Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges.

Nathalie Mayer,Stefania A Riboldi,Marielle Pasdeloup,Matteo Moretti,Arianna B Lovati,Frédéric Mallein-Gerin,Silvia Lopa,Giuseppe Talò

doi:10.1371/journal.pone.0161479

Abstract

Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer in vitro articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated in vivo by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage.

Highlights

The incidence of degenerative and traumatic cartilage lesions is increasing due to the global population ageing and to the more intensive practice of sport activities
We found that the FI-BIT combination allows high human articular chondrocytes (HACs) proliferation and cartilaginous extracellular matrix (ECM) synthesis in collagen sponges [7]
When BIT was added in static conditions, peripheral deposition of ECM was observed, but no matrix was detected in the sponge core

Summary

Introduction

The incidence of degenerative and traumatic cartilage lesions is increasing due to the global population ageing and to the more intensive practice of sport activities. Cartilage injuries are irreversible and often evolve to osteoarthritis (OA), causing pain and disability Several surgical approaches, such as mosaicplasty or microfractures, have been developed to repair damaged cartilage to avoid or at least delay joint replacement. Autologous chondrocyte implantation (ACI) [3] is performed worldwide to treat articular cartilage focal lesions. In this procedure, human articular chondrocytes (HACs) are isolated from a small cartilage biopsy and amplified in vitro, before being re-implanted back in the patient. During in vitro expansion, HACs undergo a dedifferentiation process characterized by the loss of type II collagen expression in favor of type I collagen This process is a major determinant in the formation of type I collagen-rich fibrocartilage following ACI [4,5,6]. To improve ACI clinical outcomes, research has been focused on the use of biomaterials and growth factors able to drive HAC redifferentiation and broaden ACI application to larger defects, such as early OA lesions

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Conclusion