CN113057761A - Bionic trachea and preparation method thereof - Google Patents
Bionic trachea and preparation method thereof Download PDFInfo
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- CN113057761A CN113057761A CN202110262268.2A CN202110262268A CN113057761A CN 113057761 A CN113057761 A CN 113057761A CN 202110262268 A CN202110262268 A CN 202110262268A CN 113057761 A CN113057761 A CN 113057761A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2002/046—Tracheae
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
- A61F2240/001—Designing or manufacturing processes
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- Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Pulmonology (AREA)
- Cardiology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Materials For Medical Uses (AREA)
- Prostheses (AREA)
Abstract
The invention relates to a preparation method of a bionic trachea, which comprises the following steps: s1, constructing a bionic tracheal stent by a fusion electrostatic direct writing technology; s2, planting chondrocytes to the bionic tracheal stent; s3, inducing the chondrocytes to differentiate to form intact cartilage tissue; s4, transmural vascularization to obtain the bionic trachea containing the cricoid cartilage and the transmural blood vessels. Aiming at the defects of the tracheal repair technology in the prior clinical application, the invention prepares a bionic trachea which is constructed based on an electrostatic direct-writing bracket and contains a cricoid cartilage and a transmural blood vessel, wherein the bionic trachea can stably exist in a living body and is similar to a natural tissue in structure and function; through the innovative design of the stent implantation mode, the graft generates a transmural vascular structure similar to a physiological trachea in the prevascularization process, and then provides nutrition and metabolic support for the inner layer structure of the graft.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a bionic trachea and a preparation method thereof.
Background
Tumors, wounds, and infections can all cause tracheal injury and stenosis, which can seriously endanger the life of the patient. For injured or stenosed airways, surgical resection followed by end-to-end anastomosis is one of the most effective treatment options. However, when the length of the tracheal lesion is too large (exceeding the full-length adult trachea 1/2 or the full-length child 1/3), a large resection range will result in increased stoma tension with serious postoperative complications. Therefore, patients suffering from long-segment tracheal lesions often need to implant a substitute with good biocompatibility and mechanical properties to repair the missing part of the trachea.
Currently, clinical treatment for long-segment tracheal lesions is limited, and common tracheal replacement methods include: artificial prosthesis trachea replacement, allogenic trachea transplantation and autologous tissue reconstruction trachea. The artificial prosthesis trachea is generally prepared from metal or organic materials, but because the artificial prosthesis trachea is poor in histocompatibility, lacks blood supply and epithelial tissue coverage, peripheral granulation tissue hyperplasia is easily caused, a lumen is blocked, and infection, stenosis, even collapse and rupture of an airway are caused; allogenic tracheal transplantation often faces the problems of donor shortage and long-term receiving immunosuppressive treatment after operation, and meanwhile, airway operation and blood circulation reconstruction are difficult, so that graft necrosis, infection and even life threatening are easily caused, and the clinical application of the allogenic tracheal transplantation is limited; the reconstruction of the tracheal-like tissue from autologous tissue (such as a cervical or forearm muscle flap) does not completely mimic the natural trachea in structure and function, and at the same time, introduces additional surgical trauma during the harvesting of the tissue. Therefore, the three tracheal substitutes cannot meet the actual clinical requirement.
Recently, the advent of tissue engineered trachea has brought new hopes for long-segment tracheal repair. However, since the trachea does not have separate supply arteries and drainage veins, the blood supply is mainly derived from the capillary network of the thyroid and periesophageal tissues. Therefore, how to maintain an adequate blood supply after the transplantation into the body becomes a major factor limiting the development thereof. Most of the tissue engineering tracheas reported at present are formed in vitro by a simple compact cartilage tubular structure, and then are placed under the skin or muscle flap of an organism to complete prevascularization by means of tissue blood transportation in the region. However, the blood vessels generated by the method are mostly distributed on the outer side wall of the cartilaginous tube and can not penetrate through compact cartilaginous tissues to reach the inner wall of the trachea covered by the epithelial tissues of the trachea. When the graft is short, the blood vessel can cover the inner wall of the trachea by extending the two ends of the transplanted trachea so as to provide nutrition metabolism support for the inner wall structure of the lumen. However, for a long section of trachea, nutrition and metabolism of part of epithelium which can not be reached by blood vessels can not be ensured, so that the trachea loses cleaning and defense functions, further infection, inflammation and collapse are caused, and even the life is threatened in severe cases.
With the development of Melt electro-static direct writing (MEW) technology, the advantages of both the electro-spinning technology and the 3D printing technology are integrated, so that the directional construction of the personalized tissue engineering scaffold through the superfine fiber scaffold becomes possible. The scale of the superfine fiber produced by MEW is similar to that of the cell, and the cell is easier to adhere to the fiber silk, thereby better growing and keeping higher proliferation rate. On the other hand, with the support of the 3D printing technology, the superfine fiber filaments can directionally construct cell scaffolds with different forms, and can be individually designed according to parameters such as the length, thickness and the like of a patient's missing trachea. Compared with the traditional support, the MEW technology can provide a more favorable microenvironment for cells while accurately controlling the geometric shape and the internal structure of the support, and has wide application prospect in the field of tissue engineering.
In summary, the patent forms a bionic trachea which can stably exist in a living body and is similar to natural tissues in structure and function and contains cricoid cartilage and transmural blood vessels based on an electrostatic direct writing bracket through the technologies of primary cartilage extraction, chondrocyte matrix secretion promotion and transmural angiogenesis.
Disclosure of Invention
The invention aims to provide a bionic trachea and a preparation method thereof aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the preparation method of the bionic trachea is provided, and comprises the following steps:
s1, constructing a bionic tracheal stent by a fusion electrostatic direct writing technology;
s2, planting chondrocytes to the bionic tracheal stent;
s3, inducing the chondrocytes to differentiate to form intact cartilage tissue;
s4, transmural vascularization to obtain the bionic trachea containing the cricoid cartilage and the transmural blood vessels.
Preferably, the bionic tracheal stent is prepared from polycaprolactone.
Preferably, the bionic tracheal stent is internally provided with a triangular stable supporting structure.
Preferably, the step S2 further includes:
s2-1, soaking the bionic tracheal stent in 75% alcohol for at least 30 minutes, and irradiating by using ultraviolet light for at least 30 minutes;
s2-2, washing the bionic tracheal stent for 2-3 times by PBS, and then washing for 1-2 times by DMEM culture solution containing serum;
s2-3, after the culture solution attached to the bionic tracheal stent is sucked to be dry by using an aspirator, sleeving the bionic tracheal stent outside the sterilized glass tube, and uniformly binding sutures outside the bionic tracheal stent;
s2-4, collecting chondrocytes cultured for 3-5 days in the 2 nd generation, and adjusting the cell suspension concentration of the chondrocytes to 1 x 108/mL；
S2-5, uniformly adding the cell suspension of the chondrocytes among the stitches of the bionic tracheal scaffold to form an intact cartilage ring structure.
Preferably, the step S3 further includes:
s3-1, treating the chondrocytes at 37 ℃ and 5% CO2Incubating for 4-5 hours under the condition of saturated humidity, and slowly adding a culture solution for promoting cartilage differentiation after the culture solution is stably attached to the bionic tracheal stent;
s3-2, taking out the suture, adding GelMA between cartilage ring structures, and carrying out photo-crosslinking;
s3-3, 5% CO at 37 ℃2And under the conditions of saturated humidity and liquid change every other day, the culture is continued for 2 weeks to secrete sufficient substrates.
And the bionic trachea prepared by the preparation method comprises the following steps: the bionic tracheal stent comprises a bionic tracheal stent and a cartilage ring and a blood vessel ring which are sequentially and alternately arranged on the bionic tracheal stent.
Preferably, the bionic tracheal stent is internally provided with a triangular stable supporting structure.
Preferably, the cartilage ring is uniformly arranged on the bionic tracheal stent.
Preferably, the blood vessel ring is uniformly arranged on the bionic tracheal stent.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
aiming at the defects of the tracheal repair technology in the prior clinical application, the invention prepares a bionic trachea which is constructed based on an electrostatic direct-writing bracket and contains a cricoid cartilage and a transmural blood vessel, wherein the bionic trachea can stably exist in a living body and is similar to a natural tissue in structure and function; by the innovative design of the stent implantation mode, the graft generates a transmural vascular structure similar to a physiological trachea in the prevascularization process, thereby providing nutrition and metabolic support for the inner layer structure of the graft; the tissue engineering trachea stent constructed by the electrostatic direct-writing three-dimensional printing technology can also realize the adjustment of parameters such as the length, the inner diameter, the outer diameter, the thickness, the internal porosity and the like of the stent, can carry out individualized design on the trachea structure, and widens the application range of the tissue engineering trachea.
Drawings
FIG. 1 is a schematic structural view of a bionic tracheal stent of the present invention;
FIG. 2 is a schematic view of the structure of a bionic trachea according to the present invention;
FIG. 3 is a schematic structural diagram of a molten electrostatic direct write apparatus in example 3 of the present invention;
wherein the reference numerals include: a glass rod 10; a bionic tracheal stent 11; a cartilage ring 12; a vascular ring 13; an air pump device 1; a gas guide tube 2; a material cylinder 3; a heating device 4; a molten material 5; a high voltage electrode 6; a printer head 7; a cylindrical rotating shaft device 8.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Example 1
The embodiment provides a preparation method of a bionic trachea, which comprises the following steps:
s1, constructing a bionic tracheal stent by a fusion electrostatic direct writing technology;
s2-1, soaking the bionic tracheal stent in 75% alcohol for 30 minutes, and then irradiating the bionic tracheal stent for 30 minutes by using ultraviolet light;
s2-2, washing the bionic tracheal stent for 2-3 times by PBS, and then washing for 1 time by DMEM culture solution containing serum;
s2-3, after the culture solution attached to the bionic tracheal stent is sucked to be dry by using an aspirator, sleeving the bionic tracheal stent outside the sterilized glass tube, and uniformly binding sutures outside the bionic tracheal stent;
s2-4, collecting chondrocytes cultured for 3-5 days in the 2 nd generation, and adjusting the cell suspension concentration of the chondrocytes to 1 x 108/mL；
S2-5, uniformly adding cell suspension of the chondrocytes among the sutures of the bionic tracheal stent to form an intact cartilage ring structure;
s3-1, treating the chondrocytes at 37 ℃ and 5% CO2Incubating for 4-5 hours under the condition of saturated humidity, and slowly adding a culture solution for promoting cartilage differentiation after the culture solution is stably attached to the bionic tracheal stent;
s3-2, taking out the suture, adding GelMA between cartilage ring structures, and carrying out photo-crosslinking;
s3-3, 5% CO at 37 ℃2Culturing for 2 weeks under the conditions of saturated humidity and liquid change every other day to secrete sufficient matrix;
s4, transmural vascularization to obtain the bionic trachea containing the cricoid cartilage and the transmural blood vessels.
As a preferred embodiment, the biomimetic tracheal stent is made of polycaprolactone.
As a preferred embodiment, the inside of the bionic tracheal stent is a triangular stable supporting structure.
Example 2
As shown in fig. 2, this embodiment provides a bionic trachea manufactured by the method of embodiment 1, including: as shown in fig. 1, the bionic tracheal stent 11 and the cartilage ring 12 and the blood vessel ring 13 are alternately arranged on the bionic tracheal stent 11 in sequence.
As a preferred embodiment, the bionic tracheal stent 11 is internally provided with a triangular stable supporting structure.
As a preferred embodiment, the cartilage ring 12 is uniformly arranged on the bionic tracheal stent 11.
As a preferred embodiment, the blood vessel ring 13 is uniformly arranged on the bionic tracheal stent 11.
Example 3
As shown in fig. 3, the present embodiment provides a molten electrostatic direct writing apparatus, including: the air pump device 1, the air duct 2, the material barrel 3, the printer nozzle 7 and the barrel-shaped rotating shaft device 8 are connected in sequence; wherein, a heating device 4 and a high-voltage electrode 6 are arranged outside the material barrel 3, and a melting material 5 is arranged in the material barrel 3;
the air pump device 1 generates air pressure to provide pressure for extruding the molten material 5 in the material barrel 3, the air pressure determines the amount of discharged material in unit time, and the air pressure can be adjusted through a knob so as to adjust the discharged material according to actual conditions;
the air duct 2 transmits air pressure between the air pump device 1 and the material barrel 3;
the material barrel 3 is used for containing the molten material 5 and is used as a carrier device for heating the molten material 5;
the heating device 4 includes: a temperature sensor and a heating wire connected to a computer, for heating the molten material 5 at a constant temperature;
the molten material 5 is a material capable of performing electrostatic direct writing of melt electrospinning, and is preferably polycaprolactone;
the high-voltage electrode 6 forms great electric field intensity between the printer nozzle 7 and the cylindrical rotating shaft device 8, and electrostatic spinning with different diameters can be formed by adjusting the electric field intensity;
extruding the molten material 5 under the action of air pressure from the printer head 7;
the cylindrical rotating shaft device 8 can perform transverse movement and rotary movement under the control of a computer, so that the bionic tracheal stents with different parameters can be constructed.
Example 4
This embodiment provides a method for extracting and expanding chondrocytes, comprising: removing fiber membrane around cartilage tissue, cutting into 1mm × 1mm size, adding 15% type II collagenase, digesting at 37 deg.C for 24 hr, shaking and blowing for several times, filtering with 0.22 μm sterile filter screen, centrifuging, washing, resuspending cells, counting, and collecting the cells with 1.0 × 104/cm2Cell density was seeded to 10cm2A culture dish; at 37 deg.C, 5% CO2Culturing under saturated humidity condition, changing the liquid for the first time in 3 days, and changing the liquid every other day later;
when the cells are basically fully spread in the culture dish, pancreatin digestive juice is added1.5mL, after cell cytoplasm retraction and morphological rounding under the microscope, DMEM culture solution (containing serum) is added to stop digestion, the cells are collected, and after centrifugation, the cells are resuspended at 1.0X 104/cm2Cell density seeded at new 10cm2The culture dish is used for repeating the steps and expanding the cells to the 2 nd generation.
Application examples
Patients, male, 53 years old, central lung cancer tracheal infiltration, planned to resect long trachea and replace with tissue engineered trachea;
constructing a 3D model of a trachea of a patient according to CT image data of the chest of the patient, carrying out individualized design on a bionic trachea structure according to 3D modeling of the trachea, and determining relevant parameters such as length, inner diameter, outer diameter, an internal structure of a bracket and the like;
constructing a bionic tracheal stent by adopting polycaprolactone through a melting electrostatic direct writing technology, and disinfecting and sterilizing the bionic tracheal stent for later use;
extracting costal chondrocytes of a patient, and planting the expanded chondrocytes to a bionic tracheal stent;
after the chondrocytes are differentiated to form cartilage tissues, placing the cartilage tissues under the skin for prevascularization to obtain a bionic trachea which contains cricoid cartilage and transmural blood vessels;
and transplanting the bionic trachea in situ.
In conclusion, aiming at the defects of the tracheal repair technology in the prior clinical application, the invention prepares the bionic trachea which is constructed based on the electrostatic direct-writing stent and contains the cricoid cartilage and the transmural blood vessel, wherein the bionic trachea can stably exist in a living body and is similar to natural tissues in structure and function; by the innovative design of the stent implantation mode, the graft generates a transmural vascular structure similar to a physiological trachea in the prevascularization process, thereby providing nutrition and metabolic support for the inner layer structure of the graft; the tissue engineering trachea stent constructed by the electrostatic direct-writing three-dimensional printing technology can also realize the adjustment of parameters such as the length, the inner diameter, the outer diameter, the thickness, the internal porosity and the like of the stent, can carry out individualized design on the trachea structure, and widens the application range of the tissue engineering trachea.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (9)
1. A preparation method of a bionic trachea is characterized by comprising the following steps:
s1, constructing a bionic tracheal stent by a fusion electrostatic direct writing technology;
s2, planting chondrocytes to the bionic tracheal stent;
s3, inducing the chondrocytes to differentiate to form intact cartilage tissue;
s4, transmural vascularization to obtain the bionic trachea containing the cricoid cartilage and the transmural blood vessels.
2. The method of claim 1, wherein the biomimetic tracheal stent is made of polycaprolactone.
3. The method for preparing the bionic tracheal stent, according to claim 1, wherein the bionic tracheal stent is internally provided with a triangular stable support structure.
4. The method for preparing a composite material according to claim 1, wherein the step S2 further includes:
s2-1, soaking the bionic tracheal stent in 75% alcohol for at least 30 minutes, and irradiating by using ultraviolet light for at least 30 minutes;
s2-2, washing the bionic tracheal stent for 2-3 times by PBS, and then washing for 1-2 times by DMEM culture solution containing serum;
s2-3, after the culture solution attached to the bionic tracheal stent is sucked to be dry by using an aspirator, sleeving the bionic tracheal stent outside the sterilized glass tube, and uniformly binding sutures outside the bionic tracheal stent;
s2-4, collecting chondrocytes cultured for 3-5 days in the 2 nd generation, and adjusting the cell suspension concentration of the chondrocytes to 1 x 108/mL；
S2-5, uniformly adding the cell suspension of the chondrocytes among the stitches of the bionic tracheal scaffold to form an intact cartilage ring structure.
5. The method for preparing a composite material according to claim 1, wherein the step S3 further includes:
s3-1, treating the chondrocytes at 37 ℃ and 5% CO2Incubating for 4-5 hours under the condition of saturated humidity, and slowly adding a culture solution for promoting cartilage differentiation after the culture solution is stably attached to the bionic tracheal stent;
s3-2, taking out the suture, adding GelMA between cartilage ring structures, and carrying out photo-crosslinking;
s3-3, 5% CO at 37 ℃2And under the conditions of saturated humidity and liquid change every other day, the culture is continued for 2 weeks to secrete sufficient substrates.
6. A biomimetic trachea prepared by the preparation method of any one of claims 1-5, comprising: the bionic tracheal stent comprises a bionic tracheal stent (11) and cartilage rings (12) and blood vessel rings (13) which are sequentially and alternately arranged on the bionic tracheal stent (11).
7. The bionic trachea according to claim 6, wherein the bionic trachea support (11) is internally provided with a stable triangular support structure.
8. The biomimetic trachea according to claim 6, wherein the cartilage ring (12) is uniformly disposed on the biomimetic trachea scaffold (11).
9. The biomimetic trachea according to claim 6, wherein the blood vessel ring (13) is uniformly disposed on the biomimetic trachea stent (11).
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