CN105169479A - Preparation method of externally constructed tissue engineering trachea matrix - Google Patents

Abstract

The invention discloses a method for preparing a tissue engineered trachea matrix constructed in vitro. The method is to pretreat fresh allogeneic tracheal tissue or heterogeneous tracheal tissue including removing fat and cleaning blood; Bioenzyme method removes tracheal tissue impurities and tissue cells, and finally uses photo-oxidative cross-linking reaction to adjust the cross-linking degree of the tracheal matrix skeleton from which tissue cells have been removed; Proliferating tissue-engineered tracheal matrix.

Description

A preparation method of tissue engineered trachea matrix constructed in vitro

technical field

The invention relates to a preparation method of decellularized tissue engineering trachea matrix, which belongs to the field of biomedical engineering.

Background technique

The trachea often causes airway obstruction due to factors such as tumors or inflammatory lesions, which seriously endangers the lives of patients. Surgical resection of the diseased trachea is the most effective clinical treatment. However, due to the limitation of the extensibility of the trachea itself, long-segment tracheectomy must be performed with the help of tracheal substitutes for airway reconstruction. At present, there are mainly three kinds of tracheal substitutes, namely, allogeneic tracheal transplantation, autologous tissue reconstruction, and artificial trachea. The trachea is a hollow and elastic vital organ that maintains ventilation, cleaning and defense functions of the human body, and has unique anatomical and physiological characteristics. Tracheal transplantation mostly fails due to insufficient blood supply and immune rejection; in addition, the allogeneic tracheal material is limited by many factors, and it is difficult to preserve and cannot be widely used in clinical practice. . Autologous tissue reconstruction is to use autologous fascia, pericardium, pleura, small intestine, cartilage, or composite tissue flaps to reconstruct the respiratory tract. The repair effect after tracheectomy is not ideal. The artificial trachea is manufactured by combining one or more artificial composite materials. However, due to material selection and design reasons, serious complications such as anastomotic stenosis, infection, and dehiscence after transplantation cannot be resolved. At present, no ideal artificial trachea has been successfully developed. So far, large tracheal tumors, long-segment tracheal stenosis or softening of the trachea are still very difficult clinical problems, and have become a bottleneck restricting the development of tracheal surgery techniques.

With the rise of biological tissue engineering and its successful application in different clinical fields, the use of biological tissue engineering technology to construct tracheal substitutes provides a new idea for tissue defect repair and is expected to become the most ideal treatment method. Tissue engineering matrix scaffold is one of the three core elements (cells, materials and biological factors) of biological tissue engineering. Tissue engineering matrix provides tissue structure scaffold and temporary carrier for the growth, proliferation and differentiation of seed cells in vitro, or assists seed cells to proliferate and differentiate in vivo to form new tissues or organs that are suitable for their own function and shape, so as to repair tissues or purpose of the organ. The components, structure, interface, pore size, strength, and elasticity of matrix scaffolds determine the biological characteristics of pre-constructed tissue engineered organs, and material selection is one of the key factors for the success of tissue engineering. An ideal tissue engineering matrix scaffold should have the following properties: (1) an interconnected porous network structure capable of enabling cells to migrate, proliferate, and deeply adhere to the interior of the scaffold; (2) provide the necessary oxygen and nutrients for cells inside the scaffold, and bring (3) Good biocompatibility for cell adhesion and proliferation; (4) Shape constructed according to surgical requirements; (5) Appropriate mechanical strength and degradation performance.

Commonly used materials for tissue engineering tracheal matrix scaffolds include synthetic polymers, such as polylactic acid and polyglycolic acid, and natural polymers, such as collagen, fibrin, and alginate. Artificial synthetic materials have poor hydrophilicity, weak cell affinity, and can cause aseptic inflammation. The mechanical properties of cell-material composites are weak, and support molds are often required. Compared with synthetic materials, natural polymers are closer to the living environment of cells in the body, and the surface properties of natural materials such as collagen and chitosan are more conducive to cell adhesion and chemotaxis to cells. It can be used alone as a scaffold material or used to construct a composite scaffold material in multiple tissue engineering fields such as soft tissue repair, cell differentiation, capillary engineering, dermis engineering, and vascularized adipose tissue. But lack of mechanical strength, the degradation time is difficult to control.

The original cellular components in biomaterials are the main factor that induces receptor rejection, but biomaterial matrices manufactured by decellularization technology have the advantages of good histocompatibility and affinity, and can be used as tissue fillers for a long time . In addition, there may be some complex growth factors in the complete natural matrix, which can induce and regulate the growth, reproduction and differentiation of cells. The application of physical, chemical, enzymatic or a combination of the above methods removes the antigenic components in the matrix and retains the three-dimensional microstructure and biomechanical properties of the matrix. The obtained decellularized matrix has more advantages in tissue engineered organ substitutes and has become New trends in the development of tissue engineering materials in the future. Various decellularized natural biological tissue materials have been widely used in the construction of various related tissues, and are a good choice for tissue engineering materials.

After decellularization of tracheal tissue, the remaining extracellular matrix material has low immunogenicity, and the complete extracellular matrix structure is conducive to the growth of host cells. Using decellularized homogeneous or heterogeneous biological tracheal components to tissue-engineer tracheal matrix scaffolds as a substitute for tracheal reconstruction can not only solve the shortage of artificial trachea donors, but also provide new ideas for the construction of other tissue-engineered organs. There are many existing methods for decellularization of tracheal tissue, including detergent TritonX-100, surfactant sodium dodecyl sulfate (SDS), trypsin and nuclease, etc. Various decellularization techniques have advantages , also have their own defects. However, there are also differences in the effects. The existing treatment methods are difficult to remove the tracheal cells and retain the complete tracheal matrix scaffolds such as cartilage scaffolds, collagen fibers, and elastic fibers. Tracheal mucosal epithelial cells are the main factor causing immune rejection of tracheal transplantation, so the existing decellularization methods are difficult to meet the requirements. In particular, the anti-degradation ability of extracellular matrix components is low, the tissue stability is poor, and it is easy to be absorbed in a short period of time after implantation, which is not conducive to the growth and shaping of host cells.

Contents of the invention

In view of the defects in the existing preparation methods of tissue engineering tracheal matrix, the purpose of the present invention is to provide a scaffold that can effectively remove tracheal cells to obtain a complete tracheal matrix, and then construct a scaffold with good stability through photo-oxidative cross-linking, which is conducive to seeding cells. A method of implanting, attaching and proliferating a tissue-engineered tracheal matrix.

In order to solve the above problems, the present invention provides a method for preparing an in vitro tissue engineered tracheal matrix, the method comprising the following steps:

Step 1: Preprocessing

Pretreatment of fresh allogeneic tracheal tissue or xenogeneic tracheal tissue including fat removal and blood washing;

Step 2: Decellularization

The pretreated tracheal tissue in Step 1 was first soaked in dodecyldimethylbenzyl ammonium bromide solution, then incubated in polyethylene glycol octylphenyl ether solution, placed in trypsin and Incubate in EDTA mixed solution, and incubate in DNase-1 and RNase-A mixed solution to obtain decellularized tracheal tissue;

Step 3: Photo-oxidative cross-linking treatment

Soak the decellularized tracheal tissue obtained in step 2 in a hypertonic PBS solution with a pH of 7.5-7.8 and an Osm of 660-700 mOsm, and then place it in a solution containing 0.8-1.2 wt% methylene blue at a pH of 7.5-7.8 and an Osm of 660-700 mOsm. After equilibrating in 310-330mOsm PBS solution, maintain the temperature of the equilibrium system at 5-15°C, pH 7.5-7.8, and carry out photo-oxidative cross-linking reaction under the conditions of light and oxygen, to obtain the product.

In the technical solution of the present invention, the tracheal tissue is firstly treated with a chemical method combined with a biological enzyme method, which can effectively remove the tracheal cells, and the tracheal matrix scaffolds, such as cartilage scaffolds, collagen fibers, and elastic fibers, can be relatively completely preserved; On the basis of combining with the photo-oxidation cross-linking method, the degree of cross-linking of the tracheal matrix scaffold is adjusted to obtain a tissue-engineered tracheal matrix with good stability, which is conducive to the planting, attachment and proliferation of seed cells. The method of the invention adopts the methylene blue cross-linking agent and the photo-oxidation method for cross-linking, the cross-linking effect is good, and the cross-linking agent has low toxicity and basically no residue, which is beneficial to the application of tissue engineering trachea matrix.

The preparation method of the tissue engineered trachea matrix constructed in vitro of the present invention also includes the following preferred options:

In a preferred solution, fresh allogeneic tracheal tissue or heterogeneous tracheal tissue is placed in phosphate buffered saline at 0-4°C in a sterile environment, and then the connective tissue including adjacent fat is removed by cutting, The tracheal tube is then flushed with phosphate buffered saline containing antibiotics to remove blood from the tracheal tube. Pretreatment of tracheal tissue can fully remove connective tissue such as fat and residual blood, which is conducive to the subsequent removal of other impurities and tissue cells in the trachea.

In a more preferred solution, the heterogeneous tracheal tissue is porcine tracheal tissue or canine tracheal tissue. The preferred tracheal tissue has the advantages of better bio-histocompatibility and affinity.

In the preferred scheme, the pretreated tracheal tissue is first soaked in a dodecyldimethylbenzyl ammonium bromide solution with a concentration of 0.08 to 0.12wt% for 25 to 35 minutes, and then placed in a temperature of 35 to 39 ℃, the concentration is 0.4～0.6wt% polyethylene glycol octyl phenyl ether solution and incubated for 45～55h, and the temperature is 35～39℃, the concentration is 0.20～0.28wt% trypsin and the concentration is 0.015 ～0.025wt% EDTA is incubated in a mixed solution composed of 0.8～1.2:1 by volume ratio for 25～35min, and the temperature is 35～39°C, the concentration of Dnase-1 is 28～32μL/mL and the concentration of Rnase-A is Incubate in a mixed solution containing DNase-1 and RNase-A at 0.25-0.35 mg/mL for 22-25 hours to obtain decellularized tracheal tissue.

In a further preferred scheme, the pretreated tracheal tissue is first soaked in a 0.1 wt% dodecyldimethylbenzyl ammonium bromide solution (brogeramine) for 30 minutes, and then placed in a temperature of 37 ° C, Concentration is 0.5wt% polyethylene glycol octyl phenyl ether solution (Trinaton X-100) incubate 48h, place temperature is 37 ℃, the concentration is 0.25wt% trypsin and concentration is 0.02wt% EDTA at a volume ratio of 1:1 and incubated for 30 min in a mixed solution containing DNase-1 and RNase- Incubate in the mixed solution of A for 24 hours to obtain decellularized tracheal tissue. In this preferred scheme, optimizing the treatment conditions is more conducive to effectively removing the tracheal cells, while retaining the tracheal stromal scaffold intact.

In the preferred scheme, the decellularized tracheal tissue is soaked in hypertonic PBS solution for 3 to 5 hours (4 hours is the best), and then placed in PBS solution containing methylene blue to equilibrate for 3 to 5 hours (4 hours is the best). .

In a preferred solution, the photo-oxidative crosslinking reaction takes 45 to 50 hours, and the most optimal time is 48 hours. Appropriate photo-oxidative crosslinking reaction time in the preferred scheme is conducive to controlling the degree of crosslinking reaction.

In the preferred solution, the photo-oxidative crosslinking reaction is realized by irradiation with a 400-600W incandescent lamp, and the incandescent lamp is 18-22 cm away from the liquid surface. Using a 500W incandescent lamp for irradiation, and the incandescent lamp is 20cm away from the liquid surface, the photooxidative crosslinking reaction effect is the best.

In the technical solution of the present invention, the introduction of oxygen continues during the photo-oxidative crosslinking reaction, and the oxygen is far in excess relative to the theoretical amount.

In a preferred solution, the tracheal tissue obtained through the photooxidative cross-linking reaction is repeatedly washed with PBS solution, sterilized by γ-rays, placed in sterile PBS solution, and stored at a temperature not higher than -20°C.

Compared with the prior art, the beneficial effects brought by the technical solution of the present invention are:

The present invention uses the combination of detergent and protease to completely remove components such as cell tracheal mucosa in fresh trachea tissue, thereby relatively completely retaining the trachea of the main extracellular interstitial components of the trachea (cartilage scaffold, collagen fibers and elastic fibers, etc.) The matrix scaffold avoids the calcification and immune reaction caused by the cells; on this basis, the photooxidative cross-linking treatment of the decellularized tracheal pipeline can adjust the degree of cross-linking of the tracheal matrix scaffold and prevent premature degradation of the tissue, thereby It is possible to form a biological material that is planted by seed cells in vitro or attached and proliferated by autologous tissue in vivo, the original structure is degraded, and finally remodeled into a part of the body and can grow; in addition, cross-linking treatment can further reduce the structure Protein-induced heterogeneous reactions. The method of the invention provides a new method and source for constructing a tissue-engineered tracheal matrix scaffold with physiological functions.

Description of drawings

[Figure 1] HE staining (150X) photo of pig tracheal matrix treated with decellularization combined with photooxidative cross-linking;

[Figure 2] It is a cross-sectional electron microscope scanning image of porcine tracheal matrix treated with decellularization combined with photooxidative cross-linking;

[Figure 3] It is a scanning electron microscope image of porcine tracheal stroma endometrium treated with decellularization combined with photooxidative cross-linking;

[Fig. 4] Schematic diagram of mechanical property testing of porcine tracheal matrix treated with decellularization combined with photooxidative cross-linking.

Detailed ways

The following examples are intended to further illustrate the content of the present invention, rather than limit the scope of the claims of the present invention.

Example 1

(1) Under sterile conditions, obtain fresh allogeneic or heterogeneous trachea (such as porcine trachea) tissues that meet the requirements, and store them in phosphate buffered saline at 0-4°C.

(2) Trim under aseptic operation, remove adjacent fat and other connective tissue, and lavage with phosphate buffer solution containing antibiotics to remove blood on the tracheal tube.

(3) decellularization, the steps include:

① Soak in 0.1wt% brogeramine for 30 minutes; place the trachea in 0.5wt% Triton X-100 (TritonX-100), and incubate at 37°C for 48 hours;

② Place in 0.25wt% trypsin and 0.02wt% EDTA (volume ratio 1:1) and incubate for 30 minutes.

③Incubate at 37°C for 24 hours with 30 μL/ml DNase-1 and 0.3 mg/mL RNase-A.

(4) photooxidative cross-linking treatment, the steps include:

① Soak the decellularized trachea in hypertonic PBS solution (pH7.6, Osm: 680 mosm) for 4 hours;

② Place in PBS solution containing 0.1wt% methylene blue (pH7.6, Osm: 320mosm) to equilibrate for 4 hours.

③Irradiate with a 500W incandescent lamp, control the temperature of the photo-oxidation cross-linking system at about 10°C, keep the light source at a distance of 20cm from the liquid surface, keep stirring and blowing in oxygen during the irradiation process, and control the pH in the reaction system to about 7.6, take out the tracheal matrix after 48 hours, Bacteria were washed repeatedly with PBS solution.

(5) The trachea treated with decellularization combined with photooxidation is bagged after passing the histological test. Sterilize and sterilize with 25KGY gamma rays, and then put them in sterile PBS solution and store them in a -20°C refrigerator for later use.

It can be seen from Figure 1 that the tracheal matrix treated with cells combined with photooxidation retains the main extracellular stroma components of the trachea (cartilage scaffold, collagen fibers and elastic fibers, etc.).

It can be seen from Figures 2 and 3 that the photooxidative cross-linking treatment of the decellularized tracheal ducts adjusted the degree of cross-linking of the extracellular matrix, and the structure of the tracheal scaffold was basically maintained, forming an early stage that can be planted by seed cells or attached to and proliferated by autologous tissues. The original structure is degraded and finally remodeled into a biomaterial that is part of the body and can grow.

It can be seen from Figure 4 that the tissue engineered tracheal matrix scaffold constructed by cells combined with photooxidation treatment is difficult to deform under the action of external force, and its biomechanical properties are similar to those of fresh tracheal tissue without treatment.

Claims (8)

1. a preparation method of the tissue engineered tracheal matrix constructed in vitro, is characterized in that, comprises the following steps: Step 1: Preprocessing Pretreatment of fresh allogeneic tracheal tissue or xenogeneic tracheal tissue including fat removal and blood washing; Step 2: Decellularization The pretreated tracheal tissue in Step 1 was first soaked in dodecyldimethylbenzyl ammonium bromide solution, then incubated in polyethylene glycol octylphenyl ether solution, placed in trypsin and Incubate in EDTA mixed solution, and incubate in DNase-1 and RNase-A mixed solution to obtain decellularized tracheal tissue; Step 3: Photo-oxidative cross-linking treatment Soak the decellularized tracheal tissue obtained in step 2 in a hypertonic PBS solution with a pH of 7.5-7.8 and an Osm of 660-700 mOsm, and then place it in a solution containing 0.8-1.2 wt% methylene blue at a pH of 7.5-7.8 and an Osm of 660-700 mOsm. After equilibrating in 310-330mOsm PBS solution, maintain the temperature of the equilibrium system at 5-15°C, pH 7.5-7.8, and carry out photo-oxidative cross-linking reaction under the conditions of light and oxygen, to obtain the product. 2. The preparation method of the tissue-engineered tracheal matrix constructed in vitro according to claim 1, characterized in that, in a sterile environment, fresh allogeneic tracheal tissue or heterogeneous tracheal tissue is placed in 0-4°C phosphoric acid After immersion in saline buffer, the connective tissue including adjacent fat was removed by cutting, and then the residual blood in the tracheal tube was removed by lavage with phosphate buffer saline containing antibiotics. 3. The preparation method of the tissue-engineered tracheal matrix constructed in vitro according to claim 1 or 2, characterized in that, the heterogeneous tracheal tissue is porcine tracheal tissue or canine tracheal tissue. 4. The preparation method of the tissue engineered trachea matrix constructed in vitro according to claim 1, characterized in that the pretreated tracheal tissue is first placed in a concentration of 0.08～0.12wt% dodecyldimethylbenzyl Soak in ammonium bromide solution for 25 to 35 minutes, then incubate in polyethylene glycol octyl phenyl ether solution at a temperature of 35 to 39 °C and a concentration of 0.4 to 0.6 wt% for 45 to 55 hours, and place at a temperature of 35 ~39°C, incubate for 25~35min in a mixed solution composed of trypsin with a concentration of 0.20~0.28wt% and EDTA with a concentration of 0.015~0.025wt% in a volume ratio of 0.8~1.2:1, and place at a temperature of 35~ Incubate in a mixed solution containing DNase-1 and RNase-A at 39°C with a concentration of DNase-1 of 28-32 μL/mL and a concentration of RNase-A of 0.25-0.35 mg/mL for 22-25 hours to obtain decellularized tracheal tissue . 5. The preparation method of the tissue-engineered tracheal matrix constructed in vitro according to claim 1, wherein the decellularized tracheal tissue is soaked in hypertonic PBS solution for 3 to 5 hours, and then placed in PBS containing methylene blue Equilibrate in the solution for 3 to 5 hours. 6 . The method for preparing an in vitro tissue-engineered tracheal matrix according to claim 1 , characterized in that, the photo-oxidative cross-linking reaction takes 45 to 50 hours. 7. The method for preparing the tissue-engineered tracheal matrix constructed in vitro according to claim 1, wherein the photo-oxidative cross-linking reaction is realized by irradiation with a 400-600W incandescent lamp, and the distance between the incandescent lamp and the liquid surface is 18-22cm . 8. The preparation method of the tissue-engineered trachea matrix constructed in vitro according to claim 1, characterized in that, the trachea tissue obtained through the photo-oxidative cross-linking reaction is repeatedly washed with PBS liquid, then sterilized by gamma rays, and then Place in sterile PBS solution and store at a temperature not higher than -20°C.