| Objects:The cartilage defect repair is still a clinical problem.Bottom-up cartilage tissue engineering provides new hope.for the regeneration and repair of the cartilage defects.Scaffolds with the structure of natural extracellular matrix for tissue engineering can promote cell adhesion,proliferation,migration and tissue formation.Therefore,in this study,by means of microfluidics equipment and supercritical drying technology,we have successfully produced the uniform chitosan micropheres with nanofibrious network mimicking the cartilage extracellular matrix for the first time.Through bottom-up cartilage tissue engineering technique,we have engineered a cartilage-like composite in vitro with the chitosan micropheres,and the new tissue was evalutated histologically and mechanical properties were tested.Materials and methods:1.Assemble microfluidics equipment.2.Inject the chitosan solution and cyclohexane solution into the microfluidic device inside the pipe and keep flowing.Collect the chitosan emulsion by sodium hydroxide solution.Dry the chitosan microgels with supercritical carbon dioxide imitating the extracellular matrix.Observe the structure characteristics by scanning electron microscopy.3.Change the microfluidics device parameters to prepare chitosan microspheres with different size.4.Change the concentration of the chitosan solution or the sodium hydroxide solution to produce chitosan microspheres with different thickness of nano fiber.5.Isolate and culture chondrocytes from rabbit ear cartilage.Combine the chondrocytes with microspheres and culture with a rotating flask for 5 days,cell adhesion rate and cell proliferation rate were determined.Cytoskeleton staining was performed to evaluate the cell growth condition.The extracellular matrix of GAG content was measured.Implant the cell-microphere composite into a mold and culture for 2 weeks.Results:1.We successfully made the microfluidic device.2.By supercritical carbon dioxide drying method,we have produced chitosan microsphere with uniform nanofiber network structure for the first time.3.The size of the chitosan microspheres can be precisely controlled,Chitosan nanofiber diameter can also be changed by regulating the concentration of the chitosan solution.4.We have successfully cultured rabbit chondrocyte and identified it with immunohistochemistry Assay of aggrecan and collagen II expression.5.Chondrocytes cultured on the microspheres(150nm)have the highest adhesion rate,cell proliferation rate and the most gag production amount.Laser confocal microscope was used to observe the chondrocytes on the surface of the material.After 7 days,chondrocytes were covered the entire surface of the material and the materials can be connected with each other through the intercellular bridge.We have obtained a tissue-like composte with smooth surface and translucent appearance after putting the cellmicrophere into the mold by bottom-up tissue engineering.HE staining and Safranin-O staining showed there was the cartilage extracellular matrix precipitation in the space among the micropheres.SEM picture indicated that the nanofibrous struction of the composte was similar to that of the normal cartilage tissues of the porcine knee.Mechanical property test confirmed the tissue-like structure holds a good modulus of elasticity.Conclusions:1.We have successfully fabricated the chitosan microsphere with the structure of the extracellular matrix.The size and structure of the material can be precisely controlled.2.The increase of the concentration of chitosan solution and sodium hydroxide solution leads to the increase of the diameter of the nanofibers.3.Chitosan microspheres with extracellular matrix-mimicking nanofibrous structure could promote the adhesion,proliferation and extracellular matrix secretion of chondrocytes.4.By bottom-up cartilage tissue engineering,we have created a cartilage-like tissue after seeding chondrocytes on the chitosan microspheres with extracellular matrix mimicking structure. |
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