| Objective:Full-thickness cartilage defect is a common refractory disease in orthopedics.In this study,a new type of silk fibroin-chitosan combined with porous tantalum composite scaffold was designed and carried with bone marrow stromal stem cell exosomes to evaluate its effect on repairing full-thickness cartilage defects.Method:(1)Extract human bone marrow stromal cells for culture,identification and passage,extract exocrine body and identify exocrine body by transmission electron microscopy,nanoparticle size analysis and protein blotting;(2)The silk fibroin-chitosan scaffold was prepared and its performance was evaluated;Porous tantalum was prepared by 3D printing technology and integrated with silk fibroin-chitosan scaffold to prepare composite scaffold;(3)Extract rat chondrocytes for culture and identification;(4)The secretions,fibroin-chitosan scaffolds and chondrocytes were co-cultured,and the proliferative ability of chondrocytes was detected by CCK-8 method after 1,4,7 and 10days,respectively,and the data were collected and statistically analyzed;(5)Thirty-six adult male SPF rats were randomly divided into four groups according to different surgical methods:Group I:pure full-thickness cartilage defect(n=12);Group II:full-thickness cartilage defect+composite scaffold implantation(n=12);GroupⅢ:full-thickness cartilage defect+composite scaffold with exocrine implant(n=12).Six rats in each group were killed at the time point of the 3rd and 6th week after operation,and the samples of the implanted area were taken out for gross observation and micro-CT scanning to observe the repair of full-thickness cartilage defects.Result:(1)The purified and cultured bone marrow stromal stem cells in rat bone marrow were uniformly distributed in the shape of long spindle or spindle under the light microscope.The flow cytometry identified that CD44 and CD90 were highly expressed,and the hematopoietic cell surface marker antigens CD45 and CD105 were low expressed.The transmission electron microscope showed that the exocrine body was a complete double-layer capsule structure with a uniform distribution and a saucer-like shape,which was the typical morphology of exocrine body under the electron microscope;The concentration of exocrine body measured by nanoparticle diameter analysis was 1.63×10~8/m L,with an average particle size of 174.2 nm,the results of protein blotting for the detection of the surface marker proteins CD9,HSP70 and TSG101 on the secretion were all positive,indicating that the purity and concentration of the obtained secretion were very high,and the structure was complete;(2)Chondrocytes from the third generation showed irregular polygonal shape under light microscope,and the cells were in good condition.Toluidine blue staining showed that the cell morphology was complete,and immunofluorescence showed that the cells were highly expressed for COLⅡ;(3)Silk fibroin-chitosan scaffold is non-toxic to chondrocytes,can support cell adhesion and growth,and the effect of cell proliferation is better after adding exocrine body;(4)Postoperative gross observation and micro-CT image analysis showed that silk fibroin-chitosan and porous tantalum combined with bone marrow stromal cell secretions could repair the full-thickness cartilage defect to a certain extent.Conclusion:Silk fibroin-chitosan scaffold has good biocompatibility and supports the adhesion and growth of chondrocytes and the formation of cartilage tissue.The exocrine body shows good biological activity,which is conducive to the regeneration of bone and cartilage.In the experiment of repairing full-thickness cartilage defects in rats by implanting silk fibroin-chitosan and porous tantalum composite scaffold with the secretion of bone marrow stromal cells,the composite scaffold was well integrated with the osteochondral tissue in the implanted area,promoting the repair and regeneration of bone and cartilage. |
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