Basic Research On The Application Of Biomechanical Adaptability Principle To The Regeneration And Repair Of Intervertebral Disc Degeneration

Part 1:Axial loading mechanical environment-Micro-Nano biomechanical environment in degenerative intervertebral discObjective:Changes of biomechanical environment of intervertebral disc degeneration induced by axial loading were observed at the micro-nano levelMethods:In this study,3-month-old adult male Sprague-Dawley rats were divided randomly into five groups:control(no intervention to the intervertebral disc of the tail)and four intervention groups that all had caudal vertebrae immobilized using a custom-made external device to fix four caudal vertebrae(Co7-Co10)but with variable subsequent compression of Co8 and Co9 for 2,4,6,or 8 weeks.Magnetic resonance imaging detection of rat coccygeal vertebrae was conducted at each time node of the experiment,and the T2 signal intensity and disc space were evaluated.Animals were euthanized and the caudal vertebrae were harvested for further analysis.Histopathology,glycosaminoglycan(GAG)content,histologic score,end plate structure,and elastic modulus of the intervertebral discs were evaluated.Results:Intervertebral disc degeneration was observed at an earlier Pfirrmann grade(Pfirrmann ?)under the microscope.With an increase in Pfirrmann grade to ?-?,the pore structure of the bony end plate changed significantly and the number of pores decreased gradually.Furthermore,the total GAG content of the nucleus pulposus decreased from an average of 640.33 mg GAG/ng DNA in Pfirrmann grade I to 271.33 mg GAG/ng DNA in Pfirrmann grade V(p<0.0001).At the early stage of clinical degeneration of intervertebral discs(Pfirrmann grades ? and ?),there were significant changes in mechanical properties of the outer annulus fibrosus compared with the inner layer(p<0.05).Further,the fibril diameters exhibited significant changes compared with the control group(p<0.05).Conclusions:Disc degeneration is initially a silent subclinical process.According to the classical Pfirrmann grading criteria,the micro-nano structure of the disc has undergone early degeneration and changes in the clinical normal state(Pfirrmann ? and ?),indicating that the Pfirrmann grading is not synchronized with the changes in the micro-nano environment of the disc.Our study found that the Pfirrmann grading system combined with intervertebral disc micro-nano structural changes more comprehensively reflected the extent of disc degeneration.These data may help improve our understanding of the pathogenesis and process of clinical disc degeneration.Part 2:In situ immobilization mechanical environment-Intervertebral disc degeneration induced by long-segment in situ immobilization:a macro,micro,and nanoscale analysisObjective:The goal of this study was to evaluate the effects of long-segment in situ immobilization of intervertebral discs of the caudal vertebra.Methods:Thirty-five fully grown,male Sprague-Dawley rats were used.Rats were randomly assigned to one of five groups:Group A,which served as controls,and Groups B,C,D,and E,in which the caudal vertebrae were in situ immobilized using a custom-made external device that fixed four caudal vertebrae(Co7-Co 10).After 2 weeks,4 weeks,6 weeks,and 8 weeks of in situ immobilization,the caudal vertebrae were harvested,and the disc height,the T2 signal intensity of the discs,disc morphology,the gene expression of discs,and the structure and the elastic modulus of discs was measured.Results:The intervertebral disc height progressively decreased,starting at the 6th week.At week 6 and week 8,disc degeneration was classified as grade ?,according to the modified Pfirrmann grading system criteria.Long-segment immobilization altered the gene expression of discs.The nucleus pulposus showed a typical cell cluster phenomenon over time.The annulus fibrosus inner layer began to appear disordered with fissure formation.The elastic modulus of collagen fibrils within the nucleus pulposus was significantly decreased in rats in group E compared to rats in group A(p<0.05).On the contrary,the elastic modulus within the annulus was significantly increased in rats in group E compared to rats in group A(p<0.05).Conclusions:Long-segment in situ immobilization caused target disc degeneration,and positively correlated with fixation time.The degeneration was not only associated with changes at the macroscale and microscale,but also indicated changes in collagen fibrils at the nanoscale.Long-segment immobilization of the spine(cervical spine)does not seem to be an innocuous strategy for the treatment of spine-related diseases and may be a predisposing factor in the development of the symptomatic spine.Part 3:Controlled immobilization-traction based on intervertebral stability is conducive to the regeneration or repair of the degenerative discObjective:Controlled immobilization-traction based on intervertebral stability is conducive to the regeneration or repair of the degenerative disc.Methods:In this study,49,6-month-old male Sprague-Dawley rats were randomly assigned to one of seven groups.Group A was the sham control group in which caudal vertebrae were instrumented with Kwires only.In Group B(model group),caudal vertebrae were immobilized using a custom-made external device to fix four caudal vertebrae(Co7-Co 10)and Co8-Co9 underwent 4 weeks of compression to induce moderate disc degeneration.In Group C,vertebrae Co8-Co9 underwent 4 weeks of compression to induce moderate disc degeneration,followed by removal of the external apparatus.Rats in the other four groups(Groups D-G),Co8-Co9 underwent 4 weeks of compression to induce moderate disc degeneration followed by 2 weeks,4 weeks,6 weeks,and 8 weeks of distraction,respectively.Caudal vertebrae were harvested and disc height,T2 signal intensity of the discs,disc morphology,total glycosaminoglycan content of the nucleus pulposus and the structure of the Co8-Co9 end plate were evaluated.Results:After 4 weeks of compression,the intervertebral height and T2 signal intensity of Co8-Co9 vertebrae of rats in Groups B to G were significantly reduced compared with Group A(sham group,all p<0.0001).Histological scores of rats in Group B averaged 10.14 and the total glycosaminoglycan(GAG)of nucleus pulposus averaged 238.21mg GAG/ng DNA.The bony end plate structure showed significant changes in comparison with the control Group.After 2 weeks to 8 weeks of traction,the disc space and T2 signal intensity of Co8-Co9 vertebrae in Group E were significantly recovered compared to that of rats in Group B(p<0.0001),and the intervertebral height of the Co8-Co9 in Group D,Group F,and Group G when compared with Group B(p<0.0001).Meanwhile,the T2 signal intensity of Co8-Co9 in Group D,F,and G when compared with Group B(p<0.001).Histological scores dropped from an average of 10.14 in Group B to 5.57 in Group E,and 5.86 in Group F(all p<0.0001).Furthermore,the total GAG content of the nucleus pulposus increased from an average of 238.21 ?g GAG/ng DNA in Group B to 601.02 ?g GAG/ng DNA in Group E(p<0.0001).The number of pores of end plates in rats in Groups D and E both were significantly increased when compared to that of rats in Group B(Groups D vs Groups B,p<0.05;Groups E vs Groups B,p<0.0001).Conclusions:A mechanical degenerative model was successfully established by using a custom-made device.We demonstrated that disc degeneration is a cascade of biochemical,mechanical,and structural changes mediated by cells in an abnormal mechanical environment.Not all levels of disc degeneration can be regenerated or repaired.Regeneration or recovery of disc degeneration requires specific conditions.Based on the immobilization-traction mode,the cascade cycle of disc degeneration is interrupted.Traction of 2 to 6 weeks is a sensitive period for regeneration of the degenerative disc.Moreover,the duration and extent of the traction loading must be moderately controllable,and beyond the limits that can lead to significant degeneration.These data may help improve our understanding of the pathogenesis of clinical disc degeneration and how to optimize the use of traction devices for possible regeneration.Part 4:Low tension traction mode actively reshaped the degenerative intervertebral disc micro-nano environmentObjective:To evaluate the possibility and effectiveness of disc regeneration or repair through low-tension traction of degenerated discs and to explore its possible mechanism.Methods:In this study,42,6-month-old male Sprague-Dawley rats were randomly divided into six groups:Group A:Model group(caudal vertebrae immobilized using a custom-made external device to fix four caudal vertebrae(Co7-Co 10),while Co8-Co9 vertebrae underwent 4 weeks of compression to induce disc degeneration);Group B:experimental control group(devices removed after the 4 week compression described in Group A).The remaining four groups represented intervention groups(Groups C and E:Co8-Co9 vertebrae compressed for 4 weeks followed by 2 or 4 weeks of excessive traction(high tension traction,HTT),respectively;Groups D and F:vertebrae compressed for 4 weeks followed by 2 or 4 weeks of in suit traction(low-tension traction,LTT),respectively.X-ray and magnetic resonance imaging(MRI)were performed at each time point to measure disc height and T2 signal intensity.At the end of the experiment,the animals were euthanized and tail vertebrae harvested for analysis of intervertebral disc histopathology,proteoglycan content,elastic modulus of fibers of the annulus fibrosus(AF)and nucleus pulposus(NP),and microstructure of the bony endplate.Results:After 2 to 4 weeks of continuous traction(HTT and LTT),the Co8-Co9 intervertebral disc space of rats in Groups C to F increased significantly compared with Groups A and B(p<0.05).In addition,signs of tissue regeneration were apparent in all four intervention groups(C-F).In addition,histological scores of the intervention groups(C-F)were significantly lower than those in the model and experimental control groups(Groups A and B,respectively),the LTT group(2w and 4w)was better than the HTT group(2w and 4w).Compared with the model group(Group A),total proteoglycan content of the NP in the intervention groups(C-F)increased significantly(p<0.05).After 2-4 weeks of intervention(HTT and LTT),the morphology of pores in the bony endplate,their number,and the diameter had recovered significantly compared with those in Group A.The LTT group was superior to the HTT group,and 4w in situ group significantly superior to the 2w group.In all intervention groups,in both the inner and outer AF,mean fibril diameter decreased significantly(p<0.05),although they remained larger in the HTT group than that in the LTT group.Consistent with trend in collagen fiber diameter,the modulus of outer AF was stiffer than the inner,and the LTT group was superior to HTT group,and LTT-4w group significantly superior to the LTT-2w group.In addition,within the NP,the variation in trend in diameter and modulus of collagen fibers was essentially consistent with that of the AF.However,the difference was that there was no significant difference in the elastic modulus of NP among the four groups(p>0.05).Conclusions:This study reconfirmed that the immobilization-traction mode blocked the cascade reaction of intervertebral disc degeneration to some extent.Low-tension traction reduced overstressed repair and osteophyte formation,and promoted synthesis of extracellular matrix in discs compared with high-tension traction.At equal time and amplitude,low-tension traction better promotes active reconstruction of bony endplates and improves the elastic modulus and micro-nano structure of the disc.Part 5:Low intensity extracorporeal shock wave therapy combined with low tension traction is beneficial to the regeneration and repair of moderate and severe degenerated discObjective:To evaluate the effectiveness of low-intensity extracorporeal shock wave therapy(ESWT)combined with low tension traction for regeneration and repair of moderately and severely degenerated discs,and to explore the possible mechanism of action.Methods:A total of 35 6-month old male Sprague-Dawley rats were randomly assigned to one of five groups(n=7,each group).In Group A(model group),caudal vertebrae were immobilized using a custom-made external device to fix four caudal vertebrae(Co7-Co 10)while Co8-Co9 underwent 4 weeks of compression to induce moderate disc degeneration.In Group B(experimental control group),as in Group A,disc degeneration was successfully induced after which the fixed device was removed for 8 weeks of self-recovery.The remaining three groups of rats represented the intervention Groups(C-E):after successful generation of disc degeneration in Group C(com-4w/tra-4w)and Group D(com-4w/ESWT),as described for group A,low-tension traction(in-situ traction)or low-energy ESWT was administered for 4 weeks(ESWT parameters:intensity:0.15Mpa;frequency:1Hz;impact:1000 each time;once/week,4 times in total);Group E(com-4w/tra-4w/ESWT):disc degeneration as described for group A,low-tension traction combined with low-energy ESWT was conducted(ESWT parameters as Group D).After experimentation,caudal vertebrae were harvested and disc height,T2 signal intensity,disc morphology,total glycosaminoglycan content,gene expression,structure of the Co8-Co9 bony endplates and elastic moduli of the discs were measured.Results:After continuous low-tension traction,low energy ESWT intervention or combined intervention,the degenerated discs effectively recovered their height and became rehydrated.However,the response in Group D was weaker than in the other intervention groups in terms of restoration of intervertebral disc height,while Group E was superior in disc rehydration.Tissue regeneration was evident in Groups C-E using different interventions.However,no apparent tissue regeneration was observed in the experimental control group(Group B).The histological scores of the three intervention groups(Groups C-E)were lower than those of Groups A or B(p<0.0001),and the scores of Groups C and E were significantly lower than those of Group D(p<0.05),but not Group C vs Group E(P>0.05).Compared with the intervention groups(Groups C-E),total GAG content of the nucleus pulposus(NP)in Group B did not increase significantly(p>0.05).There was also no significant difference in the total GAG content between Groups A and B(p>0.05).Of the three intervention groups,the recovery of NP GAG content was greatest in Group E.The expression of collagen ? and ?,and aggrecan in the annulus fibrosus(AF)was up-regulated(p<0.05),while the expression of MMP-3,MMP-13,and AD AMTS-4 was down-regulated(p<0.05).Of the groups,Group E displayed the greatest degree of regulation.The trend in regulation of gene expression in the NP was essentially consistent with that of the AF,of which Group E was the greatest.In the intervention groups(Groups C-E),compared with Group A,the pore structure of the bony endplate displayed clear changes.The number of pores in the endplate in Groups C-E was significantly higher than in Group A(p<0.0001),among which Group C vs Group D(p=0.9724),and Group C vs Group E(p=0.0116).There was no significant difference between Groups A and B(p=0.5261).In addition,the pore diameter also increased,the trend essentially the same as that of pore density.However,there was no significant difference between the three intervention groups(p=0.7213).It is worth noting that,compared with Groups A and B,peripheral pore density and size in Groups D and E of the three intervention groups recovered significantly.The elastic modulus and diameter of collagen fibers in the AF and NP varied with the type of intervention.It is clear that low tension traction combined with ESWT resulted in the greatest impact on the diameter and modulus of collagen fibers.Conclusions:Low energy ESWT combined with low tension traction provided a more stable intervertebral environment for the regeneration and repair of moderate and severe degenerative discs.Low energy ESWT promoted the regeneration of disc matrix by reducing MMP-3,MMP-13,and AD AMTS-4 resulting in inhibition of collagen degradation.While axial traction promoted the recovery of height and rehydration of the IVD,combined with low energy ESWT,the micro-nano structure of the bony endplate underwent positive reconstruction,tension in the annulus of the AF and nuclear stress of the NP declined,and the biomechanical microenvironment required for IVD regeneration and repair was reshaped.