ENGINEERING 2D AND 3D CELLULAR MICROENVIRONMENTS USING LASER DIRECT WRITE - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 107

the focal contact point takes place, and there is a greater stress on the cytoskeleton elements causing
cell changes. Significant differences in substrate stiffness manifest in different cell morphologies,
cytoskeletal reorganization, gene expression, and fate decisions. The pioneering work of Dennis
Discher's group ( Engler et al., 2006 ) showed that mesenchymal stem cells (MSCs), when cultured on
substrates of different elastic moduli, exhibited different morphologies and gene profiles; substrate
stiffness could be used to direct their differentiation ( Engler et al., 2006 ). Thus, the substrate stiffness
is an important factor to consider when engineering the cellular microenvironment.
Functional groups present and their relative concentration in the ECM also play a pivotal role
in the overall behavior of the cell. The presentation and binding of ligands is necessary to induce
adhesion, motility, survival, differentiation, and other specific cell functions. For example, human
embryonic stem cell (hESC) culture differentiation typically requires the use of a Matrigel ® sub-
strate, a hydrogel derived from mouse tumor basement membrane. However, synthetic substrates
can be functionalized with the integrin ligands found within Matrigel ® to support hESC attachment
and growth ( Liu et al., 2011 ). In addition to the presence of the specific functional groups, their rela-
tive concentrations also play a key role in cell behavior. Substrates modified with different surface
concentrations of the arginine-glycine-aspartic acid (RGD) motif exhibit differing degrees of cel-
lular attachment, affecting the cytoskeletal structure, its organization, and the cellular morphology
( Massia et al., 1991 ). Another feature of engineered motifs is that binding affinity is different from
that of the same motif within a natural protein, such as collagen or fibronectin. As a result, engineered
substrates may have different binding kinetics than natural substrates. However, the surface-coated
RGD can be engineered to contain additional peptide sequences that enhance its specificity or binding
kinetics to a ligand ( Petrie et al., 2006 ). Overall, mechanical properties of the material, such as elastic
modulus, and biochemical properties of the material, such as functionalization, can have a profound
influence on cells within the microenvironment.
5.2.2 MATRIGEL-BASED LDW
Initial studies of cell printing by LDW utilized Matrigel ® as a coating on the receiving substrate, as well
as on the ribbon as a sacrificial layer/transfer material ( Figure 5.3 ). Matrigel ® is a soluble tumor extract
comprised of an assortment of proteins, and it undergoes thermal gelation at 37°C, creating a 3D gel
( Kleinman et al., 1986 ). There are several features that make Matrigel ® a good candidate material for
the receiving substrate and as a transfer material for LDW studies. Matrigel ® contains large quantities
of all the essential structural proteins along with many of the other proteins, proteases, and growth fac-
tors found in the basement membrane ( Kleinman et al., 2005 ). Matrigel ® has been used widely for in
vitro cell culture to differentiate a variety of cells and is currently used almost exclusively as the scaf-
folding material for maintaining undifferentiated human embryonic stem cell culture ( Xu et al., 2001 ).
Additionally, Matrigel ® provides a permanent matrix to immobilize printed material on the receiving
substrate, contributing to the high pattern fidelity.
However, despite these many benefits, Matrigel ® has a number of shortcomings that may preclude
or limit its use in certain applications, causing some researchers to seek alternative ribbon and sub-
strate materials. As a transfer material, Matrigel ® requires a cell-based biopayload to loosely attach
to the Matrigel ® matrix. Cellular attachment times vary depending on the cell type, making coculture
transfers difficult and limited to adherent cell types. As an ECM-mimetic substrate material, Matrigel ®
has an inherent batch-to-batch variability because it is grown and extracted from a mouse tumor. This