Study On Peptide-containing Temperature-sensitive Hydrogel Scaffold For Regeneration Of Spinal Cord Injury

As the structure and function of the spinal nerve tissue is complicated,if the injury occurs,will cause the patient has the limb dysfunction and even falling a lifelong disability.Preventing the secondary nerve injury and promoting the nerve regeneration are effective strategies for the treatment of spinal cord injury currently.The temperature-sensitive hydrogel has become an important matrix material in the field of spinal cord repair due to its good biocompatibility and tissue regeneration ability.However,the low biological activity and the poor temperature sensitive response will limit its wide application.Taking all of these into consideration,this study used Nisopropylacrylamide(NIPAM)monomer and polyethylene glycol(PEG)as raw materials to synthesize a temperature-sensitive hydrogel with rapid temperature response properties by using the reversible addition-fragmentation chain transfer(RAFT)polymerization method.At the same time,the peptide IKVAV with specific biological activity for spinal cord repair was introduced to improve the biological activity of the material.Herein,we developed a temperature-sensitive hydrogel grafted with a bioactive peptide IKVAV.At first,we modified the two kinds of the polyethylene glycols(PEG)with different molecular weights,obtained two different product.One kind of it was arboxylated at one end while vinylated at another end,referred to as AC-PEG-COOH.Another kind of the product was vinylated on both ends,referred to as AC-PEG-AC,which abbreviated as PEGDA.Then the AC-PEG-IKVAV conjugates were prepared by coupling reaction of the amino group of IKVAV to the carboxyl group of AC-PEGCOOH.Finally,The hydrogel was prepared by copolymerization of N-propan-2-ylprop-2-enamide(NIPAM)and AC-PEG-IKVAV copolymers via RAFT polymerization,using polyethylene glycol(PEGDA)and N,N’-Methylenebisacrylamide(BISAM)as cross-linking agents.After that,the test mothods NMR and FTIR were used to verify whether the IKVAV peptide was grafted to the hydrogel grid successfully.At the same time,the formation mechanism and swelling properties of the gel were discussed.The results showed that the prepared hydrogel scaffold demonstrates a regular three-dimensional porous structure;a good swelling performance which can reach 85% of the swelling degree within 5 minutes and a rapid deswelling performance which can achieve 80%of the dehydration within 20 minutes;the low critical solution temperature(LCST)of the hydrogel was from 35 to 40 °C.Futher,the neural stem cells(NSCs)was selected as the cell model in order to know the cell compatibility of gel scaffold in vitro.The results showed that the NSCs can grow well and proliferation rapid on it.In addition,the scaffold can promoted the adhesion of nerve cells on it,promoted the expression of adhesion gene Lamb2,and guiden the NSCs to differentiate into neuronal cells effectively.In summary,the prepared hydrogel has a good cell compatibility.Finally,we built a spinal cord injury(SCI)animal trancection model by using the SD rats,after 2 weeks,the Basso-Beattle-Bresnahan(BBB)score and weight assessment were used to evaluating the motor function of the rats.The injured spinal cord tissue was obtained by cardiac perfusion,observe the general appearance of the obtained spinal cord specimens and HE staining were used for further evaluate the biocompatibility and nerve repair ability of the gel scaffold in vivo.The results showed that the BBB score of the gel scaffold can reach 8-10 points,HE staining result display the gel scaffold can effectively reduce the formation of glial scars,the area of lesions and cystic cavity,so as to better reconstruct the injured neural network and accelerate the recovery of nerve function.The peptide containing temperature-sensitive hydrogel provides a new idea for the regeneration and repairability of spinal cord.