A method for preparing a bionic chondrogenic 3D printed tissue engineering trachea

Abstract

The present invention relates to a preparation method of a bionic chondrogenic 3D printed tissue engineering trachea, comprising the following steps: step (1), 3D printing polycaprolactone (PCL) tracheal stent; step (2), obtaining xiphoid cartilage decellularized matrix (DCM); step (3), preparing a composite stent; first preparing a methacrylylated hyaluronic acid (HAMA) solution, then immersing the PCL stent printed in step (1) into the solution, immersing at 37°C for 15 minutes, and fully cross-linking under blue light for 5 minutes, so that HAMA uniformly wraps the PCL stent; step (4), culturing and identifying bone marrow mesenchymal stem cells (BMSCs); step (5), preparing biological ink; step (6), constructing a C-shaped cartilage ring bionic structure by light-curing three-dimensional bioprinting technology. Through the present invention, a bionic chondrogenic tracheal substitute is prepared, which provides a new idea for clinical tracheal replacement therapy, especially tissue engineering tracheal chondrogenic treatment.

Description

Technical Field

[0001] The invention relates to a method for preparing a bionic cartilage-promoting 3D printed tissue engineering trachea, and belongs to the field of biotechnology. Background Art

[0002] 3D printing technology, especially 3D bioprinting that can directly load cells / growth factors, is expected to develop tracheal stents that can support multiple cell types and are reproducible and patient-specific. Decellularized matrix removes autologous cells and antigen molecules that may cause immune rejection reactions to the greatest extent, while retaining other active ingredients and three-dimensional structures to the greatest extent. It is an excellent natural material for tissue and organ repair. Hyaluronic acid (HA) is a natural glycosaminoglycan polymer and a component of the extracellular matrix of animal tissues. It plays an important role in many biological processes such as cell proliferation, differentiation, morphogenesis, inflammation and wound healing, and cartilage regeneration. Methacryloyl hyaluronic acid (HAMA) is a hyaluronic acid molecular chain that introduces methacrylic groups to give it photocuring ability. While retaining its original properties, it solidifies into a gel within 10 seconds under visible light irradiation, and has strong cell-loading capacity and material scalability. Kartogenin (KGN), as a newly discovered small molecule, has the advantages of good stability, low immunogenicity, and easy regeneration compared with growth factors. It has broad application prospects in cartilage regeneration and protection. Based on the above background, the present invention utilizes 3D printing technology, combines xiphoid cartilage decellularized matrix and light-cured hyaluronic acid hydrogel, loads bone marrow mesenchymal stem cells and Kartogenin, a small molecule drug that induces cartilage differentiation, to construct a cartilage-promoting tissue engineering trachea with a cartilage ring bionic structure. Summary of the invention

[0003] The purpose of the present invention is to target chondrogenesis, one of the difficulties of tissue engineering trachea, by making use of the good biomechanical properties and biocompatibility of polycaprolactone, utilizing the good cartilage repair and regeneration properties of xiphoid cartilage decellularized matrix and methacryloyl hyaluronic acid, and organically combining the three; preparing DCM-HAMA bio-ink loaded with BMSCs / KGN, and constructing a C-shaped cartilage ring bionic structure on the composite scaffold through photocuring 3D bioprinting technology, giving full play to the multidirectional differentiation potential of bone marrow mesenchymal stem cells and the stable cartilage differentiation inducing performance of Kartogenin, thereby providing a preparation method of a bionic chondrogenesis-promoting 3D printed tissue engineering trachea.

[0004] The object of the present invention is achieved by: a method for preparing a bionic cartilage-promoting 3D printed tissue engineering trachea, characterized in that it comprises the following steps:

[0005] Step (1), 3D printing polycaprolactone PCL tracheal stent;

[0006] Add PCL material into the barrel of the 3D printer to 3D print the PCL bracket;

[0007] Step (2), obtaining xiphoid cartilage decellularized matrix DCM;

[0008] The rabbit xiphoid cartilage was taken, and the surface muscles, mucosa and other redundant tissues were removed, and then the cartilage was cut into pieces. Under vacuum conditions, the decellularized xiphoid cartilage was obtained by a detergent combined with an enzyme method. The cartilage was washed with distilled water, and the surface moisture was absorbed by filter paper. The cartilage was added into a mortar and ground into powder with liquid nitrogen to obtain a powdered xiphoid cartilage decellularized matrix. The cartilage was then placed in a pepsin solution for digestion, and finally a gel-like DCM was obtained.

[0009] Step (3), preparation of the composite scaffold;

[0010] First, prepare a methacryloyl hyaluronic acid HAMA solution, then soak the PCL scaffold printed in step (1) in the methacryloyl hyaluronic acid HAMA solution at 37° C. for 15 minutes, and fully cross-link under blue light for 5 minutes to allow the HAMA to evenly wrap the PCL scaffold to obtain a HAMA / PCL composite scaffold;

[0011] Step (4), culturing and identifying bone marrow mesenchymal stem cells (BMSCs);

[0012] BMSCs were cultured by whole bone marrow adherent culture method; BMSCs were identified by tri-lineage differentiation and flow cytometry analysis;

[0013] Step (5), preparation of bio-ink;

[0014] The xiphoid cartilage decellularized matrix obtained in step (2) is added to the HAMA solution and stirred to mix; the extracted BMSCs and Kartogenin (KGN) are added to the DCM-HAMA composite solution and transferred to the barrel of a light-curing 3D printer as bio-ink for subsequent printing;

[0015] Step (6), constructing a C-shaped cartilage ring bionic structure by using photocuring 3D bioprinting technology;

[0016] The bio-ink obtained in step (5) is applied to the surface of the HAMA / PCL composite scaffold obtained in step (3) through photocuring three-dimensional bioprinting to print a C-shaped cartilage ring with a biomimetic structure.

[0017] In step (2), the decellularized matrix is ​​obtained by vacuum detergent combined with enzyme method, specifically 1% SD + 1% Triton X-100, 37°C, 100r, shaking for 24 hours; 2 ku DNAse, 37°C, 100r, shaking for 36 hours. To achieve the decellularization effect, other concentrations, other times, other detergents, and even other decellularization methods can also be selected.

[0018] In step (3), the methacryloyl hyaluronic acid can also be combined with methacryloyl chitosan, methacryloyl chondroitin sulfate and other light-curable hydrogels that are beneficial to cartilage repair and regeneration.

[0019] In step (5), the small molecule drug Kartogenin that induces cartilage differentiation can also be selected from other growth factors, such as TGF-β1.

[0020] In step (6), the method for constructing the C-shaped cartilage ring bionic structure is:

[0021] The HAMA / PCL composite scaffold prepared in step (3) was placed on the rotating axis of the photocurable 3D bioprinter, and the bio-ink containing DCM-HAMA loaded with BMSCs and Kartogenin, a small molecule drug that induces cartilage differentiation, was added into the barrel. The printing parameters were set, and the DCM-HAMA / BMSCs / KGN was extruded from the nozzle while being photocured. A C-shaped cartilage ring structure was printed on the composite scaffold, and the gap between the cartilage rings was set to 5 mm.

[0022] The method of the present invention is advanced and scientific. The present invention provides a method for preparing a bionic chondrogenic 3D printed tissue engineering trachea, which comprises the following steps: step (1), 3D printing of polycaprolactone (PCL) tracheal stent; step (2), obtaining xiphoid cartilage decellularized matrix (DCM); step (3), preparing a composite stent; first preparing a methacrylylated hyaluronic acid (HAMA) solution, then immersing the PCL stent printed in step (1) into the solution, immersing at 37°C for 15 minutes, and fully cross-linking under blue light for 5 minutes, so that HAMA uniformly wraps the PCL stent; step (4), culturing and identifying bone marrow mesenchymal stem cells (BMSCs); step (5), preparing biological ink; and step (6), constructing a C-shaped cartilage ring bionic structure by light-curing three-dimensional bioprinting technology. Through the present invention, a bionic chondrogenic tracheal substitute is prepared. PCL can provide good mechanical properties; the decellularized matrix derived from natural cartilage tissue is more suitable for cartilage regeneration in terms of structure and composition as a biomaterial; HAMA has good biocompatibility, strong cell-loading capacity and material scalability, is cheap and degradable, and is green and pollution-free; 3D printing can achieve individual customization and accurately construct a bionic C-shaped cartilage ring structure. The present invention provides a new idea for clinical tracheal replacement therapy, especially tissue engineering tracheal chondrogenesis.

[0023] The present invention combines natural materials and synthetic materials to prepare a composite scaffold, which becomes a better strategy and provides a place and environment for cell adhesion, growth, proliferation and differentiation. The ultimate purpose of the tracheal stent is not for permanent implantation. As the stent is gradually degraded and absorbed in the body, the seed cells continue to proliferate and differentiate in the body under the action of cytokines and microenvironment, and finally form the tissue or organ required by the body, thereby achieving the purpose of damaged tissue repair and organ function reconstruction. Therefore, constructing a bionic structure and promoting its chondrogenesis is the long-term solution to ensure the mechanical properties of implanted trachea. The present invention is reasonably designed and bionic in structure. It makes full use of the characteristics of the decellularized matrix of xiphoid cartilage and methacryloyl hyaluronic acid, and exerts the role of stem cells and differentiation-inducing drugs, so that the bionic chondrogenic tracheal substitute prepared by the present invention provides a new idea for clinical tracheal replacement therapy and is expected to achieve clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a scanning electron micrograph of the microstructure of DCM-HAMA composite hydrogel;

[0025] Figure 2 This is a scanning electron micrograph of the microstructure of methacryloylated hyaluronic acid loaded cells;

[0026] Figure 3 It is a bionic composite trachea substitute. Implementation

[0027] The present invention is further described below in conjunction with specific implementation modes.

[0028] A method for preparing a bionic cartilage-promoting 3D printed tissue engineering trachea, specifically:

[0029] 1. 3D printed polycaprolactone (PCL) tracheal stent;

[0030] PCL material was added into the barrel, and the printing nozzle was used to print on the rotating shaft. The printing temperature was set to 90°C: the rotating shaft rotated at a certain speed, and the printing nozzle moved back and forth along the rotating shaft. The printing speed was 5 mm / s. The bottom layer of tracheal stent was first printed on the rotating shaft; then the rotating shaft rotated in the opposite direction to the bottom layer, and the second layer of tracheal stent was printed, and then a porous structure was formed with the previous layer. This was repeated for 6 layers with a layer thickness of 0.1 mm.

[0031] 2. Obtaining xiphoid cartilage decellularized matrix (DCM);

[0032] The rabbit xiphoid cartilage was obtained, and the surface muscles, mucosa and other redundant tissues were removed, and then cut into pieces and put into ddH 2 O and wash several times. Transfer to a vacuum blood collection tube and add ddHO. 2 O, 4°C, 24h; aspirate ddH 2 O, add 1% SD + 1% Triton X-100, aspirate to vacuum state, put into shaker, 37℃, 100r, 24h; aspirate the detergent, use ddH 2 O for several times, add 2 ku DNAse, aspirate to vacuum state, put into shaker, 37℃, 100r, 36h. Aspirate DNAse to obtain decellularized xiphoid cartilage; wash with distilled water, remove surface moisture with filter paper, add into mortar, grind into powder with liquid nitrogen, obtain powdered xiphoid cartilage decellularized matrix; prepare pepsin solution, dissolve 2 mg pepsin in 1mL 0.1M HCL, dissolve 0.1 g powder in 1mL pepsin solution, put into shaker, 37℃, 100r, 24h; add 1 / 10 volume of 10×PBS to the dissolved decellularized xiphoid cartilage solution to terminate digestion, adjust pH to about 7.4 with 1.0M NaOH, leave at room temperature for half an hour, and finally obtain gel-like DCM.

[0033] 3. Preparation of composite scaffolds;

[0034] First, prepare the methacryloyl hyaluronic acid (HAMA) solution: First, prepare a 0.25% (w / v) initiator standard solution: Take 20 mL of PBS, add it to a brown bottle containing 0.05g of initiator LAP, and heat it in a 40-50℃ water bath to dissolve it for 15 min, shaking it several times during the process. Then, weigh the required mass of HAMA solid at a concentration of 2% and put it into a centrifuge tube. Add the initiator standard solution to the above centrifuge tube, stir / shake it to dissolve it at room temperature for 0.5-1 h, and place it at 4℃ for later use. Then, immerse the PCL scaffold printed in step (1) in the solution, immerse it at 37℃ for 15 minutes, and fully cross-link it under blue light for 5 minutes to make the HAMA evenly wrap the PCL scaffold to obtain a HAMA / PCL composite scaffold.

[0035] 4. Cultivation and identification of bone marrow mesenchymal stem cells (BMSCs);

[0036] The rabbit was anesthetized, and the bone marrow puncture needle was slowly inserted into the left and right tibial plates. When there was a clear breakthrough, the puncture needle was fixed, and the bone marrow (about 2 ml) was extracted with a 5 ml syringe containing 1 ml of heparin. In the clean bench, the mixture was injected into a 15 ml centrifuge tube, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded; PBS was added to mix, and the mixture was centrifuged at 1000 r / min for 5 min, and the supernatant was discarded; 10 ml of F12 medium was added for resuspending, and the mixture was inoculated into a culture bottle and placed in a 37°C, 5% CO 2 Incubator. After that, the medium was changed every 48 h, and other cells were removed by the adherent screening method using the characteristics of BMSCs to grow on the wall. When the cells in the culture bottle grew close to 80% confluence, they were passaged at a ratio of 1:2-1:3, and the second generation was used for experiments. Cell identification was performed through three-lineage induction experiments of adipogenesis, chondrogenesis and osteogenesis, as well as conventional flow cytometry analysis.

[0037] 5. Preparation of bio-ink;

[0038] The xiphoid cartilage decellularized matrix obtained in step (2) was added to the HAMA solution at a volume ratio of 1:1, and stirred to obtain a DCM-HAMA composite solution; Kartogenin (KGN) was prepared at a concentration of 10 mM, and a small amount of HAMA solution was blown and mixed with the BMSCs precipitate obtained by passage centrifugation. The precipitate was added together with KGN to the DCM-HAMA composite solution to prepare bio-ink, which was quickly transferred to the barrel of a light-curing 3D printer for subsequent printing.

[0039] 6. Constructing C-shaped cartilage ring biomimetic structure through photocuring 3D bioprinting technology;

[0040] The HAMA / PCL composite scaffold prepared in step (3) was placed on the rotating shaft of the photocuring 3D bioprinter, the bio-ink prepared in step (5) was added to the barrel, the printing parameters were set, and the DCM-HAMA / BMSCs / KGN was extruded from the nozzle while being photocured, and a C-shaped cartilage ring structure was printed on the composite scaffold, and the gap between the cartilage rings was set to 5 mm. The hardness of the hydrogel can be adjusted by adjusting the photocuring time.

Claims

1. A method for preparing a bionic cartilage-promoting 3D printed tissue engineering trachea, characterized in that: The following steps are involved: Step (1), 3D printing polycaprolactone PCL tracheal stent; Add PCL material into the barrel of the 3D printer and 3D print the PCL bracket; Step (2), obtaining xiphoid cartilage decellularized matrix DCM; The rabbit xiphoid cartilage was taken, and the surface muscles, mucosa and other redundant tissues were removed, and then the cartilage was cut into pieces. Under vacuum conditions, the decellularized xiphoid cartilage was obtained by a detergent combined with an enzyme method. The cartilage was washed with distilled water, and the surface moisture was absorbed by filter paper. The cartilage was added into a mortar and ground into powder with liquid nitrogen to obtain a powdered xiphoid cartilage decellularized matrix. The cartilage was then placed in a pepsin solution for digestion, and finally a gel-like DCM was obtained. Step (3), preparation of the composite scaffold; First, prepare a methacryloyl hyaluronic acid HAMA solution, then soak the PCL scaffold printed in step (1) in the methacryloyl hyaluronic acid HAMA solution at 37° C. for 15 minutes, and fully cross-link under blue light for 5 minutes to allow the HAMA to evenly wrap the PCL scaffold to obtain a HAMA / PCL composite scaffold; Step (4), culturing and identifying bone marrow mesenchymal stem cells (BMSCs); BMSCs were cultured by whole bone marrow adherent culture method; BMSCs were identified by tri-lineage differentiation and flow cytometry analysis; Step (5), preparation of bio-ink; The xiphoid cartilage decellularized matrix obtained in step (2) is added to the HAMA solution and stirred to mix; the extracted BMSCs and Kartogenin (KGN) are added to the DCM-HAMA composite solution and transferred to the barrel of a light-curing 3D printer as bio-ink for subsequent printing; Step (6), constructing a C-shaped cartilage ring biomimetic structure by using photocuring 3D bioprinting technology; The bio-ink obtained in step (5) is applied to the surface of the HAMA / PCL composite scaffold obtained in step (3) by photocuring three-dimensional bioprinting to print a C-shaped cartilage ring with a biomimetic structure; In step (2), the decellularized matrix was obtained by vacuum detergent combined with enzyme method, specifically 1% SD + 1% TritonX-100, 37°C, 100r, shaking for 24 hours; 2 ku DNAse, 37°C, 100r, shaking for 36 hours; In step (6), the method for constructing the C-shaped cartilage ring bionic structure is: The HAMA / PCL composite scaffold prepared in step (3) was placed on the rotating axis of the photocurable 3D bioprinter, and the bio-ink containing DCM-HAMA loaded with BMSCs and Kartogenin, a small molecule drug that induces cartilage differentiation, was added into the barrel. The printing parameters were set, and the DCM-HAMA / BMSCs / KGN was extruded from the nozzle while being photocured. A C-shaped cartilage ring structure was printed on the composite scaffold, and the gap between the cartilage rings was set to 5 mm.

2. The method for preparing a bionic cartilage-promoting 3D printed tissue engineering trachea according to claim 1, characterized in that: In step (3), methacryloyl hyaluronic acid is replaced by a photocurable hydrogel which is combined with methacryloyl chitosan and methacryloyl chondroitin sulfate to facilitate cartilage repair and regeneration.

3. The method for preparing a bionic cartilage-promoting 3D printed tissue engineering trachea according to claim 1, characterized in that: In step (5), Kartogenin, a small molecule drug that induces cartilage differentiation, is replaced by TGF-β1.

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