heparin = “bioactive” part of the material:

binding of growth factors and adhesion ligands

• covalent decoration of heparin with cell-adhesive ligand

structures, e.g. cyclic RGD peptides

• loading with heparin-binding chemokines and growth

factors, e.g. SDF1, FGF2, VEGF

• uptake of non-heparin-binding biomolecules, e.g.

albumin

Macroporous starPEG-heparin cryogel scaffolds

and microcarriers for cell culture studies in three-

dimensional environment

Preparation and characterization of macroporous scaffolds and microcarriers

Properties of the biohybrid hydrogel system

Motivation

Summary and Outlook

Claudia Renneberg, Carla Günther, Petra B. Welzel, Karolina Chwalek, Andrea Zieris, Uwe Freudenberg, Carsten Werner 1 Leibniz Institute of Polymer Research and Max Bergmann Centre of Biomaterials Dresden, ² Dresden University of Technology, ³ Center of Regenerative Therapies Dresden

Cryogels are macroporous hydrogel scaffolds that are gaining increased interest for tissue engineering applications as three-dimensional (3D) analogues of the extracellular matrix

(ECM). Due to their sponge-like structure and their large interconnected pores, cryogels not only allow for a three-dimensional organisation of cells and for a sufficient nutrient supply

and waste disposal but also facilitate blood vessel ingrowth. However, the lack of blood vessels and resulting hypoxia is still a fundamental challenge in regenerative medicine. The

present study investigates the potential of a novel type of cryogel based on a modular starPEG-heparin hydrogel platform with adaptable physical and biomolecular properties to

induce in vitro pre-vascularization.

Network formation

• MMP-cleavable gels: gel formation via

starPEG- peptide conjugates susceptible to

cell-triggered proteolysis

• In situ assembling gels

• Directly crosslinked gels: activation of

carboxylic acid groups of heparin, conversion

with (amino-) end-functionalized multi-arm

(star=) polyethylene glycol (PEG)

Secondary biofunctionalization Structure-property characterization

Physical network properties

tunable by

starPEG to heparin

molar ratio γ

… at constant

heparin concentration

and

biomolecular composition

of the swollen gels

NH2O

O OO

NH2

O

O

NH2

O

O

NH2

n n

n

n

heparin

Cryogenic treatment

scaffold

freezing lyophilization

pore ice crystal (porogen) aqueous reaction mixture

non-frozen liquid phase

dispersion polymerization Subzero temperature treatment of the gel formation reaction mixture and subsequent

lyophilization of the incompletly frozen gel resulted in macroporous biohybrid cryogels.

standard polymerization

1,5 2 3 4 6 0

2

4

6

8

10

12

14

16

18

molar ratio PEG:heparin

sto

rage m

odulu

s [ kP

a ]

0,0

0,2

0,4

0,6

molar ratio PEG:heparin

g

RG

D/h

ep

ari

n

[m

ol/m

ol]

1,5 2 3 4 6

1,5 2 3 4 6 0

5

10

15

20

25

30

35

40

45

molar ratio PEG:heparin

he

pa

rin

or

PE

G c

on

c.

[ m g/ m

l ]

heparin PEG 10,000 g/mol

Cell cultivation

[1] U. Freudenberg et al. A starPEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases. Biomaterials 30 (2009) 5049-5060 [2] P.B. Welzel et al. Macroporous starPEG-heparin cryogels. Biomacromolecules 13 (2012) 2349-2358 [3] A. Zieris et al. FGF-2 and VEGF functionalization of starPEG-heparin hydrogels to modulate biomolecular and physical cues of angiogenesis. Biomaterials 31 (2010) 7985-7994

SEM image of the

microcarrier surface

Swollen cryogel

microcarrier (cLSM)

The morphology of the dry and swollen cryogel scaffolds and microcarrier was characterized by

scanning electron microscopy (SEM) and confocal laser scanning microscopy (cLSM). The pore

size of the in PBS swollen materials varied between about 20 and 300 µm.

For the preparation of cryogel-microcarriers the

principle of dispersion polymerization was used.

The gel formation mixture was added to a

nonpolar solvent and agitated continuously while

cryogenic treatment at -20 °C.

The diameters of the

achieved microcarrier

ranges from 100 µm

up to 800 µm in

swollen state with

irregular macropores

of 20 to 200 µm in

diameter.

bio-

compatible

tunable network

properties

adaptable

biomolecular

properties

interconnected

macropores:

20 -300 µm

advantageous

mechanical properties

rapid

swelling

mesh size:

5 – 30 nm

bio-

responsive

high

porosity

about 90%

‘direct’ covalent bond

Leucine- zipper

MMP-cleavable peptides

The cells completely overgrow small pores and mimic the structure of large pores. Since pores are

overall rather small, no tube formation in pores is expected. HUVECs will rather coat pores with

endothelium like in capillaries. The pores‘ and resulting „vasculature‘s“ densitiy is similiar to vasculature

in brain and liver.

Swelling behavior

The applicability of the cryostructured material after secondary biofunctionalization with an RGD-

containing peptide was shown by co-culturing human umbilical vein endothelial cells (HUVECs) and

human mesenchymal stem cells (hMSCs) in vitro within the 3D-scaffolds and microcarriers. The

appropriate size and interconnectivity of the pores enabled HUVECs and hMSCs to migrate into the

cryogel materials. Viability tests and cLSM analysis of fluorescence labelled samples indicated three-

dimensional spreading and good survival of the cells colonizing the cryogel materials.

7 day cultivation of 1·106 HUVECs and 10 % MSCs on macroporous

cryogel-scaffolds; fluorescent labeling: green = hydrogel matrix, red = aktin

(cytoskeleton), yellow = CD 31 (endothelia cell marker)

3 day (left<) and 5 day (right) cultivation of 1·106 HuVECS and 10 %

MSCs on macroporous cryogel-microcarrier; fluorescent labeling: green

= hydrogel matrix, red = aktin (cytoskeleton)

0

50

100

150

200

250

300

350

400

450

0 20 40 60 80 100

strain [%]

str

es

s [

kP

a]

0

1

2

3

4

5

0 20 40 60 80 100

strain [%]

str

es

s [

kP

a]

the swollen

materials are

rather soft but

very tough and

can withstand

large deformation

without losing

integrity

Uniaxial compression stress–strain curves obtained for the different cryogel types.

Black: g = 3. Dark grey: g = 2. Light grey: g = 1.25.

Mechanical properties

rapid swelling in aqueous solution (PBS)

polymer regions of cryogels swell less than corresponding

homogeneous hydrogels

44.1 ± 1.07.2 ± 0.43

59.2 ± 3.18.8 ± 1.42

88.1 ± 0.610.5 ± 1.81.5

hydrogelcryogel

volume swelling ratio

γ

44.1 ± 1.07.2 ± 0.43

59.2 ± 3.18.8 ± 1.42

88.1 ± 0.610.5 ± 1.81.5

hydrogelcryogel

volume swelling ratio

γ

For the preparation of cryogel-scaffolds gel

mixture was pipetted into a multiwell plate and

frozen at -20°C. This method enables easy

production of scaffolds with interconnected

pores of 30 – 300 µm sizes. Scaffolds can be

labeld with Alexa dyes and feature user

friendly treatment.

light microskop image

of a dry microcarrier

light microskop

image of a swollen

microcarrier

By cultivating HUVECs on our 3D macroporous scaffolds and microcarrier cell proliferation was directed to form an ensemble of similar cells which spreads over the whole scaffold and mimics the scaffolds structure. Further impact on the ensemble is possible by variation of the scaffolds stiffness, incorporation of VEGF and by coculture with supporting MSCs. These results are encouraging steps on the way to taylor made vascularized tissue which represents a stepping stone to tissue engineering of complex organs like liver and brain. However in order to demonstrate the engineering of a tissue like structure cells have to carry out a function. This has to be achieved in further experiments.

SEM Image of

scaffold surface Swollen Scaffold its

porestructure through cLSM

Scaffolds and

microcarrier

allow simple

analyzation via

different

methods