Résumé
Objective:To explore the possibility of constructing tissue-engineered cartilage three-dimensional nanoscaffolds with collagen Ⅱ (COLⅡ), hyaluronic acid (HA) and chondroitin sulfate (CS).Methods:The tissue-engineered cartilage scaffolds were prepared by electrospinning techniques with the mixture COLⅡ-HA-CS solvent, which dissolved by 3-trifluoroethanol-water. The surface topography was observed under electron microscope (SEM). And the diameter of nanofibers, the water absorption rate, contact angle and degradation rate were also detected. Generation 2 rabbit chondrocytes were seeded into the scaffold. The cell survival rate and proliferation were evaluated by Cell Counting Kit-8.Results:When the concentration range of electrospinning was 80-120 mg/ml and the mixing ratio of Col, HA and CS was 6-8∶1∶1-2, the tissue engineered cartilage nanoscaffolds could be successfully prepared. Their diameters were mainly distributed between 126.5±23.3 nm and 374.7±14.1 nm. The scaffolds had satisfactory hydrophilicity and degradability. The chondrocytes could well adhere and proliferate on the scaffold.Conclusions:The COLⅡ-HA-CS tissue-engineered cartilage nanoscaffolds have good physical and biological properties, which suggests its promising application in tissue-engineered cartilage.
Résumé
[Objective]To prepare a cartilage acellular matrix scaffold and to explore its feasibility in cartilage tissue engineering. [Methods]Microparticles about 100 ?m~154 ?m were prepared after calf cartilage physically shattered and experienced gradient centrifugation,and then treated by a modified Courtman's four-step method which was improved to produce acellular cartilage matrix.After this treatment the microparticles were made into 3% suspension which was placed into moulds.With the freeze-drying method,3-D cartilage acellular matrix (CACM) was prepared.The scaffolds were cross-linked by a neotype crosslinking agent genepin for 48h,and then placed into glycine solution server times for removing redundant genepin.The freeze-drying method was used to prepare CACM.The scaffolds were investigated by gross observation,histological staining (haematoxylin-eosin,toluidine blue) ,scanning electron microscope (SEM) observation and porosity measurement,water absorption rate and degradation rate analysis.After being cultivated for ten days,bone marrow stranal cells (BMSCs) of rabbit were seeded into the scaffold.MTT test and SEM were done to assess the growth and proliferation of BMSCs.[Results]Gross observation showed the scaffolds had a loosely porous and dark blue appearance after being cross-linked by genepin.The histological staining (haematoxylin-eosin,toluidine blue staining) showed that there were no chondrocyte fragments in the scaffold.The CACM scaffold had 90% porosity,(1314?337) % water absorption rate,and (13.69?7.3)% or (25.99?8.9) % degradation rate at 2 or 4 weeks.MTT test showed that BMSCs grew well in the 3-D CACM scaffolds of logarithmic trend,supporting that the scaffolds had no cytotoxic effect on BMSCs.SEM micrographs indicated that the scaffolds were porous and the cells covered the scaffolds firmly with cell processes.[Conclusion]The improved Courtman's four-step method makes a more thoroughly acellular scaffold.The 3-D CACM scaffold retains most of extracelluar matrix.After being cross-linked by genepin,the 3-D CACM scaffold has good biocompatibility and degradation rate of the scaffolds is decreased,which makes it a suitable carrier for cartilage tissue engineering.
Résumé
The purpose of this study was to examine the feasibility of using fibrin glue polymer designed to produce tissue-engineered cartilage by in vitro culture and to determine the optimal fibrinogen and chondrocyte concentrations required to generate cartilage and to evaluate the biomechanical properties of the generated cartilages. The most favorable fibrinogen and chondrocyte concentrations were determined by measuring the volume and weight gains, evaluating the histologic changes and measuring the biomechanical properties after construction of 12 different chondrocyte-fibrin glue constructs. The results are as follows. 1. The fibrin glues without chondrocytes degraded during 8 weeks of culture period. 2. The construct gains more weight in those with higher chondrocyte and fibrinogen concentration. 3. Histologic analysis of the generated cartilage showed nearly homogeneous cartilage when using 80 mg/cc fibrinogen and 4 x 10(7) chondrocyte/cc at 8 weeks in vitro culture. 4. The compressive modulus is higher in constructs with higher fibrinogen concentration and is 35.4 -36.8% of the normal joint cartilage. 5. The yield stress is higher in constructs with higher fibrinogen concentration and is 11% of the normal joint cartilage. This study demonstrates that fibrin glue is a suitable polymer for generation of cartilage by in vitro culture method. The construct with 80 mg/cc fibrinogen and 4 x 10(7) chondrocyte/cc yields the best quality of cartilage at 8 weeks when analyzed by weight and volume changes, histology and biomechanical properties. The compressive modulus and yield stress of the generated cartilage are lower than those of normal joint cartilage, and may still be adequate for the purpose of craniofacial reconstruction which seeks more pliant cartilage not requiring weight bearing strength.