Proliferation of human articular chondrocytes in 3D culture systems


Moreno Masip, Marc

Osteoarthritis is a degenerative disease affecting mainly the joins by progressively deteriorating the articular cartilage and its associated bone tissue.
Our knowledge about this particular pathology has increased dramatically over the decades but nevertheless we are still in a very early therapeutic stage. This disease affects many the elderly fraction of population, but it can be diagnosed in younger people. The origin of osteoarthritis is related to multiple factors but in general the presence of non-well healed articular injuries, the presence of senescent cartilage cells (chondrocytes), a genetic predisposition and the poor regenerative capacity of cartilage tissue might influence the initiation and progression of the disease. An interesting approach to overcome these drawbacks is the use of cartilage tissue engineering strategies to assist repair and regenerate the articular cartilage. One of the most widely used approaches is the implantation of autologous chondrocytes (ACI) at the injured site. Briefly, this methodology consists in the isolation of the patient's own chondrocytes from a small biopsy, an in vitro expansion, a cartilage construct development in vitro and a reimplantation. However, there are still some obstacles that limit the use of this therapy. The main issue is the use of classical culture dishes to expand the cells outside the body, what it is known as 2D culture technique. In this conditions chondrocyte undergo rapid proliferation (expansion), cell de-differentiation or loss of the tissue commitment and most importantly accelerated ageing (turning into senescent cells). In order to overcome these obstacles, three-dimensional cultures (3D) have emerged. Such cultures are able to mimic the microenvironment found in native tissues and offer a more realistic option than 2D cultures. Specifically, in this work, RAD16-I, which is a self-assembling peptide hydrogel, was employed to create a scaffold that will be used as a 3D culture system. Different types of cells have been cultured in this 3D scaffold and their proliferative capacity have been evaluated. Further, to test whether or not the 3D culture system reduces the cellular senescence rate during cell expansion different assays were designed to run side by side 2D versus 3D cultures. The degree of senescence was evaluated by measuring the levels of β-galactosidase activity, which is a senescent biomarker, and the proliferation rate was measured by MTT assay. Moreover, Immunoassays and western blots were performed to identify the presence of telomerase reverse transcriptase (TERT). Finally, differentiation capacity of expanded chondrocytes was also evaluated after expansion in 2D and 3D culture systems.



Semino Margrett, Carlos


IQS SE - Master’s Degree in Bioengineering