Chondrogenic Commitment of human Bone  Marrow Mesenchymal Stem Cells cultured under perfusion within a 3D collagen  environment releasing hTGF\(\beta\)1

Abstract

The optimal growth, maturation and function of bioengineered tissues are mediated by both biochemical and physical cues. We here describe a 3D biomimetic environment directing stem cells towards a chondrogenic phenotype. This system comprises a collagen hydrogel and poly-lactic-co-glycolic acid microcarriers (PLGA-MCs) engineered to protect, carry and release a human Transforming Growth Factor b1 (hTFGb1) payload. PLGA-MCs were prepared using supercritical emulsion extraction technology and integrated into a collagen hydrogel co-seeded with human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs). Testing different concentrations of hTFGb1 supplemented to cell monolayer cultures suggested 10 ng/mL as the most appropriate concentration to promote upregulation of SRY-Related HMG-BOXGene 9 (4-fold) and collagen type II (2-fold) specific markers, at Day 16. A similar growth factor concentration was delivered within the 3D bioengineered environment cultured in a dynamic via a custom perfusion bioreactor. A chondrogenic commitment was obtained as indicated by upregulation of collagen type II (5-fold) and downregulation of collagen types I and III (both 0.1-fold) at Day 16. Histological analysis confirmed the remodeling of the synthetic extracellular matrix in where an enhanced mass exchange was described by FEM analysis of fluid-dynamics and related nutrient mass transfer within the 3D construct. This study supports the use of 3D bioengineered scaffolds cultured in a dynamic environment as a suitable tissue engineered model to study chondrogenic differentiation in vitro and opens perspectives for an injectable collagen-based advanced therapy system.