1. 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 biofunctionalizationStructure-property characterization
Physical network properties
tunable by
starPEG to heparin
molar ratio γ
… at constant
heparin concentration
and
biomolecular composition
of the swollen gels
NH2
O
O O
O
NH2
O
O
NH2
O
O
NH2
n n
n
n
heparin
Cryogenic treatment
scaffold
freezing lyophilization
poreice 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
storagemodulus[kPa]
0,0
0,2
0,4
0,6
molar ratio PEG:heparin
g
RGD/heparin[mol/mol]
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
heparinorPEGconc.[mg/ml]
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 [%]
stress[kPa]
0
1
2
3
4
5
0 20 40 60 80 100
strain [%]
stress[kPa]
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