Bone repair biological scaffold material and preparation method thereof

Abstract

The present invention relates to a bone repair biological scaffold material and a preparation method thereof, which is prepared from raw materials comprising vascular endothelial growth factor VEGF loaded by black phosphorus nanoparticles BPNMs, urine-derived stem cell-derived exosomes USC-Exos, carboxymethyl chitosan CMC, and polyethylene glycol diacrylate PEGDA. It not only has immunomodulatory function and angiogenic function, but also has a dual sustained-release function. On the one hand, cytokines are released by degradation of the material, and on the other hand, the cytokines loaded by black phosphorus are slowly released, thereby achieving a long-lasting release function and solving the shortcomings of fast degradation and short action of active cytokines. The composite gel scaffold prepared by the two-step gelation method in the present application has a double network structure. Compared with the simultaneous gelation method after mixing, it has more complete molecular polymerization efficiency, more complete gelation, greater elasticity and toughness of the gel, and stronger strength.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a bone repair biological scaffold material and a preparation method thereof. Background Art

[0002] Large bone defects caused by trauma, congenital diseases, infections and tumors pose a great challenge to clinical treatment. Currently, the main methods for repairing large bone defects in clinical practice are autologous bone transplantation, allogeneic bone transplantation and tissue-engineered bone. Autologous bone transplantation is still the preferred source of bone transplantation in current clinical treatment, but it often faces a series of problems such as limited number of donor sites, postoperative infection, and pain. Due to the rich source and wide range of materials available for allogeneic bone transplantation, it is widely used in the reconstruction of large bone defects after tumor resection. However, problems such as late rejection reactions and the possible risk of disease transmission have limited its application in clinical large bone defects.

[0003] Biomaterial-based tissue engineering has emerged as a promising approach for repairing bone defects in recent years. Traditional biomaterial design primarily focuses on accelerating the osteogenic differentiation of seeded cells, with little consideration given to the immunomodulatory properties of bone biomaterials. It is well known that when biomaterials are implanted into the body to repair damage, they first interact with the body's immune system. Implants may be treated as foreign matter, generating a strong inflammatory response and requiring clearance, which in turn affects the healing and repair process. Recent research results indicate that this biomaterial-immune system interaction plays a crucial role in material-mediated tissue regeneration.

[0004] Bioscaffold materials are an important component of tissue engineering. Their application in tissue engineering should possess good biocompatibility and physicochemical properties, and be able to provide a suitable microenvironment for cell adhesion, proliferation, and differentiation. In the field of tissue engineering, ideal scaffold materials should have the following characteristics: 1. Good biocompatibility, without causing immune rejection reactions; 2. Good mechanical properties, able to maintain the structural support required for tissue growth; 3. Good biodegradability, with the degradation rate of the scaffold material roughly balanced with the rate of tissue regeneration; 4. Reasonable microstructure and growth factors, providing a good microenvironment for cell adhesion and proliferation; 5. Strong plasticity, allowing for personalized repair of different locations and defects; 6. Simple and economical manufacturing processes, etc. Summary of the Invention

[0005] In order to solve the above technical problems, the present application provides a bone repair biological scaffold material and a preparation method thereof.

[0006] This application is implemented through the following technical solutions:

[0007] The bone repair biological scaffold material provided in the present application is prepared from raw materials including VEGF, USC-Exos, CMC and PEGDA.

[0008] Optionally, the weight ratio of the raw materials is: 3 parts of CMC, 4 parts of PEGDA, 0.005 parts of VEGF, and 0.3 parts of USC-Exos.

[0009] Particularly, the vascular endothelial growth factor is loaded on BPNMs.

[0010] Preferably, the weight ratio of the raw materials is: 3 parts of CMC, 4 parts of PEGDA, 0.005 parts of VEGF, 0.3 parts of USC-Exos, and 0.05 parts of BPNMs.

[0011] The method for preparing a bone repair biological scaffold material provided in this application comprises the following steps:

[0012] Prepare CMC solution and PEDGA solution respectively, and add photoinitiator 2959;

[0013] Take a certain amount of BPNMs solution, add VEGF, and stir thoroughly;

[0014] Take an appropriate amount of PEG-PE I-FA and add it to the BPNMs solution containing VEGF, and stir it thoroughly to obtain the BPNMs solution loaded with VEGF;

[0015] The VEGF-loaded BPNMs solution was mixed with the CMC solution, and then the USCs-Exos solution was added. After shaking evenly, 0.1 M EDC and 0.025 M NHS were added, and then allowed to stand to form a gel.

[0016] The gel is immersed in a PBS solution and allowed to stand for a period of time. The gel is then taken out and placed in a PEGDA solution, which is then placed in a constant temperature water bath and shaken for a period of time. The gel is then dried to allow the PEGDA to gel, thereby finally obtaining the bone repair biological scaffold material.

[0017] Optionally, the drying treatment refers to irradiation with 365nm ultraviolet light for 10-15min.

[0018] Optionally, the preparation of the USCs-Exos solution comprises the following steps:

[0019] Collect human urine, remove the supernatant after centrifugation, and retain the bottom liquid;

[0020] Add PBS to the base solution, resuspend, and centrifuge again. Repeat this process several times, and finally retain the base solution.

[0021] Add USCs basal growth medium to the droplet, resuspend, transfer to a culture flask, and culture in a cell culture incubator;

[0022] The culture medium of the above cells is collected and dispensed into ultracentrifuge tubes. After serial centrifugation, the obtained supernatant is filtered to obtain a filtrate;

[0023] The filtrate was then transferred to an ultracentrifuge tube and subjected to ultrahigh-speed centrifugation to obtain a precipitate rich in USC-Exos;

[0024] PBS was added to the precipitate and mixed, followed by ultrahigh-speed centrifugation. Subsequently, the supernatant was removed, and PBS was added to resuspend the USCs-Exos precipitate to obtain a USCs-Exos solution, which was finally stored at low temperature.

[0025] Compared with the prior art, this application has the following beneficial effects:

[0026] 1. Urine-derived stem cell-derived exosomes (USCs-Exos) are easy, quick, and non-invasive to obtain. They also have excellent stem cell proliferation and differentiation capabilities. More importantly, USCs-Exos have excellent immunomodulatory functions. When combined with materials and implanted into the body, they can reduce inflammatory responses in the microenvironment and reduce rejection reactions after implantation, thereby improving tissue repair effects.

[0027] 2. The bone repair biological scaffold material of the present application has immunomodulatory and angiogenic functions;

[0028] 3. CMC, PEGDA and black phosphorus have good biocompatibility and are non-toxic, and can be used well in tissue engineering bone repair materials;

[0029] 4. Black phosphorus has a huge surface-to-volume ratio, surface functional groups, and modification sites, resulting in a large drug loading capacity and high loading rate, making it an excellent drug carrier in the biological field. It also has good biocompatibility and targeting, and its degradation products are non-toxic and harmless to the body. The bioactive material of this application increases the vascularization process and accelerates tissue repair through the sustained and slow release of VEGF loaded by black phosphorus nanoparticles. At the same time, it combines immune regulation to reduce inflammatory responses and improve the effect of bone defect repair. It provides a good treatment strategy, especially for the problem of large bone defects.

[0030] 5. The biomaterial scaffold provided in this application has a dual sustained-release function. On the one hand, cytokines are released through degradation of the material, and on the other hand, cytokines loaded with black phosphorus are slowly released, thereby achieving a long-lasting release function, solving the shortcomings of active cytokines that degrade quickly and have a short effect.

[0031] 6. The composite gel scaffold prepared by the two-step gelation method in this application has a double network structure. Compared with the simultaneous gelation method after mixing, it has more complete molecular polymerization efficiency, more complete gelation, greater elasticity, toughness and strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings described herein are used to provide a further understanding of the embodiments of the present application, constitute a part of this application, and do not constitute a limitation on the embodiments of the present invention.

[0033] Figure 1 This is an appearance diagram of the bone repair biological scaffold material in the embodiment;

[0034] Figure 2 is a scanning electron microscope image of the bone repair biological scaffold material in the embodiment;

[0035] Figure 3 is a sustained release curve diagram of the bone repair biological scaffold material in the embodiment;

[0036] Figure 4 The immunomodulation map, angiogenesis map and bone repair map of the bone repair biological scaffold material in the embodiment are shown;

[0037] Figure 5 is the CD31 immunohistochemical staining display;

[0038] Figure 6 This is a diagram showing the results of a three-dimensional CT scan of a rat skull. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in a variety of different configurations.

[0040] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0041] Example 1

[0042] The present embodiment discloses a bone repair bioscaffold material with immunomodulatory and angiogenic functions, which is prepared from raw materials including black phosphorus nanoparticles (hereinafter referred to as BPNMs) loaded with vascular endothelial growth factor (hereinafter referred to as VEGF), urine-derived stem cell-derived exosomes (hereinafter referred to as USC-Exos), carboxymethyl chitosan (hereinafter referred to as CMC), and polyethylene glycol diacrylate (hereinafter referred to as PEGDA).

[0043] Preparation of USC-Exos: Collect 50 ml of human urine in a sterile test tube, centrifuge for 5 minutes (1200 rpm), remove the supernatant, retain about 5 ml of liquid at the bottom, add PBS to resuspend, and centrifuge again. Repeat 3 times, and finally retain about 1 ml of liquid at the bottom; add 5 mL of USCs basal growth medium, resuspend, and transfer to a 25T culture flask, and culture in a cell culture incubator at 37°C, 5% CO2, and 95% humidity.

[0044] The culture medium collected from these cells was aliquoted into ultracentrifuge tubes and centrifuged sequentially at 300g for 10 min at 4°C, 2000g for 10 min at 4°C, and 10,000g for 30 min at 4°C. The resulting supernatant was filtered through a 0.22 μm filter and then transferred to an ultracentrifuge tube. Ultracentrifugation at 100,000g for 70 min at 4°C yielded a precipitate rich in USC-Exos. To further remove residual protein impurities, PBS was added and mixed, followed by another ultracentrifugation at 100,000g at 4°C. The supernatant was removed, and the USC-Exos pellet was resuspended in PBS. The pellet was then transferred to a sterile 1.5 ml EP tube and stored at -80°C.

[0045] Other material components can be purchased.

[0046] The preparation method of the bone repair biological scaffold material comprises the following steps:

[0047] Prepare 5% w / v CMC, 10% w / v PEDGA solution and add 0.4% w / v photoinitiator 2959;

[0048] A certain amount of ultrasonically dispersed BPNMs solution was adjusted to pH 8, and VEGF (5 μg / mL) was added. The solution was stirred at room temperature overnight to allow the BPNMs to fully adsorb VEGF. An appropriate amount of polyethylene glycol-polyethyleneimine-folic acid (PEG-PE I-FA) was added to the BPNMs solution, stirred in a water bath for 30 min, and then stirred at room temperature overnight to obtain a VEGF-loaded BPNMs solution.

[0049] The BPNMs solution loaded with VEGF was mixed with a certain amount of 5% w / v CMC solution, and then the USCs-Exos solution was added and shaken evenly; each ml of the mixed solution was rich in CMC 3mg, PEGDA 4mg, VEGF 0.005mg, USC-Exos 0.3mg, and BPNMs 0.05mg.

[0050] 0.1 M EDC and 0.025 M NHS were then added to the mixture, which was then transferred to a 96-well plate and allowed to stand for 30 min to allow it to fully form a gel (first network);

[0051] The gel was immersed in PBS solution at room temperature and allowed to stand for 24 hours. Then, a certain amount of 10% w / v PEGDA solution was taken and a 4% w / v PEGDA solution was prepared according to the dosage of the above experimental solution. The gel was taken out and placed in the 4% w / v PEGDA solution to fully cover the gel. The gel was placed in a 37° constant temperature water bath and shaken for 24 hours. The gel was then exposed to 365nm ultraviolet light for 10-15min to form PEGDA into a gel (second network). Finally, a composite double network gel scaffold was obtained. The composite double network gel scaffold is a bone repair biological scaffold material and its preparation method (hereinafter referred to as BPNMs-CMC / PEGDA). Figure 1 , Figure 2 shown.

[0052] Optionally, the concentrations of the bone repair biological scaffold materials prepared by the method of this embodiment are: CMC 3% w / v, USCs-Exos 300ug / ml, VEGF 5ug / ml, BPNMs 50ug / ml, PEGDA 4% w / v.

[0053] The properties of BPNMs-CMC / PEGDA are shown in Table 1:

[0054] Table 1: Properties of BPNMs-CMC / PEGDA

[0055]

[0056] The drug loading efficiency W1 of black phosphorus was 100%, and the encapsulation efficiency W2 (chemical coupling) was 47.49±0.54%.

[0057] like Figure 3 As shown, the release factor of the bone repair biological scaffold material of the present application is released slowly and gradually, and the sustained release rate of the material is 48.36±1.08% after 56 days.

[0058] Among them, the rheological properties of BPNMs-CMC / PEGDA are shown in Table 2:

[0059] Table 2: Rheological test results of materials at different frequencies

[0060]

[0061] The elastic modulus of BPNMs-CMC / PEGDA in this example is: 13.89±0.16 kPa.

[0062] like Figure 4 As shown in the data, in terms of the immunoregulatory aspects of the materials: the expression of CD68 positive cells in each group was relatively low, the expression of CD206 positive cells in the USC-Exos group (containing USC-Exos) was higher than that in the control group; and the expression of iNOS positive cells in the control group (without USC-Exos) was higher than that in the USC-Exos group, indicating that the USC-Exos group has the effect of regulating immune expression toward M2 type (i.e., anti-inflammatory type).

[0063] like Figure 5 As shown in the figure, CD31 immunohistochemical staining showed that a large number of vascular cavity-like structures were formed in the control group (containing VEGF but not USC-Exos) and the USC-Exos group (containing VEGF and USC-Exos), while very few were seen in the blank group, indicating that a large number of new blood vessels were formed under the action of VEGF and USC-Exos, which is conducive to new bone formation.

[0064] like Figure 6 As shown, the three-dimensional CT scan results of rat skulls showed that a large amount of new bone formation was observed in the control group and the USC-Exos group, and the amount of new bone formed in the USC-Exos group was the largest. The bone repair ability of the surface material under the action of VEGF and USC-Exos was the strongest.

[0065] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of this application in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bone repair biological scaffold material, characterized by: It is prepared from raw materials containing VEGF, USC-Exos, CMC, and PEGDA; The VEGF is loaded on BPNMs; The weight ratio of the raw materials is: 3 parts of CMC, 4 parts of PEGDA, 0.005 parts of VEGF, 0.3 parts of USC-Exos, and 0.05 parts of BPNMs.

2. The method for preparing a bone repair biological scaffold material according to claim 1, characterized in that: The following steps are involved: Prepare CMC solution and PEDGA solution respectively, and add photoinitiator 2959; Take a certain amount of BPNMs solution, add VEGF, and stir thoroughly; Add an appropriate amount of PEG-PEI-FA to the BPNMs solution containing VEGF and stir thoroughly to obtain a BPNMs solution loaded with VEGF; The VEGF-loaded BPNMs solution was mixed with the CMC solution, and then the USCs-Exos solution was added. After shaking, the mixture was evenly mixed with 0.1 M EDC and 0.025 M NHS, and then allowed to stand to form a gel. The gel is immersed in a PBS solution and allowed to stand for a period of time. The gel is then taken out and placed in a PEGDA solution, which is then placed in a constant temperature water bath and shaken for a period of time. The gel is then dried to allow the PEGDA to gel, thereby finally obtaining the bone repair biological scaffold material.

3. The preparation method according to claim 2, wherein: The pH value of the BPNMs solution is 8.

4. The preparation method according to claim 2, wherein: The concentration of the CMC solution is 5% w / v; The concentration of the PEDGA solution is 10% w / v; The concentration of photoinitiator 2959 was 0.4% w / v; In the step of taking a certain amount of BPNMs solution, adding VEGF, and fully stirring, the VEGF concentration is 5 μg / mL.

5. The preparation method according to claim 2, wherein: The drying treatment refers to irradiating with 365 nm ultraviolet light for 10-15 minutes.

6. The preparation method according to claim 2, wherein: The temperature of the constant temperature water bath was 37°C.

7. The preparation method according to claim 2, characterized in that: The preparation of the USCs-Exos solution comprises the following steps: Collect human urine, remove the supernatant after centrifugation, and retain the bottom liquid; Add PBS to the base solution, resuspend, and centrifuge again. Repeat this process several times, and finally retain the base solution. Add USCs basal growth medium to the base solution, resuspend and transfer to a culture flask, and culture in a cell culture incubator; The culture medium of the above cells is collected and dispensed into ultracentrifuge tubes. After serial centrifugation, the obtained supernatant is filtered to obtain a filtrate; The filtrate was then transferred to an ultracentrifuge tube and subjected to ultrahigh-speed centrifugation to obtain a precipitate rich in USC-Exos; PBS was added to the precipitate and mixed, and then ultra-high-speed centrifugation was performed; subsequently, the supernatant was removed, and PBS was added to resuspend the USCs-Exos precipitate to obtain a USCs-Exos solution, which was finally stored at low temperature.

8. The preparation method according to claim 7, characterized in that: The preparation of the USCs-Exos solution comprises the following steps: Collect human urine in a sterile test tube, centrifuge at 1200 rpm for 5 minutes, remove the supernatant, and retain about 5 ml of liquid at the bottom; Add PBS to resuspend and centrifuge again, repeat 3 times, and finally retain 1 ml of liquid at the bottom; Add 5 mL of USCs basal growth medium, resuspend, and transfer to a 25T culture flask. Culture in a cell culture incubator at 37°C, 5% CO2, and 95% humidity. The culture medium of the above cells was collected and divided into ultracentrifuge tubes, and the supernatant was filtered through a 0.22 μm filter to obtain a filtrate after sequential centrifugation at 4°C, 300 g for 10 min, 4°C, 2000 g for 10 min, and 4°C, 10,000 g for 30 min. The filtrate was then transferred to an ultracentrifuge tube and ultracentrifuged at 100,000 g for 70 min at 4°C to obtain a precipitate rich in USC-Exos. PBS was added to the precipitate and mixed, and ultracentrifuged again at 100,000 g at 4°C; After obtaining the USCs-Exos solution, transfer it to a 1.5 ml sterile EP tube and store it in a -80°C refrigerator.

9. A composite gel scaffold with a double network structure, characterized in that: The method is prepared according to any one of claims 2 to 8.

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