Preservation of the Native Phenotype of Human Meniscus Fibrochondrocytes Using an Organ-on-a-Chip

Abstract

Introduction: The meniscus is a fibrocartilaginous structure essential to knee joint biomechanics. Meniscal injuries are highly prevalent, and their repair is hindered by the loss of the native fibrochondrocyte (FC) phenotype during conventional in vitro culture, a process known as dedifferentiation. Organ-on-a-Chip (OoC) systems represent a cutting-edge microfluidic platform capable of replicating the cellular physiological microenvironment with greater fidelity than traditional two-dimensional models.

Objective: To develop an Organ-on-a-Chip model capable of preserving the native phenotype of human fibrochondrocytes isolated from meniscal tissue, by evaluating the expression of key phenotypic markers (COL2A1, COL1A1, SOX9) through indirect immunofluorescence.

Methods: Fibrochondrocytes were isolated from human meniscal tissue obtained at INR and cultured in PDMS chips fabricated via soft lithography. The microfluidic design was computationally validated by CFD simulation (ANSYS Fluent), ensuring physiological shear stress conditions (0.01–0.1 dyn/cm²). Culture surfaces were functionalized with Sulfo-SANPAH and coated with extracellular matrix proteins. Phenotypic characterization was performed by indirect immunofluorescence for COL2A1, COL1A1, and SOX9, with quantitative image analysis in ImageJ/Fiji using the CTCF index.

Expected outcomes: The OoC model is expected to generate flow conditions that promote retention of the fibrochondrocytic phenotype, evidenced by higher expression of COL2A1 and SOX9 compared to static 2D culture. The platform aims to establish a reproducible methodology for the investigation of regenerative therapies for the human meniscus.