CN114642520A - Functional hybrid tracheal graft and preparation method thereof - Google Patents

Abstract

The invention provides a functionalized hybrid tracheal graft, which comprises: animal trachea and Polycaprolactone (PCL); preparing an intact extracellular matrix structure from an animal trachea by adopting a vacuum-assisted decellularization technology; preparing a support structure matched with an extracellular matrix mechanism by using Polycaprolactone (PCL) as a raw material and adopting a 3D printing technology; the stent structure is arranged on the inner wall of an extracellular matrix structure, and the manufacturing method of the tracheal graft is also disclosed, wherein the tracheal graft is prepared by using sodium deoxycholate/polyethylene glycol octyl phenyl ether solution for shaking table incubation, washing with sterile distilled water, then immersing into NaCl solution containing 1-4kU/mL deoxyribonuclease I and 2-8U/mL ribonuclease, and carrying out shaking table incubation to prepare a decellularized tracheal extracellular matrix structure with low immunogenicity; the high-temperature screw nozzle of the 3D bioprinter and the rotating shaft of the bionic trachea structure are selected, the rotating shaft is C-shaped, and the good biocompatibility, low immunogenicity, angiogenesis induction performance of the acellular trachea matrix and good biomechanical performance of the synthetic material are integrated.

Description

Functional hybrid tracheal graft and preparation method thereof

Technical Field

The invention relates to the field of tissue engineering and regenerative medicine, in particular to a functionalized hybrid tracheal graft and a preparation method thereof.

Background

Trachea transplantation is still a clinical difficult problem in the world at present, and the reasons for the difficult clinical problem are mainly the shortage of ideal trachea substitutes and the formation obstacle of graft vascularization caused by special anatomical structures. Severe tracheal injury is often caused by tumors, trauma, infection, malacia, congenital stenosis, etc., and excision of lesions with end-to-end anastomosis is the standard surgical procedure for tracheal reconstruction. When the trachea lesion exceeds 50% of adults or 30% of children, trachea replacement therapy is required. Bioprostheses, allografts, and autologous tissue reconstruction have been used for tracheal repair, but have a number of drawbacks: such as material degradation, graft necrosis, chronic bacterial infection, mediastinitis, granulation tissue proliferation, lack of effective vascularization and epithelialization, need for immunosuppressants, etc.

Regenerative medicine is based on the combination of natural or synthetic materials, living cells and cell signaling systems to repair, replace or enhance tissue function, promote cell and tissue growth. The tissue engineering technology is an effective trachea reconstruction method by combining cells with a 3D scaffold to construct a highly bionic physiological and biochemical microenvironment to promote tissue growth. The tissue engineering trachea substitute meeting the clinical requirements simultaneously meets the following conditions: 1) can provide a good microenvironment for cell growth, 2) can induce the formation of functional epithelial cells, and 3) can form a rich microvascular network for clinical transplantation needs.

An ideal tracheal substitute would simultaneously meet the following requirements: the material has proper elasticity and compression resistance, good biocompatibility, no toxic or side effect, cell adhesion and proliferation support, and can also quickly form a micro-vascular network to promote cell growth and prevent necrosis of a graft. The structure and the components of the scaffold material play an important role in the growth and the migration of seed cells and the formation of functional structures. The extracellular matrix (ECM) is an important component of tissues, and the natural ECM (such as fibrin, collagen and acellular matrix) has good biocompatibility, biodegradability and angiogenesis promotion performance, is a main component of a cell growth microenvironment in an organism, and is superior to a polymer material in the aspects of regulating biological behaviors of cells and promoting tissue regeneration. The decellularized ECM has the functions of promoting in-vivo tissue repair and regeneration, can also guide and regulate cell (proliferation and differentiation) reaction, supports differentiation of bone marrow Mesenchymal Stem Cells (MSCs) to chondrocytes and proliferation of epithelial cells, and can provide a relatively ideal microenvironment for cell growth.

In recent years, a series of works are carried out in the fields of searching for proper tracheal graft, improving the performance of a stent and constructing graft microvasculature, and the optimal cycle period of rabbit tracheal decellularization treatment is selected by adopting a detergent-combined enzyme method through decellularization treatment with different cycle periods, and comparing and verifying multiple dimensions of immunogenicity removal, tissue structure integrity and in vivo biocompatibility. In the research, compared with the primary trachea, the acellular treatment has no influence on the transverse tensile property of the stent, but causes the longitudinal compression mechanical property of the stent to be obviously reduced.

Disclosure of Invention

The invention aims to provide a functional hybrid tracheal graft;

the invention also aims to provide a preparation method of the functionalized hybrid tracheal graft, which has the advantages of simple method, wide raw material source and low cost, and the prepared tracheal graft is made of natural substances and has biomechanical properties completely matched with the original trachea so as to solve the defects in the prior art.

In order to achieve the above object, the present invention is realized by: a functionalized hybrid tracheal graft comprising: a acellular tracheal matrix structure and a C-shaped reticular tracheal stent;

the C-shaped reticular tracheal stent is arranged on the inner wall of the acellular tracheal matrix structure.

The C-shaped reticular tracheal stent is made of polycaprolactone.

The preparation method of the functionalized hybrid tracheal graft comprises the following steps,

the method comprises the following steps: carrying out acellular treatment on an isolated trachea to prepare an acellular trachea matrix structure;

step two: manufacturing a tracheal stent;

step three: a simulated body trachea graft was prepared.

The preparation of the acellular tracheal matrix structure in the first step comprises the following steps:

preparing a complete and immunogenicity-removed tracheal extracellular matrix structure by adopting a vacuum-assisted decellularization technology, firstly obtaining a matched animal trachea, removing connective tissues around the trachea and phlegm in a lumen, and placing the trachea in sterile distilled water at 0-40 ℃ for infiltration and dissolution; washing with sterile distilled water, adding 0.1-4 wt% sodium deoxycholate/polyethylene glycol octyl phenyl ether solution, and incubating in a shaking table; washing with sterile distilled water again, then soaking in NaCl solution containing 1-4kU/mL DNase I and 2-8U/mL RNase, incubating in shaking table, and maintaining vacuum state during cell removal.

The manufacturing of the tracheal stent in the second step comprises the following steps:

preparing a trachea support matched with the structure in the step one by adopting a 3D printing technology, selecting a high-temperature screw spray head of a 3D biological printer and a bionic trachea structure rotating shaft by taking polycaprolactone as a raw material, wherein the rotating shaft is C-shaped, the filling pattern is sine filling, the number of filling lines in one circle is 12, and printing the C-shaped netted trachea support structure.

The preparation of the simulated body trachea graft in the third step comprises the following steps:

and (3) placing the C-shaped reticular tracheal stent knot prepared in the step two into the inner wall of the acellular tracheal matrix structure prepared in the step one to construct the hybrid tracheal graft.

The invention innovatively prepares a vacuum-assisted acellular tracheal (VADT) stent, effectively shortens the acellular processing time from 9 days to 3 days, and effectively enhances the immunogenicity removal and ECM structure retention effects. In addition, besides the aspects of morphological structure bionic and micro-vascularization construction, there is still another important but easily neglected problem, namely bionic of biomechanical properties. The natural material has poor biomechanical property and risks of softening and collapsing of a lumen, and the synthetic material has the defects of stiffness of a graft and the like caused by over-strong mechanical property. Therefore, the applicant develops a 3D hybrid bionic trachea graft by combining a 3D printed Polycaprolactone (PCL) large mesh stent and VADT, and the 3D hybrid bionic trachea graft is proved to have biomechanical performance completely matched with the original trachea through multi-dimensional biomechanical performance tests.

According to the technical scheme, the invention has the beneficial effects that:

1. the vacuum-assisted acellular tracheal matrix has good cell adhesion and angiogenesis induction performance, provides an effective stent source for vascularization of transplanted tracheas, has simple and easy preparation process, effectively reduces the use concentration of medicaments, and quickly achieves ideal acellular effect.

2. According to the invention, a C-shaped large-mesh tracheal stent matched with a acellular tracheal matrix structure is constructed by adopting a 3D printing technology, and the stent has certain compression resilience and can be effectively matched with the acellular tracheal matrix structure; the unique macroporous structure does not influence the adhesion and proliferation of cells on the surface of the acellular matrix structure.

3. The acellular tracheal matrix structure is combined with a 3D printed macroporous PCL bracket to construct a hybrid tracheal graft, so that the defect of insufficient biomechanical property of the acellular tracheal matrix structure is effectively overcome.

Drawings

FIG. 1 is a schematic design diagram of the present invention.

FIG. 2 is a comparison of the HE, Masson trichrome and safranin O stained tissues of decellularized trachea and proto-histochemical trachea according to the invention.

FIG. 3 is a comparison of the tissue structure of DAPI, MHC-I, Merge fluorescence pictures of decellularized trachea and proto-histochemical trachea in accordance with the present invention.

FIG. 4 is a comparison of the tissue structure of DAPI, MHC-II, Merge fluorescence pictures of decellularized trachea and proto-group trachea in accordance with the present invention.

Fig. 5 shows that the PCL macroporous or microporous tracheal stent is prepared by adopting a 3D printing technology, and the PCL macroporous or microporous tracheal stent and the acellular tracheal matrix are used for constructing the hybrid tracheal graft.

Fig. 6 is a comparison of the biomechanical properties of different groups of tracheal grafts.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

As shown in fig. 1: the invention provides a functionalized hybrid tracheal graft, which comprises an animal trachea and Polycaprolactone (PCL), wherein the animal trachea is prepared into a complete extracellular matrix structure by adopting a vacuum-assisted decellularization technology, the Polycaprolactone (PCL) is used as a raw material, a support structure matched with the extracellular matrix mechanism is prepared by adopting a 3D printing technology, and the support structure is arranged on the inner wall of the extracellular matrix structure.

The immunogen of the native trachea is removed by the design of the scheme, an Extracellular Matrix (EMC) structure is reserved, the native ECM has good biocompatibility and biodegradability and angiogenesis promotion performance, the regeneration of cells is guaranteed, in addition, a support is prepared by using a 3D printing technology and is placed on the inner wall of the extracellular matrix removal structure, the use strength of the trachea is guaranteed, the problems that the natural material is poor in biomechanical performance, the risks of softening and collapse of a lumen exist, and the synthetic material has the defects that the mechanical performance is too strong, the graft is stiff and the like are solved.

Firstly, the method for manufacturing the tracheal graft is specifically explained in detail as follows:

1. preparation of vacuum assisted acellular tracheal matrix

The whole section of the trachea of the experimental animal is obtained by standard surgical operation, and connective tissues on the outer wall of the trachea are immediately stripped. Cutting trachea into 10 mm long trachea segment, placing into 10 mL sterile vacuum blood collection tube, and dissolving in 0-40 deg.C sterile distilled water for 24 hr; washing with sterile distilled water for half an hour, adding 0.1-4% sodium deoxycholate/Triton-X100 (polyethylene glycol octyl phenyl ether) solution, and incubating at 80rpm on a constant temperature shaking table at 0-40 deg.C for 24 hr; washing with sterile distilled water for half an hour, and incubating in 1mol/L NaCl solution containing 1-4kU/mL of Dnase-I (deoxyribonuclease I) and 2-8U/L of RNase (ribonuclease) for 24 h; the above operations were performed in a vacuum environment, and after the cell removal treatment, each group was washed with sterile distilled water and stored in 1% P/S-containing PBS buffer at 4 ℃ for further use.

2.3D prints PCL trachea support

With Polycaprolactone (PCL) as the raw materials, selecting a 3D biological printer high-temperature screw rod nozzle, setting the diameter of a nozzle to be 200 mu m, customizing a bionic trachea structure rotating shaft according to the required size of a transplanted trachea, setting a charging basket temperature control to be 90 ℃, a silk outlet speed to be 1mm/s, a printing speed to be 1mm/s, a rotating shaft pattern to be C type, a printing shaft angle to be 270, a filling pattern to be sine filling, and the number of filling lines in one circle to be 12, thereby printing a C type mesh trachea support structure.

3. Preparation of hybrid bionic tracheal graft

The 3D printed C-shaped tracheal stent is placed on the inner wall of a decellularized trachea to construct a hybrid tracheal graft, and a universal testing machine is utilized to carry out various biomechanical property tests.

Secondly, the effect of the embodiment is verified: the effect analysis of the transplanted trachea is carried out as follows;

2.1. histological routine staining analysis

Each group of samples was immersed in 4% paraformaldehyde, fixed at room temperature for 24h, paraffin-embedded, sectioned (4 μm), and the histological morphological changes of the sections in HE staining, Masson trichrome and safranin O staining were observed by an optical microscope.

2.2. Immunofluorescence assay

The expression of MHC-I, MHC-II antigen in each group of samples was detected by immunohistochemical staining, frozen sections were shake-sealed with 5% BSA at 37 ℃ for 60min, diluted primary antibodies (MHC-I, MHC-II, antibody concentration 1: 200) were added dropwise overnight at 4 ℃, fluorescent secondary antibodies were added the next day for reaction at room temperature for 30min, and the reaction was observed by DAPI-stained fluorescence microscope.

2.3. Biomechanical property testing

A fixed platen sensor of a universal testing machine is adopted, a sample to be tested is placed in the center of a disk, initial load pressure is given to be 0.01N, constant compression is started at the speed of 2 mm/min at room temperature, the strain pressure and the elastic modulus value of a sensor when a tube cavity is compressed to be half of the initial diameter are recorded, and the tube diameter size and the 0.01-0.2N compression displacement of each group of grafts are recorded.

Thirdly, please refer to fig. 2 to fig. 6 for the test results;

3.1 As shown in FIG. 2, HE staining, Masson trichrome staining, and safranin O staining showed that after decellularization, the tissue structure remained intact and nuclear material disappeared.

3.2. Acellular tracheal matrix immunogenicity analysis

As shown in FIG. 3, the results of MHC-I immunofluorescence showed near complete disappearance of MHC-I expression following decellularization.

3.3. Acellular tracheal matrix immunogenicity analysis

As shown in FIG. 4, the MHC-II immunofluorescence results showed nearly complete disappearance of MHC-II expression following decellularization.

Construction of 3.43D hybrid bionic trachea

As shown in fig. 5, a C-type tracheal stent was prepared using 3D printing technology and combined with decellularization to construct a hybrid tracheal graft.

3.53D hybrid Bionical tracheal biomechanical analysis

As shown in fig. 6, the biomechanical properties of the VADT tracheal graft are significantly reduced compared to native trachea, while the hybrid tracheal graft is perfectly matched to native tissue in terms of inner diameter of trachea, 50% stress in compression, elastic modulus, etc.

By adopting the preparation method of the functionalized 3D hybrid tissue tracheal graft, the advantages of good biocompatibility, low immunogenicity, angiogenesis inducing performance of the acellular tracheal matrix and good biomechanical performance of the synthetic material are effectively extracted, and a 3D hybrid bionic trachea is constructed; can provide an effective selection scheme for the tracheal graft. In conclusion, the method provides effective thinking and method for the construction of the current engineering trachea transplantation, has certain clinical transformation potential, and is expected to bring new gospel to patients who need trachea transplantation urgently.

It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.

Claims (6)

1. A functionalized hybrid tracheal graft, comprising: a acellular tracheal matrix structure and a C-shaped reticular tracheal stent;

the C-shaped reticular tracheal stent is arranged on the inner wall of the acellular tracheal matrix structure.

2. The functionalized hybrid tracheal graft according to claim 1, wherein the C-shaped reticular tracheal stent is made of polycaprolactone.

3. The method for preparing a functionalized hybrid tracheal graft according to claim 1 or 2, comprising the steps of,

the method comprises the following steps: carrying out acellular treatment on an isolated trachea to prepare an acellular trachea matrix structure;

step two: manufacturing a tracheal stent;

step three: a simulated body trachea graft was prepared.

4. The method for preparing a functionalized hybrid tracheal graft according to claim 3, wherein the step one for preparing the acellular tracheal matrix structure comprises the following steps:

preparing a complete trachea extracellular matrix structure without immunogenicity by adopting a vacuum-assisted decellularization technology, firstly, obtaining a matched animal trachea, removing connective tissues around the trachea and phlegm liquid in a lumen, and putting the trachea extracellular matrix structure in sterile distilled water at 0-40 ℃ for infiltration and dissolution; washing with sterile distilled water, adding 0.1-4 wt% sodium deoxycholate/polyethylene glycol octyl phenyl ether solution, and incubating in a shaking table; washing with sterile distilled water again, soaking in NaCl solution containing 1-4kU/mL deoxyribonuclease I and 2-8U/mL ribonuclease, incubating in shaking table, and maintaining vacuum state during cell removal.

5. The method for preparing a functionalized hybrid tracheal graft according to claim 3, wherein the step two of preparing a tracheal stent comprises the following steps:

preparing a trachea support matched with the structure in the step one by adopting a 3D printing technology, selecting a high-temperature screw spray head of a 3D biological printer and a bionic trachea structure rotating shaft by taking polycaprolactone as a raw material, wherein the rotating shaft is C-shaped, the filling pattern is sine filling, the number of filling lines in one circle is 12, and printing the C-shaped netted trachea support structure.

6. The method for preparing a functionalized hybrid tracheal graft according to claim 3, wherein the step three for preparing the simulated tracheal graft comprises the following steps:

and (3) placing the C-shaped reticular tracheal stent knot prepared in the step two into the inner wall of the acellular tracheal matrix structure prepared in the step one to construct the hybrid tracheal graft.

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