Transglutaminase Crosslinking of Resilin-based Proteins for Vascular Applications
Yeji Kim, Emily E. Gill, and Julie C. Liu
School of Chemical Engineering & Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907
Introduction: Protein-based biomaterials have emerged
as powerful tools for tissue engineering applications.
Recombinant DNA techniques can be used to precisely
tune material properties at the molecular level, and
multiple peptide modules can be incorporated into a
single material. In this study, we utilized recombinant
proteins that include a motif based on resilin, a highly
resilient protein. We found that hydrogels formed using
transglutaminase as a crosslinker performed better in
long-term cell attachment studies compared to a widelyused crosslinker, tris(hydroxylmethyl)phosphine (THP)
[1]. Furthermore, by varying matrix stiffness, we were
able to modulate cell spreading and endothelial
differentiation of human mesenchymal stem cells (hMSCs)
on the hydrogels [1].
Methods: Proteins containing the RGD cell-binding
domain and resilin repeats from Anopheles gambia [2]
were expressed in E. coli at 37 °C with isopropyl β-D-1thiogalactopyranoside in a fermentor. Proteins were
purified by a salting-out and heating method.
Transglutaminase (2 U/mL) was added to protein
solutions (6-12 wt%), and the solutions were placed in a
silicone mold (8 mm diameter, 250 m thick) to form
crosslinked hydrogels. The swelling ratio and water
content were calculated after 3 d of swelling. Matrix
moduli were measured using an AR2000 rheometer (TA
Instruments). hMSCs were seeded on various hydrogels
or on glass, and cell viability, metabolic activity, and
BrdU incorporation were assessed. Cell spreading was
evaluated after 10 h of attachment by calculating cell area
and circularity. For endothelial differentiation, cells were
grown in endothelial growth medium-2 (Lonza)
supplemented with 50 ng/mL VEGF for 14 d, and the
uptake of acetylated low density lipoprotein (ac-LDL)
was determined.
Results: First, we found that after 6-7 d of incubation,
significantly more cells were observed on hydrogels
formed by crosslinking with transglutaminase compared
with THP even when properties such as ligand density,
mechanical properties, and water contact angle were kept
constant (data not shown). Thus, transglutaminase
crosslinking resulted in surfaces more suitable for cell
attachment and cell studies, and we continued to examine
only these surfaces in our study. Hydrogels formed within
60-340 s. As total protein concentration increased, the
swelling ratio (ranging from 8-13) and water content
(ranging from 88-92%) decreased. By varying the total
protein concentration from 6-12 wt%, the storage moduli
of the gels were varied from 44 Pa to 7 kPa.
When hMSCs were seeded on hydrogels with various
matrix stiffness values, there were no significant
differences when evaluating the cell viability (>94% after
2 d), metabolic activity (~70% of activity compared to
glass), and BrdU incorporation (50-57%). Cells on the
softest 6 wt% hydrogels (44 Pa) displayed a rounded
morphology, had the smallest cell area, and displayed the
greatest circularity, whereas cells on the stiffest 12 wt%
matrices (7 kPa) exhibited extensive spreading, had the
largest cell area, and resulted in the lowest circularity
(Figure 1). Finally, endothelial differentiation of hMSCs
was assessed by evaluating endothelial functional
behavior. Specifically, internalization of ac-LDL is a
characteristic behavior of endothelial cells. The
percentage of cells positive for ac-LDL on the stiffest 12
wt% hydrogels was 78% and was statistically higher than
that on the softest 6 wt% gels (39% of cells) (Figure 2).
%
44
Pa
%
91
Pa
%
%
402
7
~70
Pa
kPa GPa
%
44
Pa
%
91
Pa
%
%
402
7
~70
Pa
kPa GPa
Figure 1. Cell spreading on the hydrogels was
modulated by matrix stiffness. Letters indicate
statistically different Tukey groups (p < 0.05).
%
44
Pa
%
91
Pa
%
%
402
7
~70
Pa
kPa GPa
Figure 2. Endothelial function of cells depended on
matrix stiffness. hMSCs were differentiated on surfaces
for 14 d. Endothelial differentiation was assessed by the
percentage of cells that internalized acetylated lowdensity lipoprotein (ac-LDL). Letters indicate statistically
different Tukey groups (p < 0.05).
Conclusions: Transglutaminase crosslinking created
hydrogels suitable for long-term cell adhesion and culture.
Varying the hydrogel stiffness modulated hMSC
spreading and endothelial function of cells. The stiffest
matrices increased cell spreading and endothelial function.
Overall, our tunable hydrogel system is a promising tool
for vascular applications and provides mechanical
environments that more closely mimic native
subendothelial environments compared to conventional
culture substrates.
References: [1] Kim, Y. et al., Biomacromolecules, 2016.
[2] Lyons, RE. et al, Protein Eng Des Sel, 2007.