Optimization of an injectable gel containing nasal chondrocytes for the one-stage intraoperative articular cartilage repair

Articular cartilage has a very limited self-repair capacity and once damaged, it undergoes irreversible degenerative changes. Ongoing studies are carried out in the field of tissue engineering with the goal to find an approach that will lead to a high quality and durable hyaline cartilage repair. The engraftment of a cartilage construct engineered with autologous articular chondrocyte is a novel and promising approach for the treatment of articular cartilage lesions. However, this procedure is costly (due to the necessity of two surgical interventions and long culture required to generate the graft) and results in unpredictable clinical outcome (due to the large donor variability in the performance of articular chondrocytes). Recent studies demonstrated that nasal chondrocytes (NC) represent an alternative cell source for the treatment of cartilage lesions, since they can be obtained from a compartment that is not affected by the joint trauma under local anesthesia (i.e. the nasal septum), have a superior and more reproducible capacity to generate functional cartilaginous tissues and show similar responses to mechanical and inflammatory stimuli to articular chondrocytes. With the main goal of repairing traumatic articular cartilage lesions of small-medium size (2-5 cm2), a possible therapeutic approach currently under investigation is the one-stage, intraoperative injection of a polyethylene glycol (PEG) hydrogel containing freshly isolated nasal chondrocytes. It has already been demonstrated that NC efficiently proliferate and produce cartilage matrix once cultured in a PEG hydrogel containing platelet lysate (PL-PEG). However, having in mind a future translation into the clinics, some aspects of the gel formulation should be optimized to guarantee a proper injection. Thus, I have aimed this study first at simplifying the gel formulation to achieve a more cost-effective and time-effective fabrication in the context of obtaining the marketing authorization. This was realized by removing the RGD adhesion ligand, since several studies showed that its role could be fulfilled by other ECM-derived components whose structures contain the RGD motif, like fibronectin (which is found in the platelet lysate in our gel formulation), without having a negative influence on the embedded cells functions. The second aim was reducing the polymerization time of the gel which was not suitable for intraoperative use (approx. 4 minutes), while maintaining the viability, proliferation and chondrogenic capacity of the cells. NCs from a total of five nasal septum biopsy donors were therefore rapidly isolated and cultured in PL-PEG hydrogels of discoid form at a low cell density mimicking the 5 intraoperative yield (0.3M cells/mL from a 6 mm diameter biopsy) for 21-28 days in the different experimental conditions and then biochemically and histologically analyzed. The results show that NCs embedded in PL-PEG hydrogel have remained viable, proliferated and differentiated at a similar extent in the presence or absence of RGD peptide. Moreover, the gelation time of the injectable hydrogel was successfully reduced to an intraoperative optimum (1 minute and 16 seconds) by increasing the concentration of Factor XIII without impairing with the cell proliferation and differentiation capacities. Nonetheless, in order to acquire statistically significant data, at least three additional donors are needed to confirm the observed trends. In addition, different spreading and proteoglycan accumulation patterns throughout the gels in cross sections were observed, leaving the gel with some regions of still undifferentiated fibroblastic-like cells. Mechanical tests will be necessary to evaluate the change in stiffness, which plays a key role in understanding the effect of FXIII in the gels, and consequently, the response of nasal chondrocytes to such conditions. Also, future in vivo orthotopic studies would be necessary to show the efficacy of the NC-PEG construct at articular sites, as well as the integration of the implant in a stable way with the surrounding tissue