Polysialic Acid Modified Polycaprolactone Nanofiber Scaffolds For The Treatment Of Spinal Cord Injury

Current therapeutic strategies for treatment of spinal cord injury(SCI)mainly focus on impeding further progression of the secondary injury and restoring of neuronal circuitry.Polysialic acid(PSA),a unique carbohydrate polymer located on the cell surface,has the capacity of preventing close interactions between cell-cell and cell-extracellular matrix via binding to neural cell adhension molecule,thus promoting cell migration,synapse formation,and neurons and myelin regeneration.In this study,we combined the bioactive PSA with biodegradable polycaprolactone(PCL)to fabricate methylprednisolone(MP)encapsulated nanofiber scaffolds(PCL,PCL/MP,PCL/PSA and PCL/MP/PSA respectively)using electrospinning technique.Morphologies of nanofiber scaffolds are influenced by solution viscosity,conductivity and volatility:with PCL concentration and solvent volume ratio of acetone increasing,the presence of beads would decrease and fiber diameters increase;with PSA concentration increasing,the fiber diameters would decrease.PCL concentration(150 mg/mL),PSA concentration(15 mg/mL)and solvent(DMF:Acetone = 1:1,v/v)were chosen as the optimal formulation.The diameters of PCL,PCL/MP,PCL/PSA and PCL/MP/PSA fibers were 564.59 ± 141.43 nm,512.85 ±69.85 nm,500.34 ± 150.7 nm,421.09 ± 57.4 nm respectively.The fourier transformed infrared spectroscopy(FT-IR)analysis showed that there was no chemical interaction between PCL,MP and PSA during the preparation process.Loading PSA into nanofiber scaffolds would accelerate MP release and cumulative MP release amount from PCL/MP/PSA nanofiber scaffold(PSA concentration 15 mg/ml)was 96%at 12 h.In vitro degradation characteristics suggested that degraded composition of PCL/MP/PSA nanofiber scaffold was mainly MP and PSA within 12 h,and PCL showed long-term degradation behaviour,contributing to the stability of scaffold structure.3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)assay and cell proliferation test with astrocyte cells showed that cell viability of PCL,PCL/MP,PCL/PSA and PCL/MP/PSA nanofiber scaffolds were above 80%and cells incubated on PCL/MP/PSA nanofiber scaffold grew well,extending to pore structure observed by transmission electron microscope(TEM).We estabilished the SCI rat model using spinal cord transection method and implanted nanofiber scaffolds into the lesion area.The tumor necrosis factor-?(TNF-?),interleukin-6(IL-6)and cell apotosis protein Caspase-3 were undertaken to evaluate the therapeutic effects of nanofiber scaffolds on spinal cord secondary injury.After 24 h of operation,there was no statistical difference of TNF-??IL-6 and Caspase-3 levels between PCL,PCL/PSA and SCI groups.In contrast,both the PCL/MP group and PCL/MP/PSA group inhibited TNF-??IL-6 and Caspase-3 expressions,suggesting that MP treatment significantly reduced the spinal cord tissue(lesion area)levels of TNF-?,IL-6 and Caspase-3,thus alleviating the inflammatory reponse and cell apoptosis.We further evaluated functional behavior of spinal cord injury rats using the Basso,Beattie and Bresnahan(BBB)score.After 7 w of operation,BBB scores of PCL,PCL/MP?PCL/PSA and PCL/MP/PSA groups was significantly higher than SCI group,and PCL/MP/PSA group showed significant functional improvent compared with other groups,showing that combination therapy of MP + PSA was the most effective in promoting functional recovery.After 7 w of operation,the spinal cord tissues(lesion area)were dissected and embedded in paraffin.Luxol fast blue(LFB)staining and TEM observation demonstrated that PCL/MP/PSA group significantly inhibited demyelination than other groups.Immunohistochemistry and immunofluorescense analysis of neurons,axons and astrocytes demonstrated that PCL/MP/PSA group was the most effective in promoting neuronal and axonal growth,and significantly inhibite the astrogliosis that other groups.According to immunofluorescense analysis of microglia,there was no statistical difference of Ibal expression between PCL,PCL/PSA and SCI groups.In contrast,both the PCL/MP group and PCL/MP/PSA group inhibited Ibal expressions,suggesting that MP treatment significantly inhibited activation of microglia in the spinal cord lesion area.In conclusion,our research indicated that PCL/MP/PSA nanofiber scaffold significantly contribute to the functional recovery of spinal cord injury rats via inhibiting inflammatory response,reducing cell apotosis,attenuating astrogliosis and improving neuronal and axonal survival.This research highlights a therapeutic potential of tissue engineering scaffold for the treatment of spinal cord injury.