Novel strategies have been proposed for articular cartilage damage occurring during osteoarthritis (OA) and -among these- Extracorporeal Shock Wave Therapy (ESWT), intra-articular injections of Platelet-Rich Plasma (PRP) or Hyaluronic Acid (HA) revealed encouraging results.
Thanks to our previous experience on in vitro models of human tendon derived cells, we established here primary cultures of human chondrocytes derived from cartilage explants and measured the in vitro effects of ESW, PRP and HA therapies. After molecular/morphological cell characterization, we assessed those effects on the functional activities of the chondrocyte cell cultures, at the protein and molecular levels.
Our preliminary data suggest that ESWT significantly prevent the progressive dedifferentiation that spontaneously occurs during prolonged chondrocyte culture. We then attested the efficiency of all such treatments to stimulate the expression of markers of chondrogenic potential such as SOX9 and COL2A, to increase the Ki67 proliferation index as well as to antagonize the traditional marker of chondrosenescence p16INK4a (known as Cdkn2a). Furthermore, all our samples showed an ESW- and HA-mediated enhancement of migratory and anti-inflammatory activity onto the cytokine-rich environment characterizing OA.
Taken together, if confirmed, those results suggest a regenerative effect of such therapies on primary human chondrocytes in vitro.
Moreover, we also plan to investigate the ESW treatment effects on the chondrocyte surface expression of major hyaluronan cell receptor CD44, which could enhance the HA benefits in patients affected by OA.
The last step of this project will require an application in vivo of our observations derived from this cell model. In particular, clinical protocols will be studied in order to better define the correct combination of such therapies able to provide novel cues to improve in vivo articular cartilage repair.
Osteoarthritis is traditionally classified into different stages of the disease. During the acute inflammation, anabolic molecules, including some Growth Factors and Cytokines (e.g. TGF-ß ligand signaling pathway), should protect cartilage against progressive loss, actively stimulating chondrocyte-mediated cartilage repair. In fact, in this stage, mechanical stimulation of articular cartilage may be more efficient in inducing chondrocyte synthetic activity. On the contrary, advanced osteoarthritis is characterized by a chronic inflammation that -through a panel of catabolic mediators (e.g. Inflammatory Cytokines and Proteases)- participate in progressive cartilage matrix degradation and unable to reverse cartilage damage (Brittberg NEnglJMed 1994; Zhang BoneRes 2016; Duan AmJTranslRes 2015).
Chondrocytes are directly involved in the production of cartilage ECM, but -similarly to other cell cultures (e.g. tenocytic lineage)- their expansion in vitro is unluckily associated to a natural tendency to dedifferentiate toward a fibroblast-like phenotype, which is unsuitable for cartilage production, as recently reviewed (Charlier, BiochemPharmacol 2019). Also for this reason, previous attempts to maintain the chondrocyte phenotype, to develop an optimal approach for autologous cell implantation in patients affected by severe osteoarthritis, have been partially unsuccessful (Zhang, BoneRes 2016; Cook, AmJSport Med 2016). To date, several types of research did not definitively clarify the complex molecular mechanisms underlying chondrocyte differentiation, even though it is well accepted that it is influenced by a large panel of microenvironmental mediators.
The innovative goal of this project is to provide novel strategies to better investigate the chondrocyte differentiation under mechanical and/or biological stimuli, in order to explain the clinical benefits induced by those therapies in vivo.
In particular, this work may contribute to the ambitious aim to optimize the cell culture conditions, finalized to autologous chondrocyte implantation in patients affected by advanced osteoarthritis, with very limited repair capabilities. In fact, novel approaches are diffusely required to counteract the previously described chondrocyte dedifferentiation, also for ameliorating the efficiency of cell-based cartilage therapy.
Emerging data seem to suggest a central role of several mediators of cartilage damage and repair, which can drive chondrocyte differentiation and then significantly influence the mechanisms of cartilage remodeling. In particular, during OA, quiescent chondrocytes with limited matrix turnover undergo phenotypic modulation by both inflammatory and anabolic cytokines (Goldring EurCellMater 2011; Boehme IntJMolSci 2018). Nevertheless, a better knowledge of the biological mechanisms triggered by ESWT, HA and/or PRP approaches may implement their use -particularly but not exclusively- in the early stages of OA, when an efficient cartilage repair/regeneration could be yet obtained. Exploring the molecular mechanisms underlying ESWT and PRP may contribute to elucidate which cocktail of mediators may be administered in vivo to regenerate the degraded cartilage.
Finally, as we recently published (Leone, Oncotarget 2015), human stem cell are now considered a promising tool for the management of several musculoskeletal disorders, albeit the modalities of their effective contribution to tissue regeneration in vivo are still unclear. Considering the complexity of the intracellular signaling pathways involved in cell differentiation, our work could allow new information for using satisfactory autologous stem cell therapies, especially in OA patient refractory to other treatments.
The final goal of this project is the translation of those in vitro data for establishing novel clinical protocols in vivo, able to provide a better management of OA, and also to support the potential of such combined therapies in inducing cartilage regeneration.