| BackgroundPeriodontal diseases are mainly caused by plaque and progressively destroy periodontal soft and hard tissues.The development of periodontal diseases will eventually lead to serious consequences of tooth loosening and loss,which will not only affect the beauty of patients,but also damage their important functions,such as chewing,pronunciation,etc.The quality of life will be greatly reduced,and mental health will also be affected.Up to now,the main treatment methods for periodontal disease include scaling,periodontal surgery,etc.The main purpose is to slow down or terminate the progress of lesions,but it is difficult to achieve the regeneration of periodontal tissue.In order to achieve periodontal reconstruction,tissue engineering technique will become a promising treatment method.The seed cells for tissue engineering periodontal regeneration include periodontal ligament stem cells,bone marrow mesenchymal stem cells,adipose stem cells and other stem cells.However,experiments have shown that the differentiation potential of different types of stem cells is different,and they have the tendency to differentiate into the source tissue.Therefore,we believe that periodontal ligament stem cells may be the best choice for periodontal regeneration.In addition to seed cells,biological scaffolds are also an essential part of tissue engineering.Biological scaffolds can provide suitable growth environment for seed cells and guide the growth and development of cells.The materials which make up of biological scaffolds should have good biocompatibility,appropriate mechanical properties,and can be loaded with some cytokines conducive to cell proliferation and differentiation.The best biological scaffolds are those rich in related factors.Dentin matrix is a good scaffold material because of its wide source,good biocompatibility and rich in various growth factors and cytokines.Enamel matrix derivatives are derived from developing tooth tissues and are rich in various proteins related to tooth development,which can promote the recovery of periodontal injury and regeneration of periodontal tissues.In this study,enamel matrix derivatives were loaded on dentin matrix scaffolds to modify them.The biological effects of enamel matrix derivatives modified treated dentin matrix on human periodontal ligament stem cells were studied,including cell proliferation,adhesion,osteoblastogenesis and cementogenic differentiation.At the same time,the related mechanisms were preliminarily explored.Method 1.Isolation,culture and identification of human periodontal ligament stem cellsHuman periodontal ligament stem cells were isolated and cultured.CCK-8 was used to determine the growth curve,clone formation rate.Flow cytometry was used to detect cell surface markers.ALP,alizarin red,oil red O staining was used to detect the osteogenic and adipogenic ability of cells.2.Preparation of enamel matrix derivatives modified treated dentin matrixTreated dentin matrix scaffolds were prepared.Enamel matrix derivatives were loaded on the scaffolds at different concentrations,and their morphologies were observed under scanning electron microscopy.3.Biological effects of enamel matrix derivatives modified treated dentin matrix on human periodontal ligament stem cells.CCK-8 was used to determine the growth curve of human periodontal ligament stem cells cultured on treated dentin matrix modified by enamel matrix derivatives at different concentrations,and DAPI and Phalloidin staining were used to detect cell adhesion and extension.4.Effects of enamel matrix derivatives modified treated dentin matrix on cementogenic and osteogenic differentiation of human periodontal ligament stem cellsReal-time fluorescence quantitative PCR was used to detect the expression of osteogenic genes and cementogenic genes in human periodontal ligament stem cells on composite scaffolds,and the expression level of cementoblast-related proteins was detected by wester-blot,Wnt pathway was blocked by inhibitor DKK1,and relative expression of Wnt pathway-related genes was detected by qPCR.Result 1.The surface markers of mesenchymal stem cells were detected by cultured human periodontal ligament stem cells.Mineralized nodules were observed by ALP and alizarin red staining,and lipid droplets were observed by oil red O staining.2.The surface of the treated dentin matrix scaffolds is smooth and the dentinal tubules are fully exposed.The enamel matrix derivatives are laid on the surface of the dental scaffolds in spheres of different sizes,and some of them enter the dentinal tubules.3.Compared with the control group,human periodontal ligament stem cells cultured on modified treated dentin matrix scaffolds proliferated faster,adhered faster and extended more rapidly at the same time point.4.Compared with the control group,the expression of osteogenic and cementogenic genes and the expression of cementogenic related proteins in the modified treated dentin matrix scaffold group were higher than those in the control group and the DKK1 group.The expression of Wnt pathway related genes in the modified treated dentin matrix scaffold group was also higher than that in the control group and the DKK1 group.Conclusion 1.The cultured human periodontal ligament stem cells have multidirectional differentiation potential,which conforms to the identification of mesenchymal stem cells.2.Dentin scaffolds modified by enamel matrix derivatives were successfully prepared.3.Dentin scaffolds modified by enamel matrix derivatives can promote the proliferation,adhesion,osteogenic and cementogenic differentiation of human periodontal ligament stem cells,and the mechanism may be related to the Wnt pathway. |
