A Tissue Engineered Model of Osteoarthritis Challenges and Opportunities for Applying the 3Rs in Osteoarthritis Research A...
Articular Cartilage <ul><li>Mechanically unique connective tissue designed to: </li></ul><ul><ul><li>withstand and distrib...
<ul><li>Avascular, aneural and alymphatic </li></ul><ul><li>Contains a single cell type: the chondrocyte </li></ul><ul><li...
The Chondrocyte  Nucleus Cytoplasm ECM Synthesizes a mechanically resilient extracellular matrix of collagens and aggregat...
Major Constituents of  Articular Cartilage Matrix Collagen IX Collagen II Fibronectin COMP Aggrecan Hyaluronan Chondrocyte...
Articular Cartilage Degradation <ul><li>Despite its durability, cartilage has a very limited self maintaining capability <...
Osteoarthritis (OA) <ul><li>Most common form of arthritis characterised by progressive deterioration and loss of articular...
Risk Factors for OA <ul><li>Age </li></ul><ul><li>Lifestyle/occupation </li></ul><ul><li>Joint trauma </li></ul><ul><li>Jo...
General Features of OA Synovial space (as assessed by radiography) Cartilage anabolism and impaired cartilage repair Synov...
Cartilage Fibrillation and  Loss of Proteoglycans in OA Human femoral  head H&E Safranin O Safranin-O stains  glycosaminog...
Molecular Changes in OA SYNOVIAL  MEMBRANE SYNOVIALFLUID ARTICULAR CARTILAGE SUBCHONDRAL BONE  IL-1 β  TNF- α Pro-MMP ROS ...
Rationale for Studying OA <ul><li>OA affects 1 in 6 adults </li></ul><ul><li>Most OA patients suffer from pain and disabil...
Animal Models of Osteoarthritis <ul><li>There are many animal models of osteoarthritis </li></ul>
Animal Models of OA INJECTION  INTO THE JOINT <ul><ul><li>Other related models: adjuvant injection for modelling pannus fo...
Animal Models of OA ACT Medial Meniscectomy Anterior Cruciate Transection (ACT) Meniscal Transection SURGICAL CREATION OF ...
Animal Models of OA SURGICAL REPLICATION  OF JOINT  TRAUMA Ligament Transection Carpal Chip Fragmentation ACT Canine Groov...
Animal Models of OA <ul><li>Disadvantages: </li></ul><ul><li>Ethical issues </li></ul><ul><li>Invasive nature of technique...
Alternative Models <ul><li>Researchers must constantly evaluate the relevance of their animal models to OA in humans </li>...
Important criteria for cartilage models for drug discovery <ul><li>Ability to monitor synthesis and release of inflammator...
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 Toxi...
Chondrocytes isolated by overnight collagenase digestion Cells counted and seeded into pre-gel mix  Alginate beads set by ...
In vitro  Models Culture of chondrocytes in alginate <ul><li>Advantages:  </li></ul><ul><ul><li>The preservation of the ch...
In vitro  Models The Explant Model <ul><li>Advantages:  </li></ul><ul><ul><li>Ideal for studies of extracellular matrix sy...
The Promise of Stem Cells and Tissue Engineering <ul><li>Stem cells </li></ul><ul><li>Biomaterials </li></ul><ul><li>Biore...
Mesenchymal Stem Cells from Bone Marrow Stromal Cells from Bone Marrow (Mesenchymal Stem Cells) In addition to red blood c...
Mesenchymal Stem Cells Cultured Mesenchymal Stem Cells Fully Differentiated Connective Tissue  Cells for Tissue Engineerin...
Multipotency of MSCs Osteoblasts (Von Kossa, Calcium) Adipocytes (Oil red, Fat vacuoles) Chondrocytes (Alcian blue, CSPG) ...
In vitro  Models High-Density Co-Culture System Petri dish Culture Medium Steel bridge Filter Cells (Chondrocytes + MSCs) ...
High Density Co-Cultures I  II  III MSC Chondrocyte Merge
Cartilage Formation in 3-Dimensional Co-cultures M M C C C
EM Evidence for Chondrogenesis Perichondrium Cartilage Matrix Chondrocyte
Electron Micrographs of High Density Co-cultures
Evidence for Collagen  Type II Chondrocyte IEM:
Evidence for Cartilage Specific Proteoglycans (CSPGs) Chondrocyte IEM
Western Blotting: Collagen Type II 100% MSCs+GF 100%  Chondrocytes 50%/50%MSC/ Chondrocytes 100% MSCs-GF 200 kDa 100 kDa 6...
Western Blotting: CSPGs 200 kDa 150 kDa 60 kDa 100% MSCs-GF 100% MSCs+GF 50%/50%MSCs/ Chondrocytes 100% Chondrocytes
Evidence for cell-cell contacts
Conclusions <ul><li>There are dynamic interactions between primary chondrocytes and MSCs </li></ul><ul><li>Chondrocytes an...
Prospects <ul><li>Tissue engineering cartilage using 3-dimensional cultures of chondrocytes and mesenchymal stem cells pro...
Acknowledgements Professor M. Shakibaei Munich Dr. Pat Harris (WALTHAM) Dr. David Allaway (WALTHAM) Collaborators: Dr. Ste...
Funding: Acknowledgements
Thank you for your attention
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Dr. A. Mobasheri Nc3 Rs And Bbsrc Symposium 1 2 April 2009 Final Version

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

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  • Tissue Engineering and Stem Cell Therapies for OA But can tissue engineering and mesenchymal stem cells be used to treat OA? Optimistic answer: Yes Realistic answer: Hopefully…
  • In addition to red blood cells, white blood cells and platelets bone marrow contains stromal cells which are also known as mesenchymal stem cells
  • Mesenchymal stem cells are multipotent cells with the capacity to give rise to a variety of connective tissue cell types including: bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), other types of connective tissue cells such as those found in tendons and ligaments
  • For high-density culture tenocytes were centrifuged and 10 microliters of the pellet were pipetted onto a membrane filter on the top of a stainless steel grid and cultivated at the medium air interface in a Petri dish.
  • 7 day old high density co-cultures of MSCs and primary chondrocytes (50:50) MSCs (red) Primary chondrocytes (green) Merge: Overlay of the single pictures shows even distribution of both cell types in high density co-cultures
  • EM: typical cartilage nodule formation, surrounded by a perichondrium C: IEM: The matrix contains high amounts of collagen type II (I) and CSPGs (II) (arrows)
  • Engineering cartilage with mesenchymal stem cells derived from the bone marrow of food producing animals and animals euthanased for clinical reasons unrelated to research will reduce the numbers and the variety of animal species used in osteoarthritis research
  • Dr. A. Mobasheri Nc3 Rs And Bbsrc Symposium 1 2 April 2009 Final Version

    1. 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
    2. 2. Articular Cartilage <ul><li>Mechanically unique connective tissue designed to: </li></ul><ul><ul><li>withstand and distribute load </li></ul></ul><ul><ul><li>act as an elastic shock absorber </li></ul></ul><ul><ul><li>provide a wear resistant surface to articulating joints </li></ul></ul>
    3. 3. <ul><li>Avascular, aneural and alymphatic </li></ul><ul><li>Contains a single cell type: the chondrocyte </li></ul><ul><li>Derived from mesenchymal progenitor cells </li></ul>Articular Cartilage
    4. 4. The Chondrocyte Nucleus Cytoplasm ECM Synthesizes a mechanically resilient extracellular matrix of collagens and aggregating proteoglycans
    5. 5. Major Constituents of Articular Cartilage Matrix Collagen IX Collagen II Fibronectin COMP Aggrecan Hyaluronan Chondrocyte Thrombospondin Decorin Biglycan Fibromodulin
    6. 6. Articular Cartilage Degradation <ul><li>Despite its durability, cartilage has a very limited self maintaining capability </li></ul><ul><li>Cartilage is vulnerable to mechanical injury and prone to structural damage and degradation </li></ul>
    7. 7. Osteoarthritis (OA) <ul><li>Most common form of arthritis characterised by progressive deterioration and loss of articular cartilage </li></ul><ul><li>Affects load-bearing synovial joints causing pain, inflammation and loss of mobility </li></ul><ul><li>Associated with ageing and predicted to increase as the ageing population grows </li></ul>
    8. 8. Risk Factors for OA <ul><li>Age </li></ul><ul><li>Lifestyle/occupation </li></ul><ul><li>Joint trauma </li></ul><ul><li>Joint instability </li></ul><ul><li>Genetics </li></ul><ul><li>Metabolic/endocrine disease </li></ul><ul><li>Obesity </li></ul>
    9. 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. 10. Cartilage Fibrillation and Loss of Proteoglycans in OA Human femoral head H&E Safranin O Safranin-O stains glycosaminoglycans red
    11. 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
    12. 12. Rationale for Studying OA <ul><li>OA affects 1 in 6 adults </li></ul><ul><li>Most OA patients suffer from pain and disability </li></ul><ul><li>By 2030 20% of Americans and Europeans will have OA </li></ul><ul><li>There are no disease modifying treatments for OA </li></ul><ul><li>Existing drugs (NSAIDs) only treat the symptoms of OA – reducing pain and inflammation </li></ul><ul><li>Therefore OA represents a major opportunity for basic and clinical research, drug discovery and the development of novel disease modifying agents and therapeutic approaches </li></ul>Source: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIAMS/National Institutes of Health, Bethesda, MD
    13. 13. Animal Models of Osteoarthritis <ul><li>There are many animal models of osteoarthritis </li></ul>
    14. 14. Animal Models of OA INJECTION INTO THE JOINT <ul><ul><li>Other related models: adjuvant injection for modelling pannus formation and joint inflammation in rheumatoid arthritis </li></ul></ul>Papain Injection Monoiodoacetate Injection Surgical Lesion Model Collagenase Injection LPS Injection
    15. 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. 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
    17. 17. Animal Models of OA <ul><li>Disadvantages: </li></ul><ul><li>Ethical issues </li></ul><ul><li>Invasive nature of techniques </li></ul><ul><li>Anaesthetics always required </li></ul><ul><li>Animals always sacrificed </li></ul><ul><li>Models are expensive and laborious to establish </li></ul><ul><li>Endpoint data frequently difficult to relate to humans </li></ul>
    18. 18. Alternative Models <ul><li>Researchers must constantly evaluate the relevance of their animal models to OA in humans </li></ul><ul><ul><li>Do animal models accurately represent the clinical disease of OA in humans? </li></ul></ul><ul><ul><li>Can animal models of OA be replaced with alternatives? </li></ul></ul><ul><ul><li>Would these alternatives be suitable for basic research, drug discovery and safety testing? </li></ul></ul><ul><ul><li>Can tissue engineering offer viable alternative models to study OA? </li></ul></ul>
    19. 19. Important criteria for cartilage models for drug discovery <ul><li>Ability to monitor synthesis and release of inflammatory mediators </li></ul><ul><ul><li>PGE 2 , NO, pro-inflammatory cytokines </li></ul></ul><ul><li>Markers of cartilage catabolism </li></ul><ul><ul><li>Loss of proteoglycans and GAGs </li></ul></ul><ul><ul><li>Collagen matrix degradation (neo-epitopes) </li></ul></ul><ul><li>Markers of cartilage anabolism </li></ul><ul><ul><li>De novo synthesis of proteoglycans </li></ul></ul><ul><ul><li>Synthesis of collagens (i.e. type II collagen) </li></ul></ul>
    20. 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. 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
    22. 22. In vitro Models Culture of chondrocytes in alginate <ul><li>Advantages: </li></ul><ul><ul><li>The preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro </li></ul></ul>
    23. 23. In vitro Models The Explant Model <ul><li>Advantages: </li></ul><ul><ul><li>Ideal for studies of extracellular matrix synthesis and degradation </li></ul></ul><ul><ul><li>Suitable for proteomic work and studying anti-inflammatory drugs and nutraceuticals </li></ul></ul>
    24. 24. The Promise of Stem Cells and Tissue Engineering <ul><li>Stem cells </li></ul><ul><li>Biomaterials </li></ul><ul><li>Bioreactors </li></ul><ul><ul><li>Induction media </li></ul></ul><ul><ul><li>Growth factors </li></ul></ul><ul><li>New tissues </li></ul><ul><li>Transplantation </li></ul>
    25. 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. 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. 27. Multipotency of MSCs Osteoblasts (Von Kossa, Calcium) Adipocytes (Oil red, Fat vacuoles) Chondrocytes (Alcian blue, CSPG) MSCs
    28. 28. In vitro Models High-Density Co-Culture System Petri dish Culture Medium Steel bridge Filter Cells (Chondrocytes + MSCs) + Growth Factors
    29. 29. High Density Co-Cultures I II III MSC Chondrocyte Merge
    30. 30. Cartilage Formation in 3-Dimensional Co-cultures M M C C C
    31. 31. EM Evidence for Chondrogenesis Perichondrium Cartilage Matrix Chondrocyte
    32. 32. Electron Micrographs of High Density Co-cultures
    33. 33. Evidence for Collagen Type II Chondrocyte IEM:
    34. 34. Evidence for Cartilage Specific Proteoglycans (CSPGs) Chondrocyte IEM
    35. 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. 36. Western Blotting: CSPGs 200 kDa 150 kDa 60 kDa 100% MSCs-GF 100% MSCs+GF 50%/50%MSCs/ Chondrocytes 100% Chondrocytes
    37. 37. Evidence for cell-cell contacts
    38. 38. Conclusions <ul><li>There are dynamic interactions between primary chondrocytes and MSCs </li></ul><ul><li>Chondrocytes and MSCs actively interact and communicate in culture </li></ul><ul><li>The interactions are important for the chondrogenic differentiation of MSCs </li></ul><ul><li>This approach may find future applications in cartilage tissue engineering and regenerative medicine </li></ul>
    39. 39. Prospects <ul><li>Tissue engineering cartilage using 3-dimensional cultures of chondrocytes and mesenchymal stem cells provides a realistic alternative to using animals </li></ul><ul><li>Other benefits: </li></ul><ul><ul><li>Innovative and relatively inexpensive </li></ul></ul><ul><ul><li>Uses bone marrow aspirates from fewer animals </li></ul></ul><ul><ul><li>Endpoint data may be relevant to the species used and may be applied to other species (comparative approach) </li></ul></ul>
    40. 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. 41. Funding: Acknowledgements
    42. 42. Thank you for your attention
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