CN112972760A

CN112972760A - Endothelial extracellular matrix-loaded 3D printing bone defect repair stent and preparation method thereof

Info

Publication number: CN112972760A
Application number: CN202110198400.8A
Authority: CN
Inventors: 江千舟; 涂欣冉; 郭吕华; 张阳; 郭黎洋; 谭国忠; 陈荣丰
Original assignee: Stomatological Hospital of Guangzhou Medical University
Current assignee: Stomatological Hospital of Guangzhou Medical University
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2021-06-18
Anticipated expiration: 2041-02-22
Also published as: CN112972760B

Abstract

The invention discloses a 3D printing bone defect repairing support loaded with endothelial extracellular matrix, which is prepared from raw materials including gelatin, sodium alginate and 58S bioglass, wherein 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; and S3, taking out the support, performing cell removal treatment, and performing freeze-drying to obtain the scaffold. The scaffold is made of gelatin, sodium alginate and 58S bioglass, is prepared by adopting a 3d printing technology, is simple to operate, and has controllable structure and framework; the inventor researches and discovers that the extracellular matrix loaded with endothelial cells can promote osteogenesis and angioblast differentiation, and the scaffold loaded with the extracellular matrix can promote the formation of bone tissues and vascular tissues at the defect part, thereby obviously improving the efficiency of repairing bone defects.

Description

Endothelial extracellular matrix-loaded 3D printing bone defect repair stent and preparation method thereof

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 support loaded with endothelial extracellular matrix and a preparation method thereof.

Background

With the increase of bone tissue injuries caused by traumas such as aging, joint degenerative changes, car accidents and the like, the bone defect repair is more and more emphasized, and bone grafting methods such as autologous bone grafting, allogeneic bone grafting, artificial bone grafting and the like are generally adopted in clinic. Autologous bone grafting is the 'gold standard' for defect repair, but the autologous bone has limited sources and is often in short supply, allogeneic bone grafting has the risk of infectious diseases, artificial bone grafting lacks bone induction activity and has poor osteogenesis efficiency, and the new tissue with a structure similar to that of healthy bone tissue is difficult to form. Therefore, the research on novel regenerative bone defect repair materials with high biological activity and capable of promoting bone formation efficiently becomes a difficult point and a hot point in recent years, and has huge clinical requirements and market prospects.

The bioactive glass is an important scaffold material for bone tissue engineering, can effectively promote biomineralization in vivo, and release silicon and calcium ions to promote stem cell osteogenesis and vascularization. The gelatin/sodium alginate hydrogel is formed by mixing natural polymer materials, has the advantages of good biocompatibility, tissue absorbability, low immunogenicity and the like, is particularly beneficial to being combined with inorganic powder with high biological activity for 3D printing forming, but has poor mechanical strength. The 3D printing can effectively construct the porous bioglass bone tissue engineering scaffold, accurately regulate and control parameters such as porosity and pore diameter, endow the scaffold with better bioactivity, and generally need to have good mechanical property, biocompatibility, osteoconductivity and osteoinductivity for repairing the scaffold. CN201911197154.3 discloses a preparation method of an antibacterial scaffold for bone repair, firstly, gelatin and sodium alginate are dissolved in ultrapure water to obtain gelatin/sodium alginate slurry; mixing 58s bioactive glass with gelatin/sodium alginate slurry, stirring uniformly, and placing into a 3d printing cylinder for defoaming to obtain gelatin/sodium alginate/biological glass composite slurry; printing the slurry into a three-dimensional porous structure scaffold by using a 3d printer, and performing freeze drying treatment to obtain a gelatin/sodium alginate/bioglass composite bone repair scaffold; placing the composite bone repair scaffold in polydopamine solution for reacting for one night, and placing the reacted scaffold in silver nitrate solution after the reaction is finished; washing the scaffold with deionized water, and freeze-drying to obtain the antibacterial scaffold for bone repair. The antibacterial stent of the above patent focuses on improving the antibacterial effect of the stent to reduce the probability of inflammation when applied to a human body, but the effect of the stent in promoting the osteogenesis and the angiogenesis of stem cells is not studied, so that the use effect of the stent is difficult to be expected by those skilled in the art.

On the basis of the composite material bracket, in order to further improve the osteogenesis efficiency of the bracket, the bracket is loaded with growth promoting factors or medicines, so that another effective means for improving the bone tissue repair is provided. Among them, the application of growth factors in artificially synthesized composite biological scaffolds has attracted great interest for efficient promotion of cell proliferation and differentiation and formation of functional proteins in vivo. The addition of growth factors such as bone matrix protein 2 (BMP-2) to the composite scaffold can promote osteogenic differentiation of stem cells, but because of its short half-life in vivo, in order to maintain an effective dose for a long time, a large amount of growth factors need to be added to the scaffold, exceeding a safe standard dose of 1.5mg/ml, thereby causing a series of adverse reactions such as inflammation, ectopic bone and tumor formation. Therefore, how to further improve the bone tissue formation efficiency without causing adverse reactions is a problem to be urgently solved at present.

Disclosure of Invention

The invention aims to provide a 3D printing bone defect repairing support loaded with an endothelial extracellular matrix, which is composed of gelatin, sodium alginate and 58S bioglass, wherein the gelatin and the sodium alginate have good biocompatibility and biodegradability and are beneficial to adhesion and proliferation of cells, and the 58S bioglass has a function of locally releasing calcium ions and promotes generation of bone tissues. The inventor researches and discovers that the extracellular matrix loaded with endothelial cells can promote osteogenesis and angioblast differentiation, and the scaffold loaded with the extracellular matrix can promote the formation of bone tissues and vascular tissues at the defect part, thereby obviously improving the efficiency of repairing bone defects.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

A3D printing bone defect repairing support loaded with an endothelial extracellular matrix comprises 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;

s3, taking out the support, carrying out decellularization treatment on the support through a decellularization treatment solution, and freeze-drying to obtain the scaffold; the decellularization treatment solution comprises the following components: Triton-100X at a concentration of 0.1% by volume and 20mM ammonia.

The invention adopts gelatin/sodium alginate/58S bioglass composite material as the scaffold matrix, thereby improving the osteogenesis efficiency of the scaffold. The Sodium Alginate (SA) gel has a three-dimensional culture structure suitable for cell nutrient exchange, and can maintain a specific form formed by large surface area and many pores. It has excellent biocompatibility, cell adhesion, biodegradability and bioactive factor loading capacity. Gelatin (Gel) has been widely used in the food and drug industry and medical devices for many years as a protein extracted from natural animal leather. The mechanical strength of the Gel/SA composite hydrogel is obviously improved compared with that of the hydrogel with a single component, and the internal environment required by cell growth can be well simulated. The 58S Bioglass (BG) can stably release calcium ions in the bracket, provides a local slightly alkaline microenvironment and is beneficial to the formation of mineralized nodules of new bone tissues.

Extracellular matrix (ECM) is a collective term for a series of proteins and other components secreted by cells, and plays a key role in cell signal transduction, regulation of cell physiological functions, and the like. The results of a large number of in vitro cell experiments show that ECM has been successfully used for various tissue regeneration as the optimal medium for inducing cells to perform their physiological functions. Particularly, the research of the invention discovers that the gelatin/sodium alginate/58S bioglass composite bracket is loaded with the extracellular matrix of rat vascular endothelial cells (RAOEC) and is applied to bone defect repair, the area of the formed vascular tissue and the number of branches are obviously improved, the formation of the bone tissue and the vascular tissue is effectively promoted, and the efficiency of bone defect repair is obviously improved.

The invention researches and optimizes the process parameters of the extracellular matrix loaded with rat vascular endothelial cells (RAOEC) on the stent so as to obtain good loading effect. In the preparation process of the scaffold loaded with the endothelial extracellular matrix according to the present invention, preferably, the sterilization treatment of S1 is: and (4) 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. Preferably, in S2, the specific steps of seeding the scaffold with RAOEC cells are as follows: will RAOEC cell suspension at 1X 10⁵The cells were seeded in each sterilized rack at a cell density of 5X 10⁴Per ml; the scaffolds were placed in wells of 12-well plates, and 2ml of cell suspension was instilled per well. Preferably, in S3, the specific steps of the decellularization process are as follows: soaking the scaffold in the cell-free treatment solution for 30min, sequentially washing with PBS for 3 times, treating with DNAase for 5min, washing with PBS for 3 times, and storing in a refrigerator at 4 deg.C.

In the present invention, preferably, 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, and physically crosslinking the semi-finished support by using a calcium chloride solution, and then soaking the semi-finished support in a glutaraldehyde solution for chemical crosslinking; and finally, cleaning and freeze-drying to obtain the product.

The support is prepared by a 3D printing technology, the operation is simple, and the structure and the framework of the support are controllable. The invention further researches and optimizes the parameter details in the 3D printing preparation process, such as a needle head for printing, air pressure, temperature, printing speed and the like. The support obtained by printing is better in structural stability and mechanical strength. Preferably, in S2, the 3D printing paste is injected into the 3D printing cartridge, and printing is started after bubble removal and homogenization. Preferably, in S2, the solution is stirred uniformly by magnetic stirring and/or mechanical stirring to obtain the 3D printing paste.

Preferably, the concentration of the calcium chloride solution is 5% -8%, and the calcium chloride solution is prepared by adding calcium chloride powder into distilled water for dissolving. Preferably, the glutaraldehyde solution has a concentration of 1.0% to 2.0% and is obtained by diluting a 50% glutaraldehyde solution with distilled water. The concentration of the calcium chloride solution and the concentration of the glutaraldehyde solution are selected, so that the cross-linking effect of the stent is better.

In the present invention, preferably, the chemical composition of the 58s bioglass is 58% SiO₂-33％CaO-9％P₂O₅And the particle size of the 58s bioglass is 1-3 microns. The 58s bioglass with the specification has excellent biological performance and obvious bone repair effect.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention adopts 3d printing technology to prepare the bracket, the operation is simple, and the structure and the framework of the bracket are controllable. The research optimizes the details of parameters in the 3D printing preparation process, such as a printing needle, air pressure and temperature, printing speed and the like. The support obtained by printing is better in structural stability and mechanical strength.

2. According to the invention, researches show that the extracellular matrix of rat vascular endothelial cells (RAOEC) is loaded on the gelatin/sodium alginate/58S bioglass composite scaffold, the scaffold is applied to bone defect repair, the area of formed vascular tissues and the number of branches are obviously increased, the formation of the bone tissues and the vascular tissues is effectively promoted, and the efficiency of bone defect repair is obviously improved.

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

4. The preparation method is simple in preparation process and easy to operate, the selected material is high in bioactivity, and the application prospect in the fields of regenerative medicine and bone repair is wide.

Drawings

FIG. 1 is a photograph of an extracellular matrix loaded 3D printed bone defect repair scaffold standard of the present invention (a: front photograph, b: side photograph).

FIG. 2 PCR assay of extracellular matrix-loaded 3D-printed bone defect repair scaffolds of the present invention shows the expression profiles of the relevant genes (a: BMP-2, b: CD31, c: RUNX2, D: OCN, e: VEGF).

FIG. 3 is a test chart of the compressive strength of the extracellular matrix loaded 3D printed bone defect repair scaffold of the present invention.

FIG. 4 is an animal experiment osteogenesis efficiency chart of the extracellular matrix loaded 3D printing bone defect repair scaffold (left: the scaffold group of the invention, middle: blank group, right: BIO-OSS bone meal positive control group).

FIG. 5 is a water absorption and swelling ratio test chart of the extracellular matrix loaded 3D printed bone defect repair scaffold of the present invention (a: water absorption test chart, b: swelling ratio test chart).

FIG. 6 is an experimental diagram of in vitro degradation detection of the extracellular matrix loaded 3D printed bone defect repair scaffold of the present invention.

FIG. 7DAPI staining shows the adhesion and proliferation profile of RAOEC on the surface of the scaffolds.

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

FIG. 9 the surface CD31 antigen of the scaffold was positive after RAOEC inoculation for 14 d.

FIG. 10 shows that the surface VEGF antigen of the scaffold was positive after 14 days of RAOEC inoculation.

Fig. 11 proliferative activity of RBMSC cells on extracellular matrix loaded 3D printed bone defect repair scaffolds.

Detailed Description

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

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

Wherein the chemical composition of the adopted 58s bioglass is 58 percent SiO₂-33％CaO-9％P₂O₅And the particle size of the 58s bioglass is 1-3 microns.

Example 1:

preparation of a 3D printing bone defect repair scaffold loaded with endothelial extracellular matrix:

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 gelatin, 6% of sodium alginate and 10.5% of 58S bioglass.

S2, uniformly stirring the solution in a magnetic stirring and mechanical stirring mode to obtain 3D printing slurry, injecting the 3D printing slurry into a 3D printing material cylinder, starting 3D printing after bubble removal and homogenization, and printing at the printing speed of 10mm/s by adopting a needle head with the aperture of 0.41mm in 3D printing under the conditions of 0.38Mpa of air pressure and 28 ℃.

S3, printing to obtain a semi-finished support, and physically crosslinking the semi-finished support by using a calcium chloride solution, and then soaking the semi-finished support in a glutaraldehyde solution for chemical crosslinking; and finally, cleaning and freeze-drying to obtain the scaffold. The pore diameter of the bracket is 0.6mm, and the porosity of the bracket is 68 percent; the concentration of the calcium chloride solution is 5%, and the concentration of the glutaraldehyde solution is 1.0%.

S4, disinfecting the support prepared by the 3D printing; the cells were sterilized by UV irradiation for 30min, and then rinsed 3 times with absolute ethanol, followed by 3 rinses with sterile PBS.

S5, inoculating the disinfected bracket with RAOEC cells, and enabling the suspension of the RAOEC cells to be at a ratio of 1 × 10⁵The cells were seeded in each sterilized rack at a cell density of 5X 10⁴Per ml; the scaffolds were placed in wells of 12-well plates, and 2ml of cell suspension was instilled per well. The cells were co-cultured for 14d with a change of medium every 3 d.

S6, taking out the scaffold, soaking the scaffold for 30min by using the cell-free treatment solution, then sequentially washing the scaffold for 3 times by using PBS (phosphate buffer solution), treating the scaffold for 5min by using DNAase, washing the scaffold for 3 times by using PBS, then placing the scaffold into a refrigerator at 4 ℃ for storage, and freeze-drying the scaffold to obtain the DNA. The components of the decellularization treatment solution are as follows: Triton-100X at a concentration of 0.1% by volume and 20mM ammonia.

Example 2:

S2, uniformly stirring the solution in a magnetic stirring and mechanical stirring mode to obtain 3D printing slurry, injecting the 3D printing slurry into a 3D printing material cylinder, starting 3D printing after bubble removal homogenization, and printing at the printing speed of 10mm/s by adopting a needle head with the aperture of 0.43mm in 3D printing under the conditions of 0.40Mpa of air pressure and 27 ℃.

S3, printing to obtain a semi-finished support, and physically crosslinking the semi-finished support by using a calcium chloride solution, and then soaking the semi-finished support in a glutaraldehyde solution for chemical crosslinking; and finally, cleaning and freeze-drying to obtain the scaffold. The pore diameter of the bracket is 0.7mm, and the porosity of the bracket is 60 percent; the concentration of the calcium chloride solution is 8 percent, and the concentration of the glutaraldehyde solution is 2.0 percent.

S4, disinfecting the support prepared by the 3D printing; UV radiation sterilization for 35min, followed by 3 washes with absolute ethanol, followed by 3 washes with sterile PBS.

Example 3:

S2, uniformly stirring the solution through magnetic stirring and mechanical stirring to obtain 3D printing slurry, and preparing a support through 3D printing; in the 3D printing operation, pour into 3D printing thick liquids into 3D and print the feed cylinder, begin to print after removing the bubble homogenization, adopt the syringe needle of 0.40mm aperture, under 0.35Mpa atmospheric pressure, 29 ℃ conditions, print according to 10 mm/s' printing speed.

S3, printing to obtain a semi-finished support, and physically crosslinking the semi-finished support by using a calcium chloride solution, and then soaking the semi-finished support in a glutaraldehyde solution for chemical crosslinking; and finally, cleaning and freeze-drying to obtain the scaffold. The pore diameter of the bracket is 0.5mm, and the porosity of the bracket is 75 percent; the concentration of the calcium chloride solution is 6 percent, and the concentration of the glutaraldehyde solution is 1.5 percent.

S4, disinfecting the support prepared by the 3D printing; the cells were sterilized by UV irradiation for 40min, and then washed 3 times with absolute ethanol, followed by 3 times with sterile PBS.

Performance testing

The endothelial extracellular matrix-loaded 3D-printed bone defect repair scaffold prepared in example 1 above was subjected to performance testing as follows:

1. and taking a picture, wherein the structural size of the endothelial extracellular matrix loaded 3D printing bone defect repair scaffold is shown in the attached drawing 1.

2. The expression of the genes related to osteogenesis and angiogenesis is tested, and as shown in FIG. 2, the genes related to the extracellular matrix loaded 3D printing bone defect repair scaffold (a: BMP-2, b: CD31, c: RUNX2, D: OCN, e: VEGF) are highly expressed by PCR assay.

The PCR method is as follows:

cells were washed 3 times with pre-cooled PBS and media and other impurities were removed. 1ml of Trizol lysate is added into each well, and the mixture is blown and evenly mixed for 2 min. The blown-up mixture was transferred to a 1ml EP tube (without immediate RNA extraction, it was transferred to a-80 ℃ freezer for further use). Adding 200 mul of chloroform into the mixed solution of each EP tube, shaking for 15s, and standing for 15min on ice after the mixed solution is emulsion. After the mixed solution is layered, the mixed solution is transferred into a low-temperature centrifuge for centrifugation at 12000r/min at 4 ℃ for 15 min. After centrifugation, 400. mu.l of the supernatant was carefully aspirated, transferred to a new EP tube, 500. mu.l of isopropanol was added, mixed by inversion for 10s, left to stand on ice for 10min at 12000r/min, and centrifuged at 4 ℃ for 10min, whereupon a white RNA precipitate was visible at the bottom of the EP tube. Discarding the supernatant, adding 1ml of 75% ethanol into the precipitate, shaking, 7500r/min, centrifuging at 4 deg.C for 5min, and removing the supernatant. Adding 1ml of absolute ethyl alcohol into the precipitate, shaking, 7500r/min, centrifuging at 4 ℃ for 5min, and removing the supernatant. The EP tube was inverted on filter paper and air dried for 8-10 min. To the precipitate, 15. mu.l of DEPC-treated water was added, and after being blown and sufficiently dissolved, the RNA concentration was measured.

RNA reverse transcription reaction

The RNA was diluted to 1000 ng/. mu.l, placed on ice until each sample was mixed at the volume of Table 1-1, transferred to a 200. mu.l EP tube, and blown up uniformly. The EP tube was centrifuged, placed in a conventional PCR instrument, programmed and run. The EP tube was removed and mixed well as in Table 1-2. The EP tube was centrifuged, placed in a conventional PCR instrument, programmed and run. After reverse transcription was completed, the sample was taken out and placed in a refrigerator at-20 ℃ for later use.

TABLE 1-1 reverse transcription reaction System

TABLE 1-2 qRT-PCR reaction System

qRT-PCR

Reaction plates were designed and 96-well plates were placed on ice, and each reaction well was charged with various reagents according to tables 1-3. The 96-well plate was sealed with a sealing membrane and centrifuged at 2000r/min for 2 min. The plate was placed into the reaction module of a CFX96 fluorescence quantitative PCR instrument, the program was prepared and run. And after the operation is finished, recording the data.

TABLE 1-3 qRT-PCR reaction System

3. And (4) testing the compressive strength, wherein the test piece size is 10mm multiplied by 4.5 mm. The test method comprises the following steps: and placing the fully-swollen extracellular matrix-loaded 3D printed bone defect repair support on a universal mechanical tester, so that the center of a pressure plate of the tester is coincided with the center of the support. The tester is started and stopped when the upper pressure plate is just contacted with the bracket. Debugging the parameters of the tester, loading at the speed of 1mm/min, stopping loading when the load is 1kN, calculating the Young modulus through software, drawing a load curve, and taking an average value after 3 experiments. As shown in fig. 3, the average young's modulus was measured: 265.63 MPa.

4. Animal experiments show that the 3D printed bone defect repair scaffold loaded with the endothelial cell extracellular matrix has good osteogenesis efficiency as shown in figure 4 (left: the scaffold group of example 1, middle: blank group, right: BIO-OSS bone powder positive control group).

The experimental method is as follows:

the rat is subjected to abdominal injection anesthesia in a sterile state, a 1.0-1.5 cm-centimeter incision is made on the lower edge of a parallel mandible, the mandible is exposed in a blunt separation mode after subcutaneous tissues are cut in a layering mode, a 5 mm-diameter annular bone drill is used for being matched with physiological saline to conduct perfusion and cooling, a 5 mm-diameter circular full-layer bone defect is manufactured, and a 3D printing bone defect repairing support loaded with endothelial cell extracellular matrix and BIO-OSS collagen are implanted respectively. The blank group is not provided with any material, the wound in the tissue is sutured layer by adopting a 5-0 suture line, and the penicillin sodium intramuscular injection is used for anti-infection continuously for 3 days after the operation.

Micro-CT osteogenesis analysis

The materials were taken at two time points of 4 weeks and 8 weeks, the rats were euthanized by carbon dioxide asphyxiation, the mandible within the defect area was extracted and fixed in 10% neutral buffered formalin for 24 hours, and then Micro-CT was used for scanning. And scanning and reconstructing the image file by using NReco software Skyscan, and analyzing the condition of the new bone tissue at the defect part. Fig. 4 shows the bone defect repair at 8 week time point.

5. Water absorption, swelling ratio and in vitro degradability. The swelling rate of the endothelial extracellular matrix loaded 3D printed bone defect repair scaffold reached a maximum after soaking in SBF for 2 hours (fig. 5), the scaffold morphology remained essentially stable after soaking in SBF for 4 weeks, and mass loss was approximately 16% after 16 weeks (fig. 6). SBF is a simulated body fluid, which is composed of a plurality of inorganic salt solutions and simulates the components and pH value of the body fluid of a human body.

6. The state representation of the RAOEC cells loaded on the surface of the stent, the RAOEC cells are planted on the stent for culture, and the DAPI staining result shows that the RAOEC cells can be adhered to the stent and proliferate (figure 7); SEM examination and observation of the adhesion condition of the RAOEC cells on the stent after the permanent planting culture for 14d, and as a result, the surface of the RAOEC cells can be adhered and stretched on the surface of the stent (shown in figure 8).

7. The status of the decellularization process is characterized in that after the RAOEC is inoculated on the scaffold 7/14/21D, the 3D printing bone defect repair scaffold loaded with the RAOEC is decellularized, and the immunofluorescence detection of markers CD31 and VEGF of the ECM is carried out, and the result is observed under a laser confocal microscope, and the result shows that the CD31 and VEGF on the surface of the scaffold are positive after 14D, and the ECM of the RAOEC exists on the surface of the scaffold (figures 9 and 10).

8. Testing the proliferation activity of RBMSC cells on the 3D printing bone defect repair scaffold loaded with the extracellular matrix; RBMSC cells were cultured at 10⁵And each is inoculated on a 3D printing bone defect repair bracket loaded with extracellular matrix, the sample is taken at 0/7/14/21D, the sample is replaced by complete culture medium containing 10 percent cck-8, the culture medium is incubated for 30min at 37 ℃, and then the culture medium is placed in an enzyme labeling instrument to observe the absorbance at 490nm wavelength. As shown in fig. 11, RBMSC cells had good proliferative activity on the extracellular matrix-loaded 3D-printed bone defect repair scaffold.

From the test results, the 3D printing bone defect repairing scaffold loaded with the endothelial extracellular matrix achieves the aim of the invention, the loading effect is good, the obtained composite scaffold is excellent in structural stability and mechanical strength, and the scaffold is applied to bone defect repair, can effectively promote the formation of bone tissues and vascular tissues and obviously improves the efficiency of bone defect repair.

Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

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%;

s1, disinfecting the support prepared by 3D printing;

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:

3. The endothelial extracellular matrix-loaded 3D-printed bone defect repair scaffold according to claim 1, wherein the sterilization treatment of S1 is: and (4) 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, wherein in S2, the specific steps of scaffold seeding with RAOEC cells are as follows: the RAOEC cell suspension is applied at 1X 10⁵The cells were seeded in each sterilized rack at a cell density of 5X 10⁴Per ml; the scaffolds were placed in wells of 12-well plates, and 2ml of cell suspension was instilled per well.

5. The endothelial extracellular matrix-loaded 3D-printed bone defect repair scaffold according to claim 1, wherein in S3, the decellularization process comprises the following specific steps: soaking the scaffold in the cell-free treatment solution for 30min, sequentially washing with PBS for 3 times, treating with DNAase for 5min, washing with PBS for 3 times, and storing in a refrigerator at 4 deg.C.

6. 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% SiO₂-33％CaO-9％P₂O₅And the particle size of the 58s bioglass is 1-3 microns.

7. 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% to 8% and is prepared by dissolving calcium chloride powder in distilled water.

8. 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.

9. 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 homogenization.

10. 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.