| Objective:To explore the repair effect of human umbilical cord mesenchymal stem cell exosome（Human umbilical cord mesenchymal stem cells exosomes,hucMSCs-exo）on femoral fracture in osteoporotic rats by cell experiment in vitro and animal experiment in vivo.Methods:Thirty-six 12-week-old female Sprague-Dawley（SD）rats weighing 200～250g were randomly divided into two groups:sham operation group（Sham group,n=6）,and ovariectomized group（Ovx group,n=30）.The model of osteoporosis（Osteoporosis,OP）was established by classical ovariectomy.12 weeks after operation,6 rats in Ovx group and Sham group were randomly selected for Micro-CT and HE staining,and the model was verified.After verification,bone marrow mesenchymal stem cells（Bone marrow mesenchymal stem cells,BMSCs）were extracted from osteoporotic rats by whole bone marrow adherent method,and identified by flow cytometry and osteogenic differentiation.In vitro cell experiment,BMSCs was cultured in the medium containing equal volume and different concentrations of hucMSCs-exo.CCK-8 solution was added at 0h,24h,48h and 72h,respectively.The absorbance of 450nm was measured by enzyme labeling instrument to observe the effect of hucMSCs-exo on the proliferation of BMSCs.The remaining 18 OP rats in Ovx group were used to establish the model of osteoporotic femoral fracture and were randomly divided into 3 groups（n=6）.Group A（PBS group）:100μL PBS buffer was injected into the fracture end;group B（low concentration Exo）:100μL was 1.5×1011 particles/mL hucMSCs-exo fracture end injection;group C（high concentration Exo group）:100μL concentration was 3.0×1011 particles/mL hucMSCs-exo fracture end injection.Four weeks after operation,the fracture healing was identified by X-ray,Micro-CT scan,HE staining,Masson staining and immunohistochemical staining to explore the effect of hucMSCs-exo on the repair of femoral fracture in osteoporotic rats.Results:1.12 weeks after ovariectomized osteoporosis model,Micro-CT three-dimensional scanning and bone tissue microstructure quantitative analysis showed that the bone mineral density（Bonemineraldensity,BMD）and trabecular thickness（Trabecularthickness,Tb.Th）in Ovx group were significantly lower than those in Sham group（P<0.001）.The number of trabeculae（Trabecularbonenumber,Tb.N）decreased and trabecular separation（Trabecularseparatingcapacity,Tb.Sp）increased significantly（P<0.001）.HE staining of distal femur showed that the thickness and number of trabeculae in Ovx group were lower than those in Sham group,the trabeculae were sparse,the structure was disordered,and there were a large number of lipid droplets between trabeculae.The above results confirm the successful construction of osteoporosis model.2.Under the microscope,the third generation BMSCs showed that the cells adhered to the wall,grew well,showed fusiform or polygonal shape,and gathered and grew in "vortex shape".Flow cytometry showed that the positive antigen was highly positive,CD29（97.6%）,CD90（99.47%）,negative antigen was highly negative,CD45（3.36%）.The results of osteogenic induction showed that the extracted cells could differentiate into osteoblasts and adipoblasts.The above results confirmed that the extracted cells were BMSCs.3.The results of CCK-8 experiment showed that the proliferation rate of BMSCs with hucMSCs-exo medium was significantly accelerated,and the exosomes promoted the proliferation of BMSCs in a concentration-dependent manner.4.Four weeks after operation,X-ray and Micro-CT threedimensional scan showed that the speed of fracture healing and callus formation in hucMSCs-exo group were faster than those in PBS group.X-ray score of fracture healing:the X-ray scores of PBS group,low concentration Exo group and high concentration Exo group were（0.83±0.40）,（1.67±0.51）and（3.17±0.40）,respectively.Histological analysis:HE staining showed that there were more callus formation in the exocrine group than in the PBS group,in which the callus area was the largest in the high concentration Exo group,a large number of bone trabeculae were formed in the exocrine group,and the trabecular arrangement tended to be regular.The results of Masson staining showed that compared with the PBS group,the collagen fibers in the exocrine group were denser and arranged more regularly,and there were more new bone tissues.Immunohistochemical staining showed that the positive area of CD31 in the exosomes group was larger than that in the PBS group,and the positive expression was the highest in the high concentration Exo group,indicating that there were more angiogenesis and vascular density in the exosomes group than in the PBS group,and the expression of osteocalcin in the exosomes group was significantly higher than that in the PBS group,indicating that there were more new bone tissues in the exosomes group.Conclusion:1.HucMSCs-exo can promote the proliferation of BMSCs in osteoporotic rats in a concentration-dependent manner.2.HucMSCs-exo promotes the repair of osteoporotic femoral fracture by increasing angiogenesis and osteogenesis in osteoporotic rat model of femoral fracture. |
