Bioprinting Chondrocyte Hydrogel Cultures onto Fibre Membranes for Cartilage Defect Repair

Abstract

Despite notable advancements in autologous chondrocyte implantation (ACI) over the past three decades, persistent challenges including uneven cell distribution, low seeding density, and limited extracellular matrix formation continue to hinder outcomes. This study suggests a novel approach to matrix-assisted ACI (MACI) that integrates reactive jet impingement (ReJI) bioprinting with Chondro-Gide® (Ch-G) membranes to give an in theatre bioprinting approach with the potential to control cell distribution and density at the point of implantation. A collagen–alginate–fibrin (CAF) hydrogel was formulated as a bioink to maintain high cell viability and enable accurate deposition of chondrocytes at both low and high densities. Bone marrow-derived immortalised stem cells were differentiated into chondrocytes and bioprinted using the ReJI system onto Ch-G substrates. Constructs were evaluated over 14 days for cytotoxicity, morphology, gene expression, extracellular matrix (ECM) deposition, and mechanical properties. Live/dead assays confirmed high cell viability across all conditions. Immunofluorescence and scanning electron microscope (SEM) analyses showed enhanced cell alignment, surface migration, and ECM formation, particularly in high-density Chondro-Gide®/CAF samples (Ch-G/CAF). ACAN and SOX9 showed significant upregulation from day 1 to day 14 and immunohistochemical analysis confirmed robust deposition of collagen II and sulphated glycosaminoglycans (sGAGs). Mechanical testing demonstrated increased compressive modulus over time, with Ch-G/CAF constructs exhibiting superior stiffness and volumetric stability. These findings suggest that ReJI bioprinting of CAF hydrogels onto Chondro-Gide® membranes supports enhanced chondrogenic activity and matrix formation, offering a promising strategy for next-generation MACI.