Performance of nasal chondrocytes in an osteoarthritic environment

Purpose: The implantation of autologous articular chondrocytes (AC) is an established surgical approach for the treatment of traumatic but not yet osteoarthritic (OA) lesions. This cell-based therapy indeed results in an unpredictable outcome when applied for the treatment of chronic cartilage defects, probably due to the poor quality of the isolated AC. Nasal chondrocytes (NC) could represent an attractive alternative cell source for the repair of OA cartilage defects due to their procurement from a ‘‘healthy” heterotopic compartment. In a recent first-in-human trial (ClinicalTrials.gov, N° NCT01605201) we have demonstrated that implantation of tissue engineered cartilage grafts generated with autologous NC (N-TEC) is feasible and safe. With the final goal to assess the possibility to extend our nose2knee concept towards the treatment of OA defects, we investigated the responses of N-TEC when exposed to inflammatory factors (Study1), its capacity to modulate the inflammatory profile of OA joint cells (Study2) and their integration into OA osteochondral (OC) tissues in vivo (Study3). Methods: Nasal and articular cartilages were collected from the nasal septum or the knee of healthy patients. Osteoarthritic AC, OC tissues and OA synovial cells (Syn) were collected from the joints of patients undergoing knee replacement. The corresponding cells were released through enzymatic digestion. NC and AC were expanded and re-differentiated for 2 weeks in chondrogenic medium. Study1: The resulting cartilaginous engineered cartilage graft (N-TEC and A-TEC) were exposed for 2 weeks to the pro-inflammatory cytokines IL-1β, TNFα and IL-6 [0.05–1ng/mL] and analysed (Immuno)histologically (Safranin-O, Collagen-II), biochemically (glycosaminoglycan -GAG- and DNA) and by RT-qPCR.Study2: Conditioned media (CM) from N-TEC or A-TEC was applied to (a) OA-Syn cultured in monolayer or (b) OA_AC cultured on a microfluidic device. The modulatory effects of TEC-CMs were determined immunohistologically (Collagen-II, MMP-13) and by quantification of secreted cytokines (Luminex) and expressed genes (RT-PCR). Study3: N-TEC were applied into cartilage defects of 6 mm in diameter created in OA-OC plugs prior implantation in a subcutaneous pocket of nude mice. After 8 weeks, explanted tissues were characterized (immuno)histologically. Results: Study1: GAG contents in N-TEC were not significantly affected following exposure to the tested inflammatory cytokines when compared to control condition. A superior cartilaginous matrix, however, was observed histologically and biochemically for N-TEC, as compared to A-TEC, confirming the superior cartilage forming capacity of NC. Study2a: When N-TEC_CM or A-TEC_CM was applied to OA-Syn, secretion of several pro-inflammatory cytokines was significantly reduced. However, reduced levels of TNFα and IL-6 secretion were only observed following exposure to N-TEC_CM (up to 1.6-fold). Study2b: In a microfluidic device, N-TEC_CM reduced -to a higher extend than A-TEC_CM- the expression of catabolic/inflammatory mediators such as MMP-13 (observed by immunostaining) as well as ADAMTS-5 and IL-8 (>10-fold, quantified by RT-PCR). Study3: In all explants (n = 3), N-TEC maintained their cartilaginous extracellular matrix and become well integrated to the subchondral bone and the adjacent OA native cartilage. Conclusions: Together our results indicate that N-TEC can be envisioned for the treatment of OA cartilage defects, due to their capacity to: (i) maintain cartilaginous matrix after prolonged exposure to inflammatory cytokines, (ii) damper the production of inflammatory/catabolic molecules by OA joint cells, (ii) to preserve the cartilaginous features in an in vivo OA environment where they successfully integrated to the adjacent native tissues.