A tissue engineering bionic tracheal stent with microenvironment regulation function and its preparation method

Abstract

The present invention relates to the technical field of medical materials, and provides a tissue engineering biomimetic tracheal stent with a microenvironment regulation function and a preparation method thereof. The present invention is based on a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, on which chondrocytes or quercetin are loaded, and then the obtained cartilage rings and quercetin rings are alternately stacked to form the tissue engineering biomimetic tracheal stent of the present invention. The tissue engineering biomimetic tracheal stent provided by the present invention can stably exist in a living body, and is similar to natural tissue in structure and function. During the pre-vascularization process, it can produce a transmural vascular structure similar to a physiological trachea, thereby providing nutrition and metabolic support for the inner structure of the graft. In addition, quercetin can regulate the immune microenvironment by promoting the polarization of macrophages to an anti-inflammatory phenotype, broadening the application scope of tissue engineering trachea and promoting its clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a tissue engineering biomimetic tracheal stent with a microenvironment regulating function and a preparation method thereof. Background Art

[0002] The human trachea is a hollow tube composed of cartilage tissue, fibrous connective tissue, and epithelial tissue. Cartilage tissue provides mechanical strength to prevent collapse, and the fibrous connective tissue between the cartilage rings provides the trachea with good flexibility. In addition, the tracheal lining is covered by pseudostratified ciliated epithelium, which has the function of protecting the airway and clearing airway secretions. Tracheal diseases caused by inflammation, trauma, and tumors have become increasingly common. When the lower trachea or carina is invaded, circular tracheal resection with end-to-end anastomosis is the most common treatment method. However, when performing circular tracheal resection of a long segment of the trachea, the end-to-end anastomosis has poor surgical results due to factors such as excessive tension, and complications and mortality are high, so tracheal replacement therapy is often required.

[0003] At present, there are limited clinical treatment options for long-segment tracheal lesions. Commonly used tracheal replacement methods include: artificial prosthetic trachea replacement, allogeneic tracheal transplantation, and autologous tissue reconstruction of the trachea. Artificial prosthetic trachea are generally made of metal or organic materials, but due to their poor tissue compatibility, lack of blood supply and epithelial tissue coverage, they can easily cause proliferation of surrounding granulation tissue, leading to occlusion of the lumen, causing airway infection, stenosis, or even collapse and rupture; allogeneic tracheal transplantation often faces the problems of lack of donors and the need for long-term immunosuppressive treatment after surgery. At the same time, airway surgical revascularization is difficult, which can easily lead to graft necrosis, infection, and even life-threatening conditions, which also limits its clinical application; tracheal-like tissue reconstructed with autologous tissue (such as neck or forearm myocutaneous flaps) cannot fully simulate natural trachea in structure and function, and at the same time, it will cause additional surgical trauma during the tissue acquisition process. Therefore, the above three tracheal substitutes are difficult to meet actual clinical needs.

[0004] In order to overcome the shortcomings of the above methods, tissue engineering came into being. Tissue engineering aims to combine cell biology, engineering materials and medicine, and achieve the repair of damaged tissues and the regeneration of failed organs through the integration of multidisciplinary technologies. This not only improves the cure rate, but also simplifies the treatment process and reduces medical costs. Suitable cell sources (seed cells), effective cell modification (growth factors) and appropriate scaffold support are the three foundations of tissue engineering. As the scaffold is gradually degraded and absorbed in the body, the implanted cells continue to proliferate and secrete extracellular matrix in the body under the action of cytokines and the microenvironment, and eventually form the tissues or organs required by the body, thereby achieving the purpose of repairing damaged tissues and reconstructing organ functions. In the past few decades, tissue engineering has developed rapidly. Together with regenerative medicine, it has injected new impetus into the development of clinical medicine and pointed out a new direction for tracheal replacement therapy.

[0005] The emergence of tissue-engineered tracheas has brought new hope for long-segment tracheal repair. However, because the trachea lacks distinct arterial and venous blood supply, its blood supply primarily derives from the capillary network surrounding the thyroid gland and esophagus. Therefore, maintaining an adequate blood supply after transplantation has been a major limitation to its development. Currently reported tissue-engineered tracheas are mostly simple, dense cartilage tubular structures. These tubular structures are formed in vitro and then placed subcutaneously or within a myocutaneous flap, where they are prevascularized by the blood supply of the surrounding tissue. However, the majority of blood vessels generated by this method are distributed along the outer wall of the cartilage tubules, largely unable to penetrate the dense cartilage tissue and reach the tracheal epithelial-lined lining. When the graft is short, blood vessels can extend through both ends of the graft to cover the tracheal lining, providing nutritional and metabolic support to the luminal lining. However, for long tracheal segments, the inaccessible epithelium is unable to maintain adequate nutrition and metabolism, resulting in a loss of tracheal cleansing and protective functions, leading to infection, inflammation, collapse, and even life-threatening conditions.

[0006] Traditionally, the immune system's primary role has been considered as a component of the body's defense system against the external environment and pathogens. However, it is becoming increasingly clear that an essential component of tissue repair is the effective regulation of the immune system. Tissue regeneration after severe injury requires a dynamic flux in the immune microenvironment, shifting from a pro-inflammatory to an anti-inflammatory microenvironment to mitigate damage, clear dead or injured tissue, exert protective functions, and increase angiogenesis and nutrient supply to aid tissue regeneration. Therefore, both pro-inflammatory and anti-inflammatory microenvironments are crucial to the tissue repair and regeneration processes. From initiating inflammation to eliminating pathogens, macrophages play a key role in mediating tissue repair and regeneration. The macrophage lineage is known to possess a high degree of plasticity. M1 macrophages are a pro-inflammatory subtype that overexpress inflammatory genes, while M2-polarized macrophages are beneficial. The transition from the M1 to M2 phenotype triggers the progression from the inflammatory phase to the tissue repair phase.

[0007] Quercetin is a small-molecule flavonoid polyphenol found in natural plants. Due to its anti-tumor, anti-inflammatory, antioxidant, anti-platelet aggregation, and free radical scavenging properties, there are currently few reports on its application in tissue-engineered trachea. Summary of the Invention

[0008] In light of this, the present invention provides a tissue-engineered biomimetic tracheal stent with microenvironmental regulation capabilities and a method for preparing the same. The tissue-engineered biomimetic tracheal stent provided by the present invention is structurally and functionally similar to natural trachea. During prevascularization, it generates a transmural vascular structure similar to that of a physiological trachea and can regulate the immune microenvironment.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] A tissue engineering bionic tracheal stent with microenvironment regulation function, comprising cartilage rings and quercetin rings arranged alternately;

[0011] The cartilage ring includes a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and chondrocytes loaded in the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold;

[0012] The quercetin ring comprises a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and quercetin loaded in the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold;

[0013] The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold comprises a three-dimensional silk fibroin sponge and a silk fibroin non-woven fabric scaffold located inside the three-dimensional silk fibroin sponge.

[0014] Preferably, the mass fraction of quercetin in the quercetin ring is 3-5%.

[0015] Preferably, the outer diameter of the tissue engineering bionic tracheal stent with microenvironment regulation function is 0.5-1 cm, and the inner diameter is 0.3-0.6 cm; the heights of the cartilage ring and the quercetin ring are independently 0.1-0.2 cm.

[0016] Preferably, the number of cartilage rings in the tissue engineering bionic tracheal scaffold is greater than or equal to 1, and the number of quercetin rings is greater than or equal to 2.

[0017] The present invention also provides a method for preparing the tissue engineering biomimetic tracheal stent with microenvironment regulation function described in the above scheme, comprising the following steps:

[0018] After absorbing the chondrocyte suspension onto the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, the scaffold is incubated and cultured in sequence to obtain a cartilage ring; the culture is carried out in a chondrogenic differentiation promoting culture medium;

[0019] The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold is immersed in a quercetin solution to obtain a quercetin ring;

[0020] The cartilage rings and the quercetin rings are alternately stacked to obtain a tissue engineering biomimetic tracheal stent with microenvironment regulation function.

[0021] Preferably, the preparation method of the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold comprises the following steps:

[0022] The silk fibroin fibers are combed, pinhole fixed and degummed in sequence to obtain a silk fibroin non-woven fabric scaffold;

[0023] Degumming the silk fibroin fibers and dissolving them in a lithium bromide solution to obtain a silk fibroin fiber solution, dialyzing the silk fibroin fiber solution and then freeze-drying it to obtain a silk fibroin porous foam;

[0024] dissolving the silk fibroin porous foam in water, adding the obtained silk fibroin porous foam solution to the silk fibroin non-woven fabric scaffold, degassing and freeze-drying the solution to obtain a scaffold precursor;

[0025] The scaffold precursor is immersed in isoflurane for cross-linking to obtain the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold.

[0026] Preferably, the concentration of the silk fibroin porous foam solution is 1 to 5 wt %; the cross-linking time is 8 to 12 hours, and the temperature is room temperature.

[0027] Preferably, the incubation conditions include: temperature of 37°C, CO2 concentration of 5wt%, saturated humidity, and incubation time of 4 to 5h;

[0028] The culture conditions include: temperature of 37° C., CO 2 concentration of 5 wt %, saturated humidity, and culture time of 14 to 28 days.

[0029] Preferably, the concentration of the quercetin solution is 3.0 to 5.3 mg / mL, and the soaking time in the quercetin solution is 18 to 24 hours.

[0030] Preferably, after the alternating stacking, the method further comprises performing transmural vascularization on the obtained tissue engineering biomimetic tracheal stent with microenvironment regulation function.

[0031] The present invention provides a tissue-engineered biomimetic tracheal stent with microenvironmental regulation function, comprising alternating cartilage rings and quercetin rings; the cartilage rings comprise a non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold and chondrocytes loaded within the non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold; the quercetin rings comprise a non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold and quercetin loaded within the non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold; and the non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold comprises a three-dimensional silk fibroin sponge and a silk fibroin non-woven scaffold positioned within the three-dimensional silk fibroin sponge. Silk fibroin is a unique natural protein extracted from silk, exhibiting excellent biocompatibility, biodegradability, bioabsorbability, low immunogenicity, and adjustable mechanical properties. The present invention utilizes a non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold as a basis, loads chondrocytes or quercetin thereon, and then alternately stacks the resulting cartilage rings and quercetin rings to form the tissue-engineered biomimetic tracheal stent of the present invention. The tissue engineering biomimetic tracheal stent provided by the present invention can exist stably in the body and is similar to natural tissue in structure and function. During the pre-vascularization process, it can generate a transmural vascular structure similar to the physiological trachea, thereby providing nutritional and metabolic support for the inner layer structure of the graft.

[0032] The tissue-engineered biomimetic trachea stent provided by the present invention is loaded with quercetin, which can regulate the immune microenvironment by promoting the polarization of macrophages toward an anti-inflammatory phenotype, thereby broadening the application scope of tissue-engineered trachea and promoting its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A schematic diagram of the construction method of the tissue engineering biomimetic tracheal stent with microenvironment regulation function provided by the present invention;

[0034] Figure 2 Actual images of non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and quercetin fibroin scaffold (not punched);

[0035] Figure 3 This is a real photo of the cartilage ring;

[0036] Figure 4 The histological staining results in Example 3;

[0037] Figure 5 The flow cytometry results of the in vitro study of the effect of quercetin on macrophages in Example 4 are as follows;

[0038] Figure 6 The quantitative results of toluidine blue staining in Example 5 are shown below:

[0039] Figure 7 The results of the PCR cartilage-related indicators (ColII, Acan) in Example 5 are shown;

[0040] Figure 8 The results of PCR cartilage-related markers (Sox9, ColX) in Example 5 are shown;

[0041] Figure 9 The test results of the number of tube nodes (left) and tube length (right) in Example 6 are shown;

[0042] Figure 10 The test results of the scratch migration experiment in Example 6;

[0043] Figure 11 This is the cartilage growth of the "sandwich" bionic trachea model in Example 7 after 4 weeks of implantation in rabbits;

[0044] Figure 12 Transplantation process of tissue-engineered trachea with long ring-to-ring structures. DETAILED DESCRIPTION

[0045] The present invention provides a tissue engineering bionic tracheal stent with microenvironment regulation function, comprising cartilage rings and quercetin rings arranged alternately;

[0046] The cartilage ring includes a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and chondrocytes loaded in the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold;

[0047] The quercetin ring comprises a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and quercetin loaded in the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold;

[0048] The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold comprises a three-dimensional silk fibroin sponge and a silk fibroin non-woven fabric scaffold located inside the three-dimensional silk fibroin sponge.

[0049] In the present invention, the mass fraction of quercetin in the quercetin ring is preferably 3-5%.

[0050] In the present invention, the outer diameter of the tissue engineering bionic tracheal stent with microenvironment regulation function is preferably 0.5 to 1 cm, the inner diameter is preferably 0.3 to 0.6 cm, and the wall thickness is preferably 0.2 to 0.4 cm; the heights of the cartilage ring and the quercetin ring are independently preferably 0.1 to 0.2 cm; the present invention has no special requirements for the length of the tissue engineering bionic tracheal stent, and it can be set according to actual needs.

[0051] In the present invention, the number of cartilage rings in the tissue engineering bionic trachea scaffold is preferably greater than or equal to 1, preferably greater than or equal to 3, and the number of quercetin rings is preferably greater than or equal to 2, preferably greater than or equal to 4; in a specific embodiment of the present invention, the number of cartilage rings and quercetin rings can be set according to the actual requirements of the length of the bionic trachea, such as using 2 quercetin rings and 1 cartilage ring, and arranging them in the order of quercetin ring-cartilage ring-quercetin ring to form a "sandwich" structure; or using 4 quercetin rings and 3 cartilage rings, and arranging them in the order of quercetin ring-cartilage ring-quercetin ring-cartilage ring...quercetin ring to form a long segment of tissue engineering bionic trachea. Figure 1 Schematic diagram of the construction method of the tissue engineering bionic tracheal stent with microenvironment regulation function provided by the present invention.

[0052] The present invention also provides a method for preparing the tissue engineering biomimetic tracheal stent with microenvironment regulation function described in the above scheme, comprising the following steps:

[0053] After absorbing the chondrocyte suspension onto the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, the scaffold is incubated and cultured in sequence to obtain a cartilage ring; the culture is carried out in a chondrogenic differentiation promoting culture medium;

[0054] The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold is immersed in a quercetin solution to obtain a quercetin ring;

[0055] The cartilage rings and the quercetin rings are alternately stacked to obtain a tissue engineering biomimetic tracheal stent with microenvironment regulation function.

[0056] First, the preparation method of the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold is described.

[0057] In the present invention, the preparation method of the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold preferably comprises the following steps:

[0058] The silk fibroin fibers are combed, pinhole fixed and degummed in sequence to obtain a silk fibroin non-woven fabric scaffold;

[0059] Degumming the silk fibroin fibers and dissolving them in a lithium bromide solution to obtain a silk fibroin fiber solution, dialyzing the silk fibroin fiber solution and then freeze-drying it to obtain a silk fibroin porous foam;

[0060] dissolving the silk fibroin porous foam in water, adding the obtained silk fibroin porous foam solution to the silk fibroin non-woven fabric scaffold, degassing and freeze-drying the solution to obtain a scaffold precursor;

[0061] The scaffold precursor is immersed in isoflurane for cross-linking to obtain the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold.

[0062] The present invention sequentially combs, pinhole fixes, and degummes silk fibroin fibers to obtain a silk fibroin non-woven fabric scaffold. In the present invention, the length of the silk fibroin fibers is preferably 55 mm. The combing is preferably performed using a carding machine, which cuts and combs the silk fibroin fibers and then beats them in layers to form a fiber mesh. The present invention has no special requirements for the pinhole fixation method; methods familiar to those skilled in the art can be used. Vacuum fixation generates fiber entanglements on the surface and inside of the fiber mesh, turning the fluffy fiber mesh into a reinforced non-woven fabric. The degumming solution is preferably a sodium carbonate solution, the concentration of which is preferably 0.01 to 0.03 M, more preferably 0.02 M. The degumming is preferably performed under boiling conditions, and the boiling time is preferably 1 to 3 hours, more preferably 2 hours. After degumming, the scaffold is preferably washed with distilled water and then dried. In a specific embodiment of the present invention, the dried silk fibroin non-woven fabric scaffold is preferably cut into cylindrical shapes with a diameter of 0.5 to 1 cm for later use.

[0063] The present invention degummes silk fibroin fibers and dissolves them in a lithium bromide solution to obtain a silk fibroin fiber solution, dialyzes the silk fibroin fiber solution, and then freeze-dries it to obtain a silk fibroin porous foam. In the present invention, the degumming method and conditions are the same as those in the above-mentioned scheme and will not be repeated here; the concentration of the lithium bromide solution is preferably 9-10M, more preferably 9.3M; the concentration of the silk fibroin fibers in the silk fibroin fiber solution is preferably 1-5w / v%, more preferably 1w / v%, 3w / v% and 5w / v%; the dialysis solvent is preferably distilled water, the dialysis tubing is preferably a benzoylated dialysis tubing, the dialysis time is preferably 48h, the distilled water is preferably replaced every 6h during the dialysis process, the molecular weight cut-off of the dialysis bag is preferably 14-42kDa; the freeze-drying temperature is preferably -60°C.

[0064] After obtaining the silk fibroin porous foam, the present invention dissolves the silk fibroin porous foam in water, adds the obtained silk fibroin porous foam solution to the silk fibroin non-woven fabric scaffold, degassing and freeze-drying to obtain a scaffold precursor. In the present invention, the water is preferably distilled water, and the concentration of the silk fibroin porous foam solution is preferably 1 to 5 wt%, more preferably 4 wt%; the present invention preferably places the silk fibroin non-woven fabric scaffold in a 24-well plate first, and then drops the silk fibroin porous foam solution into the 24-well plate, and the amount of the silk fibroin porous foam solution is based on the amount of the solution added to a height of 0.15 to 0.2 cm; the degassing is preferably carried out under static conditions, the degassing temperature is preferably 4°C, and the degassing time is preferably 6 hours; after degassing, the present invention preferably allows the system to stand for 12 to 24 hours, and then freeze-drying is performed, the freeze-drying temperature is preferably -60°C, and the time is preferably 48 hours.

[0065] After obtaining the scaffold precursor, the present invention immerses the scaffold precursor in isoflurane for cross-linking, thereby obtaining the non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold. In the present invention, the cross-linking time is preferably 8 to 12 hours, and the temperature is preferably room temperature. The present invention does not have any particular requirements for the amount of isoflurane used, as long as it can immerse the scaffold precursor. After cross-linking is completed, the resulting non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold is preferably washed and dried. The present invention enhances the strength of the scaffold through cross-linking.

[0066] After obtaining the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, the present invention preferably uses a puncher to punch holes in the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold to form a ring structure.

[0067] After obtaining the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, the present invention adsorbs the chondrocyte suspension onto the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, and then incubates and cultures the scaffold to obtain a cartilage ring. In the present invention, the concentration of the chondrocyte suspension is preferably 1×10 8 / mL; the present invention has no special requirements for the source of the chondrocyte suspension, and a method familiar to those skilled in the art can be used. In a specific embodiment of the present invention, the chondrocyte suspension is extracted and amplified from rabbit ear cartilage; the present invention preferably extrude the chondrocyte suspension into a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold so that the scaffold can fully absorb it.

[0068] In a specific embodiment of the present invention, before adsorbing the chondrocyte suspension, the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold is preferably soaked in alcohol, irradiated with ultraviolet light, washed with PBS buffer and rinsed with serum-containing DMEM culture medium in sequence. After rinsing with serum-containing DMEM culture medium, the culture medium attached to the scaffold is preferably sucked dry using an aspirator; the volume fraction of the alcohol is preferably 75%, the alcohol soaking time is preferably 30 minutes, and the ultraviolet tube irradiation time is preferably 30 minutes.

[0069] In the present invention, the incubation conditions preferably include: temperature of 37°C, CO2 concentration of 5wt%, saturated humidity, and incubation time of 4 to 5 hours; the present invention allows chondrocytes to stably attach to the scaffold through incubation; and the culture is carried out in a cartilage differentiation-promoting culture medium.

[0070] In the present invention, the culture conditions preferably include: temperature of 37°C, CO2 concentration of 5wt%, saturated humidity, and culture time of 14 to 28 days; the medium is preferably changed every other day during the culture process; the present invention allows chondrocytes to secrete sufficient matrix through culture.

[0071] The present invention involves soaking a non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold in a quercetin solution to produce a quercetin ring. In the present invention, the concentration of the quercetin solution is preferably 3 to 5.3 mg / mL, and the soaking time is preferably 18 to 24 hours. After soaking, the scaffold is washed and dried.

[0072] After obtaining the cartilage rings and the quercetin rings, the present invention alternately stacks the cartilage rings and the quercetin rings to obtain a tissue-engineered biomimetic tracheal stent with microenvironment regulation function. In the present invention, the cartilage rings and the quercetin rings are simply alternately placed without any other processing.

[0073] In the present invention, after the alternating stacking, the obtained tissue engineering biomimetic tracheal stent is preferably subjected to transmural vascularization; the transmural vascularization is preferably performed subcutaneously in a living body.

[0074] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0075] Example 1

[0076] Silk fibroin fibers were cut into 55 mm lengths, then cut and combed using a carding machine and layered and beaten to produce a silk fiber web. The collected fiber web was then pinned through a needle to create fiber entanglements on the surface and within the mat, transforming the fluffy mat into a reinforced nonwoven fabric. The silk fibroin nonwoven fabric was then boiled in a 0.02 M sodium carbonate solution for 2 hours to thoroughly degas, then rinsed three times with distilled water and dried in a 60°C oven to produce a silk fibroin nonwoven fabric scaffold. Finally, the silk fibroin nonwoven fabric scaffold was cut into 8 mm diameter cylinders for further use.

[0077] The silk fibroin fibers were boiled in a 0.02M sodium carbonate solution for 2 hours and then rinsed three times with distilled water. The degummed silk fibroin fibers were dissolved in a 9.3M lithium bromide solution to form a transparent solution. The solution was then dialyzed in a benzoylated dialysis tubing with distilled water for 48 hours (the distilled water was changed every 6 hours). The silk fibroin fiber solution was freeze-dried to obtain a porous silk fibroin foam.

[0078] A 4wt% aqueous solution of porous silk fibroin foam was prepared by dissolving it in distilled water. A non-woven silk fibroin scaffold was placed in a 24-well plate. The porous silk fibroin foam solution was then dripped into the plate to a liquid level of 0.15 cm and incubated at 4°C for 6 hours to release the bubbles. The mixture was then freeze-dried at -60°C for 2 days to obtain a scaffold precursor. This scaffold precursor was then cross-linked in isoflurane for 12 hours to produce a non-woven fabric-reinforced three-dimensional silk fibroin sponge scaffold.

[0079] The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold was immersed in a quercetin solution with a concentration of 5.3 mg / mL for 24 hours to obtain a quercetin scaffold.

[0080] Figure 2 This is a physical picture (unpunched) of the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and quercetin scaffold prepared in the present invention, wherein the left side is the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, which is white, and the right side is the quercetin scaffold, which is yellow.

[0081] Example 2

[0082] After the rabbits were anesthetized or killed, their ears were removed, their skin peeled off, and the fibrous membrane surrounding the cartilage tissue was removed. The ears were then minced into 1 mm × 1 mm pieces. 15% type II collagenase was added and digested at 37°C for 24 h. During the digestion, the cells were shaken several times and filtered through a 0.22 μm sterile filter. The cells were centrifuged, washed, resuspended, and counted. The cells were counted at 1.0 × 10 4 / cm 2 Cells were seeded at a density of 10 cm 2 Culture dishes were cultured at 37°C, 5% CO2, and saturated humidity. The medium was changed for the first time on the third day and every other day thereafter. When the cells were almost confluent in the culture dish, about 1.5 mL of trypsin digestion solution was added. When the cell cytoplasm shrank and the cell morphology became round under the microscope, DMEM culture medium (containing serum) was added to terminate the digestion. The cells were collected, resuspended after centrifugation, and then diluted to 1.0 × 10 4 / cm 2 The cells were seeded at a new 10 cm 2 Repeat the above steps to expand the cells to the second generation. After 3-5 days of culture, collect the cells and count the cells after the cells have covered the culture dish. Adjust the concentration of chondrocyte suspension to 1×10 8 / ml.

[0083] The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold prepared in Example 1 was punched, and the resulting scaffold ring was directly immersed in 75% alcohol for 30 minutes, irradiated with ultraviolet light for 30 minutes, rinsed repeatedly with PBS 2-3 times, and rinsed once with serum-containing DMEM culture medium (containing serum), and then the culture medium attached to the scaffold was aspirated with an aspirator;

[0084] The chondrocyte suspension was squeezed onto a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold ring to allow the scaffold to fully absorb it. The scaffold was incubated at 37°C, 5% CO2, and saturated humidity for 4 to 5 hours. After the chondrocytes and scaffold were stably attached, the cartilage differentiation-promoting culture medium was slowly added and the culture was continued at 37°C, 5% CO2, and saturated humidity. The culture medium was changed every other day. The culture was carried out in the culture medium for 2 weeks to obtain a cartilage ring.

[0085] Figure 3 This is a physical picture of the cartilage ring obtained in this example.

[0086] Example 3

[0087] Other conditions were the same as those in Example 1, except that the concentration of the silk fibroin porous foam aqueous solution was changed to 1%, 3%, and 5%, to prepare three types of non-woven fabric reinforced three-dimensional silk fibroin sponge scaffolds;

[0088] According to the method in Example 2, chondrocytes were inoculated into three non-woven fabric reinforced three-dimensional silk fibroin sponge scaffolds, cultured in vitro at 37°C and 5% CO2 for 28 days, and the effect of silk fibroin concentration on cartilage growth was observed by staining.

[0089] The research results show that when the concentration of the silk fibroin porous foam aqueous solution is 3%, it has the best effect on promoting cartilage growth, that is, 3% is the optimal concentration. Figure 4 The histological staining results of the experimental group with a 3% concentration of the silk fibroin porous foam aqueous solution. Figure 4 The staining results of HE, SO, AB, and Col2 show mature cartilage pits, the secretion of glycosaminoglycans (cartilage matrix), and type II collagen, which is characteristic of cartilage.

[0090] Example 4

[0091] In vitro verification of the regulatory effect of quercetin on the immune microenvironment: the experiment was divided into 5 groups. The first group used whole culture to treat macrophages, the second group used whole culture with LPS added (where the concentration of LPS was 1 mg / mL) to treat macrophages, the third group used whole culture with interleukin-4 and interleukin-13 added (where the concentration of interleukin-4 was 10 μM, and the concentration of interleukin-13 was 10 μM) to treat macrophages, the fourth group used whole culture with quercetin added (where the concentration of quercetin was 10 μM) to treat macrophages; the fifth group used whole culture with LPS and quercetin added (where the concentration of LPS was 1 μg / mL, and the concentration of quercetin was 20 μM) to treat macrophages.

[0092] The effects of five experimental conditions on macrophage phenotypes were analyzed by flow cytometry. Figure 5 As shown, Figure 5The figure on the left shows the proportion of M1 macrophages after treatment with different experimental conditions; the figure on the right shows the proportion of M2 macrophages after treatment with different experimental conditions. Figure 5 The results in

[15] showed that quercetin could promote the polarization of macrophages toward M2 (anti-inflammatory) phenotype when cultured in vitro.

[0093] Example 5

[0094] The effects and influences of different immune microenvironments formed by culture conditions on chondrocytes were explored as follows: using a Transwell model, macrophages were inoculated in the upper chamber and chondrocytes were inoculated in the lower chamber. Subsequently, different culture media were added to the Transwell wells (the culture media used were the same as the five culture media in Example 4) to form a macrophage-chondrocyte co-culture system. After 7 days, toluidine blue staining was performed for quantification and PCR for cartilage-related indicators (ColII, Sox9, Acan, ColX) was performed.

[0095] Figure 6 The quantitative results of toluidine blue staining are shown. Figure 6 The results in [ 14 ] indicate that the local immune microenvironment induced by quercetin can promote cartilage development.

[0096] Figure 7 The test results of PCR cartilage-related indicators (ColII, Acan) are shown. Figure 8 These are the test results of PCR cartilage-related indicators (Sox9, ColX). Figures 7-8 The results showed that the expression of cartilage-related genes was upregulated, while the expression of cartilage fibrosis and hypertrophy-related genes was downregulated, proving that the immune microenvironment formed under quercetin culture conditions promoted the secretion of cartilage matrix and inhibited chondrocyte hypertrophy and fibrosis.

[0097] Example 6

[0098] Test the effects and impacts of different immune microenvironments on endothelial cells, as follows:

[0099] Endothelial cell tube formation assay: Using a Transwell model, macrophages were seeded at a density of 200,000 / well in the upper chamber. Simultaneously, 1 ml of matrigel was plated in the lower chamber of the Transwell model. One hour later, endothelial cells were seeded in the matrigel at a density of 60,000-80,000 / well. Subsequently, different culture media (the same five culture media used in Example 4) were added to the Transwell wells. Six hours later, the angiogenesis of the endothelial cells was observed, and the number of tube nodes and tube length were quantified.

[0100] Figure 9 The test results of the number of tube nodes (left) and tube length (right). Figure 9The results in this study showed that the immune microenvironment under quercetin culture conditions promotes endothelial cell angiogenesis.

[0101] Scratch migration assay: Transwell model, macrophages were seeded at a density of 200,000 / well in the upper chamber, and endothelial cells were seeded at a density of 300,000 / well in the lower chamber. A 200 μL pipette tip was used to scratch a uniform vertical cell-free space. Subsequently, different culture media (the culture media used were the same as the five culture media in Example 4) were added to the Transwell wells. Endothelial cell migration was observed after 6, 12, and 24 hours, and the residual scratch area was quantified.

[0102] The test results of the scratch migration experiment are as follows Figure 10 shown. Figure 10 The results in the study showed that the local immune microenvironment induced by quercetin can promote endothelial cell tube formation and migration, thereby promoting angiogenesis.

[0103] Example 7

[0104] A quercetin silk ring (prepared and punched according to the method of Example 1) was placed above and below the cartilage ring (prepared according to the method of Example 2) to obtain a "sandwich" biomimetic tracheal model. The obtained "sandwich" biomimetic tracheal model was transplanted into rabbits for 2 and 4 weeks. Samples were taken to analyze cartilage growth, and the successful appearance of transmural blood vessels and the degree of inflammatory infiltration in the surrounding tissues were observed. Figure 11 The cartilage growth of the "sandwich" biomimetic trachea model was observed in rabbits 4 weeks after implantation. The results showed that the cartilage structure was well maintained after 4 weeks in vivo, and the quercetin ring promoted the infiltration of surrounding tissues for vascularization, and the retention of the ring-ring structure could be seen.

[0105] Example 8

[0106] Three cartilage rings (prepared according to the method of Example 2) and four quercetin rings (prepared and punched according to the method of Example 1) were alternately stacked to form a long segment of "ring-ring" structured tissue-engineered trachea. The long segment of "ring-ring" structured tissue-engineered trachea was embedded in the rabbit body for prevascularization for 2 weeks. After prevascularization was completed, the tracheal segment was replaced, thereby completing the in vivo feasibility verification of the tissue-engineered trachea. Figure 12 This is the process of long-segment "ring-ring" structure tissue-engineered trachea transplantation. The picture on the left shows the gross appearance of the long-segment "ring-ring" structure tissue-engineered trachea before transplantation. The picture in the middle shows the end-to-end anastomosis of the long-segment "ring-ring" structure tissue-engineered trachea in vivo during the surgery; the picture on the right shows the gross picture of the long-segment tissue-engineered trachea sampling 6 weeks after tracheal transplantation. Figure 12 The results showed that the tissue-engineered trachea was successfully transplanted, the experimental animals survived for more than one month, and the trachea had good biocompatibility and maintained relatively excellent mechanical properties.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A tissue engineering bionic tracheal stent with microenvironment regulation function, characterized in that: including alternating rings of cartilage and quercetin; The cartilage ring includes a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and chondrocytes loaded in the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold; The quercetin ring comprises a non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold and quercetin loaded in the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold; The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold comprises a three-dimensional silk fibroin sponge and a silk fibroin non-woven fabric scaffold located inside the three-dimensional silk fibroin sponge; The preparation method of the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold comprises the following steps: The silk fibroin fibers are combed, pinhole fixed and degummed in sequence to obtain a silk fibroin non-woven fabric scaffold; Degumming the silk fibroin fibers and dissolving them in a lithium bromide solution to obtain a silk fibroin fiber solution, dialyzing the silk fibroin fiber solution and then freeze-drying it to obtain a silk fibroin porous foam; dissolving the silk fibroin porous foam in water, adding the obtained silk fibroin porous foam solution to the silk fibroin non-woven fabric scaffold, degassing and freeze-drying the solution to obtain a scaffold precursor; The scaffold precursor is immersed in isoflurane for cross-linking to obtain the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold.

2. The tissue engineering biomimetic tracheal stent with microenvironment regulation function according to claim 1, characterized in that: The mass fraction of quercetin in the quercetin ring is 3-5%.

3. The tissue engineering biomimetic tracheal stent with microenvironment regulation function according to claim 1, characterized in that: The outer diameter of the tissue engineering bionic tracheal stent with microenvironment regulation function is 0.5-1 cm, and the inner diameter is 0.3-0.6 cm; the heights of the cartilage ring and the quercetin ring are independently 0.1-0.2 cm.

4. The tissue engineering biomimetic tracheal stent with microenvironment regulation function according to claim 1, characterized in that: The number of cartilage rings in the tissue engineering bionic tracheal scaffold is greater than or equal to 1, and the number of quercetin rings is greater than or equal to 2.

5. The tissue engineering biomimetic tracheal stent with microenvironment regulation function according to claim 1, characterized in that: The concentration of the silk fibroin porous foam solution is 1-5 wt %; the cross-linking time is 8-12 hours, and the temperature is room temperature.

6. The method for preparing the tissue engineering biomimetic tracheal stent with microenvironment regulation function according to any one of claims 1 to 5, characterized in that: The following steps are involved: After absorbing the chondrocyte suspension onto the non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold, the scaffold is incubated and cultured in sequence to obtain a cartilage ring; the culture is carried out in a chondrogenic differentiation promoting culture medium; The non-woven fabric reinforced three-dimensional silk fibroin sponge scaffold is immersed in a quercetin solution to obtain a quercetin ring; The cartilage rings and the quercetin rings are alternately stacked to obtain a tissue engineering biomimetic tracheal stent with microenvironment regulation function.

7. The preparation method according to claim 6, characterized in that The incubation conditions include: temperature of 37°C, CO2 concentration of 5wt%, saturated humidity, and incubation time of 4 to 5h; The culture conditions include: temperature of 37° C., CO 2 concentration of 5 wt %, saturated humidity, and culture time of 14 to 28 days.

8. The preparation method according to claim 6, characterized in that The concentration of the quercetin solution is 3.0 to 5.3 mg / mL, and the soaking time in the quercetin solution is 18 to 24 hours.

9. The preparation method according to claim 6, characterized in that After the alternating stacking, the method further includes performing transmural vascularization on the obtained tissue engineering biomimetic tracheal stent with microenvironment regulation function.

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