Wound dressing and preparation method thereof

Abstract

The invention relates to the technical field of blood products, in particular to a wound dressing and a preparation method thereof. According to the preparation method provided by the invention, the plasma matrix is jointly treated by using a freeze drying technology and collagen, so that the three-dimensional protein fiber network density of the plasma matrix can be improved, meanwhile, the mechanical strength of a plasma matrix product is improved, the degradation time is prolonged, and the prepared wound dressing has a better effect in a clinical application scene.

Description

Technical Field

[0001] The present invention relates to the technical field of blood products, and in particular to a wound dressing and a preparation method thereof. Background Art

[0002] Full-thickness skin wounds caused by trauma, burns, and chronic diseases are serious conditions that are difficult to treat. Skin grafts and substitutes are the main treatment for full-thickness skin wounds, but skin transplants can cause defects in the donor site and may differ in color and texture from the recipient skin. In addition, transplants are subject to timely limitations. Tissue-engineered skin may be an effective skin substitute, and some related products are already available, but the risk of infection and rejection remains.

[0003] Wound repair is complex, and vascularization is crucial for wound repair. Biomatrix materials can simulate extracellular matrix to repair skin damage. However, the use of a single matrix cannot induce sufficient angiogenesis. Multiple growth factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), transforming growth factor-β1, epidermal growth factor (EGF), etc., affect cell proliferation, differentiation and new blood vessel formation. After these growth factors are injected into the body by direct injection, they are usually incorporated into a controlled release system due to the lack of continuous diffusion and degradation restrictions, but the controlled release system usually encapsulates only one or two growth factors, and wound healing is driven by different growth factors, so it is not enough to repair the wound.

[0004] Plasma matrix is ​​a new generation of platelet concentrate based on platelet-rich plasma (PRP). This material is rich in a variety of growth factors, including VEGF, PDGF, bFGF and EGF, which play a key role in tissue repair and regeneration. In addition, these growth factors can be released slowly. Studies have found that the growth factors contained in the plasma matrix can be continuously released for more than 2 weeks. Other studies have also confirmed that the plasma matrix is ​​a more stable sustained-release system than PRP. However, fresh plasma matrix is ​​not easy to store and transport, has a small elastic modulus, low hardness, high immunogenicity, and fast degradation. These problems limit the application of plasma matrix in large-scale production. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a wound dressing and a preparation method thereof. The present invention adopts a method of freeze-drying a solid plasma matrix in combination with collagen, and the wound dressing prepared has the characteristics of a denser fibrin structure, higher strength, and slower degradation, which can solve the problems of insufficient strength and short degradation time of plasma matrix products in the current clinical application scenarios of skin wound dressings.

[0006] In a first aspect, the present invention provides a method for preparing a wound dressing, comprising the following steps:

[0007] (1) collecting a whole blood sample in a blood collection tube and centrifuging it;

[0008] (2) separating and collecting the upper solid plasma matrix;

[0009] (3) freeze-drying the solid plasma matrix;

[0010] (4) Mixing the freeze-dried solid plasma matrix with a collagen solution to obtain a wound dressing of the freeze-dried plasma matrix mixed with collagen.

[0011] The present invention reduces the water content of the plasma matrix to a minimum through freeze-drying technology, which is beneficial to maintaining the stability of the morphology of the plasma matrix and the biological properties of the plasma matrix protein, especially the biological activity of various growth factors.

[0012] The collagen added by the preparation method of the present invention, as a main component of the skin, has the advantages of low antigenicity, high biocompatibility, excellent biological activity, etc., and can be incorporated into the plasma matrix to make the fibrin network formed inside the plasma matrix denser, increase the mechanical strength of the plasma matrix product, and prolong the degradation time.

[0013] In some embodiments of the present invention, the centrifugal acceleration is 20-950g, for example, 50g, 150g, 300g, 500g, 700g, 900g, etc.

[0014] In some embodiments of the present invention, the centrifugation time is 2-8 min, for example, 3 min, 4 min, 5 min, 6 min, 7 min, etc.

[0015] In some embodiments of the present invention, the preparation method further comprises, before step (3), preparing the solid plasma matrix into a solid plasma matrix product having a preset shape.

[0016] In some embodiments of the present invention, the solid plasma matrix product comprises a plasma matrix membrane or a plasma matrix plug.

[0017] In some embodiments of the present invention, the concentration of the collagen solution is 0.5-2%, such as 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, etc.

[0018] In some embodiments of the present invention, the mass volume ratio of the collagen solution to the solid plasma matrix after freeze-drying is 1:1-2, such as 1:1.2, 1:1.4, 1:1.6, 1:1.8, etc.

[0019] In some embodiments of the present invention, the mixing method includes dropping the collagen liquid into the solid plasma matrix after freeze-drying.

[0020] In some embodiments of the present invention, the mixing method includes immersing the solid plasma matrix product after freeze-drying in a collagen solution.

[0021] In some embodiments of the present invention, the soaking time is 10-30 min, such as 15 min, 20 min, 25 min, etc.

[0022] In some embodiments of the present invention, the preparation method further comprises heating the freeze-dried solid plasma matrix and the collagen solution after mixing.

[0023] The heating treatment in the preparation method of the present invention is used to prolong the degradation time of the mixture of solid plasma matrix and collagen solution.

[0024] In some embodiments of the present invention, the temperature of the heating treatment is 37-70°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, etc.

[0025] In some embodiments of the present invention, the heating treatment time is 1 to 60 minutes, for example, 5 minutes, 15 minutes, 25 minutes, 35 minutes, 45 minutes, 55 minutes, etc.

[0026] In a second aspect, the present invention provides a wound dressing, which is prepared by the preparation method described in the first aspect.

[0027] The wound dressing provided by the present invention is prepared by mixing freeze-dried plasma matrix with collagen, and has a more ideal application effect in application scenarios such as clinical wound dressing.

[0028] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0029] The preparation method provided by the present invention uses freeze-drying technology to process the plasma matrix, and jointly prepares the plasma matrix with collagen to obtain a wound dressing, which can improve the density of the three-dimensional protein fiber network of the plasma matrix, better maintain the morphology of the plasma matrix and the biological stability of the plasma matrix protein, especially the biological activity of growth factors, while increasing the mechanical strength of the plasma matrix product and prolonging the degradation time, thereby achieving better therapeutic effects in application scenarios such as clinical wound dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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.

[0031] 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.

[0032] Figure 1 This is a scanning electron microscope photograph of the wound dressing of Example 1 of the present invention;

[0033] Figure 2 This is a scanning electron microscope photograph of the wound dressing of Example 2 of the present invention;

[0034] Figure 3 This is a scanning electron microscope photograph of the wound dressing of Example 3 of the present invention;

[0035] Figure 4 This is a scanning electron microscope photograph of the wound dressing of Example 4 of the present invention;

[0036] Figure 5 This is a scanning electron microscope photograph of the wound dressing of Comparative Example 1 of the present invention;

[0037] Figure 6 Result diagram of tensile strain of wound dressings of Examples 1-4 of the present invention and Comparative Example 1;

[0038] Figure 7 Result diagram of tensile stress of wound dressings of Examples 1-4 of the present invention and Comparative Example 1;

[0039] Figure 8 The degradation behavior diagram of the wound dressings of Examples 1-4 of the present invention and Comparative Example 1;

[0040] Fig. 9 This is a graph showing the cumulative release rate of growth factors of the wound dressings of Example 1 and Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] Example 1

[0044] This embodiment provides a wound dressing, and the preparation method is as follows:

[0045] (1) Collect 10 mL of whole blood sample using a hydrophilic blood collection tube and quickly centrifuge it at 700 g for 8 min;

[0046] (2) separating and collecting the upper solid plasma matrix, and pressing the solid plasma matrix into a membrane using a membrane pressing device box;

[0047] (3) The intact fresh solid plasma matrix membrane was frozen and stored at -80°C for 30 min and then freeze-dried at -51°C overnight;

[0048] (4) The solid plasma matrix after freeze-drying overnight is immersed in a 0.5% collagen solution for 30 minutes (the volume ratio of the solid plasma matrix to the collagen solution is 1:1), and then heated at 45° C. for 5 minutes to obtain the freeze-dried plasma matrix mixed with collagen wound dressing.

[0049] Example 2

[0050] This embodiment provides a wound dressing, which is different from Embodiment 1 in that the concentration of the collagen solution is 1%.

[0051] Example 3

[0052] This embodiment provides a wound dressing, which is different from Embodiment 1 in that the concentration of the collagen solution is 1.5%.

[0053] Example 4

[0054] This embodiment provides a wound dressing, which is different from Embodiment 1 in that the concentration of the collagen solution is 2%.

[0055] Comparative Example 1

[0056] This comparative example provides a wound dressing, which differs from Example 1 in that step (4) is not included.

[0057] Comparative Example 2

[0058] This comparative example provides a wound dressing, which differs from Example 1 in that step (3) is not included.

[0059] Performance Testing

[0060] 1. Detection of changes in fibrin structure

[0061] The wound dressings prepared in Examples 1-4 and Comparative Example 1 were treated in a 2.5% glutaraldehyde solution for 4 hours and observed under a scanning electron microscope. Figure 1-5 shown.

[0062] Figure 1-4 The following are scanning electron microscope photos of the wound dressings of Examples 1-4, Figure 5 The scanning electron microscope photo of the wound dressing of Comparative Example 1 is shown in FIG. Figure 1-4 and Figure 5 It can be seen from the comparison that the present invention combines collagen with freeze-dried plasma matrix membrane to significantly increase the three-dimensional protein fiber network density of the plasma matrix product.

[0063] 2. Mechanical strength test

[0064] The wound dressings prepared in Examples 1-4 and Comparative Example 1 were tested for mechanical properties using a tensile tester. The results are as follows: Figure 6 and 7 shown.

[0065] Figure 6 The result diagram of the tensile strain of the wound dressing is given by Figure 6 It can be seen that the present invention combines collagen with freeze-dried plasma matrix membrane, so that the tensile strain of the plasma matrix product is significantly improved, and within the range of 0.5-2% collagen, the higher the concentration of incorporated collagen, the greater the tensile strain of the plasma matrix product. Figure 7 The result diagram of the tensile stress of the wound dressing is given by Figure 7 It can be seen that the present invention combines collagen with freeze-dried plasma matrix membrane to significantly improve the tensile stress of the plasma matrix product. Figure 6 and Figure 7 , indicating that the combined use of collagen and freeze-dried plasma matrix membrane can significantly improve the mechanical strength of the plasma matrix membrane.

[0066] 3. Degradation effect detection

[0067] The wound dressings prepared in Examples 1-4 and Comparative Example 1 were placed in a DMEM medium containing 10% serum for degradation testing. Photos were taken at 1d, 3d, 5d, 7d, and 9d to observe the degradation. The results are as follows: Figure 8 shown.

[0068] Figure 8 The degradation behavior diagram of wound dressing is shown in Figure 2. Figure 8 It can be seen that the combined use of collagen and freeze-dried plasma matrix membrane can significantly prolong the degradation time of plasma matrix products, and within the range of 0.5-2% collagen, the higher the concentration of incorporated collagen, the slower the degradation rate of plasma matrix products.

[0069] 4. Growth Factor Release Detection

[0070] The wound dressings prepared in Example 1 and Comparative Example 2 were placed in 4 mL of DMEM and subjected to ELISA to detect PDGF. The wound dressings were collected at 1 d, 4 d, 7 d, 14 d, 21 d, and 28 d, respectively. The results are as follows: Fig. 9 shown.

[0071] Fig. 9 is the cumulative release rate of growth factors from the wound dressing (release amount of growth factors in the wound dressing / volume of the wound dressing), Fig. 9 It can be seen that the release rate of growth factors in the plasma matrix product of the freeze-dried plasma matrix membrane is significantly slowed down compared with the plasma matrix membrane that has not been freeze-dried.

[0072] In summary, the freeze-dried plasma matrix mixed with collagen wound dressing prepared by the preparation method provided by the present invention can increase the density of the three-dimensional protein fiber network of the plasma matrix, slow down the release rate of growth factors, and at the same time increase the mechanical strength of the plasma matrix product, prolong the degradation time, and provide a more effective effect for wound healing.

[0073] 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.

[0074] 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 wound dressing, characterized in that: The steps include: (1) collecting a whole blood sample in a blood collection tube and centrifuging it; (2) separating and collecting the upper solid plasma matrix; (3) freeze-drying the solid plasma matrix; (4) Mixing the freeze-dried solid plasma matrix with the collagen solution to obtain the wound dressing.

2. The preparation method according to claim 1, characterized in that: The centrifugal acceleration is 20-950g.

3. The preparation method according to claim 1 or 2, characterized in that: The centrifugation time is 2-8 min.

4. The preparation method according to any one of claims 1 to 3, characterized in that The preparation method further comprises, before step (3), preparing the solid plasma matrix into a solid plasma matrix product having a preset shape; Preferably, the solid plasma matrix preparation comprises a plasma matrix membrane or a plasma matrix plug.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The concentration of the collagen solution is 0.5-2%; Preferably, the mass volume ratio of the collagen solution to the solid plasma matrix after freeze-drying is 1:1-2.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The mixing method includes dropping the collagen liquid into the solid plasma matrix after freeze-drying.

7. The preparation method according to any one of claims 1 to 5, characterized in that: The mixing method includes immersing the freeze-dried solid plasma matrix in the collagen solution; Preferably, the soaking time is 10-30 minutes.

8. The preparation method according to any one of claims 1 to 7, characterized in that: The preparation method further comprises: mixing the freeze-dried solid plasma matrix with the collagen solution and then heating the mixture; Preferably, the temperature of the heating treatment is 37-70°C; Preferably, the heating treatment time is 1 to 60 minutes.

9. A wound dressing, characterized in that: The wound dressing is prepared by the preparation method according to any one of claims 1 to 7.

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