Maintaining the Cartilage Phenotype of Late Passage Chondrocytes Using Salidroside, TGF-β and Sulfated Alginate for Cartilage Tissue Engineering Applications

Abstract

Cartilage self-repair following injury or osteoarthritis is limited due to its low regenerative capacity, avascular and aneural nature. Therefore, a promising treatment of cartilage injury and disease may involve engineering cartilage tissue in vitro by combining cells (e.g. chondrocytes), scaffolds (e.g. alginate sulfate (AlgSulf)), and signals (e.g. Salidroside and TGF-β). Autologous chondrocyte transplantation (ACT) is one of the most commonly used techniques for the treatment of cartilage defects. In ACT, chondrocytes are expanded in vitro till passage 3 or 4 and are then injected in the defect site. However, late passage chondrocytes undergo a process called dedifferentiation whereby cells lose their phenotype assuming a fibroblastic phenotype, thereby secreting type I collagen (Col1) instead of type II collagen (Col2) among other key changes. We have previously shown that chondrocytes encapsulated in three-dimensional (3D) biomimetic sulfated polysaccharides such as AlgSulf exhibit increased proliferation while maintaining their cartilage phenotype. The effects of alginate sulfate on cell growth and matrix deposition may be linked to enhanced binding of growth factors such as TGF-β. Moreover, Salidroside was found to upregulate the expression of cartilage specific genes such as Col2, aggrecan (Acan) and Sox9, while down-regulating the expression of the fibroblastic gene Col1 by acting on the TGF-β/Smad3 signalling pathway. This thesis aims to study the effect of treatment of P2 and P4 chondrocytes seeded on AlgSulf 2D films with Salidroside, TGF-β and their combination. Therefore, P2 and P4 chondrocytes were seeded on 2D films of AlgSulf of three different degrees of sulfation (DS=1, 1.5 and 2), in the presence or absence of Salidroside, TGF-β and their combination, and then, cell viability, proliferation, phenotype, and collagen secretion were assessed. Trypan Blue was used to study cell proliferation, while a live/dead assay was performed to assess cell viability. Quantitative real time PCR and Western Blot were also performed to evaluate the expression and synthesis of chondrogenic markers. The proliferation assay showed that a combination of Salidroside and TGF-β on AlgSulf films of DS=2 (AlgSulf2.0) resulted in the highest chondrocyte proliferation compared to all other treatments (p<0.0001). Under this same treatment, chondrocytes exhibited a round morphology when observed using Ethidium homodimer and Calcein AM, and displayed the highest levels of Col2A1, Acan and Sox9, while down-regulating the expression of Col1 and MMP13 (p<0.0001). The Western Blot also showed a prominent expression of Col2 and downregulation of Col1 when tested on P2 chondrocytes subjected to Salidroside on AlgSulf2.0. Therefore, this work shows that the treatment of chondrocytes seeded on AlgSulf2.0 films with a combination of Salidroside and TGF-β significantly promoted the proliferation of late passage chondrocytes while preserving their phenotype. The results of the current study may therefore be used in cartilage engineering applications.