Studies Of Optimization Of Strategies For Seed Cells Used In Cartilage Tissue Engineering And Construction Of Ear-shaped Cartilage

Background:Cartilage tissue engineering provides an alternative solution to repair cartilage defects, but the seed cell related problems have always been the bottleneck to its further development. The approaches of chondrocytes based, chondrogenic induction of BMSCs, and co-culture of chondrocytes and BMSCs have been used to construct tissue engineered cartilage, although each approach has its own advantages and disadvantages. However, the optimal strategy remains to be identified. Besides, as remnant ear tissue is an important cell source for ear reconstruction of microtia patients, the biological characteristics of the cells from remnant ear and their abilities to form cartilage also remain to be systematically explored. Since auricular cartilage tissue engineering becomes an active area, generation of ear-shaped cartilage has always been studied with the immunodeficiency mice whereas the results in an immunocompetent animal model still remains unknown.Objectives:In the present study, different seed cell strategies of auricular chondrocytes, BMSCs induction, and co-culture to construct tissue engineered cartilage were compared in order to identify the optimal one for elastic cartilage tissue engineering. The feasibility of constructing ear-shaped cartilage in the immunocompetent animal model was investigated.The biological characteristics of the cells derived from remnant ear and their in vivo fates influenced by in vitro expansion and redifferetiation were evaluated so as to determine the optimal strategy for remnant ear chondrocytes based cartilage tissue engineering. The feasibility of constructing ear-shaped cartilage with chondrocytes from remnant ear of individual microtia patient was investigated.Methods, Result&Conclusion:1. Construction of tissue engineered cartilage with different cells and strategies1.1Comparison of tissue engineered cartilage constructed by chondrocytes derived from different type of cartilageMethods The differences between the cell-scaffold complexes constructed by cells derived from articular cartilage and auricular cartilage were evaluated by histological examinations.Result There were no differences found on the histological level between the complexes constructed by chondrocytes derived from articular cartilage and auricular cartilage in vitro. However, after6weeks subcutaneous implantation, the complex constructed by articular chondrocytes became partly ossified while those constructed by auricular chondrocytes had elastic fibers stained positive.Conclusion The chondrogenic response of chondrocytes from different types of cartilage demonstrates that an initial chondrocyte and cartilage type recapitulates the same chondrocyte and cartilage type in later tissue-engineered development.1.2Comparison of tissue engineered cartilage constructed with different strategiesMethods The three strategies of auricular chondrocytes, BMSCs induction, and co-culture to construct tissue engineered cartilage were compared using the histological assay, biomechanical evaluation and quantitative analysis of gene expression.Result The BMSCs induction group was not able to form elastic cartilage after6weeks implantation, and the complex became obviously ossified with a significant up-regulation of COL10Al, MMP13, and ALPL. Compared with the BMSCs induction group, the co-culture group not only maintained the stable chondrocytic phenotype, but also showed a denser elastic fibers and a higher Young's modulus with a significantly higher gene expression profile of COL9A1, COMP, DCN, and LOXL2than the chondrocytes only group. Moreover, the depositions of Dlkland Ki67were also heavier in the co-culture group.Conclusion Co-culture of auricular chondrocytes and BMSCs was demonstrated to be the optimal strategy for engineering elastic cartilage. The complex constructed by co-culture showed the densest elastic fibers and highest Young's modulus with better proliferative and chondrogenic potentials after implantation in vivo.1.3Construction of ear-shaped cartilage in a pig modelMethods Porcine auricular chondrocytes and BMSCs were mixed at a ratio of5:5and seeded on the PGA/PL A scaffold in the shape of human ear. The complex was cultured for10weeks in vitro and autotransplanted subcutaneously for20weeks in vivo.Result Co-culturing porcine auricular chondrocytes with BMSCs at a ratio of5:5on an ear-shaped PGA/PLA scaffold could finally form a tissue engineered cartilage with a defined auricular figure and good elasticity after10weeks in vitro culture. The neo-cartilage could survive for20weeks after autotransplantation in the immunocompetent animal, however, the precise shape of human ear was lost.Conclusion The ear-shaped cartilage engineered with co-culture strategy was able to survive for20weeks in the immunocompetent animal model.2. Construction of tissue engineered cartilage with remnant ear chondrocytes derived from microtia patients2.1The proliferation, chondrocytic phenotype alteration, and cell yield of remnant ear chondrocytesMethods The initial cell yield of remnant ear chondrocytes was calculated according to the weighing of remnant ear and cell counting after digestation. The proliferative ability and expansion efficiency of remnant ear chondrocytes were studied by MTT assays and cell counting. The chondrocytic phenotype altering after continuous passages was characterized by immunofluorence staining and PCR. The differences of the cartilage related gene profile between remnant ear chondrocytes and normal auricular chondrocytes were evaluated.Result The cell yield of remnant ear tissue was (3.90±1.27) X106/g. The proliferative abilities of remnant ear chondrocytes from P1-P4passages were enhanced by adding bFGF, and the cells expanded to P4could reach around (328.4±50.4)-fold multiplication. However, the remnant ear chondrocytes at P3showed weak staining of Safranin O and low expression of COLⅡ, and even both of them were negative in P4chondrocytes. Additionally, there were no obvious differences in the cartilage-related gene expression profile between remnant ear chondrocytes and normal auricular chondrocytes except that the expressions of COL2A1V2and COL9A1were lower in chondrocytes from some individual remnant ear samples.Conclusion Remnant ear chondrocytes could be extensively expanded in vitro with bFGF, cells passaged within P3could reach both quantity and quality for construction of a full-sized human ear cartilage.2.2Influences of in vitro expansion and chondrogenic induction on the in vivo fate of remnant ear chondrocytes to form elastic cartilage.Methods The in vitro and in vivo fates of cell-scaffold complexes constructed by P3-P8remnant ear chondrocytes under redifferetiation or not were evaluated by histological assay, quantitative analysis of gene expression and biomechanical evaluation.Result The complexes constructed by remnant ear chondrocytes within4passages were able to form elastic cartilage in vivo without in vitro redifferentiation. On the other hand, the complexes constructed by remnant ear chondrocytes from P3-P8could all form the cartilage-like tissues under redifferentiation in vitro4W. However, the expression of DLKl at4W in vitro was significantly lower than in the non-redifferentiated group, and severe ossifications were observed after8weeks implantation.Conclusion Remnant ear chondrocytes within P4could form elastic cartilage in vivo although failed to develop cartilage in vitro. Remnant ear chondrocytes from P3-P8could form cartilage-like tissues after in vitro chondrogenic induction which although easily caused ossification after in vivo implantation. DLK1might play an important role in endochondral ossification during tissue engineered cartilage construction.2.3Construction of human ear-shaped and-sized cartilage with remnant ear chondrocytes derived from individual microtia patientMethods Chondrocytes derived from individual remnant ear were expanded to P3-P4and seeded on the PGA/PLA scaffold with the shape and full size of adult human ear. The complex was cultured under a redifferentiation system composed of chondrogenic factors, alginate gel, and a rotating culture device. The complex cultured under regular condition was set as control.Results A full-sized cartilage with a defined shape of human ear could be engineered in vitro by the extensively expanded chondrocytes from autologous remnant ear under a redifferentiation culture system.Conclusion An adult-sized cartilage with a defined shape of human ear could be engineered in vitro by the expanded chondrocytes from individually autologous remnant ear under a redifferentiation culture system.