Application Of Chondrocyte-laden Injectable Microspheres In Cartilage Tissue Engineering

ObjectiveHerein,a method of microfluidics-assisted technology producing the hydrogel microspheres that capable of lading chondrocyte was ultimately generate biobrick,is presented.To explore the application of biobrick in cartilage tissue engineering.MethodsThe Chitosan(CS)and poly(ethylene glycol)diacrylate(PEGDA)microspheres(CP-MSs)based on the biomimetic material were synthesized by fabricated microfluidic flow-focusing device.The fluorescence morphology of CP-MSs stained with Fluorescein isothiocyanate(FITC)shows microspheres of different sizes in different flow rate and flow rate ratio via fluorescence microscope.In addition,the molecular structures and rheological characteristics of microspheres were tested by Fourier Transform Infrared Spectrometer(FTIR)and rheological experiment,respectively.The surface structure of microsphere was investigated and measured by scanning electron microscopy(SEM).Primary cartilage chondrocytes of this experiment were harvested from the knee-joint of one-week-old New Zealand white rabbits,and chondrocytes within 4 generations were chosen.The chondrocytes cultured in microspheres of 550μm and 220μm diameter,and then using Live/Dead staining after 1,3 and 7 days.Futhermore,both of these chondrocyte-laden microspheres also using phalloidin/DAPI staining after 3 days.After injection the biobrick,the viability and self-assembly were evaluated via Live/dead staining after 1,3 and 7 days.ResultsIn the microfluidic flow-focusing device,the hydrogel microspheres with sizes ranges from 550±9μm to 121±23 μm can be produced.FITR spectra showed that the obvious C=O stretching vibration at 1734 cm-1 for the PEGDA hydrogel.In addition,the rheological experiment indicated that the loss moduli(G")was apparently lower than the storage moduli in the 0.1-10 rad/s of experiment of frequency range.The SEM results showed the surface coarse fiber structure of microspheres,and the diameter of fiber was 397±146nm.The Live/dead staining showed the chondrocytes maintain good vitality in microspheres of 550μm and 220μm diameter with the development of culture periods,and the cell viability of>80%were kept for 7 days.The phalloidin/DAPI staining demonstrating that the chondrocytes favourable attached to the surface of the microsphere after 3 days.After injection the chondrocyte-laden microspheres of 220μm diameter,the Live/dead staining showed maintain good cell morphology and the cell viability of>80%were kept after 1,3 and 7 days.ConclusionInjectable size-controllable CP-MSs were rapidly and conveniently produced through the technique of microfluidic.Different flow rates ratio have more influence than different flow rates on the diameter distribution of the microspheres.FITR spectra confirmed that the PEGDA hydrogel has been almost entirely crosslinked.In addition,the rheological experiment indicated that the CS-PEGDA hydrogels have the rheological characteristics of the well-developed crosslinking.The surface nanofibrous structure of microspheres may obtain better adhesion ability of microspheres for chondrocytes from the SEM results.The controllability of microspheres diameter will make CP-MSs to become a cell-laden applications possesed good biocompatibility.The chondrocyte-laden microspheres as biobrick without declining the cell viability and bottom-up self-assemble into 3D macroporous scaffold through cell-cell interconnectivity after injection via Live/dead staining.