The Experimental Study Of Thermosensitive Celecoxib-loaded Injectable Chitosan Hydrogel In The Treatment Of Intervertebral Disc Degeneration

Objective: In this study,thermosensitive injectable chitosan hydrogels loaded with celecoxib were prepared independently.We explore its mechanical properties and evaluate the biocompatibility and histocompatibility of the hydrogels.To observe the restoration effect and anti-inflammatory function of hydrogel on local intervertebral disc defect,as well as the ability to delay the intervertebral disc degeneration.Methods: Thermosensitive injectable chitosan hydrogel scaffolds loaded with celecoxib were prepared,consisting of chitosan,celecoxib and β-sodium glycerophosphate,and the physical appearance and mechanical properties of the scaffolds were observed and studied.The cytocompatibility of the hydrogels was tested by CCK-8 and Hoechst staining,and the histocompatibility and degradation of the hydrogels were evaluated by subcutaneous implantation of hydrogel.Fifteen New Zealand white rabbits were selected as experimental animals to establish the intervertebral disc degeneration model of New Zealand white rabbits.The intervertebral disc degeneration models of L2/3,L3/4,L4/5 and L5/6 were respectively made by using the method of acupuncture and suction of nucleus pulposus,and divided into groups according to different interventions.L2/3,L3/4,L4/5 and L5/6 was respectively the normal control group,the degenerative group,the hydrogel group and celecoxib hydrogel loaded group.Intervertebral discs of New Zealand white rabbits were examined by X-ray and MRI at 2 weeks,1 month,and 2months postoperatively,and disc degeneration in each group was assessed by Bradner Disc Index(BDI)and modified Thompson scale,respectively.Intervertebral discs of New Zealand white rabbits were obtained 2 months after surgery,and Hematoxylin and Eosin(H&E)staining was performed after decalcification to evaluate the degeneration of intervertebral discs at different segments.Results: In this study,the thermosensitive injectable chitosan hydrogel loaded with celecoxib was successfully prepared.It was liquid at room temperature,with a p H about 7.0.After being placed at 37℃ in 12±0.84 minutes,it becomes a solid gel.According to the observation under electron microscope,the hydrogel had good porosity,and the Energy-dispersive X-ray spectroscopy(EDS)showed that the celecoxib was distributed evenly in hydrogel.In terms of biocompatibility evaluation,we used the CCK-8,Hoechst staining and hydrogel subcutaneous implantation methods to detect its cell compatibility and tissue compatibility.The results showed that the hydrogel loaded with celecoxib had no obvious inhibitory effect on cell proliferation,and had a relatively good cell compatibility and tissue compatibility.The hydrogel can be degraded in certain period of time and its degradation product was also no obvious toxicity to surrounding tissue.The animal experiments showed that the intervertebral space in the degenerative group began to narrow significantly at 2 weeks after surgery.The intervertebral space height of hydrogel group and celecoxib-loaded hydrogel group decreased compared with the normal control group,but the decreased height was less than those of the degenerative group.(P < 0.05)Slower rate of degeneration was observed in the celecoxib-loaded hydrogel group at 2 weeks,1 month,and 2 months postoperatively compared with degenerative group by using sagittal Magnetic Resonance Imaging(MRI)and the modified Thompson scale.(P < 0.05)The results of HE staining and pathological scores of intervertebral discs showed that the intervertebral discs injected with chitosan hydrogel or chitosan hydrogel loaded with celecoxib,comparing with the degenerative group,had the effect of delaying the degeneration of intervertebral discs to a certain extent.Conclusion: The injectable chitosan hydrogel loaded with celecoxib had good biocompatibility,and can be implanted by injection.The thermosensitive characteristics could turn liquid hydrogel into solid gel in local defects in short time.It can effectively repair the local defects,and relieve the local inflammation,and maintain mechanical stability of intervertebral disc,and delay the intervertebral disc degeneration.This composite hydrogel provided a new therapeutic prospect for the further methods of intervertebral disc regeneration,and the prevention of re-herniation and degeneration of intervertebral disc.