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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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adipocyte differentiation, whereas placement of the same cells on
stiff hydrogels led to osteogenic differentiation.
84,85
MANUFACTURING
Having considered the cellular and biomaterial building blocks of
tissue- engineered structures, and the morphogenic blueprints for
creating them, we now examine commonly used manufacturing
methods.
3D printing. 3D printing is a particularly versatile methodology for
tissue engineering, because it can be used to precisely fashion
biomaterial scaffolds, morphogenic proteins and RNAs, and even
cells themselves.
86,87
The basic schema for 3D printing involves
making a computer-aided design template of the structure to be
created, selection of one or more “inks” (the materials to be
deposited), and deposition of these materials in the selected order
by a programmed 3D printer. Further curing steps to lock in
intermediate and final steps are often required. A major advantage of
3D printing is the capacity to generate customized solutions, tailored
to the patient’s anatomy and the nature of the defect. 3D printing has
entered the clinical realm in the form of customized, CT-guided bone
substitutes for craniofacial and orthopedic reconstructions. However,
these manifestations only result in inert polymeric implants that do
not contain cells.
88,89
Over the last decade, however, numerous
efforts have extended 3D printing to create living, tissue-engineered
structures. For example, in a highly sophisticated application of this
approach, researchers at the Wyss Institute in Boston used three
types of iPSCs—two of which were programmed with fate-biasing
transcription factors—as inks to re-create a vascularized,
multilaminar neuronal structure reminiscent of developing
forebrain.90 Other examples are in clinical trials for plastic and
reconstructive surgery applications as discussed below.
Electrospinning. Another widely used manufacturing strategy for
tissue engineering is electrospinning.91 The approach dates back to
the early 1900s, but recent decades have seen a renaissance in
electrospinning as applied to biomaterial scaffolds. The basic
approach entails ejecting solubilized, charged biomaterials under
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high voltage from the nozzle of an electrospinning apparatus. As the
charged stream exits, it condenses into a nanofiber, which is then
collected on a grounded plate or drum. The drum rotates to create a
sheet composed of nanofibers much like woven fabric.
Electrospinning is compatible with a variety of biomaterials in
common use including PCL, PLGA, collagen, gelatin, and even
decellularized ECM. Nanofibers produced in this manner can be
highly mimetic of the native collagen and elastin fibers in ECM. Once
produced, the nanofibers can be further functionalized through
surface modifications induced by plasma treatment or wet chemistry
to improve cell adherence and enable conjugation of growth and
differentiation factors. As detailed above, electrospinning generates
flat sheets of nanofibers and is therefore particularly well suited for
engineering thinner, relatively homogenous structures like fascia and
tendon. However, more recent advancements have enabled
electrospinning of scaffolds with multiple constituents, and hybrid
approaches combining electrospinning with 3D printing are enabling
increasingly complex, and increasingly biomimetic 3D scaffolds.
Electrospun materials have been used in tissue engineering of bone,
cartilage, skin and soft tissue, nerve, cardiac, and vascular
applications.
92
Bioreactors. Development of tissues and organs entails a complex
interplay of cell-cell interactions, morphogenic instruction, and
mechanical forces. Orchestrating this process ex vivo is among the
most difficult challenges in tissue engineering. Bioreactors are
devices that enable cell expansion and differentiation while providing
metabolic support to keep developing tissues alive. Most bioreactors
used in tissue engineering are closed loop systems that isolate the
developing constructs from the outside environment. This is
particularly valuable for clinical translation as it is easier to maintain
quality and compliance with good manufacturing processes in
isolated systems. Perfusion bioreactors contain pumps for circulating
media through the developing tissues with real time monitoring of O2,
CO2, and other critical parameters to mimic the native circulatory
system93-95 (Figure 12.4). Bioreactors can also be designed to
provide mechanical cues, aiding in the differentiation of particular
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tissue types.
96,97
For example, NeoCart—an engineered hyaline
cartilage graft produced from patient-specific chondrocytes cultured
on a collagen scaffold—is generated in a high-pressure, low-oxygen
bioreactor meant to mimic differentiation conditions of native articular
cartilage. After 2 months of in vitro development in the bioreactor, the
engineered cartilage emerges with mechanical properties closely
mimicking native articular cartilage.
98
FIGURE 12.4. Perfusion bioreactor. Computer control
allows for continuous monitoring of critical tissue
parameters and alterations of media flow to mimic
physiologic developmental conditions. A. Custom
benchtop perfusion bioreactor. B. Bioreactor setup. (From
Sarkar N, Bhumiratana S, Geris L, Papantoniou I, Grayson
WL. Bioreactors for engineering patient-specific tissue
grafts. Nat Rev Bioeng. 2023;1(5):361-377.)
EXAMPLES
Tissue engineers have employed the biomaterial scaffolds, cells,
differentiation cues, and manufacturing methods highlighted above
to create ever more sophisticated, therapeutically relevant tissues. A
number of these engineered constructs can restore function in
preclinical models, and in recent years, they have reached clinical
trials. An exhaustive cataloging of these efforts is beyond the scope
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of this chapter, so below we highlight those tissues and structures
with particular relevance for reconstructive surgery, focusing on
examples with human validation or of extraordinary import.
SKIN
The restoration of cutaneous defects—from infection, trauma, burns,
oncologic resection, or congenital differences—is a daily problem in
plastic and reconstructive surgery. With extensive damage such as
total body burns, donor sites can be depleted, necessitating
engineered solutions. One of the first applications of regenerative
medicine to reach the clinic—CEA—arose from just such a
challenge. These autografts were developed by pioneering stem cell
biologist Howard Green at Harvard Medical School in the 1990s,
building off of his discovery of keratinocyte stem cells and
development of ex vivo culture conditions enabling their expansion
and differentiation into mature keratinocytes. The first application of
CEA to clinical reconstruction occurred in 1980 when Mulliken and
O’Connor99 harvested 2 cm2 of skin from two patients with 80% and
40% total body surface area burns and expanded these on a graft
bed prepared in athymic mice.
100
Multiple sets of cultured grafts were
autographed onto the patients with excellent take. Since that seminal
study, the approach has been commercialized and is now available
as Epicel. However, CEAs suffer from poor mechanical integrity and
a propensity to slough due to the lack of dermal compartment. A
promising recent approach from Meuli and colleagues in Switzerland
has addressed this issue and enabled the creation of patientspecific, bilaminar engineered skin.
101
The technology has been
licensed to the Swiss company CUTISS AG, which is currently
conducting phase 2 clinical trials on patients with severe burns. If
successful, this approach and others like it could be a watershed for
patients with major cutaneous defects. Even CUTISS AG’s
approach, however, does not create a faithful skin replica, as it lacks
skin appendages like hair follicles and sweat glands required for full
function. A promising recent approach from Boston Children’s
Hospital using organoid culture enabled the creation of multilaminar
human skin with appendages and subdermal adipocytes and
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vasculature.78 Further evolution of these approaches may lead to the
creation of true tissue-engineered skin on demand.
BREAST
With increased attention to the drawbacks of silicone implant–based
breast reconstruction including implant-associated illness and breast
implant–associated anaplastic large cell lymphoma, there is an
urgent need for true regenerative solutions. Numerous materials
have been used in animal models to create adipose tissue with and
without the addition of lipoaspirate. Morrison and colleagues
conducted a clinical trial on five postmastectomy patients treated
with a porous acrylic shell they termed the tissue engineering
chamber (TEC). The chambers had sufficient rigidity to maintain the
breast mound shape and had sizes up to 350 cm2. In each patient, a
small thoracodorsal artery perforator flap ranging from 5 to 50 mL in
volume was placed under the TEC. Patients were followed up for
6 months at which point the TEC was explanted and any
regenerated tissues examined. Of the four patients who completed
the trial, however, only one had significant tissue replacement inside
the TEC, with approximately 210 mL of tissue present. Even in this
patient, only the lateral aspect of the breast mound had a fatlike
consistency, with the central region being more fibrotic. The
remaining patients did not generate significant adipose tissue but
mainly had a fibrotic reaction to the TEC. The study highlights
challenges for breast tissue engineering of having scaffolds with
sufficient mechanical strength to maintain the pocket while
simultaneously avoiding fibrosis and encouraging soft-tissue
remodeling. An alternative approach that recently entered clinical
trials uses a highly porous 3D printed PCL breast scaffold coupled
with autologous fat grafting. The approach maintains pocket integrity
while avoiding the rigid structure of the TEC. It also enhances the
rate of soft-tissue remodeling by adding lipotransferred adipocytes.
The methodology developed by Hutmacher and colleagues and
licensed to the German company BellaSeno entered phase 1 clinical
trials in Australia with the first patient treated in 2022. Based on
preclinical studies, it is expected that the implants will fully resorb in
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2 years to be replaced with the patient’s own adipose tissue. The
initial study is expected to enroll up to 20 patients and follow them up
for 2 years.
NERVE
Peripheral nerves are among the few human tissues that retain a
significant capacity for regeneration. If the two cut ends of a
damaged nerve cannot be coapted in a tension-free manner;
however, such regeneration cannot occur, and even under optimal
circumstances, the rate of regeneration is slow. Replacement of
missing nerve segments with autograft is the standard for repair,
though limited by sacrifice of sensory function distal at the donor site,
and axonal mismatch between common sensory donors and motor
or mixed motor recipients. Given these challenges, effective tissueengineered nerve grafts would be highly valuable. Many synthetic
biomaterials described above including PCL, PGA, PVA, and type I
collagen have been used to fabricate nerve regeneration adjuncts,
102
and several are FDA approved. However, most of these consist of
hollow tubes to direct axonal regeneration and are not true
regenerative scaffolds. By contrast, decellularized and processed
human cadaveric peripheral nerve (Axogen) is a true 3D scaffold
with endoneurial tubes to direct regenerating axons. The product has
been successfully employed in clinical studies and is the subject of
ongoing clinical investigations to support a Biologics License
Application with FDA. To date, there are no rigorous studies
comparing clinical outcomes of this solution to those from patientderived autografts, however. A number of cell sources including
BMSCs, ADSCs, and iPSC-derived Schwann cells have been used
to enhance the rate of nerve regeneration on decellularized nerve
scaffolds.
102
Despite promising preclinical models, clinical superiority
to autografts has also yet to be demonstrated with these combined
approaches.
BONE
Bone tissue engineering exemplifies the full range of strategies
detailed above to create new tissues. Both biodegradable (eg, PCL,
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PGA, PLGA) and long-lived (eg, calcium hydroxyapatite and betatricalcium phosphate) scaffolds have all been used as a basis for
engineered bone.
103
Precursor cells from pluripotent ES to
multipotent BMSCs have been successfully employed as well.
104
Finally, osteogenic growth factors that can guide development of
these precursors into osteoblasts including BMP-2 and BMP-7 have
been identified and are available as FDA-approved standalone
products.
105,106
A particularly compelling use case is in craniofacial
reconstruction where the complex and personalized geometry of the
facial skeleton can be replicated in image-guided, 3D printed
scaffolds embedded with bioactive agents.89 One attempt to restore
a mandibular defect using PCL scaffolds with grafted platelet-derived
growth factor was plagued by implant extrusion and limited bone
formation over 1 year, though attempts to use more rapidly
biodegradable polymers may address some of these
shortcomings.
107
A more successful recent example in long bones
from Novadip Biosciences demonstrated nearly 90% of patients in
their phase 1/2 clinical trial successfully healing bony nonunion
following lower extremity trauma. The product which programs
autologous ADSCs to create ECM using instructive growth factors
and microRNAs illustrates the convergence of developmental biology
insights and manufacturing development required to successfully
translate tissue-engineered solutions.
CARTILAGE
Restoration of cartilaginous support is critical to nasal and auricular
reconstruction. The creation of a human auricle on a mouse 25 years
ago by Vacanti and colleagues remains a touchstone in the popular
understanding of tissue engineering capabilities (Figure 12.2).
3
Limitations of autologous chondrocytes include their poor
proliferative potential and propensity to dedifferentiate.
108
Differentiation of MSCs to chondrogenic fates represents an
alternative approach. A particular advantage of MSCs is that
differentiation protocols tend to produce fibrocartilage suitable for
structural support rather than hyaline cartilage used in articular
reconstruction.
109
A recent example of auricular reconstruction for
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microtia demonstrates the possibilities of autologous cartilage tissue
engineering. Zhou and colleagues performed CT scanning of
contralateral ears in patients with unilateral microtia to design a 3D
printed negative mold.
110
This was used to generate a composite
scaffold consisting of a PCL core for structural support surrounded
by a PGA/poly(L-lactide) (PLLA) fiber mesh. Autologous
chondrocytes from the microtia remnants were expanded and
seeded onto this custom scaffold followed by serum-free culture for
12 weeks. The resulting auricular framework was implanted under a
tissue expanded pocket with good shape and size match in 4 out of
5 patients with the longest follow up at 2 years. Engineered carriage
grafts using autologous chondrocytes for augmentation of dorsum
and ala have also shown promise in pilot clinical trials.
111,112
Finally,
an engineered autologous cartilage graft for osteoarthritis of the
knee called MACI has been approved for clinical use by the FDA and
the European Medicines Agency.
93
CHALLENGES AND FUTURE DIRECTIONS
The advances detailed above offer encouragement that tissueengineered solutions may enter into reconstructive practice in the
coming years. However, for these approaches to become
mainstream solutions like flaps and grafts, a number of challenges
need to be overcome. Below we highlight just a few of the
impediments to broad clinical implementation.
Vascularization. Unlike inert prosthetics, tissue-engineered
constructs have living cellular components, which must therefore be
within 200 mm of a vascular structure to enable nutrient and waste
exchange.
113
In native tissues, vasculature develops in close
apposition to parenchymal structures as a function of local metabolic
need. In engineered tissues by contrast, these vascular channels
must be designed into the constructs or induced to self-assemble.
This has been particularly challenging for tissues large enough to be
used in clinical settings. A number of approaches from self-assembly
to decellularization to printing of vascular networks have been
attempted to solve this issue.
113,114
Recently, Lewis and colleagues
developed a multi-ink 3D printing strategy in which fibrin-gelatin
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blends were concomitantly printed with human umbilical vein
endothelial cells, MSCs, and fibroblasts to form tissues up to 1 cm in
thickness. Subsequent osteogenic differentiation led to thick,
vascularized bone tissue that was capable of perfusion through the
developed vascular network and in vitro survival beyond 6 weeks.
115
This strategy, which uses 3D printing to specify tissue
macroarchitecture while allowing vascular networks to emerge
within, is one promising way to enable durable engineered tissue
replacements, though the scale and manufacturing standardization
needed for clinical translation remain to be demonstrated.
Immune rejection. One of the major challenges hampering survival
of tissue-engineered constructs is immune rejection of cellular
components. As highlighted above, the few clinically validated
examples have tended to use autologous cell sources to avoid
immune rejection. This strategy is burdensome however, requiring
patient-specific cell harvest, and costly, difficult to standardize
fabrication methods. The use of allogeneic cell sources with
immunosuppression is generally impractical due to the significant
infection and malignancy risk attending lifelong immunosuppressive
medication. A promising recent strategy involves “cloaking” allogenic
cell sources to prevent rejection by host immune responses.
Schrepfer and colleagues from the cell therapy company Sana
Biotechnology developed “hypoimmune” nonhuman primate
pluripotent cells missing antigen-presenting molecules major
histocompatibility complex class I and II and overexpressing the
innate immune inhibitor CD47.
116
Strikingly, these cells survived for
months in immunocompetent macaques and retained potential for
multilineage differentiation. They are extending this approach to
engineered human cells of therapeutic import including endocrine
cells of beta islets and chimeric antigen receptor (CAR)-T cells.
Clinical success of this approach would greatly expand reach of
engineered cell replacements.
Composite tissues. Many of the structures plastic surgeons
reconstruct are composites of several different tissue types, but as
detailed above, most clinical examples of tissue engineering have
focused on discrete tissues such as nerve, bone, or skin. Combining
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these elements to produce functional reconstructions like moving,
sensate extremities are beyond the reach of current approaches.
Creating even a single component of such structures—such as
vascularized and innervated functional muscle—is a significant
challenge. Gilbert-Honick and Grayson recently reviewed engineered
strategies for volumetric muscle loss, highlighting preclinical
successes with induced neurotization of prevascularized muscle
constructs.
117
Despite progress in small animal models, these
approaches have yet to be scaled to clinically relevant defects.
Another approach is to reperfuse decellularized scaffolds with varied
cellular precursors, allowing the instructive scaffold environment to
direct complex tissue formation. In a particularly striking application
of this method, Ott and colleagues produced an engineered rat
kidney capable of perfusion and urine production following
orthotropic transplantation.31 Whether such an approach can work at
the scale and complexity of composite tissue defects seen in
reconstructive surgery remains unknown.
Cost. Even if these and other technical hurdles can be overcome,
the significant cost of tissue-engineered solutions is a barrier to their
widespread use, particularly in the foreseeable reimbursement
landscape. Custom scaffold manufacturing, cell harvest and culture,
bioreactor costs, and quality validation impose a significant barrier to
entry for tissue-engineered approaches to reach the clinic. However,
as more researchers have embraced these methods and as the first
engineered cell therapies such as CAR-T cells have entered clinical
practice with attendant maturation of manufacturing methods, these
costs are coming down.
118
It is conceivable that further
improvements such as the use of allogenic cell sources mentioned
above—eliminating the need for costly, bespoke cell manufacture—
will allow some engineered solutions to reach parity with
conventional reconstruction. For example, one estimate of the cost
of producing tissue- engineered bone grafts in a bioreactor placed the
costs at $10,000 to $15,000 prior to surgical implantation, in line with
other sophisticated medical devices.
103
CONCLUSION
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