| Background:Tissue engineering has provided a promising perspective in the field of auricular reconstruction.Two of the key problems are the seed cells and the precise control of the shape of human ear cartilage tissue engineering.Microtia chondrocytes should be considered as a clinically applicable cell source with ready accessibility and no damage to healthy tissues.The microtia chondrocytes carried a genomic defect,which was often ignored by the investigators who applied it in cartilage tissue repair and regeneration.The microtia chondrocytes could form cartilage tissue in nude mice,they showed inferior levels of biochemical and biomechanical properties after 12 weeks in vivo compared to the healthy ear chondrocytes formed engineered cartilage.The weaker chondrogenic phenotype in P1 healthy chondrocyte was accompanied with its stronger migration ability.It is suggested that the migration ability of chondrocytes may be related to the quality of tissue-engineered cartilage.For human-ear-shape cartilage with delicate 3-D structures,the most widely used form of PGA material in cartilage engineering is unwoven fiber mesh,which is difficult to be initially prepared into a complicated 3D structure of human ear shape.To overcome this problem,the PGA-based scaffold should be prefabricated into the exact shape of a human ear.3D printing technology could be used to make a titanium mold of human ear.Objectives:1.To study the feasibility of gene engineering technology to promote the migration of microtia chondrocytes.2.To study the effect of chondrocytes with improved migration ability cultured alone or with adipose stem cells on the construction of cartilage tissue.3.To construct tissue-engineered cartilage of human ear shape with the help of 3D printing technology.Methods,Results and Conclusions:1.The feasibility of gene engineering technology to promote the migration of microtia chondrocytes.Methods By comparing the microtia chondrocytes and normal ear chondrocytes,we can find out the different migration related genes,and improve the migration ability of microtia chondrocytes by overexpression of the gene.Results The expression of RhoA in normal ear chondrocytes was higher than that in microtia chondrocytes.It was confirmed by scratch and Transwell experiments that overexpression of RhoA can promote the migration of chondrocytes.Conclusion Overexpression of RhoA can promote the migration of chondrocytes.2.Isolation of Human Adipose-Derived Stromal Cells Using Suction-Assisted or Third-Generation Ultrasound-Assisted Lipoaspiration for Constructing Tissue Engineered CartilageMethods There are 3 groups:Experiment group 1,microtia chondrocytes mixed with ADSCs obtained by suction-assisted lipoaspiration 1:1;Experiment group 2,microtia chondrocytes mixed with ADSCs obtained by third-generation ultrasound-assisted lipoaspiration 1:1;Control group,microtia chondrocytes.After 4 weeks in vitro and 8 weeks in vivo,the constructs were harvested for gross observation,wet weight measurement,glycosaminoglycan(GAG)quantification,histological and immunohistochemical staining and biomechanical evaluation.Results Tissue-engineered cartilage were formed in Experiment group 1,Experiment group 2 and control group 4 weeks in vitro.Histological observation showed chondrogenic ECM deposition,loosened structure and undegraded scaffolds.Histological observation of the control group showed that the cartilage tissue was not uniform,most of the regions formed mature cartilage tissue,but some regions had loose tissue structure and less cartilage specific ECM secretion 8 weeks In vivo.In Experiment group 1,Experiment group 2,very mature cartilage tissues were formed with uniform structure,and a large number of cartilage lacunae and extracellular matrix were secreted,and the scaffold materials had been completely degraded.The test results of wet weight,glycosaminoglycan and biomechanics all showed that the cartilage of experimental group 1 and 2 was better than that of control group,and there was no significant difference between experimental group 1 and 2.Conclusion There are no differences in formation of tissue engineered cartilage by co-culture of microtia chondrocytes and ADSCs obtained by SAL or third-generation UAL.3.Experimental study on the co-culture of RhoA overexpressed microtia chondrocytes and adipose stem cells to construct tissue-engineered cartilage.Methods There are six groups:Group 1,microtia chondrocytes;Group 2:microtia chondrocytes mixed with adipose stem cells 1:1;Group 3,pCDH over expressed microtia chondrocytes;Group 4:pCDH over expressed microtia chondrocytes mixed with adipose stem cells 1:1;Group 5:RhoA over expressed microtia chondrocytes;Group 6,RhoA over expressed microtia chondrocytes mixed with adipose stem cells 1:1.After 4 weeks in vitro,4 weeks in vivo and 8 weeks in vivo,the constructs were harvested for gross observation,wet weight measurement,glycosaminoglycan(GAG)quantification,histological and immunohistochemical staining,special cartilage gene(ACAN、COLⅡ)and biomechanical evaluation.Results Histology,wet weight,GAG content,biomechanics,specific cartilage genes(ACAN、COLⅡ)and biomechanical evaluation showed that the cartilage quality of RhoA over expressed microtia chondrocytes mixed with adipose stem cells 1:1 was the best.Conclusion The cartilage quality of RhoA over expressed microtia chondrocytes mixed with adipose stem cells 1:1 was the best.This provides a reference for the construction of tissue-engineered cartilage of human ear-shaped cartilage.4.Construction of human ear-shaped tissue engineering cartilage by 3D printingMethods A set of human-ear-shaped Titanium-alloy molds was made by CAD/CAM and 3D printing technology.A PLA/PGA human-ear-shaped scaffold was made with human-ear-shaped Titanium-alloy molds which replicated the exact a 3D structure symmetrical to the patient’s healthy ear.After microtia chondrocytes seeding,the cell-scaffold constructs were cultured for 4 weeks in vitro.Then it was implanted subcutaneously into nude mice with inner part of human-ear-shaped Titanium alloy molds as a stand for 16 weeks.Results A patient-specific ear-shaped cartilage-like tissues was formed at 16 weeks in vivo,which revealed a tissue structure with abundant cartilage extracellular matrices and mature lacuna.The patient-specific ear-shaped cartilage reached a similarity level of 91.42%compared with the original scaffold by the shape analysis using 3D laser scan system.Conclusion This study may provide a useful strategy for regeneration of human-ear-shaped cartilage by human microtia chondrocytes assisted with 3D printing technology in vivo.5.Construction of human ear-shaped tissue engineering cartilage by 3D printing with over expressed RhoA microtia chondrocytes mixed with ADSCs obtained by suction-assisted lipoaspiration.Methods A set of human-ear-shaped Titanium-alloy molds was made by CAD/CAM and 3D printing technology.A PLA/PGA human-ear-shaped scaffold was made with human-ear-shaped Titanium-alloy molds which replicated the exact a 3D structure symmetrical to the patient’s healthy ear.The cell-scaffold was seed with over expressed RhoA microtia chondrocytes mixed with ADSCs obtained by suction-assisted lipoaspiration.The cell-scaffold constructs were cultured for 4 weeks in vitro.Then it was implanted subcutaneously into nude mice with inner part of human-ear-shaped Titanium alloy molds as a stand for 16 weeks and examined by histology,biomechanics and three-dimensional scanning.Results A patient-specific ear-shaped cartilage-like tissues was formed at 16 weeks in vivo,which revealed a tissue structure with abundant cartilage extracellular matrices and mature lacuna.The patient-specific ear-shaped cartilage reached a similarity level of 90%compared with the original scaffold by the shape analysis using 3D laser scan system.The histology shows that the mature cartilage tissue lacuna has the biomechanical strength close to the normal ear cartilage tissue.Conclusion Regeneration of human-ear-shaped cartilage by over expressed RhoA microtia chondrocytes mixed with ADSCs obtained by suction-assisted lipoaspiration assisted with 3D printing technology in vivo. |
PDF Full Text Request