The Experimental Study Of Vertebral Cartilage Endplate Degeneration

Part One Association of Endothelin-1Expression and Cartilaginous Endplate Degeneration in HumansObjective:This objective of the study is to verify whether the expression of ET-1in human degenerated cartilage endplate (CEP) of intervertebral disc and the ET-1of different content compared with normal CEP. Next, the expression of ET-1in human cartilaginous endplate cells (CECs) and its role in disc degeneration would be explored.Methods:The CEPs used in this study was obtained from eight patients with lumbar degenerative disease. Control tissue specimens were obtained from eight patients, with acute burst fractures of lumbar vertebra but no signals of disc degenerative or Modic changes in CEPs on MRI. All participants underwent posterior discectomy and fusion for lumbar disease. The status of lumbar disc degeneration was described according to the modified Pfirrmann grading system. Gross observation and H&E staining were performed for all specimens. The expression of ET-1in degenerated CEP was analyzed by immunohistochemical staining and Western blotting compared with normal CEP.The surgically harvested endplate cartilages were carefully cleaned of nucleus pulposus and annulus fibrosus. The cartilaginous endplate cells (CECs) were isolated by type Ⅱ collagenase digestion, and cultured in regular condition. The morphological appearances and biological characteristics of CECs were evaluated by H&E and toluidine blue staining, and immunocytochemical staining for collagen type Ⅱ and aggrecan. ET-1was demonstrated in cartilaginous endplate cells (CECs) by immunofluorescent staining. The ET-1mRNA expression and protein production by CECs stimulated by tumor necrosis factor alpha (TNF-α,0-100ng/ml), a pro-inflammatory cytokine, were determined by real-time PCR analysis after24h treatment and Western blotting with the treatment of36h, respectively. The matrix metalloprotease-1(MMP-1), MMP-13and tissue inhibitor of metalloproteases-1(TIMP-1) levels in the supernatant of cultured CECs treated with ET-1(0-100nM) after36h were determined using enzyme-linked immunosorbent assays. Nitric oxide (NO) release and nitric oxide synthase (NOS) activity were measured using a spectrophotometric assay. The apoptosis of CECs by ET-1(0-100nM) after72h was measured by an Annexin V-FITC detection assay.Results:With H&E staining, normal CEP appeared as hyaline cartilage after being cleared of both annulus fibrosus and nucleus pulposus. The extracellular matrix of CEP was homogeneous, with round-shaped CECs organized into obvious layers. Degenerated CEP had obvious fibrosis, fragmentation, ossification and occasional lysis of cell nuclei, accompanied by vascular invasion. Immunohistochemical staining showed that most ET-1immunoreactivity was present in degenerated CEPs; most cells showed positive reactions in the cytoplasm. Normal CEPs contained few cells with positive ET-1staining. The production of ET-1in degenerated cartilage endplate was significantly higher than normal CEP by Western blotting assay.CECs were successfully cultured and passaged. Under the inverted microscope observing, the primary culture cells were adherent at12-24h, and began to proliferate within48h. The cultured CECs formed colonies after approximately7days. When the primary cultures reached90%confluence at about14days, the CECs exhibited various morphologies, which ranged from spindle-shaped to polygonal-shaped. H&E staining showed that CECs had a mostly polygonal appearance, with a round or elliptical nucleus. The cytoplasm appeared blue with toluidine blue staining. Immunohistochemical staining of type Ⅱ collagen and aggrecan were positive in the cytoplasm of CECs; no immunoreactivity was observed in the negative control groups. Therefore, the phenotypes and biological characteristics of CECs were similar to those of articular chondrocytes. The results of both real-time PCR and western blotting showed that ET-1was expressed by CECs and modulated by TNF-α in a dose-dependent manner. ET-1increased production of MMP-1and MMP-13, decreased TIMP-1production, and induced NO and NOS release by cultured CECs. The direct stimulation of CECs by ET-1did not promote cell apoptosis.Conclusion:There is higher level of ET-1expression in the degenerated cartilage endplate compared with normal CEP. The expression of ET-1in cartilage endplate, which is subjected to the regulation of TNF-α, a pro-inflammatory cytokine, and CECs by ET-1released MMP-1, MMP-13and NO. Therefore, ET-1played a pivotal role in human CEP degeneration, and may be a new target for development of therapies for this condition. Part Two Establishment of ischemic coccygeal vertebrae adjacent to endplate model in rat tailObjective:The objective of the study was to evaluate the effects of ischemic coccygeal vertebra adjacent to endplate, by percutaneous puncture followed by absolute alcohol injection in rat tail, on intervertebral degeneration of both vertebral endplate and nucleus pulposus/annulus fibrosus, and to mimic the pathogenesis similar to natural process of human IVD degeneration.Methods:Skeletally mature three months old, forty Sprague Dawley (SD) rats were enrolled in the study, normal intervertebral disc (IVD) was harvested from eight rats without any treatment as normal control. Under the guidance of X-ray fluoroscopy instrument, two consecutive coccygeal vertebra, the caudal eighth caudal coccygeal vertebrae (Co8) adjacent about3mm to vertebral endplate was percutaneously inserted lumbar puncture needle (7G) until marrow cavity, after the stylet was withdrawn, the coccygeal vertebrae was slowly injected30μl absolute alcohol with1-ml syringe for the purpose of microcirculation disturbance, the corresponding Co7/Co8disc level; Similarly the Co9caudal coccygeal vertebrae injected with30μl phosphate buffered saline (PBS) as the control corresponding Co8/Co9disc level. At the1,2,3and4month time point, radiographs were performed for the analysis of disc height while anesthetized. After X-ray scanning, eight rats were randomly sacrificed until four months. The changes of intervertebral disc including endplate, NP and AF were evaluated by histochemical staining. In brief, Hematoxylin and eosin (H&E), safranin O-fast green, safranin O, and toluidine staining were performed for cell morphology, ossification extent of endplate, the expression of glycosaminoglycan (GAG) as well as tidemark and cartilage matrix of endplate, respectively. In addition, immunohistochemical (IHC) staining of collage type II, aggrecan and Sox-9were used to detect IVD degeneration.Results:The disc height gradually decreased after2-month post-operation in the ethanol injection groups, especially after3-month there were subchondral bone sclerosis formation of endplate with bone proliferation detected by X-ray. H&E staining showed that the morphology of growth plate of endplate with time was destroyed due to blood shortage, cartilaginous endplate cells (CECs) of which become degenerated and gradually lost, finally the endplate got calcification until ossification. Interestingly, the cell transformation in NP happened with time, there were vacuolar cells gradually increased from one month to two months, then become chondrogenic cells after3-month, eventually into fibrocartilaginous phenotype after4-month along with fibrosis of nucleus pulposus. With progressive degenerative changes, the structure of annulus fibrosus demonstrated disorder of the morphology and rough lamellae, even rupture and fibrosis. Safranin O-fast green showed the extent of ossification increased gradually with time, especially on3-month afterward. After1-month degeneration, Safranin O staining reported the waved tidemark of growth plate regressed, and disappeared with the destruction of endplate structure. In addition, the content of GAG in NP matrix decreased regularly except for on3-month time point by Safranin O staining. Toluidine blue staining on behalf of the endplate cartilage matrix faded gradually with time, and even transformed to the bony endplate. Immunohistochemical staining of collagen type Ⅱ demonstrated the positive degree gradually decreased on the ischemic side of endplate, while collagen type Ⅱ of nucleus pulposus remained the positive degree or increased occasionally compared with control group. The immunohistochemistry for aggrecan from control group to1,2-month time point showed gradually declined, but the positive level of staining increased on3-month time point, next, the positive staining decreased significantly on4-month time point. Sox-9is a key gene in the development and differentiation of cartilage and type Ⅱ collagen. Immunohistochemical staining for Sox-9protein in NP decreased gradually except for the3-month time point, in accordance with the expression of collagen type Ⅱ compared with the control group with time.Conclusion:The establishment of microcirculatory disturbance model closed to endplate reproduced successfully intervertebral disc degeneration by absolute alcohol injection. The ischemic vertebra adjacent to endplate could be considered to play an important role in pathogenesis of IVD. Therefore, the model was suitable for the etiology study of disc degeneration.