The Study Of Histological Endplate Structure And Lubricin’ Protective Effect In Modic Changes

Objective:1):To describe the microarchitecture of the annulus-endplate region,histological features of the mice,rats,monkeys and rabbits endplate were analyzed and compared to humans.To choose an appropriate laboratory animal to establish an animal model of Modic changes.2):To explore the expression and distribution of lubricin in the lumbar endplate and its association with Modic changes(MCs).Methods:1.6 motion segments(L3-L4,L4-L5)from 3 human cadaveric lumbar spines were harvested(2male,1 female,mean age 44.1 years,range 21-57).Annulus-endplate junction section of each segment(divided as anterior,lateral and posterior)were dissected,chemically fixed and decalcified.After being routinely embedded in paraffin,sections were cut into 4μm slices,stained with HE and Masson trichrome.The annulus-endplate junction section structure were observed with light microscope.2.Histological features of the mice,rats,monkeys and rabbits endplate were analyzed and compared to humans.Rabbit was chosen to build animal model of Modic changes.forty-eight rabbits were randomly divided into four groups.The subchondral bone superior to the L4-5 and L5-6 discs was injected with 1 mL Propionibacteriumacnes,normal saline(Vehicle),or punctured percutaneously without injection(Sham).The other 12 rabbits were blank control group.The degenerative process was evaluated using MRI before surgery and 0.5,1,3,and 6 months postoperatively.Following sacrifice,cytokine expression(IL-1β、IL-4、TNF-α、IFN-γ、PDGF-β)was quantified in endplate tissues.The bone mineral density(BMD)and bone volume/tissue volume(BV/TV)by μCT,and Osteogenic gene expressions of Runx2 and OCN by immunohistochemical staining were compared between all MCs types and normal endplate.3.Human endplate specimens were harvested from patients undergoing surgery for thoracolumbar spine fractures,or lumbar interbody fusion.Two groups of endplate specimens were identified according to preoperative spine MR imaging:MCs group and normal group.Lubricin expression was examined by immunohistochemistry,and differences between the groups were analyzed using real-time PCR.Lubricin expression and distributior,as well as differences between endplates with MCs and normal endplates,were confirmed using a rabbit model where MCs were initiated by injecting Propionibacterium acnes(P.acnes)into the discs.In a final experiment,endplate chondrocytes were co-cultured with P.acnes supernatant fluid(at concentrations of 0%,1%,2%,4%)for 48h and the expression of lubricin,aggrecan,collagen-type-Ⅱ,sox9,and several matrix degrading enzymes were evaluated.Results:1.The histological endplate structure varied significantly between laboratory animals and humans.Rabbit,rat,and monkey endplates consist primarily of cartilaginous endplate(CEP),which is separated from the vertebral body(VB)by the growth plate(GP)and a robust bony endplate(BEP)with multiple vascular channels which are absent in humans.The mouse endplate consists of CEP and BEP that is separated from the VB by GP.The human endplate consists of CEP and BEP without multiple vascular channels and lacks a GP.2.At postoperative month 6,6(24)segments injected with P.acne were identified as typeⅡ MCs.And TNF-a,interleukin-1β,and interferon-γ levels in the P.acne,Vehicle,and Sham groups significantly increased compared to blank controls.The BMD and BV/TVof endplates with MCs type Ⅱ were higher than those of normal endplates.The expression of Runx2 and OCN were higher in endplates with MCs than in normal endplates.3.Lubricin was found in human endplates and its expression was lower in the MCs group compared to the normal group.In the rabbit model,lubricin was found in the bony endplate and,at higher levels in the cartilage endplate.In rabbits injected with P.acnes(the MCs group),lubricin expression was decreased compared to the normal group.In cultures of endplate chondrocytes,the expression of lubricin,aggrecan,sox9 and collagen-type-Ⅱ decreased significantly,while that of MMP-1 and ADAMTS5 increased significantly,when cells were co-cultured with p.acnes.Conclusion:1.The histological endplate structure varied significantly between laboratory animals and humans.Maybe rabbit was not the best choice for MCs model due to its special histological features of endplates,especially the robust BEP with multiple vascular channels.However,the animal model of Modic changes still can successfully be built by injecting P.acnes to rabbit endplates.2.Lubricin is present in both the bony and cartilage endplate where it may have an anti-inflammatory role.P.acnes infection inhibits lubricin expression by cartilage endplate cells and this may facilitate the progression of MCs and endplate degeneration.