Study On Osteogenic Properties Of Human Periodontal Ligament And Human Dental Pulp-derived Cells Composite Porous HA Scaffold

As the most easily obtained seed cells in human body,the research and application of human dental-derived cells in the field of oral tissue engineering is becoming more and more extensive.Researchers found that both human periodontal ligament cells and human dental pulp cells have strong osteogenic differentiation potential and similar biological characteristics.The study of the difference between the two is not clear,so this study will combine the porous hydroxyapatite scaffold to study the characteristics of osteogenic differentiation of stem cells from two sources in vitro and in vivo,providing a reference for the selection of stem cells in the field of oral regenerative medicine.Objective: Cells of two sources were combined with porous hydroxyapatite(HA)scaffolds for osteogenic induction to study the gene expression characteristics of the cells of the two sources during different stages of osteogenic differentiation.Analysis of the differences in the osteogenic differentiation of the two types of cells provides a reference for the selection of seed cells.Methods: Extract and culture human periodontal ligament cells and human dental pulp cells to verify their osteogenic and adipogenic differentiation capabilities.In vitro studies:(1)CCK-8 was used to detect the proliferation of cells from two sources on the scaffold.(2)Two kinds of cells were compounded on the scaffold,and the ALP activity on the 7th and 14 th days was detected after osteoinduction in vitro.(3)Cell composite scaffold culture,using q-PCR to detect the expression of osteogenesis-related genes on the 4th,7th and 14 th days.In vivo study: Human periodontal ligament cells and human dental pulp cells were co-cultured with HA scaffolds for 24 h.Then,the porous HA scaffold carrying human periodontal ligament cells,the porous HA scaffold carrying human dental pulp cells,and the porous HA scaffold not carrying cells were transplanted under the back skin of 4-week old nude mice.After 8 weeks of feeding,the nude mice were sacrificed and the scaffold material was removed.(1)Staining of slices to observe the growth and differentiation of cells on the scaffold.(2)Analyze the contents of related proteins ALP,OCN and VEGF in implants by WB.Results: Human periodontal ligament cells and human dental pulp cells are similar in shape and have a long fusiform shape,and are capable of osteogenic and adipogenic differentiation.In vitro study of composite porous HA scaffold: The proliferation rate of human dental pulp cells is higher than that of cells derived from human periodontal ligament(P <0.05).Human dental pulp cells make up ALP activity during the bone differentiation process is higher than that of human periodontal ligament cells(P <0.05).The expression of ALP,BMP2 and OCN genes in human dental pulp cell group was higher than that in human periodontal ligament cell group(P <0.05).There was no significant difference in the expression of COL-1 and Runx2 genes between the two.In vivo studies: Hyperplasia of fibroblasts,fibroblasts or adipocytes was observed in subcutaneous implants of human periodontal ligament-derived cell group and human dental pulp-derived cell group,with a small amount of inflammatory cell infiltration.The expression of ALP in the subcutaneous implants of the human dental pulp cell group was higher than that of the human periodontal ligament cell group(P <0.05).No significant differences were observed in the expression of OCN and VEGF in the subcutaneous implants of human dental pulp cell group(P> 0.05).Conclusion: Both human periodontal ligament cells and human dental pulp cells have multi-directional differentiation ability.When these two types of cells are co-cultured with porous HA scaffolds,human dental pulp cells have higher cell proliferation ability and osteogenic differentiation ability.In vivo transplantation experiments,the addition of these two cells can enhance the osteoinductive ability of the porous HA scaffold.From the WB results,the porous HA scaffold group carrying human dental pulp cells had better osteogenic efficiency than the porous HA scaffold group carrying human periodontal ligament cells.