Fabrication Of Decellularized Extracellular Matrix Ornamented 3D Printed Polycaprolactone Scaffolds With The Gradient Distribution Of Pore Structure For Cartilage Tissue Engineering

Cartilage tissue engineering holds great promise to regenerate cartilage defects.In recent years,researchers believe that recapitulating the zonal organization within articular cartilage represents the feasible strategy to engineer authentic cartilage.In particular,in order to achieve this,by designing the composition and structure of biomaterials scaffolds,the non-homogeneous structure can be achieved,such that cells are provided with the specific microenvironment,which eventually guides cellular behaviors.In the present study,3D printing was exploited to fabricate porous scaffolds with designed gradient distribution of pore structure.These scaffolds were then ornamented with decellularized extracellular matrix(dECM)in situ to develop highly bioactive scaffolds for cartilage tissue engineering.First,by setting up the key parameters of 3D printing,polycaprolactone(PCL)scaffolds with controlled pore structures were fabricated.In PCL scaffolds with gradient pore structure,three integrated parts were included with designed parameters,with the alignment angles between fibers in adjacent layers 30°,45°and 60°and the distances between neighboring fibers within the same layer were 450 μm,400 μm and 300 μm,respectively,where are designated in short as 30°-450 μm,45°-400 and 60°-300 μm,respectively.Each part of the scaffolds included 6 layers of deposited fibers,which resulted in a scaffold with a dimension of 10 mm×10 mm×3 mm.The scaffolds were featured with interconnecting pore structure,56.91%of porosity and 48.45 MPa of Young’s modulus.Second,the adhesion,proliferation,matrix production and gene expression of bovine articular chondrocytes(bAC)in these scaffolds were investigated.It was found that control scaffolds with homogeneous pore structures corresponding to respective parts of the gradient scaffolds better supported cell adhesion.However,compared to control scaffolds,both cell proliferation and matrix production were stimulated on the gradient scaffolds.In addition,within the gradient scaffolds,the heterogeneous distribution of cell density and matrix content was present among three parts,showing decrease from 30°-450 μm,45°-400 μm to 60°-300 μm.Then,after bAC were cultured on the scaffolds for 14 days,dECM was produced via decellularization treatment in situ on scaffolds to fabricate dECM ornamented PCL composite scaffolds(dECM/PCL).Albeit some loss of matrix composition was observed after decellularization,there was abundant matrix deposited in the gradient scaffolds,which were featured with dense,smooth,and interconnected mesh structure.In addition,matrix content and structure were different among three parts.In the end,the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells(rMSCs)was evaluated on dECM/PCL composite scaffolds.It was demonstrated that dECM/PCL scaffolds had great potential for conducting the chondrogenesis.The dECM/PCL scaffolds could significantly promote the production of cartilage specific matrix components and gene expression in rMSCs.Importantly,the gradient distribution of both matrix production and gene expression was achieved in the three parts of the scaffolds,with decrease from 30°-450 μm,45°-400 μm to 60°-300μm.Taken together,the present study provides a novel idea to fabricate bioactive cartilage engineering scaffolds,lays a solid foundation for engineering the zonal structure for native articular cartilage tissue.This will enable the rapid advancement of cartilage tissue engineering to clinical applications.