Modified plasma matrix material as well as preparation method and application thereof

Abstract

The invention relates to a modified plasma matrix material as well as a preparation method and application thereof, and the preparation method comprises the following steps: mixing osteoblast extracellular vesicles with a liquid plasma matrix, standing and pressing to obtain the modified plasma matrix material. The prepared modified plasma matrix material can slowly release osteoblast extracellular vesicles, promote osteogenesis mineralization in the vesicles, promote spontaneous mineralization in the plasma matrix and accelerate the bone regeneration process, has the effects of promoting cell proliferation, migration and osteogenesis differentiation, can be used for bone defect repair, and has a good application prospect. The problem of poor osteogenic activity of a common plasma matrix is solved, and a more effective clinical application scheme is provided for bone regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly relates to a modified plasma matrix material, a preparation method thereof, and an application thereof. Background Art

[0002] Plasma matrix is a material derived from autologous peripheral blood. By centrifugation, active cells such as platelets and white blood cells and cytokines in supra-physiological doses are enriched. It has good ability to promote the regeneration of hard and soft tissues. In recent years, it has been widely used in clinical fields such as the repair of oral hard and soft tissue defects and the healing of refractory skin wounds. By changing the centrifugation conditions and the material of the blood collection tube, plasma matrix products in different states can be prepared, and the mutual conversion of products in different states can also occur.

[0003] However, traditional plasma matrix has problems such as insufficient bone regeneration activity. Therefore, it is of great significance to provide a modified plasma matrix material to improve its bone regeneration ability. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a modified plasma matrix material, a preparation method thereof, and an application thereof. The modified plasma matrix material provided by the present invention can slowly release osteoblast extracellular vesicles, promote osteogenic mineralization inside the vesicles, and at the same time promote spontaneous mineralization inside the plasma matrix, accelerate the bone regeneration process, has the effects of promoting cell proliferation, migration, and osteogenic differentiation, can be used for bone defect repair, overcomes the problem of poor osteogenic activity of ordinary plasma matrix, and provides a more effective clinical application solution for bone regeneration.

[0005] In the first aspect, the present invention provides a preparation method of a modified plasma matrix material. The preparation method includes mixing osteoblast extracellular vesicles with liquid plasma matrix, standing and then pressing to obtain the modified plasma matrix material. The liquid plasma matrix can remain liquid for a period of time after being prepared. When it is liquid, it is mixed evenly with osteoblast extracellular vesicles, and then stands and condenses into a gel state, and is pressed to obtain the modified plasma matrix material of the present invention.

[0006] The preparation method provided by the present invention modifies the plasma matrix with osteoblast extracellular vesicles. On the one hand, it can improve the spontaneous mineralization inside the plasma matrix and accelerate the bone regeneration process. On the other hand, it can effectively improve the slow-release effect of osteoblast extracellular vesicles. The prepared modified plasma matrix material can regulate the concentration of osteoblast extracellular vesicles according to actual treatment needs, has different osteogenic regulation effects, and can promote cell proliferation, migration, and osteogenic differentiation. Specifically:

[0007] The osteoblast extracellular vesicles of the present invention are extracellular vesicles secreted during the mineralization process of osteoblasts, which play a huge role in the osteogenic process. The present invention loads them into the plasma matrix, which can promote the spontaneous mineralization inside the plasma matrix, accelerate the bone regeneration process. At the same time, the plasma matrix contains rich fibrin, which provides a scaffolding role during the osteogenic process, improves the regeneration effect, and can regulate the slow release process of extracellular vesicles. The two work together to improve the bone regeneration effect, regulate the local bone regeneration process, can promote cell proliferation, migration, and osteogenic differentiation, and play an important role in the field of tissue regeneration.

[0008] As a preferred technical solution of the present invention, the preparation method further includes centrifuging the blood to obtain the liquid plasma matrix. In the present invention, the patient's blood is collected in a liquid plasma matrix collection tube, and the liquid plasma matrix is prepared by centrifugation using a plasma matrix preparation platform. After centrifugation, it is divided into an upper light yellow liquid plasma matrix layer, a middle brown-yellow layer, and a lower red blood cell layer. In the present invention, syringes, pipettes, etc. are used to collect the upper and middle layers for use because the middle brown-yellow layer contains more immune cells that can be used.

[0009] As a preferred technical solution of the present invention, the centrifugal force of the centrifugation is 50 - 1000g, such as 100g, 200g, 400g, 600g, 800g, etc.

[0010] As a preferred technical solution of the present invention, the centrifugation time is 2 - 10 min, such as 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, etc.

[0011] As a preferred technical solution of the present invention, the mass-to-volume ratio of the osteoblast extracellular vesicles to the liquid plasma matrix is (30 - 70) μg:1 mL, such as 35 μg:1 mL, 40 μg:1 mL, 45 μg:1 mL, 50 μg:1 mL, 55 μg:1 mL, 60 μg:1 mL, 65 μg:1 mL, etc.

[0012] By regulating the different ratios of osteoblast extracellular vesicles to the liquid plasma matrix, the present invention can prepare modified plasma matrix materials that match the extracellular vesicle concentrations required for different treatment needs, thereby obtaining different osteogenic regulation effects.

[0013] As a preferred technical solution of the present invention, the preparation method further includes culturing osteoblasts in vitro, centrifuging and separating the supernatant after culturing, and extracting osteoblast extracellular vesicles. The present invention cultures osteoblasts in vitro to induce mineralization, and the obtained osteoblast extracellular vesicles play a huge role in the osteogenic process.

[0014] As a preferred technical solution of the present invention, the culture medium is DMEM medium.

[0015] As a preferred technical solution of the present invention, the culture medium is DMEM cell culture medium comprising 10 wt% FBS (fetal bovine serum), 0.1 μmol / L dexamethasone, 0.2 mmol / L vitamin C, and 10 mmol / L β-glycerophosphate.

[0016] As a preferred technical solution of the present invention, the temperature during culture is 37 °C.

[0017] As a preferred technical solution of the present invention, the CO2 content during culture is 5%.

[0018] As a preferred technical solution of the present invention, the relative humidity during culture is 95%.

[0019] As a preferred technical solution of the present invention, the culture time is 7 - 21 days, such as 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, etc.

[0020] As a preferred technical solution of the present invention, the method of centrifugal separation is to centrifuge at 300 g for 10 min, at 2000 g for 10 min, and at 18000 g for 30 min in sequence.

[0021] In the present invention, osteoblasts are cultured in vitro for mineralization induction, and after 7 - 21 days of culture, centrifugal separation is performed. The extracellular vesicles secreted by osteoblasts are concentrated in the supernatant, and then the extracellular vesicles of osteoblasts are obtained by extraction.

[0022] The present invention does not overly limit the method for extracting extracellular vesicles of osteoblasts, and as long as the extracellular vesicles of osteoblasts can be obtained, they can be applied to the present invention.

[0023] As a preferred technical solution of the present invention, the method for extracting extracellular vesicles of osteoblasts is ultracentrifugation.

[0024] As a preferred technical solution of the present invention, the centrifugal force of the ultracentrifugation is 110000 g.

[0025] As a preferred technical solution of the present invention, the time of the ultracentrifugation is 70 - 180 min, such as 80 min, 100 min, 120 min, 140 min, 160 min, etc.

[0026] As a preferred technical solution of the present invention, the temperature during the centrifugal separation and the ultracentrifugation is independently 4 °C.

[0027] As a preferred technical solution of the present invention, the preparation method further includes placing the osteoblast extracellular vesicles in a solvent and then mixing them with the liquid plasma matrix.

[0028] The osteoblast extracellular vesicles obtained in the present invention can be directly placed in the liquid plasma matrix for mixing, or can be first placed in a solvent, mixed evenly, and then mixed with the liquid plasma matrix.

[0029] The present invention does not overly limit the solvent, and any solvent that does not affect the osteoblast extracellular vesicles and the liquid plasma matrix is applicable to the present invention. Exemplarily, the solvent can be physiological saline, phosphate buffer solution, etc.

[0030] As a preferred technical solution of the present invention, the volume of the solvent is 0.05 - 0.2 mL.

[0031] As a preferred technical solution of the present invention, the standing time ≥ 10 min. After mixing the osteoblast extracellular vesicles with the liquid plasma matrix in the present invention and standing for a period of time, it can solidify into a clot, which is convenient for subsequent pressing.

[0032] As a preferred technical solution of the present invention, the pressure for pressing is 1 - 5 N, such as 2 N, 3 N, 4 N, etc.

[0033] As a preferred technical solution of the present invention, the time for pressing is 2 - 10 min, such as 4 min, 6 min, 8 min, etc.

[0034] The present invention presses the obtained clot, shapes it into a film or plug, etc., to obtain a modified plasma matrix membrane or plasma matrix plug, etc. The present invention does not overly limit the equipment used for pressing, as long as the required shape can be obtained. Exemplarily, the equipment can be a plasma matrix compression instrument box, etc.

[0035] As a specific embodiment of the present invention, the preparation method includes the following steps:

[0036] (1) Culturing osteoblasts in vitro for 7 - 21 days, after culturing, centrifuging and separating at 300 g for 10 min, at 2000 g for 10 min, and at 18000 g for 30 min in sequence, collecting the supernatant, and then extracting osteoblast extracellular vesicles by ultracentrifuging at 110000 g for 70 - 180 min;

[0037] (2) Centrifuging blood at 50 - 1000 g for 2 - 10 min to obtain a liquid plasma matrix;

[0038] (3) Mix the osteoblast extracellular vesicles with the liquid plasma matrix, let it stand for ≥10 min, and then press it at a pressure of 1-5 N for 2-10 min to obtain the modified plasma matrix material.

[0039] In a second aspect, the present invention provides a modified plasma matrix material prepared by the preparation method described in the first aspect.

[0040] The modified plasma matrix material prepared by the present invention can slowly release osteoblast extracellular vesicles, promote osteogenic mineralization inside the vesicles, and at the same time promote spontaneous mineralization inside the plasma matrix, accelerating the bone regeneration process. It has the effects of promoting cell proliferation, migration, and osteogenic differentiation. Moreover, using autologous plasma matrix and osteoblast extracellular vesicles is safer and more reliable, and it can be used for bone defect repair, improving the treatment effect and broadening the indications in the field of tissue regeneration.

[0041] In a third aspect, the present invention provides the application of the modified plasma matrix material described in the second aspect in bone defect repair materials.

[0042] The modified plasma matrix material provided by the present invention can be directly used as a bone defect repair material, or can be used in combination with a bone substitute material, and the obtained bone defect repair material can be used for bone defect repair.

[0043] The technical solutions provided by the embodiments of the present invention have the following advantages compared with the prior art:

[0044] 1. The modified plasma matrix material provided by the present invention uses osteoblast extracellular vesicles to modify the plasma matrix, which can improve the spontaneous mineralization inside the plasma matrix and accelerate the bone regeneration process.

[0045] 2. The modified plasma matrix material provided by the present invention can effectively improve the slow-release effect of osteoblast extracellular vesicles, can regulate the concentration of osteoblast extracellular vesicles according to actual treatment needs, and has different osteogenic regulation effects.

[0046] 3. The modified plasma matrix material provided by the present invention has the effects of promoting cell proliferation, migration, and osteogenic differentiation, can be used for bone defect repair, overcomes the problem of poor osteogenic activity of ordinary plasma matrix, and provides a more effective clinical application plan for bone regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The drawings herein are incorporated into the specification and form a part of this specification, showing embodiments consistent with the present invention and, together with the specification, are used to explain the principles of the present invention.

[0048] 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 in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0049] Figure 1 It is the extracellular vesicle sustained-release effect diagram of the modified plasma matrix material prepared in Example 1;

[0050] Figure 2 It is the effects of the modified plasma matrix material and osteoblast extracellular vesicles prepared in Example 1 on the osteogenic differentiation of cells respectively. Detailed implementation manners

[0051] In order to more clearly understand the above-mentioned objects, features, and advantages of the present invention, the following will further describe the solutions of the present invention. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0052] Many specific details are set forth in the following description to facilitate a thorough understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only a part of the embodiments of the present invention, rather than all the embodiments.

[0053] Example 1

[0054] This example provides a modified plasma matrix material and a preparation method thereof. The preparation method includes the following steps:

[0055] (1) Cultivate osteoblasts in vitro for 14 days. The culture medium is DMEM cell culture medium containing 10 wt% FBS (fetal bovine serum), 0.1 μmol / L dexamethasone, 0.2 mmol / L vitamin C, and 10 mmol / L β-glycerophosphate. The temperature of the incubator is 37 °C, the CO2 level is 5%, and the relative humidity is 95%. After cultivation, centrifuge the culture medium at 4 °C successively at a centrifugal force of 300 g for 10 min, at a centrifugal force of 2000 g for 10 min, and at a centrifugal force of 18000 g for 30 min to remove dead cells, cell debris, microvesicles, apoptotic bodies, etc. in the culture medium; collect the supernatant and centrifuge it at a centrifugal force of 110000 g for 70 min at 4 °C to obtain the osteoblast extracellular vesicle precipitate;

[0056] (2) Collect the patient's blood in a liquid plasma matrix blood collection tube, and use the plasma matrix preparation platform to centrifuge at a centrifugal force of 500 g for 6 min to obtain the liquid plasma matrix;

[0057] (3) Resuspend the osteoblast extracellular vesicles in 0.2 mL of phosphate buffer solution, add liquid plasma matrix, and make the mass-to-volume ratio of osteoblast extracellular vesicles to liquid plasma matrix 50 μg:1 mL. Let it stand for 10 min, and use the plasma matrix preparation kit to press it at a pressure of 1 N for 10 min to obtain the modified plasma matrix material.

[0058] Example 2

[0059] This example provides a modified plasma matrix material and a preparation method thereof. The preparation method includes the following steps:

[0060] (1) Cultivate osteoblasts in vitro for 21 days. The culture medium is DMEM cell culture medium containing 10 wt% FBS (fetal bovine serum), 0.1 μmol / L dexamethasone, 0.2 mmol / L vitamin C, and 10 mmol / L β-glycerophosphate. The temperature of the incubator is 37 °C, the CO2 level is 5%, and the relative humidity is 95%. After cultivation, centrifuge the culture medium at 4 °C successively at a centrifugal force of 300 g for 10 min, at a centrifugal force of 2000 g for 10 min, and at a centrifugal force of 18000 g for 30 min to remove dead cells, cell debris, microbubbles, apoptotic bodies, etc. in the culture medium; collect the supernatant and centrifuge it at 4 °C at a centrifugal force of 110000 g for 120 min to obtain the osteoblast extracellular vesicle precipitate;

[0061] (2) Collect the patient's blood in a liquid plasma matrix blood collection tube, and use the plasma matrix preparation platform to centrifuge it at a centrifugal force of 1000 g for 2 min to obtain the liquid plasma matrix;

[0062] (3) Place the osteoblast extracellular vesicles in the liquid plasma matrix, and make the mass-to-volume ratio of osteoblast extracellular vesicles to liquid plasma matrix 30 μg:1 mL. Let it stand for 10 min, and use the plasma matrix preparation kit to press it at a pressure of 5 N for 2 min to obtain the modified plasma matrix material.

[0063] Example 3

[0064] This example provides a modified plasma matrix material and a preparation method thereof. The preparation method includes the following steps:

[0065] (1) Osteoblasts were cultured in vitro for 7 days. The culture medium was DMEM cell culture medium containing 10 wt% FBS (fetal bovine serum), 0.1 μmol / L dexamethasone, 0.2 mmol / L vitamin C, and 10 mmol / L β-glycerophosphate. The temperature of the incubator was 37 °C, the CO2 level was 5%, and the relative humidity was 95%. After culturing, the culture medium was centrifuged at a centrifugal force of 300 g for 10 min, at a centrifugal force of 2000 g for 10 min, and at a centrifugal force of 18000 g for 30 min successively at 4 °C to remove dead cells, cell debris, microbubbles, apoptotic bodies, etc. in the culture medium; the supernatant was collected and centrifuged at a centrifugal force of 110000 g for 180 min at 4 °C to obtain the osteoblast extracellular vesicle precipitate;

[0066] (2) The patient's blood was collected in a liquid plasma matrix collection tube, and the plasma matrix preparation platform was used to centrifuge at a centrifugal force of 50 g for 10 min to obtain the liquid plasma matrix;

[0067] (3) The osteoblast extracellular vesicles were resuspended in 0.05 mL of physiological saline, and the liquid plasma matrix was added so that the mass-volume ratio of the osteoblast extracellular vesicles to the liquid plasma matrix was 70 μg:1 mL. After standing for 10 min, the plasma matrix preparation kit was used to press at a pressure of 3 N for 5 min to obtain the modified plasma matrix material.

[0068] Performance Test 1

[0069] To explore the extracellular vesicle sustained-release effect of the modified plasma matrix material prepared in Example 1, the test method was as follows: The modified plasma matrix material prepared in Example 1 and the osteoblast extracellular vesicle precipitate obtained in step (1) of Example 1 were directly placed in DMEM culture medium for testing.

[0070] The test results are as Figure 1 shown, Figure 1 which is the extracellular vesicle sustained-release effect diagram of the modified plasma matrix material prepared in Example 1. It can be seen from Figure 1 this that the release time of the osteoblast extracellular vesicles by the modified plasma matrix material prepared in the present invention is 13 days, while the osteoblast extracellular vesicles not loaded on the plasma matrix are completely released in 1 day, indicating that the modified plasma matrix material of the present invention has a significant extracellular vesicle sustained-release effect.

[0071] Performance Test 2

[0072] To investigate the effect of the modified plasma matrix material prepared in Example 1 on the osteogenic differentiation of cells, the test method was to prepare a DMEM cell culture medium containing 10 wt% FBS (fetal bovine serum), 0.1 μmol / L dexamethasone, 0.2 mmol / L vitamin C, and 10 mmol / L β-glycerophosphate, and change the medium once every 3 days. The cells were cultured using a transwell chamber. The cells were seeded in the lower chamber, and the modified plasma matrix material prepared in Example 1 or osteoblast extracellular vesicles were placed in the upper chamber. The osteoblasts were induced to mineralize. After culturing at 37 °C for 14 days, a Biyuntian alizarin red staining kit was used for detection.

[0073] The test results are as Figure 2 shown in Figure 2 Figure [specific figure number] for the effects of the modified plasma matrix material prepared in Example 1 and osteoblast extracellular vesicles on the osteogenic differentiation of cells respectively. As can be Figure 2 seen, the modified plasma matrix material prepared in the present invention has the effect of promoting the osteogenic differentiation of cells and can accelerate the bone regeneration process.

[0074] It should be noted that in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variation thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the existence of additional identical elements in the process, method, article or device comprising the element.

[0075] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments described herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a modified plasma matrix material, characterized in that The preparation method includes mixing osteoblast extracellular vesicles with a liquid plasma matrix, standing and then pressing to obtain the modified plasma matrix material.

2. The preparation method according to claim 1, characterized in that, The preparation method further includes centrifuging blood to obtain the liquid plasma matrix; Preferably, the centrifugal force for the centrifugation is 50 - 1000g; Preferably, the time for the centrifugation is 2 - 10 min.

3. The preparation method according to claim 1 or 2, characterized in that, The mass - volume ratio of the osteoblast extracellular vesicles to the liquid plasma matrix is (30 - 70) μg:1 mL.

4. The preparation method according to any one of claims 1-3, characterized in that, The preparation method further includes culturing osteoblasts in vitro, centrifuging and separating after culturing to collect the supernatant and extracting osteoblast extracellular vesicles; Preferably, the culture medium for the culture is DMEM medium; Preferably, the time for the culture is 7 - 21 days.

5. The preparation method according to claim 4, characterized in that The method for centrifugal separation is to centrifuge at 300g for 10 min, centrifuge at 2000g for 10 min, and centrifuge at 18000g for 30 min in sequence.

6. The preparation method according to claim 4 or 5, characterized in that, The method for extracting osteoblast extracellular vesicles is ultra - centrifugation; Preferably, the centrifugal force for the ultra - centrifugation is 110000g; Preferably, the time for the ultra - centrifugation is 70 - 180 min.

7. The preparation method according to claim 6, characterized in that, The temperature during the centrifugal separation and the ultra - centrifugation is independently 4°C.

8. The preparation method according to any one of claims 1-7, characterized in that, The preparation method further includes placing the osteoblast extracellular vesicles in a solvent and then mixing with the liquid plasma matrix; and / or, the standing time ≥10 min; and / or, the pressure for pressing is 1 - 5 N; and / or, the time for pressing is 2 - 10 min.

9. The modified plasma matrix material prepared by the preparation method according to any one of claims 1 - 8.

10. The application of the modified plasma matrix material according to claim 9 in bone defect repair materials.

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