| The repair of craniofacial cartilage defects has always been a great challenge in plastic surgery.At present,the mainstream repair method is autologous cartilage transplantation,which can avoid immune rejection and provide stable repair results.However,in the case of large cartilage defects with complex shapes,the application of this method will be restricted by the limited donor tissue,the severe damage in the donor site,and the difficulty of surgery.The development of three-dimensional(3D)bioprinting provides a new solution for the repair of large cartilage defects with complex shapes.In cartilage bioprinting,the most commonly used bioinks are a mixture of expanded chondrocytes and biocompatible hydrogels.However,the dispersion of cells and the dense texture of the hydrogel in the conventional bioinks may limit the cell-cell/cell-extracellular matrix(ECM)contacts and interactions,counting against cartilage regeneration and maturation.To address this issue,in this study,we developed a novel bioink for cartilage bioprinting based on chondrocyte spheroids(CSs)and microporous hydrogels.Among them,CSs are multicellular aggregates formed by the self-assembly of chondrocytes,which can provide extensive cellcell and cell-ECM contacts and interactions,mimicking the natural cartilage microenvironment;microporous hydrogels are made by introducing the polyethylene oxide(PEO)as a porogen into gelatin methacrylate(GelMA),which can provide space and channels for the growth and fusion of CSs.Objectives1.Determine the influence of CSs preparation parameters(cell number and culture time)on CSs,and evaluate the property of GelMA+PEO microporous hydrogel and its feasibility for 3D bioprinting.2.Investigate the feasibility and effectiveness of CSs-laden bioink in the bioprinting of engineered cartilage with specific shapes.Methods1.CSs with different cell numbers(200,500,1000 cells/CS)and culture times(1,7,14 days)were prepared by the non-adherent microwell array chip method,and subjected to morphological,cell viability,histological and biochemical assays.10%GelMA+1%PEO microporous hydrogels were prepared,and 10%GelMA was used as a control to compare the pore structure,rheological properties,and mechanical properties of the two groups.Then,all groups of CSs were encapsulated into the microporous hydrogel,and their growth in the hydrogels was compared.2.3D bioprinting was performed using CSs-laden microporous hydrogels,with cellladen microporous hydrogels as a control.The morphology and cell viability of the printed CSs were assessed in vitro.Then,the two groups of printed constructs were implanted into nude mice,and taken after 4 and 12 weeks in vivo for gross observation,histological staining,quantitative biochemical assays,and related gene expression analysis.Results1.The size of CSs increased with increasing cell number and culture time.The formation of 200 cells/CS was unstable,with many dispersed cells and debris around the CSs.With the extension of culture time,the number of living cells in CSs decreased slightly and the glycosaminoglycan(GAG)deposition increased.The introduction of PEO can produce a large number of highly connected micropores in GelMA without adversely affecting the rheological properties,mechanical properties,and biocompatibility of GelMA.When cultured in the microporous hydrogel,the CSs with lower cell numbers and shorter culture times had higher area and DNA content increase folds.2.The CSs-laden microporous bioink can be smoothly extruded and printed into the predefined lattice-shape construct.After printing,the CSs were intact and the cells survived well.Compared to the cell-laden constructs,the regenerated cartilage tissue in the CSsladen constructs was more mature,with typical cartilage lacuna structure and abundant cartilage-specific ECM deposition,and the chondrocyte function and phenotype were better,but the cell proliferation was slower.Conclusion1.The non-adherent microwell array chip method allows for high-throughput preparation of uniform CSs with controlled cell number and culture time.CSs with lower cell numbers and shorter culture time had better proliferation and growth potential in the hydrogel and were more suitable for cartilage bioprinting.GelMA+PEO microporous hydrogels have good microporous structure,rheological properties,mechanical properties,and biocompatibility,and can be used for extrusion 3D bioprinting.2.The CSs-laden microporous bioink has excellent printability and cartilage regeneration ability,which can rapidly regenerate mature cartilage tissue similar to natural cartilage in vivo.The 3D microenvironment within CSs is conducive to the maintenance of chondrocyte function and phenotype,but the high density of cells and ECM also impact cell proliferation which needs to be optimized in future studies. |
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