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Porous PEG-Fibrinogen
Hydrogel Scaffolds for Tissue
Engineering
Ortal Yom-Tov
The Interdepartmental Program for
Biotechnology
Technion
Supervisors: Prof. Havazelet Bianco-Peled and Prof. Dror
Seliktar December
2012
Introduction
Tissue engineering is a science of
creating new tissues in order to
regenerate an organ functionality or to
replace damaged organ parts.
1. Biocompatibility
2. Mechanical strength
3. Porosity
4. Biodegradability
5. Enabling cell development and migration
Required scaffold’s characteristics:
Motivation
Designing porous hydrogels with
controllable pore size and porosity,
which allows for the in vitro
encapsulation of cells
System Design
Fibrinogen
PEG
polyethylene glycol
PEGylated fibrinogen fragments
Concept
PEG-fibrinogen polymer solution
Oil-in-water emulsion
PEG-fibrinogen hydrogel
Emulsion-templated PF hydrogel
Encapsulation of cells prior to polymerization
Oil droplets coated with surfactant layer
Research Goals1. System design - formulations for emulsions and emulsion-
templated PF hydrogels will be obtained and manipulated in
order to control hydrogel's pore size and porosity.
2. Structure characterization – the structure of the porous PF
hydrogels will be investigated in order to evaluate its relation
to the emulsion characteristics.
3. Physical properties – Young modulus and water weight gain
of the resulted emulsion-templated hydrogels will be
investigated.
4. Cellular biocompatibility – cytotoxicity and outgrowth studies
will be performed; the relationship between porosity and pore
size to cell proliferation will be explored.
System Design
pluronic®F108
pluronic®F68
75/25 (PF/oil)
50/50 (PF/oil)
90/10 (PF/oil)
95/5 (PF/oil)
Overhead mechanical stirrer
Magnetic stirrer
Stirring method
Surfactant type
Emulsion composition
System Design
structure characterization
Determination of oil extraction
Oil droplets size analysis
Cellular biocompatibility
Cytotoxicity assays
Morphology experiments
Hypothesis: structure impacts cellular biocompatibility
F68 95/5 (PF/oil)
F68 90/10 (PF/oil)
F108 95/5 (PF/oil)
F108 90/10 (PF/oil)
Oil extraction versus surfactant conc. - Magnetic stirrer
Oil Extraction
Higher surfactant concentrations results
in higher amounts of oil extracted
5% 10% 15% 20%0
20
40
60
80
100
120
140
160
180
Surfactant conc. [%w/v]
Am
oun
t of
oil
ext
ract
ed [
mg]
Oil extraction versus surfactant conc. - Overhead mechanical stirrer
Oil Extraction
Lower oil percentages
results in lower amounts of oil
extracted
3% 7% 10%0
20
40
60
80
100
120
Surfactant concentration [%w/v]
Am
oun
t of
oil
ext
ract
ed [
mg]
F68 95/5 (PF/oil)
F68 75/25 (PF/oil)
F68 50/50 (PF/oil)
10% F68 90/10( PF/oil)
10% F68 95/5( PF/oil)
10%F108 90/10( PF/oil)
10%F108 95/5( PF/oil )
Light Microscopy Images – Magnetic Stirrer
As-prepared hydrogels
Hydrogels submerged in water for 24 hr
0 1 4 710
15
20
25
30
35
40
Days
Oil
dro
ple
t d
iam
eter
[µ
m]
Size AnalysisOil droplet diameter versus time - Magnetic stirrer
The average droplet diameter diminishes
with time, which implies that oil from
larger droplets diffuse faster
F68 95/5 (PF/oil)
F68 90/10 (PF/oil)
F108 95/5 (PF/oil)
F108 90/10 (PF/oil)
Light Microscopy Images – Overhead Mechanical Stirrer 3%,7%,10% Pluronic® F68
95/5 (PF/oil)
Incre
asin
g su
rfacta
nt
conce
ntra
tions
Incre
asin
g su
rfacta
nt
conce
ntra
tions
3%,7%,10% Pluronic® F68 75/25 (PF/oil)
3%,7%,10% Pluronic® F68 50/50 (PF/oil)
1% 2% 3% 4% 5% 6% 7% 8% 9% 10%20
25
30
35
40
45
50
55
Surfactant percentage from emulsion [%w/v]
Oil
dro
ple
t d
iam
eter
[µ
m]
Size Analysis
Oil droplet diameter versus surfactant conc. – Overhead mechanical stirrer
Oil droplet diameter decreases with
higher surfactant percentages regardless of
emulsion composition.
F68 95/5 (PF/oil)
F68 75/25 (PF/oil)
F68 50/50 (PF/oil)
Cytotoxicity Assays
5% Pluronic®F68
5% Pluronic®F108
15% Pluronic®F68
15% Pluronic®F108
HFFs embedded in emulsion solutions composed of 90/10 (DMEM:oil)
1 4 70.2
0.3
0.4
0.5
0.6
0.7
0.8
Days
Sh
ape
ind
ex
Shape index versus time – Magnetic stirrer
Cellular Morphometrics
10%Pluronic®F68 95/5 (PF/oil)
exhibits the lowest shape index for
t>4.
PF-control
F68 95/5 (PF/oil)
F68 90/10 (PF/oil)
F108 95/5 (PF/oil)
F108 90/10 (PF/oil)
1 4 70
500
1000
1500
2000
2500
3000
3500
Days
Are
a [µ
m2]
Cell area versus time – Magnetic stirrer
Cellular Morphometrics
Cell area increases as a function of
time
PF-control
F68 95/5 (PF/oil)
F68 90/10 (PF/oil)
F108 95/5 (PF/oil)
F108 90/10 (PF/oil)
Cellular Morphogenesis versus Structure
10 15 20 25 30 35 401000
1500
2000
2500
3000
3500
Oil droplet diameter [µm] C
ell a
rea
[µm
2]
Shape index and cell area at day 7 versus initial oil droplet diameter
10 15 20 25 30 35 40 45 500.2
0.3
0.4
0.5
0.6
0.7
Oil droplet diameter [µm]
Sh
ape
ind
ex
Summary and future work• Oil droplet size can be controlled through
the stirring method and the surfactant concentration.
• A relation between hydrogel structure and cellular morphogenesis exists and needs to be further investigated.
• Future work will focus on characterizing the nanostructure of the system and exploring its relationship to cell viability.
Thank you for your attention!
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