Journal of Biomechanics 115 (2021) 110134

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Journal of Biomechanics
journal homepage: www.elsevier.com/locate/jbiomech
www.JBiomech.com

Cell aggregation on nanorough surfaces

F. Gentile ⇑
Department of Electrical Engineering and Information Technology, University Federico II, 80125 Naples, Italy
Department of Experimental and Clinical Medicine, University Magna Graecia, 88100 Catanzaro, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The ability to control adhesion and the spatial organization of cells over nanoscale surfaces is essential in
Accepted 12 November 2020 tissue engineering, regenerative medicine, the growth of organoids and spheroids as an in-vitro-model of
human development and disease. Nonetheless, despite the several different works that have explored the
influence of nanotopography on cell adhesion and clustering, little is known about how the forces arising
Keywords: from membrane conformational change developing during cell adaptation to a nanorough surface, and
Cell-surface interaction the cell-cell adhesion forces, interact to guide cell assembly. Here, starting from the works of Decuzzi
Nanotopography
and Ferrari, who examined how the energy of a cell varies while adhering to a nanoscale surface, and
Cell-cell adhesion
Cell clustering
of Armstrong and collaborators, who developed a continuous model of cell-cell adhesion and morphogen-
Cell aggregation esis, we provide a description of how nanotopography can modulate cellular clustering. In simulations
Pattern formation where the parameters of the model were varied over large intervals, we found that nanoroughness
may induce cell aggregation from a homogenous, uniform state, also for weak cell-cell adhesion.
Results of the model are relevant in bio-engineering and biomedical nanotechnology, and may be of
interest for those involved in the design and fabrication of biomaterials and scaffolds for tissue formation
and repair.
Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction
Understanding and controlling the adhesion and proliferation of
cells on a substrate is essential in nanomedicine and bioengineer-
ing, especially pertaining tissue engineering, regenerative medi-
cine, the design of in-vitro-models for verifying the growth of
tumor cells and the effects that a controlled delivery of drugs
may have on them. The characteristics of systems of cells on a sub-
strate is determined by the interaction of those cells with the
material and by the cooperation between the cells forming the
originating nucleus cultured on the substrate. Thus, cell-substrate
interaction, cell-cell adhesion forces, and the competition between
the two, influence the evolution of a system of cells and may guide
cells towards ordered structures.
The adhesion of a cell on a substrate is regulated by a cascade of
chemical and mechanical signals that originate at the biointerface
and propagate through the cell membrane to the cytoskeleton
(Geiger et al., 2001; Geiger et al., 2009). Cell-adhesion-molecules
with a characteristic length scale in the low nano-meter range
mediate those signals (Kanchanawong et al., 2010), as a result
the process of adhesion and the adhesion strength can be modu-
lated by tailoring surface topography at the nanoscale (Decuzzi
⇑ Address: Viale Europa, Loc. Germaneto, Catanzaro 88100, Italy.
E-mail address: francesco.gentile@unicz.it
and Ferrari, 2010; Stevens and George, 2005), and several different
publications have experimentally demonstrated control of cell
adhesion on nano-scale surfaces (Anselme et al., 2010; Cimmino
et al., 2018; Gentile et al., 2010; Karuri et al., 2004; Lou et al.,
2019; Lynch et al., 2017; Robotti et al., 2018; Ventre et al., 2014;
Wu et al., 2018; Zhou et al., 2018). At the same time, several stud-
ies have examined how nanotopography influences the collective
behavior of cells and their spatial organization. Substrates with
nanoscale details have been used to direct cell confinement, aggre-
gation, or condensation, chondrogenesis and osteogenesis, tissue
formation and development (Baranes et al., 2012; Bettinger et al.,
2009; Brammer et al., 2011; Chen et al., 2014; Chen et al., 2012;
Dalby et al., 2007; Lee et al., 2017; Schulte et al., 2018;
Syedmoradi et al., 2017). Research studies(Marinaro et al., 2015;
Onesto et al., 2017; Onesto et al., 2019) conducted by my group
have themselves illustrated that cells cultured on surfaces with
some degree of roughness form clustered configurations, compared
to the same cells cultivated on flat surfaces (Fig. 1).
Nonetheless, despite a large body of work, little is known about
the origin or underlying mechanisms of how cells interact with
surface topographic patterns and themselves to generate networks
- and how the balance/unbalance between cell-cell interaction
forces and the forces experienced by a cell during adhesion to a
nanorough substrate, can determine the topological properties of
those networks.

https://doi.org/10.1016/j.jbiomech.2020.110134
0021-9290/Ó 2020 Elsevier Ltd. All rights reserved.
F. Gentile Journal of Biomechanics 115 (2021) 110134

Fig. 1. Neuroblastoma cell networks cultured over flat surface are compared to the same cells growing on nanoscale surfaces. Cells over substrates with values of roughness
oscillating around 30 nm aggregate into clusters.

In previous works (Decuzzi and Ferrari, 2010), Decuzzi and Fer- other cells and (ii) forces that originate because of the adhesion
rari have developed a model that estimates the adhesion energy of to the surface and the consequent release of energy initially stored
a cell on a substrate as a function of the parameters of the system, in the cell membrane. Notice that cell-cell adhesion forces are
including the specific and non-specific adhesion strength of the mediated by specific (cell adhesion molecules - CAMs) and non-
cell, the membrane stiffness, and the roughness of the substrate. specific (electrostatics, electrodynamics, van der Waals) interac-
In reference (Armstrong et al., 2006), Armstrong, Painter and Sher- tions (Bell, 1978; Evans and Calderwood, 2007; Sackmann and
ratt modeled cell-cell adhesion using a continuum approach. They Smith, 2014), while forces resulting from the adhesion of a cell to
demonstrated that under certain values of cell-cell adhesion the substrate would also depend on the geometry of the surface
strength, the originating population of cells tends to aggregation. as reported in reference(Decuzzi and Ferrari, 2010) and described
Here, we combine the models of cell-surface interaction of in the following of the paper. To describe the system, we use a
Decuzzi and Ferrari, and of cell-cell adhesion of Armstrong and col- modified form of the continuum model of cell-cell adhesion
leagues, in a single mathematical framework. We used the energy described by Armostrong and colleagues in reference (Armstrong
functional derived from Decuzzi and Ferrari to estimate the force et al., 2006):
produced by a cell during adhesion to a substrate. Then, we used
@u @2u @ / @

that term in the partial differential equation that describes the evo- ¼ D 2  ðkuÞ  F df u ð1Þ
lution of a system of cells under the effect of internal and external @t @x @x R @x
disturbances. In numerical simulations where we varied the where u ¼ uðx; tÞ is the concentration of cells, that is a function of
parameters of the model over large intervals, we observed that space (x) and time (t); D is the diffusion coefficient of cells over a
forces induced by nanoscale surface topography can serve as a surface, / is the viscosity, R is the sensing radius, i.e. the maximum
direct signal to activate cell aggregation also for weak cell-cell distance below which interaction between cells is effective. In Eq.
interaction forces. (1), k accounts for the force exerted on a cell by its nearest
neighbors:
Z
/ R
2. Materials and methods kðx; uÞ ¼ auðx þ xo ÞsignðuÞdxo ; ð2Þ
R R

2.1. The model with a a constant, while F df is the force generated on the system
because of the conformational change of cells caused by their adhe-
Consider the scheme reported in Fig. 2. Cells are positioned on sion on a nanorough surface:
an irregular substrate with a roughness in the nanometer range.
F df ¼ F o qf ðtÞ; ð3Þ
Cells are subjected to (i) forces generated by the interaction with
2
F. Gentile Journal of Biomechanics 115 (2021) 110134

Fig. 2. Cells are laid on a curvy surface with a characteristic length of l along the x coordinate, and a height h. Cells on the substrate will sense other cells in their
neighborhood, and, for certain values of the specific and non-specific adhesion energy, membrane stiffness, and curvature, will adhere to the substrate. Cell-cell attraction
forces arise from the first of these events, while a conformational change force develops from the latter. The absolute values of forces and their ratio will cause cellular
reorganization, described by the cell density variable u. Numbers in the figure refer to the temporal evolution of the cell membrane curvature due to cell-substrate
interaction.

where F o is the maximum adhesion-mediated force on a surface z. Assuming that the system is conservative and the entire process
with a specific geometry, q is a random number chosen to be either reversible, the change of energy per change of length is an estimate
1 or 1 in a sub-region of the domain of length l, and f ðt Þ describes of the force exerted on the system during adhesion:
how fast F df decays with time. Here f ðtÞ has the form of a logistic @E
function: F df ¼  : ð7Þ
@z
A The non-dimensional form of E has been derived by Decuzzi and
f ðt Þ ¼ ; ð4Þ
1 þ eBðttoÞ Ferrari (Decuzzi and Ferrari, 2010) as:
where A, B and to are constants. Equations can be rescaled to give a eC eq þ cns   p2  p 
G 2pz; h p2 þ p2 z þ sinð4pzÞ
2
non-dimensional model, let: E¼
2p 12 4
 x  D  u  a  / 1 1    2
; t ¼ t 2 ; u ¼ R/; a ¼ þ ; F df ¼ F df ; sin ð2pzÞh s þ 2 G 2pz; h p2  2pz s;
2 2 3 2
x¼ ð5Þ þ ð8Þ
R R d a D 1  2z 4p
using these positions, and dropping the marks for notational where h and s are the wavy substrate height (roughness) and thick-
convenience, after some math one can obtain the non- ness, cns is the non-dimensional non-specific energy associated to
dimensional form of Eq. (1): the dimensional one by the relationship cns ¼ cdns =ðYlÞ, where Y is
the elastic modulus of the cell-biolayer, G is the elliptic integral of
@u @ 2 u @ @

e ¼ mb K b T=ðYlÞ
¼  ðkuÞ  F df u ; the second kind. In Eq. (8), and
@t @x2 @x @x C eq ¼ C  log ðml =mr Þ, with mb , ml and mr the density of new bonds
Z 1 (mb ), and of the ligand molecules expressed on the cell surface (ml )
k¼a uðx þ xo ÞsignðuÞdxo ð6Þ and the substrate (mr ). Moreover, K b is the Boltzmann’s constant, T
1 is the temperature, and C the chemical binding energy factor. Thus
that describes the evolution of a system of cells under the action the term eC eq accounts for the specific interactions associated to the
of cell-cell (incorporated in k) and cell-surface (incorporated in F df ) formation to new bonds and c ¼ eC eq þ cns is a variable comprising
interactions. both the effects of non-specific and specific interactions. In Eq. (8),
all the lengths are rescaled to the substrate wavelength l. Differen-
2.2. The conformational-change force F df tiating E with respect to z yields the formula for the force F df :
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
In Eq. (6), the term F df describes the forces released on the cells F df ðaÞ ¼ c 1 þ p2 h sinð2pzÞ
2 2

following the process of adhesion to the substrate. During adhe-  qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

1
sion, the portion of cell-membrane in contact with the substrate þ ð2pz  GÞ 1  1 þ p2 h sinð2pzÞ s
2 2

(z) smoothly varies from 0 (no adhesion) to l=2 (full adhesion), p

where the wave-length l describes the period of the substrate that, h
2
p2 ðpð1  2zÞcosð2pzÞ þ sinð2pzÞÞ
with Decuzzi and Ferrari, is here considered sinusoidal. Stable  s3 : ð9Þ
6ð1  2zÞ2
adhesion implies that the energy of the system (E) decreases with
3
F. Gentile
The force discharged on the cell during the process of adhesion
depends on the energy density of attraction between the cell and
the surface (c), on the mechanical properties of the cell membrane
(Y), and the geometry of the system (l; h; s). Once obtained the non-
dimensional expression for the instability force F df , its dimensional
counterpart is readily derived as F dim
df ¼ ðYlbÞF df , where b is the
width of the system. Using in Eq. (9) the following values of the
variables: cd 4  105 J=m2 , Y ¼ 100kPa, l ¼ 100nm, h ¼ 0:1l,
s ¼ 0:1l, b ¼ 103 l, one can find that the maximum force unleashed


during adhesion (F o ¼ max F df ) is F o 4nN. F o can be tuned by
changing the parameters of the model, especially the energy den-
sity of adhesion (cd ) and the geometrical characteristics of the sub-
strate (l; h). F o varies from 5pN for cd 5  107 J=m2 , to 0:4nN for
cd 4  106 J=m2 , to more than 40nN for cd 40  105 J=m2 . For
cd 40  105 J=m2 and a surface height of h ¼ 0:5l, F o 60nN. The lar-
ger the surface energy of interaction cd , the larger the value of F o
and the greater the effects that the surface topography has during
the process of adhesion.
2.3. Finding a numerical solution of cell density as a function of space
and time
We constructed a finite difference scheme (FDS) in Mathemat-
ica to find an approximate solution of the Partial Differential Equa-
tion (PDE) 6. We restricted the spatial variable x to the finite
interval ½p; q of length lx ¼ 5, that is the computational domain
where the dependent variable u ¼ uðt; xÞ was calculated. We set
the initial condition as:
uð0; xÞ ¼ rp  x  q ð10Þ
The initial value of u for every x in the computational domain is
a random generated value r from the uniform distribution on the
interval ½a; b; where a and b are a down and up perturbation of
0:05 from a central value of 0:1. We used a grid of equally spaced
grid points to discretize the computational domain. The constant
grid spacing Dx is Dx ¼ lx =mx , where mx is the number of divisions
that we have chosen equal to 200. Then Dx ¼ 0:025. The spacing
between time levels is Dt ¼ 0:0005. We discretized the PDE into
a system of ODEs. We used a central differencing scheme for diffu-
sion and high order upwinding for the advection term. We calcu-
lated the integral directly by summing over the enclosed points
and the time integration uses an explicit trapezoidal scheme. So
the final FDS is:
Dt
n 
unþ1 ¼ uni þ u  2uni þ uni1
i
Dx2 iþ1
Dt
n 
 uiþ2 k þ 4uniþ1 k  3uni k
6 Dx
Dt
n  from 552 for F df ¼ 1 to 209 for F df ¼ 5. Wanting to approximate
 uiþ2 F df þ 4uniþ1 F df  3uni F df : ð11Þ
6 Dx
This is a time-marching scheme. Starting from the initial condi-
tion u0 , we used data for each grid point at time t n to find data at
each grid point at the future time t n þ Dt. In finding the solution,
since we have no expected prediction on the form of the final dis-
tribution of cells, we used periodic boundary conditions (PBC).
Solutions evolve from an initial homogeneous state to clustered
distribution depending on the intensity of k and F df .
3. Results
We solved Eq. (6) numerically (Methods) to examine how the
conformational change force F df , the cell-cell adhesion forces k,
and the combination of the two, influences cell clustering. We dis-
4
Journal of Biomechanics 115 (2021) 110134
cretized the original domain of length 5 into 200 elements so that
the space resolution was Dx ¼ 0:025, and we used a time step of
Dt ¼ 0:0005. With these positions, Dt=Dx ¼ 0:025, that assures ful-
fillment of the Courant-Friedrichs-Lewy (CFL) condition and
numerical convergence for any initial value of F df and k smaller
than about 50. We found the value of cell concentration u in the
domain as a function of time for several different values of F df
and of the adhesion strength a. Some of the values assumed by
the parameters can cause cells to condensate, from an initially uni-
form distribution of cells (Fig. 3a). The ranges of values of the
parameters over which we tested the system are reported in
Fig. 3b and Fig. 3c: for these values of the variables the conforma-
tional change force F df and the cell-cell adhesive force k fall in the
same dimensional range. Remarkably, the solution of the advec-
tion–diffusion equation (6) can be represented for each configura-
tion in a space–time diagram, where a sharp rise of concentration
in the line of time indicates cell condensation in some regions of
space (Fig. 4).
Using a similar representation, for all the possible combinations
of F df and a we derived the condensation time (t cond ), i.e. the time
necessary to the system to reach a distribution where the maxi-
mum value of concentration is 10 times above the average, that
is a hallmark of clustering.
Fig. 5a reports tcond as a function of F df for different values of a.
F df ¼ 0 represents the non-external forces case, i.e. a configuration
in which the evolution of the system is determined by the interac-
tion among cells only - embodied by a. For this configuration, the
system transitions towards clustered distributions of cells for any
a  600, and the higher the value of a the smaller the time that
the system takes to reach condensation. For values of a < 600,
the condensation time is indefinitely large, indicating that the
attraction among cells is not sufficiently strong to induce cluster-
ing. The effect of F df is to make the system reach condensation ear-
lier. At F df ¼ 1, the values of tcond are smaller compared to the
corresponding values found in absence of the force F df . The larger
values of F df the smaller the values of tcond for a specific a, and
the lower the values of cell-cell adhesion strength for which clus-
tering is possible. For F df ¼ 2, the smallest a for which cells con-
densate is a ¼ 500, that is brought to a ¼ 400 for F df ¼ 3, and to
a ¼ 300 for F df ¼ 8. A conformational change force of F df ¼ 15
would hypothetically prompt cell clustering for the remarkably
low value of cell-cell adhesion strength a ¼ 100 (Fig. 5a).
To quantify the effect of F df on the time to clustering, we repre-
sented in Fig. 5b tcond as a function of F df for different values of a.
For a fixed a, t cond decreases with F df , and more steeply the more
a is initially small. For a ¼ 900, tcond moves from 172 non-
dimensional units for F df ¼ 0 to 42 non-dimensional units for
F df ¼ 10, with a variation of about 62%. For a ¼ 500, one obtains
the same percentage change in half the force span: t cond moves
the condensation time tcond with a function of the type
tcond ¼ A þ B=F df , we found after fitting data with the model the fol-
lowing values of the constant B for different a0 s: B200
a 8446,
B300 400 500 700
a 2622, Ba 1024, Ba 654, Ba 198, Ba900 117. Thus, at
a ¼ 300 the change in time is more than 10 times higher than that
at a ¼ 700 for the same F df increment. The effects of the conforma-
tional change force are more relevant in the low cell-cell adhesion
strength range.
The data reported in the diagrams and the trend of t cond as a
function of a or F df can be used to identify for each of the consid-
ered a0 s a critical force, F crit , that is the minimal value of F df that
causes clustering. Below F crit the initial population persists in a
state of equilibrium without forming isolated groups of cells. The
plot of F crit against a (Fig. 5c) allows to identify a region where
F. Gentile Journal of Biomechanics 115 (2021) 110134

Fig. 3. The conformational change force resulting from cell adhesion and adaptation to a substrate is lumped in the variable F df . The adhesion strength a and the cell-cell
forces k describe the interactions of cells among themselves. For certain values of the parameters, the system of cells forms clusters from an initial homogenous state (a). In
the work, we have explored several different values of F df , a and k, recapitulated in inset (b) and (c).

Fig. 4. The evolution of a system of cells on a surface can be represented in a space–time diagram, where for each combination of space and time the concentration of cells is
reported in grayscale levels. From the plot, one can identify the emergence of cell clustering occurring for some values of the system’s parameters.

F crit is negative, indicating that for these values of a clustering pro-
ceeds irrelevantly of F df . The time to condensation determined for
all the combinations of a and F df is reported in the density plot of
Fig. 5d. The diagram – that enables to visualize at a glance all the
information about the system – suggests that for the considered
range of values the system is more sensitive to changes of F df than
to changes of a. For any initial point in the diagram identified
through a and F df , tcond varies more rapidly along F df than along
a. Thus, the geometry of the substrate and of the forces generated
during adhesion seem to have a significant role in cell clustering.
Notably, while the model makes an estimate of the density of
cells on a surface because of the cell-cell interaction and the force
induced by a rough surface, the decision on whether cells form
cluster is made downstream of the model output. The post hoc
hypothesis that cells form clusters is tested by comparing the max-
imum values of concentration arising in the cell population to a
threshold value. In the article, we have chosen the threshold value
to be 10, so that any variation of concentration above 10 times the
initial – uniform - value of cell density, is a hallmark of clustering.
With this condition set forth, we observe that while clustering is
facilitated by the nano-scale roughness of the surface, if cell-cell
interaction forces are very weak (a ! 0) cell condensation is pro-
hibited, whatever the levels of F df . Moreover, results of the model
and date reported in Fig. 5 indicate that, for a given value of F df ,

5
F. Gentile Journal of Biomechanics 115 (2021) 110134

Fig. 5. The time to condensation tcond , i.e. the time necessary to the cells to achieve aggregation, is reported in (a) as a function of the adhesion strength a for different values
of the conformational change force F df : for a fixed F df , tcond decreases with a. In (b), t cond is represented against F df for different values of a. Examination of the diagrams
enables to identify, for each of the a0 s, a critical value of the conformational change force F crit above which the system forms aggregates (c). List-density-plot of t cond as a
function of a and F df , from which it’s clear the relative importance of the variables on the condensation time.

the time of condensation tends asymptotically to infinity for
increasingly weaker cell-cell interaction forces (a ! 0), consistent
with the experimental observations of clustering in nano-
structured scaffolds. Further to this end, in examining results of
Fig. 5, one should consider the fact that not all the values of confor-
mational change force in the diagram are physically admissible.
Assuming a non-dimensionalization factor of 1=nN, whereby to a
dimensional force of 1nN corresponds 1 non dimensional unit, very
large values of the Decuzzi-Ferrari force (F df > 15) may be
obtained only under very special conditions and very high values
of the adhesion energy density as discussed in a separate Supple-
mentary Material section 1.
Notably, the form of the diagrams in Fig. 5 and Supplementary
Material Fig. 1.1 suggest that the dependence of the conforma-
tional change force on the roughness of the substrate is strongly
non-linear. Roughness can influence on adhesion and clustering
only in narrow dimensional intervals that depend on the cell char-
acteristics, in agreement with precedent experimental results
(Gentile Biomaterials, Onesto SR 2017).
4. Discussion and conclusions
The mathematical model that we have developed is based
partly on the interaction among cells (a) and partly on the forces
generated during the process of adhesion and spreading of a cell
on nanorough surfaces (F df ). While cell-cell adhesion forces have
been examined in previous studies (Armstrong et al., 2006),
nonetheless several different experiments where cells were
6
observed to cluster under the effect of the substrate topography
cannot be explained by simple cell-cell interaction models. Here,
building on the influential work of Decuzzi and Ferrari (Decuzzi
and Ferrari, 2010), we have estimated the forces that arise in a sys-
tem of cells that develop firm adhesion on a surface.
These forces depend on: (i) the characteristics of a cell, incorpo-
rated by the energy density of adhesion c and the stiffness Y, and
(ii) the geometry of the substrate, described for simplicity by the
wavelength l and height h. For all the values of the parameters
being held constant, the force is determined by the sole h that,
for a fixed value of l, is proportional to the roughness of the sub-
strate. As for an example, for a cell-membrane of stiffness
Y ¼ 100kPa, an initial specific and non-specific energy density of
c 40  105 J=m2 , a substrate wavelength of l ¼ 100nm, and for a
system’s thickness of b ¼ 5lm, the resulting value of F df ranges
from about 2nN for h ¼ 0:1 to more than 3nN for h ¼ 0:5.
Using nanofabrication techniques, a substrate can be easily
engineered to exhibit different geometrical configurations, rough-
ness and topography that determine, through equation (9), the
intended forces. Thus, while the characteristics of a cell cannot
be varied and are determined a priori, one can act on the substrate
and the fabrication process parameters to vary the surface topogra-
phy and obtain, within the limits of the remaining constants, the
wanted value of F df . Combined to the interaction force between
cells (a=k), it can cause cell clustering earlier than on flat surfaces
where F df is smaller (Fig. 5a-b). Remarkably, F df can induce cluster-
ing also for those cells with weak attractive interaction forces and,
for any sufficiently large value of cell-cell interaction strength (a),
F. Gentile
there is some F df (F df ¼ F crit ) for which cell-condensation is secured
(Fig. 5c-d). If cells on a substrate are in a state of unstable equilib-
rium, with cells distributed uniformly even at higher energetic
costs, F df is that disturbance that takes the system out-of-
equilibrium producing condensation.
In rescaling the conformational-change force in a non-
dimensional form (Eq. (5)) we have imposed the position
F nondim
df ¼ ð/=DÞF dim
df , where D is the diffusion coefficient of the cells
over a surface and / is the viscosity of the system. Typical values of
D for a cell fall in the 1014  109 m2 =s dimensional range, while
/ ¼ 103 Pa=s (Luby-Phelps et al., 1993; Painter and Coleman,
1997; Palsson and Othmer, 2000; Simpson et al., 2017) and, as a
consequence, the non-dimensionalization factor ð/=DÞ may range
from 103 to 102 1=nN. Fixing the value of ð/=DÞ as ð/=DÞ ¼ 1=nN,
we have that for values of F dim
df falling in the 1  20nN interval,
the corresponding non-dimensional perturbation force F nondim
df
takes values ranging from 1 to 20 non-dimensional units.
Remarkably, the forces produced by cytoskeletal remodeling
( 1nN) observed during the process of cell adaptation to soft or
rigid substrates and reported in other studies (Gupta et al.,
2015; Roca-Cusachs et al., 2017) are comparable the values that
we have found for the conformational change force F dim df . More-
over, the tensile forces measured at cell-cell junctions vary from
1 to some tens of nN (Charras and Yap, 2018; Maruthamuthu
et al., 2011; Sancho et al., 2017), coherently with the values of
a and k reported in Fig. 3 and used in the simulations and
throughout the work. Thus, the values of the forces that we have
considered in this study are of practical interest and are propor-
tionate to the true forces generated by a cell during the process
of adhesion to the substrate and to the surrounding cells. In sep-
arate Supplementary Material sections we discuss even further on
the temporal dimension of the process (Supplementary Material
section 2) and on the generality of the model (Supplementary
Material section 3).
The ability to operate on the topography of a substrate to mod-
ulate cell-clustering is remarkable, because both mathematical
models (Hopfield, 1982; Hutmacher et al., 2014; Latora and
Marchiori, 2001; Strogatz, 2001; Watts, 2003; Watts and
Strogatz, 1998) and experiments (Bullmore and Sporns, 2012;
Onesto et al., 2017; Onesto et al., 2019; Takahashi et al., 2010;
Takahashi et al., 2007) suggest that the topology of a cell-
network determines their properties. This has implications in sev-
eral different fields.
In the analysis of neurodegenerative diseases, the design and
fabrication of bio-computers, neural tissue engineering, where it
has observed that colonies of neural cells with high clustering
and short path lengths feature enhanced computational ability
compared to the same networks of cells with more balanced values
of the topological parameters. In embryogenesis and the study of
immune responses, where directed cell motion, directed cell
assembly, and collective behavior of cells determines the way the
embryo forms and develops, and the fate of the individual. In chon-
drogenesis, where the initial shape of condensed mesenchyme tis-
sue determines the properties of cartilage. In bioprinting, tissue
repair and regeneration, the formation of organs, organoids and
in vitro models, where the evolution of a tissue or organ depends
on the way the living-building blocks (in increasing order of com-
plexity: cells, spheroids, cell beads, living legos, cell fibers) are
assembled together.
The mathematical model that we have developed can explain
the tendency of cells to form clusters on nanostructured surfaces,
and provides ways to design substrates and scaffolds for cell
growth that, under certain conditions, can guide the cells into
intended structures and configurations.
7
Journal of Biomechanics 115 (2021) 110134
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jbiomech.2020.110134.
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