Plasma matrix compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a plasma matrix compound and a preparation method and application thereof. The preparation method comprises the steps that blood is centrifuged and then mixed with a biological cross-linking agent for a reaction, and the plasma matrix compound is obtained, or mixing the blood with the biological cross-linking agent in the centrifugation process for reaction to obtain the plasma matrix compound. The plasma matrix compound provided by the invention has the characteristics of high strength, long degradation time, more lasting release of active factors and the like, has excellent anti-inflammatory performance and cartilage regeneration capacity, and solves the problems that the existing plasma matrix membrane is low in strength and fast in degradation and cannot be used for promoting tissue regeneration and wound healing for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a plasma matrix complex and a preparation method and application thereof. Background Art

[0002] Plasma matrix is ​​a biomaterial derived from autologous peripheral blood. Due to its good ability to promote the regeneration of soft and hard tissues, plasma matrix has been widely used in clinical fields such as oral soft and hard tissue defects and refractory skin wound healing in recent years. The significant repair effect of plasma matrix comes from the living cells such as platelets, fibrin scaffolds and growth factors that can promote tissue healing contained in it. Recent studies have shown that a large number of white blood cells are also the main influencing factors in the process of tissue wound healing. It can further enhance angiogenesis and tissue formation, and play a good role in wound healing and repair. Liquid plasma matrix can remain liquid for a period of time after preparation and can be used for injection to the required position. It has certain applications in the treatment of intervertebral disc lesions and osteoarthritis. Liquid plasma matrix can become a gel after solidification, and can also provide some buffering effects on intervertebral discs, articular discs, etc.

[0003] However, the plasma matrix prepared under current technical conditions has a short degradation time in vivo and cannot meet the needs of long-term wound healing. Therefore, it is of great significance to provide a plasma matrix complex and a preparation method thereof. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a plasma matrix complex and a preparation method and application thereof. The plasma matrix complex provided by the present invention has the characteristics of high strength, long degradation time, more sustained release of active factors, and excellent anti-inflammatory properties and cartilage regeneration ability, which solves the current problems of low strength, rapid degradation, and inability to be used for long-term promotion of tissue regeneration and wound healing of plasma matrix membranes.

[0005] In a first aspect, the present invention provides a method for preparing a plasma matrix complex, the preparation method comprising: mixing blood with a biological cross-linking agent after centrifugation to react, thereby obtaining the plasma matrix complex;

[0006] Alternatively, the blood is mixed with a biocrosslinking agent during centrifugation to react and obtain the plasma matrix complex.

[0007] The preparation method provided by the present invention utilizes a biological cross-linking agent to react with a plasma matrix, and the prepared plasma matrix complex fibrin structure is more compact and has higher strength, has a prolonged degradation time, and releases active factors more persistently. When applied to wound healing and tissue regeneration, it can be retained for a longer time, effectively improving the therapeutic effect, and has broad application prospects in clinical practice. Specifically:

[0008] The preparation method of the present invention utilizes a biocrosslinking agent to react with a plasma matrix after or during blood centrifugation, which can form a denser fibrin network structure inside the plasma matrix, making it stronger, thereby effectively improving the strength and degradation time of the prepared plasma matrix complex, continuously providing the required growth factors and barrier protection for tissue regeneration, and solving the current problems of insufficient plasma matrix membrane strength, rapid degradation, and low bone regeneration activity.

[0009] As a preferred technical solution of the present invention, the step of mixing with a biological cross-linking agent for reaction after centrifugation comprises:

[0010] (1) Centrifuging the blood to obtain the upper plasma matrix;

[0011] (2) The upper plasma matrix or the upper plasma matrix is ​​pressed into a film and then mixed with a biocrosslinking agent solution to react to obtain the plasma matrix complex.

[0012] The upper plasma matrix may be a liquid plasma matrix or a gel-like plasma matrix solidified from the liquid plasma matrix.

[0013] As a preferred technical solution of the present invention, the upper plasma matrix is ​​mixed with the bio-crosslinker solution for a reaction time of 5-60 minutes, for example, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, etc., so that the upper plasma matrix can fully react with the bio-crosslinker, thereby effectively improving its performance.

[0014] As a preferred technical solution of the present invention, the time for the upper layer of plasma matrix to be mixed with the bio-crosslinker solution for reaction after being pressed into a membrane is 5-60 minutes, for example, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, etc., so that the plasma matrix membrane can fully react with the bio-crosslinker, thereby effectively improving its performance.

[0015] As a preferred technical solution of the present invention, the mass volume ratio of the biocrosslinking agent in step (2) to the blood in step (1) is 0.01-0.5 g:10 mL, for example, 0.01 g:10 mL, 0.1 g:10 mL, 0.2 g:10 mL, 0.3 g:10 mL, 0.4 g:10 mL, 0.5 g:10 mL, etc. The present invention adjusts the amount of the biocrosslinking agent according to different treatment needs to obtain plasma matrix complexes with different crosslinking strengths.

[0016] As a preferred technical solution of the present invention, the temperature during the reaction is 37-60°C, for example, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc.

[0017] As a preferred technical solution of the present invention, the step of mixing with the biocrosslinking agent for reaction during the centrifugation process comprises:

[0018] (1) spraying a biocrosslinking agent solution into a plasma matrix blood collection tube;

[0019] (2) placing the blood in the plasma matrix blood collection tube described in step (1) and centrifuging it to obtain an upper plasma matrix complex.

[0020] The material of the plasma matrix blood collection tube can be plastic or glass.

[0021] As a preferred technical solution of the present invention, the mass volume ratio of the biocrosslinking agent in step (1) and the blood in step (2) is 0.1-5g:100mL, for example, 0.1g:100mL, 1g:100mL, 2g:100mL, 3g:100mL, 4g:100mL, 5g:100mL, etc. The present invention adjusts the amount of the biocrosslinking agent according to different treatment needs to obtain plasma matrix complexes with different crosslinking strengths.

[0022] As a preferred technical solution of the present invention, after the biocrosslinker solution is sprayed on the plasma matrix blood collection tube and mixed with blood, the concentration of the biocrosslinker is 0.01-2mM, for example, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 2mM, etc., and the concentration of the biocrosslinker herein is the concentration of the biocrosslinker in the total volume of the solvent and blood.

[0023] As a preferred technical solution of the present invention, the biocrosslinking agent includes genipin. The present invention uses genipin as a biocrosslinking agent for the plasma matrix, which can effectively improve the strength and degradation time of the plasma matrix, and at the same time can enhance the anti-inflammatory properties of the plasma matrix and the ability to promote tissue healing and cartilage regeneration. It can be applied in the fields of wound healing and tissue regeneration to improve the therapeutic effect and broaden the indications. Specifically:

[0024] Genipin is a natural biocrosslinker with excellent biocompatibility and biodegradability. It can react with the amine groups in the plasma matrix protein components to play a crosslinking role, thereby improving the strength of the liquid plasma matrix or plasma matrix membrane and prolonging the degradation time. Moreover, the process of genipin reacting with the plasma matrix is ​​mild and green, and its cytotoxicity is much lower than that of chemical crosslinkers. In addition, genipin can significantly promote the differentiation of macrophages to the M2 phenotype. This phenotype of macrophages can secrete anti-inflammatory factors and promote tissue healing, thereby effectively improving the anti-inflammatory ability of the plasma matrix and the cartilage regeneration ability.

[0025] The present invention first sprays a biocrosslinker solution onto a plasma matrix blood collection tube, then adds blood, and during the centrifugation process, the plasma matrix and the biocrosslinker in the blood are evenly mixed and reacted, and the upper layer after centrifugation is the plasma matrix complex of the present invention, which can be post-processed according to the actual application scenario, such as pressed into a film. The film pressing of the present invention is performed using a dedicated instrument box for the plasma matrix.

[0026] As a preferred technical solution of the present invention, the centrifugation is horizontal centrifugation.

[0027] As a preferred technical solution of the present invention, the centrifugal force of the centrifugation is 500-700×g, for example, 500×g, 550×g, 600×g, 650×g, 700×g, etc.

[0028] As a preferred technical solution of the present invention, the centrifugation time is 2-8 min, for example, 2 min, 4 min, 6 min, 8 min, etc.

[0029] As a preferred technical solution of the present invention, the concentration of the biocrosslinking agent solution is 0.1-5 g / mL, for example 0.1 g / mL, 0.5 g / mL, 1 g / mL, 2 g / mL, 3 g / mL, 4 g / mL, 5 g / mL, etc.

[0030] As a preferred technical solution of the present invention, the solvent in the biocrosslinking agent solution is a phosphate buffer solution.

[0031] The present invention utilizes a biocrosslinking agent to treat plasma matrix (liquid or gel) or plasma matrix membrane, which can significantly improve the toughness, tensile strength and degradation time of the liquid plasma matrix after gelation or the plasma matrix membrane, making it more conducive to tissue regeneration.

[0032] In a second aspect, the present invention provides a plasma matrix complex prepared by the preparation method described in the first aspect.

[0033] The plasma matrix complex provided by the present invention has the characteristics of high strength, long degradation time, more sustained release of active factors, and excellent anti-inflammatory properties and cartilage regeneration ability, which solves the current problems of low strength, rapid degradation, and inability to be used for long-term promotion of tissue regeneration and wound healing of plasma matrix membranes.

[0034] In a third aspect, the present invention provides the use of the plasma matrix complex described in the second aspect in materials for promoting wound healing and tissue regeneration.

[0035] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0036] 1. The preparation method provided by the present invention utilizes a biocrosslinking agent to react with a plasma matrix, and the prepared plasma matrix complex fibrin structure is denser and stronger, has a prolonged degradation time, and releases active factors more persistently. When applied to wound healing and tissue regeneration, it can be retained for a longer time, effectively improving the therapeutic effect, and has broad application prospects in clinical practice.

[0037] 2. The present invention utilizes genipin as a biocrosslinking agent for the plasma matrix, which can effectively improve the strength and degradation time of the plasma matrix, and at the same time can enhance the anti-inflammatory properties of the plasma matrix and the ability to promote tissue healing and cartilage regeneration. Its application in the fields of wound healing and tissue regeneration can improve the therapeutic effect and broaden the indications. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0040] Figure 1 This is a comparison chart of the time taken for the products of Example 1 of the present invention and Comparative Example 1 to form gel;

[0041] Figure 2 It is a tensile performance diagram of the products of Example 2 of the present invention and Comparative Example 2;

[0042] Figure 3 It is the degradation performance diagram of the products of Example 2 of the present invention and Comparative Example 2. DETAILED DESCRIPTION

[0043] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0044] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0045] Example 1

[0046] This embodiment provides a plasma matrix complex and a preparation method thereof, wherein the preparation method comprises the following steps:

[0047] (1) Collect 10 mL of whole blood sample in a plastic plasma matrix blood collection tube, quickly place it in a horizontal centrifuge and centrifuge it at a centrifugal force of 500 × g for 8 min to obtain an upper liquid plasma matrix;

[0048] (2) collecting the obtained upper liquid plasma matrix with a syringe and mixing it with 0.5 mL of 0.3 g / mL genipin phosphate buffer for 10 minutes to obtain a bio-crosslinked plasma matrix, i.e., the plasma matrix complex;

[0049] The whole blood sample was collected from the cubital fossa of the volunteers, the plastic plasma matrix blood collection tube was purchased from Primedun, and the platelet content in the whole blood sample was 100×10 9 -300×10 9 / Lift.

[0050] Example 2

[0051] This embodiment provides a plasma matrix complex and a preparation method thereof, wherein the preparation method comprises the following steps:

[0052] (1) Collect 10 mL of whole blood sample in a glass plasma matrix blood collection tube, quickly place it in a horizontal centrifuge and centrifuge it at a centrifugal force of 700 × g for 8 min to obtain an upper solid plasma matrix clot;

[0053] (2) using scissors to bluntly separate the lower red blood cell layer from the obtained upper solid plasma matrix clot, and then using a special film pressing device box (Pramei Shield) to press it into a film shape, and then immersing the plasma matrix membrane in 0.5 mL of genipin phosphate buffer for 10 minutes to obtain a bio-crosslinked plasma matrix membrane, i.e., the plasma matrix complex;

[0054] The whole blood sample was collected from the cubital fossa of the volunteer, the glass plasma matrix blood collection tube was purchased from Primedun, the concentration of genipin phosphate buffer was 0.3 g / mL, and the platelet content in the whole blood sample was 100×10 9 -300×10 9 / Lift.

[0055] Example 3

[0056] This embodiment provides a plasma matrix complex and a preparation method thereof. The preparation method is the same as that of Example 1, except that in this embodiment, the centrifugal force is 700×g, the centrifugation time is 2 min, and the concentration of genipin phosphate buffer is 0.1 g / mL.

[0057] Example 4

[0058] This embodiment provides a plasma matrix complex and a preparation method thereof. The preparation method is the same as that of Example 1. The difference from Example 1 is that in this embodiment, the centrifugal force is 600×g, the centrifugation time is 5 min, the concentration of genipin phosphate buffer is 5 g / mL, and the volume is 0.1 mL.

[0059] Example 5

[0060] This embodiment provides a plasma matrix complex and a preparation method thereof. The preparation method is the same as that of Example 2. The difference from Example 2 is that in this embodiment, the centrifugal force is 600×g, the centrifugation time is 2 min, and the plasma matrix membrane is immersed in genipin phosphate buffer for 60 min.

[0061] Example 6

[0062] This embodiment provides a plasma matrix complex and a preparation method thereof. The preparation method is the same as that of Example 2. The difference from Example 2 is that in this embodiment, the centrifugal force is 500×g, the centrifugation time is 5 min, and the plasma matrix membrane is immersed in genipin phosphate buffer for 5 min.

[0063] Example 7

[0064] This embodiment provides a plasma matrix complex and a preparation method thereof, wherein the preparation method comprises the following steps:

[0065] (1) Spray 0.5 mL of 0.3 g / mL genipin phosphate buffer into a plastic plasma matrix blood collection tube;

[0066] (2) placing 10 mL of the whole blood sample in the plasma matrix blood collection tube described in step (1), and quickly placing the sample in a horizontal centrifuge and centrifuging it at a centrifugal force of 500×g for 8 minutes to obtain an upper layer of biologically cross-linked plasma matrix, which is the plasma matrix complex;

[0067] The whole blood sample was collected from the cubital fossa of the volunteers, the plastic plasma matrix blood collection tube was purchased from Primedun, and the platelet content in the whole blood sample was 100×10 9 -300×10 9 / Lift.

[0068] Comparative Example 1

[0069] This comparative example provides a plasma matrix and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that step (2) is not performed in this comparative example, that is, after obtaining the upper liquid plasma matrix, it is not mixed with the genipin solution for reaction.

[0070] Comparative Example 2

[0071] This comparative example provides a plasma matrix and a preparation method thereof. The preparation method is the same as that of Example 2, but differs from Example 2 in that in this comparative example, after being pressed into a film, it is not mixed with a genipin solution for reaction.

[0072] Performance Testing

[0073] 1. Observe the time for the products of Example 1 and Comparative Example 1 to form gel. The results are as follows: Figure 1 shown.

[0074] Depend on Figure 1 It can be found that the plasma matrix complex prepared by the present invention has a shorter coagulation time than that of a common liquid plasma matrix.

[0075] The present invention selects whole blood samples of 10-20 volunteers to carry out the treatment of Example 1, and the obtained genipin bio-cross-linked plasma matrix complex gel structure is more stable, has better rheological properties, slower degradation, and better bone and soft tissue formation effects.

[0076] 2. The products prepared in the examples and comparative examples were subjected to tensile tests.

[0077] Among them, the tensile properties of the products of Example 2 and Comparative Example 2 are as follows Figure 2 As shown, the fracture strength on the ordinate is the maximum strength of the material when it breaks, which is given by Figure 2 It can be found that the plasma matrix complex prepared by the present invention has better tensile properties than ordinary plasma matrix membranes.

[0078] 3. The degradation performance of the products prepared in the examples and comparative examples was tested.

[0079] The plasma matrix complex prepared in Example 2 and the common plasma matrix membrane prepared in Comparative Example 2 were placed in 10% serum DMEM for degradation test, and their masses were measured at 1d, 3d, 5d, 7d, 9d, 14d and 28d to observe the degradation.

[0080] The degradation performance of the products of Example 2 and Comparative Example 2 is as follows Figure 3 As shown. Figure 3 It can be found that the degradation time of the plasma matrix complex prepared by the present invention is significantly longer than that of the ordinary plasma matrix membrane. This shows that when the bio-crosslinked plasma matrix complex (plasma matrix composite membrane) prepared by the present invention is used to guide bone regeneration surgery, the implanted bio-crosslinked plasma matrix complex can support the defect position for a longer time and play a better soft tissue barrier role. Moreover, the plasma matrix complex in this embodiment is easy to prepare and can be easily obtained from the patient himself, so it is usually not necessary to obtain it from other individuals, avoiding the risk of immune rejection.

[0081] The present invention selects whole blood samples from 10-20 volunteers to carry out the treatment of Example 2, and the obtained bio-cross-linked plasma matrix complex (membrane) fibrin structure is more compact, and has the characteristics of higher strength, slower degradation and better osteogenesis effect.

[0082] 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 "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0083] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may 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 the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a plasma matrix complex, characterized in that: The preparation method comprises: mixing the blood with a biological cross-linking agent after centrifugation to react and obtain the plasma matrix complex; Alternatively, the blood is mixed with a biological cross-linking agent during centrifugation to react and obtain the plasma matrix complex.

2. The preparation method according to claim 1, characterized in that: The step of mixing with a biological cross-linking agent for reaction after centrifugation comprises: (1) Centrifuging the blood to obtain the upper plasma matrix; (2) The upper plasma matrix or the upper plasma matrix is ​​pressed into a film and then mixed with a biocrosslinking agent solution to react to obtain the plasma matrix complex.

3. The preparation method according to claim 2, characterized in that: The time for the upper plasma matrix and the biological cross-linking agent solution to be mixed and reacted is 5-60 minutes; Alternatively, the upper plasma matrix is ​​pressed into a membrane and then mixed with the biocrosslinking agent solution for a reaction time of 5-60 minutes.

4. The preparation method according to claim 2 or 3, characterized in that: The mass volume ratio of the biocrosslinking agent in step (2) to the blood in step (1) is 0.01-0.5 g:10 mL; And / or, the temperature during the reaction is 37-60°C.

5. The preparation method according to claim 1, characterized in that: The step of mixing with the biocrosslinking agent to react during the centrifugation process comprises: (1) spraying a biocrosslinking agent solution into a plasma matrix blood collection tube; (2) placing the blood in the plasma matrix blood collection tube described in step (1) and centrifuging it to obtain an upper plasma matrix complex.

6. The preparation method according to claim 5, characterized in that: The mass volume ratio of the biocrosslinking agent in step (1) to the blood in step (2) is 0.1-5 g:100 mL.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The biocrosslinking agent includes genipin; And / or, the centrifugal force of the centrifugation is 500-700×g; And / or, the centrifugation time is 2-8 min.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The concentration of the biocrosslinking agent solution is 0.1-5 g / mL; And / or, the solvent in the biocrosslinking agent solution is a phosphate buffer solution.

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

10. Use of the plasma matrix complex according to claim 9 in materials for promoting wound healing and tissue regeneration.

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