Stem Cells Bioengineering 21th December 2012

Diana Santos nº 72459 MEBiom

Sofia Sousa nº 54180 MEBiol

•Cellular densities similar to those in native tissues

•Diffusion limit of O2 and nutrients (Porosity and interconnectivity)

•Size, shape and material of the scaffolds

• Immune rejection in transplants

•Need for cellular expansion

“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

restore, maintain, or improve tissue function”

Langer & Vacanti

Tissue Engineering Limitations

hMSCs for Clinical Applications

•Graft-vs-Host disease treatment

•Bone grafts /Cartilage repair/Vertebral disks damage

•Coronary Heart Disease

•Parkinson’s, Alzheimer’s and epilepsy disease

• Incontinency/Renal failure/artificial bladder

•Burns

• Chron’s disease

• Myocardial ischemia

•Cornea/Retina substitution

•Cancer

• Important role in the co-transplant with HSC

Bladder Trachea

Intervertebral disk

Skin

Cornea

MSC Sources and Differentiation Process

Source: T.L. Bonfield, Discovery Medicine, 2010

T-Flask Spinner-flask Stirred Bioreactor Rotative Walls

TPS Roller Bottle Wave Bioreactor

Static Culture

•Non-homogeneous growth

•Non-homogeneous differentiation

•Low O2 and nutrients diffusion

•Difficulty of monitoring and control

•Low productivity

Dynamic Culture

•Better homogeneity

•O2 and nutrients supply during exposition to shear stress

•Higher cellular growth

•Higher control and productivity

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

TPS bioreactor for 3D

dynamic culture of

hMSCs in spherical

alginate beads

Study

Source: Yeatts, A , Tissue Engineering, 2011

Landmark studies

Sikavitsas et al. (2003)

•Alginate -> support

proliferation and

osteoblastic

differentiation of BM

stromal cells

Mauney et al. (2005)

Utting et al. (2006)

• If low oxygen levels

are combined with

nutrient deprivation,

significant cell death

occurs (48h)

Potier, et al. (2007)

• Increased

proliferation and differentiation for hMSCs exposed to 2% O2 conditions compared to 20%

Grayson et al. (2007)

•Dextran does not

influence cell

differentiation and

proliferation

Li, et al. (2008)

Li, et al. (2009)

•Low oxygen (3%) concentrations can inhibit bone formation and in vitro osteoblastic differentiation

Iida et al. (2010)

Shear Stress and O2 Levels

Source: Yeatts, A , Tissue Engineering, 2011

Middle section of TPS growth chamber, 3mL/min flow rate

O2 concentrations throughout alginate beads Alginate bead diffussion model

cm/s

m

Flow velocities

•Higher in the contact points between beads

O2 concentration on the bead

•Static cultured falls to a minimum along the distance

•TPS minimum concentration in the center

1. Expansion in DMEM 10% FBS

2. Culture flasks (Passage each 3days)

3. Incubation at 37ºC, 5% CO2 (Passage each

6-7 days)

4. Osteogenic medium

hMSCs Culture

Calibration Curve: Outer annuli -> 18min

Alginate Beads and hMSCs Isolation

Experimental Groups

TPS large beads 4mm

TPS small beads 2mm

Control Groups

Static Culture large beads 4mm

Static culture small beads 2mm

Inner and outer annuli

Inner and outer annuli

Source: Yeatts, A , Tissue Engineering, 2011

5 mm

Sourc

e:

Bio

mate

rials

II,

IST,

2011

Alginate beads on static osteogenic media and TPS Bioreactor (3ml/min)

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

Shear stress study

Experimental groups

TPS with 3% dextran

TPS with 9% dextran

Control Groups Static Culture

In 4mm beads

BMP-2

Day 1

Day 4

Day 8

Day 14

Day 21

Osteopontin (OPN)

Day 21 Day 14

Marker for osteoblastic

differentiation

Bone Morphogenetic Protein-2 and Osteopotin

BM

P-2

•Days 1,4,8 Weak correlation with shear stress

•Days 14,21 Strong correlation

OPN

•Shear stress increasing leads to higher OPN expression levels

•Day 21 shows higher [OPN] compared to day 14

Dependence of the expression levels of OPN

and BMP-2 with the shear stress

Shear stress study

For the same shear stress BMP-2 and OPN

levels are higher with each passing day

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

hMSCs Proliferation in Relation to Position Pro

life

rati

on

•Day 1 -> all cells appeared viable

•Day 7 -> Increased 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

Live dead images of entire bead, inner annuli and small bead after one day

of bioreactor culture

Live and Dead Assay

1,000 μm

hMSCs Osteoblastic

Differentation

ALP

Day 1-14 -> ALP

expression is

higher in

controls

Day 21-> High

expression in TPS

and control, in

larger beads

inner annulli

OPN

Day 7-14 -> OPN

expressed low

levels

Day 21-> High

expression in TPS

larger beads

inner annulli and

small beads

ALP

Day 1

Day 7

Day 14

Day 21

OPN

Day 7

Day 14

Day 21

Mineralized matrix production

Day 7-14 -> Higher mineralization in

control small beads

Day 21-> Higher mineralization in TPS

inner annuli

hMSCs Osteoblastic Differentation

M

Day 1

Day 7

Day 14

Day 21

Proliferation

Differentiation Differentiation

Differentiation

Conclusions

Shear stress

Involved in temporal effect on the osteoblastic differentiation

High increase in OPN and BMP-2 in latest days

Osteoblastic differentiation

hMSCs position within scaffold plays a role in the osteoblastic differentiation of cells

MSCs may directed down a specific pathway by physical factors in their environment, helping the differentiation of inner cells of large beads

Oxygen levels and shear vary throughout the scaffold

Static culture of large beads leads to reduced osteoblastic differentiation and low mineralization

Proliferation

Dynamic culture can overcome the nutrients diffusion limitation in comparison to static culture

hMSCs position within scaffold play a role in the proliferation of cells

Bioreactor cultured small beads had the highest levels of proliferation

• 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

References