The Feasibility Study Of Constructing Nucleus Pulposus Scaffold By Using Supramolecular Hydrogel

Background:Disc degenerative disease is a common clinical disease,and the current clinical treatment can not repair the lesion disc in the structural and functional level.As a new treatment of disc degeneration,nucleus pulposus tissue engineering uses well-functioning nucleus pulposus seed cells to replace the degenerated cells.It can restore the normal physiological structure and function of nucleus pulposus,and reverse the disc degeneration process.In the study of nucleus pulposus tissue engineering,cell scaffolds are essential for the activity and function of seed cells.Currently,most hydrogels belong to natural materials or synthetic high molecular polymer hydrogels.However,these two types of materials have their own shortcomings.With the development of materials chemistry,a type of supramolecular hydrogels composed of small molecular oligopeptides as the basic unit has been found,which has excellent biocompatibility,good mechanical properties and approximate extracellular matrix structure.And it may become a new choice for the cell scaffold of nucleus pulposus tissue engineering.Objective:To explore the feasibility of using supramolecular hydrogel composed of oligopeptide(Nap-F-F-Y(P))as the basic unit and compounding chondroitin sulfate to construct the cell scaffold of nucleus pulposus tissue engineering.Method:The oligopeptide(Nap-F-F-Y(P))was synthesized by solid-phase synthesis in chemical laboratory and identified by mass spectrometry.The self-assembly of oligopeptide occured under alkaline phosphatase.And chondroitin sulfate was compounded into the hydrogels.The effects of different concentrations of hydrogels on the gelation time,macroscopic morphology,microstructure and mechanical properties were discussed.Nucleus pulposus cells obtained from caudal vertebrae of 12-week-old rats were used as seed cells.Supernatotic hydrogels compounding nucleus pulposus cells were co-cultured for two weeks.The normal culture without hydrogels was used as control.Cell viability was observed by live/dead cell staining and CKK-8.The characteristic phenotypes(type II collagen and aggrecan)of nucleus pulposus cells were observed by immunofluorescence staining.Results:After mass spectrometry analysis,it was demonstrated that the molecular weight and the structure were correct and that the purity of the product was high.The oligopeptide can be further used in self-assembly into hydrogels,and the gel concentration ranges from 0.5%(w/v)to 1.5%(w/v).The transparency of the gel was decreased with the increase of the concentration.When the concentration of the oligopeptide was 1%,the gelation rate was the fastest and the macroscopic morphology was the most stable.The observation of the microstructure of the hydrogel by transmission electron microscope(TEM)showed that the nanoscale fibers were arranged criss-cross which formed a porous structure inside the gel.Dynamic strain and frequency scanning of the hydrogel found that its mechanical properties met the viscoelastic requirements of tissue engineering hydrogel.For 1%concentration of oligopeptide,compounding chondroitin sulfate can further enhance its mechanical properties.The rat nucleus pulposus cells were homogenized after primary culture,and were similar to chondrocytes.Phenotypic identification showed that the expression of type II collagen and aggrecan was intact.After 2 weeks of culture,live/dead cell staining showed no difference in the viability of the nucleus pulposus cells between two groups.CKK-8 test showed that the cell proliferation was not inhibited in the group co-cultured with hydrogels.And cell immunofluorescence found that nucleus pulposus cells were phenotypically intact.Conclusion:The supramolecular hydrogel consisting of oligopeptides(Nap-F-F-Y(P))as the basic unit and compounding chondroitin sulfate has approximate extracellular matrix structure,appropriate viscoelasticity and good biocompatibility.After co-cultured with the hydrogels,nucleus pulposus cells can maintain their viability and phenotype.The gelation process is simple and can occur in a physiological environment.In conclusion,this supramolecular hydrogel has a good feasibility to construct the cell scaffold for nucleus pulposus tissue engineering.