1. Stem Cells Bioengineering
21th December 2012
Diana Santos nº 72459 MEBiom
Sofia Sousa nº 54180 MEBiol

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. 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. MSC Sources and Differentiation Process
Source: T.L. Bonfield, Discovery Medicine, 2010

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. Study TPS bioreactor for 3D
dynamic culture of
hMSCs in spherical
alginate beads
Purpose
•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. 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% differentiation
Grayson et al. Li, et al. Li, et al. Iida et al.
(2007) (2008) (2009) (2010)

8. Shear Stress and O2 Levels
Middle 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. 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. Alginate Beads and hMSCs Isolation
Experimental Groups
Source: 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. 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. 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. Shear stress study
OPN
BMP-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. hMSCs Proliferation and Osteoblastic Differentation
in 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. hMSCs Proliferation in Relation to Position
Live and Dead Assay
•Day 1 -> all cells
Proliferation
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. hMSCs Osteoblastic
Differentation
Day 1-14 -> ALP Day 7-14 -> OPN
expression is expressed low
OPN
ALP
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. 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. Proliferation Differentiation
Differentiation Differentiation

19. Conclusions
Shear 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 specific
Proliferation 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. 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;
• http://terpconnect.umd.edu/~jpfisher/index_files/presearch.htm
• Cell and Tissue Engineering – Biomaterials 2012 IST
• Biomaterials II – 2012 IST
• Stem Cell Bioengineerging – 2012 IST