Biological Effects Of Compressive Stress On Nucleus Pulposus Tissue In The Isolated Intervertebral Disc Model And The Mechanism Behind The Compressive Stress-associated Disc Degeneration

IntroductionIntervertebral disc degeneration(IDD)is a worldwide disease with high morbidity and complex pathogenesis.Current treatments for it are mainly aimed to alleviate symptom but do not address the onset of IDD.Therefore,further research and understanding on the pathogenesis of IDD is important and will provide theoretical foundation for exploring new prevention and cure strategies of IDD.Compressive stress is an important external pathological factor in the process of IDD.Previous clinical observations and basic studies have suggested that compressive stress plays an important role in regulating the incidence of IDD and the biological behavior of disc cells.On the one hand,excessive compressive stress can promote the occurrence of IDD.On the other hand,certain specific stress stimulation can reverse the disc degeneration or maintain the health status of the disc.These positive and negative effects indicate that there may be a "window" type of biological effects of stress on the disc cells.Additionally,previous study shows that nucleus pulposus(NP)cell apoptosis and senescence may be the important reasons for the acceleration of disc degeneration caused by relevant pathological factors.Although studies have shown that high compressive stress is an important external pathological factor for the development of IDD,the role of high compressive stress in promoting the apoptosis and senescence of disc NP cells is in the preliminary research stage and its mechanism needs further researches.The in vitro disc organ culture model is a new research model in the field of intervertebral disc(IVD)research.Compared with the previous animal model and disc cell culture model,it not only retains the structure integrity of the IVD,but also precisely controls the stress conditions and other physical and chemical factors.However,it is difficult to ensure the sufficient nutrient supply into the central disc NP and to maintain the stability of the culture environment.So maintenance of the biological activities of NP is a technical bottleneck for establishing the disc organ culture model.Previously,our research team has developed an Intelligent Tissue Culture System that can real-timely-and feedback-control the culture environment and exert biomimetic mechanical stimulation with different parameters.This system provides a technology and equipment basis for establishing the disc organ culture model and makes the research background of compressive stress closer to the physiological condition.Therefore,based on the Intelligent Tissue Culture System,this study will establish the disc organ culture model by improving nutrient supply into the central disc NP tissue and optimizing the relevant culture parameters.And then,this study will observe the window effects of compressive stress on disc NP cells by applying different parameters of biomimetic compressive stress to the intact disc in the established disc organ culture model.Finally,this study will further explore the possible pathways and mechanisms of high-magnitude compression-induced IDD.MethodsPart I1.After the rabbits discs were harvested,the bone endplate was removed but the cartilage endplate(CEP)was retained.Then,the isolated discs were randomly divided into the control group and experimental group.The discs in the experimental group were treated with the pre-treatment protocol.Specifically,one-third of the outer annulus fibrosus(AF)was removed with a scalpel blade and then the discs were placed in the low concentration of trypsin to loose the residual outer AF.The discs in the control group did not undergo such treatment.2.After the discs were underwent the pre-treatment protocol,the diffusion efficiency of solute into the IVD was observed by methylene blue diffusion experiment,and the structural changes of CEP and the inner AF were observed by histomorphology staining(HE staining).3.All discs in these two groups were cultured in the Intelligent Tissue Culture System.On days 7 and 14,NP cell viability,histomorphological changes(toluidine blue and HE),NP matrix molecules(aggrecan and collagen II)expression,the biochemical components(glycosaminoglycan(GAG)and hydroxyproline(HYP))in the NP tissue were measured.Additionally,the biological activities of NP tissue in these two groups were compared with those of fresh disc NP tissue on day 14.Part II1.The porcine IVDs were separated and underwent the pre-treatment protocol as described in the Part I.2.Using the Intelligent Tissue Culture System,the porcine IVDs were cultured in different types of culture medium for 7 days,such as different glucose levels(low glucose: 1.0 g/L;placose: 2.0 g/L;high glucose: 4.5 g/L),different osmolarity levels(hypo-: 330 mOsm/kg,iso-: 430 mOsm/kg,hyper-: 550 mOsm/kg)and different serum levels(5%,10% and 20%).By evaluating NP cell viability,gene expression of matrix metabolism-related molecules in the NP cell and the content of biochemical components(GAG and HYP)in the NP tissue,the optimal levels of glucose,osmolarity and serum which could maintain NP biological activity were determined.3.The porcine IVD were further cultured in the above optimized culture environments.On day 14,some parameters were analyzed and compared with those of fresh disc NP samples,such as NP cell viability,histomorphology(alcian blue staining and HE staining),NP cell distribution,the T2 weighted MRI images of NP tissue,gene expression of matrix metabolism-related molecules in NP cells,NP matrix macromolecules(aggrecan and collagen II)expression and the content of biochemical components(GAG and HYP)in the NP Tissue.Part III1.Porcine disc isolation,pre-treatment before culture and the establishment of disc organ culture were processed as described in the part I and II.2.The perfusion-cultured discs were compressed for 7 days using the Intelligent Tissue Culture System.The biological effects of different biomimetic compressive magnitudes(0.1,0.2,0.4,0.8 and 1.3 MPa at a frequency of 1.0 Hz for 2 h per day),compressive frequencies(0.1,0.5,1.0,3.0 and 5.0 Hz at a magnitude of 0.4 MPa for 2 h per day)and compressive durations(1,2,4 and 8 hrs per day at a magnitude of 0.4 MPa and frequency of 1.0 Hz)on NP tissue were observed.The non-compressed discs were used as controls.3.At the end of culture,NP cell apoptosis,histomorphological changes(alcian blue staining and HE staining),gene expression of matrix metabolism-related molecules,protein expression of NP matrix molecule(collagen II)and the content of biochemical component(GAG)were analyzed in each group.Part IV1.The rat IVDs were isolated as described in the part I.After the central NP tissue was separated and digested with trypsin and type I collagenase,the NP cell pellets were collected and seeded into scaffold material using the method of biphasic inoculation.Then,the scaffolds seeded with NP cells were perfusion-cultured in the Intelligent Tissue Culture System for 5 days with different compressive loads for 4 h once per day.The designed groups were: control group(non-compression),low-magnitude compression group(2% compressive deformation)and high-magnitude compression group(20% compressive deformation).The frequency of compression was 1.0 Hz.At the same time,the role of p38 MAPK pathway and intracellular reactive oxygen species(ROS)accumulation in the effects of high-magnitude compression on NP cells was observed by using inhibitor SB203580 and scavenger NAC in the high-magnitude compression group.At the end of culture,NP cell proliferation,senescence-associated ?-galactosidase(SA-?-Gal)activity,the fraction of NP cells arrested in the G1 phase of cell cycle,expression of senescence markers(p16 and p53),telomerase activity,intracellular ROS accumulation,extracellular matrix metabolism and p38 MAPK pathway activity were analyzed.2.After the rat discs were isolated,the rat disc organ culture model was established using the Intelligent Tissue Culture System.All discs were perfusion-cultured for 10 days and compressed with different compressive loads for 4 h once per day.The designed groups were: control group(non-compression),low-magnitude(0.1 MPa)compression group and high-magnitude(1.3 MPa)compression group.The frequency of compression was 1.0 Hz.At the end of culture,the SA-?-Gal activity,expression of senescence markers(p16 and p53),telomerase activity,intracellular ROS accumulation,extracellular matrix metabolism and p38 MAPK pathway activity were analyzed.Part V1.The rat IVDs were isolated as described in the part I.After the central NP was separated and digested with trypsin and type I collagenase,the NP cell pellets were collected and seeded into scaffold material using the method of biphasic inoculation.2.The scaffolds seeded with NP cells were perfusion-cultured in the Intelligent Tissue Culture System for 5 days with different compressive loads for 4 h once per day.The designed groups were: control group(non-compression),low-magnitude compression group(2% compressive deformation)and high-magnitude compression group(20% compressive deformation).The frequency of compression was 1.0 Hz.At the end of culture,NP cell apoptosis,Caspase 3 activity,gene expression of apoptosis-related molecules(Bcl-2,Bax,Caspase 3),protein expression of apoptosis-related molecules(Bcl-2,Bax,Cleaved-caspase 3),gene and protein expression of N-cadherin(N-CDH),PI3K/Akt pathway activity,and downstream GSK-3? activity were analyzed.3.After N-CDH expression in the NP cells were enhanced,N-CDH overexpressed NP cells were seeded into the scaffold material.At the end of high-magnitude compression stimulation,NP cells were analyzed regarding the above parameters.4.In the high-magnitude compression group,after the PI3K/Akt pathway in the N-CDH overexpressed NP cells was inhibited using the pathway inhibitor LY294002,the above parameters were analyzed again.ResultsPart I1.After the discs were underwent this pre-treatment protocol,the outer AF became disorganized,but no specific changes occurred in the inner AF and CEP.Further more,this pre-treatment increased solute transport into the disc NP tissue.2.During the culture,by comparison with the control group,NP cell viability,gene and protein expression of matrix macromolecule,the main biochemical component within the NP tissue in the experiment group were increased,whereas the gene expression of matrix degrade enzymes was decreased.3.After 14 days of culture,when compared with the fresh discs,results showed that NP cell viability and matrix metabolism of the discs in the experiment group were more similar to the fresh discs than that of disc s in the control group.Part II1.For high-glucose(4.5 g/L),iso-osmolarity(430 mOsm/kg)or 10% serum,NP cell viability,the gene expression profile(matrix macromolecules and tissue inhibitor of metalloproteinase),and content of biochemical components(GAG and HYP)were more favorable than for other levels of glucose,osmolarity and serum.Additionally,these culture environments decreased gene expression of matrix degradation enzyme to some extent.2.Compared with the fresh NP sample,when the discs were cultured for 14 days under the optimized conditions(high glucose,iso-osmolarity and 10% serum),the disc NP tissue exhibited favorable cell viability,cell morphology and distribution,tissue hydration and matrix anabolism.Part III1.Compared between compression groups,the 1.3 MPa group,5.0 Hz group and 8 h group showed a significant increase in apoptotic cells,down-regulated matrix macromolecules expression,up-regulated matrix degradation enzymes expression,decreased GAG content and collagen II deposition in the disc NP tissue.2.Compared with the non-compression control group,the other compressive magnitude(0.1-0.4 MPa)group,compressive frequency(0.1-3.0 Hz)group and compressive duration(1-4 h)showed a healthier status of NP tissue regarding cell viability,expression of matrix metabolism-related molecules and GAG content within the NP tissue.Part IV1.In the NP cell three-demensional(3D)culture model and the disc organ culture model,compared with the low-magnitude compression and the control group,high-magnitude compression increased the SA-?-Gal activity,inhibited NP cell proliferation and telomerase activity,up-regulated the expression of senescence-related markers(p16 and p53),and reduced the matrix biosynthesis in NP cells.Additionally,high-magnitude compression significantly increased intracellular ROS accumulation and p38 MAPK pathway activity.2.Under high-magnitude compression in the NP cell 3D culture model,ROS scavenger NAC decreased SA-?-Gal activity,increased NP cell proliferation potency and telomerase activity,decreased expression of senescence-related markers(p16 and p53)expression,and enhanced NP cell matrix biosynthesis.Additionally,NAC can effectively reduce intracellular ROS accumulation,but NAC has no significant effect on p38 MAPK pathway activity under high-magnitude compression.3.Under high-magnitude compression in the NP cell 3D culture model,when the p38 MAPK pathway was inhibited by inhibitor SB203580,the intracellular ROS accumulation,SA-?-Gal activity and senescence-related markers(P16 and p53)expression were decreased,whereas NP cell proliferation potency,telomerase activity and NP cell matrix biosynthesis were increased.Part V1.Compared with the control group and the low-magnitude compression group,high-magnitude compression increased NP cell apoptotic rate and Caspase 3 activity,down-regulated anti-apoptotic molecule(Bcl-2)expression,and up-regulated pro-apoptotic molecules(Bax,Caspase 3/Cleaved-caspase 3)expression.Additionally,high-magnitude compression decreased N-CDH expression,inhibited activation of PI3K/Akt pathway and increased activity of GSK-3?.2.Under high-magnitude compression,cell apoptosis and Caspase 3 activity were decreased after N-CDH expression in NP cells was enhanced.And anti-apoptotic molecule(Bcl-2)expression and pro-apoptotic molecules(Bax,Caspase 3/Cleaved-caspase 3)expression were increased and decreased,respectively.Additionally,N-CDH overexpression in NP cells increased PI3K/Akt pathway activity but decreased activity of GSK-3? under high-magnitude compression.3.Under high-magnitude compression,when the LY294002 inhibited activation of PI3K/Akt in the N-CDH overexpressed NP cells,cell apoptosis and Caspase 3 activity were increased.And anti-apoptotic molecule(Bcl-2)expression and pro-apoptotic molecules(Bax,Caspase 3/Cleaved-caspase 3)expression were decreased and increased,respectively.Additionally,LY294002 enhanced the activity of GSK-3? but have no effects on N-CDH expression in the N-CDH overexpressed NP cells under high-magnitude compression.Conclusions1.This study established a pre-treatment protocol before culturing disc,which promoted solute transport into the central disc NP tissue and maintained a stable NP cell viability and matrix biosynthesis during a 14-days experiment period of disc perfusion culture.2.Based on the Intelligent Tissue Culture System,this study optimized the culture environments of IVD,and demonstrated that disc NP tissue maintained similar biological activities to those of fresh NP samples under the optimized culture environments(high glucose: 4.5 g/L;iso-osmolarity: 430 m Osm/kg;serum: 10%).3.In the disc perfusion culture model,high compressive magnitude or frequency and long compressive duration could promote NP cell apoptosis and destroy the matrix homeostasis within NP tissue,but certain compression magnitudes(0.1-0.4 MPa),frequencies(0.1-3.0 Hz)and durations(1-4 h)maintained relatively “healthy” biological activities of NP tissue in vitro.4.This study demonstrated that high-magnitude compression could increase intracellular ROS accumulation through activating p38 MAPK pathway,and then promote NP cell senescence.This mechanism may be a possible approach for high-magnitude compression to accelerate IDD.5.This study demonstrated that high-magnitude compression decreased PI3K/Akt pathway activity through decreasing N-CDH expression and then increased the GSK-3? activity,which finally promoted NP cell apoptosis through the mitochondria pathway.This mechanism may be another possible approach for high-magnitude compression to accelerate IDD.