| Objective To evaluate the effect of mesenchymal stem cell-derived exosomes combined with collagen peptides in the treatment of osteoarthritis,and explore the possible related mechanisms.Methods1.Isolation and identification of mesenchymal stem cells and exosomes.Mesenchymal stem cells were isolated by tissue adherence method,and cell morphology was observed by inverted microscope;surface markers CD34,CD45,CD73,CD90 and CD105 were detected by flow cytometry.The exosomes were extracted by differential centrifugation,and the morphology was observed by transmission electron microscope;the surface markers CD9,CD63 and CD81 were detected by Western Blot;the particle size and number were detected by nanoparticle tracking analyzer.2.Efficacy observation of mesenchymal stem cell-derived exosomes combined with collagen peptides on osteoarthritis.A total of 120 3-month-old BALB/c male mice were selected,with an average weight of 29.4g,and were raised in an SPF environment.（1）Design a low-temperature modeling device for rat osteoarthritis,including a temperature-controlled box and a rat fixator.Twenty mice were randomly divided into a normal control group and a low temperature treatment group,10 mice in each group.The normal control group had normal diet and drinking water.On the 31st day,the hind limbs of the mice were grossly observed;the degree of joint swelling was measured;the contents of IL-1β,TNF-α,HA,and Ig M in serum were detected by ELISA;the HE staining sections of the intact ankle joint were used to evaluate the modeling effect.（2）100 mice were randomly divided into normal control group,model group,cell low,medium and high dose groups,exosome low,medium and high dose groups,collagen peptide group,exosome combined with collagen peptide group,10 per group.The normal control group was given normal diet and water,while the other groups were given osteoarthritis modeling.On the 31st day,1×10~4,1×10~5,and 1×10~6 cells were injected in each dose group of cells,6μg,12μg,and 18μg of exosomes were injected in each dose group of exosomes,and the collagen peptide group was injected with 6.06 mg/Kg/d The dose of d was intragastrically administered.In the exosome combined with collagen peptide group,12μg of exosomes and collagen peptide were administered by intragastric administration at the same dose as above,and the model group was injected with PBS.Cells,exosomes,and PBS were injected every other day.From the day of injection,the degree of joint swelling was measured on 0d,10d,and30d.After 30d,the hind limbs of the mice were grossly observed;the contents of IL-1β,TNF-α,and HA in serum were detected by ELISA;Type II collagen immunohistochemical staining sections;OARSI histological scores were used to evaluate the efficacy.Nutritional status and body weight changes;automatic biochemical analyzer detects serum ALT,AST,GGT,TBIL,ALP,BUN,CR,UA,Cys C,CK,CK-MB,HBDH,LDH-1 content;heart,liver,Spleen,lung,kidney,testis,pancreas,intestine,stomach,brain HE stained sections were used for safety evaluation.3.Mechanism of mesenchymal stem cell-derived exosomes combined with collagen peptides on osteoarthritis.（1）Three 46-week-old BALB/c male mice were selected,with an average weight of 29.3g,and were raised in an SPF environment.Ankle chondrocytes were isolated by improved two-step enzymatic digestion method in laboratory,cultured in vitro,and cell morphology was observed by inverted microscope;immunofluorescence staining of toluidine blue and type II collagen;primary adherent cell culture was determined by dimethylmethylene blue chromogenic method The content of sulfated glycosaminoglycan in liquid and digestive juice;chondrocyte proliferation detection for chondrocyte identification.（2）The above methods were used to extract ankle chondrocytes in the normal control group,model group,high-dose cell group,high-dose exosome group,collagen peptide group,and exosome combined with collagen peptide group on the 31st day after injection.miRNA high-throughput Sequencing,screening to identify differentially expressed miRNAs,GO functional annotation and KEGG pathway enrichment analysis.Results1.Isolation and identification of mesenchymal stem cells and exosomes.Under the inverted microscope,the cells grew in spindle and swirling shapes;the mesenchymal stem cells positively expressed CD73,CD90,and CD105,but did not express CD34 and CD45,as measured by flow cytometry.Disk-shaped and cup-shaped vesicles were seen under transmission electron microscope;Western Blot showed positive expression of CD9,CD63 and CD81;particle size measured by nanoparticle tracking analyzer was between 80-200nm,and the number of particles was 1.8×10~9/m L.2.Effects of mesenchymal stem cell-derived exosomes combined with collagen peptides on osteoarthritis.（1）The results of mouse osteoarthritis modeling.The ankle joint in the low temperature treatment group had obvious swelling,the cartilage surface was damaged and degenerated,the chondrocytes were proliferated and degenerated,and the synovial tissue was degenerated and adhered,and the ankle joint swelling was higher than that in the normal control group（P<0.001）,the contents of TNF-α,IL-1βand HA in serum were higher than those in the normal control group（P<0.05）.（2）Effects of mesenchymal stem cells,exosomes,exosomes combined with collagen peptides and collagen peptides on osteoarthritis.Ankle joint hypertrophy and severe deformity in the model group,low dose of cells,and slight rollover of the joint in the collagen peptide group,Accompanied by obvious swelling,the high dose of cell neutralization,exosome combined with collagen peptide,and high dose of exosome had no deformation and slight swelling,while the low and medium dose of exosome group had no obvious deformation and slight swelling of ankle joint;The degree of joint swelling at 0d,10d,and 30d showed that mesenchymal stem cells,exosomes,collagen peptides,and exosomes combined with collagen peptides could alleviate joint swelling in mice with osteoarthritis;Mesenchymal stem cells,exosomes,collagen peptides,and exosomes combined with collagen peptides can reduce serum IL-1βand TNF-αlevels,increase HA content（P<0.05）,and decrease IL-1βlevels.The effect of exosomes combined with collagen peptides was greater than that of low-dose exosomes（P<0.05）;in the model group,the cartilage surface was severely eroded and defected,chondrocytes were greatly reduced and degenerated,and proteoglycan and type II collagen were negatively expressed at the point where the cartilage tissue was completely eroded.In the collagen peptide group,the cartilage surface was damaged,and proteoglycan and type II collagen were positively expressed in some cartilage tissues.The cartilage surface of the high-dose cell group was relatively flat,and some proteoglycans and type II collagen were positively expressed.The cartilage surface of exosome combined with collagen peptide and high-dose exosome group was complete and smooth,and proteoglycan and type II collagen showed uniform positive expression;histological scores showed that the scores of each intervention group were lower than those of the model group（P<0.05）;The safety evaluation results showed that,compared with the normal control group,the nutritional status of each intervention group was good,the difference in body weight was not statistically significant,and there was no obvious pathological change in the HE staining of organs.3.Mechanism of mesenchymal stem cell-derived exosomes combined with collagen peptides on osteoarthritis.（1）Microscopically,the cells showed spindle-shaped and paving-stone-like growth;cells stained with toluidine blue showed blue-purple easy-staining particles,and immunofluorescence staining of type II collagen showed positive expression of red fluorescence;sulfated glycosaminoglycan in the primary cell culture medium and digestion solution The contents were（42.41±15.00）and（65.63±10.00）mg/L respectively;the viability of cells in the first passage was greater than that in the second passage and the primary passage.（2）Six groups of chondrocyte miRNA high-throughput sequencing detected a total of 1122 known miRNAs and 435 new predicted miRNAs.The results of differentially expressed miRNA screening showed that the difference in the up-regulated folds of the normal control group vs collagen peptide group,normal control group vs exosome combined with collagen peptide group,normal control group vs exosome high dose group was the largest for novel-miR-223,novel-miR-127,model group vs collagen peptide group,model group vs exosome combined with collagen peptide group,model group vs exosome high-dose group with the largest up-regulated folds of novel-miR-187,novel-miR-409.The GO annotation results showed that the biological processes of differentially expressed miRNA predicted target genes in each group were mainly concentrated in detoxification,rhythmic process,growth and behavior,the cell composition was mainly concentrated in synapses,synaptic parts and supramolecular complexes,and the molecular functions were mainly concentrated in antioxidant activity,electron carrier activity and structural molecular activity.KEGG pathway enrichment showed that highly significant pathways were mainly concentrated in axon guidance,endocytosis,and lysosomes.Conclusions1.Mesenchymal stem cells and exosomes were successfully isolated and extracted.2.Mesenchymal stem cell exosomes combined with collagen peptides and high-dose exosomes can effectively inhibit synovial inflammation and cartilage tissue degeneration in osteoarthritis mice,and protect and repair the biological function of chondrocytes.3.Mesenchymal stem cell-derived exosomes combined with collagen peptides and high dose exosomes are likely to slow down osteoarthritis in mice by affecting the expressions of novel-miR-223,novel-miR-127,novel-miR-187,and novel-miR-409in the lesions of arthritis,the functions of target genes predicted by differentially expressed miRNAs are likely to involve the growth of joint tissue cells,circadian rhythms,and oxidative stress,and the pathways involved are likely to be related to axon guidance and endocytosis. |
PDF Full Text Request