Preparation Of Fluorapatite-polycaprolactone Composite Scaffolds And Induction Of Osteogenic Differentiation Of Human Periodontal Stem Cells

[Objective]To study the relationship between Fluoroapatite-Polycaprolactone(FA-PCL)composite scaffolds with different synthesis time and their biological properties,and to develop a FA-PCL composite scaffold with strong hydrophilicity,good biocompatibility and excellent osteogenic differentiation induction potential.[Methods]The PCL electrospun nanofiber membrane was prepared,and FA coating was synthesized on the surface of PCL nanofiber membrane by hydrothermal method under near physiological conditions.FA-PCL composite scaffold materials with different synthesis time were prepared according to the synthesis time.The physical and chemical properties of each group of materials were examined scanning electron microscope(SEM),energy dispersive spectroscopy(EDS),X-ray Diffraction(XRD)and water contact angle analysis(WCA).Human periodontal ligament stem cells(hPDLSCs)were isolated and extracted by tissue block method.The 2nd generation cells were labeled with antibody and analyzed by flow cytometry.Select the 3-5th generation cells,inoculate the cells on the surface of each group of materials(direct method)or co-culture with the material leaching solution(indirect method),and use CCK-8(Cell counting kit)method to detect the proliferation of cells on the surface of each group of materials.Alkaline phosphatase(ALP),Alizarin red staining(ARS)and Von Kossa staining were used on the 7th,21st,and 28th day of cell growth on the surface of the material,aim to observe the effect of each group of composite scaffolds on osteogenic differentiation of hPDLSCs.[Results]The PCL electrospun membrane was a three-dimensional network structure with smooth fibers and abundant pores.With the change of synthesis time,the FA crystals on the surface of PCL nanofibers were in different forms and quantities to form different surface morphology.In the PCL-FA-6h group,the FA crystal was fusiform and evenly covered on the surface of the fiber.At this time,some fibers were still exposed.In the PCL-FA-12h group,the fusiform crystal almost completely covered the PCL fiber.In the PCL-FA-18h group,the crystal grows into a larger columnar shape,and some of the fibers are re-exposed;the larger columnar crystals in the PCL-FA-24h group completely cover the fiber surface.The physicochemical properties of the composites show that the FA crystals are formed on the surface of the PCL nanofiber membrane at different synthesis times,but the degree of crystallization is different.The PCL-FA-24h group has the highest degree of crystallization and the best hydrophilicity.On the 7th day of primary culture,it can be seen that the cells climbed out from the tissue block and grew adherently.When the cell growth reached about 80%,it was digested and passed.Flow cytometry was performed using the 2nd generation cells,CD31,34 were negatively expressed,and CD105,STRO-1 was positively expressed,which proved to be a mesenchymal-derived stem cell.CCK-8 results demonstrated that hPDLSCs proliferated well on each group of composite scaffolds.The results of ALP staining,ARS staining and Von Kossa staining indicated that the composite scaffolds could induce the osteogenic differentiation of hPDLSCs.The ability of FA-PCL composite scaffolds to induce differentiation was better than that of PCL nanofiber membrane group.In the FA-PCL composite scaffolds of different surface morphology,with the prolongation of synthesis time,the overall trend of hPDLSCs osteogenic differentiation increased,suggesting that the composite scaffold ossification induction potential increased.[Conclusion]By controlling the synthesis time,the surface morphology of the FA-PCL composite scaffolds can be changed,and the physicochemical properties of the scaffolds can be improved.The PCL-FA-24h group has the highest degree of crystallization and the best hydrophilic properties.The FA-PCL composite scaffolds with different synthesis time have good biocompatibility and are superior to the PCL nanofiber membrane in inducing osteogenic differentiation of hPDLSCs,suggesting that FA coating can enhance the ossification-inducing potential of the material.PCL-FA-24h composite scaffolds can be preferably used for bone tissue engineering research.