Engineering bone tissue using human Embryonic Stem Cells

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Bone defects lead by traumatic injuries, congenital malformations and other surgical rescissions rises the immediate need for a more evolved and safer approaches in tissue repair at alarming rates for the downgrading issues with existing strategies which needs to be addressed. Currently practiced treatment methods addressing the issue with bone defects are invasive, traumatic and are not cost effective. Yet, issues of immune rejection either immediately or in the later stages have been reported claiming its ineffectiveness in some selective case studies.
Previous work by researchers carried out the "Biomimetic" approach to provide the cells with the microenvironment and in situ conditions for the cells seeded into the 3D Osteogenic scaffolds enriched with growth supplments. Here, we address the issue of non-availability of therapeutic cells - a major problem with current translational medicine by proposing the use of Human Embryonic Stem Cells in generating strong and structurally rigid bone tissue. Inducing the production of Mesenchymal Progenitor cells from Human Embryonic Stem cells in Serum supplemented expansion medium and elimination of bone Fibroblast growth factor produced high quality MPCs which were induced in osteogenic medium to result in bone differentiating cells. Culturing these MPCs produced from three different protocols into 3D Scaffold and 3D-Endoret Osteogenic Scaffold produced tissue constructs which are analysed both biochemically and Histologically to check for the Bone tissue differentiation parameters such as Bone sialoprotein deposition, Osteopontin accumulation and Collagen deposition. Matrix mineralization in these constructs were studied by uCT imaging and safety studies were conducted by studying Orthotopic implantation models in SCID mouse. And the results are expected to be optimistically affirmative which shall lay a new foundation and pioneer a whole new approach in the field of Tissue Engineering.

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Engineering bone tissue using human Embryonic Stem Cells

  1. 1. Engineering Bone Tissue from Human Embryonic Stem Cells Balaganesh Kuruba Department of Biological Chemical and Physical sciences Illinois Institute of Technology
  2. 2. Bone defects from Congenital and trauma malformations. Metatarsus adductus Hammer Toe Bunion’s feet
  3. 3. Current strategies in treating bone defects Illizarov frame: To treat bone deformations or fractures. Osteobridge: Stabilization of bone defects
  4. 4. Problems with conventional methods • Extremely Traumatic . • Not so economical use of mechanical and structurally competent scaffolds. • Requirement of synergistic development of vascular supply and bone to maintain viability. Alternative Use of Human Embryonic Stem cells in engineering bone tissue custom made for individuals thus easing the process in avoiding graft rejection related issues. Safe to use and time saving by generating the bone tissue on an increased time scale.
  5. 5. Hypothesis Cultivation of hESC-derived Mesenchymal Progenitor Cells (MPCs) on 3D Osteoconductive scaffolds in perfusion reactor system leads to formation of large and compact bone constructs.
  6. 6. Bone Tissue engineering protocol and timeline Fig 1: Bone Engineering protocol and time line.
  7. 7. Specific aims • Aim 1: Derivation of Mesenchymal progenitors from hESCs. • Aim 2: Culturing MPCs on 3D scaffolds in perfusion bioreactor system. • Aim 3: Effects of Reactor cultivation on MPCs in tissue development. • Aim 4: In vivo Safety and stability analysis of the engineered bone construct.
  8. 8. Aim 1: Derivation of Mesenchymal progenitors from hESCs. • Differentiation is induced in two hESC Cell lines – H9 and H13. • Mesenchymal Differentiation potential was analysed and suitable cell line was selected for further analysis. Differentiation of hESCs into different cell types.
  9. 9. • H9 and H13 cultures were found to show continuous growth. • Mesenchymal surface antigens (Blue box) were expressed on progenitors. • Further analysis on the cell lines were carried out. Fig S1C: Surface antigen expression of hESC progenitors
  10. 10. Fig : Mesenchymal differentiation potential screened in monolayer cultures.
  11. 11. Fig : Mesenchymal differentiation potential screened in monolayer cultures.
  12. 12. • From the tests performed H9 cultures showed – Lesser or zero adipogenic potential – Higher chondrogenic potential – Higher activity of Alkaline phospatase and – Increased matrix mineralization In comparison with H13 cultures and Bone Marrow derived mesenchymal stem cells (BMSCs) as positive control. Based on which, H9 cultures are selected for the long run.
  13. 13. Aim 2: Culturing MPCs on 3D scaffolds in perfusion bioreactor system. Compact Bone structure 3D Scaffold Structure
  14. 14. Perfusion Bioreactor system Criteria: Interstital media flow Concept: Channeling media into scaffolds Design: Final End product
  15. 15. Fig 2 Aim 3: Effects of Reactor cultivation on MPCs in tissue development.
  16. 16. Fig 3: uCT analysis revealing bone mineralization and maturation during in vitro and invivo conditions.
  17. 17. Aim4: In vivo Safety and stability analysis of the engineered bone construct. Fig 4
  18. 18. Fig 4B
  19. 19. And what am I proposing.. • Analysis of Bone-tissue characteristics of hESCmesenchymal progenitors generated from different tissue engineering protocols – Suggesting addition of Beta glycerol phosphate – And Ascorbic acid – 2 - phosphate. • Determination of influence of PRGF- Endoret implanted 3D scaffold system on the rate of development of MPCs into bone tissue. • Long term safety studies in orthotopic implantation models.
  20. 20. Thank you

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