The Effect Of Recombinant Human Bone Morphogenetic Protein-2to Retard Disc Degeneration In The Injured Rat Caudal Disc Model

Chapter I:Disc Degeneration Model Induced by Pereutaneous Needle Puncture in the Rat tailPURPOSETo establish a simple, reliable and efficient model of intervertebral disc degeneration in vivo, and evaluate the degeneration model induced by pereutaneous need puncture using radiography and magnetic resonance imaging examination at different time point.METHODA total of25,12-14weeks male Lewis rats were obtained (weight ranged320-360g). The coccygeal intervertbral discs at Co6-Co7and Co7-Co8were punctured by18-gauge needle and the Co8-Co9disc served as an uninjured control. The tip of the needle was placed in the center of the nucleus pulposus (NP) at a depth of5mm from the skin, turned360degree and placed for30seconds. Each rat was assessed by plain radiographs and magnetic resonance imaging examination prior to puncture,2and4weeks post-pucture. Disc height index ratio (DHI%) and ratio disc height change (RDHC) was calculated according to plain X-rays. MRI was used to grade discs I-IV and calculate discs degeneration rate, with grade I being normal and grade IV indicating severe degeneration.RESULTIn all the50punctured discs,the number of degenerated discs was26at the second week after pereutaneous needle puncture,and the rate of intervertebral disc degeneration was52%. The number of degenerative disc was42at the forth week after surgery, with the intervertebral disc degeneration rate arrived as high as84%. At the second week after disc puncture, the average DHI%was0.7, the mean RDHC was0.75, the mean score of degenerated discs was2.35. A statistically difference was observed when compared with these parameters before the piercing procedure (P<0.05). At the fourth week after disc puncture, the average DHI%was0.6, the mean RDHC was0.7, with a decreased trend when compared with these two parameters at2weeks post-pucture. The T2-Weighted magnetic resonance images demonstrated intervertebral disc structure was damaged, signal intensity decreased from white to gray, and the intervertebral disc height decreased or collapsed.The mean score of degenerated discs was3.07with a gradual worsening trend of intervertebral disc degeneration from2to4weeks post-puncture.CONCLUSIONThe rate of intervertebral disc degeneration increased with improved puncture technology.Intervertebral disc degeneration gradually increased from2to4weeks post-puncture, Pereutaneous needle puncture established a simple, reliable and efficient model of intervertebral disc degeneration in rat tail.ll Figures,8Tables and24References. Chapter II:The effect of timing of rhBMP-2Injection on Intervertebral Disc Degeneration in a Rat Tail ModelPURPOSETo evaluate the effect of timing of rhBMP-2injection on degenerated discs in a rat-tail puncture model of disc degeneration,with particular attention to the timing of BMP-2adminstration in relation to the initial injury.METHODSAt4weeks post-puncture,rats demonstrating disc degenetation were divided into three groups.Each group received7.5ug of rhBMP-2(1.5μg/μ1,2μ1) or phosphate buffered saline (PBS,2μ1) control by injection into the disc at one of three different time points:4,6or8weeks post-puncture. Each rat was assessed by radiographs and MRI prior to treatment and every two weeks thereafter. At six weeks after treatment,the animals in each group were sacrificed and the studied intervertebral discs were excised and underwent histological and immunohistochemical analysis.RESULTSThe disc grade after puncture continued to worsen with time in disc punctured injected with PBS at2,4and6weeks after treatment.Injection with BMP-2significantly improved the disc grade from a mean grade of3.3to2.5in disc that received BMP-24weeks post-puncture as assessed with MRI grading of the degenerative disc two weeks after treatment (P<0.05).However,the mean grade increased to2.8by four weeks after treatment and had returned to3.3six weeks after treatment.Discs that were injected with BMP-2at6weeks and8weeks post-puncture failed to demonstrate an improvement in disc grade. Chondrogenic differentiation was identified within moderately degenerated discs after BMP-2injection by detecting increased Sox-9positive cells compared with discs injected with phosphate buffered saline.CONCLUSIONSBMP-2injection into degenerated intervertebral discs at4weeks post-puncture promoted a transient improvement in disc grade,but was ineffective at later time points when a greater degree of degeneration had occurred. BMP-2appears to stimulate chondrogenic differentiation and promote formation of aggrecan at lower grades of disc degeneration.This data suggests that timing and severity of degeneration are important keys for the use of BMP-2in degenerative disc disease.12Figures,4Tables and41References. Chapter III:Dose-dependent effect of recombinant human bone morphogenetic protein-2to retard disc degeneration in the injured rat caudal discPURPOSETo investigate the efficacy, mechanism and dose-dependent effect of rhBMP-2in the treatment of disc degenerationMETHODSThe coccygeal intervertbral discs at Co6-Co7and Co7-Co8were punctured by18-gauge needle in36Lewis rats. Nine animals received no intervention and were sacrificed at4,6,10weeks. The remaining rats were divided equally into4groups, injected with2μl of either sterile water or one of three different doses of rhBMP-2(0.5,1and1.5μg/μ1) at2weeks after injury. Three rats from each group were euthanized at4,6and10weeks post-injury (2,4,8weeks post-treatment). Radiographic disc height and MRI were evaluated at all time points. Histological analysis and immunohistochemistry were performed to evaluate cellular and matrix changes in the area of NP.RESULTSRhBMP-2treatment groups tended to maintain better radiographic disc height when compared with PBS group, and the high dose group maintained a significant difference at all time points(P<0.05). MRI grade tended to be lower in rhBMP-2treated groups when compared to PBS and only puncture group, with significant difference in the high dose group at the8weeks after treament.Better histological score and greater proteoglycan content were also identified at various time points in the rhBMP-2groups but only the high dose group showed a significant difference at8weeks after treament. There was no difference seen in the number of nucleus pulposus cells between all groups. However, the percentage of Sox-9positive cells increased as the dose of rhBMP-2increased when compared to control at4and8weeks post-treatment.CONCLUSIONOur study shows the potential of rhBMP-2to retard disc degeneration after needle puncture in the rat caudal disc model. This effect seems to be dose-dependent as the greatest difference including radiographs, immunohistochemical and cellular analysis was seen in the high dose group. This dose dependence may be explained by the increasing chondrogenic activity that was seen in the disc cells as the dose of rhBMP-2increased.11Figures,2Tables and38References.