Effects of glycosaminoglycan supplementation in the chondrogenic differentiation of bone marrow- and synovial- derived mesenchymal stem/stromal cells on 3D-extruded poly (ε-caprolactone) scaffolds

Abstract

The lack of effective and long-term treatments for articular cartilage defects has increased the interest for innovative tissue engineering strategies. Such approaches, combining cells, biomaterial matrices and external biochemical/physical cues, hold promise for generating fully functional cartilage tissue. Herein, this study aims at exploring the use of the major cartilage glycosaminoglycans (GAGs), chondroitin sulfate (CS) and hyaluronic acid (HA), as external biochemical cues to promote the chondrogenic differentiation of human bone marrow- and synovium-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) on custom-made 3 D porous poly (ε-caprolactone) (PCL) scaffolds. The culture conditions, namely the chondrogenic medium and hypoxic environment (5% O₂ tension), were firstly optimized by culturing hBMSCs on PCL scaffolds without GAG supplementation. For both MSC sources, GAG supplemented media, particularly with HA, promoted significantly cartilage-like extracellular matrix (ECM) production (higher sulfated GAG amounts) and chondrogenic gene expression. Remarkably, in contrast to tissues generated using hBMSCs, the hSMSC-based constructs showed decreased expression of hypertrophic marker COL X. Histological, immunohistochemical and transmission electron microscopy (TEM) analysis confirmed the presence of typical articular cartilage ECM components (GAGs, aggrecan, collagen fibers) in all the tissue constructs produced. Overall, our results highlight the potential of integrating GAG supplementation, hSMSCs and customizable 3 D scaffolds toward the fabrication of bioengineered cartilage tissue substitutes with reduced hypertrophy.

GRAPHICAL ABSTRACT

Keywords:

• Articular cartilage
• chondroitin sulfate
• hyaluronic acid
• mesenchymal stem/stromal cells
• poly (ε-caprolactone) scaffolds

Acknowledgements

The authors acknowledge Pedro Henriques for the help with sample preparation for TEM analysis.

Disclosure statement

The authors confirm that there are no conflicts of interest.

Additional information

Funding

The authors acknowledge financial support from Fundação para a Ciência e Tecnologia (FCT, Portugal) through iBB – Institute for Bioengineering and Biosciences [UID/BIO/04565/2019] and from Programa Operacional Regional de Lisboa 2020 [Project N. 007317] and also through the projects PRECISE – Accelerating progress toward the new era of precision medicine [PAC-PRECISE-LISBOA-01-0145-FEDER – 016394], Stimuli2BioScaffold [FCT grant PTDC/EME-SIS/32554/2017]. João C. Silva is grateful to FCT for financial support through the scholarship [SFRH/BD/105771/2014].