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!