Development of a cartilage tissue engineering construct based on hASCs spheroids differentiated under hypoxia in a chitosan/chitin nanocrystals 3D scaffold.

Abstract

The generation of a cartilage tissue engineering (TE) construct is currently a viable strategy for the treatment of osteoarthritis. Such construct requires a biomaterial with optimal properties, including biocompatibility, biodegradability and porosity, among others. Natural polymers like, CH and CS are attractive building block for the development of biomaterials for TE applications. We investigated the role of the incorporation of chitin nanoforms (i.e., nanocrystals or nanofibers) into Genipin-chitosan crosslinked (GCS) matrices designed in two different shapes, 2D films and 3D porous scaffolds. The aim was to assess the potential of these biomaterials as support for L-929 murine fibroblast cell line and human adipose-derived stem cells (hASCs) growth.The incorporation of chitin nanocrystals or nanofibers on the GCS 2D films and 3D porous scaffolds displayed better swelling properties and enhanced the mechanical performance when compared to nanoform-free GCS materials. Furthermore, our data showed that these biomaterials provide topological cues to support hASCs growth. The incorporation of low concentration of chitin nanoforms, in particular,was found to be the most appropriate supports for the proliferation and adhesion of hASCs.As cell source, hASCs have shown potential for cartilage regeneration when cultured in the appropriate conditions. Moreover, it has been suggested that physiological low oxygen (hypoxia) can significantly improve hASCs adhesion, proliferation and chondrogenic differentiation while preventing their osteogenic differentiation. We first investigated the chondrogenic potential of the hASCs spheroids and demonstrated that were positive to cartilage-specific markers including collagen type II (COL2A1) and aggrecan (ACAN), while lacked expression in the osteogenic differentiation marker collagen type I (COL1A2). Moreover, hypoxia inducible factor 1¿, which positively directs COL2A1 and ACAN expression, was upregulated in chondrospheroids cultured under hypoxia.Finally, this 3D culture hypoxic system created a pro-chondrogenic environment that allowed hASCs to differentiate into the chitosan/chitin nanocrystals 3D porous scaffold producing a chondral extracellular matrix with a high sulphated glucosaminoglycan content, which is characteristic of articular cartilage. This 3D chondrogenic differentiation model mimics the in vivo cartilage environment during the embryonic development, which entails a further step in cartilage TE, having potential application for articular cartilage regeneration.