Sa12b-modified Functional Self-assembling Peptide Hydrogel Enhances The Biological Activity Of Nucleus Pulposus Mesenchymal Stem Cells By Inhibiting Acid-sensingion Channels

Background: Low back pain（LBP）is a common health problem worldwide.Lumbar disc degeneration is considered a major cause of chronic back pain and degenerative lumbar spine disease.The microenvironment of a degenerative intervertebral disc（IVD）is characterized by hypoxia,low p H,hypertonicity,and poor nutrient supply.Therefore,as adults age and intervertebral disc degeneration（IDD）,the internal microenvironment changes significantly.The lower p H had the most significant inhibitory effect on cells in the microenvironment.Currently,the gold standard for intervertebral disc disease（IVDD）treatment is spinal fusion,which focuses on pain relief and removal of degenerated discs.However,this treatment did not reverse the progression of degeneration or restore the normal mechanical function of the disc.Therefore,in addition to alleviating symptoms,restoring IVD function should be the focus of IVDD treatment.In recent years,tissue engineering has been successfully applied in clinical orthopaedics to treat defects of bones,skeletal muscles and tendons,and replace already damaged tissues with biomaterials and cells.Hydrogel scaffolds are one of the promising biomaterials,which have been successfully applied in tissue engineering and regenerative medicine,and have become an important topic in biomaterial research.A variety of hydrogels have been studied for nucleus pulposus regeneration,but none of them can overcome the effects of acid environment changes on cells during disc degeneration.Therefore,in this study,an acid-sensitive ion channel inhibitor,Sa12 b,was conjugated to the C-terminus of RADA16-I,and a new functionalized self-assembled peptide hydrogel scaffold material,RAD/SA1,was designed.The biological activity of environmental degenerated human nucleus pulposus mesenchymal stem cells（hNPMSCs）was observed in vitro.It is expected that this novel functionalized self-assembled peptide nanoscaffold hydrogel material can overcome the influence of the change of the acidic environment on cells during the degeneration of the intervertebral disc,and can not only provide a three-dimensional culture environment for the degenerated hNPMSCs under the acidic environment,but also possess the ability of Sa12 b and prolong its action time,thereby enhancing the biological activity of hNPMSCs in acid environment.Objective:1.In this study,a new functionalized self-assembling peptide hydrogel scaffold material RAD/SA1 was designed by conjugating the acid-sensitive ion channel inhibitor Sa12 b to the C-terminus of RADA16-I.2.The physical properties of the RAD/SA1 material were evaluated.3.Three-dimensional culture of hNPMSCs and hydrogel scaffolds was performed to evaluate the effect of hydrogels on the biological activity and biocompatibility of hNPMSCs in an acid environment.4.Three-dimensional culture of hNPMSCs and hydrogel scaffolds was carried out to explore the possible mechanism of hydrogels affecting the biological activity of hNPMSCs in acid environment.Methods:1.The physical properties of RAD/SA1 materials were detected by atomic force microscope（AFM）,scanning electron microscope（SEM）,circular dichroism spectrometer and rheometer.2.The hNPMSCs were isolated from the nucleus pulposus tissue of patients with intervertebral disc herniation surgery,and identified by flow cytometry.3.In vitro culture in an acid environment model of intervertebral disc degeneration at p H 6.2 and 6.8.Different hydrogels and P3 generation hNPMSCs were three-dimensionally cultured,and their cell proliferation ability and cell viability were tested.4.The expression levels of collagenⅡ,proteoglycan and SOX-9 in different hNPMSCs treatment groups were detected by enzyme-linked immunosorbent assay（ELISA）and Western blotting（WB）.5.WB was used to detect the expression levels of collagenⅡ and proteoglycan in different hNPMSCs treatment groups.6.Quantitative Real-time Polymerase Chain Reaction（QRT-PCR）technique was used to detect the expression of collagen I,collagen II,proteoglycan and SOX-9 in different hNPMSCs treatment groups.7.The influx of calcium ions in different hNPMSCs cell treatment groups was detected by laser confocal,and the expression of p-ERK in different hNPMSCs cell treatment groups was detected by WB.Conclusion:1.Functional self-assembled peptide nanofiber hydrogels designed with a short Sal2 b motif can be used as excellent scaffolds for nucleus pulposus tissue engineering.2.RAD/SA1 may show great potential in the treatment of IVD.