Character And Mechanism Study Associated With The Migration Of Chondrocytes From Endplate To Nucleus Pulposus In Rat Discs

1 ObjectiveIntervertebral disc is the most vulnerable organ in human skeleton muscle system degenerate with aging and intervertebral disc degeneration was the most common causes of low back pain and cervical syndrome. The changes association with biomechanical and biological factors had etiological meanings in many vertebrate column disorders. So intervertebral disc degeneration became the focus of clinical treatment and research. Though there were many unknown aspects of etiological factors and pathological changes with intervertebral disc degeneration, there were evidence indicate that intervertebral disc degeneration originated from functional disorder of disc cells. The cells in nucleus pulposus generate extracellular matrix, such as proteoglycan and type II collagen, extracellular matrix in turn determine the character of physiological and biomechanical function of intervertebral disc. So make certain of the rules and mechanisms of nucleus pulposus cells in aging and degeneration process will play an important role in etiology researching and strategy formulating of intervertebral disc disorders.The human nucleus pulposus (NP) undergoes a chronological transition from a notochordal to a fibrocartilaginous NP. This transition accompanies changes in celltypes from notochordal cells to so-called chondrocyte-like cells, and in NP tissues from highly vacuolated notochordal tissue to fibrocartilage. This cell plays a central role in the transition and ultimately comprises the major cell population of the mature or adult form of the fibrocartilaginous NP. Thus, the chondrocyte-like cell is the most important cell type of the NP. It determines the initial state of the newly formed fibrocartilaginous NP and maintains it thereafter. Therefore, depending on the origin of the chondrocyte-like cell, the mechanisms of transition from a notochordal to a fibrocartilaginous NP can be either an endogenous development or an exogenous replacement. Thus far, the origin of the chondrocyte-like cell is unknown.This study, using intact rabbit intervertebral discs (IVDs), shows the origin of the so-called "chondrocytelike cells" to be chondrocytes from the cartilage endplate. This study also documents histologic findings associated with the transition of a notochordal NP to a fibrocartilaginous NP.This study established a bipedal rat aging degeneration cervical disc model that faithfully simulate human degenerative disc disease, at the same time make the normal rats as the control groups. The origin of the cartilage-like cells during the transition process of nucleus pulposus and the migration mechanism mediated by hyaluronan-CD44 signal pathway were researched.2 Materials and MethodsThe bipedal rats model was developed in 40 infancy SD rats by forelimb amputation. Thirty-six normal rats in the same age served as the control. When the rats in the experimental group were 3 6 9 and 12 months (named E3,E6,E9 and E12group respectively) and in the control group were 4,8,12 and 16 months (named C4,C8,C12 and C16 group respectively) postoperatively, the vertebral columns fromC2~3 to T2~3 were removed and observed under radiographic and histological examination after the rats were sacrificed.The rats in the experiment groups were undergone general anaesthesia by ketamine in a dose of 0.1g/kg injected into abdominal cavity, a ring incision was made at the armpit under sterilitas condition, the shoulder bone was cut down at the junction of the superior and the middle section, the muscle of the upper limb was ligatured to stop bleeding. The rats without any treatment act as contral group. The two groups of rats feeded with the same feed and moved freely in cages. 2.1 The study of establishment cervical spine degeneration model in bipedal ratsTen of the 40 rats in the experiment group were killed 3 months after operation (E3 group) in order to observe the short-term effects of the surgical intervention, and 9 of the 36 rats in the contral group were killed and examined 4 months after operation(C4 group). Another 10 rats in the experimental group were killed 6 or 9 months after operation(E6 or E9 group) in order to observe the midterm effects of the surgical intervention, and another 9 rats in the contral group were killed 8 or 12 months after operation(C8 or C12 group). The remaining 10 rats in the experimental group were killed 12 months after operation (E12 group) in order to observe long-term effects, and the remaining 9 rats in the contral group were kill 16 months after operation (C16 group). All the animals were killed with an overdose of ether.Radiographic studies were performed to examine (1)disc-space narrowing, (2)osteophyte, and (3) changes in spinal alignment. Anteroposterior and lateral radiographs were taken with a soft radiographic machine immediately after the mice were killed. After radiographs were taken of the cervical spine, two transverse cuts were made through the vertebral column: one at the base of the skull and the other at the upper thoracic segmengt. The entire vertebral column was then quickly dissected free and the cervical and upper thoracic spine was examined radiographically alongthe anteroposterior and lateral planes.Histologic studies were performed to examine the degeneration process of the intervertebral discs and osteophyte formation. The semiquantitative scales were made based on the degeneration of cartilage endplate, annulus fibrosus and nucleus pulposus. The specimens, the entire dissected vertebral columns from C2~3 to T2~3, were fixed in 4% neutral buffered formalin (pH 7.4) 20 min in 4°C,decalcified in EDTA(20%), split midsagittally, embedded in paraffin, and sectioned at 4 um. Serial sections were stained with hematoxylin and eosin, safranin O for the light microscopic examination.2.2 The study of the rule association with the transition of the nucleus pulposus and the migration of the endplate chondrocytes to the nucleus pulposus in aging or degeneration cervical spine.Serial sections at inferior cervical spine (C4-5> Cs-6^ C5-7) were dissected and stained with hematoxylin and eosin, safration O for the light microscopic examination at different surgical intervention. The migration of endplate chondrocytes and displacement of notochordal nucleus by fibrocartilage nucleus were observed.2.3 Mechanism study associated with the migration of endplate chondrocytes to nucleus pulposusIn this study 64 male SD rats were used and separated into two group just as animal model study has done. In each surgical intervention 8 rats were killed and the HA content of cervical 5 to 6 segment was measured. The sections from the migration study were used for immunohistochemistry stain to measure the expression and distribution of CD44> ADAM 10 and ADAM 17 during the aging and cervical degeneration process.3 Results3.1 Experimental study of bipedal rat cervical spine degeneration modelIn the C4, C8, C12 groups, no disc-space narrowing, osteophyte formation, or abnormal spinal alignment was observed. In the C16 group only mild degeneration signs were observed in part of the rats. In the vertebral columns of all the animals in the E9 group, however, lesions were characterized by disc-space narrowing, osteophyte formation and anterior osteophyte formation were revealed in different degree, especially at the C4~5> Cs~* and C6~7levels of E12 group.Most of the intervertebral discs of animals in the C4> C8 and C12 groups appeared normal. Most of the intervertebral discs of rats in the C16> E3 and E6 groups exhibited shrinkage of notochordal nucleus pulposus, cartilage-like cells became the most popular cells in the nucleus pulposus, notochordal cells decreased and most appeared denaturation, the cartilage endplate became thinner and loss of lamellar structure in the anterior annulus fibrosus. Disappearance of the notochordal nucleus pulposus and clefts in the annulus fibrosus were observed in several discs. In the E9 group, disc degeneration was more advanced, and the lesions were characterized by disappearance of the nucleus pulposus, fissures in the annulus fibrosus, thinning and defection of the cartilage endplate. Herniation of disc material and anterior osteophyte formation were observed in several discs. In the E12 group, disc degeneration was most advanced, and the lesion were characterized by herniation of disc materials, disappearance of cartilage endplate and anterior osteophyte formation. To assess the morphologic state of the discs, we propose original morphologic semiquantitative grading schema for annulus fibrosus, nucleus pulposus and cartilage endplate, the results indicated that the degeneration of the cervical discs in experiment groups were more severer and earlier than control groups.3.2 The study of the rule in the transition process of the nucleus pulposus morphology and migration of the chondrocytes to nucleus pulposus in aging ordegeneration rats cervical spine3.2.1 Typical Histologic Signs Associated With the Transition of a Notochordal Nucleus Pulposus to a Fibrocartilaginous Nucleus PulposusAlong with the aging of the rat there were more and more chondrocyte-like cells and collagen fibers and the notochordal cell degenerated and became less and less. The origin of these collagen fibers, however, did not occur randomly, but proceeded from the periphery toward the center of the CE. Therefore, the newly formed fibrocartilage initially involved replacement of the peripheral regions, subsequently followed by replacement of the central region. As the centripetal origination and sequential replacement advanced, notochordal tissue became smaller and rounded, then fragmented into small pieces. The structure of the newly formed fibrocartilage nucleus pulposus act as lamellar structure of annulus fibrosus. In the aging of bipedal rats the process of this transition occurred rapider and earlier, thereafter the nucleus pulposus fibrous degenerated and cartilage endplate became thinner and defected, in some cases ossification developed. The semiquantitative scales indicates that in bipedal rats this transition completed rapider and earlier.3.2.2 The rule of the migration of the endplate chondrocyteIn the process of aging and bipedal rats cervical spine, the endplate chondrocyte migrate to nucleus pulposus and deposite collagen fibers in a centripetal manner. In different stages and zones of this transition there were different rules of migration.? The juncture of nucleus pulposus, cartilage endplate and inner annulus fibrosus: In the aging process of rats, the chondrocytes come from endplate migrate along the alignment of collagen fibers to the center of the interverberal disc. The migration manner changed from scattered in infantile stage to catenated in adult stage. The chondrocytes migrated to the margin of the notochordal nucleus generating and depositing collagen fibers, makes the notochordal nucleus shrinking centripetally anddisplaced by fibrocartilage nucleus eventually. In the same zone of the bipedal cervical spine degeneration model, the chondrocytes of endplate demonstrate a mass catenated migrated manner, so the notochordal nucleus displaced by the frbrocartilage nucleus rapidly and early.?The central zone of the intervertebral disc: In the aging process of rats, the chondrocytes come from central zone of nucleus pulposus migrate along endplate in a catenated manner to the margin of the shrinking notochordal nucleus initially, but when the transition was completed the direction of this migration was almost vertical to the surface of the cartilage endplate. In the same zone of the bipedal cervical spine degeneration model, the chondrocytes migrated in the similar manner in notochordal nucleus pulposus, but migrated in mass, catenated or flakiness manner when the notochordal nucleus was disappeared completely. The migration of chondrocytes led to a depletion of chondrocytes in the cartilage endplate, a disappearance of the zonal arrangement of the cartilage endplate, and a decrease in the thickness of the cartilage endplate. The bulk and rapid migration of the chondrocytes makes the displacement of the notochordal nucleus more rapidly and early. With advancing histologic findings of transition, chondrocyte migration occurred from the more central part of the cartilage endplate, leading to a diffuse narrowing of the cartilage endplate and fissures in the junction of cartilage endplate and nucleus pulposus regions. 3.3 The mechanism study of the endplate chondrocytes migrating to the nucleus pulposus. 3.3.1 The content of hyaluronan in the intervertebral discThe content of HA in C4 group was (343±9) ug/g and it was the highest of all the control groups; the content of HA in C8^ C12 and C16 groups, which had certain decrease, was (194±14) ug/ g, (182±14) ug/ g and (161±9) ug/g respectively. The HA content in C4 group was higher significantly than the other three control groups( P <0.001), and between C8 and C12 group the content had no different statistically( P > 0.05), but higher than C16 group( P < 0.05)oThe content of HA was (326±9)ug/g and it was the highest of all the experiment groups; the content of HA in E6, E9 and E12 groups, which had certain decrease, was (132±8)ug/g, (126±6)ug/g and (65±5)ug/g respectively. The HA content in E3 group was higher than the other three experiment groups( P < 0.001). Between E6 and E9 group the HA content had no different statistically( P > 0.1), but higher than E12 group signaficantly( P < 0.001).For the life span of the SD rat is approximately 2 years, this study separated aging groups into infant group (C4 group), adult group(C8 and C12 groups) and old group(C16 group), separated degeneration groups into early group(E3 group), intermediate group(E6 and E9 groups) and advanced group(E12 group), so it can be easily compared for corresponding stages of normal life period with degeneration course and describe the structure of intervertebral disc, The results of this study indicate that between C4 group and E3 group the HA content had appeared different significantly (P < 0.05), and in C8, C12 and C16 groups there were more higher than E6> E9 and E12 groups(P< 0.001). 3.3.2 The distribution and express intensity of CD44> ADAM 10 and ADAM 17There can be seen CD44, ADAM 10 and ADAM 17 positive cells in control and experiment groups. These three biomacromolecule positive cells had similar distribution rule, the endplate chondrocytes had deeper stain, nearer tidemark deeper stain, and the positive cells most distributed membran and nearby extracellular matrix.To compare the intensity of the distribution of immunohistochemistry positive cells, the semiquantitative stage of CD44> ADAM10 and ADAM17 express were made. The results indicate that the expression of CD44> ADAM 10 and ADAM 17 inmigration chondrocytes were increased with time, and the expression in adult and old groups were higher than infant group significantly( P < 0.001). In old group (C16 group), the expression of ADAM 10 was decreased somewhat, but when compared with adult group it had no statistically sense( P > 0.05).In cervical degeneration process, the expression of CD44> ADAM10 and AD AM 17 in early stage were lower than intermediate and advanced groups( P < 0.001), the highest expression was seen in intermediate group (E6 and E9 group) ,there were different between advanced group significantly( P < 0.001).To compare between aging groups with cervical degeneration groups revealed the expression of CD44> ADAM10> AD AMI 7 in intermediate degeneration groups was higher than adult stage rats much more significantly( P < 0.001). For CD44 expression early degeneration stage was different with infant rat significantly(P =0.01), but when compared between advanced degeneration with old rats there was no different significantly (P >0.05) . In contrast with CD44, the expression of ADAM 10 and ADAM 17 had no different between early degeneration and infant rats (P >0.05) , but there were much more higher between advanced stage between old stages( P < 0.05).Dependability analysis were made between different stages of experiment and control groups associated with expression of CD44, AD AMI 0 and AD AMI 7, the results indicated that expression of these three biomacromolecule had negative dependablity with the content of HA.,4 Conclusions4.1 In this study, we established a reproducible experimental bipedal rat model of cervical disc degeneration by means of amputated upper extremities. Although no biomechanical examination was carried out, it can be assumed that overload on thecervical spine elicited by this surgical intervention accelerated the process of cervical intervertebral disc degeneration. The availability of this experimental model should be valuable for comprehensive understanding of the pathogenesis of cervical degeneration. The degeneration process of the bipedal rats'discs is in agreement with that of human beings.4.2 In the aging and degeneration process of rat cervical discs, all forms of fibrocartilage lamellas and fibers found in the nucleus pulposus were formed by chondrocytes that had originated and migrated from the cartilage endplate. The origin of chondrocytes proceeded in a centripetal direction from the periphery toward the center of the cartilage endplate. The newly formed fibrocartilage lamellas and fibers, therefore, initially involved replacement of the peripheral regions of the nucleus pulposus, followed by replacement of the central region. This centripetal sequential replacement mechanism decreased the size of the notochordal tissue while increasing the lamellar structure of the intervertebral disc. The chondrocytes changed notochordal nucleus pulposus into fibrocartilaginous nucleus pulposus by depositing fibrocartilage lamellas and fibers in a centripetal direction. In bipeald rats model this process was shortened greatly. This transition and migration process is not only the nature component element of the cervical discs aimed to accommodate the load condition but also the start element of cervical disc degeneration. It is the view of the current research that the newly formed fibrocartilage NP is the true normal state of the adult NP, and that degeneration ensues after the end stage of transition.4.3 The content of hyaluronan has an intrinsic association with the transition of nucleus pulposus and cervical disc degeneration. The hyaluronan-CD44 signaling pathway had an important role in regulating CD44 positive endplate chondrocytes migrate to nucleus pulposus through HA contained extracellular matrix. CD44> AD AM 10 and ADAM 17 had relationship with the migration of endplate chondrocytes to nucleus pulposus, the signal transduction mediated by CD44,AD AMI 0 and AD AMI 7 participating the transition of nucleus pulposus and may play an important role in the process of endplate chondrocytes migrated to nucleus pulposus.4.4 The process of the active migration of endplate chondrocytes associated with internalization of hyaluronan rapidly, the content of the hyaluronan in intervertebral disc had a relationship with the signal transduction mechanism of endplate chondrocytes migrating to nucleus pulposus. Because the transition of the nucleus pulposus postnatal is the results of the biomechanical condition of the intervertebral disc, so it can be assumed that at the aging and cervical degeneration process the biomechanical factors would play an important role in switching of the signal transduction process of the endplate chondrocytes migrating to nucleus pulposus. The chondrocytes located at the junction of cartilage endplate and nucleus pulposus activated by the stress transferred by extracellular matrix, then activated the intracellular and extracellular signal cascade pathway and started the process of endplate chondrocytes migrating to nucleus pulposus. Apart of the interaction between extracellular matrix and cells during the migration process, signal transduction process between cells may also participate into this process. Make a profound researching of signal transduction association with this process will establish a new pathway for studying the degeneration mechanism of intervertebral disc and may offer an important theoretical base for biomedical therapy to intervertebral disc degeneration diseases.