| Three-dimensional(3D)culture of cells is a method that enables cells to grow in threedimensional space,3D culture conditions provide a superior biologically structure,enabling cells to maintain good growth,the living environment of cells is closer to that in vivo,comparing to the 2D culture system.Adipose Tissue-derived Mesenchymal Stem Cells(ADMSCs)are favorable for various research and applications because of their wide source,convenient sampling,minimal damage and discomfort to donors,less immunogenicity,and possess basic stem cell properties and functions.MSCs exert therapeutic effects mainly through paracrine mechanisms and immunomodulation in vivo,while 3D culture can increase the multiple growth factor secretion amounts of MSCs,therefore,it might be possible to achieve better therapeutic effects with 3D cultured ADMSCs.Animal alopecia can be caused by a variety of causes,among which,alopecia faced by cancer animals after chemotherapy has gradually become the focus of researchers.Clinical data showed that MSCs could also help to reduce the tissue damage caused by chemotherapy drugs,mainly by establishing a protective microenvironment for functional cells.It will be one of the methods to improve animal welfare and increase animal production by MSCs transplantation.In this study,ADMSCs were cultured in 3D using microspheres constructed from bovine acellular dermal matrix(BADM),to detect the expression of stemness genes and the secretion of growth factor related to stemness.And we also evaluted the therapeutic effects of ADMSCs in 3D BADM microspheres comparing to the 2D culture system in vivo animal models.As for animal experiments,wild type male mice aged 6-8 weeks were selected to establish a alopecia model by injecting cyclophosphamide after inducing anagen phase of hair.After detecting the adhesion factors in serum that are associated with apoptosis of hair follicle cells and hair follicle degeneration,the establishment of CIA model was determined.Subsequently,the 2D and 3D cultured ADMSCs were subcutaneously injected for transplantation treatment respectively,and the model group and3 D control group were injected with PBS and 3D scaffold for treatment respectively.By comparing the number of hair follicles and the number of proliferating cells in hair follicles,and the content of adhesion factors in the serum of each group after treatment,the therapeutic efficacy of the 2D and 3D cultured ADMSCs was evaluated.The specific results were as follows:1.ADMSCs in 3D culture have higher stemness and growth factor secretion.The expression of stemness related genes of ADMSCs in 2D and 3D group was detected at m RNA level,as well as the secretion of related growth factor,the expression of stemness genes Sox2 and Oct4 of ADMSCs were increased,and the expression of Nanog also showed a trend of increasing in 3D culture.The secretion of TSG-6 and EGF of AMDSCs increased significantly in 3D culture compared with that of 2D culture,and the secretion of TGF-β also increased,suggesting 3D cultured ADMSCs had higher stemness and growth factor secretion than 2D culture.2.ADMSCs have better transplantation therapeutic effect under 3D culture.Mice were collectively divided into control,model,2D treated,3D control and 3D treated groups.By HE histological staining and ELISA,the results showed that the control group had the highest number of hair follicles and the distribution of hair follicles was closer to the panniculus carnosus,the contents of ICAM-1 and ELAM-1 in this group were at normal levels.The model control group and 3D control group had the lowest number of hair follicles,also the content of serum adhesion factors was the highest,and there was no significant difference between the two groups,which proved that 3D microspheres had no effect on hair regeneration;The 3D treatment group had the second highest number of hair follicles and the hair follicle distribution was closer to that of the control group,and the content of serum adhesion factors was the lowest in all the model group The number of hair follicles in 2D treatment group was significantly lower than that in the 3D treatment groups,and almost no hair follicles were distributed near the panniculus carnosus,The content of serum adhesion factors in this group was lower than that in the 3D control group,but higher than that in the 3D treatment group.The results of Ki67 immunohistochemical staining also showed that there was no cell proliferation in the hair follicles of the model and 3D control group,while a large number of proliferating cells could be detected in the control group,2D and 3D treatment groups.It was proved that 3D microspheres had no therapeutic effect,and the ability of 3D cultured ADMSCs to promote hair regeneration was better than that of 2D cultured ADMSCs.Comprehensive analysis of experimental results,3D cultured ADMSCs had higher stemness as well as higher cytokine secretion amounts.After 3D culture,ADMSCs can play a significant transplantation effect in clinical treatment,which can increase the number of hair follicles in the skin and increase the proliferation of cells in hair follicles,,providing a theoretical basis for the clinical application of ADMSCs in hair follicles regeneration. |
PDF Full Text Request