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Human mesenchymal stem cell position within scaffold influences cell fate in dynamic culture


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Human mesenchymal stem cell position within scaffold influences cell fate in dynamic culture

  1. 1. Stem Cells Bioengineering 21th December 2012 Diana Santos nº 72459 MEBiom Sofia Sousa nº 54180 MEBiol
  2. 2. Tissue Engineering Limitations “Regenerative Medicine is an interdisciplinary field of research that applies the principles of engineering and the life sciences towards the development of biological substitutes that• Cellular densities similar to those in native restore, maintain, or improve tissue function” tissues Langer & Vacanti• Diffusion limit of O2 and nutrients (Porosity and interconnectivity)• Size, shape and material of the scaffolds• Immune rejection in transplants• Need for cellular expansion
  3. 3. hMSCs for Clinical Applications• Graft-vs-Host disease treatment• Bone grafts /Cartilage repair/Vertebral disks damage Bladder Trachea• Coronary Heart Disease• Parkinson’s, Alzheimer’s and epilepsy disease• Incontinency/Renal failure/artificial bladder Intervertebral disk • Burns • Chron’s disease • Myocardial ischemia • Cornea/Retina substitution Skin • Cancer • Important role in the co-transplant with HSC Cornea
  4. 4. MSC Sources and Differentiation Process Source: T.L. Bonfield, Discovery Medicine, 2010
  5. 5. Static Culture Dynamic Culture • Non-homogeneous growth •Better homogeneity • Non-homogeneous differentiation •O2 and nutrients supply during exposition to • Low O2 and nutrients diffusion shear stress• Difficulty of monitoring and control •Higher cellular growth • Low productivity •Higher control and productivity T-Flask Spinner-flask Stirred Bioreactor Rotative Walls TPS Roller Bottle Wave Bioreactor
  6. 6. Study TPS bioreactor for 3D dynamic culture of hMSCs in spherical alginate beadsPurpose •Shear stress effect on osteoblastic differentiation of bioreactor culture beads •Cellular position in a scaffold and it relation with cell proliferation •Influence of radial position in hMSC osteoblastic differentiation Source: Yeatts, A , Tissue Engineering, 2011
  7. 7. Landmark studies • Alginate -> support • If low oxygen levels proliferation and are combined with osteoblastic nutrient deprivation, differentiation of BM significant cell death stromal cells occurs (48h)Sikavitsas et al. Mauney et al. Utting et al. Potier, et al.(2003) (2005) (2006) (2007)• Increased • Dextran does not • Low oxygen (3%) proliferation and influence cell concentrations can differentiation for inhibit bone formation differentiation and and in vitro hMSCs exposed to 2% proliferation osteoblastic O2 conditions compared to 20% differentiationGrayson et al. Li, et al. Li, et al. Iida et al.(2007) (2008) (2009) (2010)
  8. 8. Shear Stress and O2 LevelsMiddle section of TPS growth chamber, 3mL/min flow rate m Flow velocities •Higher in the contact points between beads O2 concentration on the bead cm/s •Static cultured falls to a minimum along the distance •TPS minimum concentration in the center O2 concentrations throughout alginate beads Alginate bead diffussion model Source: Yeatts, A , Tissue Engineering, 2011
  9. 9. hMSCs Culture 1. Expansion in DMEM 2. Culture flasks 10% FBS (Passage each 3days) 3. Incubation at 37ºC, 4. Osteogenic 5% CO2 (Passage each medium 6-7 days)
  10. 10. Alginate Beads and hMSCs Isolation Experimental GroupsSource: Biomaterials II, IST, 2011 TPS large beads 4mm Inner and outer annuli TPS small beads 2mm Calibration Curve: Outer annuli -> 18min Control Groups Alginate beads on static osteogenic media and TPS Bioreactor (3ml/min) Static Culture large beads 4mm Inner and outer annuli Static culture small 5 mm beads 2mm Source: Yeatts, A , Tissue Engineering, 2011
  11. 11. Bioreactor Design Features Incubator at 37ºC Osteogenic media changed every 3 days 1.0 mL/min for annuli studies 3.0 mL/min for shear stress studies Growth Chamber Platinum-cured silicone tubing dinner=6.4mm, douter=11.2mm, δ=2.4mm High Permeability to O2 and CO2 Large δ -> Lower gas diffusion
  12. 12. Shear stress study Marker for osteoblastic differentiation Bone Morphogenetic Protein-2 and Osteopotin Day In 4mm beads 1 Day Day 21 4 BMP-2 TPS with 3% dextran Experimental groups Day Day 14 8 TPS with 9% dextran Osteopontin (OPN) Control Groups Static Culture Day 14 Day 21
  13. 13. Shear stress study OPNBMP-2 •Days 1,4,8 •Shear stress Weak increasing correlation leads to with shear higher OPN stress expression levels •Days 14,21 Strong •Day 21 shows correlation higher [OPN] compared to day 14 Dependence of the expression levels of OPN and BMP-2 with the shear stress For the same shear stress BMP-2 and OPN levels are higher with each passing day
  14. 14. hMSCs Proliferation and Osteoblastic Differentationin Relation to Position Experimental Groups TPS large beads 4mm TPS small beads 2mm Control Groups Static Culture large beads 4mm Static culture small beads 2mm
  15. 15. hMSCs Proliferation in Relation to Position Live and Dead Assay •Day 1 -> all cellsProliferation appeared viable 1,000 μm Live dead images of entire bead, inner annuli and small bead after one day •Day 7 -> Increased of bioreactor culture proliferation in TPS small bead •Day 14 –> Decreased proliferation in static large bead inner •Day 21 –> Control beads have less proliferation compared to TPS beads
  16. 16. hMSCs Osteoblastic Differentation Day 1-14 -> ALP Day 7-14 -> OPN expression is expressed low OPNALP higher in levels controls Day 21-> High Day 21-> High expression in TPS expression in TPS larger beads and control, in inner annulli and larger beads small beads inner annulli Day 1 Day 7 Day Day 21 ALP 7 OPN Day Day Day 21 14 14
  17. 17. hMSCs Osteoblastic Differentation Day 1 Mineralized matrix production Day M Day 21 7 Day 7-14 -> Higher mineralization in Day 14 control small beads Day 21-> Higher mineralization in TPS inner annuli
  18. 18. Proliferation DifferentiationDifferentiation Differentiation
  19. 19. ConclusionsShear stress Osteoblastic differentiation Involved in temporal effect on the osteoblastic differentiation High increase in OPN and BMP-2 in latest days hMSCs position within scaffold plays a role in the osteoblastic differentiation of cells MSCs may directed down a specificProliferation pathway by physical factors in their environment, helping the differentiation of inner cells of large beads Dynamic culture can overcome the Oxygen levels and shear vary throughout nutrients diffusion limitation in the scaffold comparison to static culture Static culture of large beads leads to reduced osteoblastic differentiation and hMSCs position within scaffold play a low mineralization role in the proliferation of cells Bioreactor cultured small beads had the highest levels of proliferation
  20. 20. References • Yeatts, Andrew B., et al (2012). “Human mesenchymal stem cell position within scaffolds influences cell fate during dynamic culture” . Biotechnology and Bioengineering 109(9): 2381- 2391; • Yeatts AB, Fisher JP. 2011b. “Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells”. Tissue Eng Part C Methods 17(3):337–348; • Yeatts AB, et al (2012). “Bioreactors to influence stem cell fate: Augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems”. Biochimica et Biophysica Acta • Salgado, A.J., O. P. Coutinho, et al (2004). “Bone and Tissue Engineerign: State of the Art and Future Trends”. MacromolecularBioscience 4(8): 743-765 • Warren L. , et al (2007). “Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells”. Biochemical and Biophysical Research 358 (3): 948 – 953; • • Cell and Tissue Engineering – Biomaterials 2012 IST • Biomaterials II – 2012 IST • Stem Cell Bioengineerging – 2012 IST