Tim Hardingham, BSc PhD DSc
We are investigating gene expression changes in human articular cartilage and fibrocartilage associated with ageing and pathology, which drive the processes that lead to tissue degeneration and arthritis and we have an active programme in cartilage tissue engineering. Array analysis of chondrocytes and OA tissue has identified genes regulated by SOX9 that are associated with osteoarthritis and we showed that a matrix forming phenotype was re-initiated in isolated chondrocytes from OA tissue when retrovirally transduced with SOX9. Increased expression of SOX9 in these cells potentiated an anabolic response to 3D cell culture, growth factors and hypoxia and showed that chondrocyte from OA joints were able to recover these responses and behave similarly to cells from healthy joints.
The scientific story
To generate cartilage we are investigating the physical cues and biological signals that drive the differentiation of chondrocytes from stem cells of different origins, including from bone marrow, infrapatellar fat pad and from human ES cells. We have shown that Notch signalling is active early in chondrogenesis of 3D cell aggregates of human bone marrow MSC. There was a transient peak of expression of the Notch ligand Jagged-1 and evidence of downstream Notch signalling. Over-expression of Jagged-1 with adenoviral transduction, which was accompanied by continued Notch signalling, completely blocked chondrogenesis. These results showed that Notch was active early, but must be switched off for chondrogenesis to proceed. We also established a new efficient culture system for MSC chondrogenesis in Transwells to form scaffold-free cartilage in just 2 weeks. All chondrogenic cultures with differentiated chondrocytes were found to respond to lowered oxygen conditions (5%), by increasing gene expression of cartilage matrix proteins, type II, IX collagen and aggrecan and increased matrix assembly. Cells from meniscus fibrocartilage also responded to low oxygen in chondrogenic culture, with increased HIF-1á expression and increased type II collagen. With low oxygen they produced a distinctive matrix, typical of fibrocartilage, with both type I and type II collagen and very little proteoglycan and this correlated with the difference in HIF-1á and type II collagen expression in the avascular (low oxygen) inner and vascular (high oxygen) outer meniscus. We are thus dissecting signaling pathways associated with chondrogenesis in order to make cartilage constructs more efficiently in vitro, or alternatively to deliver appropriate signals in vivo to drive chondrogenesis in cartilage repair.
Recent key publications
Oldershaw, R. A., Baxter, M. A., Lowe, E. T., Bates, N., Grady, L. M., Soncin, F., Brison, D. R., Hardingham, T. E. and Kimber, S. J. (2010) Directed differentiation of human embryonic stem cells toward chondrocytes. Nat Biotechnol. 28, 1187-1194.
Oldershaw, R. A. and Hardingham, T. E. (2010) Notch signaling during chondrogenesis of human bone marrow stem cells. Bone. 46, 286-293.
Jowitt, T. A., Murdoch, A. D., Baldock, C., Berry, R., Day, J. M. and Hardingham, T. E. (2010) Order within disorder: Aggrecan chondroitin sulphate-attachment region provides new structural insights into protein sequences classified as disordered. Proteins
Hardingham, T. E. (2010) Fell-Muir lecture: cartilage 2010 - the known unknowns. Int J Exp Pathol. 91, 203-209.