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Bone Presentation Final5 5 2008

Bone Presentation Final5 5 2008






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Bone Presentation Final5 5 2008 Bone Presentation Final5 5 2008 Presentation Transcript

  • CONTROL OF IN VITRO TISSUE-ENGINEERED BONE-LIKE STRUCTURES USING HUMAN MESENCHYMAL STEM CELLS AND POROUS SILK SCAFFOLDS Biomaterials 28 (2007) 1152 – 1162 S. Hoffmann, H. Hagenmuller, A. M. Koch, R. Muller, G. Vunjak-Novakovic, D. L. Kaplan, H. P. Merkle, L. Meinel Christian Britt Amy Chaibi Polliana Macedo Niyati Patel Joseph Saltzbart
  • Presentation Topics/ Agenda:
    • Introduction
    • Experimental Design
    • Discussion
    • New Design
    • References
    • Bone Structure
    • Bone Function
    • Cell Types in Remodeling & Healing
    • Musculoskeletal Injury and Disease
    • How does the experimental design presented in the article contribute to the understanding / solving of the problem?
    • Tissue Engineering Strategies
  • Bone Structure
    • Human Skelton has 206 Bones
      • Axial Flat & Irregular Bones
      • Appendicular Tubular Bones
        • Long Bones
        • Short Bone
    • Two Types of Bone Tissue
      • Cortical Bone (Compact)
      • Trabecular Bone (Cancellous, Spongy)
  • Mechanical Properties 2 – 12 .05 - .5 30 - 90 Trabecular 50 - 150 100 – 230 7 – 30 5 - 30 Cortical Bending Compressive Strength (MPa) Young’s Modulus (GPa) Porosity (%) Bone
  • Bone Function
    • Bone is a highly specialized tissue of the skeletal system that has five main functions:
      • Mechanical Support (ribs)
      • Movements, as the site of muscle attachments (long bones)
      • Protective, as encasement of organs (skull)
      • Metabolic, as a reserve pool of various ions especially calcium and phosphorous
      • Hematopoietic: bones provide host sites for the hematopoietic tissue (the bone marrow)
  • Bone Cells
    • Bone Remodeling – lifelong physiological process. Old bone is resorbed. New bone is formed.
      • Osteoblasts
        • Bone Forming
        • Arise from Osteoprogenitors & MSC
      • Osteoclasts
        • Bone Resorbing
        • Remove Debris and Pathologic Materials
      • Osteocytes
        • Mature Osteoblasts that reside within the bone matrix
        • Communicate with other cells
  • Bone Cells
  • Fracture Healing
  • Musculoskeletal Injury & Disease
    • Intrinsic Factors Affecting Remodeling
      • Vasculature (Smoking, Diabetes)
      • Metabolic Factors (Disorders)
      • Age Related Changes*
    • Extrinsic Factors Affecting Repair
      • Trauma (Periosteum, Vasculature)
      • Reduction / Fixation (Displacement / Plates, Screws)
      • Medication
    • *Osteoporosis – porous bone, bone deterioration
      • 1.5 million people suffer fractures in the US (Est.)
  • Contribution of the Experimental Design to Solving of the Problem
    • The authors note that “many groups have put effort on controlling the properties of scaffolds made out of various materials, but little work has been devoted to controlling the structure of the in vitro engineered bone (before implantation) on those scaffolds through scaffold design.”
      • Hoffmann S et al. / Biomaterials 28 (2007) 1152 – 1162.
    • Biomimetics
      • Scaffold geometry modified outside of the body is hypothesized to optimize bone growth in a novel biomimic approach that seeks to engineer a bone-like tissue in vitro in an attempt to mimic the natural tissue structure.
  • Tissue Engineering Strategies
    • Cells
      • Osteoblasts, Chondrocytes, Stem Cells
    • Growth Factors
      • BMPs, TGF-  , IGF I&II, PDGF, Fibroblasts
    • Bioreactors
      • Spinner Flasks, Rotating, Flow Perfusion
    • Scaffolds (3-D) - underlying material, macrostructure (special geometry), micro-structure (bulk versus porous), mechanical properties, and degradation characteristics
      • Biodegradable Polymers, Bioactive Ceramics, Biocomposites, Nanocomposites
    • Demonstrate the feasibility of controlling dual scaffold geometries on a single scaffold
    • Control the structure of the engineered bone through scaffold design
    • Provide an implant that would mimic tissue morphology at the site of implantation
    • Scaffold Dynamic Seeding: hMSC were suspended in liquid matrix, seeded on prewetted scaffolds, incubated (37°C, 5%, CO 2 ) and cultivated in spinner flasks stirred at 60 rpm for 24 hours.
    • Scaffold Static Seeding: P2 hMSC were suspended in a control medium, added to a spinner flask, the cell suspension was stirred at 60rpm and incubated for 24 hours (37 o C, 5%, CO 2 )
  • Findings Overview
    • The structure of tissue-engineered bone using hMSCs seeded on SF scaffolds in osteogenic medium was guided by the geometry of the scaffold
    • Optimum bone growth occurred through the use of dynamic seeding in small pore (112-224 mm) silk scaffolds
  • Silk Scaffolds Pores
  • Tissue-engineered bone on mixed pore scaffolds
  •   Biochemical parameters of osteogenic differentiation of hMSC cultured on SF scaffolds
  •   Cell proliferation and activity
  •   hMSC on scaffolds with different pore sizes as visualized by CLSM (A–F) and histology
  • hMSC grown on SF scaffolds with mixed pores
  • Did the authors succeed?
    • Goal: Engineer varying bone-like pore structures in vitro using a single scaffold
    • DID SUCCEED -Examining the effect of different pore sizes on silk scaffolds.
    • Strong Conclusion: dynamic seeding was the best way to grow bone tissue since it removed dead cells.
  • Competing Studies- Comparisons
      • Previous work - various pore sizes on a single implant shown in HA scaffolds.
      • Similar study: hMSCs derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts.
      • hMSCs alleviate the problems of immunorejection.
      • This Article : hMSC cultured on protein scaffolds (SF) for tissue engineering of bone-like structure.
      • Another Study found out -hMSC attachment, proliferation, and metabolic activity - better on slowly degrading silk than on fast-degrading collagen scaffolds
          • SILK : Better Choice
  • Competing Studies- Contrasts
      • Translation into in vivo environment.
      • Ingrowths of other tissues as a function of change in pore size and porosity
      • Impact cell survival and integration of the implant into the host tissues. E.g. BMP-2 induced in vivo bone formation demonstrated to be affected by pore geometry.
      • Different positions of the scaffolds inside the spinner flask bioreactor, in particular at the transition areas of small to large pores : VARIABILITY of the Bone formation outcome
      • Suggestion: investigate and research- vascularization behavior and type of tissue & scaffold design
  • Medical & Clinical Applications
    • Concept - advantageous in Musculoskeletal tissue engineering applications
    • Tissue engineering uses 3 components to obtain musculoskeletal healing or regeneration:
      • (1) stem cells, specifically MSCs
      • (2) scaffolds to provide a template for tissue in growth
      • (3) growth factors or morphogens (physical or chemical factors inducing tissue healing).
    • Generation of osteochondral implants- use pore geometry to target differentiation of cells
    • Evaluation of the in vivo outcome of tissue engineered bone with variable trabecular structure
    • Useful in Genetic Engineering- transfer of DNA material into the cell to produce specific GF’s : Target desired cell attachment & adhesion
  • Design of a New Strategy
    • The cell type used: as in an alternative to hMSC
    • The scaffold used in terms of material
    • The processing technique to optimize bone growth
  • Cells
    • The authors proposed the use of hMSC. However, there are advantages and disadvantages.
      • Advantages include:
        • Human material: less immuno-rejection issues
        • Well researched and practiced in the field
      • Disadvantages include:
        • Availability (and cost) of obtaining the material
        • Potential ethical hurdles
    • We propose the use of xenogeneic stem cells as an alternative. There is a plentiful supply.
  • Scaffold
    • The authors proposed the use of SF. However, there are advantages and disadvantages.
      • Advantages include:
        • Material that occurs in nature
        • Lightweight and durable
      • Disadvantages include:
        • Availability (and cost) of obtaining the material
    • We propose the use of xenogeneic collagen as an alternative. Use of the same animal source.
  • Processing
    • The study found that dynamic seeding (Using a Spinner Flask System) is superior to static seeding.
    • Spinner flask: The cells on the outer periphery of scaffolds are subjected to turbulent flow, whereas interiorly the cells exhibit static molecular diffusion.
    • We propose the use of a Perfusion Flow Bioreactor.
    •  Flow Perfusion systems overcome the flow issues because medium is flushed directly through scaffolds and, thereby providing nutrient and stimuli throughout the entire volume of the scaffold.
  • Tests to Evaluate Characteristics and Efficacy
    • Measurement of activity can be through the use of a Scanning Electron Microscope
    • Also, through the level of Alkaline Phosphatase present in the samples
    Xenogeneic Cells + Animal Collagen Flow Perfusion Bioreactor Spinner Flask hMSC + SF Flow Perfusion Bioreactor Spinner Flask
  • Improvements Upon Current Strategies
    • Xenogeneic sources:
      • More supply available
      • Ethical issues are circumvented as a result of using animal sources.
      • obtaining the cells and the scaffold from the animal is a more efficient use of animal tissue.
      • More cost effective
    • Flow perfusion bioreactor allows for better vascularization throughout the scaffold and therefore results in a faster bone growth.
  • References
    • Bronner F, Farach-Carson MC, Mikos AG, Engineering of Functional Skeletal Tissues, Springer, 2007.
    • Hollinger JO, Einhorn TA, Doll BA, Sfeir C, Bone Tissue Engineering, CRC Press, 2005.
    • Lanza R, Langer R, Vacanti J, Principles of Tissue Engineering, Academic Press, Third Edition, 2007.
    • Jackson MJ, Ahmed W, Surface Engineered Surgical Tools and Medical Devices, Springer, 2007.
    • Meyer U, Wiesmann HP, Bone and Cartilage Engineering, Springer, 2006.
    • Ratner BD, Hoffman AS, Schoen FJ, Lemons JE, Biomaterials Science, Second Edition, Elsevier Academic Press, 2004.
    • Reis RL, San Roman J, Biodegradable Systems in Tissue Engineering and Regenerative Medicine, CRC Press, 2005.
    • Thorngren K-G (Author), Kotz R (Foreward), Poitout D (Editor), Biomechanics and Biomaterials in Orthopedics, Springer-Verlag London Limited, 2004.
    • Webster TJ, Nanotechnology for the Regeneration of Hard and Soft Tissues, World Scientific, 2007.
    • Lea Bjerrea, Cody E. Büngera, Moustapha Kassemb and Tina Myginda “Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds.” Biomaterials.2008
    • Michael J. Jaasmaa, b, Niamh A. Plunketta, b and Fergal J. O’Briena, b. Design and validation of a dynamic flow perfusion bioreactor for use with compliant tissue engineering scaffolds. Journal of Biotechnology February 2008
  • References (cont’d)
    • Wany, Fu-Sheng; Trester, Cathy. Bone Marrow Cells and Myocardial Regeneration. International Journal of Hematology 79 (2004) 322-327.
    • Eridani, Sandro; Sgaramella, Vittorio; Cova, Lidia. Stem cell: From embryology to cellular therapy? An appraisal of the present state of the art. Cytotechnology44: 125-141 (2004).
    • Tuominen, et. al. Bovine bone implant with bovine bone morphogenetic protein in healing a canine with ulnar defect. International Orthopaedics (2001) 25:5-8.
    • Yang, Yong-Guang. Application of xenogeneic stem cells for induction of transplantation tolerance: present state and future direction. Springer Semin Immun (2004) 26: 187-200.
    • Dykes, Donald, et. al.Response of human tumor xenografts in athymic nude mice to docetaxel (RP 56976, Taxotere). Investigational New Drugs, 13: 1-11, 1995.
    • Bansal, M.R.; Bhagat, S.B.; Shukia, D.D. Bovine cancellous xenograft in the treatment of tibial plateau in elderly patients. International Orthopaedics (SICOT)- January, 2008
    • Tampieri A, Celotti G, Sprio S, Delcogliano A, Franzese S. Porositygraded hydroxyapatite ceramics to replace natural bone. Biomaterials 2001;22(11):1365–70.
    • Jin QM, Takita H, Kohgo T, Atsumi K, Itoh H, Kuboki Y. Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation. J Biomed Mater Res 2000; 52(4):491–9.
  • References (cont’d)..
    • Meinel L, Hofmann S, Karageorgiou V, Zichner L, Langer R, Kaplan D, Vunjak-Novakovic G. “Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds.” Division of Health Sciences & Technology, Massachusetts Institute of Technology, E25-330, 45 Carleton Street, Cambridge, Massachusetts 02139, USA.
    • [1] Sandra Hofmann, Henri Hagenmuller, Annette M. Koch, Ralph Muller, Gordana Vunjak-Novakovic, David L. Kaplan, Hans P. Merkle, Lorenz Meinel. “Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds” Science Direct: Biomaterials 28 (2007) 1152–1162.
    • Hoffmann S et al. / Biomaterials 28 (2007) 1152
    • National Osteoporosis Foundation
    • Webster TJ, Nanotechnology for the Regeneration of Hard and Soft Tissue, World Scientific, 2007.
    • The European Commission (Scientific Committee on Medical Products and Medical Devices, SCMPMD)
    • Meyer U, Wiesmann HP, Bone and Cartilage Engineering, Springer, 2007.