Invited presentation at the NC3Rs and BBSRC Symposium 1-2 April 2009, Royal College of Surgeons, London.

1. A Tissue Engineered Model of Osteoarthritis Challenges and Opportunities for Applying the 3Rs in Osteoarthritis Research Ali Mobasheri Tissue Engineering Symposium 1-2 April 2009

4. The Chondrocyte Nucleus Cytoplasm ECM Synthesizes a mechanically resilient extracellular matrix of collagens and aggregating proteoglycans

5. Major Constituents of Articular Cartilage Matrix Collagen IX Collagen II Fibronectin COMP Aggrecan Hyaluronan Chondrocyte Thrombospondin Decorin Biglycan Fibromodulin

9. General Features of OA Synovial space (as assessed by radiography) Cartilage anabolism and impaired cartilage repair Synovial inflammation and hyperplasia Proteolytic activity (collagenases, gelatinases) Cartilage degeneration

10. Cartilage Fibrillation and Loss of Proteoglycans in OA Human femoral head H&E Safranin O Safranin-O stains glycosaminoglycans red

11. Molecular Changes in OA SYNOVIAL MEMBRANE SYNOVIALFLUID ARTICULAR CARTILAGE SUBCHONDRAL BONE IL-1 β TNF- α Pro-MMP ROS IL-1 β TNF- α PGE 2 IL-1 β TNF- α Hyaluronate degradation Pro-MMP MMP ECM DEGRADATION Plasmin Plasminogen Serine proteases Cysteine proteases ↓ ECM synthesis Substance P Pain TRAUMA + INFLAMMATION Apoptosis MMP Adapted from Goodrich (2006) chondrocyte TRAUMA TRAUMA

15. Animal Models of OA ACT Medial Meniscectomy Anterior Cruciate Transection (ACT) Meniscal Transection SURGICAL CREATION OF JOINT INSTABILITY Ligament Transection Carpal Chip Fragmentation ACT Canine Groove Model

16. Animal Models of OA SURGICAL REPLICATION OF JOINT TRAUMA Ligament Transection Carpal Chip Fragmentation ACT Canine Groove Model Ovine Groove Model Surgical Lesion Model

20. In vitro Models Chondrocyte Monolayers Curcumin 6.25 μ M Curcumin 12.5 μ M Curcumin 25 μ M Curcumin 50 μ M Ideal for Toxicity testing

21. Chondrocytes isolated by overnight collagenase digestion Cells counted and seeded into pre-gel mix Alginate beads set by addition of Ca 2+ Cultured at 37 0 C, 5% CO 2 in DMEM + 10% FCS Cells released by chelating calcium with EDTA Downstream applications In vitro Models Culture of chondrocytes in alginate

25. Mesenchymal Stem Cells from Bone Marrow Stromal Cells from Bone Marrow (Mesenchymal Stem Cells) In addition to red blood cells, white blood cells and platelets bone marrow contains stromal cells which are also known as mesenchymal stem cells

26. Mesenchymal Stem Cells Cultured Mesenchymal Stem Cells Fully Differentiated Connective Tissue Cells for Tissue Engineering and Autologous Transplantation Myocytes Cardiomyopathies Tenocytes Tendonitis Osteoblasts Bone diseases Chondrocytes Osteoarthritis

27. Multipotency of MSCs Osteoblasts (Von Kossa, Calcium) Adipocytes (Oil red, Fat vacuoles) Chondrocytes (Alcian blue, CSPG) MSCs

28. In vitro Models High-Density Co-Culture System Petri dish Culture Medium Steel bridge Filter Cells (Chondrocytes + MSCs) + Growth Factors

29. High Density Co-Cultures I II III MSC Chondrocyte Merge

30. Cartilage Formation in 3-Dimensional Co-cultures M M C C C

31. EM Evidence for Chondrogenesis Perichondrium Cartilage Matrix Chondrocyte

32. Electron Micrographs of High Density Co-cultures

33. Evidence for Collagen Type II Chondrocyte IEM:

34. Evidence for Cartilage Specific Proteoglycans (CSPGs) Chondrocyte IEM

35. Western Blotting: Collagen Type II 100% MSCs+GF 100% Chondrocytes 50%/50%MSC/ Chondrocytes 100% MSCs-GF 200 kDa 100 kDa 60 kDa

36. Western Blotting: CSPGs 200 kDa 150 kDa 60 kDa 100% MSCs-GF 100% MSCs+GF 50%/50%MSCs/ Chondrocytes 100% Chondrocytes

37. Evidence for cell-cell contacts

40. Acknowledgements Professor M. Shakibaei Munich Dr. Pat Harris (WALTHAM) Dr. David Allaway (WALTHAM) Collaborators: Dr. Stephen Richardson University of Manchester Prof. Judith Hoyland University of Manchester

41. Funding: Acknowledgements

42. Thank you for your attention