BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine

14 (2018) 13 – 25

Review Article nanomedjournal.com

Polymer hydration and stiffness at biointerfaces and related cellular processes

Garry Kerch, Dr Sci Ing⁎
Riga Technical University, Riga, Latvia
Received 19 June 2017; accepted 16 August 2017

Abstract

The direct and indirect (by changing mechanical properties) effects of hydration at interfaces on cellular processes and tissue diseases are
reviewed. The essential effect of substrate stiffness on cellular processes was demonstrated in the last decade. The combined effect of surface
stiffness and hydration at interfaces has garnered much less attention, though hydration and dehydration play important roles in biological
processes. This review focuses on the studies that demonstrate how hydration affects biological processes at interfaces. Elevated sodium and
dehydration stimulate inflammatory signaling in endothelial cells and promote atherosclerosis. Various types of implant and blood contacting
device coatings with varied surface stiffness and hydration have been reported. Effect of hydration on polymer modulus of elasticity and
viscoelasticity was discussed taking into account cells adhesion, migration, proliferation, differentiation on surfaces with various degree of
hydration. Future directions of research were considered, including the use of nanotechnology to regulate the hydration degree.
© 2017 Elsevier Inc. All rights reserved.

Key words: Polymer; Hydration; Stiffness; Biointerface; Cellular processes

Water is an integral part of various biological molecules and
biopolymer systems. Water is the most abundant molecule in the
body and water is essential for life. This review focuses on effect
of polymer hydration/dehydration on the processes at the cell–
biomaterial interface and cell–extracellular matrix interactions.
Ventre and Netti 1 , 2 consider that “a thorough understanding
of the cell-material crosstalk has not been achieved yet”. It is
known that biological cues, topography, and mechanical
properties control cell fate and functions. It has been suggested
in the recent review 3 that cells respond to elasticity and
topography of biomaterials by generating tractional forces on
their adhesive contacts. Muzzio et al 4 observed that cells adhere
better on relatively smoother annealed substrates. So they
suggested that surface roughness is not the most relevant factor
in the interaction of adherent cells with solid surfaces. The
influence of surface stiffness on the adhesion of cells can be
considered as a more important factor. Balance of chemistry,
topography, and mechanical properties at the cell–biomaterial
interface was analyzed by Wong et al 5 in 2004 and the authors
stated that “effect of substrate elasticity on cell behavior is still a
relatively unexplored research field”. More knowledge has been
generated during the last decade, and at present already surface
stiffness can be considered as a more important factor compared
to a surface topography. Huang et al 6 note that “it is not clear
whether the surface chemistry or topography is the main factor
on modulating cellular behavior, because the commonly used
surface modification techniques for titanium-based implants
simultaneously modify surface topography and chemistry”.

Abbreviations: ECM, extracellular matrix; PEG, polyethylene glycol; PEO, poly(ethylene oxide); PEMs, polyelectrolyte multilayers; FN, fibronectin; PGs,
proteoglycans; BMSCs, bone marrow stromal cells; Hap, hydroxyapatite; Cap, carbonate-substituted hydroxyapatite; PEO, poly(ethylene oxide); PSS,
poly(sodium 4-styrenesulfonate); PDADMAC, poly(diallyldimethylammonium) chloride; ECs, endothelial cells; SMCs, smooth muscle cells; PRX,
polyrotaxane; iPS cells, pluripotent stem cells; α-CD, α-cyclodextrin; CNWs, cellulose nanowhiskers; SBR, poly(styrene-co-butadiene); PBD, polybutadiene;
PVOH, poly(vinyl alcohol); hMSC, human mesenchymal stem cells; ESL, endothelial surface layer; NO, nitric oxide; pSBMA, poly(sulfobetaine methacrylate);
DSC, differential scanning calorimetry; DTA, differential thermal analysis; LbL, layer-by-layer; PIPAAm, poly(N-isopropylacrylamide); PSBMAm,
poly(sulfobetaine methacrylamide); BSA, bovine serum albumin.
Conflict of interest statement: I have no conflict of interest to declare.
Funding: This review did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
⁎ Institute of Polymer Materials, Department of Materials Science and Applied Chemistry, Riga Technical University, Riga, Latvia.
E-mail address: garrykerch@inbox.lv.

https://doi.org/10.1016/j.nano.2017.08.012
1549-9634/© 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Kerch G, Polymer hydration and stiffness at biointerfaces and related cellular processes. Nanomedicine: NBM 2018;14:13-25, https://
doi.org/10.1016/j.nano.2017.08.012
14 G. Kerch / Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 13–25
Human bone marrow derived mesenchymal stem cells (BMSCs)
were cultured to form cell sheets by researchers at Nanyang
Technological University, Singapore. 7 Collagen formation
within the cell sheet was enhanced on substrates with lower
stiffness, higher hydrophobicity and roughness. Li et al 8 in
experiments with rat bone marrow mesenchymal stem cells
found that substrate stiffness is the main factor in regulating cell
proliferation and differentiation, but topography plays a lesser
role in directing cell differentiation. No statistically significant
influence of surface roughness on osteoblast proliferation and
cell viability was detected by Cai et al. 9
Layer-by-layer (LbL) assembly based on the alternating
adsorption of polyelectrolytes on surfaces allows for designing
multilayer architectures with nanometer precision. Nanocoatings
can be used in biomedical applications. The role of the surface
stiffness of polyelectrolyte multilayers (PEMs) in interaction with
cells is generally recognized. 10–13 It is important to control the
adhesion processes of mammalian and bacterial cells at interfaces
in such biomedical applications as polymer coatings of implants
and biosensors, antifouling and antibacterial coatings, tissue
engineering. 14 Adhesion affects other cell functions, such as
spreading, migration, proliferation and differentiation. 15 The
correlation between cell viability and surface energy (wettability),
modulus (matrix stiffness), and surface charge of the polyelectro-
lyte multilayer coatings has been studied and the authors at McGill
University, Montreal, Quebec, Canada concluded that stiffness has
the highest impact on cells survival. 16 In other publication it was
observed that surface hydration can play a stronger role in
comparison with surface stiffness in the control of platelet
adhesion. 17
The mechanical properties of extracellular matrix (ECM) to a
great extent depend on properties of ECM component collagen,
and the conformation and mechanical properties of collagen
depend on water molecules that through water bridges stabilize
triple-helical conformation of collagen. 18 , 19 It has been widely
believed for the past several decades that structure of water in
collagen fibrils is very different from bulk water. 20–23
This review focuses on the effect of combination of surface
stiffness with surface hydration, which is still underestimated
aspect with a direct effect on cells adhesion, migration,
proliferation, growth and differentiation.
States of water in polymers
Hydration water structure depends on the architecture of
hydrated polymer chain. 24 , 25 Dynamics of hydration water plays
an active role for proper functions of proteins. 26 Non-freezing
water, freezing water and free water can be observed by the
methods of differential scanning calorimetry (DSC) and differen-
tial thermal analysis (DTA). 27, 28 The states of water have been
reported as a tightly bound water, loosely bound water and bulk
water. The concept of intermediate water, with the properties
similar to freezing water, or loosely bound water, has been also
proposed. 29 The water is tightly bound to macromolecules
provided the water content in a polymer is lower than a certain
water content threshold. 30–32 The value of this threshold depends
on polymer macromolecule chemical structure.
The macromolecules change the properties of water. Water is
an integral part of various biomolecular complexes. 33 The
distribution and binding energy of water molecules to biopoly-
mers was investigated for food products, such as bread 28 , 34–44
and meat. 45–47 Water-holding capacity of pork increases during
aging due to degradation of the cell cytoskeleton. 47 Protein
oxidation increases hydration but decreases water binding in
pork. 48 Hydration water is essential in biological processes and
plays a major role in actin–myosin binding. 49–51
There is essential change of water properties in confined
conditions. Goertz et al 52 observed effective viscosities that are
∼10 6 times greater than that of bulk water for nanometer-scale
interfacial separations.
The influence of polymer chain architecture of polysaccha-
rides on the structuring of confined hydration water has been
reported by researchers of Dutcher Lab at the University of
Guelph, Canada. 24 , 53 They studied phytoglycogen, a highly
branched, water-soluble polymer of glucose with dendrimeric or
tree-like structure and remarkable capacity to retain water. They
found similarities between water structuring in two linear
polysaccharides, hyaluronic acid and chitosan, and significant
differences between the linear molecules and highly branched
phytoglycogen. Quasi-elastic neutron scattering measurements
revealed a significant slowing down or retardation of the
hydration water relative to bulk water by an average factor of
~5.8. The hydration water in the phytoglycogen nanoparticles is
significantly more highly ordered and tightly bound than in the
linear polysaccharides, as indicated by the large peak at
~3200 cm −1 in the phytoglycogen infrared spectra. 24 , 54 They
found a correlation between the structural rearrangement of the
hydrogen-bonding network of the tightly bound hydration water
and the interchain separation in the highly branched phytoglyco-
gen nanoparticles. 25
It was reported that polyvalent dendrimer glucosamine
conjugates prevent scar tissue formation. 55 So it would be
interesting to confirm the relation of water retention to ability to
prevent scar formation or to show that such processes are not
interdependent.
Design principles and clinical applications of dendrimers in
nanomedicine have been recently reviewed. 56
Hydrated polymer coatings and surfaces
Cell biology and biomaterials science are related. Polymer
materials are used in implants and in many cases polymers mimic
the properties of living tissues. Biopolymers are used in the
design of scaffolds for regenerative medicine. Similar behavior
of cells can be expected on the surfaces of biocompatible
polymers and on the surfaces of living tissues with the similar
physical properties.
PEG coatings
Hydrophilic polyethylene glycol (PEG) with molecular structure
H–(O–CH2–CH2)n–OH can tightly bind water molecules to form a
surface hydration layer, which can resist nonspecific protein
adsorption and platelet adhesion. 57 PEG can also affect protein
conformational changes. 58 Molecular simulations demonstrated the
 G. Kerch / Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 13–25
correlation between oligo (ethylene glycol) (OEG) surface resistance
to protein adsorption and the surface density of OEG chains. The
increase in surface density increases a number of tightly bound water
molecules around OEG chains. 59 Various techniques intended to
improve surface endothelialization on vascular grafts for various
polymer coatings have been recently reviewed. 60
Zwitterionic coatings
Zwitterionic polymers have both cationic and anionic
moieties on the same side chain while maintaining overall
charge neutrality. 57 , 61 Surface hydration is the key nonfouling
mechanism of zwitterionic materials due to the strong hydrogen
bonding of the interfacial water molecules. 62 The formation of a
strong hydration layer prevents protein adsorption at the polymer
interfaces. 63 State-of-the-art in the design of protein resistant
polymer coatings and their applications in biomedicine have
been recently reviewed. 64
Multilayer polyelectrolyte coatings
Polyelectrolyte multilayers (PEMs) are built up via subse-
quent adsorption of oppositely charged polycations and poly-
anions. The swelling behavior of PEMs is very important in
various potential applications. 65 The influence of different types
of water on the water distribution and the swelling behavior of
polyelectrolyte multilayers have been recently discussed in the
Ph.D. thesis by Zerball. 66
Sulfated polymers that have properties similar to the
properties of natural glycosaminoglycan heparin, can be used
as polyanions in PEMs. 67 It has been shown that heparin-like
PEMs based on fungal sulfated chitosan decrease platelet
adhesion due to the increased hydration and reduced stiffness. 68
Zhang and coworkers 69 demonstrated the interplay of salt and
water in the plasticization of polyelectrolyte assemblies and dual
nature of salt both as plasticizer and hardener of PEMs. Water
decreases the stiffness of PEMs. The salt ions on the one hand
provide additional free volume for polymer chain motion and
weaken interaction between polyanion and polycation layers due
to electrostatic screening. On the other hand, salt ions immobilize
water in their hydration shells reducing the plasticization by
hydration.
Hydrated extracellular matrix (ECM)
The ECM is composed of water, proteins and polysaccharides, 70
more specifically Bosman and Stamenkovic71 suggested that ECM is
composed of type I and III collagens and elastin that, together with
fibronectin (FN), form a relaxed network of fibers that are surrounded
by and embedded in a hydrogel of glycosaminoglycan-chain-
containing proteoglycans (PGs).
Collagen crosslinking leads to ECM stiffening. Dehydration
also can lead to collagen stiffening 72 , 73 and can influence
vascular smooth muscle cell phenotype. Cells spreading
increases on dehydrated collagen fibrils compared to cells on
fully hydrated fibrils. Dehydration also essentially increased the
rate of cell proliferation. 73 Molecular dynamic simulations of a
collagen-like peptide have shown that the absence of water
results in increase in the number of intra-molecular hydrogen
bonds. 74 Babaei and coauthors 75 reported that remodeling by
15
fibroblasts alters the rate-dependent mechanical properties of
collagen and changes stress relaxation behavior. Collagen
molecules are surrounded by a hydration layer. 36 , 76 Dehydration
leads to the tighter packing of collagen fibrils and enhances
stiffness by increasing intra-molecular hydrogen bonds. 73 , 74 , 77
As a result of dehydration at nanolevel deformation mechanism
shifted from molecular sliding to molecular stretching. 78
It has been also found that breast tumorigenesis is
accompanied by collagen crosslinking and ECM stiffening. 79
Hydrogels
Hydrogels can be used as native ECM mimics. 80–82 It has
been reported that biomimetic poly(HEMA)-based hydrogel
influences growth and proliferation of anchorage-dependent
human muscle fibroblasts (RMS 13) cells. Hydrogels that have
lower elastic modulus and lower bulk-to-bound water ratio
preferentially support cell attachment. 83 It has been reported that
coupling chitosan with polyethylene oxide through “click
chemistry” results in development of hydrogel that shows a
great amount of absorbed water and a rapid swelling kinetics. 84
Effect of hydrated and dry collagen on cellular processes
Mechanical properties of polymer structures, including ECM
and tissues, depend on the content of water. Collagen is one of
the main components of ECM and various tissues. So the
mechanical properties of dry and hydrated collagen play an
important role in many biological processes and can be linked
with various age-related diseases. Tissue stiffness can control a
number of normal and pathological processes that involve cell
locomotion. 85–87 It is well known that mechanical stress
regulates gene expression. It has been reported that integrins,
the transmembrane adhesion and signaling receptors which
physically link ECM to the cytoskeleton transduce mechanical
signals presumably via MAP kinase and NF-κB pathways. 88
Mechanical stress also regulates cell adhesion and cytoskeletal
organization. 89 The connections between stress fibers and the
cell nucleus have been recently reviewed in relation to cell
migration and nuclear function. 90
Effect of hydration on cell adhesion
Detachment of endothelial cells and hepatocytes from
poly(N-isopropylacrylamide) (PIPAAm) surface caused by the
spontaneous hydration of PIPAAm chains was observed in 1995
by Okano et al. 91 The effects of hydration and collagen
cross-linking treatment on biomechanical properties of the
cornea have been recently reported. 92 Chen et al 93 concluded
that highly hydrated chemical groups are the keys to developing
effective nonfouling materials. Researchers at the University of
Washington, Seattle, USA concluded that zwitterions with a
balanced charge and minimized dipole are excellent candidates
as nonfouling materials due to their strong hydration capacity. 94
Leng et al 63 reported that strong hydrogen bonds in the strong
hydration layers prevent protein fouling at the polymer
interfaces. Strong surface hydration has been proposed as a
significant contributor to the nonfouling mechanism. 62 , 95 , 96
Wang et al97 investigated the anion specificity of poly(sulfobetaine
methacrylamide) (PSBMAm) brushes with different carbon spacer
16 G. Kerch / Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 13–25
lengths and demonstrated that carbon spacer length can control
protein adsorption on the surface of the PSBMAm brushes. The
addition of anions can more effectively enhance the hydration of
the PSBMAm brushes with a medium-length carbon spacer
compared with that of the PSBMAm brushes with either shorter or
longer carbon spacer.
Kiroshka et al 98 investigated the influence of chitosan–chitin
nanofiber composites on cytoskeleton structure and the prolif-
eration of rat bone marrow stromal cells (BMSCs). They found
that changes in properties of the chitosan–chitin whiskers
nanocomposite films give rise to changes in the cytoskeleton
structure of the rat bone marrow stromal cells that control the
behavior of cells in culture (adhesion, spreading, and prolifer-
ation). Lower swelling of the films facilitated adhesion, and
increased spreading area of BMSCs. The presence of hydrated
domains, and decreases in rigidity due to swelling of chitosan/
chitin matrices decreased the spreading area of BMSCs.
Immune responses to synthetic hydroxyapatite (HAp) and
carbonate-substituted hydroxyapatite (CAp) have been recently
investigated by culturing RAW264 cells (macrophage precur-
sors) on their surfaces. It has been reported that greater surface
hydration of CAp surface can attenuate adverse inflammatory
responses to implanted bone prostheses. Cell spreading on the
CAp was decreased and the secretion of inflammatory cytokines
was reduced as compared to cells grown on Hap. 99
Researchers at Purdue University, West Lafayette, USA,
investigated fibroblast cells seeded on the photocrosslinked
poly(ethylene glycol) (PEG)-silica hydrogel surface and found
that PEG hydrogels had minimum cell adhesion, spreading and
proliferation, but the addition of silica nanospheres significantly
decreased the hydration degree, increased the mechanical
strength and the toughness of the PEG hydrogels, and increased
cell adhesion, cell spreading and the metabolic activity. 100
Addition of silicate reduces hydration and swelling of
cross-linked poly(ethylene oxide) (PEO) and enhances mouse
preosteoblast cells adhesion, spreading and proliferation. A
change in cytoskeleton organization was observed when the
silicate concentration was increased from 40% to 70%—at
higher silicate concentrations actin stress fibers were very well
organized in thick bundles that were stretched inside the cells. 101
Cell adhesion is reduced at room and lower temperatures as
compared with physiological temperature. Cell attachment is a
temperature-dependent process. 102–104
Effect of hydration on cells migration
Migration of vascular smooth muscle cells to the low hydration
side was observed by the researchers at Zhejiang University,
Hangzhou, China in poly(sodium 4-styrenesulfonate) (PSS)/
poly(diallyldimethylammonium) chloride (PDADMAC) multi-
layers with swelling gradients. 105 The cell migration rates on the
gradient surface were significantly larger than migration rates on
the uniform surfaces. The mobility of smooth muscle cells was
controlled by the hydration ratio of the multilayers. 106 It has been
also reported that surface swelling gradient rather than topography
affected cell migration rate. 107 The migration rate of endothelial
cells (ECs) over smooth muscle cells (SMCs) can play an
important role in the acceleration of endothelialization of
Hydration at biointerfaces
Prevents protein adsorption Prevents inflammation
Prevents platelets and cells Increase of cells migration rate
adhesion
Prevents thrombus formation Cancer metastasis
Figure 1. The effect of hydration at biointerfaces on protein adsorption,
platelet adhesion and related diseases.
blood-contacting implants and in prevention of vascular
restenosis. 108
Rates of cells migration can also be related to the rates of
cancer metastasis. The migration rate of cancer cells is higher on
surfaces with lower stiffness where adhesion of cells is low.
Cancer cells are more compliant than normal cells. 109–112 Cancer
cells contain more free water molecules than normal cells. 113 , 114
But the stiffness of ECM is higher in tumor tissue. 115 Collagen is
the dominating material in the extracellular matrix and its
stiffness controls cell migration, differentiation, growth, and
pathology. 78 Dehydration increases the stiffness of collagen. 73
The migration of cells can be governed by several simulta-
neously acting stimuli—it has been demonstrated that opposing
rigidity-protein gradients reverse fibroblast durotaxis. 116
The effect of hydration at biointerfaces on protein adsorption,
platelet adhesion and related diseases is schematically repre-
sented in Figure 1.
Effect of hydration on cells proliferation
Stiffness increases with dehydration and decreases as a result
of hydration. 73 The increased stiffness was reported to be an
additional mechanical stimulus to promote chondrocyte growth
and proliferation. 117 Kiroshka et al 98 found that lower swelling
of chitosan/chitin films promoted the proliferation of cultured
BMSCs. The presence of hydrated domains, and decreases in
rigidity due to swelling under conditions of culture on the
chitosan/chitin matrices decreased the proliferation of BMSCs.
Effect of hydration on cells differentiation
Increase of water content commonly leads to the increase of
molecular mobility. It has been reported that molecular mobility
of polyrotaxane (PRX) supramolecular surfaces directs stem
cells differentiation. 118 Seo et al 119 investigated the effect of
PRX hydrated surface molecular mobility on differentiation of
mouse induced pluripotent stem cells (iPS cells) into cardio-
myocytes. PRX was composed of α-cyclodextrin (α-CD)
threaded on linear poly(ethylene glycol) (PEG)-capped terminals
with bulky end-groups. The degree of molecular mobility at the
hydrated state on the PRX surfaces can be varied by changing the
number of threaded α-CDs. They found that expression of
N-cadherin and Rac1 was significantly upregulated for adhering
iPS cells on the PRX surface with high molecular mobility, and it
was downregulated on surfaces with low molecular mobility.
The PRX surface with higher molecular mobility was found to be
 G. Kerch / Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 13–25
higher in cardiomyogenesis. The authors concluded that surface
hydrated molecular mobility, varied by changes in a supramo-
lecular PRX architecture, plays a significant role in controlling
cytoskeletal signaling pathways that contributed to the direction
of stem cell commitment.
Effect of hydration on tissue stiffness
The sorption of water by polymers results in the increase of
free volume and mobility of polymer chains, and related decrease
of glass transition temperature and elastic modulus. A limited
equivalence between the action of water on the polymer and the
action of temperature was proposed. 33 The hydration resulted in
Young's modulus decrease of silk fibroin films. 120 The water is
tightly bound to macromolecules provided the water content in a
polymer is lower than a certain water content threshold. 33–35
A hydration increase in bone samples prepared with different
water content was associated with a decrease in bone stiffness
determined by nanoindentation. 121 Hydroxyapatite, collagen,
and water are the main components of bone as a composite
material. Nyman et al 122 concluded that loss of water in the
collagen phase decreases the toughness of bone, whereas loss of
water associated with the mineral phase decreases both bone
strength and toughness. It was reported that stress relaxation rate
of bovine femur decreases with water content. 123 Water in bone
exists in two compartments: within pores and bound to the
matrix. Bound water concentration, measured using 1H nuclear
magnetic resonance (NMR), is positively correlated with both
the strength and toughness of hydrated bone. 124 Mechanical
properties of tissues depend on extracellular matrix (ECM)
hydration and composition both in vivo and in vitro. 125
Physiologically responsive, mechanically adaptive polymer
nanocomposites have a great potential for biomedical applica-
tions. The mechanical contrast between “stiff” microelectrodes
and “soft” brain tissue results in neuroinflammatory and
neurodegenerative responses. 126 The nanocomposite implants
soften considerably under physiological and in vivo conditions
after implantation due to plasticization upon absorption of water
at interfaces. 127–130 New biomimetic nanocomposites, which
change their mechanical properties upon exposure to water and
display a water-activated shape-memory effect, were described
in a number of publications—polyurethane films with cotton
cellulose nanowhiskers (CNWs), 131 poly(styrene-co-butadiene)
(SBR) or polybutadiene (PBD) with cellulose whiskers isolated
from tunicates, 132 water-responsive bioderived rubber–cellulose
nanocrystal composites, 133 styrene–butadiene rubber and cellu-
lose nanocrystals, 134 elastomeric thermoplastic polyurethane
with hydrophilic chitosan-treated clay, 135 elastomeric thermo-
plastic polyurethane with chitosan modified cellulose
whisker, 136 and poly(vinyl alcohol) (PVOH) with cellulose
nanocrystals derived from tunicates and cotton. 137 Competitive
hydrogen bonding between water and CNWs reduces the
hydrogen bonding between the CNWs and weakens the CNW
network.
Protein conformational changes and related diseases
Many cellular processes are regulated by the changes in
protein conformation and may be linked to various diseases. 138
17
Water not only surrounds proteins but is also an integral part of
proteins. Conformational changes of proteins are closely related
to behavior of hydration water. Water is tightly coupled to
protein conformations and dynamics. 139–141 Interior water
molecules affect conformations and mechanical characteristics
of various amyloid fibrils. 142 It has been reported that water
molecules in the amorphous regions of the silk films acted as a
plasticizer and induced β-sheet crystallization. 143 It is generally
recognized that β-sheet formation is associated with Alzheimer
disease.
Protein adsorption—the effect of surface stiffness and hydration
Cells do not interact with a bare surface, but with surfaces on
which the proteins from biological fluids have already
adsorbed. 15 The design of protein-resistant surfaces is a
challenge in the current state of the art. It has been demonstrated
that the presence of water at the interface plays an important role
in the mechanism of protein adsorption to the surface. 144
Protein adsorption and conformation depends on substrate
stiffness. 145–150 Beilis et al 151 studied surface-induced confor-
mational changes in doped bovine serum albumin self-assembled
monolayers. The bovine serum albumin unfolding phenomenon
is correlated with dehydration.
Dehydration induces aggregation of albumin and formation
of fibers. 152 Dehydration of bovine serum albumin (BSA)
solutions results in spontaneous formation of densely packed
fibrils with the majority of proteins converted to parallel
β-sheets. Wu et al suggested that removing the inner water
shell surrounding globular proteins leads to protein unfolding
and aggregation at the water/air interface as a result of
interactions between BSA and salts. Sfera et al 153 hypothesized
that dehydration facilitates protein misfolding and aggregation,
predisposing to neurocognitive disorders. Chong and Ham 154 at
Sookmyung Women's University, Seoul, Korea, demonstrated
that water molecules play a crucial role in determining the
protein aggregation propensity. It is known that protein
aggregation is linked to Alzheimer's disease, Parkinson's
disease, and type II diabetes. Researchers at Max Planck Institute
for Human Cognitive and Brain Sciences, Leipzig, Germany,
investigated brain structural changes and found a significant
decrease of gray matter and white matter volume associated with
dehydration. 155 Similar degrees of shrinkage in white matter
volume have been reported in studies of mild cognitive
impairment or Alzheimer's disease during disease progression.
Link between protein dehydration and protein conformational
diseases is presented in Figure 2.
The effect of water on the stability, structure, dynamics, and
function of proteins and other biomolecules has been recently
reviewed. 156
Platelet adhesion is linked with age-related cardiovascular
diseases. Platelet adhesion and activation due to interaction with
solid surface depends on the proteins adsorption and
surface-induced conformational changes at interface. 157–159
Viscoelasticity
The polymer materials are viscoelastic. In many publications
the effect of substrate elasticity on cell behavior was studied.
18 G. Kerch / Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 13–25
PROTEIN DEHYDRATION
PROTEIN CONFORMATIONAL CHANGES
PROTEIN AGGREGATION
PROTEIN CONFORMATIONAL DISEASES
Figure 2. Link between protein dehydration and protein conformational
diseases.
Evidently, the effect of substrate time-dependent behavior on cells
must be taken into account. The interesting viscoelastic processes
at substrate/cell interface have been recently observed. 160–163
Substrate stress relaxation regulates cell spreading. Increased
spreading and stress fiber formations were observed on viscoelastic
substrates in comparison with elastic substrates. So it can be
concluded that dissipative effects in microenvironment can play an
important role in affecting cell behavior.
Chaudhuri and coauthors suggested that “parallel to stiffness,
substrate stress relaxation is a fundamental physical property of
model ECM that has a substantial impact on cell behavior and
function. The effect of stress relaxation was mediated through
integrin adhesions and actomyosin-based contractility, and
increased stress relaxation drives nuclear translocation of YAP
(Yes-associated protein)”. Cell spreading and proliferation were
higher on substrates with high rate of stress relaxation, when
compared with elastic substrates with the same initial elastic
modulus. They demonstrated that cell spreading, proliferation, and
osteogenic differentiation of mesenchymal stem cells are all
enhanced in cells cultured in gels with faster relaxation. Relaxation
times of viscoelastic polymers depend on applied mechanical
stress and determine the long-term behavior of polymer materials.
These findings highlight stress relaxation as a key characteristic of
cell–ECM interactions. It has been reported yet in 2001 that
dehydration of porcine aortic valve cusps can cause increased
hysteresis and stress relaxation. 164 Remodeling by fibroblasts also
alters the rate-dependent mechanical properties of collagen and
changes stress relaxation behavior. 75 Stress relaxation rate of
bovine femur decreases with water content. 123
It has been also reported that dehydrating the tissue makes the
tissue stiffer and increasing the stretch in the native tissue results
in a faster relaxation. 125 Stress relaxation behavior in coatings
depends on water absorption, 165 it means, that cells spreading
also must depend on water absorption in a substrate.
It has been also demonstrated that substrate fluidity regulates
cell adhesion and morphology on poly (ε-caprolactone)-based
materials. 162 The authors suggested that cells sense more
dynamic nature of their surrounding environment and the
adhesion and morphology of NIH 3 T3 fibroblasts were
regulated by the fluidity, rather than by the substrate elasticity.
Increasing the fluidity decreased cell spreading area.
Cameron et al 166 , 167 demonstrated the different behavior of
mesenchymal stem cells on elastic and viscoelastic hydrogels.
They found that creep-induced loss of cytoskeletal tension is
compensated by an increase of Rac1 activity. Rho and rac are
members of the superfamily of small GTPases and regulate the
polymerization of actin to produce stress fibers and lamellipodia,
and filopodia. 168 The Rho GTPase family members RhoA,
Cdc42 and Rac1 are the key regulators in the dynamics of the
actin cytoskeleton and play key roles in platelet aggregation,
secretion, spreading and thrombus formation. 169
Evidently, that physiological extracellular matrix is not
elastic, but viscoelastic with time dependent modulus, and
exhibits stress relaxation. 170 Viscoelastic properties of polymeric
hydrogels and their experimental determination have been
described in a review by Anseth and co-authors. 171
Viscoelastic properties of various tissues have been discussed
in a number of publications. The researchers at The University of
Queensland, Australia, using a polyacrlyamide gel system with
constant compressive modulus and varying loss modulus
determined that changes to substrate loss modulus substantially
affected human mesenchymal stem cells morphology, prolifer-
ation and differentiation 166 and showed that the effect of
substrate loss modulus on hMSC behavior is due to a reduction
in both passive and actively generated isometric cytoskeletal
tension caused by the inherent creep of substrates with a high
loss modulus. These findings highlight substrate creep, or more
explicitly substrate loss modulus, as an important mechanical
property of a biomaterial system that can be tailored to encourage
the growth and differentiation of specific cell types.
Earlier dynamic mechanical behavior of starch-based scaf-
folds in dry and physiologically simulated conditions was
studied 172 and for the immersed scaffolds under physiological
conditions the loss factor increased significantly compared with
dry state measurements. It was concluded that the mechanical
performance of porous polymeric constructs in dry and in
immersed state is completely different, and for comparison with
biomechanical performance of tissues, the tests should ideally be
performed in immersed state. The biomechanical properties of
the bovine cornea have been found to be closely related with
tissue hydration. 173
Glycocalyx
The gel-like layer “glycocalyx”, or “endothelial surface layer”
(ESL) modulates a number of biological processes, such as
inflammation, vascular permeability and atherosclerosis. 174 The
reduction of ESL layer thickness brings platelets and leukocytes
into close proximity with the vessel wall, as it was observed in
European FP7 PRESTIGE project 175 , 176 and results in the
formation of a more prothrombogenic surface. Antifouling effect
of glycocalyx can be explained by the presence of both a water
barrier effect associated with the hydrated saccharide residues and
steric hindrance from the polymer backbone. 177 Well-hydrated
glycocalyx increases nitric oxide (NO) formation. 178 Plasma
sodium stiffens vascular endothelium and reduces nitric oxide
release 179 (Figure 3). Salt overload damages the glycocalyx
sodium barrier of vascular endothelium and leads to hardening and
flattering of endothelial glycocalyx. 180 It is interesting that in
polyelectrolyte multilayers the interpenetration of polyanion,
polycation and NaCl due to mutual diffusion results in charge
compensation. 181
 G. Kerch / Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 13–25 19

DEHYDRATION

Elevation of plasma sodium content

Endothelial cells stiffening Increased Na-induced arterial stiffness

Shear stress Increased VCAM-1, Increased endothelial layer

downregulates E-selectin and MCP- permeability due to disrupted
VCAM-1 expression cell-cell junctions on stiffer
expression matrices

Increased leukocyte adhesion and transmigration

Atherosclerotic plague formation

Figure 3. Effect of dehydration on plasma sodium concentration, endothelium stiffening and related atherosclerotic plague formation.

Polyphenol-rich compounds can induce a swelling of the
endothelial glycocalyx. Researchers at University of Münster,
Germany, reported that polyphenols resveratrol, (−)-epicatechin,
and quercetin lead to endothelial glycocalyx swelling with a
simultaneous decrease in cortical stiffness. 182 The causal role of
sodium in arterial stiffness has been reported. 183–185
Weinbaum and co-authors 186 reviewed the role of the ESL in
transducing fluid shear stress into the intracellular cytoskeleton of
endothelial cells, in the initiation of intracellular signaling, and in
the inflammatory response. Tarbell and Cancel 187 concluded that
glycocalyx provides a protective surface on ECs that suppresses
leucocyte adhesion and associated inflammation, supports the
vascular transport barrier and mediates critical mechanotransduc-
tion processes such as flow-mediated vasodilation. It is important
that for the treatment of vascular disease the glycocalyx should be
enhanced, whereas for the treatment of cancer/metastasis it is often
preferable to suppress the glycocalyx.
Hydration status is associated with aortic stiffness, but not with
peripheral arterial stiffness, in chronically hemodialyzed patients. 188
Researchers at National Yang-Ming University, Taiwan, 189
identified water hydrated states of poly(sulfobetaine methacrylate)
pSBMA by DSC at various salt concentrations. At low NaCl
concentration, salt ions bind to the charged groups of PSBMA and
promote the incorporation of water into polymer chains. At higher
NaCl concentration the excess ions are binding with free water
molecules, which imposes an osmotic pressure on the hydrated
pSBMA molecules and causes the shrinkage of the molecules.
Cardiovascular diseases and cancer
Increase in matrix stiffness promotes cardiovascular diseases
as a result of enhanced endothelial permeability and leukocyte
transmigration. 190 Thrombosis formation depends on platelets
adhesion and activation. 191–193
Metastasis is the major reason for the mortality of patients
with cancer. Figure 1 shows that cancer metastasis is promoted
by the increase in cell migration rate, and the increase in
migration rate can be due to the decrease of cell adhesion at the
hydrated biointerfaces. Figure 1 illustrates that hydration can
prevent thrombus formation but also can promote cancer
metastasis. The hydration states of cancer cells and tissues may
differ from hydration of healthy cells. Greater hydration state of
cancerous breast tissue was reported. Increased hydration levels
may be associated with a higher content of hyaluronan in the
ECM of migrating and metastatic cells. 194 Similarly to other
glycosaminoglycans, hyaluronan binds large amounts of water
and has lubricating properties. Hyaluronan is highly hygroscopic
and controls tissue hydration and water transport. Due to its
viscoelastic properties hyaluronan can form highly hydrated,
expanded matrices. Increased tissue hydration supports malig-
nant cell migration.
The effect of arterial stiffness and mechanical stresses on the
development of thrombosis and restenosis is represented in
Figure 4.
Hydration and cellular processes
Direct effect of hydration on cellular processes has been
demonstrated for skin tissue. Decreased levels of hydration
resulted in an increased expression of proinflammatory genes,
such as IL-1β, IL-8, TNF-α (tumor necrosis factor-α), and
COX-2 (cyclooxygenase 2), in human ex vivo skin culture. 195 It
has been also reported recently that S100 protein family
members S100A8 and S100A9 are induced by decreased
20 G. Kerch / Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 13–25

Endothelial cells are mechanosensitive

Increase of mechanical stress in Increase of arterial stiffness Aging

endothelial cells

Impaired Compromised Increase in

shear stress- cell-cell oxidative
induced nitric junction stress
oxide integrity

Decrease in Leukocyte and neutrophils

Inflammation
smooth muscle transmigration into the intima,
relaxation and greater cholesterol uptake
vasodilation

Restenosis Atherosclerotic plaque

formation. Thrombosis

Figure 4. Effect of arterial stiffness on biological processes in vascular walls.

hydration in the epidermis and promote fibroblast activation and
fibrosis in the dermis. S100A8 and S100A9 have been
considered to be novel targets in preventing scarring. 196
Dehydration can increase tissue stiffness. Substrate stiffness
regulates expression of inflammatory genes via NF-κB activa-
tion. A stiff substrate induces actomyosin contractions, which
triggers NF-κB activation. NF-κB activation was induced in
H1299 lung adenocarcinoma cells grown on a stiff substrate but
not in cells grown on a soft substrate. 197
Conclusion and future perspectives
The influence of the composition and structure of ECM and
polymeric surfaces on interactions with cells can be used to
regulate the biological processes at biointerfaces. Mechanical
properties of ECM depend on collagen and water content.
Collagen concentration increases with aging. It is well
established now that mechanical properties of ECM and implant
surfaces can affect biochemical processes in adherent cells and
can be linked to various age-related diseases. Hydration/
dehydration of macromolecules in ECM and in polymer coatings
of implants can change surface stiffness and lead to related
changes in cellular processes. It would be interesting to confirm
the relation of water retention to ability to prevent inflammation
and scar formation in the processes of wound healing or to show
that such processes are not interdependent. Degree of hydration
and water molecule binding energy depend on macromolecular
chains architecture, determine the mobility of polymer chains
and surface stiffness at biointerfaces, and play an important role
in controlling cytoskeletal signaling pathways in cellular
processes.
The mechanical properties of biological tissues and bioma-
terials are not elastic, but viscoelastic. The influence of
viscoelastic time-dependent properties of ECM and polymer
coatings, such as stress relaxation time and loss moduli, on
time-dependent biological processes will be studied in more
detail in the nearest future taking into account the degree of
hydration of macromolecules. The relaxation time depends on
mechanical stress and the value of internal mechanical stresses
depends on temperature and hydration. The knowledge about
how hydration of microenvironment affects biological processes
in cells and tissues is very important in designing biomaterials
for tissue engineering and regenerative medicine. For example,
the changes of concentration of nanofiller in nanocomposite
biomaterials allow for regulating hydration and mechanical
properties of substrate and in such a way for regulating cell
biological behavior at interfaces.
The increased attention to the effect of the degree of
macromolecular hydration in combination with mechanical
properties at biointerfaces on cellular processes in further studies
has potential to essentially improve current state-of-the-art in
biomedical applications of polymer materials. The knowledge
about polymer hydration and mechanical properties can be
translated and applied in cell biology, tissue engineering,
regenerative medicine and nanomedicine in future development.
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