Articular cartilage regenerative strategies based on platelet derivatives with perspective view in 3D-bioprinting technology
Creators: name: CARLUCCIO, SIMONETTA
Publisher: name: Universit� degli studi di Genova
Year: 2020

description: Articular cartilage is an unique and highly specialized connective tissue adapted to bear compressive loads and shear forces in synovial joints, which is a crucial function during body?s motion. In adult joints, articular cartilage is composed by chondrocytes immersed in an intricate extracellular matrix, displaying a complex multi-zonal organization. Despite a relatively low metabolic activity within a harsh physical environment, chondrocytes are active in maintaining the matrix, thus allowing healthy tissue to sustain itself and carry out its functions. 
Damage to tissue high level of organization and molecular architecture is a major source of morbidity for articular cartilage, thus it is usually susceptible to malfunction following acute injury or chronic diseases such as osteoarthritis. Cartilage has poor intrinsic repair and regenerative capacity, although it has been demonstrated to contain a subset of progenitors even in adults. These cells have shown to react to injury, but do not seem to be able to mount an effective tissue reparative or regenerative action when needed. 
Hence, much effort has been devoted to finding ways by which articular cartilage repair could be induced and enhanced. Surgical techniques and bioengineered treatment options developed over recent decades have led to several clinical treatment modalities for acutely injured or osteoarthritic joints. To prevent progressive cartilage degeneration or to replace damaged tissue, the surgical treatment is often the only option, but it does not ensure full tissue function recovery. Therefore, regenerative medicine has emerged as an important field of research in the treatment of cartilage disorders. In this context, the medical community have shown great interest in therapeutic strategies based on the use of platelet-derived products, such as platelet rich plasma (PRP) and platelet lysate (PL). Since these derivatives are a mix of growth factors, cytokines and chemokines normally involved in tissue healing, the rationale behind their application is the re-activation of latent endogenous regenerative mechanisms.
PRP therapeutic use in musculoskeletal disorders have led to promising outcomes, although mechanism of action and efficacy of platelet products in orthopaedics still need to be elucidate. 
The main objective of this PhD thesis is to study the effects of platelet derivatives on cartilage cell behaviour, including chondrocytes and chondro-progenitors, in order to highlight potentialities and even identify limits concerning their use, both useful in better direct biomedical applications in the field of articular cartilage therapy. Indeed, a better understanding of the events that could induce cartilage repair by PRP or PL may allow to clarify current experimental outcomes and offer the opportunity to conceive innovative strategies or tools in cell therapy approach and in the latest tissue engineering technologies.
In this regard, it will be beneficial to find alternative options to cell transplantation (based on mesenchymal stem cells or chondrocytes) aimed in achieving cartilage regeneration, planning interventions focused on in situ stimulation of resident cell population or local progenitor niche in the joint, that are developmentally endowed with greater chondrogenic potential than traditional cell sources. 
In parallel, the research in the field of tissue engineering continues to be active and innovative strategies for the fabrication of enhanced bioengineered grafts are recently emerging by the spreading of 3D-printing technology. 3D-bioprinting especially represents a developmental biology inspired alternative to classic scaffold-based approaches in the field, since it shows the ability to assemble biological components replicating complex native-like tissue architecture more faithfully than traditional methods of assembly as well as patient customization. In this context, bioprinted constructs may provide a solution for cartilage injuries and defects, despite 3D-bioprinting is still a technology in progress and consequently some challenges have to be overcome before its translation to clinical applications.
The research tasks of this work and the main obtained results are: 
1) the in vitro characterization of human articular chondrocyte responses at PL treatment with focus on the recruitment/re-activation of a chondro-progenitor cell population from PL-treated cartilage explant cultures. 
Stem cell-based therapies to achieve articular cartilage regeneration attract great interest. Stem cell niches are located within the joint, where they could participate directly in tissue homeostasis and repair processes. It is reasonable that exploiting local chondro-progenitors for cartilage repair may be a better and more efficient cell-based therapeutic strategy compared to the use of mesenchymal stem cell from different sources, given that they are developmentally primed for differentiation into chondrocytes. Furthermore, in the field of regenerative medicine therapeutic strategies targeting stem cells in situ could be more attractive and more advantageous than stem cell transplantation. According to this approach, endogenous stem cells could be recruited to the injury site by administration of bioactive factors. Thus, in the last decades, among a wide range of products, PRP has spread as a clinical treatment tool for musculoskeletal diseases. Several studies have investigated PRP or PL roles both in vitro and in vivo, highlighting their capacity to exert anti-inflammatory and proliferating effects on cells, as well as to stimulate resident progenitors or to recruit circulating ones.
Primary cultures of human articular chondrocytes and cartilage chips were set up from donor biopsies and were treated in vitro with PL. Proliferation, clonogenic potential and phenotype of chondrocytes and chondro-progenitor cells derived from explant cultures in PL were characterized. Tri-lineage differentiation potential were tested in vitro and scaffold-assisted chondrogenesis of these cells were studied in nude mice. Moreover, secretory profile of chondro-progenitors were analysed together with their migratory capabilities by mimic osteoarthritis in vitro. Finally, it was reported that ex vivo treatment of human articular cartilage with PL induced activation and outgrowth of cells showing features of stemness, such as clonogenicity and expression of nestin. The stimulation of nestin-positive progenitor cells induced by PL in articular cartilage is of particular interest for the future development of therapeutic strategies given the involvement of these cells in tissue regenerative processes. In addition, their high proliferation capacity with concurrent chondrogenic potential maintenance further sustain the potential of PL-mobilized chondro-progenitor cells as promising tool in the field of cartilage tissue engineering. Moreover, PL-induced effects on phenotype of mature articular chondrocytes were further characterized, showing that they can revert to an earlier stage similar to chondro-progenitor one.
2) Embedding and re-differentiation of primary human articular chondrocytes in PRP-based hydrogel suitable for 3D-bioprinting. 
Although the implantation of cultured chondrocytes intended for injured cartilage therapy is performed worldwide, there are still unresolved challenges associated with the maintenance of their chondrogenic phenotype. The expansion of chondrocytes in vitro is associated with de-differentiation, which is a reduction in the expression of cartilage-specific markers. Accordingly, such cells often produce fibrocartilage rather than native hyaline cartilage when used in clinical procedures. Several strategies to counteract this phenomenon have been adopted, such as 3D-culture of chondrocytes encapsulated in biomaterials. Adoption of hydrogels attracts particular interest in cartilage regeneration since they provide a highly hydrated environment similar to that of native tissue. In this context, progresses are expected thanks to the application of the 3D-bioprinting. However, the field of biofabrication often strongly focuses on the biomaterial rheology to allow controlled production, taking less care of its inherent impact on cellular phenotype. The combination of both aspects can be achieved by incorporation of biological components in printable hydrogels that often lack of bioactivity. 
Primary human articular chondrocytes derived from previous monolayer culture expansion were bioprinted by embedding them in a commercial available alginate-based ink and their ability to regain the chondrogenic potential both in vitro and in vivo was evaluated. Interestingly, an improved ability to sustain cell viability, proliferation and a certain degree of chondrogenic phenotype rescue were found inside the bioprointed constructs by adding PRP as a source of biological agents in the ink formulation.