"Functional" will mean different things to different labs depending on their research focus. There are a lot of metrics people use; it depends on what you want to study. To show extent of differentiation, most folks like to use Alcian blue staining in conjuction with qPCR or Western blot measurements of chondrocytic marker genes such as collagen IIa1, aggregan, sox9, and Gli1. In the context of implantable constructs for tissue engineering, mechanical testing is a critical assay.

I agree with Dr. Cohen. Please note that the components of the media used for chondrogenic differentiation really matter. Also, the markers should show sustained expression. Primary chondrocytes easily loose Collagen ii a and become fibroblastic with collagen I after 304 days in culture. M. Goldman has many good papers on this. She used C3H10T1/2 cells. Hope this helps!

Error--it should say 3-4 days (not 304) days. Sorry!

Thanks Dr.Cohen...By saying functional i meant if i could possibly do some kind of co-culture or others using mesenchymal stem cells maybe with articular chondrocytes to see if mesenchymal stem cells can differentiate with the paracrine signals to hypothesise their differentiation and engraftment in-vivo.

Charan- qPCR would be a good approach in theory; however, it may be difficult to apply in your experimental system. Assuming the articular chondrocytes are from the same species as your mesenchymal stem cells, any qPCR study of the population would be dominated by signal from your chondrocytes (unless you could separate the two populations of cells prior to lysis). Is there any way you can separate the two populations (physically or based on species identity? If not, you will have to prelabel your hMSCs and perform immunostaining or in situ hybridization for chondrocytic markers, and then score differentiation based on fluorescence microscopy images.

If you are considering co-culture, you will find that explants of articular hyaline cartilage are preferred to articular chondrocytes-I gave the reason above. We and others have published data showing that explant cultures work v. well for evaluating anti-arthritic and anti-inflammatory drugs. This suggests that the explants are giving physiologically meaningful results which in turn means that the paracrine signalling in intact. This is not surprising because the ECM of explants is undisturbed. In the case of Articular chondrocytes, there is no authentic ECM and they rapidly de-differentiate in vitro. Hope this helps!

Thank you so much for your valuable inputs.I have another query...Could quantification of collagen or proteoglycans relate to higher levels in differentiated cells compared to the control(MSC)?Could there be more collagen type 2 or sGAG deposition in the differentiated cells when compared to the undifferentiated cells?There could aslo be a lot of ECM release from the undifferentiated MSCs also right?

Charan- I think both cell types could contribute. MSCs could differentiate and produce these marker genes. Chondrocytes might increase their degree of differentiation in the co-culture system.

Degradation of cartilage or rapid turnover of cartilage also causes release of collagen type 2 or sGAG. So, finding a marker of differentiated cartilage is tricky. Pl. check on the other types of collagen (23 in total !). A profile of collagen isoforms and particular Proteoglycans may be indicative of differentiated cells. There are other PCR markers which we did not try. Pl. check M.Godring's (not M.Goldman) work. Hope this helps!

Charan, Glad to be of help. The C3H10T1/2 cells are used by M. Goldring for differentiation assays. Rabbit chondrocytes were used in chondrogenesis assays with alcian blue for identifying drugs which can harm cartilage development. Hope this helps!