Transcript Mechanical Strain and Growth Factors?

Mechanical Strain and
Growth Factors To
Stimulate SMC Growth
Prepared by Seth Hills
Introduction
 Atherosclerosis
 Costs-Treatments
 Artery
Composition
Our Goal:

Biocompatible
tissue engineered
vascular graft
Our Strategy:
Natural Materials
 Growth Factors
 Cyclic Strain
 Use them
together to
enhance SMC
growth

Why are we studying this?
1.
2.
3.
4.
5.
They haven’t been studied together.
Growth factors (GFs) stimulate SMC growth.
Similar to in vivo
Potential additive effects
Understand mechanism of atherosclerosis
Methods:
 Tissue
Culturing Substrates
 Cell Number vs. Cell DNA
 GF Concentrations vs. Cell
Proliferation
Growth Factors
TGFβ (Transforming Growth Factor Beta)
 PDGF (AA, AB, BB)

(Platelet Derived Growth Factor)
FGF (Fibroblast Growth Factor)
 VEGF (Vascular Endothelial Growth Factor)
 IL-2 (Interleukin 2)

Results:
Cell Count vs. Cell DNA
Concentration
Cell Count (Thousands)
10
y = 0.0964x2 - 35.205x + 60.068
R2 = 0.9702
5
0
0
100
200
300
400
-5
Cell DNA Concentration (ng/mL)
500
600
Cell DNA vs. GF Concentration
700
Cell DNA (ng/mL)
650
600
550
IL-2
500
FGF
450
TGFβ
400
350
300
0
15
Growth Factor Conc. (ng/mL)
30
Cell DNA (ng/mL)
Cell DNA Concentration as
Percentage of Baseline
125
120
115
110
PDGF-AA
105
PDGF-AB
100
PDGF-BB
95
90
5
30
55
80
GF Concentration (ng/mL)
Percentage Cell
Growth from Baseline
PicoGreen-Percentage Cell growth from Baseline vs.
Substrate Type
250
200
150
100
50
0
Amino
Collagen I Collagen
IV
Laminin
Substrate
Control
Pronectin
Future Work
 Mold
design for vascular graft
 Factorial Designs
 Design pulsatile flow system
Vascular Graft Molds
Factorial Designs
1.
Mechanical Testing
–

Vascular Graft Composition:
–

Study the effect of cyclic strain, compared to
static controls, on SMCs with and without GFs.
Optimize for good mechanical properties
Cell encapsulation
Cyclic
Strain
• Vacuum provides
stretch
• Induces biochemical
changes in response to
strain
Mechanical
Testing
Vascular Graft Composition
Full Factorial
Design Parameters
Run
A
B
C
D
Side*
1
+
+
+
+
R
2
+
+
+
-
R
3
+
+
-
-
R
4
+
-
-
-
R
5
-
-
-
-
L
6
-
-
-
+
L
7
-
-
+
+
L
8
-
+
+
+
L
Run
A
B
C
D
Side
9
-
+
-
+
L
1
+
+
+
+
R
10
+
-
+
-
L
2
+
+
+
-
R
11
-
-
+
-
L
3
+
+
-
-
R
12
+
+
-
+
R
4
+
-
-
-
R
13
+
-
-
+
R
5
+
+
-
+
R
14
-
+
+
-
R
6
+
-
-
+
R
15
+
-
+
+
R
7
-
+
+
-
R
16
-
+
-
-
L
8
+
-
+
+
R
*for splitting into a half factorial
Levels:
Control Factors:
2µg/mL (-)
4µg/mL (+)
A Collagen
B Chitosan
C Elastin
D Fibrinogen
Half Factorial
Flow System
(Jeng-Jiann Chiu)
Vascular Graft
(Cummings)
Methods:
Substrate Study
•
•
Seed cells at 96,000 cells/well in
BioFlex™ 6 well plates of different
substrate along with a control.
When cells are confluent in one of the
substrates Lyse the cells and perform DNA
assay.
Methods:
Cell Number vs. Cell DNA
•
•
Day 1: Seed cells at different
concentrations in 24 well plate
Day 2: Lyse cells and perform DNA
assay
Methods:
GF Concentration Study
•
•
•
•
•
Day 1: Seed cells in 24 well plates at 20,000
cells/well.
Day 2: Feed cells with quiescent media.
Day 3: Begin feeding cells with different GF
concentrations.
Feed every other day
Lyse cells when they are confluent in one of
the concentrations and perform DNA assay.