CN112972760B - Endothelial extracellular matrix loaded 3D printing bone defect repair support and preparation method thereof - Google Patents

Abstract

The invention discloses a 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix. The preparation raw materials of the scaffold include gelatin, sodium alginate and 58S bioglass, the pore diameter of the scaffold is 0.5-0.7 mm, and the porosity of the scaffold is 0.5-0.7 mm. 60-75%; the preparation process of the scaffold loaded with endothelial extracellular matrix is as follows: S1. Sterilize the scaffold prepared by 3D printing; S2. Inoculate the sterilized scaffold with RAOEC cells, change the medium every 3 days, and co-culture the cells 14d; S3. The scaffold was taken out, decellularized, and lyophilized. The scaffold of the present invention is composed of gelatin, sodium alginate, and 58S bioglass, and is prepared by 3D printing technology. Angiogenic differentiation, the scaffold loaded with the above-mentioned extracellular matrix can promote the formation of bone tissue and vascular tissue at the defect site, and significantly improve the efficiency of bone defect repair.

Description

A 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix and its preparation method

technical field

The invention belongs to the technical field of bone tissue engineering repair and reconstruction, and mainly relates to a 3D printing bone defect repair scaffold loaded with endothelial extracellular matrix and a preparation method thereof.

Background technique

With the increase of bone tissue damage caused by trauma such as aging, joint degenerative diseases, and traffic accidents, more and more attention has been paid to the repair of bone defects. Autologous bone transplantation is the "gold standard" for defect repair, but the source of autologous bone is limited, and the supply is often in short supply. Allogeneic bone transplantation has the risk of infectious diseases, while artificial bone transplantation lacks osteoinductive activity and has poor osteogenesis efficiency. Organizing nascent tissues with similar structures. Therefore, the research on new regenerative bone defect repair materials with high biological activity and efficient osteogenesis has become a difficult and hot spot in recent years, and has huge clinical needs and market prospects.

Bioactive glass is an important scaffold material for bone tissue engineering, which can effectively promote biomineralization in vivo, release silicon and calcium ions, and promote stem cell osteogenesis and angiogenesis. Gelatin/sodium alginate hydrogel is a mixture of natural polymer materials, which has the advantages of good biocompatibility, tissue absorbability, low immunogenicity, etc., especially it is beneficial to combine with highly biologically active inorganic powders 3D printing is carried out, but its mechanical strength is relatively poor. 3D printing can effectively construct porous bioglass bone tissue engineering scaffolds, precisely control parameters such as porosity and pore size, and endow them with better biological activity. Repair scaffolds usually need to have good mechanical properties, biocompatibility, osteoconductivity, bone inductive. CN201911197154.3 discloses a preparation method of an antibacterial scaffold for bone repair. First, dissolve gelatin and sodium alginate in ultrapure water to obtain gelatin/sodium alginate slurry; mix 58s bioactive glass with gelatin/ The sodium alginate slurry was mixed, stirred evenly, and then put into the 3D printing barrel for defoaming to obtain a gelatin/sodium alginate/bioglass composite slurry; the slurry was printed into a three-dimensional porous structure with a 3D printer, and freeze-dried The gelatin/sodium alginate/bioglass composite bone repair scaffold was obtained; the composite bone repair scaffold was placed in a polydopamine solution to react overnight, and after the reaction was completed, it was placed in a silver nitrate solution; the scaffold was rinsed with deionized water and freeze-dried to obtain Antimicrobial scaffolds that can be used for bone repair. The antibacterial stent of the above-mentioned patent focuses on improving the antibacterial effect of the stent to reduce the probability of inflammation when used in the human body, but the effect of the stent on promoting stem cell osteogenesis and angiogenesis has not been studied, so those skilled in the art use it. The effect is unpredictable.

On the basis of the composite scaffold, in order to further improve the osteogenic efficiency of the scaffold, loading growth-promoting factors or drugs on the scaffold has become another effective means to improve bone tissue repair. Among them, the application of growth factors in artificially synthesized composite bioscaffolds has aroused great interest of researchers because of its efficient promotion of cell proliferation and differentiation and the formation of functional proteins in vivo. Adding growth factors such as bone matrix protein-2 (BMP-2) to the composite scaffold can promote the osteogenic differentiation of stem cells, but due to its short half-life in vivo, in order to maintain an effective dose for a long time, it needs to be added in the scaffold. It was added in large quantities, exceeding the safe standard dose of 1.5 mg/ml, thus causing a series of adverse reactions, such as inflammation, ectopic bone and tumor formation. Therefore, how to further improve the efficiency of bone tissue formation without causing adverse reactions has become an urgent problem to be solved.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix, which is composed of gelatin, sodium alginate, and 58S bioglass, wherein gelatin and sodium alginate have good biocompatibility and biodegradability 58S bioglass has the function of local release of calcium ions and promotes the formation of bone tissue. Furthermore, the inventors found that the extracellular matrix loaded with endothelial cells can promote osteogenic and angiogenic differentiation, and the scaffold loaded with the above-mentioned extracellular matrix can promote the formation of bone tissue and vascular tissue at the defect site, and significantly improve the efficiency of bone defect repair.

For achieving the above object, the technical scheme provided by the invention is as follows:

A 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix, comprising a scaffold and an endothelial extracellular matrix loaded on the scaffold, the preparation raw materials of the scaffold include gelatin, sodium alginate and 58S bioglass, and the pore size of the scaffold is 0.5 ~0.7mm, the porosity of the stent is 60-75%;

The preparation process of the scaffold-loaded endothelial extracellular matrix is as follows:

S1. Sterilize the scaffold prepared by 3D printing;

S2. The sterilized scaffold was inoculated with RAOEC cells, the medium was changed every 3 days, and the cells were co-cultured for 14 days;

S3. Take out the scaffold, decellularize it with a decellularization treatment solution, and freeze-dry it; the decellularization treatment solution includes the following components: Triton-100X with a volume percent concentration of 0.1% and 20 mM ammonia water.

The present invention adopts gelatin/sodium alginate/58S bioglass composite material as the scaffold matrix, thereby improving the osteogenic efficiency of the scaffold. Sodium alginate (SA) gel has a three-dimensional culture structure suitable for cell nutrient exchange, and can maintain a specific morphology due to its large surface area and many pores. It has good biocompatibility, cell adhesion, biodegradability and bioactive factor loading capacity. As a protein extracted from natural animal leather, gelatin (Gel) has been widely used in the food and drug industry and medical devices for many years. The mechanical strength of Gel/SA composite hydrogel is significantly improved compared with that of single-component hydrogel, and it can better simulate the internal environment required for cell growth. 58S bioglass (BG) can stably release calcium ions in the scaffold, providing a local weak alkaline microenvironment, which is conducive to the formation of mineralized nodules of new bone tissue.

Extracellular matrix (ECM) is a general term for a series of proteins and other components secreted by cells, which play a key role in cell signal transduction and regulation of cell physiological functions. At present, the results of a large number of in vitro cell experiments show that ECM, as the most ideal medium for inducing cells to perform their physiological functions, has been successfully used in various tissue regeneration. In particular, the present invention has found through research that the extracellular matrix of rat vascular endothelial cells (RAOEC) is loaded on the above-mentioned gelatin/sodium alginate/58S bioglass composite scaffold and applied to the repair of bone defects. The number of branches is significantly increased, which effectively promotes the formation of bone tissue and vascular tissue, and significantly improves the efficiency of bone defect repair.

The invention researches and optimizes the process parameters of the extracellular matrix loaded with rat vascular endothelial cells (RAOEC) on the scaffold to obtain a good loading effect. In the preparation process of the stent-loaded endothelial extracellular matrix of the present invention, preferably, the disinfection treatment described in S1 is: ultraviolet radiation disinfection for 25-40 minutes, then rinsed with absolute ethanol for several times, and then rinsed with sterile PBS for several times. Preferably, in S2, the specific steps of seeding the scaffolds with RAOEC cells are as follows: seed the RAOEC cell suspension on each sterilized scaffold at 1×10 ⁵ /well, and the cell density is 5×10 ⁴ /ml; 2ml of cell suspension was dripped into each well of a 12-well plate. Preferably, in S3, the specific steps of decellularization treatment are as follows: soak the scaffold with decellularization treatment solution for 30 minutes, then sequentially wash with PBS for 3 times, treat with DNAase for 5 minutes, wash with PBS for 3 times and store in a 4°C refrigerator.

In the present invention, preferably, the preparation of the stent comprises the following steps:

S1. Dissolve gelatin, sodium alginate and 58S bioglass in distilled water to obtain a solution, wherein the mass/volume concentration of each component in the solution is gelatin 15%, sodium alginate 6%, 58S bioglass 10.5%;

S2. Stir the solution evenly to obtain a 3D printing paste, and prepare a scaffold by 3D printing; in the 3D printing operation, a needle with a diameter of 0.41mm is used, under the conditions of 0.38Mpa air pressure and 28°C, at a printing speed of 10mm/s Print;

S3. The semi-finished stent is obtained after printing. The semi-finished stent is first physically cross-linked with calcium chloride solution, and then immersed in glutaraldehyde solution for chemical cross-linking. Finally, it is washed and freeze-dried.

The above bracket is prepared by 3D printing technology, the operation is simple, and the structure and structure of the bracket are controllable. The present invention further studies and optimizes the parameter details in the 3D printing preparation process, such as the needle used for printing, as well as air pressure and temperature, printing speed, and the like. The printed scaffolds are superior in structural stability and mechanical strength. Preferably, in S2, the 3D printing paste is injected into the 3D printing barrel, and the printing is started after de-foaming and homogenization. Preferably, in S2, the solution is stirred uniformly by magnetic stirring and/or mechanical stirring to obtain a 3D printing slurry.

Preferably, the concentration of the calcium chloride solution is 5% to 8%, which is obtained by adding calcium chloride powder into distilled water to dissolve. Preferably, the concentration of the glutaraldehyde solution is 1.0% to 2.0%, which is obtained by diluting a 50% glutaraldehyde solution with distilled water. The choice of the concentration of calcium chloride solution and glutaraldehyde solution makes the cross-linking effect of the scaffold better.

In the present invention, preferably, the chemical composition of the 58S bioglass is 58% SiO ₂ -33% CaO-9% P ₂ O ₅ , and the particle size of the 58S bioglass is 1-3 microns. The 58S bioglass with the above specifications has excellent biological properties and has a remarkable effect in bone repair.

Compared with the prior art, the beneficial effects of the present invention:

1. The present invention adopts the 3D printing technology to prepare the bracket, the operation is simple, and the structure and structure of the bracket are controllable. The research optimizes the details of parameters in the 3D printing preparation process, such as the needle used for printing, as well as air pressure and temperature, printing speed, etc. The printed scaffolds are superior in structural stability and mechanical strength.

2. After research in the present invention, it is found that the extracellular matrix of rat vascular endothelial cells (RAOEC) is loaded on the gelatin/sodium alginate/58S bioglass composite scaffold and applied to the repair of bone defects, and the area of the formed vascular tissue and the number of branches are significant. It can effectively promote the formation of bone tissue and vascular tissue, and significantly improve the efficiency of bone defect repair.

3. The present invention studies and optimizes the preparation process parameters of the extracellular matrix loaded with rat vascular endothelial cells (RAOEC) on the gelatin/sodium alginate/58S bioglass scaffold, and obtains a good loading effect.

4. The preparation process of the present invention is simple and easy to operate, and the selected materials have high biological activity, and have broad application prospects in the fields of regenerative medicine and bone repair.

Description of drawings

Figure 1 is a photo of the 3D printed bone defect repair scaffold standard part loaded with extracellular matrix of the present invention (a: front photo, b: side photo).

Figure 2 Gene expression map related to PCR determination of the 3D printed bone defect repair scaffold loaded with extracellular matrix of the present invention (a: BMP-2, b: CD31, c: RUNX2, d: OCN, e: VEGF).

Fig. 3 Compressive strength test chart of the 3D printed bone defect repair scaffold loaded with extracellular matrix of the present invention.

4 is a graph of the osteogenesis efficiency of the animal experiment of the 3D printed bone defect repair scaffold loaded with extracellular matrix of the present invention (left: scaffold group of the present invention, middle: blank group, right: BIO-OSS bone powder positive control group).

Figure 5. The water absorption and swelling rate test diagrams of the 3D printed bone defect repair scaffold loaded with extracellular matrix of the present invention (a: water absorption test diagram, b: swelling rate test diagram).

Figure 6 Experiment diagram of in vitro degradation detection of the 3D printed bone defect repair scaffold loaded with extracellular matrix of the present invention.

Figure 7 DAPI staining showing the adhesion and proliferation of RAOEC on the scaffold surface.

Fig. 8 SEM observation of the surface microstructure of the 3D printed bone defect repair scaffold loaded with extracellular matrix and the adhesion and extension of RAOEC on the scaffold (arrows indicate RAOEC).

Fig. 9 The surface of the scaffold was positive for CD31 antigen 14 days after RAOEC inoculation.

Fig. 10 The surface of the scaffold was positive for VEGF antigen 14 days after RAOEC inoculation.

Figure 11. Proliferation activity of RBMSC cells on 3D printed bone defect repair scaffolds loaded with extracellular matrix.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments of the description, but the protection scope of the present invention is not limited to the embodiments.

The raw materials used in the following examples are commercially available unless otherwise specified.

The chemical composition of the 58S bioglass used is 58% SiO ₂ -33% CaO-9% P ₂ O ₅ , and the particle size of the 58S bioglass is 1-3 microns.

Embodiment 1:

Preparation of a 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix:

S1. Dissolve gelatin, sodium alginate and 58S bioglass in distilled water to obtain a solution, wherein the mass/volume concentration of each component in the solution is 15% gelatin, 6% sodium alginate, and 10.5% 58S bioglass.

S2. The solution is stirred evenly by magnetic stirring and mechanical stirring to obtain a 3D printing slurry. The 3D printing slurry is injected into the 3D printing barrel, and the 3D printing starts after de-foaming and homogenization. Under the conditions of 0.38Mpa air pressure and 28°C, print at a printing speed of 10mm/s.

S3. The semi-finished stent is obtained after printing. The semi-finished stent is first physically cross-linked with calcium chloride solution, and then immersed in glutaraldehyde solution for chemical cross-linking. Finally, the stent is obtained by washing and freeze-drying. The pore diameter of the stent is 0.6 mm, and the porosity of the stent is 68%; the concentration of the calcium chloride solution is 5%, and the concentration of the glutaraldehyde solution is 1.0%.

S4. Sterilize the scaffold prepared by 3D printing; sterilize by ultraviolet radiation for 30 minutes, then rinse with absolute ethanol for 3 times, and then rinse with sterile PBS for 3 times.

S5. The sterilized scaffolds were seeded with RAOEC cells, and the RAOEC cell suspension was seeded on each sterilized scaffold at 1×10 ⁵ /well, and the cell density was 5×10 ⁴ /ml; the scaffolds were placed in a 12-well plate 2 ml of cell suspension was dripped into each well. The medium was changed every 3 days, and the cells were co-cultured for 14 days.

S6. Take out the scaffold, soak the scaffold in the decellularization treatment solution for 30 minutes, then wash it with PBS for 3 times, treat it with DNAase for 5 minutes, wash it with PBS for 3 times, store it in a 4°C refrigerator, and freeze-dry it. The components of the decellularization treatment solution are as follows: 0.1% Triton-100X and 20mM ammonia water.

Embodiment 2:

Preparation of a 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix:

S1. Dissolve gelatin, sodium alginate and 58S bioglass in distilled water to obtain a solution, wherein the mass/volume concentration of each component in the solution is 15% gelatin, 6% sodium alginate, and 10.5% 58S bioglass.

S2. The solution is stirred evenly by magnetic stirring and mechanical stirring to obtain the 3D printing slurry. The 3D printing slurry is injected into the 3D printing barrel, and the 3D printing starts after de-foaming and homogenization. The 3D printing uses a needle with a 0.43mm aperture. Under the conditions of 0.40Mpa air pressure and 27°C, print at a printing speed of 10mm/s.

S3. The semi-finished stent is obtained after printing. The semi-finished stent is first physically cross-linked with calcium chloride solution, and then immersed in glutaraldehyde solution for chemical cross-linking. Finally, the stent is obtained by washing and freeze-drying. The pore diameter of the stent is 0.7 mm, and the porosity of the stent is 60%; the concentration of the calcium chloride solution is 8%, and the concentration of the glutaraldehyde solution is 2.0%.

S4. Sterilize the scaffold prepared by 3D printing; sterilize by ultraviolet radiation for 35 minutes, then rinse with absolute ethanol for 3 times, and then rinse with sterile PBS for 3 times.

S5. The sterilized scaffolds were seeded with RAOEC cells, and the RAOEC cell suspension was seeded on each sterilized scaffold at 1×10 ⁵ /well, and the cell density was 5×10 ⁴ /ml; the scaffolds were placed in a 12-well plate 2 ml of cell suspension was dripped into each well. The medium was changed every 3 days, and the cells were co-cultured for 14 days.

S6. Take out the scaffold, soak the scaffold in the decellularization treatment solution for 30 minutes, then wash it with PBS for 3 times, treat it with DNAase for 5 minutes, wash it with PBS for 3 times, store it in a 4°C refrigerator, and freeze-dry it. The components of the decellularization treatment solution are as follows: 0.1% Triton-100X and 20mM ammonia water.

Embodiment 3:

Preparation of a 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix:

S1. Dissolve gelatin, sodium alginate and 58S bioglass in distilled water to obtain a solution, wherein the mass/volume concentration of each component in the solution is 15% gelatin, 6% sodium alginate, and 10.5% 58S bioglass.

S2. Stir the solution uniformly by magnetic stirring and mechanical stirring to obtain a 3D printing slurry, and prepare a bracket by 3D printing; during the 3D printing operation, inject the 3D printing slurry into the 3D printing barrel, and start printing after de-foaming and homogenization. Using a needle with a 0.40mm aperture, under the conditions of 0.35Mpa air pressure and 29°C, print at a printing speed of 10mm/s.

S3. The semi-finished stent is obtained after printing. The semi-finished stent is first physically cross-linked with calcium chloride solution, and then immersed in glutaraldehyde solution for chemical cross-linking. Finally, the stent is obtained by washing and freeze-drying. The pore diameter of the stent is 0.5 mm, and the porosity of the stent is 75%; the concentration of the calcium chloride solution is 6%, and the concentration of the glutaraldehyde solution is 1.5%.

S4. Sterilize the scaffold prepared by 3D printing; sterilize by ultraviolet radiation for 40 minutes, then rinse with absolute ethanol for 3 times, and then rinse with sterile PBS for 3 times.

S5. The sterilized scaffolds were seeded with RAOEC cells, and the RAOEC cell suspension was seeded on each sterilized scaffold at 1×10 ⁵ /well, and the cell density was 5×10 ⁴ /ml; the scaffolds were placed in a 12-well plate 2 ml of cell suspension was dripped into each well. The medium was changed every 3 days, and the cells were co-cultured for 14 days.

S6. Take out the scaffold, soak the scaffold in the decellularization treatment solution for 30 minutes, then wash it with PBS for 3 times, treat it with DNAase for 5 minutes, wash it with PBS for 3 times, store it in a 4°C refrigerator, and freeze-dry it. The components of the decellularization treatment solution are as follows: 0.1% Triton-100X and 20mM ammonia water.

Performance Testing

The performance test of the 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix prepared in Example 1 above is as follows:

1. Taking pictures, the structural dimensions of the 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix are shown in Figure 1.

2. Test the expression of genes related to osteogenesis and angiogenesis. As shown in Figure 2, the related genes (a: BMP-2, b: CD31, c: RUNX2, d) were determined by PCR of the 3D printed bone defect repair scaffold loaded with extracellular matrix. : OCN, e: VEGF) were highly expressed.

The PCR method is as follows:

Cells were washed 3 times with pre-cooled PBS to remove medium and other impurities. Add 1 ml of Trizol lysis buffer to each well, and mix by pipetting for 2 min. Transfer the pipetting mixture to a 1ml EP tube (transfer to a -80°C refrigerator for later use if RNA is not to be extracted immediately). Add 200 μl of chloroform to the mixture in each EP tube, shake for 15 s, and place the mixture on ice for 15 min after it becomes an emulsion. After the mixed liquid was separated into layers, it was transferred to a low temperature centrifuge at 12000 r/min and centrifuged at 4°C for 15 min. After centrifugation, carefully aspirate 400 μl of supernatant, transfer it to a new EP tube, add 500 μl of isopropanol, invert and mix for 10 s, stand on ice for 10 min, centrifuge at 12000 r/min for 10 min at 4°C, and at this time, it can be seen that the bottom of the EP tube has White RNA pellet. Discard the supernatant, add 1 ml of 75% ethanol to the pellet, shake, centrifuge at 7500 r/min for 5 min at 4°C, and remove the supernatant. Add 1 ml of absolute ethanol to the precipitate, shake at 7500 r/min, centrifuge at 4°C for 5 min, and remove the supernatant. Put the EP tube upside down on the filter paper and air dry for 8-10min. Add 15 μl of DEPC-treated water to the pellet, pipet it, and detect the RNA concentration after it is fully dissolved.

RNA reverse transcription reaction

Dilute the RNA to 1000ng/μl, put it on ice for use, mix each sample according to the volume in Table 1-1, transfer it into a 200μl EP tube, and pipette evenly. The EP tube was centrifuged, placed in a conventional PCR machine, programmed and run. Take out the EP tube and mix well according to Table 1-2. The EP tube was centrifuged, placed in a conventional PCR machine, programmed and run. After the reverse transcription is completed, the samples are taken out and placed in a -20°C refrigerator for later use.

Table 1-1 Reverse transcription reaction system

Table 1-2 qRT-PCR reaction system

qRT-PCR

Design the reaction plate, put the 96-well plate on ice, and add various reagents to each reaction well according to Table 1-3. The 96-well plate was sealed with a blocking membrane and centrifuged at 2000 r/min for 2 min. Put the plate into the reaction module of the CFX96 real-time PCR instrument, adjust the program and run. After the run is complete, record the data.

Table 1-3 qRT-PCR reaction system

3. Compressive strength test, the size of the specimen is 10mm×10mm×4.5mm. Test method: Place the fully swollen 3D printed bone defect repair scaffold loaded with extracellular matrix on the universal chemical tester, so that the center of the tester's platen and the center of the scaffold coincide. Turn on the tester and stop when the upper platen just touches the bracket. Adjust the parameters of the tester, load at a speed of 1mm/min, stop the load when the load is 1kN, calculate the Young's modulus through the software, draw the load curve, and take the average value of 3 experiments. As shown in Figure 3, the average Young's modulus was measured: 265.63 MPa.

4. Animal experiments, the bone defect repair situation after implanting the 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix in the rat jaw defect model is shown in Figure 4 (left: the scaffold group of Example 1, middle: blank group, Right: BIO-OSS bone powder positive control group), experiments show that the 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix has good osteogenic efficiency.

The experimental method is as follows:

The rats were anesthetized by intraperitoneal injection under aseptic state, and a 1.0-1.5 cm incision was made on the lower edge of the parallel mandible. The subcutaneous tissue was incised in layers and then bluntly dissected to expose the mandible. A 5mm diameter trephine drill was used with saline perfusion for cooling. A circular full-thickness bone defect with a diameter of 5 mm was fabricated, and 3D printed bone defect repair scaffolds loaded with endothelial extracellular matrix and BIO-OSS collagen were implanted respectively. In the blank group, no material was placed, the wounds in the tissue were sutured with 5-0 suture in layers, and intramuscular injection of penicillin sodium was given for 3 consecutive days after the operation.

Micro-CT osteogenesis analysis

The samples were collected at 4 weeks and 8 weeks, and the rats were euthanized by carbon dioxide asphyxiation. The mandibles including the defect area were excised and fixed in 10% neutral buffered formalin for 24 hours, and then scanned by Micro-CT. The NRecon software Skyscan was used to scan and reconstruct the image files to analyze the new bone tissue in the defect site. Figure 4 shows the bone defect repair at the 8-week time point.

5. Detection of water absorption, swelling rate and in vitro degradability. The 3D-printed bone defect repair scaffold loaded with endothelial extracellular matrix reached the maximum swelling rate after soaking in SBF for 2 hours (Fig. 5), and the shape of the scaffold remained basically stable after soaking in SBF for 4 weeks. was 16% (Figure 6). SBF is a simulated body fluid, which is composed of a variety of inorganic salt solutions and simulates the composition and pH of human body fluids.

6. Characterization of the state of RAOEC cells loaded on the surface of the scaffold. The RAOEC cells were colonized on the scaffold for culture. The DAPI staining results showed that RAOEC could adhere and proliferate on the scaffold (Fig. 7). As a result, the surface RAOECs could adhere and stretch on the surface of the stent (Fig. 8).

7. Characterization of the state of decellularization. After the RAOECs were seeded on the scaffolds on 7/14/21d, the 3D printed bone defect repair scaffolds loaded with RAOECs were decellularized and the ECM markers CD31 and VEGF were detected by immunofluorescence. Observation under the laser confocal microscope showed that CD31 and VEGF on the scaffold surface were positive after 14 days, indicating the existence of RAOEC ECM on the scaffold surface (Figures 9 and 10).

8. Test the proliferation activity of RBMSC cells on the 3D printed bone defect repair scaffold loaded with extracellular matrix; 10 ⁵ RBMSC cells were seeded on the 3D printed bone defect repair scaffold loaded with extracellular matrix, at 0/7/ Samples were taken on 14/21d, replaced with complete medium containing 10% cck-8, incubated at 37 °C for 30 min, placed on a microplate reader, and the absorbance at 490 nm was observed. As shown in Figure 11, the proliferation activity of RBMSC cells on the 3D printed bone defect repair scaffold loaded with extracellular matrix was good.

From the above test results, the 3D printed bone defect repair scaffold loaded with endothelial extracellular matrix prepared by the present invention achieves the purpose of the present invention, the loading effect is good, and the obtained composite scaffold has better structural stability and mechanical strength. Applied to bone defect repair, it can effectively promote the formation of bone tissue and vascular tissue, and significantly improve the efficiency of bone defect repair.

Based on the disclosure and teaching of the above specification, those skilled in the art to which the present invention pertains can also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the protection scope of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims (8)

1. The 3D printing bone defect repairing support loaded with the endothelial extracellular matrix is characterized by comprising a support and the endothelial extracellular matrix loaded on the support, wherein the preparation raw materials of the support comprise gelatin, sodium alginate and 58S bioglass, the pore diameter of the support is 0.5-0.7 mm, and the porosity of the support is 60-75%;

the preparation process of the stent loaded with the endothelial extracellular matrix comprises the following steps:

s1, disinfecting the support prepared by 3D printing;

s2, inoculating the disinfected bracket with RAOEC cells, changing the liquid every 3 days, and co-culturing the cells for 14 days;

the specific steps for inoculating the RAOEC cells on the scaffold are as follows: the RAOEC cell suspension is diluted at 1X 10 ⁵ The cells were seeded at 5X 10 density per sterilized scaffold ⁴ Per ml; placing the scaffold in the wells of a 12-well plate, and dripping 2ml of cell suspension into each well;

s3, taking out the bracket, carrying out decellularization treatment on the bracket by using a decellularization treatment solution, and freeze-drying to obtain the scaffold; the decellularization treatment solution comprises the following components: triton-100X with the volume percentage concentration of 0.1% and 20mM ammonia water;

the specific steps of the decellularization treatment are as follows: soaking the scaffold for 30min by using the cell-free treatment solution, then sequentially washing the scaffold for 3 times by using PBS, treating the scaffold for 5min by using DNAase, washing the scaffold for 3 times by using PBS, and then putting the scaffold into a refrigerator at 4 ℃ for storage.

2. The endothelial extracellular matrix-loaded 3D printed bone defect repair scaffold according to claim 1, wherein the preparation of the scaffold comprises the steps of:

s1, dissolving gelatin, sodium alginate and 58S bioglass in distilled water to obtain a solution, wherein the mass/volume concentration of each component in the solution is 15% of the gelatin, 6% of the sodium alginate and 10.5% of the 58S bioglass;

s2, uniformly stirring the solution to obtain 3D printing slurry, and preparing a support through 3D printing; in the 3D printing operation, a needle head with the aperture of 0.41mm is adopted, and printing is carried out at the printing speed of 10mm/s under the conditions of 0.38Mpa and 28 ℃;

s3, printing to obtain a semi-finished support, wherein the semi-finished support is physically crosslinked by using a calcium chloride solution and then is soaked in a glutaraldehyde solution for chemical crosslinking; and finally, cleaning and freeze-drying to obtain the product.

3. The endothelial extracellular matrix-loaded 3D-printed bone defect repair scaffold according to claim 1, wherein the sterilization treatment S1 is: sterilizing by ultraviolet radiation for 25-40 min, washing with absolute ethyl alcohol for multiple times, and then washing with sterile PBS for multiple times.

4. The endothelial extracellular matrix-loaded 3D-printed bone defect repair scaffold according to claim 1 or 2, wherein the chemical composition of the 58S bioglass is 58% SiO% ₂ -33％CaO-9％P ₂ O ₅ The particle size of the 58S bioglass is 1-3 microns.

5. The endothelial extracellular matrix-loaded 3D printed bone defect repair scaffold according to claim 2, wherein the calcium chloride solution has a concentration of 5-8% and is prepared by dissolving calcium chloride powder in distilled water.

6. The endothelial extracellular matrix-loaded 3D-printed bone defect repair scaffold according to claim 2, wherein the glutaraldehyde solution has a concentration of 1.0% to 2.0% obtained by diluting a 50% glutaraldehyde solution with distilled water.

7. The endothelial extracellular matrix-loaded 3D printed bone defect repair scaffold according to claim 2, wherein in S2, a 3D printing slurry is injected into the 3D printing cartridge and printing is started after bubble removal and homogenization.

8. The endothelial extracellular matrix-loaded 3D printed bone defect repair scaffold according to claim 2, wherein in S2, the solution is stirred uniformly by magnetic stirring and/or mechanical stirring to obtain 3D printed slurry.

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