Preservation method of plasma matrix
By preserving the plasma matrix in a constant temperature metal bath device, the problems of coagulation and collapse at room temperature are solved, their biological activity and mechanical properties are maintained, and clinical applications are expanded.
Patent Information
- Application Number
- CN202411290692.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-17
AI Technical Summary
Plasma matrix is prone to coagulation or collapse after being placed at room temperature for a long time, resulting in limited clinical use scenarios.
The plasma matrix is stored using a constant temperature metal bath device, and the temperature is controlled at 0-8°C to maintain its biological activity and mechanical properties.
It effectively prevents the coagulation of liquid plasma matrix and the collapse of solid plasma matrix, broadening its clinical use scenarios.
Smart Images
Figure CN119138405A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomaterials, and in particular to a method for preserving a plasma matrix. 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 the healing of refractory skin wounds 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 greater 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 gel-like after solidification, and can also provide some buffering effects for intervertebral discs, articular discs, etc. At present, in the clinical use scenario of plasma matrix, it is necessary to draw blood from patients before surgery for preparation. However, due to the complexity and time-consuming nature of oral soft and hard tissue surgery, long-term waiting at room temperature will cause the plasma matrix product to have problems such as liquid plasma matrix coagulation and solid plasma matrix collapse.
[0003] Based on this, the present invention provides a method for preserving a plasma matrix. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a method for preserving plasma matrix, which effectively solves the problems of liquid plasma matrix coagulation and solid plasma matrix collapse after long-term storage at room temperature while ensuring that the biological activity and mechanical properties of the plasma matrix are not affected, and at the same time provides a new idea for long-term preservation of plasma matrix products.
[0005] In a first aspect, the present invention provides a method for preserving a plasma matrix, the method comprising: immediately placing the upper plasma matrix obtained by centrifuging blood in a constant temperature metal bath device for preservation until it is taken out for use.
[0006] The present invention uses a constant temperature metal bath device to replace the traditional room temperature storage condition as the plasma matrix storage condition. The operation is simple and convenient, and can ensure that the biological activity and mechanical properties of the plasma matrix are not affected. It effectively solves the problems of liquid plasma matrix coagulation and solid plasma matrix collapse after long-term storage at room temperature, and broadens the clinical application scenarios of plasma matrix products.
[0007] As a preferred technical solution of the present invention, the temperature of the constant temperature metal bath device is 0-8°C, such as 0°C, 2°C, 4°C, 6°C, 8°C, etc. When stored at this temperature, the plasma matrix is more stable, and the biological activity, mechanical properties, etc. are not affected.
[0008] As a preferred technical solution of the present invention, the upper plasma matrix includes liquid plasma matrix or solid plasma matrix gel.
[0009] As a preferred technical solution of the present invention, the centrifugal force during centrifugation is 500-700×g, for example, 500×g, 550×g, 600×g, 650×g, 700×g, etc.
[0010] 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.
[0011] As a preferred technical solution of the present invention, the blood centrifugation is to place the plasma matrix blood collection tube containing blood in a centrifugal device for centrifugation, and the preservation is to place the plasma matrix blood collection tube obtained by obtaining the upper plasma matrix in a constant temperature metal bath device for preservation.
[0012] In the present invention, before blood coagulation, the blood is placed in a plasma matrix blood collection tube and centrifuged to obtain an upper plasma matrix. The plasma matrix blood collection tube is immediately placed in a constant temperature metal bath device for storage. It is understood that during the storage process, the blood collection tube should not be opened to keep the plasma matrix product isolated from the outside world, thereby avoiding the influence of the external environment on it. The material of the plasma matrix blood collection tube is plastic or glass.
[0013] As a preferred technical solution of the present invention, the amount of blood in the plasma matrix blood collection tube is 10 mL.
[0014] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:
[0015] The present invention uses a constant temperature metal bath device to replace the traditional room temperature storage condition as the plasma matrix storage condition. The operation is simple and convenient, and can ensure that the biological activity and mechanical properties of the plasma matrix are not affected. It effectively solves the problems of liquid plasma matrix coagulation and solid plasma matrix collapse after long-term storage at room temperature, and broadens the clinical application scenarios of plasma matrix products. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] 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.
[0017] 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.
[0018] Figure 1 This is a schematic diagram of the preparation and storage process of the plasma matrix in Example 1 of the present invention;
[0019] Figure 2 This is a comparison chart of the coagulation time of the liquid plasma matrix prepared in Example 1 of the present invention and Comparative Example 1 respectively;
[0020] Figure 3 This is a comparison chart of the cell activities of the plasma matrix gel after being stored in a 4° C. constant temperature metal bath according to Example 1 of the present invention and the liquid plasma matrix prepared in Comparative Example 1;
[0021] Figure 4 This is a SEM image of the plasma matrix gel after preservation in Example 1 of the present invention;
[0022] Figure 5 This is a SEM image of the liquid plasma matrix prepared in Comparative Example 1 of the present invention;
[0023] Figure 6 The volume comparison diagram of the plasma matrix gel obtained by storing Example 2 of the present invention and Comparative Example 2 respectively;
[0024] Figure 7 These are the degradation performance diagrams of the plasma matrix membranes prepared in Example 2 of the present invention and Comparative Example 2 respectively;
[0025] Figure 8 This is a comparison diagram of the areas of the plasma matrix membranes prepared in Example 2 of the present invention and Comparative Example 2 respectively;
[0026] Fig. 9 This is a SEM image of the plasma matrix membrane prepared in Example 2 of the present invention;
[0027] Fig.10 This is a SEM image of the plasma matrix membrane prepared in Comparative Example 2 of the present invention;
[0028] Fig.11 The maximum strain comparison diagram of the plasma matrix membranes prepared in Example 2 of the present invention and Comparative Example 2 respectively;
[0029] Fig.12 The graph is a comparison of the breaking strength of the plasma matrix membranes prepared in Example 2 of the present invention and Comparative Example 2, respectively. DETAILED DESCRIPTION
[0030] 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.
[0031] 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.
[0032] Example 1
[0033] This embodiment provides a method for preserving a plasma matrix. The schematic diagram of the preparation and preservation process of the plasma matrix is as follows: Figure 1 As shown, the specific steps include:
[0034] (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;
[0035] (2) Immediately place the plastic plasma matrix blood collection tube described in step (1) into a 4°C constant temperature metal bath for storage.
[0036] The whole blood sample was collected from the cubital fossa of the volunteer. The plastic plasma matrix blood collection tube was purchased from Primedun. The platelet content in the whole blood sample was 100×10 9 ~300×10 9 / Lift.
[0037] Example 2
[0038] This embodiment provides a method for preserving a plasma matrix, comprising the following steps:
[0039] (1) Collect 10 mL of whole blood sample into 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 a solid plasma matrix clot on the upper layer;
[0040] (2) Immediately place the glass plasma matrix blood collection tube described in step (1) into a 4°C constant temperature metal bath for storage for 30 minutes.
[0041] (3) using scissors to bluntly separate the upper solid plasma matrix clot placed in the blood collection tube in step (2) from the red blood cell layer below, and then using a special film pressing device box (Plemedon) to press it into a film shape to obtain a plasma matrix membrane;
[0042] 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 platelet content in the whole blood sample was 100×10 9 ~300×10 9 / Lift.
[0043] Example 3
[0044] This embodiment provides a method for preserving a plasma matrix, which is the same as that of Embodiment 1. The difference from Embodiment 1 is that the centrifugal force in this embodiment is 700×g and the centrifugation time is 2 min.
[0045] Example 4
[0046] This embodiment provides a method for preserving a plasma matrix, which is the same as that of Embodiment 1. The difference from Embodiment 1 is that the centrifugal force in this embodiment is 600×g and the centrifugation time is 5 min.
[0047] Example 5
[0048] This embodiment provides a method for preserving a plasma matrix. The method is the same as that in Embodiment 2. The difference from Embodiment 2 is that the centrifugation time in this embodiment is 2 minutes.
[0049] Example 6
[0050] This embodiment provides a method for preserving a plasma matrix. The method is the same as that in Embodiment 1. The difference from Embodiment 1 is that the centrifugation time in this embodiment is 2 minutes.
[0051] Example 7
[0052] This embodiment provides a method for preserving a plasma matrix, which is the same as that of Embodiment 2. The difference from Embodiment 2 is that the centrifugal force in this embodiment is 600×g and the centrifugation time is 5 min.
[0053] Comparative Example 1
[0054] This comparative example provides a method for preserving a plasma matrix, which is the same as that of Example 1. The difference from Example 1 is that in this comparative example, the plastic plasma matrix blood collection tube described in step (1) is stored at room temperature.
[0055] Comparative Example 2
[0056] This comparative example provides a method for preserving a plasma matrix, which is the same as that of Example 2. The difference from Example 2 is that in this comparative example, step (2) is replaced by preserving the glass plasma matrix blood collection tube described in step (1) at room temperature for 30 minutes, and then performing step (3).
[0057] Performance Testing
[0058] 1. Observe the coagulation time of the liquid plasma matrix prepared in Example 1 and Comparative Example 1 respectively. The results are as follows: Figure 2 shown.
[0059] Depend on Figure 2 It can be found that in Example 1 of the present invention, by immediately placing the prepared liquid plasma matrix into a 4° C. constant temperature metal bath for storage, the time for the liquid plasma matrix to form a gel can be effectively prolonged.
[0060] 2. After culturing HGF cells for 72 hours using the plasma matrix gel preserved in Example 1 and the liquid plasma matrix prepared in Comparative Example 1, the number of cells was measured. The results were as follows: Figure 3 .
[0061] Depend on Figure 3 It can be found that there is no significant difference in the number of cells after the plasma matrix of Example 1 of the present invention is stored in a 4°C constant temperature metal bath and that of cells cultured in a common liquid plasma matrix, indicating that the storage of the plasma matrix in a constant temperature metal bath will not affect its biological activity.
[0062] 3. The plasma matrix gel after storage in Example 1 and the liquid plasma matrix prepared in Comparative Example 1 were subjected to SEM tests. The SEM images of the plasma matrix gel after storage in Example 1 are as follows: Figure 4 As shown, the SEM image of the liquid plasma matrix prepared in Comparative Example 1 is as follows Figure 5 shown.
[0063] Depend on Figure 4 and Figure 5 It can be found that there is no significant difference in the fibrin structure density between the plasma matrix of Example 1 of the present invention after being stored in a 4°C constant temperature metal bath and the ordinary liquid plasma matrix, indicating that the mechanical properties of the plasma matrix will not be affected after being stored in a constant temperature metal bath.
[0064] 4. Observe the volume of the plasma matrix gel obtained by storing Example 2 and Comparative Example 2, and the results are as follows: Figure 6 shown.
[0065] Depend on Figure 6 It can be found that there is no significant difference in the volume of the plasma matrix gel after being stored in a 4°C constant temperature metal bath in Example 2 of the present invention and the plasma matrix gel after being stored at room temperature in Comparative Example 2.
[0066] 5. Investigate the degradation performance of the plasma matrix membrane prepared in Example 2 and Comparative Example 2.
[0067] The plasma matrix membrane obtained in Example 2 and the plasma matrix membrane obtained in Comparative Example 2 were respectively placed in 10% serum DMEM for degradation test, and the mass was measured at 1d, 3d, 5d, 7d, 14d, 21d, and 28d to observe the degradation. The results are as follows: Figure 7 shown.
[0068] Depend on Figure 7 It can be found that the degradation time of the plasma matrix after being stored in a 4° C. constant temperature metal bath according to Example 2 of the present invention after being pressed into film is no different from that of the common plasma matrix membrane.
[0069] 6. The areas of the plasma matrix membranes prepared in Example 2 and Comparative Example 2 were observed. Figure 8 shown.
[0070] Depend on Figure 8 It can be found that there is no significant difference in the area of the plasma matrix after being pressed after being stored in a 4°C constant temperature metal bath in Example 2 of the present invention and the area of the plasma matrix after being pressed after being stored at a normal temperature.
[0071] 7. The plasma matrix membrane prepared in Example 2 and the plasma matrix membrane prepared in Comparative Example 2 were subjected to SEM tests. The SEM image of the plasma matrix membrane prepared in Example 2 is as follows: Fig. 9 As shown, the SEM image of the plasma matrix membrane prepared in Comparative Example 2 is as follows Fig.10 shown.
[0072] Depend on Fig. 9 and Fig.10 It can be found that there is no significant difference in the ultrastructure of the plasma matrix membrane prepared in Example 2 and the plasma matrix membrane prepared in Comparative Example 2.
[0073] 8. The plasma matrix membrane prepared in Example 2 and the plasma matrix membrane prepared in Comparative Example 2 were subjected to tensile tests. The maximum strain results are as follows: Fig.11 The breaking strength results are shown in Fig.12 shown.
[0074] Depend on Fig.11 and Fig.12 It can be found that there is no significant difference in the mechanical strength of the plasma matrix membrane after being pressed after being stored in a 4°C constant temperature metal bath in Example 2 of the present invention and the plasma matrix membrane prepared by being stored at room temperature in Comparative Example 2, indicating that the storage of the plasma matrix in a constant temperature metal bath will not affect its mechanical properties.
[0075] 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.
[0076] 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 preserving a plasma matrix, It is characterized in that The storage method comprises: immediately placing the upper plasma matrix obtained by centrifuging the blood in a constant temperature metal bath device for storage until it is taken out for use.
2. The storage method according to claim 1, It is characterized in that The temperature of the thermostatic metal bath device is 0-8°C.
3. The storage method according to claim 1 or 2, It is characterized in that The upper plasma matrix includes a liquid plasma matrix or a solid plasma matrix gel.
4. The storage method according to any one of claims 1 to 3, It is characterized in that The centrifugal force during the centrifugation is 500-700×g; And / or, the centrifugation time is 2-8 min.
5. The storage method according to any one of claims 1 to 4, It is characterized in that The blood centrifugation is to place the plasma matrix blood collection tube containing blood in a centrifugal device for centrifugation, and the preservation is to place the plasma matrix blood collection tube obtained by obtaining the upper plasma matrix in a constant temperature metal bath device for preservation.
6. The storage method according to claim 5, It is characterized in that The amount of blood in the plasma matrix blood collection tube is 10 mL.