Biologically-crosslinked solid plasma matrix as well as preparation method and application thereof
By cross-linking treatment with magnesium ions and strontium ions in solid plasma matrix, the problems of insufficient mechanical strength and short degradation time of solid plasma matrix are solved, and a stronger and longer-lasting tissue regeneration effect is achieved.
Patent Information
- Application Number
- CN202411484128.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2024-12-17
AI Technical Summary
The existing solid plasma matrix has insufficient mechanical strength and short degradation time, which cannot meet the needs of long-term wound healing.
Using metal ions (such as magnesium ions and strontium ions) as crosslinking agents, biocrosslinked solid plasma matrix is prepared by centrifugation and immersion treatment, enhancing its fibrin network, improving mechanical strength and degradation time.
It significantly improves the mechanical strength and degradation time of solid plasma matrix, extends its duration in the body, and enhances anti-inflammatory and promotes tissue regeneration capabilities.
Smart Images
Figure HDA0005098274700000011 
Figure HDA0005098274700000012
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomaterial histological engineering, and in particular to a bio-cross-linked solid plasma matrix and a preparation method and application thereof. Background Art
[0002] Plasma matrix is a kind of active cells and cytokines derived from autologous peripheral blood, which is enriched with supraphysiological doses of platelets, leukocytes, etc. through centrifugation. It has a good ability to promote the regeneration of soft and hard tissues. In recent years, it has been widely used in clinical fields such as the healing of oral soft and hard tissue defects and refractory skin wounds.
[0003] By changing the centrifugal conditions and the material of the blood collection tube, plasma matrix products in different states can be prepared. By using a hydrophilic surface blood collection tube, a solid plasma matrix can be prepared, which can be subsequently pressed into a plasma matrix membrane as a barrier membrane for application in the field of guided bone regeneration / guided tissue regeneration. However, the solid plasma matrix prepared under current technical conditions often has the disadvantages of insufficient mechanical strength and short in vivo degradation time, which cannot meet the needs of long-term wound healing. Summary of the invention
[0004] In order to solve at least one of the above technical problems, the present invention provides a bio-crosslinked solid plasma matrix and a preparation method and application thereof. By using metal ions as crosslinking agents of the solid plasma matrix, the anti-inflammatory properties and the ability to promote tissue healing of the solid plasma matrix are improved.
[0005] As one aspect, the present invention provides a method for preparing a bio-crosslinked solid plasma matrix, and the preparation method is selected from any one of the following methods: Method I: placing a first volume of blood in a blood collection tube, centrifuging and stripping to obtain a solid plasma matrix; soaking the solid plasma matrix in a solution containing metal ions, and then washing to obtain a bio-crosslinked solid plasma matrix; Method II: spraying or adding a solution containing metal ions into a blood collection tube to obtain a blood collection tube containing metal ions; placing a second volume of blood in a blood collection tube containing metal ions, centrifuging and stripping to obtain a bio-crosslinked solid plasma matrix; the metal ions include magnesium ions and / or strontium ions.
[0006] In some embodiments of the present invention, the mass concentration of metal ions in the solution containing metal ions is 0.1% to 10%, for example, it can be 0.1%, 1.3%, 2.6%, 3.9%, 5.7%, 7.4%, 8.1%, 8.9%, 9.3%, etc.
[0007] In some embodiments of the present invention, the solute of the solution containing metal ions is at least one of chloride, sulfate, nitrate or acetate of the metal ions; and / or the solvent of the solution containing metal ions is phosphate buffer; and / or the preparation material of the blood collection tube is selected from plastic or glass.
[0008] In some embodiments of the present invention, the chloride of the metal ion includes at least one of magnesium chloride and strontium chloride.
[0009] In some embodiments of the present invention, the centrifugal acceleration is 30-1000g, for example, it can be 58g, 83g, 169g, 265g, 388g, 498g, 589g, 623g, 769g, 835g, 968g, 998g, etc., and the centrifugal time is 1-30min, for example, it can be 1.8min, 3.7min, 12.6min, 17.3min, 19.6min, 21.6min, 23.6min, 26.6min, 29.6min, etc.
[0010] In some embodiments of the present invention, in method I, the soaking temperature is 37°C to 60°C, for example, it can be 39°C, 43°C, 49°C, 53°C, 56°C, 59°C, etc.; and / or the soaking time is 1 to 30 min, for example, it can be 2.8 min, 9.7 min, 13.6 min, 21.8 min, 25.9 min, 29.7 min, etc.
[0011] In some embodiments of the present invention, in method I, the solution used for washing is physiological saline.
[0012] In some embodiments of the present invention, in method II, the ratio of the volume of the solution containing metal ions to the second volume is in the range of 1% to 5%, for example, it can be 1.6%, 2.3%, 3.2%, 4.1%, 4.6%, 4.9%, etc.
[0013] As another aspect, the present invention provides a bio-crosslinked solid plasma matrix, which is obtained by the above-mentioned preparation method.
[0014] As yet another aspect, the present invention provides a use of the bio-cross-linked solid plasma matrix as described above in bone regeneration materials.
[0015] The technical solution of the embodiment of the present invention has the following advantages:
[0016] The preparation method of the bio-crosslinked solid plasma matrix of the embodiment of the present invention can enhance the fibrin network inside the solid plasma matrix by using the metal ions in the cross-linked drug as the bio-crosslinking agent of the solid plasma matrix, making it stronger and resistant to degradation. When applied to tissue regeneration, it can be retained for a longer time and improve the effect. In addition, the preparation method of the bio-crosslinked solid plasma matrix can adjust the concentration of the metal ion solution used according to the needs of treatment, thereby obtaining a solid plasma matrix with different crosslinking strengths.
[0017] The bio-cross-linked solid plasma matrix obtained by the preparation method of the embodiment of the present invention has the characteristics of higher strength, longer degradation time, more sustained ability to release active factors, and more optimized anti-inflammatory ability; it solves the problems of low strength, rapid degradation, and inability to be used for long-term promotion of tissue regeneration and wound healing of the current solid plasma matrix membrane. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] 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.
[0019] 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.
[0020] Figure 1 This is a comparison chart of the biocompatibility of the bio-cross-linked solid plasma matrix prepared in Example 1 of the present invention and the solid plasma matrix prepared in Comparative Example 1;
[0021] Figure 2 This is a comparison diagram of the fibrin structure of the bio-cross-linked solid plasma matrix prepared in Example 2 of the present invention and the solid plasma matrix prepared in Comparative Example 2. DETAILED DESCRIPTION
[0022] 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.
[0023] 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.
[0024] Metal compounds in cross-linking drugs (such as strontium chloride, magnesium chloride, etc.) have been extensively studied in the field of hard tissue regeneration and have good biocompatibility.
[0025] Studies have shown that strontium ions can prolong the survival time of osteoblasts, promote early adhesion, proliferation, differentiation and matrix mineralization of osteoblasts, and inhibit the expression of proinflammatory factors tumor necrosis factor-α, IL-6, and IL-1β in macrophages, reduce the differentiation of macrophages into osteoclasts, and reduce the number of osteoclasts, thereby promoting new bone formation and promoting osteogenesis.
[0026] Studies have shown that magnesium ions significantly promote the proliferation, migration and adhesion, and osteogenic differentiation of mesenchymal stem cells, especially directly promoting osteogenic differentiation through the classical Wnt pathway, MAPK / ERK pathway, etc., and indirectly promoting osteogenic differentiation by promoting ganglion cells to secrete CGRP, macrophages to secrete bone morphogenetic protein and other cytokines. Magnesium ions mainly promote their proliferation, migration and adhesion, and osteogenic activity through the TRPM / PI3K signaling pathway, inhibit their apoptosis, and can inhibit osteoclast formation and bone resorption activity through the nuclear transcription factor κB signaling pathway. Magnesium ions can regulate the immune response in the bone environment, transform macrophage phenotypes, promote the secretion of osteogenic factors, reduce the secretion of inflammatory factors, reduce inflammatory responses, and promote the proliferation, migration and adhesion of vascular endothelial cells, angiogenesis, and promote angiogenesis.
[0027] According to an embodiment of the present invention, a method for preparing a bio-crosslinked solid plasma matrix is provided, and the preparation method is selected from any one of the following methods: Method I: placing a first volume of blood in a blood collection tube, centrifuging and peeling, to obtain a solid plasma matrix; immersing the solid plasma matrix in a solution containing metal ions, and then washing to obtain a bio-crosslinked solid plasma matrix; Method II: spraying or adding a solution containing metal ions into a blood collection tube to obtain a blood collection tube containing metal ions; placing a second volume of blood in a blood collection tube containing metal ions, centrifuging and peeling, to obtain a bio-crosslinked solid plasma matrix; the metal ions include magnesium ions and / or strontium ions.
[0028] In some embodiments of the present invention, the solution containing metal ions includes the following situations: the solute in the solution is only a compound containing metal ions (in this case, the solution containing metal ions is a metal ion solution), the solute in the solution includes a compound containing metal ions, and other compounds that do not react with the compound containing metal ions.
[0029] In some embodiments of the present invention, a method for preparing a bio-cross-linked solid plasma matrix comprises the following steps:
[0030] (1) placing a first volume of blood in a blood collection tube, and centrifuging at a centrifugal acceleration of 30 to 1000 g for 1 to 30 minutes to obtain a first centrifugation product;
[0031] Preferably, the material used to prepare the blood collection tube is selected from plastic or glass.
[0032] (2) Using a stripper to separate the solid plasma matrix from the interface between the red blood cells in the first centrifugation product obtained in step (1), thereby obtaining a solid plasma matrix.
[0033] (3) soaking the solid plasma matrix obtained in step (2) in a solution containing metal ions at a temperature of 37° C. to 60° C., and then washing to obtain a bio-crosslinked solid plasma matrix;
[0034] Preferably, the solution used for washing is physiological saline; the mass concentration of the solution containing metal ions is 0.1% to 10%; and the solvent of the solution containing metal ions is phosphate buffer.
[0035] In some embodiments of the present invention, a method for preparing a bio-cross-linked solid plasma matrix comprises the following steps:
[0036] (1) Spray or add a solution containing metal ions into the blood collection tube;
[0037] Preferably, the material for preparing the blood collection tube is selected from plastic or glass; the solute of the solution containing metal ions is a metal ion compound, and the metal ion compound is selected from at least one of magnesium chloride and strontium chloride.
[0038] (2) collecting a second volume of blood into the blood collection tube of step (1);
[0039] Preferably, the ratio of the mass of the metal ion-containing solution sprayed in step (1) to the second volume collected in step (2) is in the range of 0.1% to 5%.
[0040] (3) centrifuging the blood collection tube containing blood obtained in step (2) at a centrifugal acceleration of 30 to 1000 g for 1 to 30 min to obtain a centrifugal product;
[0041] (4) Separating the solid plasma matrix from the interface between the solid plasma matrix and the red blood cells from the centrifugal product obtained in step (3) using a stripper to obtain a bio-cross-linked solid plasma matrix.
[0042] In order to further illustrate the effect of the present invention, specific embodiments are used for illustration below.
[0043] Example 1
[0044] This embodiment provides a method for preparing a bio-cross-linked solid plasma matrix, comprising the following steps:
[0045] (1) collecting a first volume of 10 mL of venous blood in a glass blood collection tube and placing the blood in a horizontal centrifuge; centrifuging the blood at 700 g for 8 minutes, and taking out the centrifuged blood to obtain a first centrifugation product;
[0046] (2) using a stripper to separate the solid plasma matrix from the interface of red blood cells from the first centrifugation product obtained in step (1) to obtain a solid plasma matrix;
[0047] (3) Pressing 2 g of the solid plasma matrix obtained in step (2) into a film, soaking it in 20 mL of a strontium chloride solution having a corresponding mass volume ratio of 10% (relative to the "first volume 10 mL") (wherein the mass volume ratio of the strontium chloride solution is 10%), and soaking it at 37° C. for 72 hours; then washing away the metal ion solution with physiological saline to obtain a bio-cross-linked solid plasma matrix;
[0048] (4) The bio-cross-linked solid plasma matrix obtained in step (3) was immersed in a DMEM culture medium solution. After three days, the immersed solution was filtered through a 0.22 μm filter to prepare an exudate. 20% of the exudate was used to prepare an osteoblast culture medium, and osteoblasts were cultured. The activity of osteoblasts was measured on the first, third, and fifth days. The specific test results are shown in Figure 1 middle.
[0049] Comparative Example 1
[0050] (1) collecting a first volume of 10 mL of venous blood in a glass blood collection tube and placing the blood in a horizontal centrifuge; centrifuging the blood at 700 g for 8 minutes, and taking out the centrifuged blood to obtain a centrifugation product;
[0051] (2) separating the solid plasma matrix from the interface of red blood cells using a stripper to obtain a solid plasma matrix;
[0052] (3) Pressing 2 g of the solid plasma matrix obtained in step (2) into a film, soaking it in a phosphate buffer solution at 37° C. for 72 hours; then washing away the metal ion solution with physiological saline to obtain a normal solid plasma matrix;
[0053] (4) The common solid plasma matrix obtained in step (3) was immersed in a DMEM culture medium solution. After three days, the immersion solution was filtered through a 0.22 μm filter to prepare an exudate. 20% of the exudate was used to prepare an osteoblast culture medium, and osteoblasts were cultured. The activity of osteoblasts was measured on the first, third, and fifth days. The specific test results are shown in Figure 1 Among them, Figure 1 In the table, “*” indicates p<0.0332; “**” indicates p<0.0021; “***” indicates p<0.0002; “****” indicates p<0.0001.
[0054] according to Figure 1 The data analysis in the results showed that the bio-cross-linked solid plasma matrix membrane obtained by immersion in metal ion solution had good biocompatibility.
[0055] Example 2
[0056] This embodiment provides a method for preparing a bio-cross-linked solid plasma matrix, comprising the following steps:
[0057] (1) Adding 0.5% mass volume ratio (relative to 10 mL volume) of magnesium metal ion solution into a glass blood collection tube;
[0058] (2) collecting 10 mL of venous blood and placing it in the blood collection tube obtained in step (1); centrifuging at 700 g for 8 minutes, and taking out the centrifuged material to obtain a centrifugation product;
[0059] (3) separating the solid plasma matrix from the interface of red blood cells using a stripper to obtain a bio-cross-linked solid plasma matrix;
[0060] (4) pressing the bio-cross-linked solid plasma matrix obtained in step (3) into a membrane to obtain a bio-cross-linked solid plasma matrix membrane;
[0061] (5) The bio-crosslinked solid plasma matrix membrane obtained in step (4) was treated in a 2.5% glutaraldehyde solution for 4 hours and observed under a scanning electron microscope. The test results are as follows: Figure 2 shown.
[0062] Comparative Example 2
[0063] (1) Collect 10 mL of venous blood into a glass blood collection tube; centrifuge at 700 g for 8 minutes, and take out the centrifuged material to obtain a centrifugation product;
[0064] (2) separating the solid plasma matrix from the interface of red blood cells using a stripper to obtain a common solid plasma matrix;
[0065] (4) pressing the common solid plasma matrix obtained in step (3) into a membrane to obtain a common solid plasma matrix membrane;
[0066] (5) The ordinary solid plasma matrix membrane obtained in step (4) was treated in a 2.5% glutaraldehyde solution for 4 hours and observed by scanning electron microscopy. The test results are as follows: Figure 2 shown.
[0067] according to Figure 2 From the data analysis in, we can draw the following conclusion: the fibrin network structure of the bio-cross-linked solid plasma matrix membrane obtained by immersion in magnesium metal ion solution is more dense.
[0068] In summary, the bio-crosslinked solid plasma matrix and its preparation method and application provided by the embodiment of the present invention; by using a metal ion solution to treat the solid plasma matrix, the mechanical strength of the solid plasma matrix membrane can be significantly improved, the degradation time can be prolonged, and the corresponding inflammation regulation ability or osteogenic ability of the metal ions can be combined to make it more conducive to anti-inflammatory and tissue regeneration. In addition, the bio-crosslinked solid plasma matrix and its preparation method and application according to the embodiment of the present invention can also effectively solve the problems of insufficient strength, too fast degradation time, too low biological activity, etc. of the current plasma matrix preparation products, and continuously provide the growth factors and barrier protection required for tissue regeneration. The bio-crosslinked solid plasma matrix obtained by the preparation method of the embodiment of the present invention has a broader application prospect in clinical practice.
[0069] 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.
[0070] 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 bio-crosslinked solid plasma matrix, characterized in that: The preparation method is selected from any one of the following methods: Method I: placing a first volume of blood in a blood collection tube, centrifuging and stripping, and obtaining a solid plasma matrix; soaking the solid plasma matrix in a solution containing metal ions, and then washing to obtain a bio-crosslinked solid plasma matrix; Method II: Spraying or adding a solution containing metal ions into a blood collection tube to obtain a blood collection tube containing metal ions; placing the second volume of blood in the blood collection tube containing metal ions, centrifuging and stripping, and obtaining a bio-cross-linked solid plasma matrix; The metal ions include magnesium ions and / or strontium ions.
2. The preparation method according to claim 1, characterized in that: The mass concentration of metal ions in the metal ion-containing solution is 0.1% to 10%.
3. The preparation method according to claim 1 or 2, characterized in that: The solute of the solution containing metal ions is at least one of chloride, sulfate, nitrate or acetate of the metal ions; And / or, the solvent of the solution containing metal ions is phosphate buffer; And / or, the blood collection tube is made of a material selected from plastic or glass.
4. The preparation method according to claim 3, characterized in that: The chloride of the metal ion includes at least one of magnesium chloride and strontium chloride.
5. The preparation method according to any one of claims 1 to 4, characterized in that: The centrifugal acceleration is 30-1000g, and the centrifugal time is 1-30min.
6. The preparation method according to any one of claims 1 to 5, characterized in that: In the method I, The soaking temperature is 37°C to 60°C; And / or, the soaking time is 1 to 30 minutes.
7. The preparation method according to any one of claims 1 to 6, characterized in that: In the method I, The solution used for the washing is physiological saline.
8. The preparation method according to any one of claims 1 to 7, characterized in that: In the method II, The ratio of the volume of the solution containing metal ions to the second volume is in the range of 1% to 5%.
9. A bio-cross-linked solid plasma matrix, characterized in that: The bio-cross-linked solid plasma matrix is obtained by the preparation method according to any one of claims 1 to 8.
10. Use of the bio-cross-linked solid plasma matrix according to claim 9 in bone regeneration materials.