By Dr. Megha Agrawal, Dr. Shyamasri Biswas, Dr.

Kim Van Vliet, Contributing Editors

Thin Films of Extracellular Matrix

Proteins and their Applications

he extracellular matrix (ECM) corresponds to the extracellular component of various multicellular structures
such as organisms, tissues and biofilms. The functional
role of ECM is to provide structural support and biochemical
stability to the surrounding cells for cell adhesion, cell-to-cell
communication and differentiation. A significant part of the extracellular region is filled by a complex network of proteins and
polysaccharides (macromolecules). Such a network is closely
attached to the surface of the surrounding cells and forms a spatially homogeneous thin film matrix structure.
The role of ECM proteins is diverse and critical to maintain
the important intracellular signals by providing both mechanical
and chemical stimuli to cells. It is possible that various sources of
different cell signals may interfere and affect the intracellular signals. Thus, to maintain the integrity of the intracellular signalling
pathways, the ability to control the ECM is very important and
a matter of high research interest for biotechnologists for studies
on various cellular responses to pharmaceuticals [1].
Many collaborative opportunities exist for thin film scientists
and engineers, vacuum technologists and biotechnologists working in the area of ECM proteins and applications for designing
thin film ECM proteins for new applications in therapeutics and
the pharmaceutical sector. For example, ultra-high vacuum technology based automated microscopy and quantitative determination can be employed together with highly reproducible and
spatially homogeneous thin film extracellular protein matrices
to investigate the response of hundreds of cells in a population.
In this regard, using thin films of extracellular matrix proteins,
researchers have quantified, on a cell-by-cell basis, phenotypic
parameters of cells on different extracellular matrices for new
applications in therapeutics [1].
The transmission of the extracellular forces through the ECM
works in the following way (see Figure 1) At first, the external cell signal is received by the ECM components [2], these are
then transmitted through the cytoskeleton network (adhesion
complexes, integrins, talin, vinculin and other proteins, connect
the ECM to the actin filaments). In the case of skeletal muscle,

dystrophin-associated protein complex connects the ECM to actin filaments. The intra- and extracellular signaling can modulate the configuration and binding affinity of these complexes.
Next, the transmission of intracellular forces occurs through the
cytoskeletal network. Nesprins and other proteins present on the
outer nuclear membrane help couple the cytoskeleton. Nesprin 1
and nesprin 2 are the two main isoforms that bind to actin filaments. In the case of nesprin 3, it can associate with intermediate

Figure 1. A schematic illustration of how extracellular forces are transmitted through the cell. The ECM is composed of laminin, collagen
and fibronectin. Components of ECM are connected to the actin filaments which is a part of the cytoskeletal network. The intracellular
forces are transmitted through the cytoskeletal components to the nucleus. [Credit: Diana E. Jaalouk & Jan Lammerding, Nature Reviews
Molecular Cell Biology 10, 63-73 (January 2009)].

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filaments through plectin. Finally, the inner nuclear membrane

proteins (e.g. SUN1 and SUN2) that are retained there by interaction with other nuclear envelope proteins such as lamins and
emerin are attracted by Nesprins. The nuclear lamin structures
can bind DNA and helps to complete the force transmission from
the ECM to the nucleus [2].
As mentioned previously, combining thin film science and
technology with biotechnology processes, researchers demonstrated the use of highly homogeneous and reproducible thin
films of collagen and thin films of fibronectin, to provide different extracellular matrix signals to mouse embryonic fibroblast
(NIH3T3) cells. The advantage of having highly homogeneous
thin films facilitates each cell in a population to have the same
microenvironment. This allows each cell to be exposed to very
similar stimuli [1]. Researchers employed automated microscopy for the quantification of cellular parameters. These included
use of intracellular green fluorescent protein (GFP) to measure
fluorescence intensity, on a cell by cell basis, for large numbers
of adherent cells. The advantage of having thin films was obvious as it facilitated the collection of unbiased and quantitative
data from many types of cells in an automated fashion. The
researchers also noted that since the films were very homogeneous in thickness, it allowed a more reliable autofocusing with
minimal scattered light as the films were thin [1]. The authors
concluded that the use of thin film technologies helped produce
an ECM protein that could be quantitatively characterized and
systematically modified to vary the characteristics of the ECM
presented to cells [1].
Scientists have used high vacuum (HV) environments for
surface sensitive microscopy for investigating the structural and
chemical properties of ECMs. The effects of HV conditions
on the bioactivity and mechanical properties of type I collagen
fibrillar ECMs were investigated. HV exposure was found to
have an unappreciable effect on the cell spreading response and
mechanical properties of these collagen fibril matrices. When
using low vacuum environment, atomic force microscopy data
indicated that the fibrils were mechanically more rigid this
made them more conducive to greater cell spreading. Time-offlight secondary ion mass spectrometry was also employed and
the results showed no noticeable spectral differences between
HV-treated and dehydrated matrices. The authors concluded that
unlike previous reports that showed that HV can denature proteins in monolayers, their investigation and results indicated that
HV-exposure did not mechanically or biochemically alter collagen in its supramolecular configuration. These results may have
technological implications for complex ECM protein matrices
such as decellularized scaffolds for applications in regenerative
medicine [3].
Stem cell biology is a new field of research where biotechnologists are trying to create new approaches to employ stem cells
for their applications in the treatment of complex diseases [4].
The organization of stem cells corresponds to a subset of tissue
cells and ECM that can support stem cells and control in-vivo
their self-regeneration. The role of ECM is to aid this organization spatially and control adhesive concentration and signaling
molecules locally. Thus, the ECM components control the ad-

hesion when the majority of cells interact and they maintain the
embryonic induction during the stages of stem cell in-vitro differentiation and their development. It is therefore of significant
interest for biotechnologists to develop the capability that allows
modulating all local changes in ECM that can dramatically control the proliferation and migration of stem cells [4].
In the field of regenerative medicine, the maintenance and differentiation of human stem cells and developing the human extracellular matrix have been the research focus to facilitate clinical
applications of human extracellular matrix based technologies.
Such an animal-free culture matrix is considered immensely advantageous for applications in cell-based clinical therapies this is
due to the fact that animal based cultures often lead to infections
or tissue rejection during the process of transplantation [4]. By
combining with thin film technologies, advanced human ECMs
could be developed that could impact all future cell-based clinical therapies for many complex health issues. For example, the
application of thin film technology would allow development of
a highly homogeneous ECM protein matrix that could facilitate
real-time quantification of cells and allow high-resolution optical imaging. Figure 2 shows immunohistochemical analysis of
a human foreskin fibroblast ECM complex when left untreated
(left panel) and when treated with lysis buffer (right panel). In
this report researchers have demonstrated that even after hypotonic lysis of human fibroblast cells, most of the human ECM
component proteins required for cell surface attachment and
cell-cell interaction are retained. As observed in the figure (right
panel) cell nuclei were missing after lysis but the overall organization of the ECM is intact. Cell nuclei were detected using
DAPI staining. All cells were visualized using a Leica confocal
microscope. [4].
In other studies, the application of thin film technology in
designing new ECM proteins was demonstrated where the researchers visualized cell ECM deposited by cells cultured on
aligned bacteriophage M13 thin films [5]. In thin film technolo-

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Figure 2. Immunohistochemical analysis of human foreskin fibroblast ECMs when left untreated (left panel) and treated with lysis
buffer (right panel). The cells were immunostained by using antibodies against fibronectin (top panel), collagen type I (middle panel) and
laminin (bottom panel). [Credit: Carmen Escobedo-Lucea, Angel Ayuso-Sacido, Chen Xiong, Sonia Prado-Lpez, Manuel Sanchez del Pino,
Dario Melguizo, Carmen Bellver-Estells, Susana Gonzalez-Granero,
M. Luz Valero, Rubn Moreno, Deborah J. Burks, and Miodrag Stojkovic, Stem Cell Rev. Mar 2012; 8(1): 170183].

gy, topographical features can be tailored according to the application. For example, features that range from micro to nanometers can be designed and developed. Such a feature is immensely
important in cell orientation and migratory pathways that can be
applied in tissue engineering and tumor migration. Researchers
showed that a convective assembly of bacteriophage M13 resulted in thin films that could be used to control the alignment of
cells [5]. Using topographical features generated by the bacteriophage M13 crystalline film, researchers were able to align the
cells and ECM proteins. Scanning electron microscopy and immunofluorescence microscopy were employed for the sequential
imaging analysis at structural levels of aligned cells and fibrillar
matrix protein. The authors observed baby hamster kidney cells
with higher degree of alignment on the ordered M13 substrates
than NIH-3T3 fibroblasts. The authors attributed this difference
to the intrinsic nature of the cells production of ECM proteins.
This study provides an important insight into the biological thin
films topological features that can be employed to control the
cell orientation and the architecture of ECM proteins [5]. Figure
3 schematically illustrates the routes based on thin film technology to generate ECM proteins deposited by cells cultured on
aligned bacteriophage M13 thin films.
Atomic force microscopy (AFM)-based single-cell force
spectroscopy (SCFS) was employed for the quantitative study
of cell adhesion. The researchers used SCFS probes for adhesive
interactions of single living cells with substrates such as extracellular matrix (ECM) proteins and other cells. They developed a
protocol to quantitatively study the adhesion of HeLa cells to covalently immobilized fibronectin and Matrigel using SCFS and
described procedures for following (a) functionalization of AFM
cantilevers, (b) preparation of maleic anhydride copolymer thin
films, (c) covalent immobilization of ECM proteins on the thin
films, (d) cell handling and attachment to the AFM cantilever,
and (e) measurement of adhesion forces [6].
In important studies that employed thin film technology to
generate superior ECM proteins, thin films of ECM protein (collagen) were prepared by adsorbing native or heat-denatured type
I collagen onto hexadecanethiol self-assembled monolayers [7].

The authors characterized the resulting films by atomic force microscopy, ellipsometry, and light microscopy. The results showed
that denatured collagen formed a topographically smooth 3.6 nm
thick film, consistent with an adsorbed protein monolayer compared to the native collagen thin film that showed supramolecular collagen fibrils. The researchers could vary the density of the
large fibrils just by changing the native collagen concentration in
the solution from which the films were prepared. They assessed
the biomimetic nature of the thin collagen films by examining
their effects on vascular smooth muscle cells [7].
Automated quantitative analysis was employed which indicated the dependence of the morphology of smooth muscle cells
on the thin films and whether the collagen was heat-denatured
or was in its native fibrillar form. The area of cells on denatured
collagen films was observed to be significantly larger than that of
cells on thin films of native fibrillar collagen. Further examination of the relationship between collagen fibril density and cell
area showed that cells responding to collagen large fibrils are determined by the cell area [7].
In related studies, researchers used vascular smooth muscle
cells (vSMC) cultured on gels of fibrillar type I collagen or denatured collagen (gelatin) that comprised a model system. Such
a model system has been employed for studies on the role of the
extracellular matrix in vascular diseases such as on hypertension,
restenosis and athrosclerosis [8]. However, this model system
has certain limitations. These include poor optical characteristics
for microscopy and difficulty in verifying the properties of the
preparation. The other limitations are heterogeneity within the
gels, and difficulty in handling the gels because they are fragile [8]. The authors proposed a more robust approach based on
thin film technology to overcome these limitations. They developed an alternative collagen matrix by forming thin films of
native fibrillar collagen or denatured collagen on self-assembled
monolayers of alkanethiols, and the researchers found that the
substrates were more robust and could be characterized by surface analytical techniques. This allowed both verification of the
reproducibility of the preparation and high-resolution analysis
of the collagen structure. With this approach, the ECM proteins

Figure 3. A schematic illustration showing the routes of generating ECM proteins deposited by cells cultured on aligned bacteriophage M13
thin films [Credit: Laying Wu, L. Andrew Lee, Zhongwei Niu, Soumitra Ghosh Roy, and Qian Wang, Langmuir, 2011, 27 (15), pp 94909496].

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were found to have excellent optical properties that allowed

more details of the cell-matrix interactions to be observed by microscopy [8]. The researchers further performed a side-by-side
structural and functional comparison of collagen gels with thin
films of collagen and they concluded that such robust thin films
of ECM proteins could be useful for elucidating the features of
the collagen matrix [8].
Scientists made thin films of artificial extracellular matrix
(aECM) proteins with controlled elastic modulus. These films
were fabricated by photo-cross-linking containing the photosensitive amino acid p-azidophenylalanine (pN3Phe) [9]. They
employed nanoindentation measurements to study the elastic
moduli of the films and they demonstrated tunable modulus in
the range 0.31.0 MPa just by controlling the irradiation time or
by varying the level of pN3Phe in the protein [9]. It is believed
that substrate mechanical properties strongly inuence cell behavior. Substrate stiffness can affect cell adhesion, morphology,
traction forces and migration rate, growth and differentiation.
Thus, the ability to modulate mechanical properties of cell culture substrates is an important property that allows for the study
of cell-matrix interactions. The thin lms of aECM opens new
avenues for the study of mechanosensitive cell behavior in the
context of coincident biological signals [9]. Figure 4 shows a
typical atomic force topography image of the protein extracellular thin film and the nanoindentation data of the tunable elastic
moduli of the prepared film [9].
In separate studies, researchers demonstrated the assembly of
hybrid materials from engineered tissues and synthetic polymer
thin films. They fabricated the assembly by culturing neonatal rat
ventricular cardiomyocytes on polydimethylsiloxane thin films
that were micropatterned with extracellular matrix proteins to
promote spatially ordered, two-dimensional myogenesis [10].
The researchers named the constructs as muscular thin films.
Such thin films adopted functional, three-dimensional conformations when released from a thermally sensitive polymer
substrate. They were successfully tested for their biomimetic
tasks by varying tissue architecture, thin-film shape, and elec-

trical-pacing protocols. These centimeter-scale films micropatterned with ECM proteins performed diverse functions such as
gripping, pumping, walking, and swimming with fine spatial and
temporal control and generated specific forces as high as 4 millinewtons per square millimeter [10].
In the ECM arena, an important component is the elastic
matrix that constitutes a specialized part of the ECM which
allows resiliency to tissues and organs subjected to repeated deformations under stress. The role of the elastic matrix in living
organisms is very critical. It is known that expression of defective inherited genes that characterize diseases gets converted into
components of the elastic matrix that leads to premature death
[11]. So, understanding the elastic matrix in the ECM is of immense interest. Scientists performed an immunohistochemical
study of the distribution of elastin and three additional components associated with elastic matrices in adult tissues (i.e., fibrillin, emilin, and type VI collagen) during the development of the
chicken embryonic heart [11]. Their results revealed three different periods of heart development with respect to the composition
of the elastic matrix of the ECM [11].
By employing thin film technology, researchers also studied
the influence of a combination of exogenous coating of ECM
proteins (fibronectin-collagen) with substratum topography. The
investigation was conducted on cytoskeletal architecture as well
as alignment and migration of immortalized corneal epithelial
cells, and the authors concluded that effects of topographic cues
on cells could be modulated by the presence of surface-associated ECM proteins [12].
Concluding Remarks
Recent research on thin film ECM proteins has demonstrated
that applying thin film technology to the design and development
of advanced ECMs is immensely helpful and technologically
generates better products for clinical trials. Future research needs
to be directed at assembling collaborative research teams comprising vacuum technologists, microscope and thin film experts

Figure 4. Atomic force topography image of the protein extracellular thin film (left panel) and the nanoindentation data of the elastic moduli of
the film (right panel) [Credit: Paul J. Nowatzki, Christian Franck, Stacey A. Maskarinec, Guruswami Ravichandran, and David A. Tirrell, Macromolecules, 2008, 41 (5), pp 18391845].

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May 2014 Vacuum Technology & Coating

and biotechnologists to overcome the challenges of generating

advanced ECM proteins for new cell-based treatments.
References
1. Kurt J Langenbach, John T Elliott, Alex Tona, Dennis McDaniel and
Anne L Plant, BMC Biotechnology 2006, 6:14 doi:10.1186/14726750-6-14.
2. Diana E. Jaalouk & Jan Lammerding, Nature Reviews Molecular
Cell Biology 10, 63-73 (January 2009).
3. 
Christopher R Anderton, Frank W DelRio, Kiran Bhadriraju and Anne L Plant, Biointerphases 8, 2 (2013); http://dx.doi.
org/10.1186/1559-4106-8-2.
4. Carmen Escobedo-Lucea, Angel Ayuso-Sacido, Chen Xiong, Sonia Prado-Lpez, Manuel Sanchez del Pino, Dario Melguizo, Carmen Bellver-Estells, Susana Gonzalez-Granero, M. Luz Valero,
Rubn Moreno, Deborah J. Burks, and Miodrag Stojkovic, Stem
Cell Rev. Mar 2012; 8(1): 170183.
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and Qian Wang, Langmuir, 2011, 27 (15), pp 94909496.
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Paul J. Nowatzki, Christian Franck, Stacey A. Maskarinec,
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Sheehy, George M. Whitesides, Kevin Kit Parker, Science 2007,
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