003cspan style=\"color: black;\"\u003ein addition, though beagles are quadrupeds animals, they like squatting for most time, such mechanical property was of the same status in human \u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[22, 24]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. We force beagles stay in erect position more than 8 hours per day after surgery by place food and water in a high place to reach, which would facilitate the progression of degeneration. A variety of methods have been conceived to achieve the establishment of the model of IVD degeneration,\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003escalpel blade and gauge needle were used in those classic models \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[21]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. According to these previous study , the velocity of the progression of disc is determined by the extent of damage to the annulus pulposus, using scalpel blade can cause an acute disc degeneration within 2 weeks, while a 16 or 18-gauge needle would make it happen in a gradually, relatively slow pattern,\u0026nbsp; which is similar to how its work in human IVD degeneration \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[25\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e, \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e26]\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e. As the purpose of this study is to clarify the ability of Coflex to decelerate or prevent ASD, which is considered as a slow progressive pathological change, a 16-gauge needle was used to develop the IVD degeneration model. In this study, we arranged control and experimental segments in the same dog,\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e \u003cspan style=\"color: black;\"\u003ewhich can eliminate individual differences such as age, weight, pre degeneration status.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eThe present study has some limitations. It has been reported that the IVD of beagles have a tendency of degeneration at an earlier age, which might cause difference of existed among objective IVDs to some extent. Therefore, this might bring potential bias to the present study.\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e \u003cspan style=\"color: black;\"\u003eIn addition, Pfirrmann classification is a non-quantitative analysis, quantitative evaluation of MRI was not performed, and the number of animals for histological and immunohistochemically analysis was small. Nevertheless, the study demonstrated the\u003c/span\u003e \u003cspan style=\"color: black;\"\u003einterspinous spacer device Coflex can protect adjacent segment after spinal fusion and decelerate the progression of adjacent IVD degeneration. The protect role can both exhibited well in adjacent segment and the middle segment in the treatment of \"skipped\" level (nonconsecutive) disc degeneration (SLDD) after fusion were delivered at two \u0026ldquo;skipped\u0026rdquo; level.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Conclusions ","content":"\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eThe present study demonstrated that the implantation of Coflex could decelerate the progression of adjacent IVD degeneration after spinal fusion. The results support the premises that Coflex is an effective interspinous device in the prevention or deceleration of ASD after spinal fusion, both in adjacent segment and middle segment. \u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eISPs: Interspinous spacer;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eASD: adjacent segment degeneration;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eIF: interbody fusion;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eCof1: L4-5 lumbar interbody fusion +L5-6 interspinous Coflex implantation\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eCof2: L4-5 and L6-7 interbody fusion+ L5-6 interspinous Coflex implantation\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eCon group: noninjuries control\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eSLDD: \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e\"skipped\" level (nonconsecutive) disc degeneration\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eMRI: magnetic resonance imaging\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eIVD: Intervertebral disc\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eNP: nucleus pulposus\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAF: anulus fibrosus \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eLDH: lumbar disk herniation\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eEthics approval and consent to participate\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eExperiments of animals were approved by the Animal Research Committee of Sixth People\u0026rsquo;s Hospital, Shanghai Jiao Tong University School of Medicine. \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAcknowledgements\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eThis study was supported by the \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eDepartment of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People\u0026rsquo;s Hospital\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e. The authors would like to thank the personnel from the spine surgery clinical team leading by prof. \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eXJG\u003c/span\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003efor participating in the surgery and data collection, and the discussion of this project.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eFunding\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eThis study was financially supported by Science and Technology Commission of Shanghai Municipality (NO. SHDC2014102). The funding \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eplays important roles in the design of the study; collection, analysis, and interpretation of data. It also helps in supplying the \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eexperimental animals; purchasing the experimental materials etc.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAvailability of data and materials\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAuthors' contributions\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eXJG conceived the initial idea and the conceptualization, chose the animal model and assisted in the surgery. LJM, LFQ and DJN conceived and participated in its design, searched databases, participated in the operation, extracted and assessed studies, participated in the data extraction and drafted the manuscript. LJM wrote and revised the manuscript. All authors read and approved the final manuscript.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eConsent for publication\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eNot applicable.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eCompeting interests\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eThe authors declare that they have no competing interests\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e Fischer CR, Cassilly R, Cantor W, Edusei E, Hammouri Q, Errico T. 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Clin Orthop Relat Res. 2015;473(6):1903-12.\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e Singh K, Masuda K, An HS. Animal models for human disc degeneration. Spine J. 2005;5(6 Suppl):267S-79S.\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e Chen CH, Chiang CJ, Wu LC, Yang CH, Kuo YJ, Tsuang YH, et al. Time course investigation of intervertebral disc degeneration in a rat-tail puncture model. Life Sci. 2016;156:15-20.\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e Zhang Y, Tao H, Gu T, Zhou M, Jia Z, Jiang G, et al. The effects of human Wharton's jelly cell transplantation on the intervertebral disc in a canine disc degeneration model. Stem Cell Res Ther. 2015;6:154.\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e Lei T, Zhang Y, Zhou Q, Luo X, Tang K, Chen R, et al. A novel approach for the annulus needle puncture model of intervertebral disc degeneration in rabbits. Am J Transl Res. 2017;9(3):900-9.\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e Mwale F, Masuda K, Grant MP, Epure LM, Kato K, Miyazaki S, et al. Short Link N promotes disc repair in a rabbit model of disc degeneration. Arthritis Res Ther. 2018;20(1):201.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp style=\"line-height: 200%; tab-stops: right 415.3pt;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cspan style=\"font-size: 10.0pt;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin: 8.85pt 0in .0001pt 11.55pt;\"\u003e\u003cspan style=\"font-size: 10.5pt; font-family: 'Tahoma',sans-serif;\"\u003eTable 1 Primers designed using Primer Premier 6.0 software\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top: .55pt;\"\u003e\u003cspan style=\"font-size: 6.0pt; 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border: none; border-left: solid black 1.0pt; padding: 0in 0in 0in 0in; height: 35.95pt;\" width=\"151\"\u003e\n\u003cp style=\"margin: 3.45pt 0in .0001pt 5.35pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003eCol 1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 191.8pt; padding: 0in 0in 0in 0in; height: 35.95pt;\" width=\"256\"\u003e\n\u003cp style=\"margin: 3.45pt 0in .0001pt 71.8pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003eNM_001003090.1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 233.8pt; border: none; border-right: solid black 1.0pt; padding: 0in 0in 0in 0in; height: 35.95pt;\" width=\"312\"\u003e\n\u003cp style=\"margin-top: 3.45pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003e5\u0026rsquo;- GTAGACACCACCCTCAAG-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top: 3.55pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003e5\u0026rsquo;-GGAAGAGCGGAGAATACT-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35.95pt;\"\u003e\n\u003ctd style=\"width: 113.15pt; 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border-top: none; border-left: solid black 1.0pt; border-bottom: solid black 1.0pt; border-right: none; padding: 0in 0in 0in 0in; height: 36.65pt;\" width=\"151\"\u003e\n\u003cp style=\"margin: 3.45pt 0in .0001pt 5.35pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003eGAPDH\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 191.8pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 0in 0in 0in; height: 36.65pt;\" width=\"256\"\u003e\n\u003cp style=\"margin: 3.45pt 0in .0001pt 71.8pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003eNM_001003142.2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 233.8pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0in 0in 0in 0in; height: 36.65pt;\" width=\"312\"\u003e\n\u003cp style=\"margin-top: 3.45pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003e5\u0026rsquo;- ATTCCACGGCACAGTCAAG-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top: 3.4pt;\"\u003e\u003cspan style=\"font-size: 10.5pt;\"\u003e5\u0026rsquo;- GGTGATGCTGGTGCTGAG-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin: 5.85pt 0in .0001pt 10.7pt;\"\u003e\u003cspan style=\"color: white;\"\u003ePrimer Premier 6.0 software from PREMIER Biosoft (palo alto, California, USA)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin: 3.05pt 0in .0001pt 10.7pt;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 11.0pt; font-family: 'Trebuchet MS',sans-serif;\"\u003eTIMP1 \u003c/span\u003e\u003c/em\u003einhibitors of matrix metalloproteinases 1\u003cem\u003e\u003cspan style=\"font-size: 11.0pt; font-family: 'Trebuchet MS',sans-serif;\"\u003e, BMP2 \u003c/span\u003e\u003c/em\u003eBone morphogenetic protein 2\u003c/p\u003e\n\u003cp style=\"margin: 2.85pt 0in .0001pt 10.7pt;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Trebuchet MS',sans-serif;\"\u003eCol 1 \u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.5pt; font-family: 'Arial',sans-serif;\"\u003etype I collagen\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Trebuchet MS',sans-serif;\"\u003e, Col 2 \u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.5pt; font-family: 'Arial',sans-serif;\"\u003etype II collagen, \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Trebuchet MS',sans-serif;\"\u003eGAPDH \u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.5pt; font-family: 'Arial',sans-serif;\"\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Coflex, interspinous spacer, adjacent segmental degeneration, spinal fusion, canine model","lastPublishedDoi":"10.21203/rs.2.12549/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.12549/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background\n\nInterspinous spacer (ISPs) was a promising treatment method for adjacent segment degeneration (ASD) after spinal fusion. Coflex, one of ISPs, has been deceived to prevent or decelerate ASD after spinal fusion, while the proof of the effectiveness of such device is still very limited. The purpose of this study was further to investigate the protection role of Coflex in vivo after spinal fusion, when implanted in adjacent segment and middle segment.\n\nMethods\n\nThree groups of beagles were allocated as follows (n=6): (1)L4-5 lumbar interbody fusion(IF). (2) L4-5 lumbar interbody fusion +L5-6 interspinous Coflex implantation(Cof1).(3) L4-5 and L6-7 interbody fusion+ L5-6 interspinous Coflex implantation (Cof2). In all animals, L5-6 discs were punctured to generate degeneration, and the intact L2–3 disc served as a noninjuries control (Con group). The effectiveness of Coflex on the prevention or deceleration of the progression of ASD was determined by magnetic resonance imaging, gross anatomical observation, histological and immunohistochemically analysis, and Real-time PCR analysis of gene expression.\n\nResults\n\nThe objective disc in every group showed degeneration, however, the degeneration was more significant in IF group than Cof1 and Cof2 groups. MRI and histologic assay demonstrated that the discs of Cof1 and Cof2 groups maintained a relatively well-preserved structure as compared to the discs of IF group. Furthermore, immunohistochemistry analysis and real-time PCR demonstrated that the indicators of disc degeneration, TIMP1, BMP2, Col I, were up-regulated and disc matrix gene, Col II was down-regulated in IF group significantly\n\nConclusions\n\nCoflex could decelerate the progression of ASD after spinal fusion, and it holds the same value not only at adjacent segment after single level spinal fusion, but also dose at the middle segment after “skipped” level (nonconsecutive) fusion","manuscriptTitle":"Effect of Coflex interspinous stabilization on the prevention of progression of adjacent segment degeneration after single level and skipped level spinal fusion in a canine model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-08-09 01:59:35","doi":"10.21203/rs.2.12549/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b9b4d60-2c78-455a-8650-e8834634a52b","owner":[],"postedDate":"August 9th, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":20216,"name":"Orthopedics"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-08-09 01:59:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-3305","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}