From Electrospinning to Protein Engineering: Novel Approaches for Cartilage Repair

Abstract

Normal joint function is dependent on the integrity of a thin layer of connective tissue that covers the articulating surfaces of the bone: cartilage. Its exceptional mechanical and biochemical properties allow the articulation to subsist in a mechanically demanding environment. However, this tissue has its limits and can deteriorate. An injury can induce acute tissue damage and lead to osteoarthritis if the lesion is left untreated. This inflammatory disease can also be favored by obesity or age-associated cellular disorders in conjugation with joint overuse, among other factors. To date, no optimal solution has been discovered. This thesis aims at exploring novel ways to promote cartilage repair by developing brand new materials or increasing the efficacy of existing treatments. In the first part of this thesis, Chapter 1 introduces various aspects of cartilage engineering and Chapter 2 describes the scope of the thesis into more details. Chapter 3 focuses on the creation of a true biomimetic of the cartilage extracellular matrix (ECM). A scaffold that replicates the biological structure of the tissue could provide great advances in cartilage repair. Articular cartilage is made of a hydrophilic matrix in which two networks interpenetrate one another. Fibrillar collagen type II proteins are embedded in a dense, negatively-charged meshwork of sulfated glycosaminoglycans (GAGs). Chondrocytes are sparsely distributed within this matrix and take care of tissue homeostasis. To recreate this architecture, two biomaterials were used in which chondrocytes were encapsulated. Alginate sulfate was used to mimic the glycosaminoglycan component, and a fibrous layer of poly(e-caprolactone) (PCL) was used as a reinforcement. A novel electrospinning technique was developed, which allowed the creation of fibrous, ultraporous and hydrophilic scaffolds. Mechanical, biochemical and biological properties of this scaffold were characterized and showed potential for cartilage repair. In Chapter 4, strategies to improve the efficacy of intra-articular injections of dexamethasone, commonly used to treat inflammation and pain linked to cartilage damage, were explored. Currently, multiple injections are generally required for a moderately effective treatment. This can be explained by the slow drug diffusion rate into cartilage and the fast turnover rate of the synovial fluid, thus rapidly clearing the drug from the joint. It was hypothesized that by specifically targeting dexamethasone to cartilage, the cellular uptake and therapeutic efficacy of the drug could be improved. Therefore, prodrugs were created that target the two components of cartilage (a collagen type II network inside a highly negatively-charged glycosaminoglycan-rich meshwork) and increase the intra-cartilage retention of dexamethasone. A prodrug is the combination of a therapeutic molecule and a drug carrier, designed to help the active drug to cross a physical barrier before releasing it. To target the glycosaminoglycan component via electrostatic interactions, dexamethasone was conjugated to polycationic chitosan. To target the collagen network, a peptide retained the drug within cartilage by specific interactions with collagen type II fibrils. The prodrugs were characterized and could show that they offer a promising and highly efficient alternative to repetitive injections of unmodified dexamethasone. Finally in Chapter 5, the role of the cytoskeleton in chondrogenesis was investigated, as well as the regulation of a key signaling pathway to stimulate the production of cartilaginous extracellular matrix. Shortly, the chondrogenic phenotype is characterized by a round cell morphology and a cortical arrangement of actin fibers, while dedifferentiated cells adopt an elongated fibroblastic morphology, with thicker actin stress fibers being a prominent characteristic. In particular, the Rho signaling pathway is known to orchestrate the (re)organization of the cytoskeleton, and the inhibition of RhoA, a key member of this pathway, was shown to stimulate chondrogenesis. Specifically, C3 transferase is a bacterial enzyme known to be a potent RhoA inhibitor. This protein was genetically engineered to allow its conjugation to a hydrogel. By immobilizing the C3 transferase variant on a modified form of alginate, alginate vinyl sulfone, safe, sustained and localized RhoA inhibition was achieved. The functionalized hydrogel was mechanically stable, had a long-lasting enzymatic activity and bovine chondrocytes encapsulated in this biocompatible hydrogel produced a collagen type II-rich extracellular matrix in vitro and in vivo. This cell-instructive hydrogel could have the potential to treat several pathologies where RhoA dysregulation plays a role, including osteoarthritis. Throughout this thesis, several approaches were investigated to provide solutions to the debilitating cartilage-related pathologies. Towards this aim, promising tissue-engineered scaffolds and strategies to improve current drugs were developed.