Osteogenesis Promotion By Injectable Methacryloylated Gelatin Containing Psoralen And Its Bacteriostatic Properties

Objective In this study,we aimed to prepare a Psoralen/GelMA(Pso-GelMA)material that can be injected,and to explore its potential for inducing bone differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in vitro,as well as its antibacterial effects against Staphylococcus aureus and Fusobacterium nucleatum.Method Part 1: Prepare 1mmol/L Pso solution and 5% GelMA precursor solution separately,mix them in a certain proportion to obtain Pso-GelMA material containing 100 μmol/L Pso,and irradiate under a 405 nm light source for 20 s to solidify the material.Evaluate the injectability,self-repairing ability,degradation,and swelling of Pso-GelMA material in vitro,and observe the morphology of the material by scanning electron microscopy.Part 2: Isolate and culture BMSCs using the whole bone marrow adherent method and identify them.Evaluate cell adhesion and proliferation in Pso-GelMA material using Live/Dead cell staining.Use CCK-8 assay to screen the optimal concentration of Pso-GelMA material extract that promotes BMSCs proliferation from different dilutions.After inducing for 12 d,study the effect of the material extract on early osteogenic marker ALP expression in BMSCs by ALP staining and activity detection.After inducing for 21 d,study the expression of matrix mineralization ability in BMSCs by alizarin red staining.After inducing for 12 d,using real-time quantitative RT-PCR and western blotting to study the effect of the material extract on osteogenic differentiation genes/proteins TGF-β1 and Smad4 in BMSCs.Test the antibacterial effect of Pso-GelMA against Staphylococcus aureus and Fusobacterium nucleatum using inhibition zone assay and colony counting method.Results(1)The uncrosslinked Pso-GelMA material could be continuously injected through a 1m L syringe needle,and scanning electron microscopy showed that the internal pore size of the solidified Pso-GelMA material was uneven,with an average of 146.33 ± 43.77 μm,while the GelMA pore size was about 149.17 ± 34.24 μm.There was no significant difference in pore size before and after loading Pso.The loose and porous structure was conducive to cell adhesion and nutrient passage.Two separate hydrogels could be well bonded together within 20 minutes,and Pso-GelMA material could self-repair well after being cut or damaged.(2)The cultured third-generation cells were mainly spindle-shaped,and after induction of adipogenesis or osteogenesis,lipid droplets,and red calcium nodules were visible.The expression rate of the CD90 positive marker on the BMSCs surface was greater than 99%,while the expression rate of the negative marker CD45 was about 0.05%.Live/Dead cell staining showed that cells could be evenly dispersed in the material,and with time,the number of live cells gradually increased while the number of dead cells remained relatively low.Compared with the control group,Pso-GelMA at a concentration of 10μmol/L could promote BMSCs proliferation(P<0.05).Alizarin red staining,ALP staining,and activity data showed that compared with the control group,the Pso-GelMA group had deeper osteogenic differentiation staining and stronger ALP activity(P<0.05).After inducing osteogenesis for 12 d,the expression levels of TGF-β1 and Smad4 genes in the Pso-GelMA group were higher than those in the control group(P<0.05),while Western blotting showed higher expression levels of osteogenic-related proteins TGF-β1 and Smad4 in the Pso-GelMA group.(3)The inhibition zone diameter of Pso-GelMA material against Staphylococcus aureus was about 23.67 ± 0.09 mm,with an inhibition rate of 86.40%,while the inhibition circle diameter of Pso-GelMA material against Fusobacterium nucleatum was 23.93 ± 0.04 mm,with an inhibition rate of 72.15%,demonstrating good antibacterial properties of Pso-GelMA.Conclusion(1)Pso-GelMA material has good biocompatibility and can promote BMSCs proliferation.(2)Pso-GelMA material can effectively up-regulate the expression of osteogenic-related genes and proteins,increase ALP activity,and promote extracellular matrix mineralization in rat bone marrow mesenchymal stem cells.(3)Pso-GelMA material has a significant antibacterial effect against Staphylococcus aureus and Fusobacterium nucleatum.