Effects Of Fucoidan-functionalized GelMA Microspheres On The Treatment Of Intervertebral Disc Degeneration

Background:Intervertebral disc degeneration(IVDD)is a degenerative disease characterized by the loss of functional cells and extracellular matrix(ECM)in the intervertebral disc(IVD).It is a common cause of low back pain and musculoskeletal atrophy.Nucleus pulposus(NP)is an important functional unit of intervertebral disc to resist spinal load.Degeneration of nucleus pulposus is one of the important characteristics of IVDD.Oxidative stress is a kind of cell or matrix damage response caused by excessive production of reactive oxygen species(ROS)or insufficient activity of antioxidant enzymes.Oxidative stress can promote the produce of inflammatory factors and matrix degrading enzymes,accelerating the cell apoptosis and matrix degradation.Nuclear factor erythroid-2 related factor 2(NRF2)is an important transcription factor that regulates the activities of antioxidant enzymes.The insufficient expression of NRF2 has been shown to be related to the aggravation of IVDD.Fucoidan(Fu)is a kind of sulfated polysaccharide with a variety of biological activities.It has strong anti-inflammatory and antioxidant potential,it can significantly remove intracellular ROS and improve the activity of antioxidant related enzymes.Gelatin methacryloyl(GelMA)is kind of hydrogel functional modificated from gelatin.It has been widely used in biological tissue engineering research because of its good biocompatibility.Microspheres(MS)based therapeutics are often used for the delivery of therapeutic molecules or cells in vivo due to their injectability,high surface area and porous structure.Objective:To explore the regulatory role of fucoidan in nucleus pulposus matrix metabolism,to clarify the effect of fucoidan-functionalized GelMA microspheres on IVDD in vivo,and to reveal the regulatory mechanism of fucoidan in protecting nucleus pulposus matrix synthesis in vitro.Methods:(1)To study the effects of fucoidan on the proliferation and matrix metabolism of NP cells:NP cells were treated with different concentrations of fucoidan.CCK-8 method was used to detect the proliferation ability of NP cells,RT-PCR and Western Blot were used to detect the expression levels of Collagen II and Aggrecan.Then,IL-1βwas used to simulate the degenerative environment of NP,and the effect of fucoidan on the synthesis and degradation of NP matrix in the degenerative environment was detected.(2)To study the repair effect of fucoidan-functionalized GelMA microspheres(Fu@GelMA-MS)on IVDD after implantation in vivo:GelMA microspheres(GelMA-MS)and Fu@GelMA-MS were prepared by microfluidic method,after that,the material characterization and biocompatibility of the two kinds of microspheres were tested.Thirty 8-week-old male Sprague-Dawley rats were randomly divided into 5 groups,6 in each group.They were Sham operation(Sham)group,IVDD group,GelMA-MS group,Fu group and Fu@GelMA-MS group.The model of IVDD was established by acupuncture in the caudal vertebra of rats,and then different interventions were injected into the disc(IVDD group:5 μl normal saline;GelMA-MS group:5 μl GelMA-MS;Fu group:5 μl fucoidan;Fu@GelMA-MS group:5 μl Fu@GelMA-MS).At 4 and 8 weeks after operation,X-ray and magnetic resonance imaging(MRI)of the rat tail vertebra were performed to evaluate the effect of IVD repair.Histological sections were stained to observe the morphological changes of the IVD.(3)To study the regulatory effect of fucoidan on the antioxidant function of NP cells in inflammatory environment:the NP cells was treated with different concentrations of fucoidan.The intracellular ROS level was detected by fluorescent probe method,and the expression levels of NRF2,HO-1 and SOD1 were detected by RT-PCR and Western Blot.Results:(1)Fucoidan promoted cell proliferation and matrix synthesis in NP under normal growth conditions.Under inflammatory conditions,fucoidan made the protein expression of MMP13 and ADAMTS5 decrease separately by 128.0%and 134.9%.The matrix synthesis capacity was also greatly improved.(2)Fu@GelMA-MS significantly improved IVDD in vivo.Fu@GelMA-MS increased the height of the IVD by 29.5%.Histological analysis showed that Fu@GelMA-MS significantly preserved the NP tissue and alleviated the damage of the IVD structure.(3)The antioxidant capacity of NP cells in inflammatory environment was improved by fucoidan.In the inflammatory environment,the ROS level in NP cells decreased by 53.8%after the intervention of fucoidan,and the protein levels of NRF2,HO-1 and SOD1 were up-regulated by 44.5%,52.3%and 30.6%,respectively.Conclusion:Fucoidan has the ability to promote the synthesis of ECM in NP.In inflammatory environment,fucoidan can not only protect matrix synthesis,but also inhibit the produce of matrix-degrading enzymes.Intradiscal injection of Fu@GelMA-MS significantly delay the progress of IVDD.In addition,further study found that fucoidan could significantly improve the antioxidant function of NP cells and eliminate ROS accumulation in NP cells.