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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_612_Библиотеки_им_академика_М_И_Перельмана
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polymers to soft-tissue engineering applications like vascular
repair.
17
Fibrin is another natural biopolymer that has been used in a
variety of tissue engineering applications including nerve, cardiac,
cartilage, and bone.18-21 As a key component of the clotting cascade,
fibrin is polymerized to form an insoluble clot for tissue repair. As
with collagen, fibrin-based products have garnered FDA approval—
in this case, as tissue adhesives—conferring added comfort with this
biomaterial as a tissue engineering substrate.
Silk-based scaffolds are another biodegradable option for soft
tissue engineering. Silk protein derived from silkworms is a
composite of fibroin decorated with sericin proteins. Processing and
solubilization allows for the protein to be manufactured into porous
scaffolds that support cell infiltration. A surgical mesh created from
processed silk—the SERI scaffold—was FDA approved in early
2009 but was later withdrawn from the market due to inflammatory
reactions and other complications.
22,23
In addition to the fibrillar biopolymers mentioned above, cell
carriers can be made from biodegradable hydrogels such as HA,
cellulose, and alginate. HA and its derivatives have been employed
in numerous tissue engineering applications. As the principal
hydrogel component of native tissue ECM, HA has excellent
biocompatibility. HA is readily produced by bacterial fermentation
and can be obtained with different molecular weights to support
engineering of tissues with varied physical properties. Further
expanding its versatility, HA is a glycosaminoglycan with several
reactive positions on the sugar backbone enabling functionalization.
These functional groups can be used for cross-linking HA polymers
to each other, to other scaffolding elements, or to bioactive stimuli.
When implanted in the body, HA undergoes slow hydrolysis by
naturally occurring hyaluronidases. A further advantage of this
biomaterial is the availability of FDA-approved hyaluronidase, which
can be used to rapidly degrade HA polymers in the event of adverse
reactions. However, like other hydrogel biomaterials, HA polymers
are easily compressible, with limited mechanical strength. To
overcome this, HA molecular weight and cross-linking can be
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increased, with the trade-off of decreased porosity and impaired cell
infiltration. Cellulose and alginate are other classes of naturally
occurring, biodegradable hydrogels that can support tissue
engineering. As with HA, these must be modified—in the case of
cellulose most commonly to carboxymethyl cellulose—to gain
sufficient mechanical integrity to make durable constructs.
The last category of biodegradable biomaterials combines aspects
of the polymers and hydrogels described above, more closely
mimicking the hybrid nature of native ECMs. These are composite
biomaterials that are either biologically derived or synthetic.
Biologically derived composites are created by removing cellular
components from native organs and tissues through the use of
detergents or other means and stabilizing the remaining
proteinaceous materials through chemical cross-linking. The
resulting structures can be hydrated and infused with new cells to
create tissue-engineered constructs. Examples include acellular
dermal matrices commonly used in soft-tissue reinforcement
(AlloDerm, DermACELL, SurgiMend) and decellularized bladder
(Acell) and placental derivatives used in wound repair in skin and
cornea.24-29 In a particularly striking application of this approach,
Atala and colleagues decellularized porcine bladder constructs and
infused them with human bladder precursors. Subsequent in vitro
development of these constructs led to an implantable bladder
capable of storing urine upon transplantation.30 Similar approaches
have been used to create functional kidney, heart, and lungs in
preclinical models.31-33 Decellularized materials can also be further
processed and incorporated into hydrogels, to more closely mimic
native ECM structure.
In addition to these biologically derived composites, a number of
synthetic composites have entered into development and practice in
recent years. A particularly useful example is Integra bilateral wound
matrix, a synthetic bilayer sheet made of shark cartilage
glycosaminoglycan and cross-linked bovine tendon collagen. This is
FDA-approved for treatment of life-threatening burn injuries and
reconstruction of burn scars. When placed on wounds, the
glycosaminoglycan-collagen layer acts as a dermal mimetic enabling
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vascularization and cellular ingrowth. Following a period of
maturation, the silicone top layer can be removed and the resulting
vascularized neodermis serves as a substitute for skin grafting. Mao
and colleagues recently reported on a fully synthetic nanofiberhydrogel composite consisting of chemically cross-linked PCL
nanofibers and HA hydrogels.34 The composite structure bears a
striking resemblance to native tissue ECM, with a fibrillar architecture
provided by the nanofibers and a high degree of porosity (Figure
12.3).35 When implanted in experimental animals, these materials
encouraged a high degree of vascularization and soft-tissue
remodeling.
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FIGURE 12.3 Biomimetic, synthetic scaffold for soft-
tissue engineering. The nanofiber-hydrogel composite
material (A) has a microarchitecture similar to extracellular
matrix taken from rodent fat pad (B) as illustrated in this
scanning electron micrograph. Arrowheads show fibers
are embedded into the HA hydrogel network. (Used with
permission of American Association for the Advancement
of Science, from Chang A, Botteri E, Gillis RD, et al. Betablockade enhances anthracycline control of metastasis in
triple-negative breast cancer. Sci Transl Med.
2023;15(693):eadf1147; permission conveyed through
Copyright Clearance Center, Inc.)
CELL SOURCES
Cells are the second critical component of tissue-engineered
structures, interacting with scaffolds to create functional, living
tissues. Cellular building blocks for tissue engineering consist of
either differentiated cells characteristic of the tissue to be replaced or
immature precursors that can be induced to form desired cell types.
Differentiated cells have the advantage of possessing key attributes
or functionalities of the target tissue. For example, mature
adipocytes used in adipose tissue engineering, or keratinocytes and
fibroblasts used to make tissue-engineered skin, have the critical
structural and physiological features of their native in vivo
counterparts. However, differentiated cells can be difficult to obtain,
and many have exited the cell cycle and therefore cannot be
expanded ex vivo. For example, under most circumstances, neurons
are postmitotic and terminally differentiated, making them too scarce
for robust nerve tissue engineering.
When mature cells cannot be obtained or are incompatible with
growth on scaffolds, tissue engineers use immature precursors such
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as stem cells that are capable of differentiation into the target cell
type. Stem cells are defined operationally as cells capable of selfrenewal (to create more stem cells) as well as differentiation (to
create specialized effectors). Stem cells can be subdivided by their
origins and capacities for differentiation. The cells with the greatest
capacity for differentiation are embryonic stem cells (ES cells)
derived from the inner cell mass of the blastocyst. These highly
specialized stem cells are capable of creating cell types from all
three germ layers—mesoderm, ectoderm, and endoderm—making
them extremely versatile for tissue engineering applications. Over
the past 2 decades, researchers have succeeded in creating many
therapeutically desirable cell types from ES cells—beta cells,36-
38
cardiomyocytes,
39,40
blood cells,
41,42
vascular endothelia,
43
neurons,
44,45
hepatocytes,46 gut,47 skin,48 and lung epithelia
49,50
and
many others. ES cells are also immortal, so once created, they can
theoretically be an unlimited source of cellular building blocks. In
actual practice, however, different ES lines vary in their propagation
and differentiation capacities.
51,52
Further, repeated passages have
led to genetic alterations in older ES cell lines that may impair
performance or hamper safety.
53,54
However, because creation of
new ES lines entails the destruction of human embryos, there are
practical limitations to the use of these lines as building blocks for
tissue engineering. Further, the high capacity of ES cells for
differentiation can be a liability as well as a virtue. Left unchecked,
ES cell differentiation can lead to tumorigenesis; indeed, one of the
functional tests of ES cells is their capacity to form teratomas in
immunocompromised animals. Therefore, in tissue-engineered
constructs generated from ES, extreme care must be taken to
ensure that any undifferentiated ES cells are removed from the final
product.
55
While ES cells come from a transient developmental stage of the
early embryo, many adult tissues maintain more specialized,
lineage- restricted stem cells throughout life. These cells lack the
broad differentiation potential of ES cells but can rather be
differentiated into one or more cell types characteristic of the tissue
from which they are derived. Because these cell types are harvested
from adult tissues, they circumvent the ethical concerns raised by
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the use of ES cells. Lineage-restricted stem cells exist to renew
tissues during homeostatic maintenance or to rebuild them after
damage. A classic example are the stem cells present in
hierarchically organized epithelia like those of skin, gut, and lung. In
skin, keratinocyte stem cells present on the basement membrane
either replicate or differentiate into mature keratinocytes, undergoing
a stereotypical pattern of maturation that leads to their ultimate
desquamation from the skin surface.
56,57
The existence of such
keratinocyte stem cells led to the first significant regenerative cell
therapy to be adopted in clinical use—cultured epithelial autografts
(CEA). Similar epithelial stem cell populations exist in the colonic
crypt and in alveoli. Other examples of lineage-restricted stem cells
used in tissue engineering include satellite cells in skeletal muscle.
Mesenchymal stem cells (MSCs) are a particular type of lineagerestricted stem cell that has garnered major interest in tissue
engineering. These stem cells are mesodermal in origin and are
defined by their ability to adhere to tissue culture plastic, expression
of various surface markers, and capacity for multilineage
differentiation into adipocytes, chondrocytes, and osteocytes, though
they can adopt other fates as well with appropriate stimulation.58 Two
of the most commonly used types include bone marrow–derived
stem cells (BMSCs) and adipose-derived stem cells (ADSCs). While
BMSCs can be isolated from medullary bone, they can also be
harvested (albeit less efficiently) from circulating blood. These can
be subsequently expanded ex vivo and differentiated. More familiar
to plastic surgeons are ADSCs, derived from the stromal vascular
fraction of lipoaspirate. These cells are obtained by centrifugation
and enzymatic digestion of lipoaspirate followed by cell culture. They
are therefore an easy-to-obtain, patient-specific, versatile cell source
for tissue engineering. In addition to their capacity for differentiation
into therapeutically relevant cell types, MSCs in their undifferentiated
state can modulate host interactions with tissue-engineered
constructs. In particular, these cells encourage angiogenesis to
support vascularization of constructs and they reduce host fibrotic
responses to improve functional integration.
59,60
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Induced pluripotent stem cells (iPSCs) are a particularly exciting
stem cell source for tissue engineering. Takahashi and Yamanaka
originally described iPSCs in 2006, a discovery for which he won the
Nobel Prize in 2012.61 These cells retain the broad differentiation
capacity of ES cells but can be derived from readily available cell
types, obviating the need to manipulate human embryos. The
original iPSCs were created from fibroblasts, and subsequent
studies have demonstrated iPSC creation from numerous cell lines
including hepatocytes, gastric epithelial cells,62 pancreatic β cells,
63
mature B cells,64 and keratinocytes.65 The initial creation of iPSCs
entailed the induced expression of four key ES cell genes in
fibroblasts—Oct4, Klf4, Sox2, and c-Myc. The expression of these
genes activates transcriptional programs that cause dedifferentiation
of mature cell types and acquisition of an ES cell–like phenotype.
While this is a low efficiency process, the successfully induced cells
can be selected, purified, and subsequently expanded to create
abundant starting material for tissue engineering applications. These
cells can then be differentiated into target tissues in the same
manner as ES cells. In the nearly 2 decades since their discovery,
iPSCs have been used to create dozens of therapeutically relevant
cell types and tissues and several iPSC-derived constructs are
currently in clinical trials.
55,66
In addition to avoiding the need for
embryonic cell harvesting, iPSCs have another major advantage.
When obtained in a patient-specific manner, these cells retain their
native human leukocyte antigen and antigen profile. Therefore, when
differentiated and placed back in the host as part of tissueengineered constructs, they are not rejected by the immune system.
MORPHOGENIC INSTRUCTION
As detailed above, scaffolds and cells are the key building blocks of
engineered tissues and organs. However, simply choosing apposite
starting materials and combining them with biomaterial scaffolds
rarely leads to functional structures. Rather, the third critical factor in
tissue engineering is provisioning of developmental instructions to
achieve desired fates. This is particularly true when precursor cells
are combined with biomaterials, because in situ development and
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maturation are necessary to create final cell types. Below we review
some of the strategies used to direct stem cell differentiation and
complex tissue formation in engineered constructs.
During normal development, precursor cells undergo cascades of
proliferation, differentiation, and morphogenesis to build functional
tissue and organs.
67,68
Many of these instructive cues are provided
by growth factors and other proteins embedded in the ECM, which
alter the fate of cells that come into contact with them. Other protein
growth factors known as morphogens are secreted by one cell
population and act at a distance on target cells. These bound and
soluble factors activate conserved intracellular signal transduction
pathways that govern cell fate. Remarkably, these pathways are
conserved across millions of years of development, allowing
developmental insights to be gleaned from model systems such as
fruit flies, zebra fish, frogs, chicks, and mice.69 Examples of these
canonical pathways include Wnt, Hedgehog, Notch, Hippo, fibroblast
growth factors, and BMPs. Ultimately, these factors alter intracellular
transcriptional programs to induce characteristic cell fates that are
further locked in through epigenetic modifications. Decades of
developmental biology studies in these model systems as well as
more recent work with human stem cells in culture have provided
tissue engineers with biological blueprints for directing cell fate.
70
One way to employ these insights is to embed growth factors in
biomaterials scaffolds. For example, recombinant BMP-2 has been
conjugated to electrospun PCL–gelatin–biphasic calcium phosphate
scaffolds to direct differentiation of BMSCs to osteoclast fates.
71
When embedded in femoral and calvarial defects in preclinical
models, these constructs further matured into functional trabecular
bone that integrated into host tissue.
72,73
A second approach uses
native growth factors remaining after decellularization of native
tissues. Scaffolds processed in this way such as decellularized
adipose tissue retain instructive growth factors which when
combined with homologous cells like adipocytes and ADSCs can
generate de novo adipose tissue.74 In addition to using protein
growth factors, core signaling pathways can be activated through the
use of small molecules.75 Differentiation cocktails combining proteins
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and small molecules have been used to mimic in vitro the successive
stages of differentiation cells undergo in vivo to achieve complex cell
fates. For example, Melton and colleagues used a combination of
growth factors and chemical agonists to take ES cells in culture
through successive stages of definitive endoderm, pancreatic
progenitors, endocrine progenitors, and finally beta cells.36 The first
products engineered in this fashion were recently implanted in two
patients, one of whom who achieved several months of glycemic
control free of exogenous insulin. The company producing the
therapy—Vertex Pharmaceuticals—plans to enroll 16 patients in a
phase 1 clinical trial in 2023. Similar directed differentiation cocktails
have been used to generate a number of therapeutically desirable
cell types including neurons, cardiomyocytes, muscle, hepatocytes,
and many others.36-50 Because the final product of these signaling
cascades is the activation of cell type–specific gene expression by
transcription factors, more recent attempts at instruction have
skipped addition of upstream growth factors and just transduced the
transcription factors themselves. For example, endothelial cells were
created by adding modified mRNAs encoding the transcription factor
ETV2.76 This modified mRNA technology (which also underlies
recent COVID-19 vaccines) enables programming of cell fate without
risk of genetic integration or DNA damage that attends virus-based
transduction methods.
Although the previously mentioned methods for morphogenic
instruction have been successful at generating individual cell types,
they struggle to re-create the complex networks of differentiated cells
that constitute tissues and organs. In recent years, organoid culture
has emerged as a solution to this challenge. Under certain in vitro
culture conditions, lineage-restricted stem cells treated with
appropriate differentiation factors can self-assemble into miniature
organs.77 The approach has been used to create bilaminar skin with
hair follicles,
78,79
intestinal villi,68 alveoli,80 liver,81 cortical
substructures,82 and even optic cup.83 Finally, activation of fate
determining signaling pathways can also be affected by the scaffolds
themselves. Altering scaffold physical properties biases
differentiation of embedded cells.84 Placing MSCs on soft hydrogel
scaffolds for example has been shown to encourage neuronal and
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