Human Degenerative Nucleus Pulposus Cell Culture In Vitro: Investigation Of The Responsibility To Tissue Growth Factor-β1 And Insulin-like Growth Factor-1

Background: Low back pain is one of the major causes of disability and, thus, has a major socioeconomic impact. The lifetime prevalence of low back pain is 75% to 80% of the population. Many of these spinal impairments are the direct or indirect result of degenerative disc disease (DDD). Current treatment options, including both conservative and surgical measures, target the clinical symptoms of intervertebral disc degeneration, as opposed to addressing the pathologic processes occurring early in the course of degeneration. However, recent molecular biology technology development may allow for the treatments focusing on the cellular changes that occur in this disease.The precise etiology and pathophysiology of DDD remains to be clearly delineated. It is known that the matrix of a healthy nucleus pulposus is rich in proteoglycans and type II collagen, whereas the annulus fibrosus is rich in type I collagen. The progressive decline in proteoglycan of the nucleus pulposus is known to be a significant and characteristic factor of the early stages of DDD. At the biochemical level, the diminished proteoglycan content reflects an imbalance in the normal anabolic and catabolic functions of the nucleus pulposus cells. With reductions in proteoglycan content of the intervertebral matrix, the nucleus pulposus dehydrates, decreasing disc height and altering its load-bearing capacity. This alteration may directly affect overall spinal biomechanical function. Although disc degeneration most probably evolves in response to a complex interplay of multiple biochemical and biomechanical factors, the ability to restore proteoglycan content may have therapeutic benefit by increasing disc hydration and potentially improving biomechanics. In 1991, Thompson et al reported that human recombinant insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor(FGF), and transforming growth factorβ(TGF-β) could increase proteoglycan synthesis in canine intervertebral disc in vitro. They suggested that these growth factors may have future therapeutic potential in the treatment of disc degeneration.Objective:1. use percutaneous aspiration operation induce rabbit disc degeneration to probation the theory that structure deletion lead to DDD.2. to find a simple and secure way to isolate nucleus pulposus(NP) cell from intervertebral disc tissue.3. to procure the NP cell growth information in vitro culture.4. stimulate the NP cell by TGF-β1 and IGF-1, to observe the alterations of cell's biological vigor.Methods: We removed New Zealand Rabbit's Nucleus pulposus tissue from healthy rabbits by percutaneous aspiration operation to make the disc degeneration. After 2 weeks, Rabbit's Nucleus pulposus cells were isolated from these discs by Trypsin plus collagenaseⅡandⅣdigestion. Rabbit's nucleus pulposus cells were cultured in monolayer in vitro to set up a cell culturing model in DMEM/F12 with 20% FBS.Cells viability were determined by trypan blue exclusion test. Then we recorded the cell number double time.Human degenerative nucleus pulposus cells were isolated from degenerated disc by Trypsin plus collagenaseⅡandⅣdigestion as talked above. Then they were cultured in monolayer in vitro to set up a cell culturing model and the cells increased slowly. Cells viability were determined by trypan blue exclusion test. Then we recorded the cell number double time. andThen we added TGF-β1 and IGF-1 to stimulate cells'biological vigor. We used the immunohistochemistry and Coomassie brilliant blue staining to detecte typeⅡcollagen produced by NP cell.Results: The morphology of the cultured cell both from rabbit and human are affinis. The double time of NP cell are 38.5 days in rabbit and 34-45days in human. The initial cell viability after isolation was about 90~95%. The viability of the cells declined after subculture was about 85～88%. TypeⅡcollagen staining showed the growth factors'stimulation to the cells'biological vigor. The positive staining of collagen typeⅡin human nucleus pulposus cell are significant differented from the group which added growth factors to the group which did not add. And we found the effectiveness is better in TGF-β1 plus IGF-1 group.Conclusion:⑴rabbit disc degeneration can be induced by percutaneous aspiration nucleus pulposus tissue probation the theory that structure deletion lead to DDD⑵NP cell can be harvested simple and quickly by Trypsin plus collagenaseⅡandⅣdigestion and probation the composition alteration in degenerated IVD tissue.⑶TGF-β1 and IGF-1 can enhance the cells'biological vigor which from human degeneration disc, effectiveness is better in TGF-β1 plus IGF-1 group.