Effect of Fluffy-PLGA Porosity on hMSC Chondrogenic Differentiation in 3D-Printed Scaffolds

The inability of cartilage to regenerate naturally or repair itself when damaged makes it a primary target of regenerative medicine. Current approaches focus on fabricating scaffolds of appropriate mechanical, physical, and biological properties to enhance cellular differentiation and cartilage regeneration. In cartilage tissue engineering, three-dimensional (3D) scaffolds attempt to provide native extracellular matrix (ECM) conditions with the aim of inducing tissue ingrowth and ECM deposition, by manipulating material properties and structure. In this study we employed a 3D-Painting process, that combines additive manufacturing of advanced 3D ink systems and salt-leaching techniques, to fabricate highly porous Fluffy-PLGA scaffolds with tunable mechanical and physical properties. Fluffy PLGA is a 3D-printable salt-laden ink that could be manipulated to form various porosities based on material composition. By manipulating salt-polymer composition, we were able to produce scaffolds of varying porosity and mechanical properties. We examined the effect of different porosities on the chondrogenic differentiation of human MSCs cultured in 3D printed scaffolds composed of 50, 60, and 70% Fluffy-PLGA. Chondrogenic differentiation was induced by culture in chondrogenic differentiation medium. Cell viability, proliferation, and chondrogenic differentiation were evaluated by Live/Dead cell assay and confocal microscopy, dsDNA quantification, and gene expression of the primary chondrogenic markers (COLI, COLII, ACAN, and SOX9), respectively. Compressive and tensile tests were performed on the scaffolds to determine and compare their mechanical properties and examine how they are influenced by material composition and porosity. We were successful in fabricating material of varying porosities with a tensile elastic modulus ranging between 0.42-5.3 MPa. In vitro experiments showed that Fluffy-PLGA scaffolds exhibit adequate porosity and mechanical properties to promote cell adhesion, proliferation, and MSC differentiation significantly on 60 and 70% Fluffy PLGA scaffolds. Cell viability assessment showed that all material compositions of Fluffy PLGA support cell viability and proliferation for at least 21 days. Mechanical testing results show that chondrogenic differentiation of MSCs is more favorable on 3D scaffolds of higher porosity and lower elastic modulus. These results indicate that 3D printed Fluffy-PLGA is a promising new biomaterial for cartilage tissue engineering that warrants further evaluation in in-vivo studies.