Another relevant ECM fibrillar component of the TMJ disc is coll II. Strikingly, the expression of this protein was low at all passages, which is in agreement with previous reports, and it did not vary along subculturing. Other less abundant collagen fibers such as coll III, IV, V, VI, IX, XII, XV and XVI also tended to decrease with sequential passaging. In general, these collagens form a 3-D structure that associates with coll I and II to constitute the main scaffold of the cartilage TMJ. Taking together, these results imply that most fibrillar ECM components did not vary with sequential culturing and, in the cases that tended to decrease, the expression levels are relatively high at most subcultures. In the second place, the analysis of non-fibrillar ECM components confirmed that the majority of genes did not decrease along consecutive cell passaging, although some specific genes did significantly vary upon subculturing. Non-fibrillar components of the ECM play an important role in cartilage homeostasis, cell adhesion and hydrostatic balance. One of the most important nonfibrillar ECM components are glucosaminoglycans and mucopolysaccharides that tend to associate to proteins to form proteoglycans, which are able to attract water molecules via osmosis to keep the ECM hydrated, as well as growth factors. In this regard, it is important to note that several relevant GAGs and PGs maintained their expression during sequential culturing including, versican, lumican, dermatan sulfate, etc. However, our results reveal that some genes could diminish their expression upon subculturing, including biglycan, decorin aggrecan and some genes encoding for chondroitin-sulfate, hyaluronic acid and heparan sulfate. This finding suggests that TMJF cells could not be able to generate an efficient fibrocartilage ECM at advanced cell passages. Chondroitin-sulfate may be the predominant proteoglycan present in cartilage. Interestingly, the highest intracellular sulfur concentrations correlated with the highest expression of chondroitin-sulfate genes, with the most functional levels found at P4 and P5. Hyaluronan Cinoxacin synthase 1 plays a role in of hyaluronan/hyaluronic acid synthesis and may be involved in prevention of cartilage destruction by the continuous production of HA. However, the tendency to decrease that we found here reveals that these cultured cells should be used at the earliest cell passages. On the other hand, biglycan, aggrecan and decorin are crucial components of the ECM. These proteins are involved in collagen fiber assembly and play an important role in the organization of the fibrillar ECM. Although the decreasing trend of these three proteins suggests that they should be used at the first cell passages, the gene expression levels of these elements were high at P5 as determined by microarray. Regarding other ECM proteins of interest, including glycoproteins our results showed that some Mechlorethamine hydrochloride cartilage-related genes decreased with culturing including chondrolectin, cartilage intermediate layer protein and cartilage oligomeric matrix protein, whereas two laminin genes tended to increase. Notwithstanding the role of these ECM components is less known, these results point out the need to use early cell passages in regenerative protocols. All our findings related to ECM components expression could suggest that TMJF cells could be functionally adequate until at least P5. The behavior from P5 to P9 could be associated to response mechanism to the dedifferentiation effects caused by ex vivo adaptative conditions.