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From In Vitro to In Situ Tissue Engineering
DEBANTI SENGUPTA,
1
STEPHEN D. WALDMAN,
2
and SONG LI
1
1
Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA; and
2
Department of Chemical
Engineering, Department of Mechanical and Materials Engineering, Queen’s University, Kingston, ON, Canada
(Received 10 February 2014; accepted 29 April 2014; published online 9 May 2014)
Associate Editor Nenad Bursac oversaw the review of this article.
Abstract—In vitro tissue engineering enables the fabrication
of functional tissues for tissue replacement. In addition, it
allows us to build useful physiological and pathological
models for mechanistic studies. However, the translation of
in vitro tissue engineering into clinical therapies presents a
number of technical and regulatory challenges. It is possible
to circumvent the complexity of developing functional tissues
in vitro by taking an in situ tissue engineering approach that
uses the body as a native bioreactor to regenerate tissues.
This approach harnesses the innate regenerative potential of
the body and directs the appropriate cells to the site of injury.
This review surveys the biomaterial-, cell-, and chemical
factor-based strategies to engineer tissue in vitro and in situ.
Keywords—Biomaterials, In situ,In vitro, Tissue engineering,
Translational medicine.
INTRODUCTION
In the past three decades, tissue engineering, which
combines elements from biology, materials science,
medicine, and engineering, has developed into an
interdisciplinary field that produces new approaches
and therapies for tissue and organ regeneration. This
field refers to the use of building blocks comprised of
cells and scaffolds, either derived from extracellular
matrix (ECM) or synthetic materials, to repair tis-
sues.
58
Examples such as skin substitutes,
7,82
micro-
encapsulated pancreatic islets,
46
replacement blood
vessels
41,59
and tissue-engineered bladders
60
clearly
demonstrate that tissue can be recreated, or ‘‘engi-
neered’’, in the laboratory, and these engineered tissues
can then be implanted into the body to restore function
of tissues and organs.
Tissue engineers have recognized, however, that
tissue reconstruction is extremely complex, and cannot
be recapitulated simply by bringing cells and ECM
together.
63
While cells constitute a crucial component
of tissue, tissue and organs are also characterized by
the existence of organized ECM, growth factors, bio-
physical factors, cell–cell interactions and vasculature
that provide instructive microenvironmental cues.
50
Many extracellular factors are therefore important
considerations in order to develop a tissue engineered
construct. The importance of the ECM has led to the
development of biomaterials that more accurately
replicate the in vivo extracellular environment experi-
enced by the cells. Similarly, bioreactors have been
used to replicate the different types of forces experi-
enced by cells, including cyclic stretch,
5
breath-induced
stretch,
29
and pulsatile flow.
25
A variety of chemical
4,14
and biophysical
21
factors have been used to engineer
tissue constructs in order to replicate the chemical
environment in vivo. A reductionist approach is valu-
able in understanding how various factors, including
the ECM, chemical factors, and mechanical factors,
impact the development of new tissue; the tissue
engineering challenge lies in bringing these distinct
elements together and recreating the multitude of sig-
nals that are found in vivo in an in vitro setting. While
functional tissues can be generated in vitro, the
understanding of tissue development also allows us to
take advantage of human body’s potential to promote
tissue regeneration in situ. Here we will review the
advancement and the evolution of the field from
in vitro tissue engineering to in situ tissue engineering.
IN VITRO AND IN SITU TISSUE ENGINEERING
For the purposes of this review, we broadly classify
tissue engineering into two separate categories—in vitro
Address correspondence to Song Li, Department of Bioengi-
neering, University of California, Berkeley, Berkeley, CA, USA.
Electronic mail: debanti@berkeley.edu, song_li@berkeley.edu
Annals of Biomedical Engineering, Vol. 42, No. 7, July 2014 (Ó2014) pp. 1537–1545
DOI: 10.1007/s10439-014-1022-8
0090-6964/14/0700-1537/0 Ó2014 Biomedical Engineering Society
1537
and in situ tissue engineering. In vitro tissue engineering
combines biomaterial scaffolds, cells, and chemical
factors outside of the body. Importantly, a developed,
functional tissue construct is produced prior to
implantation (Fig. 1). In contrast, in situ tissue engi-
neering harnesses the body’s regenerative capacities to
rebuild lost tissues. In situ tissue engineering refers to
tissue regeneration at the site of injury using extracel-
lular factors to guide functional restoration (Fig. 1).
This review will discuss the differences between in vitro
and in situ tissue engineered approaches, which are
compared in Table 1.
IN VITRO TISSUE ENGINEERING
In vitro tissue engineering has produced many
important advances in the field of regenerative medicine.
This approach aims to reproduce functionally mature
tissue structures in a dish, allowing us to build models for
the in vivo environment. For example, cardiac tissues can
be developed in culture dishes by engineering scaffolds
and applying electrical stimulation.
45,66
Hepatocytes in
hollow fiber perfusion bioreactors have been used to
develop the extracorporeal Liver Assist device.
20
An-
other example of successful in vitro tissue engineering is
the development of the artificial bladder,
2
where bladder
biopsies from patients were grown in culture, seeded
onto a collagen-PLGA scaffold, and combined in a
bioreactor. After 7 weeks of growth, these structures
were then implanted into patients and were found to
restore bladder function. The use of autologous tissue
circumvents the problem of immune rejection; in cases
where such autologous tissue is available, this type of
in vitro tissue engineering remains a feasible and suc-
cessful approach towards the development of regener-
ative therapies.
Stem cells have also been used to derive specific cell
types for in vitro tissue engineering. It is possible to
extensively differentiate these types of cells and test for
functional maturity prior to implantation. This
approach has been utilized to generate human
embryonic stem cell-derived pancreatic endoderm.
36
In
this particular example, human embryonic stem cells
were differentiated extensively until they matched the
transcription factor profile of committed pancreatic
embryonic endoderm. These cells were further com-
bined with a gel–foam biomaterial, overlaid with
FIGURE 1. A pictorial representation of in vitro and in situ tissue engineering. In vitro tissue engineering (arrows in blue)
commonly involves the use of cells, growth factors, and scaffolds combined ex vivo and cultured until functional maturation (often
achieved in a bioreactor), whereupon the construct is implanted. In situ tissue engineering (arrows in red) uses a combination of
scaffolds and growth factors that are specifically chosen to elicit a regenerative response in the body. Cells are sometimes
implanted in an in situ tissue engineering approach, but the tissue is not extensively cultured ex vivo to functional maturation. The
combination of biomaterials, growth factors, and cells is used to stimulate tissue regeneration in situ as opposed to developing
functional tissue in vitro.
SENGUPTA et al.1538
Matrigel, and implanted into mice. Functional analysis
of the grafts revealed the presence of human insulin
and C-peptide, further suggesting maturity of the tis-
sue. It is to be noted that the animals used were im-
mune-compromised; any translation to the clinic may
require immunosuppressive therapy or the use of
autologous cells.
In vitro tissue engineering is also valuable in devel-
oping prevascularized constructs. Vascularized tissue
has been hypothesized to shorten the duration of
ischemia.
48,49
Using an in vitro approach, it is possible
to develop vascular networks by pre-seeding vascular
cells in hydrogels. For example, human umbilical vein
endothelial cells (ECs) and fibroblasts were co-cultured
in fibrin gels,
13
resulting in gels with prevascularized
capillary networks. When prevascularized gels were
compared to nonvascularized controls, it was found
that the prevascularized gels exhibited more and larger
perfused lumens than the controls, and hosted larger
numbers of proliferating cells.
Another approach to developing perfusable vascu-
lature is to engineer angiogenesis in vitro using a
micropatterning method. For example, carbohydrate
glass was developed to print 3D vascular networks in a
variety of biomaterials, including natural materials
such as Matrigel and fibrin, as well as synthetic
materials such as poly(ethyleneglycol).
55
A mixture of
biomaterials and ECs was cast around the vascular
network, and the glass was dissolved away to produce
perfused vascular networks. These perfused channels
were able to functionally sustain rat hepatocytes that
would undergo suppressed function in constructs
without the channel.
Organ decellularization represents a state-of-art
approach to in vitro tissue engineering. A number of
different organs have been decellularized by removing
the living cellular matter with detergents.
62
These de-
cellularized organs retain the original vascular network
structures and ECM organization, allowing reperfu-
sion with vascular ECs and specific cell types to
repopulate the organs. Research is currently underway
to re-transplant a variety of organs, including bioarti-
ficial livers,
78
hearts,
62
and lungs,
61
where the organs
are decellularized and then recellularized in vitro prior
to transplantation. It is recognized that preservation of
tissue structures, surface composition and architecture
require further optimization.
17
Furthermore, decellu-
larized materials can provoke immune reactions,
1,32
and can be difficult to recellularize efficiently.
73
An-
other major challenge is to prevent clotting and
maintain the patency of the vascular network upon
in vivo implantation.
In vitro tissue engineering approaches make it pos-
sible to recapitulate physiological and pathological
conditions outside the body; these approaches also
enable the development of tissue-on-the-chip and or-
gan-on-the-chip platforms. However, the in vitro tissue
engineering approach still faces technical and regula-
tory obstacles prior to in vivo translation. Translational
concerns include immune-acceptable cell sources,
challenges with off-the-shelf availability and scaling up
capability, cost effectiveness, as well as preservation
and handling issues. An emerging body of research
suggests that these challenges faced in traditional
in vitro tissue engineering can be mitigated by the use
of in situ tissue engineering.
IN SITU TISSUE ENGINEERING: REMODELING
IN THE BODY
In situ tissue engineering attempts to regenerate tis-
sues by harnessing the native regenerative potential of
the body. While cells can be transplanted for in situ tissue
regeneration, in situ tissue engineering approaches often
focus on the recruitment of endogenous stem cells to the
site of injury by using biomaterial- or growth factor-
based cues in order to enhance healing.
27,43,69
A dis-
tinctive feature of in situ tissue engineering is that any
engineered constructs that are implanted are not func-
tionally mature. In contrast, in vitro tissue engineering
focuses on the functional development of a tissue prior
TABLE 1. A comparison of in situ and in vitro tissue engineering approaches.
In vitro tissue engineering In situ tissue engineering
Availability off-the-shelf Possible More likely
Scalability Difficult Easier
Ease of clinical translation Complex May be easier
Biomaterials Extensively used Extensively used
Bioreactors Used Not commonly used
Chemical factors Used Used
Cells Used Not commonly used
Cost-effectiveness Less More
Disease modeling Yes N/A
Drug screening modeling Yes N/A
From In Vitro to In Situ Tissue Engineering 1539
to implantation. The in situ approach has several
advantages—in situ tissue-engineered products offer
improved off-the-shelf availability of the finalized pro-
ducts, because in situ tissue engineering often does not
involve extensive manipulation of cells and materials
outside of the body to create functionalized tissue. Be-
cause in situ tissue engineering often relies on extracel-
lular components to stimulate native regeneration, this
approach offers the opportunity to bypass some (but not
all) of the regulatory issues inherent in in vitro tissue
engineering-based approaches. Table 1summarizes key
differences between in vitro and in situ engineering
strategies.
Extracellular factors play a critical role in situ tissue
engineering strategies. Biomaterials have been most
commonly employed to mimic or facilitate the various
interactions that the tissue microenvironment is com-
prised of. While the immune response varies from tis-
sue to tissue and material to material,
3
many
biomaterials have been FDA approved for clinical use.
A biomaterial can be synthetic or naturally derived,
and is used to augment physiology either as part of a
medical device or as part of regenerative therapy.
81
Synthetic materials are derived from inorganic or or-
ganic materials. Commonly used synthetic biomateri-
als include poly(L-lactic acid) (PLLA), poly(lactic-co-
glycolic acid) (PLGA), polyurethane, poly(c-caprolac-
tone) (PCL), and so on. Many other biomaterials are
built out of naturally occurring components of tissue,
such as decellularized ECM and biologically inspired
protein-based materials inspired by naturally occurring
proteins such as elastin
72
and silk.
53
Peptide-based
materials have also been developed for drug delivery
56
and tissue engineering.
26
Recently, self-assembling
peptides were engineered to contain a proteolysis site
in order to serve as a controlled-release drug delivery
vehicle.
39
In general, both the structure and bioactivity
of biomaterials can be engineered to promote tissue
regeneration, which will be discussed below. It is
important to note that techniques used for in situ tissue
engineering may also be used in in vitro tissue engi-
neering applications. However, particular bioengi-
neering methods may be used to enhance in situ tissue
engineering outcomes by incorporating signals that
enhance native regeneration.
Structure and Porosity
In general, biomaterial structure is an important
aspect of in situ tissue engineering, because structure
can be used to mimic the in vivo environment, guide
cell growth and infiltration, and recruit cells to the site
of injury. The porosity and microstructure of bioma-
terials can regulate cell infiltration and inflammatory
responses. A variety of techniques currently exist to
manipulate biomaterial structure and porosity, and
will be discussed in this section.
Hydrogel-based biomaterials have porous structures
that allow for the diffusion of signaling molecules and
for cell remodeling.
18
For example, in vivo implanta-
tion of chondroitin sulfate-functionalized photopoly-
merizable poly(ethylene glycol diacrylate)hydrogels
resulted in the swift filling of cartilage defects.
80
There
is evidence that a variety of hydrogel-based materials
can be implanted over an extended period of time
without causing significant toxicity.
10,16
Biomaterials
can also be electrospun into nanofibers or microfibers
to form fibrous structure similar to that in native
ECM, which provides contact guidance to cells and a
porous structure conducive to remodeling.
15,28,42,44,47
Electrospun fibers have been widely used in tissue
engineering applications. For example, electrospun fi-
brous scaffolds were implanted into rats, and were
found to attract dense infiltration by interstitial and
ECs, producing functional blood vessels in 7 days.
77
It
has been demonstrated that aligned electrospun fibers
can preferentially induce cell infiltration in vivo as
compared to unaligned fibers.
12
Fiber alignment, sac-
rificial fibers and laser ablation can also be used to
increase biomaterial porosity and enhance cell infil-
tration.
38,40,65
In addition, biomaterial foams can be used for tissue
engineering purposes. A PLLA-based foaming method
has been previously described,
54
where polymer-salt
composites are cast to introduce interconnected pores
within the biomaterial. The salt may later be dissolved
away, producing a microporous, degradable biomate-
rial. It is also possible to use poly (methyl methacry-
late) microspheres to template pore size in
biomaterials; the microspheres can be solubilized away
to build a microporous system with precise control
over pore size.
51
Interestingly, biomaterial structure can also facili-
tate immune response activation. It has been demon-
strated that when acellular grafts are implanted into
rats, a specific inflammatory response known as the
M2 type macrophage response is initiated, which leads
to constructive remodeling. In contrast, any implants
that contain a cellular component, even if autologous,
result in an M1 macrophage response, which causes
scarring and dense connective tissue formation.
9
This
work lends credence to the idea that tissue may be
successfully engineered in situ, where native cells are
recruited rather than implanted. Further, it has been
found that macrophages seeded on rough surfaces se-
crete more pro-inflammatory cytokines as compared to
when they are seeded on smooth surfaces.
67
Given that
proper management of the inflammatory response can
significantly impact tissue regeneration,
8
the ability to
control macrophage response through biomaterial
SENGUPTA et al.1540
structure represents a powerful in situ engineering tool.
Indeed, it has even been demonstrated recently that
when porous biomaterials are implanted subcutane-
ously in mice, microporous biomaterials with an opti-
mized pore size can direct the spatial organization of
an M1 (pro-inflammatory) or M2 (pro-healing) mac-
rophage response.
75
Given that micro- and nanopatterned biomaterials
have long been used for a number of tissue engineering
applications, and that a number of techniques are al-
ready in place to control microtopography (including
soft lithography,
31,79
photopatterning,
33
microcontact
printing,
30
and so on), micropatterning represents an-
other viable approach in controlling tissue architecture
and structure. It was demonstrated that when macro-
phages are cultured on a variety of topographically
patterned and smooth surfaces, they respond to topo-
graphical features on the micro- but not the nano-
scale.
64
Finally, 3D bioprinting represents an exciting
new opportunity to develop personalized, customized
biomaterial structure for in situ tissue engineering. For
example, implantable structures have already been
created using 3D printing technology for bone regen-
eration, and have been used to study osseoinduction.
23
Bioactivity
Beyond engineering structure and mechanical integ-
rity, biomaterials can also aid in tissue engineering by
serving as vehicles for components such as chemical
growth factors and drugs in addition to cell adhesion
molecules. These biomaterial-based modifications can
facilitate the recruitment of cells to the injury site, deliver
chemical factors that can decrease detrimental inflam-
mation, and promote healing. Growth factors are
commonly incorporated into biomaterials to enable
in situ tissue engineering by promoting the recruitment
and growth of particular cells. For example, a degrad-
able, acidic gelatin-based hydrogel that contained basic
fibroblast growth factor (bFGF) was able to produce
prolonged vascularization when implanted subcutane-
ously into mice.
76
bFGF has also been used in con-
junction with injectable biomaterials that are
crosslinkable in situ,
11
resulting in a striking improve-
ment in neovascularization. It has previously been
demonstrated that the delivery of FGF and VEGF, two
angiogenic factors, can promote angiogenesis in clinical
trials.
24,70
However, the delivery of growth factors is
often hampered by the short half-life of these factors
in vivo. Controlled release of angiogenic factors by using
engineered biomaterials provides a solution.
22,83
A
mineralized poly(lactide-co-glycolide) based scaffold
was used to release VEGF for up to 15 days to determine
release kinetics in an osteoconductive scaffold.
57
A
similar strategy was also used in a rat model, where
VEGF was released from alginate microspheres, and
demonstrated increased vascularization as compared to
controls without released VEGF.
19
VEGF and plate-
let-derived growth factor were also used in combination
and were released with distinct kinetics to promote
angiogenesis
68
using a PLG scaffold. These results
demonstrate the potential uses of biomaterials-based
drug delivery to promote angiogenesis in tissue regen-
eration. Cell surface signaling factors can also be
incorporated into the biomaterial to modulate cell
function. For example, Jagged-1, a Notch ligand, can be
immobilized in its active conformation to a biomaterial
surface, resulting in enhanced cell differentiation.
6,37
Release rates, the amount of delivery, as well as other
factors that may contribute to absorption and tissue
response will all play a role in tailoring this type of
biomaterial technology for future in situ tissue engi-
neering applications.
Biodegradability
Biomaterial biodegradability as well as the ability to
control biodegradability are important aspects of
in situ tissue engineering. An ideal in situ biomaterial
can be tailored to provide the maximum in situ tissue
engineering support while also ensuring that there is
efficient biological clearing of the material in order to
minimize a foreign body response.
3
This helps ensure
that in situ tissue engineering occurs where needed, and
ends once the regenerative process is complete. The
degradation of biomaterials can be tailored to
dynamically control their porosity, mechanical prop-
erties and drug release rates. Materials such as PLGA,
dextran, and PLLA are known to degrade globally by
hydrolysis. In addition to global degradation, specific
enzyme-based degradation sequences can also be
incorporated into the biomaterial. Matrix metallo-
proteinase (MMP)-cleavable materials are often used
because of the ubiquity of this enzyme in various
biological systems. For example, VEGF in MMP-2
degradable PEG-based hydrogels can be released by
EC-mediated degradation of the matrix to enhance
vascular healing.
71
In this example, engineered bio-
material degradation assists in the release of in situ
tissue engineering mediating growth factors and allows
for greater vascular infiltration. Multiple degradation
mechanisms can be incorporated into the materials,
and a variety of technologies enable degradation.
Specifically, enzymatic degradation can be tailored to
respond to in situ tissue remodeling. For example,
photopolymerizable PEG-based hydrogels can be
engineered to be degraded by collagenase and elastase,
allowing for cell migration and proliferation.
52
Hyal-
uronic acid-based photopolymerizable hydrogels can
also be enzymatically degraded, thus altering
From In Vitro to In Situ Tissue Engineering 1541
degradation-mediated cellular traction and directing
differentiation.
34
Another example of enzyme-cleav-
able biomaterials is an elastomeric protein-based bio-
material that contains plasminogen activator-cleavable
sequences. Using a protein-library, biomaterials with
multiple different degradation rates were engineered
such that multiple drug depots may potentially be
created within the same biomaterial construct by
modifying the specific protein-based biomaterial se-
quences used.
74
Biomaterial degradation may also be
triggered by using photosensitive materials. For
example, it has been demonstrated that PEG-based
hydrogels can be designed to be photodegradable, thus
affording dynamic, temporal and spatial biomaterial
control.
35
These advances in biomaterial degradability
open the door to in situ tissue engineering technologies
that employ responsive, dynamic remodeling of the
tissue’s microenvironment, where biomaterial clearing
time specifically depends on tissue healing and regen-
eration.
FUTURE DIRECTIONS
A variety of techniques exist to facilitate in situ
engineering, which complement existing in vitro tissue
engineering methods. In addition to in vivo therapies,
in vitro tissue engineering will be an invaluable tool to
establish models of tissues and organs for mechanistic
studies and drug screening. Prevascularized tissue
constructs, decellularized organs and organ-on-a-chip
will be further developed for these applications. On the
other hand, bioactive and tunable materials with
incorporated adhesion molecules, growth factors and
drugs present a bright future for in situ tissue engi-
neering, especially for the regeneration of connective
tissues. The advancement of micro- and nano-fabri-
cation technologies will provide the platforms to
engineer biomaterials structures at resolutions never
achieved previously. The development of various smart
materials is another important area of future research.
Biomaterials that are dynamically tunable by chemis-
try, enzymes, light, mechanics etc. can be tailored for
specific applications. An emerging and exciting area
related to in situ tissue engineering is immuno-engi-
neering, in which biomaterials can be used to regulate
immune responses and used as vaccines or for thera-
pies. The combination of in situ tissue engineering and
immuno-engineering approaches may lead to more
effective and new treatments to repair tissues and or-
gans. In situ tissue engineering thus represents a
promising new avenue of regenerative therapy
research, and will continue to provide important
solutions to the clinical problems we are facing today.
ACKNOWLEDGMENTS
This work is supported in part by a Siebel Post-
doctoral Fellowship (to D.S.), a grant from the Na-
tional Institute of Health (EB012240 to S.L.), and a
grant from the California Institute of Regenerative
Medicine (RB3-05232to S.L.).
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