Innovative strategies for articular cartilage repair

Tissue engineering strategies for clinical treatments of cartilage defects are mainly based on transplantation of in vitro expanded autologous chondrocytes in the patient damaged tissue. However, the chondrocytes dedifferentiation during in vitro expansion often leads to suboptimal outcomes in the therapeutic intervention. Three-dimensional (3D) culture systems can revert this dedifferentiated state and re-establish chondrogenic phenotype. In this scenario, 3D-bioprinting is a technology suitable to create a patient-shaped grafts starting from cell-laden bioink by layer-by-layer fabrication. This allows to obtain a more physiological environment for transplanted cells, promoting tissue regeneration and repair. Since 3D printed scaffolds often lack reactivity, it was decided to functionalize a commercial bioink using Platelet Rich Plasma (PRP), a cocktail of platelet-derived growth factors involved in healing and tissue regeneration processes, with beneficial effects on proliferation and maintenance of differentiation of many cell types including chondrocytes. Thus, human articular chondrocytes (HACs) were isolated from patient tissue biopsies and expanded in vitro in monolayer (2D culture). 3D-bioprinting was performed embedding expanded HACs in Alginate or Alginate Platelet Rich Plasma (PRP)-mixed bioinks. The aim of this study is to evaluate the potential of 3D-bioprinting to improve cartilage tissue regeneration, exploring the use of different combinations of biomaterials, factors, and cells, reconstructing a cellular microenvironment as close to physiological as possible, creating an advanced, translatable and customized 3D-system. The purpose of this work is also to test the ability of primary chondrocytes, previously expanded in 2D monolayer, to regain their chondrogenic potential. By adding PRP as a source of biological agents, we want to test the ability of the enriched and functionalized ink to support cell vitality, as well as the recovery of the chondrogenic phenotype of dedifferentiated chondrocytes. Finally, data suggest that the 3D-bioprinting of HACs allowed them to recover chondrogenic phenotype and the embedding in PRP supplemented bioink supported chondrogenic culture inside the printed constructs. In view of future clinic translation, the choice of cell source for 3D-bioprinting of patient-specific grafts will have to be oriented towards cell types with higher potential, such as chondro-progenitors, than mature HACs in order to ensure a successful cartilage regeneration and repair. Furthermore, a support for 3D cell culture can be provided by decellularized extracellular matrix-based bioinks, able to engineer more the cartilage tissue and provide organ-specific microenvironment. Moreover, cells produce lipid-bound vesicles into the extracellular space known as Extracellular Vesicles (EVs). Exosomes and microvesicles, which come from the endosomal system or the plasma membrane, respectively, are two types of EVs that represent a diverse group of cell-derived membranous structures. They are a part of numerous physiological and pathological processes and are found in body fluids. EVs are currently thought to be an alternative method of intercellular communication that enables the flow of genetic material, proteins, and lipids between cells. To better understand the physiological and pathological roles of these vesicles as well as therapeutic applications requiring their usage and/or analysis, in this study a protocol for the isolation of extracellular vesicles directly from cartilaginous tissue and from conditioned medium has been studied and optimized. Currently, traumatic and degenerative pathologies involving articular cartilage are treated through symptomatic treatments (systemic pharmacological treatments and local intra-articular injections) and clinical repair treatments (surgical approaches such as microfracture and regenerative medicine). These different types of treatment, however, present difficulties that limit their application in the patient. Consequently, we set out a study of a functional protocol that would allow for the isolation of extracellular vesicles, both directly from cartilaginous tissue and from primary cultures of human chondrocytes, to search for a new therapeutic approach that would allow us not to use cells directly and overcome the limitations of other currently available therapies. Following the isolation of the EVs, both the characterization of HACs and of EVs isolated from conditioned medium and cartilage tissue were performed. Aware that the therapeutic effects of EVs depend on the load they have carried, and that tissue regeneration is also based on the healing factors brought by the EVs to generate a trophic microenvironment suitable for chondrogenesis and the maintenance of hyaline ECM, we think that the EVs used as therapeutic agents in their natural state after isolation from biological samples or conditioned medium as regenerative medicine tools to repair and restoration of cartilage.