Effects Of Mitochondrial Autophagy On The Differentiation Of Bmscs Into Cartilage Induced By Collagen-based Hydrogel

Objective: To explore mitophagy during the differentiation of bone marrow mesenchymal stem cells(BMSCs)into cartilage by simulating the microenvironment of cartilage development.To demonstrate the relationship between mitophagy and the aging of chondrocytes,to reflect the relationship between stem cell differentiation and mitophagy,and to evaluate the influence of mitophagy on stem cell differentiation.By inducing and inhibiting the regulation of mitophagy,we explore its effect on the differentiation of BMSCs.Finally,we constructe a three-dimensional system of hydrogel-based cells to further explore the role of mitophagy in the differentiation of BMSCs into cartilage by collagen-based hydrogels.Methods: In the first part,we stained mitochondria of chondrocytes from different rabbit ages,and then observed the changes of mitophagy during the differentiation of BMSCs into chondrocytes;finally,we detected the expression of mitochondrial autophagy gene BNIP3 L and cartilage gene Col2 A by PCR.In the second part,the experiment is divided into three groups: normal cartilage induction culture group,rapamycin induction group,and chloroquine inhibition group.First,we stained mitochondria,then the autophagy-related protein LC3 was detected by immunoblotting,and finally the expressions of BNIP3 L and Col2 A in the three groups were detected by immunofluorescence staining.In the third part,we modified collagen hydrogel with methacrylic acid dry,and measured the oxygen content inside the hydrogel with invasive oxygen measuring instrument,and observed the degree of autophagy by mitochondrial staining,we observed cell proliferation through cytoskeletal staining,cell death staining,and CCK8.At the same time,we observed the secretion of the cell matrix by section staining.Results: The mitochondrial staining results showed that the number of mitochondria in chondrocytes of young rabbits was more than that of old rabbits,and the cytoskeletal microfilament structure that provided power for autophagy was more obvious than that of old rabbits.During the chondrogenic differentiation of BMSCs,PCR found the expression of BNIP3 L and Col2 A in the cartilage induction group was significantly higher than the basic culture group.Mitochondrial autophagy staining found that mitochondrial autophagy was active in the autophagy inducer rapamycin group,the cytoskeletal microfilament structure gradually weakened,and the cell morphology gradually changed from long fusiform to oval in the autophagy inducer rapamycin group;the microfilament structure has not changed in autophagy inhibitor chloroquine group,and the cell morphology remained a long spindle.WB detection found that LC3 BII accumulated in the chloroquine inhibition group.Immunofluorescence showed that compared with the normal group and the chloroquine inhibition group,the expression levels of BNIP3 L and Col2 A increased in the autophagy inducer rapamycin group.Mitochondrial staining found that the mitochondrial state gradually split into dots in collagen-based hydrogel.HE staining found that the cell morphology gradually became elliptical or round in the later period of culture.TB staining showed obvious blue-purple meta-stained areas at 21 days.Cell activity FDA/PI staining found that the number of cells in the collagen hydrogel gradually increased with the number of days of culture,and cytoskeletal F-actin staining found that the spread of cells in the late stage of culture increased.Conclusion: Mitophagy is related to the development of chondrocytes,to some extent,mitophagy participates in the differentiation of BMSCs into chondrocytes;mitophagy may promote the differentiation of BMSCs into chondrocytes;the microenvironment of collagen hydrogels activates mitophagy to a certain extent,and influences the differentiation of BMSCs into chondrocytes through related factors such as hypoxia and mechanical stimulation.