ArticlePDF AvailableLiterature Review

From In Vitro to In Situ Tissue Engineering


Abstract and Figures

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.
No caption available
Content may be subject to copyright.
From In Vitro to In Situ Tissue Engineering
Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA; and
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.
AbstractIn 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.
KeywordsBiomaterials, In situ,In vitro, Tissue engineering,
Translational medicine.
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-
Examples such as skin substitutes,
encapsulated pancreatic islets,
replacement blood
and tissue-engineered bladders
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
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.
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,
and pulsatile flow.
A variety of chemical
and biophysical
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.
For the purposes of this review, we broadly classify
tissue engineering into two separate categoriesin vitro
Address correspondence to Song Li, Department of Bioengi-
neering, University of California, Berkeley, Berkeley, CA, USA.
Electronic mail:,
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
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 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.
Hepatocytes in
hollow fiber perfusion bioreactors have been used to
develop the extracorporeal Liver Assist device.
other example of successful in vitro tissue engineering is
the development of the artificial bladder,
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.
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
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,
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).
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.
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,
and lungs,
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.
Furthermore, decellu-
larized materials can provoke immune reactions,
and can be difficult to recellularize efficiently.
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 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.
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
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,
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.
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
and silk.
materials have also been developed for drug delivery
and tissue engineering.
Recently, self-assembling
peptides were engineered to contain a proteolysis site
in order to serve as a controlled-release drug delivery
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.
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.
is evidence that a variety of hydrogel-based materials
can be implanted over an extended period of time
without causing significant toxicity.
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.
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.
has been demonstrated that aligned electrospun fibers
can preferentially induce cell infiltration in vivo as
compared to unaligned fibers.
Fiber alignment, sac-
rificial fibers and laser ablation can also be used to
increase biomaterial porosity and enhance cell infil-
In addition, biomaterial foams can be used for tissue
engineering purposes. A PLLA-based foaming method
has been previously described,
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.
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.
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.
Given that
proper management of the inflammatory response can
significantly impact tissue regeneration,
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.
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,
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-
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.
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.
bFGF has also been used in con-
junction with injectable biomaterials that are
crosslinkable in situ,
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
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.
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.
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.
VEGF and plate-
let-derived growth factor were also used in combination
and were released with distinct kinetics to promote
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.
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.
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.
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.
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.
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
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.
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
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-
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.
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.).
Allaire, E., P. Bruneval, C. Mandet, J.-P. Becquemin, and
J.-B. Michel. The immunogenicity of the extracellular
matrix in arterial xenografts. Surgery 122:73–81, 1997.
Atala, A., S. B. Bauer, S. Soker, J. J. Yoo, and A. B. Retik.
Tissue-engineered autologous bladders for patients needing
cystoplasty. Lancet 367:1241–1246, 2006.
Babensee, J. E., J. M. Anderson, L. V. McIntire, and A. G.
Mikos. Host response to tissue engineered devices. Adv.
Drug Del. Rev. 33:111–139, 1998.
Babensee, J. E., L. V. McIntire, and A. G. Mikos. Growth
factor delivery for tissue engineering. Pharm. Res. 17:497–
504, 2000.
Balachandran, K., S. Konduri, P. Sucosky, H. Jo, and A.
P. Yoganathan. An ex vivo study of the biological prop-
erties of porcine aortic valves in response to circumferential
cyclic stretch. Ann. Biomed. Eng. 34:1655–1665, 2006.
Beckstead, B. L., D. M. Santosa, and C. M. Giachelli.
Mimicking cell–cell interactions at the biomaterial–cell
interface for control of stem cell differentiation. J. Biomed.
Mater. Res. A. 79:94–103, 2006.
Billingham, R., and J. Reynolds. Transplantation studies
on sheets of pure epidermal epithelium and on epidermal
cell suspensions. Br. J. Plast. Surg. 5:25–36, 1952.
Brown, B. N., R. Londono, S. Tottey, L. Zhang, K. A.
Kukla, M. T. Wolf, K. A. Daly, J. E. Reing, and S. F.
Badylak. Macrophage phenotype as a predictor of con-
structive remodeling following the implantation of bio-
logically derived surgical mesh materials. Acta Biomater.
8:978–987, 2012.
Brown, B. N., J. E. Valentin, A. M. Stewart-Akers, G. P.
McCabe, and S. F. Badylak. Macrophage phenotype and
remodeling outcomes in response to biologic scaffolds with
and without a cellular component. Biomaterials 30:1482–
1491, 2009.
Burczak, K., E. Gamian, and A. Kochman. Long-term
in vivo performance and biocompatibility of poly(vinyl
alcohol) hydrogel macrocapsules for hybrid-type artificial
pancreas. Biomaterials 17:2351–2356, 1996.
Cai, S., Y. Liu, X. Zheng Shu, and G. D. Prestwich.
Injectable glycosaminoglycan hydrogels for controlled re-
lease of human basic fibroblast growth factor. Biomaterials
26:6054–6067, 2005.
Cao, H., K. Mchugh, S. Y. Chew, and J. M. Anderson. The
topographical effect of electrospun nanofibrous scaffolds
on the in vivo and in vitro foreign body reaction. J. Biomed.
Mater. Res. Part A. 93:1151–1159, 2010.
Chen, X., A. S. Aledia, C. M. Ghajar, C. K. Griffith, A. J.
Putnam, C. C. Hughes, and S. C. George. Prevasculariza-
tion of a fibrin-based tissue construct accelerates the for-
mation of functional anastomosis with host vasculature.
Tissue Eng. Part A. 15:1363–1371, 2009.
SENGUPTA et al.1542
Chen, L., Z. He, B. Chen, M. Yang, Y. Zhao, W. Sun, Z.
Xiao, J. Zhang, and J. Dai. Loading of VEGF to the
heparin cross-linked demineralized bone matrix improves
vascularization of the scaffold. J. Mater. Sci. Mater. Med.
21:309–317, 2010.
Chew, S. Y., J. Wen, E. K. Yim, and K. W. Leong. Sus-
tained release of proteins from electrospun biodegradable
fibers. Biomacromolecules 6:2017–2024, 2005.
Christensen, L. H., V. B. Breiting, A. Aasted, A. Jørgensen,
and I. Kebuladze. Long-term effects of polyacrylamide
hydrogel on human breast tissue. Plast. Reconstr. Surg.
111:1883–1890, 2003.
Crapo, P. M., T. W. Gilbert, and S. F. Badylak. An
overview of tissue and whole organ decellularization pro-
cesses. Biomaterials 32:3233–3243, 2011.
DeForest, C. A., and K. S. Anseth. Advances in bioactive
hydrogels to probe and direct cell fate. Annu. Rev. Chem.
Biomol. Eng. 3:421–444, 2012.
Elcin, Y. M., V. Dixit, and G. Gitnick. Extensive in vivo
angiogenesis following controlled release of human vascu-
lar endothelial cell growth factor: implications for tissue
engineering and wound healing. Artif. Organs 25:558–565,
Ellis, A. J., R. D. Hughes, J. A. Wendon, J. Dunne, P. G.
Langley, J. H. Kelly, G. T. Gislason, N. L. Sussman, and
R. Williams. Pilot-controlled trial of the extracorporeal li-
ver assist device in acute liver failure. Hepatology 24:1446–
1451, 1996.
Engler, A. J., Berry M. F., Sweeney, H. L., Discher, D. In:
Biomedical Engineering Society Annual Fall Meeting.
Baltimore, MD, 2005.
Fischbach, C., and D. J. Mooney. Polymers for pro- and
anti-angiogenic therapy. Biomaterials 28:2069–2076, 2007.
Habibovic, P., U. Gbureck, C. J. Doillon, D. C. Bassett, C.
A. van Blitterswijk, and J. E. Barralet. Osteoconduction
and osteoinduction of low-temperature 3D printed bioce-
ramic implants. Biomaterials 29:944–953, 2008.
Henry, T. D., K. Rocha-Singh, J. M. Isner, D. J. Kereiakes,
F. J. Giordano, M. Simons, D. W. Losordo, R. C. Hendel,
R. O. Bonow, S. M. Eppler, T. F. Zioncheck, E. B.
Holmgren, and E. R. McCluskey. Intracoronary adminis-
tration of recombinant human vascular endothelial growth
factor to patients with coronary artery disease. Am. Heart
J. 142:872–880, 2001.
Hoerstrup, S. P., R. Sodian, J. S. Sperling, J. P. Vacanti,
and J. E. Mayer, Jr. New pulsatile bioreactor for in vitro
formation of tissue engineered heart valves. Tissue Eng.
6:75–79, 2000.
Holmes, T. C. Novel peptide-based biomaterial scaffolds
for tissue engineering. Trends Biotechnol. 20:16–21, 2002.
Huang, Q., J. Goh, D. Hutmacher, and E. H. Lee. In vivo
mesenchymal cell recruitment by a scaffold loaded with
transforming growth factor b1 and the potential for in situ
chondrogenesis. Tissue Eng. 8:469–482, 2002.
Huang, N. F., S. Patel, R. G. Thakar, J. Wu, B. S. Hsiao,
B. Chu, R. J. Lee, and S. Li. Myotube assembly on
nanofibrous and micropatterned polymers. Nano Lett.
6:537–542, 2006.
Huh, D., B. D. Matthews, A. Mammoto, M. Montoya-
Zavala, H. Y. Hsin, and D. E. Ingber. Reconstituting or-
gan-level lung functions on a chip. Science 328:1662–1668,
Jackman, R. J., J. L. Wilbur, and G. M. Whitesides.
Fabrication of submicrometer features on curved
substrates by microcontact printing. Science 269:664–666,
Karp, J. M., J. Yeh, G. Eng, J. Fukuda, J. Blumling, K.-Y.
Suh, J. Cheng, A. Mahdavi, J. Borenstein, and R. Langer.
Controlling size, shape and homogeneity of embryoid
bodies using poly (ethylene glycol) microwells. Lab Chip
7:786–794, 2007.
Kasimir, M., E. Rieder, G. Seebacher, A. Nigisch, B. De-
kan, E. Wolner, G. Weigel, and P. Simon. Decellularization
does not eliminate thrombogenicity and inflammatory
stimulation in tissue-engineered porcine heart valves. J.
Heart Valve Dis. 15:278, 2006.
Khetan, S., and J. A. Burdick. Patterning network struc-
ture to spatially control cellular remodeling and stem cell
fate within 3-dimensional hydrogels. Biomaterials 31:8228–
8234, 2010.
Khetan, S., M. Guvendiren, W. R. Legant, D. M. Cohen, C.
S. Chen, and J. A. Burdick. Degradation-mediated cellular
traction directs stem cell fate in covalently crosslinked three-
dimensional hydrogels. Nat. Mater. 12:458–465, 2013.
Kloxin, A. M., A. M. Kasko, C. N. Salinas, and K. S.
Anseth. Photodegradable hydrogels for dynamic tuning of
physical and chemical properties. Science 324:59–63, 2009.
Kroon, E., L. A. Martinson, K. Kadoya, A. G. Bang, O. G.
Kelly, S. Eliazer, H. Young, M. Richardson, N. G. Smart,
and J. Cunningham. Pancreatic endoderm derived from
human embryonic stem cells generates glucose-responsive
insulin-secreting cells in vivo.Nat. Biotechnol. 26:443–452,
Kurpinski, K., H. Lam, J. Chu, A. Wang, A. Kim, E. Tsay,
S. Agrawal, D. V. Schaffer, and S. Li. Transforming
growth factor-beta and notch signaling mediate stem cell
differentiation into smooth muscle cells. Stem Cells 28:734–
742, 2010.
Kurpinski, K. T., J. T. Stephenson, R. R. Janairo, H. Lee,
and S. Li. The effect of fiber alignment and heparin coating
on cell infiltration into nanofibrous PLLA scaffolds. Bio-
materials 31:3536–3542, 2010.
Law, B., R. Weissleder, and C.-H. Tung. Peptide-based
biomaterials for protease-enhanced drug delivery. Biomac-
romolecules 7:1261–1265, 2006.
Lee, B. L.-P., H. Jeon, A. Wang, Z. Yan, J. Yu, C. Grig-
oropoulos, and S. Li. Femtosecond laser ablation enhances
cell infiltration into three-dimensional electrospun scaf-
folds. Acta Biomater. 8:2648–2658, 2012.
L’Heureux, N., N. Dusserre, G. Konig, B. Victor, P. Keire,
T. N. Wight, N. A. Chronos, A. E. Kyles, C. R. Gregory,
G. Hoyt, R. C. Robbins, and T. N. McAllister. Human
tissue-engineered blood vessels for adult arterial revascu-
larization. Nat. Med. 12:361–365, 2006.
Li, W. J., C. T. Laurencin, E. J. Caterson, R. S. Tuan, and
F. K. Ko. Electrospun nanofibrous structure: a novel
scaffold for tissue engineering. J. Biomed. Mater. Res.
60:613–621, 2002.
Li, S., D. Sengupta, and S. Chien. Vascular tissue engi-
neering: from in vitro to in situ.Wiley Interdiscip. Rev. Syst.
Biol. Med. 6:61–76, 2014.
Liang, D., B. S. Hsiao, and B. Chu. Functional electrospun
nanofibrous scaffolds for biomedical applications. Adv
Drug Deliv Rev. 59:1392–1412, 2007.
Liau, B., N. Christoforou, K. W. Leong, and N. Bursac.
Pluripotent stem cell-derived cardiac tissue patch with ad-
vanced structure and function. Biomaterials 32:9180–9187,
From In Vitro to In Situ Tissue Engineering 1543
Lim, F., and A. M. Sun. Microencapsulated islets as bio-
artificial endocrine pancreas. Science. 210:908–910, 1980.
Liu, W., S. Thomopoulos, and Y. Xia. Electrospun
nanofibers for regenerative medicine. Adv. Healthc. Mater.
1:10–25, 2012.
Losordo, D. W., and S. Dimmeler. Therapeutic angiogen-
esis and vasculogenesis for ischemic disease part I: angio-
genic cytokines. Circulation 109:2487–2491, 2004.
Losordo, D. W., and S. Dimmeler. Therapeutic angiogen-
esis and vasculogenesis for ischemic disease Part II: cell-
based therapies. Circulation 109:2692–2697, 2004.
Lutolf, M., and J. Hubbell. Synthetic biomaterials as
instructive extracellular microenvironments for morpho-
genesis in tissue engineering. Nat. Biotechnol. 23:47–55,
Madden, L. R., D. J. Mortisen, E. M. Sussman, S. K.
Dupras, J. A. Fugate, J. L. Cuy, K. D. Hauch, M. A.
Laflamme, C. E. Murry, and B. D. Ratner. Proangiogenic
scaffolds as functional templates for cardiac tissue engi-
neering. Proc. Natl. Acad. Sci. USA 107:15211–15216,
Mann, B. K., A. S. Gobin, A. T. Tsai, R. H. Schmedlen,
and J. L. West. Smooth muscle cell growth in photopoly-
merized hydrogels with cell adhesive and proteolytically
degradable domains: synthetic ECM analogs for tissue
engineering. Biomaterials 22:3045–3051, 2001.
Meinel, L., S. Hofmann, V. Karageorgiou, C. Kirker-
Head, J. McCool, G. Gronowicz, L. Zichner, R. Langer, G.
Vunjak-Novakovic, and D. L. Kaplan. The inflammatory
responses to silk films in vitro and in vivo.Biomaterials
26:147–155, 2005.
Mikos, A. G., A. J. Thorsen, L. A. Czerwonka, Y. Bao, R.
Langer, D. N. Winslow, and J. P. Vacanti. Preparation and
characterization of poly (L-lactic acid) foams. Polymer
35:1068–1077, 1994.
Miller, J. S., K. R. Stevens, M. T. Yang, B. M. Baker, D.-
H. T. Nguyen, D. M. Cohen, E. Toro, A. A. Chen, P. A.
Galie, and X. Yu. Rapid casting of patterned vascular
networks for perfusable engineered three-dimensional tis-
sues. Nat. Mater. 11:768–774, 2012.
Morishita, M., N. Kamei, J. Ehara, K. Isowa, and K.
Takayama. A novel approach using functional peptides for
efficient intestinal absorption of insulin. J. Control Releas.
118:177–184, 2007.
Murphy, W. L., M. C. Peters, D. H. Kohn, and D. J.
Mooney. Sustained release of vascular endothelial growth
factor from mineralized poly(lactide-co-glycolide) scaffolds
for tissue engineering. Biomaterials 21:2521–2527, 2000.
Nerem, R. M., and A. Sambanis. Tissue engineering: from
biology to biological substitutes. Tissue Eng. 1:3–13, 1995.
Niklason, L. E., J. Gao, W. M. Abbott, K. K. Hirschi, S.
Houser, R. Marini, and R. Langer. Functional arteries
grown in vitro.Science 284:489–493, 1999.
Oberpenning, F., J. Meng, J. J. Yoo, and A. Atala. De
novo reconstitution of a functional mammalian urinary
bladder by tissue engineering. Nat. Biotechnol. 17:149–155,
Ott, H. C., B. Clippinger, C. Conrad, C. Schuetz, I. Pom-
erantseva, L. Ikonomou, D. Kotton, and J. P. Vacanti.
Regeneration and orthotopic transplantation of a bioarti-
ficial lung. Nat. Med. 16:927–933, 2010.
Ott, H. C., T. S. Matthiesen, S.-K. Goh, L. D. Black, S. M.
Kren, T. I. Netoff, and D. A. Taylor. Perfusion-decellu-
larized matrix: using nature’s platform to engineer a bio-
artificial heart. Nat. Med. 14:213–221, 2008.
Pashuck, E. T., and M. M. Stevens. Designing regenerative
biomaterial therapies for the clinic. Sci. Transl. Med.
4:160rs164, 2012.
Paul, N. E., C. Skazik, M. Harwardt, M. Bartneck, B.
Denecke, D. Klee, J. Salber, and G. Zwadlo-Klarwasser.
Topographical control of human macrophages by a regu-
larly microstructured polyvinylidene fluoride surface. Bio-
materials 29:4056–4064, 2008.
Phipps, M. C., W. C. Clem, J. M. Grunda, G. A. Clines,
and S. L. Bellis. Increasing the pore sizes of bone-mimetic
electrospun scaffolds comprised of polycaprolactone, col-
lagen I and hydroxyapatite to enhance cell infiltration.
Biomaterials 33:524–534, 2012.
Radisic, M., H. Park, H. Shing, T. Consi, F. J. Schoen, R.
Langer, L. E. Freed, and G. Vunjak-Novakovic. Func-
tional assembly of engineered myocardium by electrical
stimulation of cardiac myocytes cultured on scaffolds.
Proc. Natl. Acad. Sci. USA 101:18129–18134, 2004.
Refai, A. K., M. Textor, D. M. Brunette, and J. D. Wa-
terfield. Effect of titanium surface topography on macro-
phage activation and secretion of proinflammatory
cytokines and chemokines. J. Biomed. Mater. Res. Part A.
70:194–205, 2004.
Richardson, T. P., M. C. Peters, A. B. Ennett, and D. J.
Mooney. Polymeric system for dual growth factor delivery.
Nat. Biotechnol. 19:1029–1034, 2001.
Ruan, M. Z., A. Erez, K. Guse, B. Dawson, T. Bertin, Y.
Chen, M.-M. Jiang, J. Yustein, F. Gannon, and B. H. Lee.
Proteoglycan 4 expression protects against the development
of osteoarthritis. Sci. Transl. Med. 5:176ra134, 2013.
Schumacher, B., P. Pecher, B. U. von Specht, and T.
Stegmann. Induction of neoangiogenesis in ischemic myo-
cardium by human growth factors: first clinical results of a
new treatment of coronary heart disease. Circulation
97:645–650, 1998.
Seliktar, D., A. Zisch, M. Lutolf, J. Wrana, and J. Hubbell.
MMP-2 sensitive, VEGF-bearing bioactive hydrogels for
promotion of vascular healing. J. Biomed. Mater. Res. A.
68:704–716, 2004.
Sengupta, D., P. M. Gilbert, K. J. Johnson, H. M. Blau,
and S. C. Heilshorn. Protein-engineered biomaterials to
generate human skeletal muscle mimics. Adv. Healthc.
Mater. 1:785–789, 2012.
Stock, U. A., and K. Schenke-Layland. Performance of
decellularized xenogeneic tissue in heart valve replacement.
Biomaterials 27:1–2, 2006.
Straley, K. S., and S. C. Heilshorn. Dynamic, 3D-pattern
formation within enzyme-responsive hydrogels. Adv. Ma-
ter. 21:4148–4152, 2009.
Sussman, E. M., M. C. Halpin, J. Muster, R. T. Moon, and B.
D. Ratner. Porous implants modulate healing and induce
shifts in local macrophage polarization in the foreign body
reaction. Ann. Biomed. Eng. 2013. doi:10.1007/s10439-013-
Tabata, Y., and Y. Ikada. Vascularization effect of basic
fibroblast growth factor released from gelatin hydrogels with
different biodegradabilities. Biomaterials 20:2169–2175, 1999.
Telemeco, T., C. Ayres, G. Bowlin, G. Wnek, E. Boland,
N. Cohen, C. Baumgarten, J. Mathews, and D. Simpson.
Regulation of cellular infiltration into tissue engineering
scaffolds composed of submicron diameter fibrils produced
by electrospinning. Acta Biomater. 1:377–385, 2005.
Uygun, B. E., A. Soto-Gutierrez, H. Yagi, M.-L. Izamis, M.
A. Guzzardi, C. Shulman, J. Milwid,N. Kobayashi, A. Tilles,
and F. Berthiaume. Organ reengineering through development
SENGUPTA et al.1544
of a transplantable recellularized liver graft using decellular-
ized liver matrix. Nat. Med. 16:814–820, 2010.
Vozzi, G., C. Flaim, A. Ahluwalia, and S. Bhatia. Fabri-
cation of PLGA scaffolds using soft lithography and
microsyringe deposition. Biomaterials 24:2533–2540, 2003.
Wang, D.-A., S. Varghese, B. Sharma, I. Strehin, S. Fer-
manian, J. Gorham, D. H. Fairbrother, B. Cascio, and J. H.
Elisseeff. Multifunctional chondroitin sulphate for cartilage
tissue–biomaterial integration. Nat. Mater. 6:385–392, 2007.
Williams, D. F. On the nature of biomaterials. Biomaterials
30:5897–5909, 2009.
Yannas, J. B. I., W. Quinby, Jr., C. Bondoc, and W. Jung.
Successful use of a physiologically acceptable artificial skin
in the treatment of extensive burn injury. Ann. Surg.
194:413, 1981.
Zisch, A. H., M. P. Lutolf, and J. A. Hubbell. Biopolymeric
delivery matrices for angiogenic growth factors. Cardio-
vasc. Pathol. 12:295–310, 2003.
From In Vitro to In Situ Tissue Engineering 1545
... Intraoperative tissue engineering in situ demonstrated its effectiveness in recent clinical studies [4]. Traditionally, the term 'tissue engineering in situ' means different approaches to creating grafts that will mature into functionally active tissue inside the recipient's body using its own regenerative potential [5]. However, we believe that the term 'tissue engineering in situ' should be applied to the process of intraoperative creation of tissue-engineered grafts with their implantation during the same surgical procedure. ...
... Importantly, tissue-engineered grafts created during surgical procedures can be enriched with cells isolated intraoperatively with minimal manipulation. Traditional tissue engineering in situ does not include scaffold enrichment with cells since it requires in vitro culturing [5]. However, existing techniques and devices allow intraoperative cell isolation and cell seeding on scaffolds. ...
... Traditional tissue engineering in situ does not include scaffold enrichment with cells since it requires in vitro culturing [5]. However, existing techniques and devices allow intraoperative cell isolation and cell seeding on scaffolds. ...
Full-text available
Transfer of regenerative approaches into clinical practice is limited by strict legal regulation of in vitro expanded cells and risks associated with substantial manipulations. Isolation of cells for the enrichment of bone grafts directly in the Operating Room appears to be a promising solution for the translation of biomedical technologies into clinical practice. These intraoperative approaches could be generally characterized as a joint concept of tissue engineering in situ. Our review covers techniques of intraoperative cell isolation and seeding for the creation of tissue-engineered grafts in situ, that is, directly in the Operating Room. Up-to-date, the clinical use of tissue-engineered grafts created in vitro remains a highly inaccessible option. Fortunately, intraoperative tissue engineering in situ is already available for patients who need advanced treatment modalities.
... Tissue engineering (TE) aims to overcome these limitations, developing a vascular construct which is biologically and mechanically close to native vessels [12]; specifically, in situ TE relies on the implantation of degradable or semi-degradable scaffolds as they are in the destination site [13]. Due to their specific structure and composition, they are able to guide and control cell recruitment, differentiation, and tissue formation at the site of implantation [14,15], turning into functional endogenous living tissues [10]. ...
... This behaviour is in line with literature data showing that, considering the same anatomical district, vascular compliance decreases with increasing animal size [55]. As concerns the venous compartment, VC compliance values cannot be directly compared to those of AA nor to 3L Silkothane® and 1L SF electrospun grafts, since analysed pressure ranges are different (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). In fact, VC was evaluated among lower pressure ranges that better mimic the in vivo conditions [43] ( figure 5(b)). ...
Full-text available
To address the need of alternatives to autologous vessels for small-calibre vascular applications (e.g. cardiac surgery), a hybrid semi-degradable material composed of silk fibroin and polyurethane (Silkothane®) was herein used to fabricate very small-calibre grafts (innner diameter = 1.5 mm) via electrospinning. Hybrid grafts were in vitro characterized in terms of morphology and mechanical behaviour, and compared to similar grafts of pure silk fibroin. Similarly, two native vessels from a rodent model (abdominal aorta and vena cava) were harvested and characterized. Preliminary implants were performed on Lewis rats to confirm the suitability of Silkothane® grafts for small-calibre applications, specifically as aortic insertion and femoral shunt. The manufacturing process generated pliable grafts consisting of a randomized fibrous mesh and exhibiting similar geometrical features to rat aortas. Both Silkothane® and pure silk fibroin grafts showed radial compliances in the range from 1.37 ± 0.86 to 1.88 ± 1.01 % 10-2 mmHg-1, lower than that of native vessels. The Silkothane® small-calibre devices were also implanted in rats demonstrating to be adequate for vascular applications; all the treated rats survived the surgery for 3 months after implantation, and 16 rats out of 17 (94%) still showed blood flow inside the graft at sacrifice. The obtained results lay the basis for a deeper investigation of the interaction between the Silktohane® graft and the implant site, which may deal with further analysis on the potentialities in terms of degradability and tissue formation, on longer time-points.
... A major concern is the need for large quantities of exogenous cells, which are difficult to reproduce and maintain cellular phenotypes during in vitro cell expansion. To eliminate the need for harvested cells, reduce regulatory hurdles and simplify the approval process, in situ BTE strategies have been introduced to guide functional regeneration to the site of the bone defect [6,7]. ...
Magnesium cation (Mg2+) has been an emerging therapeutic agent for inducing vascularized bone regeneration. However, the therapeutic effects of current magnesium (Mg) -containing biomaterials are controversial due to the concentration- and stage-dependent behavior of Mg2+. Here, we first provide an overview of biochemical mechanism of Mg2+ in various concentrations and suggest that 2-10 mM Mg2+in vitro may be optimized. This review systematically summarizes and discusses several types of controlled Mg2+ delivery systems based on polymer-Mg composite scaffolds and Mg-containing hydrogels, as well as their design philosophy and several parameters that regulate Mg2+ release. Given that the continuous supply of Mg2+ may prevent biomineral deposition in the later stage of bone regeneration and maturation, we highlight the controlled delivery of Mg2+ based dual- or multi-ions system, especially for the hierarchical therapeutic ion release system, which shows enhanced biomineralization. Finally, the remaining challenges and perspectives of Mg-containing biomaterials for future in situ bone tissue engineering are discussed as well.
... Tissue engineering (TE) seeks to harness cells, engineering and materials together with biochemical and physico-chemical cues to restore, maintain, improve or replace tissues of interest. Cells in combination with 3D scaffolds offer a physiologically relevant environment to examine cell fate, tissue maturation or in situ regeneration as well as drug screening in vitro, ex vivo and in vivo [39,40]. Cell approaches include incorporation of primary cells (terminally differentiated or stem cells) from patients that can be expanded in vitro and encapsulated in biocompatible water-based matrices, called hydrogels. ...
Full-text available
Bone pain typically occurs immediately following skeletal damage with mechanical distortion or rupture of nociceptive fibres. The pain mechanism is also associated with chronic pain conditions where the healing process is impaired. Any load impacting on the area of the fractured bone will stimulate the nociceptive response, necessitating rapid clinical intervention to relieve pain associated with the bone damage and appropriate mitigation of any processes involved with the loss of bone mass, muscle, and mobility and to prevent death. The following review has examined the mechanisms of pain associated with trauma or cancer-related skeletal damage focusing on new approaches for the development of innovative therapeutic interventions. In particular, the review highlights tissue engineering approaches that offer considerable promise in the application of functional biomimetic fabrication of bone and nerve tissues. The strategic combination of bone and nerve tissue engineered models provides significant potential to develop a new class of in vitro platforms, capable of replacing in vivo models and testing the safety and efficacy of novel drug treatments aimed at the resolution of bone-associated pain. To date, the field of bone pain research has centred on animal models, with a paucity of data correlating to the human physiological response. This review explores the evident gap in pain drug development research and suggests a step change in approach to harness tissue engineering technologies to recapitulate the complex pathophysiological environment of the damaged bone tissue enabling evaluation of the associated pain-mimicking mechanism with significant therapeutic potential therein for improved patient quality of life. Graphical abstract Rationale underlying novel drug testing platform development. Pain detected by the central nervous system and following bone fracture cannot be treated or exclusively alleviated using standardised methods. The pain mechanism and specificity/efficacy of pain reduction drugs remain poorly understood. In vivo and ex vivo models are not yet able to recapitulate the various pain events associated with skeletal damage. In vitro models are currently limited by their inability to fully mimic the complex physiological mechanisms at play between nervous and skeletal tissue and any disruption in pathological states. Robust innovative tissue engineering models are needed to better understand pain events and to investigate therapeutic regimes
Melt electrowriting (MEW) enables the electric field‐assisted digital fabrication of precisely defined scaffold architectures of micron‐sized fibers. However, charge accumulation and consequent disruption of the precoded pattern by fiber bridging prevents controlled printing at small interfiber distances. This, together with the periodical layer stacking characteristic for additive manufacturing, typically results in scaffolds with channel‐like macroporosity, which need to be combined with other biofabrication techniques to achieve the desired microporosity for cellular infiltration. Therefore, a design strategy is devised to introduce controlled interconnected microporosity directly in MEW scaffolds by an algorithm that creates arrays of bridging‐free parallel fibers, angularly shifted from layer to layer and starting at a random point to avoid periodical fiber stacking, and hence channel‐like pores while defining micropores. This work hypothesizes that pore size can be controlled, decoupled from fiber diameter, and the mechanical properties, including anisotropy ratio, can be tuned. The authors demonstrate this while leveraging the platform for both flat and seamless tubular scaffolds and characterize them via micro‐computed tomography and tensile loading. Lastly, successful cell ingrowth into the micropores and extracellular matrix formation are shown. This platform enables microporous scaffolds entirely via MEW that can be tailored to the architectural and mechanical requirements of the target tissues. This work introduces controlled microporosity to fibrous scaffolds fabricated via melt electrowriting. A software platform enables the multifaceted design of both flat and seamless tubular scaffold architectures that can be independently controlled in terms of their fiber diameter, pore size, and shape, as well as their structural anisotropy, while enabling efficient cell infiltration.
Tissue engineering offers novel solutions to overcome the limitations of currently used heart valve substitutes and small vessel bypass grafts. The main benefit of tissue-engineered substitutes is their intrinsic potential to grow and remodel in response to changing environmental conditions, which is particularly important for cardiovascular tissues, such as blood vessels and heart valves. The traditional tissue engineering triad of cells, scaffolds and stimuli to culture living replacement tissues in the lab has been nuanced with more recent strategies of pure cell-based or pure scaffold-based tissue engineering scaffolds, and the role of patient-specific microenvironmental stimuli (hemodynamic, immunological) is gaining attention as the focus shifts from creating the perfect tissue in the lab to creating adequate integration of tissue-engineered grafts in the body. Considerable progress has been made in recent years with the development of tissue engineered heart valves and blood vessels. As these laboratory-based projects make translational steps towards the clinic, a new set of hurdles need to be negotiated. These include the choice between, in vitro, in vivo and in situ strategies, immunogenicity of implanted material, tissue growth and adaptation and use of percutaneous implantation of tissue engineered constructs. In addition, bioengineered tissues may serve as in vitro platforms to model vascular and valvular diseases, investigate underlying pathomechanisms and develop more efficient and/or novel therapeutic strategies. In this research topic, we aim to address several of the current scientific and translational questions: what inspiration can we draw from native valvular and vascular development? How is the integration of a tissue engineered graft influenced by patient-specific conditions, such as immunological or hemodynamic conditions? Can we use tissue-engineered (disease) models to predict this?
Critical bone defects are the result of traumatic, infection- or tumor-induced segmental bone loss and represent a therapeutic problem that has not been solved by current reconstructive or regenerative strategies yet. Scaffolds functionalized with naturally occurring bioactive factor mixtures show a promising chemotactic and angiogenic potential in vitro and therefore might stimulate bone regeneration in vivo. To assess this prospect, the study targets at heparin-modified mineralized collagen scaffolds functionalized with naturally occurring bioactive factor mixtures and/or rhBMP-2. These scaffolds were implanted into a 2-mm segmental femoral defect in mice and analyzed in respect to newly formed bone volume (BV) and bone mineral density (BMD) by micro-computed tomography scans after an observation period of 6 weeks. To rate the degree of defect healing, the number of vessels, and the activity of osteoclasts and osteoblasts were analyzed histologically. The sole application of bioactive factor mixtures is inferior to the use of the recombinant growth factor rhBMP-2 regarding BV and degree of defect healing. A higher rhBMP-2 concentration or the combination with bioactive factor mixtures does not lead to a further enhancement in defect healing. Possibly, a synergistic effect can be achieved by further concentration or a prolonged release of bioactive factor mixtures. Statement of significance : The successful therapy of extended bone defects is still a major challenge in clinical routine. In this study we investigated the bone regenerative potential of naturally occuring bioactive factor mixtures derived from platelet concentrates, adipose tissue and cell secretomes as a cheap and promising alternative to recombinant growth factors in a murine segmental bone defect model. The mixtures alone were not able to induce complete bridging of the bone defect, but in combination with bone morphogenetic protein 2 bone healing seemed to be more physiological. The results show that naturally occuring bioactive factor mixtures are a promising add-on in a clinical setting.
DESCRIPTION Tissue Engineering is a research field within the life sciences focused on developing engineered tissue units. These tissue-like systems can be used in vitro as a platform for drug testing and developmental research and can ultimately be used in vivo to repair damaged tissues and organs. Electrical signaling is crucial for cell communication in certain tissues, such as the heart, the brain, and the pancreas. In this case, to mimic the organ's full biological functionality, the engineered tissue unit must conduct electrical signals. Thus, the tissue-like system must be equipped with bioelectronic devices capable of coordinating, controlling, and registering its electrical activity. The merge between Tissue Engineering and bioelectronics can give rise to ‘smart’ tissue-like structures, capable of controlling the system function and even modifying its performance when needed. This chapter will review some fundamental concepts related to the Bioelectronics/Tissue Engineering field and provide an overview of the current related research while offering insights into future approaches.
Biodegradable porous scaffolds with different structure, porosity, and strength play a critical role in the repair and regeneration of defects in bone tissue engineering by changing the proliferation condition for cell. In this study, polylactic acid (PLA) scaffold with directional porous structure is designed and fabricated using the method of ice template and phase inversion for speeding up bone repair by promoting the growth and proliferation of bone cells. The morphology, mechanical properties, hydrophilicity, and wicking properties of PLA scaffolds were characterized by scanning electron microscope, universal testing machine, contact angle tester and wicking rate test, respectively. In vitro biocompatibility has been investigated through measuring cell adhesion, proliferation, and viability on PLA scaffold with directional porous structure. Prepared PLA scaffold was implanted into animals to observe the repair mechanism of large-sized bone defects. This study proposes a novel bioporous scaffold design to induce osteocyte growth at the rat calvaria with a directional pore structure, and the scaffold edges were integrated with the calvaria at week 12, effectively promoting the repair and regeneration of defective bone tissue.
In situ tissue engineering strategies are a promising approach to activate the endogenous regenerative potential of the cardiac tissue helping the heart to heal itself after an injury. However, the current use of complex reprogramming vectors for the activation of reparative pathways challenges the easy translation of these therapies into the clinic. Here, we evaluated the response of mouse neonatal and human induced pluripotent stem cell-derived cardiomyocytes to the presence of exogenous lactate, thus mimicking the metabolic environment of the fetal heart. An increase in cardiomyocyte cell cycle activity was observed in the presence of lactate, as determined through Ki67 and Aurora-B kinase. Gene expression and RNA-sequencing data revealed that cardiomyocytes incubated with lactate showed upregulation of BMP10, LIN28 or TCIM in tandem with downregulation of GRIK1 or DGKK among others. Lactate also demonstrated a capability to modulate the production of inflammatory cytokines on cardiac fibroblasts, reducing the production of Fas, Fraktalkine or IL-12p40, while stimulating IL-13 and SDF1a. In addition, the generation of a lactate-rich environment improved ex vivo neonatal heart culture, by affecting the contractile activity and sarcomeric structures and inhibiting epicardial cell spreading. Our results also suggested a common link between the effect of lactate and the activation of hypoxia signaling pathways. These findings support a novel use of lactate in cardiac tissue engineering, modulating the metabolic environment of the heart and thus paving the way to the development of lactate-releasing platforms for in situ cardiac regeneration.
Full-text available
Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.
Full-text available
The ability to regenerate damaged tissue is one of the great challenges in biomaterials and medicine. Successful treatments will require advances in areas ranging from basic cell biology to materials synthesis, but there have been major hurdles in translating the biomedical advances, such as scaffolds that direct stem cell differentiation, into marketed products. Careful consideration of the challenges going from bench to bedside is paramount in maximizing the chances that a good idea becomes a good treatment. We look at a variety of material-based platforms that have made it into the clinic, from biodegradable polymers for wound healing to organs grown ex vivo, and how they have been able to navigate the scientific, regulatory, and business hurdles into the market place.
The objective of this pilot controlled study was to evaluate the extracorporeal liver assist device (ELAD) in patients with acute liver failure who were judged to still have a significant chance of survival (approximately 50%) and in those who had already fulfilled criteria for transplantation. Twenty-four patients were divided into two groups, 17 with a potentially recoverable lesion (group I) and 7 listed for transplantation (group II), and then randomly allocated to ELAD haemoperfusion or control. The median period of ELAD haemoperfusion was 72 hours (range 3-168 h). Biocompatibility of the device was good, with no acceleration in platelet consumption, and haemodynamic stability was maintained. Two patients were withdrawn from the study because of worsening of preexisting disseminated intravascular coagulation in one case and a hypersensitivity reaction in the other. Deterioration with respect to encephalopathy grade was more frequent in the control patients, 7 of 12 (58%), than in the ELAD-treated patients, 3 of 12 (25%). In group I where survival for the ELAD cases was 7 of 9 (78%), there was a higher than expected survival in the controls, 6 of 8 (75%). For group II cases, survival was 1 of 3 (33%) for the ELAD- treated patients, and 1 of 4 (25%) for the controls. Both of the survivors underwent transplantation. Assessment of additive function for the device revealed an improvement in galactose elimination capacity after 6 hours of haemoperfusion. Based on the results of this pilot-controlled trial, better indices of prognosis will be required, in addition to those used to select for transplantation, if patients at an earlier stage of clinical deterioration are to be included in future studies. (Hepatology 1996 Dec;24(6):1446-51)
Recent progress in applying electrospun nanofibers to regenerative medicine is reviewed by Younan Xia and co-workers on page 10. Following a brief introduction to electrospinning, they discuss how scaffolds are fabricated from electrospun nanofibers with well-controlled compositions, structures, and alignments. Applications of the scaffolds in four specific areas are highlighted: nerves, dural tissues, tendons, and the tendon-to-bone insertion site.
The foreign body reaction (FBR) to implanted materials is of critical importance when medical devices require biological integration and vascularization to support their proper function (e.g., transcutaneous devices, implanted drug delivery systems, tissue replacements, and sensors). One class of materials that improves FBR outcomes is made by sphere-templating, resulting in porous structures with uniform, interconnected 34 μm pores. With these materials we observe reduced fibrosis and increased vascularization. We hypothesized that improved healing is a result of a shift in macrophage polarization, often measured as the ratio of M1 pro-inflammatory cells to M2 pro-healing cells. In this study, macrophage polarity of 34 μm porous implants was compared to non-porous and 160 μm porous implants in subcutaneous mouse tissue. Immunohistochemistry revealed that macrophages in implant pores displayed a shift towards an M1 phenotype compared to externalized cells. Macrophages in 34 μm porous implants had up to 63% greater expression of M1 markers and up to 85% reduction in M2 marker expression (p < 0.05). Macrophages immediately outside the porous structure, in contrast, showed a significant enrichment in M2 phenotypic cells. This study supports a role for macrophage polarization in driving the FBR to implanted materials.
Blood vessels transport blood to deliver oxygen and nutrients. Vascular diseases such as atherosclerosis may result in obstruction of blood vessels and tissue ischemia. These conditions require blood vessel replacement to restore blood flow at the macrocirculatory level, and angiogenesis is critical for tissue regeneration and remodeling at the microcirculatory level. Vascular tissue engineering has focused on addressing these two major challenges. We provide a systematic review on various approaches for vascular graft tissue engineering. To create blood vessel substitutes, bioengineers and clinicians have explored technologies in cell engineering, materials science, stem cell biology, and medicine. The scaffolds for vascular grafts can be made from native matrix, synthetic polymers, or other biological materials. Besides endothelial cells, smooth muscle cells, and fibroblasts, expandable cells types such as adult stem cells, pluripotent stem cells, and reprogrammed cells have also been used for vascular tissue engineering. Cell-seeded functional tissue-engineered vascular grafts can be constructed in bioreactors in vitro. Alternatively, an autologous vascular graft can be generated in vivo by harvesting the capsule layer formed around a rod implanted in soft tissues. To overcome the scalability issue and make the grafts available off-the-shelf, nonthrombogenic vascular grafts have been engineered that rely on the host cells to regenerate blood vessels in situ. The rapid progress in the field of vascular tissue engineering has led to exciting preclinical and clinical trials. The advancement of micro-/nanotechnology and stem cell engineering, together with in-depth understanding of vascular regeneration mechanisms, will enable the development of new strategies for innovative therapies. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.
Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.
Protein-engineered biomaterials are designed to enable culture and differentiation of human myoblasts (isolated from skeletal muscle biopsies) into functionally contractile myotubes ex vivo. Individual myoblasts align with microtopographical features (white dashed lines), fuse to form myotubes with several nuclei (blue), and form sarcomeric structures (α-actinin, green) that enable electrical pacing of contractions.
Technical Report
This Progress Report reviews recent progress in applying electrospun nanofibers to the emerging field of regenerative medicine. It begins with a brief introduction to electrospinning and nanofibers, with a focus on issues related to the selection of materials, incorporation of bioactive molecules, degradation characteristics, control of mechanical properties, and facilitation of cell infiltration. Next, a number of approaches to fabricate scaffolds from electrospun nanofibers are discussed, including techniques for controlling the alignment of nanofibers and for producing scaffolds with complex architectures. The article also highlights applications of the nanofiber-based scaffolds in four areas of regenerative medicine that involve nerves, dural tissues, tendons, and the tendon-to-bone insertion site. The Progress Report concludes with perspectives on challenges and future directions for design, fabrication, and utilization of scaffolds based on electrospun nanofibers.