Effects Of PGSA Scaffolds With Different Apertures On Adhesion And Proliferation Of Goat Temporomandibular Joint Disc Cells

Background:Temporomandibular Joint Disorder(TMD)refers to a series of diseases characterized by pain in the temporomandibular joint and surrounding tissues,limited mandibular function,or bounce of the temporomandibular joint during movement.The rise and development of temporomandibular joint disc tissue engineering provides better choices for the fields of joint disc replacement materials and joint replacement surgery.Polyglycerol sebacate(PGS)is a chemical polymer formed by the addition and condensation of glycerol and sebacate in an equimolar ratio.Preliminary in vitro and vivo biocompatibility studies showed that PGS is a promising scaffold material for tissue engineering.However,PGS still has some problems and deficiencies:Firstly,the reaction conditions for synthesis are relatively harsh,and there are disadvantages such as high temperature,high vacuum,and long duration required for the curing reaction.Moreover,as a scaffold material,the adhesion and proliferation of cells on PGS scaffold still have problems.Purpose:In this study,we are aim to provide some new ideas for modification of PGS scaffold materials,and synthesis of PGSA scaffold materials with good mechanical properties and abilities to promote growth and proliferation of goat temporomandibular joint disc cells,we also hypothesize that in the future,apply them for temporomandibular joint disc tissue regeneration engineering supporting structure will have good performance.Methods:The sebacic acid and glycerol were mixed at a molar ratio of 2:1,and then have been heaten for 12 hours to synthetize PGS,performed acryloyl acylation to synthesize PGSA,and obtained a PGSA scaffold with a pore size of 150μm and a pore size of 75μm through the salting-out method.The goat temporomandibular joint disc cells were isolated and cultured to P2,and then seeded on the scaffold for culture.Through SEM(Scanning Electron Microscope),FTIR(Fourier Infrared Spectroscopy),~1H NMR(Proton Nuclear Magnetic Resonance Spectroscopy),XPS(X-ray Photoelectron Spectroscopy),compression/tension capability testing,hydrophilicity analysis and other methods to analyze morphology,element composition,molecular structure,surface hydrophilicity,compression,extension and other mechanical properties of the construct;then cytotoxicity of the materials was detected by methods such as living dead cell staining,CCK-8 staining,HE staining,and RT-PCR,Cell adhesion,proliferation on the surface of scaffold,and related gene expression levels of cells planted on the scaffold.Results:Firstly,it is proved that when the molar ratio of sebacic acid to glycerol is increased to 2:1,reaction heating time can be reduced correspondingly to 12h,and synthesized PGS has no obvious difference in element composition and structure from the original traditional method.Then the synthesis of PGSA reduces its curing conditions and makes the operation easier.SEM results showed that surface of PGSA with a pore size of 150μm is rougher and has a larger pore structure.In cell adhesion experiment,SEM scanning of cells’adhesion on the stent surface found that more goat articular disc cells were attached to surface of the 150μm PGSA scaffold.HE staining results also showed that more cells adhered to the PGSA scaffold with 150μm pore size,and then grew,proliferated;CCK-8 results showed that goat articular disc cells on 150μm pore size PGSA grew and proliferated more;the living dead cell staining results showed that PGSA,150μm pore size PGSA,75μm pore size PGSA scaffold materials were non-cytotoxic,meanwhile150μm pore size PGSA scaffolds had more cells to adhere and proliferate on it.The RT-PCR results showed that Aggrecan and COL-Ⅰgene expression level was higher in cells on 150μm PGSA scaffold,and COL-Ⅱgene expression was higher in cells on 75μm PGSA scaffold.Conclusions:The synthesis method with a molar ratio of sebacic acid and glycerol of 2:1and a heating time of 12 hours can successfully synthesize PGS.After adding acrylic acid groups,it can synthesize PGSA with thermal curing and light curing capabilities.The 150μm pore size PGSA is more conducive to cells’adhesion,growth and proliferation.It can be seen that hydrophilicity and mechanical properties of 150μm pore PGSA scaffold and 75μm PGSA scaffold are superior to those of PGSA.PGSA stent with 150μm pore size has higher compressive elastic modulus,it helps cells to synthesize Aggrecan and COL-Ⅰ,while PGSA stent with 75μm pore size has higher tensile elastic modulus and is more conducive to tensile stress,it is more helpful to synthesize COL-II.Finally,we explored and completed the production of 3D printing PGSA scaffold.