4. Discussion
Effective repair of tracheal diseases caused by tumor, trauma, infection
or congenital abnormalities is still an urgent clinical demand. When the
length of trachea lesions is less than half of the total length of
adults or one-third of children, end-to-end anastomosis is feasible.
However, when the length exceeds this proportion, trachea replacement
therapy is often required.[7, 21] Although the
reported incidence rate of tracheal diseases seems to be low, factors
such as prehospital death and common symptoms similar to other diseases,
for example asthma, conceal the true incidence rate of tracheal diseases
worldwide. In addition, this misdiagnosis often leads to high
mortality.[22] Therefore, the study of tracheal
transplantation and regeneration has important clinical significance.
The first attempt of tracheal transplantation and regeneration can be
traced back to the end of the 19th century, and in long-term research,
the replacement treatment of long segment tracheal defects is still
unsatisfactory.[23] Serious postoperative
complications and difficulties in obtaining substitutes need to be
solved urgently in clinical treatment. Postoperative infection,
necrosis, collapse, stenosis, air leakage, rejection and other problems
are important reasons for transplantation failure. In recent years,
tracheal replacement research has turned to the field of tissue
engineering, which has developed rapidly in the past decades. Together
with regenerative medicine, they have injected new impetus into the
development of clinical medicine and pointed out new directions for
tracheal replacement therapy.[24, 25]
As is known to all, scaffold, seed cell and cytokine are the three
elements
of tissue engineering, and the same is true for tissue engineering
trachea. On this basis, three major research directions are mainly
focused on epithelization, vascularization and cartilaginization. The
ideal tissue engineering tracheal scaffold should be equipped with the
following capabilities: 1) Specific tubular structure to maintain
ventilation; 2) Sufficient mechanical properties to prevent collapse; 3)
Biomimetic extracellular matrix structure for cell adhesion; 4)
Bioactive environment for epithelial regeneration, cartilage
regeneration and angiogenesis.[26] Until now, a
variety of scaffolds, including synthetic scaffolds and natural
scaffolds, have been used for tissue engineering trachea. However,
natural derived materials are limited by mismatched shapes, poor
mechanical properties and rapid degradation
rate.[27] While the use of synthetic materials is
hindered by their low adhesion rate, limited biological activity and
aseptic inflammation.[28] Therefore, the composite
scaffold prepared by combining natural materials with synthetic
materials has become a better strategy.
Silk fibroin, a natural biological material, has attracted extensive
attention in the fields of regenerative medicine and tissue engineering.
Various forms of scaffolds, including foam, film, electrospun fibers and
hydrogels, all can be prepared via SF.[13] Among
them, the application of hydrogel is gradually increasing for its
three-dimensional structures, which are equipped with suitable porosity
and proper pore size for cell migration, survival and tissue
regeneration and repair.[29] Kim’s resarch group
developed a methacrylated photocurable silk fibroin (SilMA) bioink,
which can be modulated by digital light processing (DLP) 3D bioprinting.
3D hydrogels using the bioink are characterized in terms of
printability, mechanical and rheological properties, and
biocompatibility. The versatile bioink can be used broadly in a range of
applications, including trachea tissue
engineering.[30, 31] Hong’s study promised that
the fabricated SilMA hydrogel using DLP 3D printer played an important
role in ensuring of viability, proliferation and differentiation to
chondrogenesis of encapsulated cells and could be applied to the fields
of tissue engineering needing mechanical properties like cartilage
regeneration.[32] Wu’s and Rajput’s research both
demonstrated that the scaffolds prepared by silk fibroin-derived bioinks
for DLP-based 3D bioprinting could be applied for tissue
engineering.[33, 34] However, the disadvantage
that the mechanical properties are insufficient to support the
load-bearing structure has become an obstacle to its application under
specific requirements.[35] PCL has been approved
by the Food and Drug Administration for internal use in the human body
and been widely used in various fields due to its several
advantages;[36] our research group has also
fulfilled some researches in tracheal scaffold using
PCL.[5, 9, 10, 37] In this study, the hybrid
scaffold was prepared by 3D printed polycaprolatone coated with Silk
Fibroin Methacryloyl hydrogel, which was also used as carrier of seed
cells and growth factors.
Cell adhesion, an index of biocompatibility, is the basis of cell
proliferation, migration and differentiation. The pore size, mesh
distribution and other properties of hydrogel, which are related to
concentration, can affect the cell adhesion. As a result, the reasonable
concentration of SilMA hydrogel should be selected first. The swelling
and degradation test, CCK-8 test and H&E staining of 3D co-cultured
test all suggested that the 20% SilMA hydrogel was a better choice. The
cytocompatibility of biomaterials, another component of
biocompatibility, is usually tested by the method of co-culturing with
cells in vitro. The result in this study indicated satisfactory
cytocompatibility of the 20% SilMA hydrogel.
The mechanical property of biomaterials is an important indicator to
evaluate whether they can be used for tracheal reconstruction in vivo.
The maintenance of longitudinal tension
and
radial compression mechanical properties is an important factor to
prevent airway collapse.[38] The stretched and
compression test results of this study showed that the longitudinal and
radial mechanical properties of 3D printed PCL scaffold and the hybrid
scaffold were significantly better than native trachea. However, in the
stretched process, the longitudinal ductility of native trachea was
better than PCL and the hybrid scaffold, which may be related to the own
physical properties of the material. The trachea scaffold should also
have a certain degree of toughness on the side to bear the pressure
during neck movement, so three-point bending test was carried out in
this study. There is no doubt that the
hybrid scaffold showed better
mechanical properties. Therefore, it is considered that the
hybrid
scaffold can bear the changes of intrathoracic pressure and the movement
of surrounding muscles during respiratory movement in vivo, so as to
maintain the patency of the tracheal lumen.
It is not sufficient to examine the properties in vitro, so the tracheal
partial window-shape defect and repair was constructed by using the
hybrid scaffold, which was loaded with seed cells and small molecule
drug, KGN. Although the trachea seems to be a simple tube, it actually
has complex biological and anatomical characteristics. The innermost
layer at the junction between the trachea wall and the lumen is the
mucous membrane, which is composed of respiratory epithelium and lamina
propria. The ciliated pseudostratified columnar epithelium is
responsible for clearing mucus, attracting inflammatory cells and
secreting various mediators when the airway suffers injury, and is the
first barrier against pathogens and particles when
breathing.[23] For this reason, the potential to
regenerate respiratory epithelium is an understood
thing.[39] In this study, autologous epithelial
cells was cultivated in the hybrid scaffold. Bronchoscopic images and
gross view of specimen two months after transplantation showed that this
hybrid scaffold maintains the luminal structure. At two months, the
results suggested that the structure around the patch was complete
without pale necrosis, purulent exudation, and obvious stenosis and
collapse. The overall structure could be clearly detected under H&E
staining, including the native area and anastomosis area. At the same
time, it could be observed that the cilia were arranged neatly on the
inner surface, similar to the native cilia in morphology, even with
inflammatory cell infiltration. Furthermore, IHC and IF
staining
of CK-18 confirmed the result again. Obvious deepening of the
brown-color at the anastomotic region and obvious enhancement of antigen
expression could be observed in IHC staining. Similarly, IF staining
indicated that the intensity of green fluorescence in the anastomotic
region is similar to that in the primary region. Taken together, this
hybrid scaffold is conducive to the growth and crawling of epithelial
cells and regeneration of ciliary structure.
The mechanical support of native trachea is mainly provided by C-shaped
cartilage ring. The implanted tracheal scaffold possesses better
mechanical properties, which is beyond doubt, but it cannot be implanted
permanently. Therefore, when the stent is gradually degraded and
absorbed in the body, the regenerated cartilage structure becomes more
and more significant. Therefore, the SilMA hydrogel containing BMSCs and
KGN were dropped on the outer surface of the patch in this study to
promote cartilaginization. Alcian blue staining, modified Saffron-O and
fast green staining and IHC staining of type II collagen were performed
on the postoperative specimens, but the results were a pity (Not shown
in the results section). Therefore, the following research will focus on
how to promote chondrogenesis, including biomimetic structure, selection
of hydrogel, optimization of experimental plan, etc. In addition, how to
achieve long tubular orthotopic transplantation based on the patch is
another challenge.
In conclusion, Silk Fibroin Methacryloyl is seized of good gelling
property and the SilMA hydrogel can maintain a stable status. Meanwhile,
SilMA hydrogel possesses appropriate 3D pore structures and the
concentration of 20% is more suitable for cells adhesion and
proliferation.
In
vitro cytotoxicity test showed that 20% SilMA has good
cytocompatibility and the mechanical test suggested the hybrid scaffold
prepared by PCL coated with 20% SilMA own better biomechanical
properties than native trachea. What is more important, the cell loaded
hybrid scaffold could be applied for tracheal window-shape defect repair
to successfully realize the crawling of epithelial cells at the
transplantation site.