A method for preparing a 3D bioprinted tracheal substitute loaded with EPCs

Abstract

The present invention provides a method for preparing a 3D bioprinted tracheal substitute loaded with EPCs, comprising the following steps: S1, preparing a complete and immunogenic decellularized tracheal matrix; S2, preparing a LAP‑NC‑dtECM hydrogel; S3, isolating and culturing EPCs; S4, preparing a LAP‑NC‑dtECM bioprinting ink loaded with EPCs; S5, preparing a 3D bioprinted tracheal substitute loaded with EPCs. The present invention combines the decellularized trachea to retain the complete natural extracellular matrix components, effectively removes the cell nucleus and immune substances, has excellent characteristics such as non-immunogenicity, good biocompatibility, and can effectively induce angiogenesis in vivo, and prepares it into a hydrogel, adds bone marrow EPCs to prepare a printable bio-ink, and combines 3D bioprinting technology to reconstruct a biomimetic tracheal stent with good biomechanical properties and angiogenesis properties.

Description

Technical Field

[0001] The present invention belongs to the field of tissue engineering and regenerative medicine, and specifically relates to a method for preparing a 3D bioprinted trachea substitute loaded with EPCs. Background Art

[0002] 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 and promote cell and tissue growth. Tissue engineering technology combines cells with 3D scaffolds to construct a highly bionic physiological and biochemical microenvironment to promote tissue growth, which is an effective method for tracheal reconstruction. However, tracheal transplantation is still a difficult clinical problem worldwide. The main reasons are the lack of ideal tracheal substitutes and the obstacles to graft vascularization caused by special anatomical structures. The ideal tracheal substitute must meet the following requirements at the same time: it has appropriate elasticity and pressure resistance, good biocompatibility, no toxic side effects, supports cell adhesion and proliferation, and can quickly form a microvascular network to promote cell growth and prevent graft necrosis. The structure and composition of the scaffold material play a vital role in the growth and migration of seed cells and the formation of functional structures.

[0003] Decellularized tracheal extracellular matrix (dtECM) has the function of promoting tissue repair and regeneration in vivo, supporting the adhesion and proliferation of endothelial progenitor cells (EPCs), and has a good effect of inducing angiogenesis in vivo. It can also guide and regulate cell (proliferation, differentiation) responses, and can provide a more ideal microenvironment for cell growth. The biomechanical properties of pure natural decellularized tracheal matrix are reduced, and cells mainly adhere and grow on the surface of the matrix. There is not enough cell growth and angiogenesis inside the graft. 3D printing technology is a new manufacturing technology that has been applied to the personalized printing of bionic tissues and organs. It has the advantages of high manufacturing precision, clear spatial hierarchy, high production efficiency, and good personalized adaptability. Based on the technical bottleneck problems of severe shortage of tracheal grafts in the current reconstructive surgery field, strong immunogenicity of native trachea, and difficulty in obtaining ideal tracheal grafts, the decellularized trachea retains the complete natural extracellular matrix components while effectively removing the cell nucleus and immune substances, and has excellent characteristics such as non-immunogenicity, good biocompatibility, and the ability to effectively induce angiogenesis in vivo. It is prepared into hydrogel through a special process, and bone marrow EPCs are added to prepare printable bio-ink. Combined with 3D bioprinting technology, it is expected to reconstruct a biomimetic tracheal scaffold with non-immunogenicity, good biomechanical properties, and angiogenesis properties, providing an effective source of graft materials for tracheal reconstructive surgery. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a 3D bioprinted tracheal substitute loaded with EPCs, combining the excellent characteristics of decellularized trachea such as extremely low immunogenicity, good biocompatibility, and the ability to effectively induce angiogenesis in vivo, as well as high-temperature 3D printing technology and low-temperature 3D bioprinting technology, to construct a fully biomimetic tracheal stent with angiogenesis performance.

[0005] In order to solve the above technical problems, an embodiment of the present invention provides a method for preparing a 3D bioprinted tracheal substitute loaded with EPCs, comprising the following steps:

[0006] S1. Preparation of intact and immunogenic decellularized tracheal matrix;

[0007] S2, preparation of LAP-NC-dtECM hydrogel;

[0008] S3, isolation and culture of EPCs;

[0009] S4, preparation of LAP-NC-dtECM bioprinting ink loaded with EPCs;

[0010] S5. Preparation of 3D bioprinted tracheal stents;

[0011] S6. Preparation of EPCs-loaded 3D bioprinted tracheal substitutes.

[0012] Wherein, the preparation of the decellularized tracheal matrix in step S1 comprises the following steps:

[0013] First, the fresh animal trachea was frozen in a deep-freezer, then thawed at room temperature, swollen in sterile distilled water under a hypotonic environment, then immersed in sodium deoxycholate and Triton X-100, washed with sterile distilled water, and dissolved in a hypertonic solution of deoxyribonuclease and ribonuclease to obtain a decellularized tracheal matrix without immunogenicity.

[0014] Wherein, step S2 comprises the following steps:

[0015] S2.1. Preparation of NC-dtECM powder: removing cartilage tissue from the decellularized tracheal matrix obtained in step S1, grinding with liquid nitrogen to obtain NC-dtECM powder, freeze-drying and storing at -80°C for later use;

[0016] S2.2, preparation of NC-dtECM hydrogel: the NC-dtECM powder obtained in step S2.1 is added to a 0.1M HCl solution at a concentration of 2% to 20% by mass, and pepsin is added at a weight ratio of 1 / 10 of the NC-dtECM powder. After digestion at 37°C for a certain period of time, 1M NaOH is added to adjust the pH to 7.4 to terminate the digestion, thereby forming a NC-dtECM hydrogel;

[0017] S2.3. Preparation of LAP-NC-dtECM hydrogel: Add photocrosslinker LAP at a concentration of 0.1% to 0.4% by mass to the NC-dtECM hydrogel obtained in step S2.2 to obtain LAP-NC-dtECM hydrogel.

[0018] Wherein, isolating and culturing EPCs in step S3 comprises the following steps:

[0019] The whole bone marrow differential adhesion method was used to isolate and culture EPCs. First, the bone marrow of the matched animal was obtained, placed in a 15 mL sterile centrifuge tube and centrifuged at 800 RPM for 5 min, the supernatant was discarded, fetal bovine serum was added to the DMEM / F12 medium with a content of 10%, and the cells were resuspended. The cells were placed in a cell culture flask and placed in an incubator and cultured at 37°C and 5% CO2 for 20 h. Subsequently, the non-adherent cells were collected and centrifuged at 800 RPM for 5 min, resuspended with EBM-2MV medium, placed in a new cell culture flask and placed in an incubator for culture, and the obtained cells were EPCs.

[0020] Step S4 includes the following steps: dividing the EPCs obtained in step S3 by 1×10 7 / mL was added to the LAP-NC-dtECM hydrogel obtained in step S2 to obtain the EPCs-loaded LAP-NC-dtECM bioprinting ink.

[0021] Wherein, the preparation of the 3D bioprinted tracheal stent in step S5 comprises the following steps:

[0022] S5.1. Preparation of 3D printed PCL tracheal stent: adding PCL material into a 3D bioprinter, and using a high-temperature nozzle to print a single-layer mesh stent on a rotating axis to obtain a 3D printed PCL tracheal stent;

[0023] S5.2. Preparation of 3D bioprinted double-layer tracheal stent: switch the low-temperature nozzle, use the bioprinting ink obtained in step S4 as the material, perform double-layer printing on the basis of PCL, and obtain a double-layer bionic tracheal stent.

[0024] Wherein, in step S5.1, the PCL material is one of polycaprolactone, polylactic acid-co-glycolic acid, polycarbonate, poly-L-lactic acid-caprolactone or poly-L-lactic acid.

[0025] Among them, in step S5.1, a 3D printed PCL tracheal stent is prepared; wherein the process parameters of 3D printing are: barrel temperature is 90°C, print nozzle diameter is 200µm, print speed is 2mm / s, print length is 10mm, and filling mode is sinusoidal filling.

[0026] Among them, in step S5.2, a 3D bioprinted double-layer tracheal stent is prepared, wherein the process parameters of 3D printing are: barrel temperature is 37°C, print nozzle diameter is 200µm, print speed is 2mm / s, print length is 10mm / s, and filling mode is linear filling.

[0027] The beneficial effects of the above technical solution of the present invention are as follows:

[0028] The LAP-NC-dtECM in the present invention is a natural hydrogel with good cell adhesion and angiogenesis induction properties, providing an effective scaffold source for transplanted tracheal vascularization, and the preparation process is simple and easy. Extracting primary animal bone marrow EPCs as vascularization seed cells can further enhance the microvascular formation effect of the transplant in vivo. At the same time, 3D printing technology is used to construct a mesh tracheal scaffold with good biomechanical properties, so that the scaffold has certain compression rebound properties and can effectively match the decellularized tracheal matrix structure. Its unique macroporous structure does not affect the adhesion and proliferation of cells on the surface of the decellularized matrix structure, and synthetic materials and natural materials are effectively integrated to construct a tracheal substitute with good biomechanical properties, in vivo biocompatibility and angiogenesis properties, thereby providing a solid foundation for industrial production and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparative diagram of the quantitative analysis results of DNA between native trachea and decellularized trachea in the present invention;

[0030] Figure 2 This is an observation diagram of NC-dtECM powder in the present invention;

[0031] Figure 3 This is an observation diagram of the gelling properties of NC-dtECM hydrogel at different concentrations in the present invention;

[0032] Figure 4 This is a scanning electron microscope image of the NC-dtECM hydrogel in the present invention;

[0033] Figure 5 This is an optical microscope morphological observation diagram of EPCs in the present invention;

[0034] Figure 6 This is a microscope observation picture of the EPCs in vitro tube formation experiment in the present invention;

[0035] Figure 7 A 3D bioprinted tracheal substitute loaded with EPCs in the present invention;

[0036] Figure 8 This is a comparative analysis diagram of the biomechanical properties of the 3D bioprinted trachea, the native trachea, and the decellularized trachea in the present invention;

[0037] Fig. 9 HE staining microscope observation images of the native trachea (A) and the 3D bioprinted trachea (B) in the present invention after being embedded in vivo for 2 weeks. DETAILED DESCRIPTION

[0038] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0039] The embodiment of the present invention provides a preparation method of a 3D bioprinted tracheal substitute loaded with EPCs, which prepares a decellularized tracheal matrix into a hydrogel, introduces a photoinitiator LAP and vascularized seed cells EPCs, and constructs a bioprinting ink with angiogenesis and 3D printing properties; and then combines high-temperature 3D printing technology and low-temperature 3D bioprinting technology to construct a fully biomimetic tracheal stent with angiogenesis. The following is a detailed description of the biomimetic tracheal preparation method:

[0040] S1. Preparation of complete and immunogenic decellularized tracheal matrix:

[0041] First, the fresh animal trachea was frozen in a deep-freezer, then thawed at room temperature, swollen in sterile distilled water under a hypotonic environment, then soaked in sodium deoxycholate and Triton X-100, washed with sterile distilled water, and dissolved in a hypertonic solution of deoxyribonuclease and ribonuclease. The immunogenicity was removed by histological analysis and DNA quantitative analysis, and finally, a decellularized tracheal matrix with immunogenicity removed was obtained.

[0042] Among them, the DNA quantitative analysis proves that the process of removing immunogenicity is as follows:

[0043] The original tracheal tissue and decellularized matrix samples were ground with liquid nitrogen, dried in a vacuum freeze dryer, and weighed 10 mg each. The sample tissue DNA was extracted using an animal tissue DNA isolation kit, and DNA quantitative analysis was performed using an ELISA instrument. Figure 1 As shown, the DNA content of the decellularized trachea group was 26.78±1.67ng / mg dry weight, which was much lower than that of the native trachea group (641.55±8.55ng / mg dry weight), so it was theoretically considered to have eliminated immunogenicity.

[0044] S2. Preparation of LAP-NC-dtECM hydrogel:

[0045] S2.1. Preparation of NC-dtECM powder: Remove cartilage tissue from the decellularized tracheal matrix obtained in step S1, grind with liquid nitrogen to obtain NC-dtECM powder (eg Figure 2 As shown), freeze-dried and stored at -80°C for later use;

[0046] S2.2, preparation of NC-dtECM hydrogel: 2% to 20% by mass of the NC-dtECM powder obtained in step S2.1 was added to a 0.1M HCl solution, and pepsin was added at a weight ratio of 1 / 10 of the NC-dtECM powder. After digestion at 37°C for a certain period of time, 1M NaOH was added to adjust the pH to 7.4 to terminate the digestion, and the NC-dtECM hydrogel (such as Figure 3 shown);

[0047] S2.3, preparation of LAP-NC-dtECM hydrogel: Adding the photocrosslinker LAP with a concentration of 0.1% to 0.4% by mass to the NC-dtECM hydrogel obtained in step S2.2, further improving the photocuring gelation performance and biomechanical properties of the hydrogel, to obtain LAP-NC-dtECM hydrogel (Scanning electron microscopy observation as shown in the figure) Figure 4 shown);

[0048] S3. Isolation and culture of EPCs:

[0049] The whole bone marrow differential adherence method was used to isolate and culture EPCs: bone marrow was isolated from the femur of a 3-week-old female rabbit, placed in a 15 mL sterile centrifuge tube and centrifuged at 800 RPM for 5 min, the supernatant was discarded and resuspended in DMEM / F12 medium containing 10% fetal bovine serum, added to a cell culture bottle and placed in an incubator for culture at 37°C and 5% CO2 for 20 h, then non-adherent cells were collected and centrifuged at 800 revolutions per minute (RPM) for 5 min, resuspended in EBM-2MV medium, added to a new cell culture bottle and placed in an incubator for culture, and the cells were obtained as EPCs; thereafter, the medium was changed every 48 h, and the second-generation cells were obtained for optical microscopy observation (such as Figure 5 shown);

[0050] In vitro tube formation experiment: Matrigel was placed on ice overnight, and after liquefaction, it was slowly added dropwise to a 48-well plate, 100 μL per well. The 48-well plate with Matrigel was incubated in a 37°C cell incubator for 1 hour to form a gel. 200 μL of 2 × 10 4 The 48-well plate was cultured in a cell culture incubator at 37°C for 24 h, and the formation of cell tubular structures (e.g. Figure 6 as shown).

[0051] S4. Preparation of LAP-NC-dtECM bioprinting ink loaded with EPCs:

[0052] EPCs were divided into 1×10 7 / mL was added to LAP-NC-dtECM hydrogel to prepare bioprinting ink;

[0053] S5. Preparation of 3D bioprinted tracheal stents:

[0054] S5.1. Preparation of 3D printed PCL tracheal stent: According to the dynamic CT image data of the trachea of ​​the experimental animal, a 3D printing rotating shaft matching the original trachea size was customized, PCL material was added to the 3D bioprinter, and a single-layer mesh stent was printed on the rotating shaft using a high-temperature nozzle to obtain a 3D printed PCL tracheal stent. The process parameters of 3D printing were: barrel temperature of 90°C, print nozzle diameter of 200µm, print speed of 2mm / s, print length of 10mm, and filling mode of sinusoidal filling;

[0055] In this embodiment, the PCL material is one of polycaprolactone, polycarbonate, poly-L-lactic acid-caprolactone or poly-L-lactic acid.

[0056] S5.2. Preparation of 3D bioprinted double-layer tracheal stent: Switch the high-temperature nozzle of the 3D bioprinter to a low-temperature nozzle, use the bioprinting ink obtained in step S4 as the material, perform double-layer printing on the PCL basis, enable the UV curing module during the printing process, and obtain a double-layer biomimetic tracheal stent, i.e., a 3D bioprinted tracheal substitute loaded with EPCs (e.g. Figure 7 ), where the 3D printing process parameters are: barrel temperature is 37°C, print nozzle diameter is 200µm, print speed is 2mm / s, print length is 10mm / s, and filling mode is linear filling.

[0057] In order to further explore the biomechanical properties of 3D bioprinted tracheal substitutes loaded with EPCs, the native trachea group (Native), the decellularized trachea group (dtECM), and the 3D bioprinted trachea group loaded with EPCs (3D bioprinting) were tested for biomechanical properties:

[0058] The compression test was conducted using a universal tensile / compression testing machine (Instron, USA). The parameters were set as follows: displacement origin load of 0.01 N, displacement speed of 2 mm / min, maximum displacement limit of 6 mm, maximum load of 2 N, and stress of 50% compression of the tracheal lumen. Figure 8 As shown, compared with the native trachea group (0.45±0.04 N), the 50% compression stress in the decellularized trachea group (0.02±0.0009N) was significantly reduced (P<0.01), while there was no significant statistical difference in the 50% compression stress in the 3D bioprinted trachea group (0.56±0.06 N) (P<0.05).

[0059] To further explore the in vivo angiogenic properties of 3D bioprinted tracheal substitutes loaded with EPCs, they were implanted into Wistar rats, and the grafts were obtained 7 days after surgery for gross observation and histological analysis. Fig. 9 As shown, after 2 weeks of implantation, the original trachea ( Fig. 9 A) Due to its strong immunogenicity, a large number of inflammatory cells infiltrate and no angiogenesis; while the 3D bioprinted tracheal substitute loaded with EPCs ( Fig. 9 B) No obvious inflammatory cell infiltration was observed, but there was a significant angiogenesis effect.

[0060] The present invention innovatively prepares 3D bioprinted vascularized tracheal substitutes. Although there are a variety of methods for preparing decellularized trachea in domestic and foreign studies, there are no reports on the use of the non-cartilage region (NC-dtECM) of the decellularized trachea to prepare hydrogels. In addition, the present invention is the first to prepare and select NC-dtECM hydrogels with different concentrations and add a suitable proportion of photoinitiator (LAP) to prepare LAP-NC-dtECM, which can further improve the gelation and printability of the hydrogel. The hydrogel has good cell adhesion and angiogenesis induction properties, providing an effective scaffold for vascularization of transplanted trachea. Source, in addition, primary animal bone marrow EPCs are extracted as vascularization seed cells, and bio-ink is constructed with the prepared LAP-NC-dtECM in a certain ratio. There is no related report, which will be able to further enhance the microvascular formation effect of the graft in vivo. In terms of structure, a combination of high-temperature printing and low-temperature printing is used to carry out integrated 3D bio-printed hybrid tracheal grafts, which contain 3D printed PCL scaffolds to provide good biomechanical properties. The prepared LAP-NC-dtECM is a natural hydrogel with good biocompatibility, cell adhesion and proliferation properties, and angiogenesis induction properties. The present invention effectively integrates the three, complements each other's advantages, and produces a 3D bio-printed tracheal substitute loaded with EPCs, which effectively promotes the formation of vascularization and functionalization of tracheal grafts after in situ transplantation in vivo, thereby providing a solid foundation for industrial production and clinical application.

[0061] The above is 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 the scope of protection of the present invention.

Claims

1. A method for preparing a 3D bioprinted tracheal substitute loaded with EPCs, characterized in that: The following steps are involved: S1. Preparation of intact and immunogenic decellularized tracheal matrix; S2, preparation of LAP-NC-dtECM hydrogel; S3, isolation and culture of EPCs; S4, preparation of LAP-NC-dtECM bioprinting ink loaded with EPCs; S5. Preparation of 3D bioprinted tracheal substitutes loaded with EPCs by 3D bioprinting; Step S2 includes the following steps: S2.

1. Preparation of NC-dtECM powder: removing cartilage tissue from the decellularized tracheal matrix obtained in step S1, grinding with liquid nitrogen to obtain NC-dtECM powder, freeze-drying and storing at -80°C for later use; S2.2, preparation of NC-dtECM hydrogel: the NC-dtECM powder obtained in step S2.1 is added to a 0.1M HCl solution at a concentration of 2% to 20% by mass, and pepsin is added at a weight ratio of 1 / 10 of the NC-dtECM powder. After digestion at 37°C for a certain period of time, 1M NaOH is added to adjust the pH to 7.4 to terminate the digestion, thereby forming a NC-dtECM hydrogel; S2.3, preparing LAP-NC-dtECM hydrogel: adding a photocrosslinker LAP with a concentration of 0.1% to 0.4% by mass to the NC-dtECM hydrogel obtained in step S2.2 to obtain LAP-NC-dtECM hydrogel; In step S5, preparing the 3D bioprinted tracheal stent includes the following steps: S5.

1. Preparation of 3D printed PCL tracheal stent: adding PCL material into a 3D bioprinter, and using a high-temperature nozzle to print a single-layer mesh stent on a rotating axis to obtain a 3D printed PCL tracheal stent; S5.

2. Preparation of 3D bioprinted double-layer tracheal stent: switch the low-temperature nozzle, use the bioprinting ink obtained in step S4 as the material, perform double-layer printing on the basis of PCL, and obtain a 3D bioprinted tracheal substitute loaded with EPCs.

2. The method for preparing a 3D bioprinted tracheal substitute loaded with EPCs according to claim 1, characterized in that: The preparation of the decellularized tracheal matrix in step S1 includes the following steps: First, the fresh animal trachea was frozen in a deep-freezer, then thawed at room temperature, swollen in sterile distilled water under a hypotonic environment, then immersed in sodium deoxycholate and Triton X-100, washed with sterile distilled water, and dissolved in a hypertonic solution of deoxyribonuclease and ribonuclease to obtain a decellularized tracheal matrix without immunogenicity.

3. The method for preparing a 3D bioprinted tracheal substitute loaded with EPCs according to claim 1, characterized in that: The isolation and cultivation of EPCs in step S3 includes the following steps: The whole bone marrow differential adhesion method was used to isolate and culture EPCs. First, the bone marrow of the matched animal was obtained, placed in a 15 mL sterile centrifuge tube and centrifuged at 800 RPM for 5 min, the supernatant was discarded, fetal bovine serum was added to the DMEM / F12 medium with a content of 10%, and the cells were resuspended. The cells were placed in a cell culture flask and placed in an incubator and cultured at 37°C and 5% CO2 for 20 h. Subsequently, the non-adherent cells were collected and centrifuged at 800 RPM for 5 min, resuspended with EBM-2MV medium, placed in a new cell culture flask and placed in an incubator for culture, and the obtained cells were EPCs.

4. The method for preparing a 3D bioprinted tracheal substitute loaded with EPCs according to claim 1, characterized in that: Step S4 includes the following steps: The EPCs obtained in step S3 were divided into 1×10 7 / mL was added to the LAP-NC-dtECM hydrogel obtained in step S2 to obtain the EPCs-loaded LAP-NC-dtECM bioprinting ink.

5. The method for preparing a 3D bioprinted tracheal substitute loaded with EPCs according to claim 1, characterized in that: In step S5.1, a 3D printed PCL tracheal stent is prepared; wherein the process parameters of 3D printing are: barrel temperature is 90°C, print nozzle diameter is 200µm, print speed is 2mm / s, print length is 10mm, and filling mode is sinusoidal filling.

6. The method for preparing a 3D bioprinted tracheal substitute loaded with EPCs according to claim 1, characterized in that: In step S5.2, a 3D bioprinted double-layer tracheal stent was prepared, wherein the process parameters of 3D printing were: barrel temperature of 37°C, print nozzle diameter of 200µm, print speed of 2mm / s, print length of 10mm / s, and filling mode of linear filling.

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