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.