The Application Strategy For Extra Cellular Matrix In Tissue Engineering

BackgroundThe auricular cartilage is an avascular,aneural,and alymotpa structure.Its ability to self-renew and regenerate is absent or very limited.Autologous costal cartilage,as current clinical standard,is limited by limited tissue source,extensive surgical injury,and donor site morbidity.Reconstruction of auricle remains a challenge and hotspot to surgeons and researchers.The extracellular matrix(ECM)of cartilage has been proved to facilitate cell attachment,proliferation,and differentiation.But the mechanical strength of pure ECM is poor.Silk fibroin(SF)and three-dimensional printing technique(3D printing)are added to improve the properties of the scaffoldObjective1.To study the feasibility of constructing cartilage scaffold by ycross-linking ECM and SF.And explore whether the ECM-SF scaffold can promote adipose-derived stem cells(ADSCs)adhesion,proliferation,and differentiation,and form cartilage-like tissue.2.Using 3D printing technique and inverted model to construct i3D scaffold with high internal connectivity.And examine whether the i3D scaffold is better than a none-i3D scaffold with closer similarity to natural cartilage3.To explore whether the chondrocytes from auricular cartilage and costal cartilage can be used to construct acellular cartilage scaffold.Methods1.The porcine ear cartilage was decellularized to gain ECM.ECM and SF were cross-linked by γ radiation to form the ECM-SF scaffold and pure SF scaffold was also produced to serve as a control.The scaffold structure was observed by SEM Chondrocytes(Chon)and ADSC were isolated.ADSC(P2)was cultured with the leaching medium from the scaffold to evaluate the cytotoxicity.Chon(P2)and ADSC(P2)were seeded into ECM-SF and SF scaffolds respectively.Cell viability was observed by SEM,immunofluorescence.The formation of cartilage was observed by immunohistochemistry2.Sacrificial PCL scaffolds were printed by the 3D printing system.ECM and SF were mixed and injected into PCL scaffolds.After y crosslinking and freeze-drying,PCL scaffolds were dissolved and left indirect 3D scaffolds(indirect 3D ECM-SF scaffold,i3D ECM-SF).At the same time,the i3D SF scaffolds were produced with a similar method.The scaffold structure was observed by SEM.ADSC(P2)was cultured with the leaching medium from the scaffold to evaluate the cytotoxicity.Chon(P2)and ADSC(P2)were seeded into i3D ECM-SF and i3D SF scaffolds respectively.Cell viability was observed by SEM,immunofluorescence.The deposition of cartilage-specific extracellular matrix was observed by immunohistochemistry3.The auricular chondrocytes(AuChon)and costal chondrocytes(CoChon)of pigs,and human adipose-derived stem cells(hADSCs)were isolated.PCL scaffolds produced by 3D printing were seeded with AuChon(P2)and CoChon(P2)respectively,which,after 10 days of culturing,were then decellularized to gain deAuECM-PCL scaffolds and deCoECM-PCL.The two scaffolds were seeded with hADSC.Cell viability was observed by immunofluorescence.Results1.The scaffold prepared by the y-crosslinking method has no cytotoxicity.The ECM-SF scaffolds are more conducive to ADSC adhesion and growth than SF scaffolds.ADSCs differentiated into chondrocytes,secret cartilage-specific extracellular matrix.But there remains hollow in these two scaffolds2.The scaffolds prepared by the reverse modeling method showed good biocompatibility.Mature cartilage tissue and cartilage-specific extracellular matrix were found in i3D ECM-SF+ADSC group.The tissue of the i3D SF+ADSC group grew well,but there was little evidence of chondrocytes differentiation.While the cells in the i3D SF+Chon group grew poorly and did not fill up the scaffold3.Pig auricular chondrocytes and costal chondrocytes were seeded onto the 3D PCL scaffolds.After acellular treatment,immunogenicity was reduced while the ECM was retained.Compared with untreated PCL scaffolds,deECM-PCL scaffolds can promote hADSC adhesion,proliferationConclusions1.The ECM-SF scaffold prepared by γ-cross-linking method shows no cytotoxicity,which is conducive to cell adhesion and proliferation and contribute to the formation of mature cartilage tissue.However,due to limited communication within the scaffold,cells can hardly grow into the center of the scaffold,which limits the transport of nutrients,oxygen,and metabolic waste2.The i3D ECM-SF scaffold prepared by the reverse modeling method shows no cytotoxicity,which is conducive to cell adhesion and proliferation and can form mature cartilage tissue.Compared with the common scaffolds,the i3D scaffolds are more conducive to the growth of cells by allowing the transport of oxygen,nutrients and metabolic waste.Cells grow into the center of the scaffolds and form the homogeneous and mature cartilage3.After the chondrocytes were seeded onto the PCL scaffold and treated with acellular treatment,the PCL scaffold is coated with cell-derived ECM.Compared with untreated PCL scaffolds,PCL scaffolds wrapped in ECM are more conducive to adhesion of ADSC.