| Background:Alveolar bone defects caused by tumors,injury,and periodontal diseases in the oral and maxillofacial region are relatively common,and alveolar bone defects are also a common phenomenon after tooth loss in patients,which often leads to functional impairments such as aesthetics,pronunciation,and mastication of patients,which seriously affects the patient’s physical and mental health.At present,the repair methods for alveolar bone defects include autologous bone transplantation and bone substitute material transplantation.Autologous bone transplantation has good osteo-conductivity and osteo-inductivity and is the "gold standard" for repairing bone defects,but there are certain drawbacks,such as open the second operating area,insufficient bone mass.Bone substitute materials lack osteo-inductivity and can cause immune rejection.The emergence of tissue engineering technology brings hope for the repair of oral and maxillofacial bone defects.However,as one of the three elements of tissue engineering,seed cells still face many problems in the application,such as poor survival rate after transplantation,low efficiency of directional differentiation,and difficult to control the direction of differentiation.Previous studies have used various growth factors or drugs to induce osteogenic differentiation of stem cells.However,a certain growth factor alone has limited ability to induce differentiation of stem cells,and may produce certain side effects,such as excessive osteogenesis and potential tumorigenic risk.More and more studies have shown that numbers of bioactive molecules contained in the conditioned medium（CM）of mesenchymal stem cells can stimulate cell proliferation,migration,and differentiation in the bone defect area.The CM can make up for many shortcomings of traditional single growth factors and drugs.However,insufficient effective concentrations of growth factors in the CM of mesenchymal stem cell also limit their clinical translation.Some scholars often optimize the concentration and types of growth factors secreted by stem cells through directional induction and inflammatory stimulation.Dental follicle stem cell（DFSC）is derived from ectodermal mesenchyme and is a newly studied dental stem cell.DFSC and its CM have unique advantages in tooth and periodontal tissue regeneration,nerve tissue regeneration,and immune regulation with broadly application foreground.However,DFSC has complex components and secreted various cytokines.How to regulate DFSC and optimize the directional function of its secreted bioactive factors is an urgent problem to be solved.Icariin（ICA）is an effective monomer of traditional Chinese medicine Epimedium.As a natural estrogen,ICA has been proven to promote bone formation and inhibit bone resorption.ICA is widely used in the field of bone tissue engineering and is often loaded into tissue engineering scaffolds as an osteo-inductive factor.Recent studies have shown that ICA can stimulate bone marrow mesenchymal stem cells secrete special cytokines via Wnt/β-catenin signaling pathway,MARK signaling pathway,Notch signaling pathway and other pathways.However,as an osteo-inductive factor,it is rarely reported whether ICA can optimize the cytokines of DFSC.Objective:To investigate the effect of icariin-induced human dental follicle stem cell conditioned medium on the proliferation,migration and osteogenic differentiation of mouse embryonic osteoblast precursor cells（MC3T3-E1）.To explore the feasibility of ICA-induced DFSC-CM as an inducing factor for directed osteogenic differentiation of stem cells.Methods:Dental follicle tissue was provided by 5 donors（aged 3 to 9 years）.Five patients underwent "extraction of supernumerary anterior maxillary teeth" in the Department of Oral and Maxillofacial Surgery of the Stomatological Hospital of Jilin University.During the operation,the operator removed the supernumerary teeth and scraped the dental follicle tissue,and transported it to the laboratory on ice.DFSCs were isolated and cultured by enzymatic digestion and tissue block methods,and the source of DFSCs was identified by immunocytochemistry and flow cytometry;the multi-directional differentiation ability of DFSCs was identified by osteogenic and lipogenic induction;DFSCs were treated with 0 μM,1 μM and 10 μM of ICA,respectively.After 24 h of serum deprivation,the culture supernatant was collected to prepare conditioned medium.The experiment was divided into 6 groups,namely blank group（complete medium containing 10% FBS,1% penicillin and streptomycin）,0ICA-CM group（DFSC-CM containing 0μM ICA）,and 1ICA-CM group（DFSC-CM containing 1 μM ICA）,10ICA-CM group（DFSC-CM with 10 μM ICA）,1ICA group（complete medium with 1 μM ICA）,10 ICA group（complete medium with 10 μM ICA）.CCK-8 method and crystal violet staining to detect the proliferation activity of MC3T3-E1;scratch test and migrated chamber test to detect the migration ability of MC3T3-E1;real-time quantitative polymerase chain reaction to detect the expression of osteogenic genes in MC3T3-E1.Results:1.DFSCs positively express mesenchymal-derived vimentin,and negatively expresses epithelial-derived cytokeratin;DFSCs surface markers positively express mesenchymal stem cell surface antigens CD90,CD105;negatively express hematopoietic stem cell surface antigens CD34,CD45;DFSCs can differentiate into osteogenesis and adipogenesis.2.In the proliferation experiment,compared with the blank group,the 0ICA-CM group and the 1ICA-CM group promoted the proliferation of MC3T3-E1 at the first day,and the 1ICA-CM group had a more significant promotion effect,with a statistical difference;on the third and 5th day,both the 0ICA-CM group and the 1ICA-CM group significantly promoted the proliferation of MC3T3-E1,but there was no statistical difference between them.3.In the migration experiment,the 0ICA-CM group and the 10ICA-CM group promoted the lateral migration of MC3T3-E1,with a statistical difference;and the1ICA-CM group significantly promoted the longitudinal migration of MC3T3-E1.4.In the qRT-PCR experiment,compared with the other groups,the 1ICA-CM group up-regulated the m RNA expression levels of Runx2,Ocn and Opn.Conclusion:DFSCs derived from supernumerary teeth have the characteristics of mesenchymal stem cell and may become candidate seed cells for clinical application of mesenchymal stem cells;ICA-induced DFSC-CM promotes the proliferation,migration and osteogenic differentiation of MC3T3-E1,and ICA-induced DFSC-CM can be used as an inducing factor for the directed osteogenic differentiation of stem cells,which is providing a basis for the osteogenic differentiation of other stem cells.ICA may serve as an osteo-inductive factor to optimize the secretion of bioactive factors from DFSCs and play a role in inducing osteogenesis. |
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