Multilayer dual-porosity 3D printed scaffolds to recreate the anisotropic microenvironment of the hyaline cartilage

Articular cartilage accounts for a multizonal structure with distinct matrix composition and chondrogenic phenotypes, responsible for the tissue's load-bearing ability. Upon damage, cartilage is clinically treated by microfracture, which allows bone marrow exudation to the previously abraded zone. However, mesenchymal stem cells (hMSC) of the marrow cannot differentiate into specific chondrogenic phenotypes and the resulting tissue is isotropic and non-functional. Here, we developed multilayer dual-porosity scaffolds with defined in-fiber and structural porosities that were able to steer hMSC's differentiation into specific chondrogenic phenotypes. A library of inks prepared from poly-(L)lactide-co-caprolactone and sacrificial gelatine microspheres of three different diameters (13 ± 8 μm, 24 ± 14 μm, and 47 ± 27 μm) were used to 3D print structures with different patterns (90°, 60° and 45°), giving rise to dual-porosity structures of tunable in-fiber and structural porosities. This pallet of structures allowed control over porosity, topography and mechanical properties (ranging from 3.1 ± 0.1 to 9.1 ± 1.8 kPa), which modulated cell adhesion, proliferation and differentiation. Multilayer scaffolds were fabricated from selected structures that promoted chondrogenic differentiation with distinct expression of collagen type I, type II (up to 9.9 fold-increase), aggrecan and versican genes, resulting on a tissue with characteristic collagen I and II deposition patterns, abundant glycosaminoglycan deposition (15.4 ± 2.0 μg _GAG · μg⁻¹ _DNA) and similar compression modulus to native cartilage (501.5 ± 72.7 kPa).