Possible that the cartilage pellet is contaminated with bone or calcified cartilage, the celltypes within each of the contaminants may produce type I collagen- perhaps a labelling experiment to see if any of the cells are transforming - I did some research on chondrocytes in the long bone fracture of rodents and observed transformation into osteoblasts.

Thanks for your answer, Shepard,

This micromass cartilage or pellet culture originated from Mesenchymal stem cells under chondrogenic medium containing TGFB and we did not isolate chondrocytes from cartilage.

Mesenchymal stem cells are likely pre-programmed to go down the pathway toward osteoblasts, while the environment the ex-vivo cells are grown is most likely to maintain a carilage phenotype, some cells due to local facotrs (pH, O2 tension, autocrine factors) may be differentiating into cells that produce type I. A protein array may discover proteins suggesting other cells liens developing.

Thanks, Shepard, for your help,

Best regards,

Subhash

We actually see types I, II, VI, and X when we chondrogenically induce MSCs (and ASCs).  It's not uncommon.  One thought is that during development there is a cellular condensation event prior to joint development that has been shown to be rich in type I collagen.  Maybe micromass culture is recapitulating that environment, and due to this specific environment, type I collagen synthesis is stimulated.  This is also why I never use the the term "cartilage" when differentiating stem cells.  It would be impossible to prove that it's cartilage, and certainly there would be "contaminating" tissues, hence the reason I focus on function of the tissue engineered "cartilage-like" material.

Thanks, Bradley, 

Subhash

What concentration of TGF are you using? If you use too much TGF (more than 10ng/ml) it will induce terminal differentiation of MSC. There will be no certain point of your differentiation on which you can stop and stay these are exactly matured chondrocytes. I would also adive to use combination of TGF and BMP (something like 50 ng/ml of BMP2). BMP2 signaling via ALK1 is necessary for proper chondrogenic differentiation at the early stage. Good luck!

Thanks, Dr. Madej,

I used 10ng/ml. Your advice is valuable. 

Thanks,

Subhash

We obtain chondrocytes in primary cultures, from articular cartilage, and also observe some positive staining for type I collagen. It is described that cells de-differentiate in culture and express different proteins than in vivo.

Thanks, Maria, sharing your important observation,

It is becoming clear (as described by earlier scientists) that conditions in making of native cartilage can not match exactly that of in vitro, at least for now till we are able to get rid of COL I, Dr. Madej made additional suggestions.

Thanks, again,

Subhash

Hi,

One noticeable additional thought is that in ATDC5 cells which are supposedly a good representation of the develomental differentiation of chondrocytes, type I is strongly expressed in initial phases of the differentiation process. However, this is mainly, in my opinion, because at this point they are NOT differentiated. (they already express it highly in growing phases without insulin). at D14 of differentiation, there is almost NO type I remaining in their ECM. Farther, chondrocytes actually expressed very little type I in cartilage (bearely detectable) and then quickly turn it on when cultured. altogether, my main impression is that type I is primarily a sign of imperfect differentiation/dedifferentiation. (in agreement with several above comments).

regarding MSCs and the use that people try to make of it for cartilage repair, type I expression is a major limitation and they actually try to find ways to avoid it. without such success so far...

cheers

JBV

Thanks, Dr. Vincourt, for sharing your experience and your thoughts,

Best regards,

Subhash

Hi Subhash,

I agree with the other researchers. At the initial phases of chondrogenesis, the primitive matrix that enables condensation of cells is rich in collagen type I. During normal development this should be downregulated and cells should start expressing collagen type II. However, as others have pointed out, MSCs seem to follow an “unnatural” chondrogenic differentiation pathway in vitro, and maintain collagen type I expression. They also seem to follow their “pre-programmed” way of differentiating towards hypertrophic chondrocytes.

Embryonic limb bud-derived microbes cultures also seem to maintain a steady level of collagen type I mRNA expression as we have described in the following paper.

Hope it helps.

BW,

Csaba

Article Comparative Analysis of Osteogenic/Chondrogenic Differentiat...

Thanks a lot, Csaba,

Thanks for sharing your experience and your paper, that is helpful,

Best regards,

Subhash

Hi, 

in addition, one should consider, that in vitro cultured differentiating chondrocytes may produce not only hyaline cartilage matrix but fibrous cartilage as well, which normally is rich in type I collagen fibers. Monitoring of the ratio of type I and type II collagen expression is helpful to distingushe the tissues.

Formation of fibrous cartiilage is parricularly prominent in case of articular cartilage regeneration, when formation of fibrous cartilage, mechanically inferior to hyaline cartilage, is one of the problematic issues of application of hMSCs.

best regards,

Roza

Thanks, Roza, Good point to think about. 

Best,

Subhash