The Construction And Stability Study Of Tissue Engineered Cartilage In Large Animals

Backgroud:Applying tissue engineered cartilage to repair craniofacial cartilage defect has wide prospect of clinical application. However, this technique still has not realized the clinical translation mainly due to the poor stability of engineering cartilage in large animals with complete immunity. The PGA/PLA polymer scaffold in the cell-scaffold construct would trigger the strong inflammation which impede the cartilage formation. Moreover, it is an innate immune response that macrophages predominantly participate in. Therefore, it is important that how to effectively resist or avoid this immune response. Firstly, lots literatures reported that BMSCs not only have the property of sternness and low immunogenicity, but also have immunomodulatory roles on a variety of immune cells. Whether engineering cartilage based on BMSCs still can inhibit the innate immune response, thus improving the quality of cartilage formation in the subcutaneous of large animals needs further exploration. Secondly, optimization the approach for the cartilage construction by extending the time of in vitro culture or using cell-sheet without scaffold, to improve the engineering cartilage formation in large animals also needs further exploration.Objectives:1. To compare tissue engineered cartilage in the subcutaneous environment of pig models with different cells and strategies.2. To explore the role of BMSC-engineered cartilage on macrophages and further elucidate the immunoregulation mechanism.3. To investigate the effects of residual of PGA/PLA in vitro on the regeneration of engineered cartilage in the subcutaneous environment of pig models.4. To use cell-sheet technology to construct engineered cartilage in the subcutaneous environment of pig models.Methods and Results1. Stability study of tissue engineered cartilage based on PGA/PLA in pigs1.1 The regeneration of engineered cartilage based on different seed cells in pigsMethods:Three strategies of auricular chondrocytes (ACs), BMSCs and 1:1 (ACs: BMSCs) to construct tissue engineered cartilage after in vitro 8 weeks culture were compared using histological, biology, biomechanical evaluation. The cartilage-forming capacity of three groups and inflammatory responses in the subcutaneous of the pig were evaluated.Results:After culturing for 8 weeks in vitro, all cell-scaffold constructs in the BMSCs, ACs, and 1:1 groups showed typical cartilaginous features. No significant differences were found among the three groups in total collagen, Young’s modulus, residual PG/PLA. After 2 weeks post-implantation, only BMSCs group maintained complete cartilage structure, part cartilage tissue were left in 1:1 group and few chondrocytes survived in the ACs group. After 8 weeks in vivo, the constructs in the three groups were damaged to varying degrees.1.2 The role of BMSC-based engineered cartilage on macrophages polarization in pigsMethods:The differences of multinucleated foreign body giant cells (FBGCs) and the expression of CD68 and CD206 on macrophages among BMSCs, ACs,1:1, and PGA/PLA groups in vivo were evaluated. Furthermore, to verify the macrophages phenotype after co-culture with the engineered cartilage by immunofluorescence, real time PCR, Elisa in a transwell system.Results:Samples in the BMSCs group caused the least infiltration of FBGCs and CD68 positive macrophages than in the other three groups in vivo, PGA/PLA group was the highest, no significant difference between 1:1 and ACs groups. M2 macrophages indicated by CD206-positive cells maintained the lowest numbers in the PGA/PLA group, but highest in the BMSCs group. In the in vitro co-culture system, the anti-inflammatory factor CD206, IL-10, Arg-1 were higher in the BMSCs/MΦ group, while the pro-inflammatory factor IL-1(3, TNF-a were lowest in the BMSCs/MΦ group than any other groups.1.3 The effects of residual PGA/PLA on the regeneration of engineered cartilage in pig models.Methods:To construct engineered cartilages based on chondrocytes or BMSCs with PGA/PLA scaffold for 8,12,16 weeks in vitro. Then to compare the engineered cartilage in different time points in histology, biology, the residual PGA/PLA and the PGA diameter. The cartilage-forming capacity of the engineered cartilage in different time points and inflammatory responses in the subcutaneous of the pig were evaluated.Results:By extending the time of in vitro culture, the residual PGA/PLA and PGA diameter of engineered cartilage became less and thinner. Two weeks after auto-transplantation, the cartilage-forming capacity of the engineered cartilage was improved by extending the in vitro culture time.2. To study the cartilage regeneration of chondrocyte cell-sheets in pig models.Methods:Cell-sheets were formed with porcine auricular chondrocytes in high density and cultured in chondrogenesis medium for 12 weeks in vitro, and then were auto-transplanted in the subcutaneous for 2,8,24 weeks.Results:The chondrocytes could finally form cell-sheets with a certain thickness and homogeneous tissue. After auto-transplantation, the cell-sheets triggered no obvious inflammation and became gradually mature and could survive for a long time.Conclusion:1. After culturing for 8 weeks in vitro, all cell-PGA/PLA constructs in the ACs, BMSCs, and 1:1 groups exhibited high quality cartilages. Two weeks after subcutaneous auto-transplantation, only BMSCs group maintained the complete cartilage structure.2. BMSC-based engineered cartilage could suppress in vivo inflammation by increasing M2 polarization of macrophages, resulting in better tissue survival in subcutaneous implantation in pigs. This was further supported by the in vitro co-culture findings including the increased releasing of CD206, IL-10, Arg-1, IL-10 and the decreasing of IL-1(3 and TNF-a in alignment with M1 to M2 transition in macrophages co-cultured with BMSC-engineered cartilage.3. The PGA was not fully degradable even the engineered cartilage cultured for 16 weeks in vitro. The residual PGA/PLA could affect the cartilage forming in subcutaneous environment. Less residual PGA/PLA of constructs, better cartilage formation after 2 weeks of implantation in pigs.4. The cell-sheet technology could avoid the disturbances of the scaffold materials on cartilage regeneration. After auto-transplantation, the cell-sheets became gradually mature and could survive well in pigs.