| Objective: Hydrogel scaffolds are often used in the field of spinal cord injury(SCI)repair because of their good biocompatibility,mechanical properties and degradability.However,single natural hydrogel materials have many problems in practical application,such as weak cell adhesion,high degradation rate and low efficiency of loading biomolecules,which affect the efficacy of SCI repair.In this project,we aim to develop an effective,simple method of constructing composite hydrogel scaffolds and an efficient loading method for neurotrophin-3(NT-3),as well as to study the adhesion and proliferation of bone marrow mesenchymal stem cells(BMSCs)and the feasibility of SCI repair using NT-3 loaded composite hydrogel scaffolds.Methods:(1)Hyaluronic acid(HA)/silk fibroin(SF)hydrogel scaffold was synthesized by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)/N-hydroxysuccinimide(NHS)method;(2)The composite hydrogel scaffold was characterized by scanning electron microscopy(SEM)and Fourier transform infrared spectroscopy(FTIR);(3)The physicochemical properties of the composite hydrogel scaffold were tested in vitro;(4)The cell compatibility in vitro was evaluated by CCK-8and fluorescence staining;(5)The apoptosis behavior of BMSCs on hydrogel scaffold was evaluated by flow cytometry;(6)The release behavior of NT-3 in vitro was determined by enzyme-linked immunosorbent assay(ELISA);(7)The rat model of thoracic 10 segment spinal cord transection was constructed and the composite hydrogel scaffold was implanted;(8)The tissue integrity and cavity formation of the damaged area were evaluated by hematoxylin-eosin(HE)staining;(9)Immunofluorescence staining and Western blot were used to detect the specific protein expression in the injured area and adjacent areas;(10)Basso – Beattie – Bresnahan(BBB)score was used to assess the recovery of motor function of rats’ hind limbs.Results:(1)HA/SF,HA/SF/p DA and HA/SF/p DA-NT3 hydrogel scaffolds were successfully prepared;In vitro performance test demonstrated that the hydrogel scaffolds in each group had appropriate water expansion rate,degradation rate,porosity and ideal mechanical properties;(2)SEM showed that all groups of hydrogel scaffolds formed an interconnected reticular porous structure with appropriate pore size.FTIR confirmed the construction of p DA coating and the loading of NT-3;(3)The results of CCK-8 and fluorescent staining indicated that BMSCs had good growth potential on HA/SF hydrogel scaffolds in different proportion groups,and the group with HA/SF ratio of 6/4 achieved the greatest proliferation result.Based on this ratio,HA/SF/p DA and HA/SF/p DA-NT3 hydrogel scaffolds were also found to support adhesion and proliferation of BMSCs.The results of flow cytometry showed that the construction of p DA coating and the loading of NT-3 had no significant effect on the apoptosis of BMSCs;(4)ELISA results confirmed that p DA coating not only increased the load of NT-3,but also prolonged the release of NT-3 to 28 days;(5)HE results indicated that compared with 6/4-p DA and SCI group,the cavity area of 6/4-NT3-p DA group was the smallest(P<0.05);(6)The results of immunofluorescence and Western-blot showed that compared with 6/4-p DA and SCI groups,the 6/4-NT3-p DA group had high expression of neurofilament protein 200(NF200),β-Ⅲ tubulin(β-Ⅲ Tubulin),myelin basic protein(MBP)and low expression of macrophage marker(CD68)and glial fibrillary acidic protein(GFAP)(P<0.05);(7)The BBB score results revealed that from the 4th week,the scores of rats in the 6/4-p DA and 6/4-NT3-p DA groups were significantly higher than those in the SCI group,and the scores in the 6/4-NT3-p DA group increased the most significantly among the three groups(P<0.05).Conclusion: In this study,HA/SF/p DA-NT3 hydrogel scaffolds were prepared to have excellent physical and chemical properties,good biocompatibility,and ideal sustained NT-3 release effects,which can reduce inflammation,cavity and scar formation in the injured area,facilitate spinal nerve regeneration,and ultimately improve rat hind limb motor function. |
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