Method for optimizing preparation of solid plasma matrix by ultraviolet irradiation and application thereof

Abstract

The application discloses a method for preparing a solid-state plasma matrix by optimizing ultraviolet irradiation, which comprises the following steps: loading a whole blood sample into a blood collection tube, and then centrifuging; separating a yellow gel solid from the top layer after centrifugation; and irradiating the obtained yellow gel solid from the top layer under ultraviolet light to obtain a solid-state plasma matrix; and the obtained solid-state plasma matrix is used for facial beautification and hair regeneration. The solid-state plasma matrix prepared by the method has the characteristics of higher strength and longer degradation time, improves the clinical application effect in oral tissue regeneration, widens the application scene, and solves the problems of insufficient strength and fast degradation speed of the current solid-state plasma matrix product.

Description

Technical Field

[0001] This invention belongs to the field of blood products technology, specifically relating to a method and application of optimizing the preparation of solid plasma matrix using ultraviolet irradiation. Background Technology

[0002] Wound healing is a complex biological process, mainly comprising four phases: the bleeding phase, the inflammation phase, the proliferative phase, and the remodeling phase. Platelet aggregation is a crucial step in these processes. Platelet concentrates, which concentrate supraphysiological doses of platelets to promote wound healing, have emerged. With technological and economic advancements, the plasma matrix has evolved over decades, and platelet concentrates, as a source of regenerative autologous growth factors, have been widely used in the treatment of soft and hard tissue regeneration, facial aesthetics, and hair regeneration.

[0003] Plasma matrix contains three basic elements required for tissue regeneration: (1) a three-dimensional scaffold: a scaffold formed by the fibrin matrix in the blood; (2) autologous living cells: autologous cells such as leukocytes, neutrophils, and platelets contained in the blood; and (3) growth factors: concentrated growth factors that may be released over time (10 to 14 days). The content of these three elements in plasma matrix may vary depending on the preparation method.

[0004] However, under current plasma matrix preparation mechanisms, the resulting products have low strength and rapid degradation, thus limiting their applications. Therefore, developing a method for preparing a solid plasma matrix with a dense structure, better tensile strength, and slower degradation is of great significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for preparing solid plasma matrix by using ultraviolet irradiation to optimize the preparation of solid plasma matrix, which addresses the shortcomings of the prior art. The solid plasma matrix prepared by this method has the characteristics of more compact structure, better tensile strength and longer degradation time, thus solving the problems of insufficient strength and fast degradation rate of current solid plasma matrix products.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for optimizing the preparation of solid plasma matrix using ultraviolet irradiation, characterized in that the method includes the following steps:

[0007] S1. Load the whole blood sample into the blood collection tube, then centrifuge for 5 min to 10 min. After centrifugation, separate the upper yellow gel-like solid.

[0008] S2. The upper yellow gel-like solid obtained in S1 is irradiated under ultraviolet light with a wavelength of 240nm to 280nm to obtain a solid plasma matrix.

[0009] Preferably, the amount of whole blood sample used in S1 is 10 mL.

[0010] Preferably, the centrifugal force in S1 is 700×g to 1000×g.

[0011] Preferably, the radiation time in S2 is 20s to 80s, and the radiation dose is 8J / cm² to 12J / cm².

[0012] The present invention also provides an application of the above-mentioned method for preparing solid plasma matrix by ultraviolet irradiation optimization, characterized in that the solid plasma matrix is ​​used for facial beauty and hair regeneration.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. Existing plasma matrices do not have a fully formed internal fibrous network after preparation. Therefore, this invention applies ultraviolet (UV) irradiation after centrifugation to alter the mechanical strength and composition of the product. During UV irradiation, its effect is primarily achieved by activating fibrin binding sites on platelets. Thus, by incorporating UV irradiation during preparation, this invention can more effectively activate the coagulation cascade reaction, promoting the formation of a three-dimensional fibrous network structure within the plasma matrix. This results in a more densely structured solid plasma matrix product. In clinical applications, the improved tensile strength and longer degradation time contribute to more ideal therapeutic effects.

[0015] 2. The method of the present invention can activate platelets in the blood by irradiating with ultraviolet light, and prepare a larger and more solid fibrin matrix network. The solid platelet-rich fibrin membrane encapsulates more platelets, white blood cells and growth factors, thereby expanding its clinical application scenarios and achieving better regenerative effects.

[0016] 3. By adjusting the irradiation time and controlling the radiation dose, this invention can flexibly regulate the binding of platelets to fibrinogen and the cross-linking of fibrin, thereby forming a fibrin matrix network with a better three-dimensional structure, increasing the bioactivity and degradation time of solid platelet-rich fibrin.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of ultraviolet irradiation of the plasma matrix in this invention.

[0019] Figure 2 The diagram shows the fracture strength of the solid plasma matrix membrane treated with ultraviolet light for different times in Example 1 and the solid plasma matrix membrane treated in the dark in Comparative Example 1.

[0020] Figure 3 This is a membrane area diagram of the solid plasma matrix membrane treated with ultraviolet light in Example 1 and the solid plasma matrix membrane treated with light protection in Comparative Example 1.

[0021] Figure 4 This is a graph showing the degradation rate of the solid plasma matrix prepared in Example 1 and the solid plasma matrix prepared in Comparative Example 1 in 0.25% pancreatic enzyme.

[0022] Figure 5 This is a graph showing the leukocyte concentration in the solid plasma matrix prepared in Example 1 and the solid plasma matrix prepared in Comparative Example 1.

[0023] Figure 6 This is a graph showing the platelet concentration in the solid plasma matrix prepared in Example 1 and the solid plasma matrix prepared in Comparative Example 1.

[0024] Figure 7 This is a graph showing the concentration of vascular endothelial growth factor (VEGF) in the solid plasma matrix prepared in Example 1 and the solid plasma matrix prepared in Comparative Example 1.

[0025] Figure 8 These are scanning electron microscope images of fibrin in the solid plasma matrix prepared by ultraviolet irradiation for different times in Example 1 and the solid plasma matrix prepared in Comparative Example 1. Detailed Implementation

[0026] Example 1

[0027] This embodiment utilizes ultraviolet irradiation to optimize the preparation of solid plasma matrix, including the following steps:

[0028] S1. Put 10 mL of whole blood sample into a blood collection tube, and then quickly put it into a horizontal centrifuge and centrifuge for 8 min at a centrifugal force of 700×g. After centrifugation, the upper yellow gel-like solid is separated.

[0029] The whole blood sample was obtained from the volunteer's elbow crease using a disposable intravenous blood collection tube, and the platelet count in the whole blood sample was 100 × 10⁻⁶. 9 / liter ~300×10 9 / Lift;

[0030] S2. The upper yellow gel-like solid obtained in S1 is irradiated with ultraviolet light at a wavelength of 240 nm for 20 seconds at a dose of 12 J / cm² to obtain a solid plasma matrix; in this embodiment, the ultraviolet irradiation of the plasma matrix is ​​as follows: Figure 1 As shown.

[0031] Comparative Example 1

[0032] The method for preparing the solid plasma matrix in this comparative example is the same as that in Example 1, except that the upper yellow gel-like solid obtained in S1 is protected from light in this comparative example.

[0033] The solid plasma matrices prepared in Example 1 and Comparative Example 1 were pressed into films using a dedicated solid plasma matrix membrane pressing instrument, resulting in ultraviolet-irradiated solid plasma matrix membranes and light-shielded solid plasma matrix membranes, respectively. The tensile properties of the light-shielded and ultraviolet-irradiated solid plasma matrix membranes were then measured using a tensile testing instrument. The ultraviolet-irradiated solid plasma matrix membranes were irradiated with ultraviolet light for 20s, 40s, 60s, and 80s, respectively. The results are as follows: Figure 2 As shown, group c represents the fracture strength of solid plasma matrix membranes treated with light-shielding. It can be seen that solid plasma matrix membranes prepared by ultraviolet irradiation have enhanced elastic modulus, tensile strain, tensile stress and maximum tensile force of solid PRF membranes (solid plasma matrix membranes). The fracture strength is the highest after 60s of ultraviolet irradiation.

[0034] Example 1: Ultraviolet irradiation treatment of solid plasma matrix membranes can better resist the stretching and tearing caused by external mechanical stress, and has broader application prospects in clinical practice. It can be used for:

[0035] (1) In soft tissue augmentation, the mucosal contour can be better maintained; in gingival papilla reconstruction, the negative impact of complex oral mastication movements on the reconstructed area can be resisted.

[0036] (2) Guiding a longer degradation time in bone tissue regeneration;

[0037] (3) Provide stronger mechanical protection for bone augmentation areas;

[0038] (4) Better stretchability allows the membrane to be applied to defective areas with complex surface morphology.

[0039] The membrane area of ​​the solid plasma matrix membrane treated with ultraviolet light in Example 1 and the solid plasma matrix membrane treated with light-shielded light in Comparative Example 1 were calculated using the software ImageJ. Figure 3 As shown, ultraviolet irradiation treatment of solid plasma matrix membranes increases the membrane area compared to light-protected solid plasma matrix membranes; therefore, the method of this embodiment can prepare larger plasma matrix products with the same blood collection volume.

[0040] The solid plasma matrices prepared in Example 1 and Comparative Example 1 were respectively placed in 0.25% trypsin for degradation testing. Data were recorded at 0h, 3h, 12h, and 24h to observe the degradation process, and degradation rate curves were plotted. The results are as follows: Figure 4As shown in the figure, the solid plasma matrix group prepared in Comparative Example 1 degraded faster than the group treated with ultraviolet light. This means that the degradation experiment of plasma matrix products was prolonged after ultraviolet light treatment, which is more conducive to their retention in the body and provides a longer-lasting barrier support for healing processes such as bone regeneration.

[0041] The concentrations of white blood cells, platelets, and vascular endothelial growth factor (VEGF) in the solid plasma matrix prepared in Example 1 and Comparative Example 1 were measured respectively. White blood cell and platelet concentrations were detected using blood biochemistry, and VEGF concentrations were detected using an ELISA kit. The concentrations were calculated. The results are as follows: Figure 5 , Figure 6 and Figure 7 As shown, the concentrations of leukocytes, platelets, and vascular endothelial growth factor (VEGF) in the solid plasma matrix prepared in Example 1 are all higher than those in the solid plasma matrix prepared in Comparative Example 1.

[0042] Electron microscopy was used to display the fibrous network in the solid plasma matrix prepared in Example 1 and the solid plasma matrix prepared in Comparative Example 1. The solid plasma matrix prepared in Example 1 was subjected to ultraviolet radiation for 20 s, 40 s, 60 s, and 80 s, respectively. The results are as follows. Figure 8 As shown, the solid plasma matrix irradiated by ultraviolet light has a larger and more robust fibrin matrix network. By adjusting the ultraviolet irradiation time, the binding of platelets to fibrinogen and the cross-linking of fibrin can be flexibly controlled to be more compact, thereby forming a fibrin matrix network with a better three-dimensional structure.

[0043] In this invention, whole blood samples from 2,000 to 3,000 volunteers were processed in steps S1-S2 of this embodiment, resulting in solid plasma matrices that exhibited more compact structures, better tensile strength, and longer degradation times.

[0044] Example 2

[0045] This embodiment utilizes ultraviolet irradiation to optimize the preparation of solid plasma matrix, including the following steps:

[0046] S1. Put 10 mL of whole blood sample into a blood collection tube, and then quickly put it into a horizontal centrifuge and centrifuge for 5 min at a centrifugal force of 1000×g. After centrifugation, the upper yellow gel-like solid is separated.

[0047] The whole blood sample was obtained from the volunteer's elbow crease using a disposable intravenous blood collection tube, and the platelet count in the whole blood sample was 100 × 10⁻⁶. 9 / liter ~300×10 9 / Lift;

[0048] S2. The upper yellow gel-like solid obtained in S1 is irradiated under ultraviolet light with a wavelength of 280nm for 80s and a radiation dose of 8J / cm2 to obtain a solid plasma matrix.

[0049] Example 3

[0050] This embodiment utilizes ultraviolet irradiation to optimize the preparation of solid plasma matrix, including the following steps:

[0051] S1. Put 10 mL of whole blood sample into a blood collection tube, and then quickly put it into a horizontal centrifuge and centrifuge for 10 min at a centrifugal force of 800×g. After centrifugation, the upper yellow gel-like solid is separated.

[0052] The whole blood sample was obtained from the volunteer's elbow crease using a disposable intravenous blood collection tube, and the platelet count in the whole blood sample was 100 × 10⁻⁶. 9 / liter ~300×10 9 / Lift;

[0053] S2. The upper yellow gel-like solid obtained in S1 is irradiated under ultraviolet light with a wavelength of 250nm for 60s and a radiation dose of 9J / cm2 to obtain a solid plasma matrix.

[0054] Example 4

[0055] This embodiment utilizes ultraviolet irradiation to optimize the preparation of solid plasma matrix, including the following steps:

[0056] S1. Put 10 mL of whole blood sample into a blood collection tube, and then quickly put it into a horizontal centrifuge and centrifuge for 9 min at a centrifugal force of 900×g. After centrifugation, the upper yellow gel-like solid is separated.

[0057] The whole blood sample was obtained from the volunteer's elbow crease using a disposable intravenous blood collection tube, and the platelet count in the whole blood sample was 100 × 10⁻⁶. 9 / liter ~300×10 9 / Lift;

[0058] S2. The upper yellow gel-like solid obtained in S1 is irradiated under ultraviolet light with a wavelength of 260nm for 40s and a radiation dose of 10J / cm2 to obtain a solid plasma matrix.

[0059] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for optimizing the preparation of solid plasma matrix using ultraviolet irradiation, characterized in that, The method includes the following steps: S1. Place the whole blood sample into a blood collection tube, then centrifuge for 5 to 10 minutes. After centrifugation, separate the upper yellow gel-like solid. The volume of the whole blood sample is 10 mL. The centrifugal force is 700 × g to 1000 × g. S2. The upper yellow gel-like solid obtained in S1 is irradiated under ultraviolet light with a wavelength of 240 nm to obtain a solid plasma matrix; the irradiation time is 60 s and the radiation dose is 12 J / cm². 2 ; The ultraviolet irradiation activates platelets in the blood, thereby activating the coagulation cascade reaction and promoting the formation of a three-dimensional fibrous network structure within the plasma matrix, thus producing a more compact solid plasma matrix product. Through ultraviolet irradiation, platelets in the blood can be activated to produce a larger and more robust fibrin matrix network, with more platelets, leukocytes, and growth factors encapsulated in the solid platelet-rich fibrin membrane.

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