| BACKGROUNDEar reconstruction for microtia is one of the clinical challenges faced by plastic surgeons.The regeneration and repair ability of cartilage tissue is poor.The use of autologous rib cartilage or artificial materials to prepare auricular scaffolds have many problems such as donor complications,limited access to cells,infection and immune rejection.3D bioprinting is a newly emerged additive manufacturing technology in the development of tissue engineering,which can apply cells and biomaterials to build biologically active three-dimensional tissue structures with computer assistance,and it is a new hope for ear reconstruction.The viability of cells,proliferation and differentiation ability in the printed structures and the efficiency of cartilage tissue formation are key concerns in studies on the construction of tissue-engineered cartilage based on 3D bioprinting technology.Adipose-derived stem cells(ADSCs)are considered as excellent seed cells for cartilage tissue engineering because of their multidirectional differentiation ability,easy access,rapid in vitro expansion and low immunogenicity.At present,there are few studies using human ADSCs for 3D bioprinting to construct tissue-engineered cartilage,and there is a lack of comparative studies on the different application strategies;therefore,there is still a lack of strong evidence to support their applicability for 3D bioprinting to prepare regenerative cartilage on key issues such as biological activity and tissue formation efficiency.OBJECTIVE1.Preparation of "bio-ink" from human adipose-derived stem cells compounded with photocrosslinkable hydrogel to produce biologically active three-dimensional tissue structures in vitro by 3D bioprinting technology.2.To compare the advantages and shortcomings of three different seed cell application strategies using human adipose-derived stem cells(1)hADSCs(2)hADSCs co-cultured with chondrocytes(3)predifferentiated hADSCs,and to select a more suitable solution for the application of 3D bioprinting for cartilage tissue engineering.3.The 3D bioprinted cell-hydrogel composite scaffolds were transplanted into subcutaneous of nude mice to observe their efficiency in forming cartilage tissue and to investigate the feasibility of this method for cartilage tissue engineering.METHODS1.Adipose tissue voluntarily donated by liposuction patients was collected to isolate and culture adipose-derived stem cells.Comparing the chongdrogenetic ability of hADSCs cultured by monolayer,hanging-drop and microcarrier method.2.Experimental group 1(hADSCs),experimental group 2(hADSCs and co-cultured with chondrocytes),experimental group 3(predifferentiated hADSCs)and control group(chondrocytes)were established.Take methacrylate-based gelatin(Gelatin methacryloyl,GelMA)and cells to make "bioink",and the cell-GelMA composite scaffolds were prepared by 3D bioprinting technology.Live/dead cells staining were taken to observe the distribution and viability of cells.CCK-8 experiment was taken to detect cell proliferation,and real-time fluorescent quantitative PCR was used to detect the expression of cartilage-related genes.3.The printed scaffolds of each group were transplanted subcutaneously on the back of nude mice,and the scaffolds were removed after 8 weeks to observe the gross morphology,and the cartilage formation was observed by histological staining and type ? collagen immunohistochemical staining.RESULTS1.Human adipose-derived stem cells were successfully isolated,extracted and characterized.hADSCs cultured with microcarriers for chondrogenesis had the highest expression of chondrogenic genes compared to monolayer and hanging-drop cultures,and can be used as a cell source for 3D bioprinting of predifferentiated hADSCs to construct tissue-engineered cartilage.2.hADSCs encapsulated by GelMA hydrogel were bioprinted in a micro-extrusion based principle,which can form a 3D morphologically stable scaffold in vitro.3.The cell viability and proliferation rate were highest in the co-culture group within one week after printing.Although the cell viability was higher in the hADSCs group and predifferentiated hADSCs group than the control group,the cell proliferation rate was low in these two groups.4.After 14 days of induced differentiation culture of each group,the relative expression of cartilage-related genes in the co-culture group was higher than that in the hADSCs group,and the expression of cartilage hypertrophy marker genes appeared higher in the induced predifferentiated hADSCs group.5.The 3D bioprinted cell-GelMA composite scaffolds transplanted to the subcutis of nude mice could form cartilage tissues,and the efficiency of cartilage tissue formation in the co-culture group was better than hADSCs group and predifferentiated hADSCs group,and has no significant difference from the control group.CONCLUSIONThe 3D bioprinted cell-GelMA composite scaffold has a stable three-dimensional structure and can form cartilage tissue when transplanted into the subcutis of nude mice.The scaffolds of co-culture group were superior to the control group,the hADSCs group and predifferentiated hADSCs group in terms of in vitro cell viability,cell proliferation and chondrogenic gene expression,and the efficiency of cartilage formation in vivo was also better than the hADSCs group and predifferentiated hADSCs group,and has no significant difference from the control group,which can be further applied to 3D bioprinting for cartilage tissue engineering. |
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