Review Article

Hindawi Publishing Corporation
BioMed Research International

Volume 2014, Article ID 921905, 18 pages
http://dx.doi.org/10.1155/2014/921905
Review Article
Cell Microenvironment Engineering and Monitoring for Tissue
Engineering and Regenerative Medicine: The Recent Advances
Julien Barthes,1 Hayriye Özçelik,1 Mathilde Hindié,2 Albana Ndreu-Halili,3

Anwarul Hasan,4,5,6 and Nihal Engin Vrana1,7
1
Institut National de la Santé et de la Recherche Médicale (INSERM) UMR-S 1121, “Biomatériaux et Bioingénierie”,
11 rue Humann, 67085 Strasbourg Cedex, France
2
Equipe de Recherche sur les Relations Matrice Extracellulaire-Cellules, Université de Cergy-Pontoise,
2 Avenue Adolphe Chauvin, 95302 Cergy Pontoise, France
3
Department of Computer Engineering, Epoka University, Tirana, Albania
4
Biomedical Engineering and Department of Mechanical Engineering, American University of Beirut, Beirut 1107 2020, Lebanon
5
Center for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School,
Cambridge, MA 02139, USA
6
Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
7
Protip SAS, 8 Place de l’Hôpital, 67000, Strasbourg, France
Correspondence should be addressed to Nihal Engin Vrana; e.vrana@protipmedical.com
Received 23 April 2014; Accepted 15 June 2014; Published 20 July 2014
Academic Editor: Vasif Hasirci
Copyright © 2014 Julien Barthes et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In tissue engineering and regenerative medicine, the conditions in the immediate vicinity of the cells have a direct effect on cells’
behaviour and subsequently on clinical outcomes. Physical, chemical, and biological control of cell microenvironment are of crucial
importance for the ability to direct and control cell behaviour in 3-dimensional tissue engineering scaffolds spatially and temporally.
In this review, we will focus on the different aspects of cell microenvironment such as surface micro-, nanotopography, extracellular
matrix composition and distribution, controlled release of soluble factors, and mechanical stress/strain conditions and how these
aspects and their interactions can be used to achieve a higher degree of control over cellular activities. The effect of these parameters
on the cellular behaviour within tissue engineering context is discussed and how these parameters are used to develop engineered
tissues is elaborated. Also, recent techniques developed for the monitoring of the cell microenvironment in vitro and in vivo are
reviewed, together with recent tissue engineering applications where the control of cell microenvironment has been exploited. Cell
microenvironment engineering and monitoring are crucial parts of tissue engineering efforts and systems which utilize different
components of the cell microenvironment simultaneously can provide more functional engineered tissues in the near future.
1. What is Cell Microenvironment? to their environment, it can be said that the control over
cell microenvironment is a fundamental aspect of tissue
Tissue engineering and regenerative medicine fields aim to engineering and regenerative medicine.
produce artificial tissues or whole organs for both clini- Cell microenvironment is constituted by factors that
cal applications and drug testing, disease models, and cell directly affect conditions around a cell or group of cells
based biosensors. Even though there are several methods which have direct or indirect effect on cell behavior via
to approach tissue engineering, whether scaffold/biomaterial biophysical, biochemical, or other routes. When considered
based approaches, utilization of decellularized natural mate- for a single cell in vivo, cell microenvironment is composed of
rials, or scaffold-free methods, presence of the cellular com- (i) extracellular matrix (ECM), (ii) homotypic or heterotypic
ponent is inevitable [1]. As the advances in the different fields cells surrounding the single cell, (iii) cytokines, hormones,
of biology demonstrated well that cells are highly sensitive and other bioactive agents around the cells due to autocrine,
2 BioMed Research International
Qin Qout
Papplied
M M
(a) (b)
E = 130 kPa or 1 MPa Papplied
PDMS variable E
15 𝜇m
Δy
Seeding Confinement Attachment

Δy = 3 𝜇m or 7 𝜇m
(c)
Figure 1: The effect of microlevel mechanical confinement on the division of HeLa cells. (a) and (b) show the macroscopic structure of the
microfluidic system and the cross-section of PDMS posts. By the application of pressure on the posts, cells can be confined within the area
between the posts (the distance between the posts is 40 𝜇m) (c). The confinement caused significant changes in the behavior of the cells during
mitosis, such as delays in mitosis, and led to daughter cells of different sizes and multidaughter cells following mitosis. Reproduced from [4].
endocrine, and paracrine secretions, (iv) nano/microscale molecules can be significantly improved for corneal fibro-
topography and physical properties of the adjuvant cells and blasts [2]. Another recent work shows that nanoscale, micro-
the ECM, and (v) mechanical forces caused by the movement patterned, and highly flexible membranes can be used to
of the organism or the movement of the physiological fluids develop retinal pigment epithelium layers for minimally inva-
such as blood. All these have a compound effect on the behav- sive implantation within the eye [3]. By mechanically con-
ior of the cells, where the relative importance of each com- fining cells in a microfluidic platform, researchers were also
ponent is tissue and cell type dependent, and the next gen- able to control the mitotic processes (Figure 1) [4].
eration of engineered tissues must imitate these effects as Microenvironment of stem cells is a particularly impor-
much as possible to be functional in their target areas as a tant topic in tissue engineering and regenerative medicine, as
long term clinical solution. they are currently the most technically feasible source which
Cell microenvironment has many aspects and their can provide the large amount of cells needed for engineer-
control can result in substantial changes in cell behavior. ing clinically relevant amounts of tissue. Stem cell reservoirs
For example, Satyam et al. showed that by macromolecular are available for replenishment of the tissue in tissues in
crowding in the cell microenvironment the secretion of ECM human body. The microenvironmental control over how
BioMed Research International 3
these cells can keep their plasticity [5], that is, how they can It is well known that cells can align along micro- and
stay quiescent and be utilized by the body only in case of nanosized parallel grooves/ridges patterns [12–19]. Several
necessity under healthy conditions, is a benchmark that needs studies indicate that alignment occurs when the periodicity
to be met by engineered tissues. Moreover, failure to con- and dimensions of the patterns are above a critical value.
trol the microenvironment of stem cells can also have For example, Loesberg et al. [20] have shown that the
deleterious effects such as dedifferentiation and subsequent fibroblasts did not show noticeable alignment with groove
tumor growth. depths around 35 nm and ridges narrower than 100 nm. In
Another important concept related to the mimicking another study, 100 nm depth was determined as a threshold
of tissue microenvironment is multidimensionality as most for alignment of cardiomyocytes [21], osteoblast-like cells
of the components of tissues have multidimensional order [16, 22], and hepatoblastoma cells [23]. On microgrooved sur-
and orientation, which necessitates mimicry to achieve their faces, groove depth is one of the most important parameters
function [6]. Multidimensionality is also an important aspect in defining cell alignment. The degree of alignment of the
of other uses of tissue engineering, namely, model tissues cells along the microscale grooves is generally proportional
and organs for pharmaceutical testing and also fundamental to groove depth and inversely proportional to groove/ridge
research. These microorgan structures should match the width if the other parameters are fixed [24]. On the other
dimensional properties of the tissue and the organ they hand, Glawe et al. [25] studied that effect of high aspect ratio
represent [7]. (aspect ratio = groove depth/groove width) microchannels
In this review, we will focus on different aspects of cell with varying widths (20–60 𝜇m) on the alignment of smooth
microenvironment and their direct effects on tissue engineer- muscle cells. It was observed that alignment was dependent
ing applications with particular focus on osteogenesis and on the channel width, and narrow microchannels (20 𝜇m
angiogenesis. Each component of the cell microenvironment and 30 𝜇m) promote alignment of smooth muscle cells. On
will be discussed separately and also in conjunction with the nanogrooved substrates, cell orientation was also found to
other components. be also less sensitive to groove width (90 to 500 nm) with
MG-63 cells and C3A cells [16, 22]. When the ridges are
2. Micro/Nanopatterning for smaller than that of focal adhesions (0.25–0.5 𝜇m wide and
Microenvironment Engineering 2.0–10.0 𝜇m long), cell alignment is inhibited. Nanogrooves
were too narrow for the cells to descend into the bottom of
All the cells in the human body are surrounded by topograph- grooves. Thus, the focal adhesions and actin filaments are
ical and biochemical signals. The physical structures com- localized on the ridges. However, for vascular smooth muscle
prise nanopores, nanofibers, and nanocrystals. Some exam- cells, channel widths as small as 332.5 nm have been shown
ples of such structures in physiological settings are nanopores to induce alignment and subsequent mechanical property
in capillaries, nanofibers in the basement membrane, and improvement in the direction of alignment [26].
nanocrystals in the form of hydroxyapatite in the bone micro- The lack of data on how height and groove width or
structure. Aligned cells are very prevalent in the tissue. For quantitative interaction of these parameters which deter-
instance, maintenance of cell alignment is essential for mus- mine the degree of cell orientation have forced researchers
cle, cardiovascular/blood vessel, and corneal and nerve tissue to establish aspect ratio dependent models. For example,
engineering in which the controlling tissue microarchitecture Kemkemer et al. [27] developed a model for predicting the
and biological function are tightly connected. Various strate- cell orientation for cases where the cell is larger than the
gies have been developed to induce cell alignment, including grooves. According to this, the square of the product of
topographical patterning (e.g., micro- and nanogrooves and groove depth and spatial frequency or the aspect ratio for
aligned nanofibers), chemical treatment (patterns with cell- symmetric grooves were found to be the important features
adhesive or repellent chemistries), controlled stress/strain for alignment. In another study, Crouch et al. [28] proposed a
conditions (e.g., stretching, fluid shear stress, and compres- simple model to explain the relationship between aspect ratio
sion), and a combination of these methods. and cell behavior on gratings with varying widths and depths.
From topography point of view, recent advances in micro- They observed a direct relationship between the alignment
and nanofabrication enabled development of complex surface of human dermal fibroblasts and aspect ratios of the channel
features by controlling their pattern, periodicity, shape, and type patterns. While aspect ratios as small as 0.01 induced
dimensional properties. Today, design and construction of significant alignment (60%), 80% alignment was achieved
substrates with well-controlled physical and chemical proper- with an aspect ratio of 0.05. The maximum aspect ratio
ties and micro- and nanoarchitecture have become an impor- required for 95% alignment was 0.16. This study indicates
tant tool in the construction of tissue engineered replace- that within a certain range the aspect ratio can be used for
ments. Several top-down and bottom-up techniques such as controlling cell response to substrate topography without
phase separation, self-assembly, thin film deposition, chem- distinguishing the effects of width and depth. However, it
ical vapor deposition, chemical etching, nanoimprinting, is important to point out that when the grating surface is
photolithography, scanning probe lithography, and electron wider than cell width, the probability of lateral cell spreading
beam lithography [11] can be used in order to tailor micro- is high. Thus, obtaining cell-type-specific contact guidance
and nanoscale structured environments (scaffolds/surfaces) thresholds by the help of the abovementioned prediction
to stimulate cell growth and guide tissue regeneration in theories can be useful to tailor the cellular microenviron-
much the same way the extracellular matrix (ECM) does. ment.
Cell alignment on physically patterned surfaces is a growth [15, 42] and differentiation [19, 32, 43, 44] of neurites.
widely used strategy, in some cases together with chemical In one of the recent studies, Migliorini et al. [45] analyzed the
patterns. The effect of chemical patterns [29, 30] or synergistic effect of nanopillars on differentiation of embryonic stem cell
effects of physical and chemical patterning [31–34] were also derived progenitors in the absence of biochemical factors and
studied intensely. Generally, in order to control cell adhesion observed an increase in the neuronal yield with increasing
and alignment, molecules such as poly(-L-lysine) (PLL), pep- pillar height from 35 to 400 nm. Pan et al. [46] tested the
tides, fibronectin, laminin, collagen, bovine serum albumin, effects of nanograting substrates with different widths on
and SAM (self-assembled monolayers) are patterned by soft human induced pluripotent stem cells (hiPSCs). Gene expres-
lithography techniques. In some cases, instead of synergistic sion profiling by real-time PCR and immunostaining showed
effects of chemical and physical patterning, one of the cues significant upregulation of neuronal markers on nanostruc-
can overcome the other one. For example, when Charest tured substrates either with solely topographical cues or
et al. [33] used grooves (4 𝜇m depth, 8 𝜇m width) overlaid combined with preneuronal induction. A width of 350 nm, in
with an orthogonal chemical pattern (10 𝜇m adhesive lanes particular, induced the highest neuronal marker expression.
with spacing ranged from 10 to 100 𝜇m), physical topography The responsiveness of the cells to nanometer scale cues stems
determined the alignment of osteoblast-like cells. Another from their specific interactions with extracellular matrix
means to produce patterned surfaces is to use thermorespon- (ECM), which is covered in the following section.
sive polymers. These “smart” polymers, based, for example,
on a poly(N-isopropylacrylamide) backbone with n-butyl 3. Microenvironmental Effects of
methacrylate side chains, are capable of a reversible transition Extracellular Matrix
from hydrophilic to hydrophobic state when their temper-
ature is lowered by a few degrees (around its low critical The ECM comprises of a wide range of molecules including
solution temperature of 32∘ C). Thermoresponsive polymers collagen, elastin, laminin, fibronectin, various glycoproteins,
can be used for cell sheet engineering to treat a wide range proteoglycans, and polysaccharides [47, 48]. Each ECM com-
of diseases, from corneal dysfunction to oesophageal cancer, ponent provides different functionalities to this complex net-
tracheal resection, and cardiac failure as growth substrates for work made of structural proteins (e.g., collagen and elastin),
cells [35]. By using patterned thermoresponsive surfaces, it is adhesive proteins (e.g., fibronectin and vitronectin), and gly-
possible to pattern cell sheets when the application requires cosaminoglycans (e.g., hyaluronic acid and heparin sulphate)
cellular alignment. by presenting different structural and biochemical properties
Biological tissues are hierarchically organized from nano- [49]. The microenvironment created by ECM surrounding
meter-to-centimeter scale. For instance, the average rough- adherent cells is essential to their survival. ECM is a physical
ness of bone tissue is 32 nm and bone tissue has a hierarchical support to physiological cells; the signals for functional ori-
structure composed of collagen and hydroxyapatite [36]. entations such as migration, proliferation, and even survival
During bone mineralization, the hydroxyapatite crystals are transduced from ECM. The absence of cell adhesion to
form micro- and nanocomposites with collagen fibers. Thus, ECM induces cell death by apoptosis [50]. This particular
preparing biomimetic surfaces which present synergistic type of apoptosis is named anoı̈kis (Greek word which means
effects of micro- and nanostructures is expected to provide “homelessness” or loss of home). This phenomenon was first
additional advantages. Two separate studies have examined described in epithelial cells [51] and contributes to maintain
the behavior of osteoblasts derived from bone marrow stro- tissue homeostasis [52]. In physiological conditions, adherent
mal cells on micropit and nanonodule hybrid topography of cells are protected from anoı̂kis by the binding to ECM and
TiO2 [37, 38]. They created nanonodules with diameters of the resulting activation of intracellular survival signalling
100 nm, 300 nm, and 500 nm by self-assembly technique and pathways. The loss of anoikis induction signal constitutes a
demonstrated that 300 nm nanonodule containing substrates hallmark of cancerous cells and contributes to the formation
created the most promising environment for osteoblast of metastasis [53, 54]. Thus, presentation of an ECM mimic to
differentiation and bone-titanium integration. These results the cells in tissue engineering applications is important. The
are in agreement with more recent studies [39, 40], which three-dimensional organization of the ECM has a regulatory
have indicated that the presence of micron and submicron- effect on cell cycle as seen in mammary epithelial cells as the
scale surface roughness on the same surface can accelerate ECM suppresses apoptosis, suggesting that ECM signaling is
bone differentiation. In another study, surface features which defined by the organization of the cells within a tissue, that
are similar in scale to osteoclast resorption pits were used to is, cell shape, intercellular spacing, and 3D position. These
study in vitro bone formation in basal medium [41] Here, the factors determine cellular response to signals.
pit dimensions were as follows: depth: 330 nm, diameters: 20, The microenvironment created by ECM components
30, and 40 𝜇m, and centre-centre spacing: 50, 60, and 90 𝜇m. such as adhesive proteins or glycosaminoglycans maintains
Osteopontin expression was relatively high in the human tissue stability and cell behavior. Bone matrix, for instance,
osteoblasts grown on the larger diameter (30 and 40 𝜇m) contains 90% of collagen type I and only 5% of noncol-
pits. In addition to expression of osteogenic markers, mature lagenous proteins like osteocalcin, osteonectin, fibronectin,
calcium depositions were shown by alizarin red staining on or hyaluronan and mineral compounds which are essential
these substrates. to conserve osteoblasts phenotype [55], whereas culturing
In the last decade, several studies reported that micro- chondrocytes on type I collagen induces their dediffer-
and nanopatterned surfaces can be a valuable tool for directed entiation [56]. Furthermore ECM components selectively
influence cell adhesion and shape as described by Schlie- also stimulates cancerous cell proliferation and dissemination
Wolter et al. [57]. Hence, cell morphology directed by [72].
the interaction with ECM induces modifications of their All differentiated cells have a cell type specific protein
behaviour and subsequently their fate [58]. expression profile with multifunctional criteria that is respon-
One of the main examples of cell signaling is integrin- sible for development and protein regulation. This protein
mediated signaling for cell adhesion where the connection expression creates an output signal to be used by cells
requires structures of focal adhesion that contain complex to control their roles. Changes in the protein expression
mixture of proteins. Cell adhesion to ECM is led by trans- profile or mutations that result in down- or upregulation of
membrane heterodimeric integrin receptors. During devel- specific proteins can be the causes of cardiac, muscular, or
opment, integrins facilitate tissue morphogenesis by deter-
mental illnesses. Hyper- and hyposensitivity responses to the
mining which ECM components the cell would bind to. Inte-
strength of stimuli can also cause sickness in the body. In
grins are the major mediators of cell-ECM contacts and they
order to model such illnesses, tissue engineering and bio-
are essential to the outside-in transmission of signals from
cell microenvironment [59]. Integrin and ligand bindings materials studies concentrated on producing artificial ECM
lead to the formation of focal adhesion complexes which networks that are made up of synthetic polypeptides or
are linked to the intracellular actin cytoskeleton [60, 61]. peptide-conjugated synthetic polymers that present bioactive
Another example of this structure-dependent ECM signaling ligands and respond to cell-secreted signals to enable prote-
pathway is in tyrosine kinases [62]. For cell binding and olytic remodeling. The production of such biomaterials can
migration, integrin signaling modulates the cell signaling be used in differentiating stem cells into neurons [73]. The
pathways of transmembrane protein kinases such as receptor goal of this approach is to mimic the properties of ECM. The
tyrosine kinases (RTK). RTK are high-affinity cell surface areas of biomimicry are specific cell adhesion, degradation
receptors for many polypeptide growth factors, cytokines, by proteolytic processes involved in cell migration and tissue
and hormones. The study of receptor tyrosine kinase (RTK) remodeling, and the ability to control cellular functions such
signaling led to the understanding of how an extracellular as ECM synthesis. An example of such systems is the use of
signal is transmitted to the nucleus to induce a transcriptional photopolymerizable polyethylene glycol (PEG) based hydro-
response [63]. Other nonintegrin adhesion receptor families gels as tissue engineering scaffolds. This material showed,
include selectins, cadherins, immunoglobulins, proteogly- when modified with necessary signals, that it can interact
cans, and some other laminin-binding proteins. In short, with cells in a manner similar to that of natural ECM, especi-
this mode of interaction conveys biochemical and positional ally in transmitting bioactive signals that control tissue for-
information by which the cell can know how and when it mation and cell phenotype.
should undertake a particular activity. ECM microenvironment is not permanent; changes
ECM is coupled to cytoskeletal and signalling effec- during aging were observed in different organs with variable
tor elements which direct crucial downstream functions, times of onset. Due to the specific interactions between dif-
such as cell growth, survival, and transcriptional activity ferent tissues, cells, and their surroundings, the cells modify
[64]. Biochemical and biomechanical modifications of ECM their own environment by reshaping their ECM components
microenvironment are transmitted to cells and induce the into the correct configuration that allows the growth of the
resulting changes in their behaviour [65]. Cell mechanosens- functioning tissues which have specific architecture and
ing is mediated by focal adhesion contacts [66]. Indeed, characteristics. ECM components are essential to stem cell
physical and mechanical forces in cell microenvironment maintenance and subsequently to support tissue regeneration
lead to changes in cell morphology and differentiation. Not [74].
only the composition of the ECM has direct effects on cell
behaviour, but also its physical properties. Stiffness of bone 4. Phenotype Control and
ECM is essential to maintain osteoblast phenotype whereas Stem Cell Differentiation via
chondrocytes dedifferentiate where they are cultured on a Microenvironmental Cues
rigid matrix [67]. Elasticity of ECM determines also the
differentiation of progenitor cells [68]. Furthermore, physical Tissue homeostasis requires a certain level of phenotypic
modifications of an adhesive protein such as fibronectin plasticity from resident cells and also the involvement of cir-
are sufficient to influence cell activities. The stretching of culating cells. The most apparent manifestation of this need is
fibronectin alters its binding to ligands [69] and more observed upon injury where the inflammatory reaction medi-
importantly fibronectin conformation regulates integrins ated by immune cells, such as neutrophils and macrophages,
attachment which controls downstream cell behaviour [70]. decides how an implant, transplant, or an engineered tissue
Another type of ECM component variation is biochemical. integrates with the body. The phenotypes of the immune cells
Glycosylation is one of the most abundant protein modifica- in the microenvironment have a significant effect on the final
tions having a role in protein stability, secretion, and function. outcome. Moreover, many tissues depend on several different
The O-glycosylation in particular is essential in cell adhesion. cell types with given phenotypes. The quality of bone tissue
Zhang and Hagen demonstrated that loss of glycosylation depends on the interaction between osteoblasts, osteoclasts,
disrupts adhesion of epithelial cells and more generally and osteocytes. Respiratory epithelium not only has a ciliary
influences cellular microenvironment [71]. Moreover, gly- epithelium layer but also requires basal cells and glandular
cosylation of adhesive proteins like laminin or fibronectin cell components.
One of the new paradigms in tissue engineering is the observe cell behavior at single-cell level. The degree of spread-
utilization of developmental pathways for engineering tissues ing of human epidermal stem cells was observed by Connelly
[75]. In one of the recent demonstrations of “developmental et al. [81]. The authors stated that when the shape of the island
tissue engineering” [76], Scotti et al. were able to produce was changed from elongated to circular, epidermal stem cells
a bone organ with functioning bone marrow by putting the showed an increase in their differentiation ability. On the
human mesenchymal stem cells through an endochondral other hand, human MSCs revealed a dependence on the area
bone formation route, that is, formation of bone organ via a of the island, that is, while round cells favoured adipogenesis,
cartilaginous tissue step [8]. This was achieved via production whereas cell spreading resulted in osteogenesis [82].
of hypertrophic cartilage tissue, by application of IL-1𝛽 and In addition to topographical cues, soluble factors like
subsequent subcutaneous implantation. By applying both growth factors and cytokines are very important in initiation
physical and chemical microenvironmental controls, they and control of SC differentiation [83]. Tissue engineering has
were able to push the initial hypertrophic cartilage structure become an important stem cell application field with the aim
to produce several cell types with their proper phenotypes, of increasing the quality of life. Therefore, researchers have
which demonstrates the strength of the developmental tissue focused more on finding appropriate cues via utilization of
engineering methods (Figure 2) [8]. biomaterials that could control the cellular environment and
The most active literature concerning cell phenotype in monitoring complex cellular levels. Both natural and syn-
tissue engineering is the research on stem cells. Stem cells thetic materials based biomaterial scaffolds have served to
(SCs), a subset of cells with replenishing ability and the understand the role of chemical cues in controlling stem cell
potential of differentiation into various types of mature cells, behaviour. It is crucial to direct SCs to differentiate into the
are categorized into two main groups, namely, embryonic right cell type, at the right time and location; therefore, spe-
stem cells (ESCs) and adult stem cells (ASCs). It has been cific cues have been investigated in the in vivo microenviron-
shown that the intrinsic genetic programs within these cells ment and have been studied in the in vitro systems that mim-
and some extracellular regulatory signals control the ability icked the natural conditions. Controlled microenvironments
of SCs to proliferate and differentiate into different functional have been designed to direct stem cell differentiation into the
mature cell types [77]. desired mature cell type. Stem cell researchers emphasize the
Stem cells reside in a specialized microenvironment need of a 3D environment instead of 2D since differences have
called stem cell niche which provides the stem cells with been observed in their self-renewal capacity, differentiation,
extracellular cues to allow their survival and identity. This adhesion, and migration ability. Cellular morphology has
niche is a key regulator to the stem cell behavior because been shown to vary depending on the biomaterials structure
it ensures a quiescent and low metabolic environment to (2D or 3D) and material type. Human mesenchymal stem
prevent exhaustion. It is believed that microenvironmental cell shape was observed to be round when entrapped in 3D
properties of the niche provide a good balance between hyaluronic acid hydrogel [84] and elongated when seeded
the ability of SCs to renew themselves and the ability to onto fibrous scaffolds or 2D biodegradable elastomer [85].
differentiate into mature cells so that continuous tissue There are other effects regarding the encapsulation of cells
regeneration occurs. A major part of the cell niche is the ECM as demonstrated by encapsulation of prostate cancer cells
(extracellular matrix) which possesses the specific mechani- (LNCaP) in polyethylene glycol (PEG) hydrogels, which
cal, biochemical, and biophysical properties for tissues and changed their cell-cell contact formation and response to
controls the overall cell behavior [78]. The composition of the androgen stimulation where these effects are also relevant to
ECM provides full support to the niche through its physical the differentiation of stem cells within confined environments
and structural properties. The main extrinsic signals that [9] (Figure 3).
regulate stem cell behavior are those coming from ECM.
Given its three-dimensional organization, the ECM pro-
vides an environment that aids in the integration of the sig- 5. Cell Microenvironment Control via
nals derived from the cell-ECM interactions in order to allow Delivery of Soluble Bioactive Agents
proper “maintenance of stem cell homeostasis” [78]. The cell-
ECM interactions are basically triggered by receptors present Another way to control the cell microenvironment is via
on the cell membrane, like integrin as described before. delivery of bioactive molecules such as drugs, hormones, or
However, studies found that the nonintegrin receptors are the growth factors. Variation in the signaling microenvironment
ones that contribute the most to stem cells homing during might cause perturbations in the signaling processes which
transplantation. Novel techniques have been developed to are at the root of multiple pathologies, including cancers, dia-
observe the interaction between stem cells and ECM proteins betes, and many other diseases [63]. Growth factors can regu-
and how this interaction influences their fate. Among the late activation, growth, proliferation, migration, and differen-
factors that influence stem cell fate are ECM adhesion, its tiation of cells which are crucial for events such as angio-
stiffness, and its topography [79]. For example, the effect genesis or osteogenesis [86]. Recent studies have focused
of micro-/nanotopography on stem cells has been recently on inserting signaling molecules such as growth factors
demonstrated. Oh et al. [80] demonstrated that human MSCs and cytokines into biomaterials (Figure 4). Some examples
can differentiate into osteoblasts under the influence of of altering cell behaviour to such molecules are induced
only nanotopography of culture substrates. Another example vascularisation (new blood vessel formation from fibroblast
is micropatterned islands, created with specific shapes to growth factor 2—FGF-2), regeneration of neurons (from
Safranin O Masson’s trichrome 3D 𝜇CT
In vitro
In vivo 5 w
In vivo 12 w
Figure 2: Bone formation via endochondral pathway. An in vitro formed artificial cartilage successfully forms a bone containing bone marrow
within 12 weeks. The in vitro grown tissue is a cartilaginous one as evidenced by the extensive safranin O staining; over time, the cartilaginous
tissue has been gradually replaced by bone tissue, as can be seen by the extensive Masson’s Trichrome staining. Micro-CT images also showed
the development of a bone like structure within 12 weeks. Reproduced from [8].
nerve growth factor, NGF), retention of stem cell pheno- vital roles in shaping the developmental processes [91]. The
type (from immobilization of cytokines to maleic anhydride binding of growth factors to the ECM is regulated by the
copolymer thin-film coating), and providing an environ- GAG side chains. One important application of this is the
ment that helps cell survival and proliferation. Epidermal regulation of specific gene expression which is done by using
growth factor (EGF), incorporated with the matrix mate- growth factor-ECM interaction, that is, by controlling the
rial, increases cell attachment to the implanted matrix and growth factor presence via their interaction with ECM [92].
increases the spreading of mesenchymal stem cells [10]. Incorporation of bioactive factors into scaffold can be
However, these molecules have high instability and very achieved by two different ways, mainly through covalent
short biological half time and can be enzymatically digested and noncovalent immobilization [93–95]. The first approach
or deactivated while in physiological fluids [87, 88]. Besides, is based on the covalent binding of the molecule to the
growth factors or drugs need to reach specific location to be scaffold via chemical reaction such as immobilization of
effective; thus, their systemic introduction is not a viable way VEGF using N-(3-dimethylaminopropyl)-N 󸀠 -ethylcarbodi-
to control their concentration in specific target areas [89]. imide hydrochloride chemistry (EDC) [96]. The noncovalent
To overcome these limitations, it was necessary to develop approach is based on interaction of the molecule with the
delivery vehicles with growth factors or drugs incorporated polymer matrix such as electrostatic interaction, hydrogen
within tissue engineering scaffolds. To illustrate this fact, bonding, or physical entrapment. In the case of incorporation
it has been shown that bolus injection of growth factors via electrostatic interaction, charged material is required
such as VEGF is less effective than a sustained and localized such as polyelectrolytes or gelatin hydrogels. In all these
delivery via biodegradable hydrogels to achieve blood vessel different approaches, the release will be triggered by scaffold
formation [90]. In the case of bolus injection, VEGF was not degradation, diffusion of the molecule through the material,
localized in the target area and stayed only for 72 h, whereas or cleavage (enzymatic or hydrolysis) of the covalent bond
with delivery from biodegradable alginate hydrogel 95% of between the molecule and the scaffold material. For example,
the growth factor was at the ischemic site (improvement of with the addition of bioactive motifs (from bone morpho-
biodistribution), and it stayed at that location for more than genetic protein 2 or osteopontin), it was shown that osteoblast
15 days and its bioactivity was higher (possibly due to the adhesion and the responsiveness to the protein were depen-
protection from denaturation). dent on the cell adhesive motif from osteopontin. The cell
One way to achieve control over local bioactive molecule interaction with the protein demonstrated in vitro bone
concentrations is the immobilization of growth or differenti- formation in a month [97].
ation factors. Mainly, the ECM harbors a lot of growth factor These delivery systems were developed with different
binding proteins. This localization of growth factors by the kind of materials. Two main categories can be identified: (i)
ECM and their signaling contributes to the establishment of natural materials such as collagen, alginate, gelatin, and poly-
a gradient for the soluble, diffusible morphogens, which play L-lysine; (ii) synthetic materials such as PLLA, PEG, and PCL
2D 3D
(a)
(b)
Figure 3: Manipulating the cell microenvironment in 3D via encapsulation within hydrogels. Encapsulation of prostate cancer cells within
PEG hydrogels resulted in more pronounced cell-cell contacts as evidenced by E-cadherin staining (a) and also formation of a necrotic core
within the cell aggregates as shown by pimonidazole staining (b). All scale bars are 75 𝜇m for (a) and 100 𝜇m for (b). Reproduced from [9].
Signaling molecules in hydrogels. In this case, a sustained release of 8 days was

achieved mainly through hydrolysis of ester backbone [106].
Neurotransmitters Cytokines Growth factors Polyelectrolyte multilayer structures (L-b-L, layer by
layer) are also used to design delivery systems because L-b-
Dopamine LIF VEGF BMP-2 L films are easy to produce; they can act as a reservoir for
bioactive molecules [107] and their properties such as perme-
ability, thickness, and charge density can be easily changed
and they can be easily coated on implants [108]. The only
Maintenance of Vascularization problem with these films for drug delivery application is the
Neuron outgrowth undifferentiated state Bone
formation fast release of molecule due to the mobility of polyelectrolyte
chains within the film. To solve this problem, recently, a
Figure 4: The main types of soluble factors that have distinct double entrapment system has been developed for VEGF to
effects on the cellular behaviour at both single cell and tissue level. achieve a long term release [109]. This strategy was based
Controlled delivery of such factors and their regulated presence in on the twofold control over the release by VEGF containing
cell microenvironment is an indispensable tool in tissue engineering PCL nanoparticles loaded in polyelectrolyte multilayer film.
research. Reproduced from [10]. The mechanism of the release was the following: either PCL
nanoparticles containing VEGF were hydrolyzed and then
VEGF diffused in the LBL film and then out; or the particle
[95]. To be an efficient carrier system, these materials must will diffuse out of the film and then hydrolyzed. With this
fulfill some requirements: (i) biocompatibility, (ii) biodegrad- system, a sustained release of 7 days was achieved [109]. To
ability, and (iii) release of active factors in a controlled spatio- prevent the fast release of drug out of LBL film, another
temporal way [98]. Release of growth factors from scaffolds system was developed by adding a mechanosensitive cap as
is mainly governed by two mechanisms: (i) diffusion through a barrier on top of LBL reservoir films. A bioactive agent
the material and (ii) degradation rate of the material [86, 99]. was loaded in PLL/HA film and a PAH/PSS barrier was
Release profile of bioactive molecules is a key parameter to built on top of it. Barrier is cracked under stretch which
control cell microenvironment. Depending on the applica- enables the diffusion of an enzyme (trypsin) within the
tion, such as enhancement of angiogenesis, stem cell dif- reservoir and the PLL/HA is enzymatically degraded leading
ferentiation, or disease treatment, bioactive factors need to to the release of drug [110]. Layer by layer technique (LBL)
be released for specific time points at specific rates. In the with polyelectrolytes can also be used in particle form (i.e.,
case of degradable scaffolds, the release of molecules can be particles formed by polyelectrolyte multilayers) [111]. Using
tuned by varying the degradation profile of the material or this technique, a stimuli-responsive controlled drug release
the molecule diffusion. The degradation rate of the scaffold has been developed in order to release bioactive agents. This
can be changed by crosslinking to reinforce the structure system was based on the absorption of the agent on meso-
and delay the release. Gelatin is a biomaterial obtained by porous silica sphere and then the deposition of a multilayer
denaturing collagen. It is a good ECM mimicking material for capping barrier PAH/PSS. The release of the encapsulated
cells [100]. Moreover, gelatin is biodegradable and has been molecule was further triggered by change of pH (pH = 1.4)
used for a long time in medical field. Gelatin is also a very use- or by change of ionic strength through NaCl concentration
ful material for drug incorporation because it can be pos- of the release media (10 mM NaCl). At higher pH value or
itively (basic gelatin) or negatively charged (acidic gelatin) lower ionic strength, the PAH/PSS layer acted as a capping
depending on collagen processing method (acid or alkaline barrier since it does not allow bioactive agent diffusion and
process) so it can complex both positively or negatively that explains why this system is appropriate for a controlled
charged molecules [101, 102]. This provides a level of ver- and sustained release of bioactive molecules [112].
satility which is not available with other commonly used In some other applications, the delivery of multiple bio-
biomaterials. Gelatin hydrogels are systemically cross-linked active factors with different release kinetics is required. In tis-
with different agents such as genipin, transglutaminase, or sue engineering, for example, angiogenesis and osteogenesis
EDC/NHS because, unless cross-linked, the structure of the are regulated by the action of multiple growth factors and
physically gelled gelatin hydrogels or films is too weak and all of them need to be released in a specific temporal way.
the degradation is too fast [103–105]. For other materials, Richardson et al. have investigated the dual delivery of VEGF
different techniques are available. For example, to control and PDGF, two growth factors necessary for blood vessels
degradation rate of alginate hydrogels, a partial oxidation formation. PLG particle with lyophilized VEGF and PLG
of the polymer chains rendered the hydrogel degradable by microspheres containing encapsulated PDGF were used [113].
hydrolysis [90]. This strategy has been used to create a deliv- All these particles were mixed together and a porous PLG
ery vehicle for VEGF. Synthetic hydrogels can also be used to scaffold was made using high pressure carbon dioxide fabri-
encapsulate and release bioactive molecules. Hyperbranched cation process. These growth factors release profiles were
polyester hydrogels capable of encapsulating hydrophobic not the same: 1.7 pmol/day for VEGF for the first seven
molecules such as growth factors or specific drugs (e.g., days mainly due to VEGF diffusion out of the scaffold
dexamethasone) have been developed. These hydrogels are and from 0.10 to 4.7 pmol/day for PDGF depending on
photocross-linkable via incorporation of methacrylate group. degradation of polymer particle using different formulations
Normally, it is very difficult to entrap hydrophobic agents [113]. In the field of regenerative periodontal therapy, an
interconnected macroporous Dex-GMA (glycidyl methacry- stimuli to the cells in tissue engineering constructs. Biore-
late dextran)/gelatin hydrogel scaffold was developed for actors are systems which enable the continuous replacement
the dual delivery of two different growth factors: BMP- of nutrients and gases either by constant agitation (as in the
2 and IGF-1. These growth factors were encapsulated in case of spinner flasks) or via perfusion. Most of the bioreactor
Dex-GMA/gelatin microparticles; basic gelatin (negatively systems have several entry ports which enables the intro-
charged) was used to encapsulate BMP-2 and acidic gelatin duction of different bioactive agents in a controlled manner.
(positively charged) for IGF-1 encapsulation. As Dex-GMA is Moreover, many bioreactors have the capacity to directly
enzymatically degradable but not hydrolytically degradable, apply physiologically relevant mechanical stress/strain con-
other Dex-GMA microparticles were prepared with neutral ditions (such as tensile, compressive shear stress according
gelatin to encapsulate dextranase in order to further trigger to the target tissue). They mimic the mechanical microen-
the release of growth factors by enzymatic degradation of vironment, for functional engineered tissues that undergo
the Dex-GMA microparticles. All these particles were mixed mechanical loading (articular cartilage, tendons, heart valves,
together and cross-linked via irradiation in order to make etc.) under in vivo conditions. They are designed to increase
the interconnected macroporous scaffold. This mechanism of the efficiency of exchange of metabolites, oxygen, nutrients,
delivery based on the degradation of microparticle followed and waste removal within the cell microenvironment and as
by degradation of the scaffold enables the sustained release of a result to enhance cellular penetration in 3D scaffolds and to
growth factors for a period of 20 days [114]. Some disease like have a better and rapid cellular expansion [118]. For this aim,
periodontitis required the multiple deliveries of antibacterial, there exist perfusion bioreactors for culturing encapsulated
anti-inflammatory, antiresorptive, and osteogenic agents. stem cells and those for cell cultivation on 3D scaffolds.
These molecules must be delivered in a very specific order Mimicking the dynamic mechanical environment is also
to be effective. To fulfill this requirement, a multilayered considered in designing functional bone implants in conjunc-
device was designed. This laminate structure is made by the tion with micro-/nanotopographical features. These studies
association of cellulose acetate phthalate (CAP) and Pluronic pointed out that osteoblasts change their morphology, gene
F-127(P) and these polymers can be further eroded. The expression, and matrix mineralization by either introducing
different drugs have been incorporated in a specific order surface topography on biomaterials or mechanical stimula-
in every stratum by mixing them with the polymer solution tion [119, 120]. Prodanov et al. [121] tested the simultaneous
and every layer is separated by one or two CAPP blank effects of nanotextured surface (300 nm wide and 60 nm deep
layers in order to slow the erosion of the structure and delay grooves) and mechanical stretching in terms of cell attach-
the release of molecules. This system was able to perform ment, ECM formation, and osteoblast differentiation. It was
the release of four different drugs in a specific temporal shown that by dual stimulation (nanogrooved surface and 8%
sequence just by unidirectional erosion of the structure for of strain) the expression of fibronectin and Cfba synergisti-
more than 120 hours depending on the condition used [115]. cally increased 2-fold in comparison to nanotextured surface
The other important cell microenvironment parameter that alone. Such combined effects of topography and mechanical
needs to be mimicked is the changes induced on cells via stimulation were also observed by other groups [41, 122–124].
dynamic stimuli, such as mechanical stimuli. The spatial and Mechanical stimulus by cyclic stretching, fluid flow [121],
temporal variations of cell microenvironment play vital roles and hydrostatic compressive pressure has also been used to
in various paracrine and endocrine cell signaling, 3D tissue align cells. In several studies, cardiac, ligament, and tendon
remodeling, alteration in stem cell niches cancer progression, derived fibroblasts, myoblasts, vascular smooth muscle cells,
and migration of various cells. For these reasons, the dynamic and osteoblasts were subjected to mechanical forces under in
nature of the cell microenvironment is vitally important as vivo conditions. Regardless of cell types, cells cultured on a
discussed below. 2D substrate that is subjected to uniaxial cyclic stretch tend
to align perpendicular to the direction of principal cyclic
6. Dynamic Aspects of Cell Microenvironment strain [122, 125, 126]. There are several studies that have used
mechanical stimuli together with micro/nanopatterning for
The native ECM microenvironments of cells are highly het- cell alignment. It has been shown that when cells grown
erogeneous in three-dimensional space [116] and they go whether on patterned surfaces with micron sized, or nano-
through continuous and dynamic remodeling with time [117]. sized groove/ridge patterns or on unpatterned [127] sub-
The interactions of the ECM components with cells are of strates subjected to cyclic stretching orient themselves in the
reciprocal nature, that is, while cells constantly produce, direction perpendicular to the applied strain. It should be
break down, rearrange, and realign components of ECM noted that applied strains in these examples were parallel to
to change its properties, and any change in ECM in turn the direction of the groove axis. However, there are studies
regulates the activities and the behavior of the cells [47]. showing that tendon fibroblasts and osteoblasts aligned along
The cell-ECM interactions are highly dynamic and complex; the direction of micro grooves regardless of the stretching
hence, a detailed understanding of the dynamic aspects of direction, which suggests that the topographical cues at
cellular microenvironment in terms of spatial and temporal this scale might be a more important determinant for the
variations of different chemical, mechanical, and biological cell alignment direction [122, 128]. For instance, mechani-
stimuli is highly important in tissue or organ engineering. cal loading, topographical patterning, and surface chemical
Bioreactors are a crucial part of tissue engineering treatment can also be combined to engineer cell alignment
research as they are the main means to exert mechanical in vitro. The combined effects of cyclic strain and substrate
microtopography on the alignment of bovine vascular SMCs and diffusion mediated, gradient based microfluidic device
have been investigated by Ahmed et al. [124], where they to infect the cells at many different concentrations of virus
observed that the organisation of actin fibers was dominated simultaneously within a single microfluidic channel. The
by cyclic strain application and the shape of cell nuclei was laminar flow and diffusion have been used for establishing
controlled by the patterns. gradient in many other studies as well [147–150].
Xu et al. [151] used a three-layer microfluidic device for in
6.1. Dynamic Control of Cell Microenvironment Using Micro- situ monitoring of the infection process of cells by a recom-
fluidics. Researchers have designed cell-laden matrices in 3D binant virus in real time. They also performed drug screening
space to mimic functions of human tissues and organs in assays on the microfluidic chip with a tree-like concentration
vitro. Many of these structures also change over time (4D gradient.
biology) [117]. Pioneering work by Petersen et al. revealed that Na et al. [152] used soft lithography based technique
mammary epithelial cells formed a normal acinus structure to create cell adherent and repellent areas on a substrate,
when encapsulated in a 3D material but aberrantly displayed thereby depositing cells in desired micropatterns and forming
cancerous phenotypes when cultured on a 2D substrate plaques of controlled size, shape, and cells number. Microflu-
[129]. Other examples [130, 131] revealed that the materials idic platforms have also been used as bioreactors [153]
based presentation and timed removal of the peptide RGDs containing separate compartments for production, preserva-
can enhance differentiation of mesenchymal stem cells into tion, and transduction of viruses or compounds on a single
chondrocytes. Thus, the spatial and temporal control of microfluidic device. Thus, microfluidic systems and micro-
microenvironment has been implemented in various studies. scale technologies present novel platforms for controlling cell
The synergistic effects of chemical factor gradients, cell- microenvironment for various cell and tissue engineering
cell-interactions, mechanical sensing, and coordinated cell applications.
movements in tissue formation can be achieved through var-
ious microscale and microfluidic technologies. Microfluidic 7. Microenvironment Monitoring
devices offer novel platforms for precise control and varia-
tion of cellular microenvironments in dynamic, automated, The level of control over microenvironment is directly related
and reproducible ways. The use of microfluidic systems in to our level of understanding the mechanisms underlying the
controlling the cellular microenvironment offers numerous dynamic processes. One of the challenges in tissue engineer-
advantages, such as the following: (i) they have the potential ing is continuous monitoring of cellular activities within 3D,
to simulate real tissue microenvironments including multiple generally opaque, thick structures. There are several exciting
cell types and ECM proteins into a 3D structure, (ii) they technologies that have been developed for visualisation of
use a very small number of cells and small quantities of 3D structures that are currently being applied to tissue
reagents, typically in the nanoliter to microliter range, (iii) engineered scaffold.
they allow precise control over cell density and cell shape as For screening purposes of biomaterials microenviron-
well as environmental cues such as attachment matrices con- ment on cells, microelectromechanical systems (MEMS) have
taining self-assembling proteins and gel based substances, been utilized. Features at length scales from 1 𝜇m to 1 cm can
(iv) they provide the ability to precisely control the mechan- be controlled with this technique for stem cell analysis [154].
ical properties (e.g., elasticity, rigidity, and strain), chemical Response of stem cells toward different microenvironmental
properties (e.g., ligand density and orientation), and topo- signals has been studied by using robotic spotters, which
graphic properties (e.g., patterning of surfaces with sub- can test cell-matrix interactions with a very high throughput
stances having different cell-substrate affinity), and (v) they [155, 156]. Another possibility to monitor the cell behaviour
allow high throughput analysis and complete automation of at process level is the real-time imaging of cell microenviron-
the processes. Due to these advantages, recently microfluidic ment in microfluidic chambers [135].
devices have been widely used in controlling the cell/tissue For direct real-time monitoring of the processes within
microenvironment in tissue engineering applications. The engineered tissues, one proposed method is the incorpora-
variation of the local mechanical properties [132], chemical tion of biosensors within the artificial tissues. This is a direct
properties, and topographic features [133, 134] has been extension of implantable biosensors for clinical applications,
achieved using microfluidic platforms. The control over loca- which can be generalized under continuous monitoring of
lized ECM [135–138], chemical gradient [139, 140], and fluid metabolites such as glucose [157]. Currently, such systems are
flow [141, 142] has also been achieved. nearly available and only hindered by the long-term problems
The applications of microfluidic technologies in tissue of foreign body reaction and biofouling which impede their
engineering and biomedical engineering, in general, have reliability and precision [158]. In addition to these problems,
become widespread, such as for development of blood vessels a remodellable tissue engineering scaffold provides a complex
and 3D vascularized tissues [143] and use of microfluidic microenvironment which also has degradation byproducts
platforms in controlling the cell microenvironment for gene of the scaffold material, host cells, implanted cells, and their
therapies [144]. secretions. Recently, a three-parameter in vivo biosensor sys-
Gilmore et al. [145] used an affinity capture technique tem was proposed by Kubon et al. [159] which can simultane-
in a microfluidic chamber for capturing and maintaining ously measure oxygen, pH, and electrical impedance to access
rotavirus double-layered particles (DLPs) in a liquid environ- the reaction to a given biomaterial. Such a system would
ment. In another study, Walker et al. [146] used a laminar flow provide the necessary information concerning oxygenation
levels, infection, and level of integration for a given volume of generation biomaterials. Moreover, for regenerative medi-
the engineered tissue microenvironment. Although this sys- cine and tissue engineering applications, understanding the
tem has not been used in vivo yet, it has been validated in an behaviours of cells in 3D is going to move the field forward.
ex vivo chorioallantoic membrane assay (CAM assay) system Immunomodulation via modulation of macrophage pheno-
[160]. type or via design of biomaterials, bottom-up techniques for
Noninvasive visualisation techniques are another way to production of multifunctional, multicellular structures, real
monitor cell microenvironment. Techniques such as optical time biosensing and linked bioactive agent delivery systems
coherence tomography [161] or nonlinear microscopy tech- within the engineered scaffolds will improve the control of
niques [162, 163] can provide relevant information about the biomedical engineers on artificial tissues further.
scaffold microenvironment and its interaction with the cells.
For assessment of the implanted cell activity within the host, Conflict of Interests
modified signal producing cells can be utilised. By using
firefly luciferase (ffLuc-MSC) expressing MSCs, Kidd et al. The authors declare that there is no conflict of interests
[164] were able to monitor the dispersion of the cells in vivo regarding the publication of this paper.
and found out that the MSCs show a preference to accumulate
if a tumor or an inflammation site is present in the host Acknowledgments
mice. This tropism is related to the presence of a cytokine
microenvironment which is more permissive and chemoat- This work has been supported by EuroTransBio BiMOT
tractive for their incorporation, which provides a guideline to Project (ETB-2012-32) and has received funding from
understand how to control the interaction of the host tissue the European Union’s Seventh Framework Programme for
with the implanted engineered tissue. Aside from cellular research and technological development and demonstration
localization, another crucial information for thick engineered under Grant Agreement no. 602694 (IMMODGEL).
tissues is the level of oxygenation, particularly within the
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Julien Barthes,1 Hayriye Özçelik,1 Mathilde Hindié,2 Albana Ndreu-Halili,3

Correspondence should be addressed to Nihal Engin Vrana; e.vrana@protipmedical.com

Received 23 April 2014; Accepted 15 June 2014; Published 20 July 2014

Academic Editor: Vasif Hasirci

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