Construction Of Engineered Elastic Cartilage And Non-invasive Assessment With MRI T2 Mapping In Vivo

BackgroundReconstructing cartilage defects have been a challenge for surgeons.Developments in cartilage tissue engineering in the past few decades have provided a promising approach.Although several cases of tissue-engineered cartilage have been reported in clinic,there is still a long way to go to realize its clinical translation.One issue should be considered is that so far,histopathological examination is still the clinical gold standard for evaluating the engineered cartilage maturation.However,this detection method is invasive and cannot monitor the in vivo outcome of tissue-engineered cartilage at any time.Thus,a systematic,properly quantitative,and noninvasive evaluating technique is sorely needed.Magnetic resonance imaging(MRI)provides high resolution in vivo images with excellent soft-tissue contrast without invasive operations.Inspired by the utilize of MRI T2 mapping in quantifying water contents and extracellular components of cartilage in the osteoarthritis model,here we want to confirm whether this technique effectively indicates the in vivo maturation of engineered elastic cartilage.Objectives1.To evaluate the construction of engineered elastic cartilage construction based on rabbit ear chondrocytes-silk fibroin bracket2.To investigate the in vivo maturation of engineered cartilage in the subcutaneous environment of immunocompetent animals.3.To clarify the feasibility of using MRI T2 mapping in evaluating engineered elastic cartilage.4.To further explore the feasibility of using MRI T2 values to indicate the biochemical index of the engineered cartilage.Methods and results1 The regeneration of engineered elastic cartilage based on auricular chondrocytes and silk fibroin(SF)scaffolds in rabbit.1.1 Fabrication of the chondrocytes-SF scaffold in vitro.Methods:P2 rabbit auricular chondrocytes were seeded into the SF scaffolds.After culturing for 4 weeks in vitro,the samples were evaluated by HE,Masson's trichrome(MT)staining,safranin-O fast green staining,and electronical microscope scanning.The concentration of sulfated glycosaminoglycan(GAG),elastin(ELN),collagen type I(COL I),collagen type ?(COL ?),and total hydroxyproline(HYP)content were quantified then.Results:After 28 days'of in vitro culturing,samples formed white cartilage-like tissue.SEM revealed that chondrocytes attached,grew and proliferated well inside the SF scaffold.HE,safranin-O fast green and MT staining confirmed the chondrocytes were surrounded by much ECM.1.2 Maturation of engineered elastic cartilage in subcutaneous environment of rabbits.Methods:Three experimental groups were included in this part:chondrocytes-SF scaffold after 28 days' of in vitro culturing(EC),autologous auricular cartilage(AC)and cell free SF scaffold(SF).The samples were implanted subcutaneously in the donor rabbit,and the contents were harvested at 2,4 and 8 weeks after implantation for assessment.Results:Histological,immunohistochemical(IHC)staining,biochemical analysis and RT-PCR results revealed that the EC group displayed inflammatory reaction at the first 2 weeks in vivo,then the chondrocytes in EC construct retained their phenotype and firmed well ECM at 4 weeks in vivo.At 8 weeks in vivo,the collagen fiber showed a trend of re-location which may eventually led to fibrosis.2 Feasibility of using T2 mapping and T2 values to monitor engineered elastic cartilage maturation.Methods:After implanting the AC,EC and SF constructs into the donor back of the rabbit,take them to MRI scanning and quantify the mean T2 values at 1,2,4 and 8 weeks in vivo.After that,harvest the samples for further assessment.Results:The 2D MIXED-T2 Multislice(T2 mapping)provided the most satisfied images among the tested sequenced.The change of mean T2 values was in line with the histological and biochemical results.Pearson correlation analysis confirmed that mean T2 was significantly correlated with total collagen,ELN and GAG contents(collagen:r=-0.946,P<0.001;ELN:r=-0.939,P<0.001;GAG:r=-0.933,P<0.001).And there were also moderate correlations found between T2 and specific COL 1 or COL 2 content(COL 1:r=-0.837,P=0.005;COL 2r=-0.797,P=0.01).Conclusion1.After 28 days'of in vitro culturing,P2 auricular chondrocytes and SF scaffold firmed an engineered elastic cartilage.2.The engineered cartilage displayed acute inflammatory reaction at the first 2 weeks in vivo,then the chondrocytes in EC construct retained their phenotype and firmed well ECM at 4 weeks in vivo.At 8 weeks in vivo,the collagen fiber showed a trend of re-location which may eventually led to fibrosis.3.MRI T2 mapping in feasible for distinguishing the native cartilage,engineered cartilage and fibrous tissue,and can be further used to monitor the maturation process of engineered elastic cartilage in immunocompetent animals.4.We clarified the linear correlations between T2 relaxation times and cartilage-specific proteins,especially GAG and elastin contents in engineered elastic cartilage.