Nerves And Blood Veseels Ingrowth Into In A New Anular-injury Rabbit Model And Nucleus Pulposus Cells Culture In Vitro

Back pain is one of the most common causes of pain and disability.There are many causes of back pain,and determining the source of the pain can help guide treatment of this common problem.Disorders of the lumbar intervertebral disc generate discogenic pain,which is considered to be a major source of low back pain.Sometimes the cause of back pain is thought to be due to degeneration of the lumber intervertebral discs.This condition is called discogenic back pain.Exactly what causes lumbar disc pain is not well understood.There are differences that can be seen between a normal lumbar disc and a degenerative lumber disc. Generally,disc degeneration is thought to be one of the causes of chronic discogenic low back pain.Because a tissue cannot generate pain without nerve structures,it is important to know the neuropathology of a painful disc to shed light on the mechanism of discogenic pain.Pain sensation is generated when nociceptive nerve endings are subject to noxious stimuli.Therefore,if an annular tear reaches nociceptive nerve endings,it may cause acute discogenic low back pain.The innervation of the normal lumbar intervertebral disc must firstly be understood. Then,it is necessary to determine whether the innervation of the painful disc is different from that of the normal disc.With long term of pain symptoms and systemic symptoms, the quality of life and social function of patients are also affected severely.The research of discogenic low back pain is a hot topic nowadays.Adequate animal model of DDD(Disc Degenerative Disease) is a useful tool of research on the therapeutic agents.Several models of experimental injured-anular resembling DDD have been reported previously.The most widely used models are induced by injuring anular such as stabbing the anular,puncturing the anualr.The histopathology of this model showed some characteristics resemblance to DDD, enabling research into the pathogenesis and therapy of this disease.Anular injury-induced DDD is a traditional model of disc degenerative disease and is widely used.In order to investigate degenerative intervertebral disc and examine nervers and blood vessels ingrowth into degenerative discs,we established the rabbit anular-injury model of disc degenerative disease model to doublely check its effect.In addition, further to research the biological feature of nucleus pulposus cells(NPCs) cluture in porous PLGA scaffolds in vitro.First we examined the effect of injury anular on innervation in a rabbit model. The results showed the loss of height in the injury groups was significantly higher than that of normal group(P<0.01).This part showed that anular-injury induced innervation. The extent of injury anular is related to nerves ingrowth into scar tissue.Then we examined the effect of administration of the porous PLGA in an anular injury rabbit model.The porous PLGA scaffold was fabricated by a porogen-leaching technique.The changes in disc height index,macroscopic damage and histologic scores were subsequently evaluated.This part showed that administration of PLGA scaffold could affect on innervations in a rabbit disc degeneration model.Based on these observations we proved that anular-injury is useful for the induction of disc degeneration.Further experiments were also done to ascertain the possible mechanisms involved in the reduction of proteoglycan effect on innervation. Lyophilization was employed in the process of Chondroitinase ABC incorporation into PLGA scaffold.Three different magnitudes of anular injury,11 blade and 16 gague needles and 3-mm diameter micro-trephine at 5 mm depth were performed at the L3/4 or L5/6 discs in New Zealand white rabbits.With or without a PLGA scaffold(+/-Chondroitinase ABC) was then carefully inserted into the defect.Disc degeneration was evaluated by radiographic,MRI and histologic and biochemical examine at different time after surgery.1,9-Dimethylmethylene blue(DMMB) staining for s-GAG was used to measure proteoglycan content in the nucleus pulposus.Distribution of chondroitin sulfate in the tissues was immunolabeled using CS56 antibody.The level of colⅠ,Ⅱand aggrecan message was detected by RT-PCR.To identify nerve ingrowth into disc, PGP9.5 and GAP-43,two general markers for nerve fibers were immunostained.The results showed that significant decreases in disc height and signal intensity in magnetic resonance imaging were observed in three injury groups.The scaffold group and 16G puncture group induced a more slower and more progressive disc degeneration than that in the control group.At the 12-week time point,nucleus pulposus tissues were extruded and scar tissues formed outside the disc.The s-GAG content of PLGA/ChABC group decreased significantly following ChABC enzymic deglycosylation at postoperative different time-point.RT-PCR demonstrated that typeⅡcollagen and aggrean mRNA was significantly down-regulated,whereas typeⅠcollagen mRNA was up-regulated after surgery.The scaffold groups had slower but more progressive disc degeneration and innervation of degenerated disc was more evident.The new tissue accompanying the ingrowth of nerve fibers extended into the deeper part of the annulus fibrosus.In the PLGA/ChABC discs,newly formed tissue accompanied with many small blood vessels appeared in the regenerated area.And the undegraded scaffolds were surrounded by newly formed tissues,where more nerve fibers grew further into the inner AF following reduction of proteoglycan.In punctured discs,PGP9.5 and GAP-43-immunoreative fibers were only observed in the outmost part of the scar tissues and superficial area. This part showed loss of PG facilicated the nervers and blood vessels ingrowth into the degenerative disc.In the end,the capacity of NP cells in vitro was determine by measuring.Rabbit NP cells grouped into 2 groups were cultured in vitro for three weeks.Group 1, monolayer culture;Group 2,three dimensional culture in PLGA scaffold. Morphological changes were observed under inverted microscope and scanning electron microscope.The proliferation of the NP cells in the each group was tested by MTT.The expression of type collagen in each group was analyzed by HE-staining and immunohistochemistry.The glycosaminoglycan(GAG) content synthesized by the NP cells in the culture medium was measured by DMMB staining.After three weeks,the proliferation of the NP cells in both monolayer and PLGA culture system was observed. Compared with the monolayer culture group,the proliferation of the NP cells,the expression of the synthesis of GAG increased significantly(P＜0.01) in the PLGA group.In conclusion,we successfully established a model of injury anular degenerative model.The study demonstrated that in the rabbit anular-injury model,a degenerative trend was evidently developed by anular injury.Total anulus fibrosus injury may play a critical role in inducing nerve ingrowth.The injury degree may directly affect such ingrowth.A porous PLGA scaffold provides an important biological channel to induce nerve fiber ingrowth into the disc.More nerve fibers ingrowth into the scar tissue was observed in the PLGA/ChABC group following ChABC enzymic deglycosylation.Loss of proteoglycan in the extracellular matrix could facilitate nerve and blood vessels ingrowth into the degenerated disc.Nerve ingrowth may act as a source of pain and strongly associated with discogenic pain.Studying this new innervation could facilitate the understanding and treatment of discogenic pain.And rabbit NP cells culture in PLGA scaffold can promote the proliferation and increase the synthesis of type collagen and GAG.This method may construct intervertebral disc tissue engineering.