(386f) Graduate Student Award Session: Tissue Guided Design of a Brain ECM Mimicking Hydrogel

Tissue Guided
Design of a Brain ECM mimicking Hydrogel

Sualyneth
Galarza¹, Shelly R. Peyton¹

¹Chemical
Engineering Department, University of Massachusetts, Amherst

1.0 Background

Recently, studies
have highlighted the role of the extracellular matrix (ECM) in dictating cell
response and disease progression.¹ To assess the role of the brain
ECM, 3-dimensional in vitro models
have been designed to represent matrix-mimicking biomaterials as seen in vivo.² Within the context
of the brain, cell culture platforms derived from tissue, such as collagen and hyaluronan hydrogels, cortical slices, de-cellularized tissues, and cerebral organoids mimic some
aspects of the brain architecture but do not readily allow investigators to
identify the individual ECM-drivers responsible for observed cell behaviors.^2-4
Synthetic hydrogels, in contrast, have the potential to provide tight control
over finite variables like stiffness and bioactive ligand density, with high
reproducibility.⁵ However, current hydrogel systems tend to
oversimplify the native ECM and neglect many complex arrays of proteins present
in real tissue.

To augment these in vitro efforts, we have designed a
brain tissue mimicking hydrogel that captures a brain-specific adhesive and
degradable ECM alongside the appropriate brain viscoelasticity.

2.0 Methods

Using
a top-down approach we have screened the human cerebral cortex for ECM tissue
features that could be incorporated into a synthetic PEG-based hydrogel to
capture mechanical and biochemical properties of the brain. The cerebral cortex
accounts for 77% of the brain volume⁶, and we assessed the brain
cortex ECM composition via histology-based bioinformatics and proteomics.
Histology data from the Human Protein Atlas⁷ was used to identify
cortex enriched proteins, their expression, and local distribution. In
addition, human cortex samples from 4 healthy donors were analyzed by LC-MS to
identify relative protein abundance and a consistent ECM signature across
donors (fig 1a). From the list of ECM proteins, those with integrin-binding
capabilities and susceptibility to matrix metalloproteinase (MMP) cleavage were
chosen to allow for cell adhesion and hydrogel degradation. Bioactive motifs of
the selected proteins previously identified in literature were synthesized by
solid phase peptide synthesis and incorporated in a poly(ethylene) glycol
hydrogel via Michael type addition reaction. Nine cell-adhesive ligand peptides
and five MMP-degradable sequences represent the synthetic brain ECM. To
modulate the physical properties of the system we characterized porcine brain
tissue and tuned our hydrogel to the same modulus (fig 1b). Our hydrogel system
is fully synthetic, comprised of PEG and peptides and allows us to tune the mechanical
properties independent of ligand density (fig 1c).

3.0
Results

In 2D, we validated integrin-peptide
affinity by conducting a competition assay between individual peptides and the
signature peptide mixture. Human cell-line derived neurons interacted with
soluble peptides, each in the same concentration as in the peptide mixture,
before being seeded on a surface covalently coupled with the same
integrin-binding peptides. Individual components of the peptide mixture were
found to prevent cell adhesion on the functionalized coverslip. The mixture of
peptides prevented adhesion better than the commonly used RGD peptide alone. In
3D, incorporation of the enzyme-sensitive peptides alongside integrin-binding
peptides followed cell degradation after 12 days in culture, validating this
fully synthetic system can be degraded by cells (fig 1d).

To assess the
effect of the synthetic brain hydrogel composition on cell behavior we cultured
human primary astrocytes, which showed processes emanating from the cell soma
reminiscent of in vivo like
morphology. To investigate levels of activation in human astrocytes when
cultured in the brain hydrogel, we screened for markers upregulated upon brain
injury. After 24 hours of culture, primary cells expressed low levels of
activation of markers identified in reactive astrocytes like GFAP, in the brain
hydrogel system compared to a collagen gel control (fig 1e). Currently, further
validation entails levels of expression for n-cadherin and TGF-beta, additional
reactive and scar-forming astrocyte markers⁸. Identification of
region-specific markers in astrocytes cultured in the brain hydrogel will be
conducted to validate the cerebral cortex-inspired hydrogel design.

4.0
Conclusions

We have design a brain tissue-inspired
hydrogel that captures the brain’s unique ECM protein signature and mechanics.
The materials used in this system, PEG and peptides, are fully synthetic and
easily tunable. We foresee the use of this platform to identify tissue-specific
features driving homeostasis or disease progression.

References: ¹Egervari,
K. et al., Glia. 2016; 64, 3. ²Hopkins, A. M. et al., Prog. Neurobiology. 2015; 125, 1-25. ³Qian, X.,
et al., Cell 2016: 165, 1238-1254. ⁴Li, Y., et al., Cell stem cell.
2017; 20, 385-396. ⁵Kyburz, K.K.A, Ann. Biomed. Eng. 2015; 43,
489-500. ⁶Swanson, L.W. et al., Trends in Neurosci.
1995, 18, 471-474. ⁷Uhlén, M. et al., Science. 2015; 347, 6220. ⁸Hara,
M., et al., Nature Medicine, 2017, 23(7), 818–828.