| BACKGROUND:The treatment of cartilage defects and cartilage-related cosmetology are important parts of plastic surgery.There are many problems in the traditional autotransplantation methods,such as limited source,donor site damage,different degrees of cartilage absorption and so on.The emergence of tissue engineering technology offered a new way.Cartilage tissue engineering technology is in the research stage and has not yet been widely carried out in clinical practice.Because there are still many technical problems to be solved,such as how to obtain a large number of seed cells with good cartilage phenotype in vitro.How to find scaffolds with simple fabrication and good biocompatibility.Microcarrier technology has been used to amplify adherent cells on a large scale for decades,and the three-dimensional culture environment is closer to the environment in vivo,which is more conducive to cell growth and function.With the continuous development of microcarrier materials,In addition to culturing cells,microcarriers can also be used as scaffolds for tissue engineering.Porous microsphere carriers made of synthetic polymer poly-L-lactic acid(PLLA)materials have insufficient affinity to cells.Studies have shown that silk fibroin(SF)can promote the growth of chondrocytes.PLLA porous microsphere was modified by SF to improve the properties and provide a new carrier and scaffold for chondrocyte culture and cartilage tissue engineering.OBJECTIVE:1.To clarify the effects of microcarrier three-dimensional dynamic culture on the proliferation and phenotype of chondrocytes,providing a technical reference for the establishment of large-scale amplification.2.To compare of the effect of poly-L-lactic acid(PLLA)porous microsphere,silk fibroin(SF)modified poly-L-lactic acid porous microsphere and commercial Cultispher G porous microsphere on the biological behavior of auricular chondrocytes in vitro.To find a suitable microsphere scaffold for the three-dimensional dynamic culture of chondrocytes.3.To construct injectable tissue-engineered cartilage with porous microspheres in nasal dorsum of rabbit and explore the effect of augmentation rhinoplasty with this method.METHODS:1.Auricular chondrocytes were isolated from swine ear by method of trypsin and collagenase digestion.The chondrocytes were divided into three groups to culture:monolayer culture in petri dish,three dimensional static culture with microcarrier,and three dimensional dynamic culture with microcarrier.Growth of chondrocytes on the microcarriers was observed by using scanning electron microscope(SEM),fluorescence inverted microscope and frozen section DAPI staining.Proliferation of chondrocytes was measured by DNA quantitative examination.Expression of cartilage relevant genes was measured by real-time PCR.2.Aricular chondrocytes were isolated from rabbit ear by method of trypsin and collagenase digestion.P1 chondrocytes were inoculated on PLLA,SF modified PLLA and Cultispher G porous microspheres,and cultured in a rotary cell culture system(RCCS).Growth of chondrocytes on the microcarriers was observed by using scanning electron microscope(SEM)and frozen section DAPI staining.Proliferation of chondrocytes was measured by DNA quantitative examination.Expression of cartilage relevant genes were measured by real-time PCR.Extracellular matrix secretion of chondrocytes were detected by histological examination.3.Auricular chondrocytes of rabbit were inoculated on SF modified PLLA porous microsphere and cultured in vitro for 1 week,then they were injected into the nasal dorsum of rabbits with hyaluronic acid and chondrocytes.The effect of augmentation rhinoplasty was observed and the formation of tissue engineering cartilage was detected by histological examination.Hyaluronic acid mixed with chondrocytes and simple hyaluronic acid were used as control.RESULTS:1.With monolayer culture in petri dish,growth of chondrocytes entered the platform stage at day4.In microcarrier dynamic culture group,the logarithmic growth phase of chondrocytes was prolonged as day2-10,and the number of chondrocytes reached the peak at day14.In microcarrier static culture group,the proliferation of the chondrocytes was slow.Chondrocytes lost phenotype in two-dimensional monolayer group and microcarrier static group for long-term culture in vitro,while three-dimensional dynamic culture with microcarriers can promote chondrocytes proliferation and maintain cartilage phenotype at the same time.2.Inoculation rates were 37.67%±2.33%,53.67%±3.69%and 73.67%±3.53%on PLLA、SF-PLLA and Cultispher G microsphere respectively.Cell proliferation of PLLA porous microspheres and SF modified PLLA porous microspheres were higher than that of Cultispher G porous microspheres.Real-time PCR showed that the maintenance of cartilage phenotype of SF modified PLLA porous microspheres was better than the other two groups.Histological examination showed that the secretion of glycosaminoglycan and collagen II was the highest in SF modified PLLA porous microspheres group.3.Porous microspheres+hyaluronic acid+chondrocytes group and hyaluronic acid+chondrocytes group could form cartilage-like tissue.Histological staining showed that there was no significant difference between the two groups,however the addition of microspheres could improve the hardness and elasticity of cartilage mass.The effect of augmentation rhinoplasty in porous microspheres+hyaluronic acid+cells group last longest,hyaluronic acid+cell group secondly,and simple hyaluronic acid shortest.CONCLUSION1.Three-dimensional dynamic culture of microcarriers combined with rotating cell culture system can maintain cartilage phenotype while expanding large numbers of chondrocytes,which can be used as a method for large-scale expansion of auricular chondrocytes.2.Compared with PLLA porous microspheres and Cultispher G porous microspheres,SF modified PLLA porous microspheres are more suitable for three-dimensional culture of chondrocytes in vitro.3.Addition of microspheres into hyaluronic acid can improve the mechanical strength of hydrogel scaffold,and the construction of injectable tissue-engineered cartilage with microsphere and hydrogel can be an alternative of augmentation rhinoplasty. |
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