Engineered SVF extracellular vesicle-loaded electrostatic spinning membrane for inducing bone regeneration and application of engineered SVF extracellular vesicle-loaded electrostatic spinning membrane

Abstract

The invention discloses an engineering SVF extracellular vesicle-loaded electrostatic spinning membrane for inducing bone regeneration and application thereof, and relates to the technical field of biology. The preparation method of the electrospinning membrane loaded with the engineered SVF extracellular vesicles comprises the following steps: preparing a polycaprolactone nanofiber membrane through an electrospinning technology, performing plasma treatment to form a single-sided hydrophilic nanofiber membrane material, and then incubating the nanofiber membrane material with the engineered SVF extracellular vesicles together to obtain the electrospinning membrane loaded with the engineered SVF extracellular vesicles. The electrospun membrane loaded with the engineering SVF extracellular vesicles is prepared. The engineered SVF extracellular vesicle-loaded electrostatic spinning membrane can effectively promote the regeneration of early bone defects in an in-vivo environment, accelerates the bone tissue repair process, and shows a good treatment effect and a potential application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an electrospun membrane loaded with engineered SVF extracellular vesicles for inducing bone regeneration and its application. Background Art

[0002] As a novel biomaterial, extracellular vesicles (EVs) have shown great potential in the fields of tissue repair, drug delivery, and regenerative medicine. However, the existing methods for preparing and applying EVs have the following deficiencies:

[0003] The traditional method for preparing EVs is complex, time-consuming, and requires ultracentrifugation: The traditional extrusion method for preparing EVs involves multiple steps, including cell culture, collection, and multiple passes through a filter membrane for extrusion. These operations are not only cumbersome but also prone to introducing contamination risks. The entire preparation process may take several days or even longer, resulting in low production efficiency and difficulty in meeting the rapid response requirements of the clinic. To obtain high-purity EVs, existing methods usually rely on ultracentrifugation technology. This technology is expensive, complex to operate, and has strict requirements for the experimental environment, increasing the cost and technical threshold. For example, the extrusion method for preparing engineered vesicles proposed in 2013 involves passing cells through a large-pore filter membrane (1 μm) and then separating the vesicles with a diameter of about 100 nm by ultracentrifugation (see the literature: Jang S C, Kim O Y, Yoon C M, et al. Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors [J]. ACS Nano, 2013, 7(9): 7698-710). The subsequent methods for preparing extruded vesicles also follow the method proposed in this literature, but this method is complex, requires an ultracentrifuge, has high requirements for instruments, and has a long preparation time.

[0004] Unstable loading: The traditional EVs loading method is difficult to ensure the uniform distribution and long-term stable attachment of extracellular vesicles on the carrier material, resulting in a significant reduction in their biological activity and functionality in practical applications.

[0005] Poor biocompatibility: The surface properties of some carrier materials are not conducive to cell adhesion and proliferation, restricting their application effects in bone defect repair.

[0006] Lack of effective microenvironment regulation: The existing technologies fail to fully simulate the in vivo microenvironment, affecting the effect and speed of bone tissue repair.

[0007] In addition, materials currently used for bone defect repair, such as polycaprolactone (PCL), although having good mechanical properties, their hydrophobicity and poor cell adhesion properties limit their application effects in regenerative medicine.

[0008] To solve the above problems, the present invention intends to develop a new method for preparing EVs, and co-incubate engineered vesicles with a hydrophilic PCL membrane treated by plasma and loaded on one side through electrospinning technology, so as to achieve stable loading of the vesicles and significantly improve their application effects in bone defect repair. Summary of the Invention

[0009] The object of the present invention is to provide a plasma-treated electrospun membrane loaded with engineered SVF extracellular vesicles for inducing bone regeneration and its application, so as to solve the problems existing in the above-mentioned prior art. This plasma-treated electrospun membrane loaded with engineered SVF extracellular vesicles can effectively promote the regeneration of early bone defects in the in vivo environment, accelerate the bone tissue repair process, and show good therapeutic effects and potential application prospects.

[0010] To achieve the above object, the present invention provides the following solutions:

[0011] The present invention provides a method for preparing a plasma-treated electrospun membrane loaded with engineered SVF extracellular vesicles for inducing bone regeneration, comprising the following steps:

[0012] Prepare a polycaprolactone nanofiber membrane by electrospinning technology, and form a nanofiber membrane material with hydrophilicity on one side through plasma treatment, and then co-incubate it with engineered SVF extracellular vesicles to prepare the plasma-treated electrospun membrane loaded with engineered SVF extracellular vesicles.

[0013] Further, the method for preparing the engineered SVF extracellular vesicles comprises the following steps:

[0014] After subjecting stromal vascular fraction cells to ultrasonic membrane rupture treatment, sequentially extrude them through 800 nm and 100 nm filters to obtain extracellular vesicles in the range greater than 100 nm and less than 800 nm, which are the engineered SVF extracellular vesicles.

[0015] Further, the frequency of the ultrasonic membrane rupture treatment is 40 KHz and the time is 10 min.

[0016] Further, the stromal vascular fraction cells are first dispersed in PBS buffer solution and then subjected to the ultrasonic membrane rupture treatment.

[0017] Further, the temperature of the incubation is 37 °C.

[0018] The present invention also provides a plasma-treated electrospun membrane loaded with engineered SVF extracellular vesicles prepared according to the above preparation method.

[0019] The present invention also provides the application of the above-mentioned electrostatic spinning membrane loaded with engineered SVF extracellular vesicles in the preparation of a bone defect repair product with the function of inducing bone regeneration.

[0020] The present invention also provides a method for preparing engineered SVF extracellular vesicles, comprising the following steps:

[0021] After subjecting stromal vascular fraction cells to ultrasonic membrane disruption treatment, they are sequentially extruded through 800 nm and 100 nm filter membranes to obtain extracellular vesicles in the range greater than 100 nm and less than 800 nm, which are the engineered SVF extracellular vesicles.

[0022] Further, the frequency of the ultrasonic membrane disruption treatment is 40 KHz and the time is 10 min.

[0023] Further, the stromal vascular fraction cells are first dispersed in PBS buffer and then subjected to the ultrasonic membrane disruption treatment.

[0024] The present invention discloses the following technical effects:

[0025] The present invention solves the problem of the need for ultracentrifugation in the traditional process of preparing engineered extracellular vesicles, realizes more efficient and convenient preparation of engineered vesicles, and facilitates the transformation of clinical applications.

[0026] The present invention co-incubates engineered vesicles with a hydrophilic PCL membrane treated with plasma by electrospinning technology on one side to prepare an electrostatic spinning membrane loaded with engineered SVF extracellular vesicles for inducing bone regeneration. Through the incubation process, EVs can be stably loaded onto the PCL membrane to form a composite material with excellent biocompatibility and bioactivity. This not only improves the loading efficiency of vesicles but also ensures their long-term stability in the in vivo environment. After the PCL membrane is treated with plasma, its surface hydrophilicity is significantly improved, enhancing cell adhesion and proliferation ability, and further improving the microenvironment for bone tissue repair. Experimental results show that the electrostatic spinning membrane loaded with engineered SVF extracellular vesicles prepared by the present invention can effectively promote the regeneration of early bone defects in the in vivo environment, accelerate the bone tissue repair process, and show good therapeutic effects and potential application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 Electron microscopy observation image of the extracellular vesicles prepared in Example 1;

[0029] Figure 2 Particle size detection results of the extracellular vesicles prepared in Example 1;

[0030] Figure 3 Contact angle measurement results of the droplet with the PCL material and the electrospun plasma-loaded material; wherein, A is the schematic diagram of the contact angle measurement; B is the statistical chart of the contact angle;

[0031] Figure 4 Electron microscopy observation images of the PCL material (A) and the electrospun plasma-loaded material (B);

[0032] Figure 5 In vivo experimental results of the electrospun membrane material loaded with SVF-engineered vesicles in a rat femoral defect model;

[0033] Figure 6 Statistical chart of the average trabecular thickness (Tb.Th);

[0034] Figure 7 Statistical chart of the average trabecular number (Tb.N). Detailed implementation manners

[0035] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0038] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0039] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] Disperse the stromal vascular fraction (SVF) cells in PBS buffer. After diluting 50 times, rupture the cell membranes in an ultrasonic instrument for 10 min (ultrasonic parameters: 40 KHz, 80 W). Load the dispersed solution after ultrasonic fragmentation into an extruder, and make the cells pass through 800 nm and 100 nm filters in sequence to obtain extracellular vesicles in the range greater than 100 nm and less than 800 nm.

[0042] Perform electron microscopy observation and particle size detection on the extracellular vesicles prepared in this example. The results are shown in Figure 1 and Figure 2 .

[0043] Example 2

[0044] Preparation of an electrospun membrane loaded with engineered SVF extracellular vesicles for inducing bone regeneration:

[0045] Prepare a polycaprolactone (PCL) nanofiber membrane by electrospinning technology. Use a plasma generator to perform plasma treatment on the nanofiber membrane to prepare a membrane material with a hydrophilic surface on one side (PCL + plasma). Then incubate it with the extracellular vesicles at a concentration of 2×10 6 cells / mL prepared in Example 1 overnight at 37 °C to obtain a biomimetic guided bone regeneration membrane (PCL + plasma + EVs).

[0046] Take SEM pictures after spraying, with magnifications of 10,000 times and 50,000 times. Use a high-resolution camera to cooperate with uniform backlighting to take side-view images of the droplets to measure the water contact angle of the membrane material before and after electrospinning, and calculate the contact angle between the droplets and the membrane material.

[0047] It is proved by contact angle detection and quantitative analysis that after loading plasma by electrospinning, the hydrophilicity of the PCL material is greatly improved, showing significant statistical differences compared with the unloaded group. The PCL material and the material obtained by incubating the electrospun and loaded material with the engineered vesicle dispersion overnight and then drying were subjected to scanning electron microscopy (SEM), which proved that the vesicles were loaded on the electrospun material and aggregated in clusters on the material. This proves that successful loading of vesicles on the material can be achieved simply by improving the hydrophilicity of the material, and the preparation method is simple and fast.

[0048] Example 3

[0049] Male Sprague-Dawley rats (6 weeks old) were used in the experiment and allowed to adaptively feed for 1 week. Bone defect modeling: The rats were anesthetized and fixed in the supine position on the operating table. The skin was pre-incised in the surgical area and disinfected with iodine tincture. The skin was incised at the distal ends of both femurs, and the subcutaneous tissue was bluntly dissected to reach the metaphyseal end of the femur. A drill bit with a diameter of 2 mm was used to connect to a drill to drill holes. The experiment was divided into a blank control group (denoted as group B), a PCL+plasma group, and a PCL+plasma+EVs group. Except for the blank control group, membrane materials were placed at the corresponding defect sites in the other two groups, and then the soft tissues and skin were sutured in layers. Samples were taken 2 weeks after implantation, and the rats were sacrificed after anesthesia for sampling and subsequent tests.

[0050] As Figures 5 - 7 shown, by analyzing the CT and tissue section results of the 2-mm defect modeling site of the rat femur 2 weeks after bone defect modeling, the bone tissue in the experimental group was closer to the cortical bone morphology than that in the control group, with a smooth and flat surface and a denser structure. Statistical results showed that the average trabecular thickness (Tb.Th) in the experimental group was larger and the average trabecular number (Tb.N) was smaller, showing significant statistical differences from the control group. This preliminarily proves that the SVF-ECVs group formed a bone tissue structure closer to lamellar bone with fewer gaps. At the same time, the present invention noted the SMI index in the micro CT analysis results that represents the proportion of rod-shaped and lamellar bone trabeculae in the analyzed area. The value in the experimental group was close to 0, indicating a higher proportion of lamellar bone trabeculae and being closer to mature cortical bone, while the control group was close to 3, indicating a higher proportion of rod-shaped bone trabeculae.

[0051] Early regeneration of bone defect areas is a difficult task. The literature shows that non-critical defect regeneration of the rat femur can form bone tissue without load-bearing function at 4-6 weeks, and it takes 8-12 weeks to gradually form half-layer bone, with the load-bearing performance restored to 60%-80%. However, the induced bone regeneration membrane loaded with SVF-engineered cell vesicles prepared in the present invention forms a bone tissue structure closer to mature bone with a higher proportion of lamellar bone trabeculae at the two-week time point, and at the same time proves that the bone tissue density is also higher and closer to mature cortical bone compared with the control group.

[0052] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing an electrospinning membrane of engineered SVF extracellular vesicles loaded with bone regeneration, characterized in that: The following steps are involved: The polycaprolactone nanofiber membrane is prepared by electrospinning technology, and is treated by plasma to form a single-sided hydrophilic nanofiber membrane material, which is then co-incubated with engineered SVF extracellular vesicles to prepare the electrospinning membrane loaded with engineered SVF extracellular vesicles.

2. The preparation method according to claim 1, characterized in that: The method for preparing the engineered SVF extracellular vesicles comprises the following steps: After the stromal vascular component cells are subjected to ultrasonic membrane permeation, they are sequentially squeezed through 800nm ​​and 100nm filter membranes to obtain extracellular vesicles larger than 100nm and smaller than 800nm, which are the engineered SVF extracellular vesicles.

3. The preparation method according to claim 2, characterized in that: The frequency of the ultrasonic membrane rupture treatment is 40KHz and the time is 10min.

4. The preparation method according to claim 2, characterized in that: The stromal vascular fraction cells are first dispersed in PBS buffer and then subjected to the ultrasonic membrane disruption treatment.

5. The preparation method according to claim 1, characterized in that: The incubation temperature was 37°C.

6. An electrospinning membrane loaded with engineered SVF extracellular vesicles prepared according to the preparation method according to any one of claims 1 to 5.

7. Use of the electrospinning membrane loaded with engineered SVF extracellular vesicles as claimed in claim 6 in the preparation of a bone defect repair product with the function of inducing bone regeneration.

8. A method for preparing engineered SVF extracellular vesicles, characterized in that: The following steps are involved: After the stromal vascular component cells are subjected to ultrasonic membrane permeation, they are sequentially squeezed through 800nm ​​and 100nm filter membranes to obtain extracellular vesicles larger than 100nm and smaller than 800nm, which are the engineered SVF extracellular vesicles.

9. The preparation method according to claim 8, characterized in that: The frequency of the ultrasonic membrane rupture treatment is 40KHz and the time is 10min.

10. The preparation method according to claim 8, characterized in that: The stromal vascular fraction cells are first dispersed in PBS buffer and then subjected to the ultrasonic membrane disruption treatment.

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