PRP loaded dopamine modified 3D nanofiber sponge and preparation method thereof

Abstract

This invention belongs to the field of biomaterials and tissue engineering technology. It relates to a PRP-loaded dopamine-modified 3D nanofiber sponge and its preparation method. The PRP-loaded dopamine-modified 3D nanofiber sponge prepared by this invention utilizes a biomimetic design strategy, inspired by the adhesion mechanism of marine mussels, and employs PDA (polydopamine) surface modification technology to form a bioactive interface layer on the nanofiber surface. The active functional groups on the PDA surface bind with growth factors in PRP, forming a stable PDA-PRP composite functional layer. While maintaining the high porosity and suitable mechanical properties of the 3D nanofiber sponge, the biomimetic modification with polydopamine improves the loading efficiency of PRP and the interfacial bonding strength, thus extending the sustained-release period of growth factors.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and tissue engineering technology, and specifically relates to a PRP-loaded dopamine-modified 3D nanofiber sponge and its preparation method. Background Technology

[0002] As the body's largest physical barrier, the skin's integrity effectively resists external bacterial invasion and maintains internal environmental stability. Wound healing is a complex repair process initiated by the body after skin damage—normally involving four stages: hemostasis, inflammation, proliferation, and remodeling, ultimately achieving tissue regeneration and functional recovery. However, some chronic, difficult-to-heal wounds, such as diabetic wounds, deep burns / scalds, and pressure ulcers, are often stalled in the inflammation or proliferation stage due to multiple pathological factors, failing to naturally transition to the remodeling stage. This becomes a major challenge in clinical treatment, not only prolonging the patient's illness but also causing significant physical and psychological suffering and financial burden.

[0003] Platelet-rich plasma (PRP) is derived from the patient's own blood and is a plasma component with a high concentration of platelets obtained through centrifugation of autologous plasma. Due to its high platelet concentration, PRP is considered a safe and effective "repair agent" for promoting wound healing. PRP is rich in growth factors, which can promote tissue repair; however, when used directly in vitro, it is prone to loss and difficult to form a stable structure, and its short duration of action makes it difficult to maintain its effect, thus affecting treatment efficacy. Studies have found that PRP can be loaded onto traditional nanofiber materials to avoid the problem of easy loss when used directly in vitro. However, the interfacial bonding between traditional nanofiber materials and PRP is weak, limiting the loading capacity of traditional nanofiber materials for PRP, and the release of growth factors is uncontrollable. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the present invention aims to provide a PRP-loaded dopamine-modified 3D nanofiber sponge and its preparation method, thereby improving the problems of weak interfacial bonding between traditional nanofiber materials and PRP and uneven PRP loading. This enhances the surface activity of the 3D nanofiber sponge, improves its PRP loading efficiency, and enables controlled and sustained release of growth factors.

[0005] To address the aforementioned technical problems, this invention provides a method for preparing PRP-loaded dopamine-modified 3D nanofiber sponges, comprising the following steps: A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge, characterized by comprising the following steps: Preparation of 3D nanofiber sponges; Dopamine was dissolved in a buffer solution with a pH of 7.0 to 9.2 to prepare a dopamine solution with a concentration of 5 mg / mL to 50 mg / mL. The 3D nanofiber sponge was then immersed in the dopamine solution for 2 h to 12 h to form a uniform coating of dopamine on the surface of the 3D nanofiber sponge, thus obtaining a dopamine-modified 3D nanofiber sponge. PRP is loaded onto dopamine-modified 3D nanofiber sponge by immersion or surface adsorption to obtain PRP-loaded dopamine-modified 3D nanofiber sponge. The thickness of the dopamine coating on the surface of the dopamine-modified 3D nanofiber sponge is 10nm~50nm.

[0006] The buffer solution provided here offers a neutral, slightly alkaline environment to promote the self-polymerization of dopamine into polydopamine. This buffer solution can be any of Tris-HCl buffer, carbonate buffer, borax buffer, or phosphate buffer.

[0007] 3D nanofiber sponges can only form a uniform and stable polydopamine coating by soaking them in a dopamine solution with a concentration of 5 mg / mL to 50 mg / mL for 2 to 12 hours. The formed polydopamine coating is the basis for achieving subsequent controllable loading and release.

[0008] A 10nm–50nm dopamine coating provides abundant active functional groups. Within this thickness range, the PDA coating can form a relatively uniform, dense, and stable structure on the nanofiber surface. A 10nm–50nm PDA coating can form a uniform "adhesion layer" on the nanofiber sponge surface, with a uniform distribution of active functional groups, providing numerous uniform adsorption and binding sites for growth factors and other components in the PRP. If the coating is too thin, it may not completely cover the nanofiber surface, resulting in some areas lacking sufficient active sites, affecting the loading and binding of the PRP; while if the coating is too thick, it may clog the pores of the nanofiber sponge, reducing its specific surface area, while increasing stress concentration in the coating and reducing the overall performance of the composite material.

[0009] The 10nm~50nm dopamine coating can significantly enhance the interfacial bonding between nanofiber materials and PRP, and can avoid local enrichment or absence of PRP on the nanofiber surface, so that PRP can be loaded more evenly on the nanofiber sponge, thereby effectively improving the problem of uneven PRP loading.

[0010] The thickness of the dopamine coating is crucial, as no single thickness optimally enhances interfacial adhesion. A thickness range of 10 nm to 50 nm ensures uniform coating coverage on the nanofiber surface, providing sufficient functional groups to form chemical bonds with growth factors or platelets in the PRP, while maintaining nanofiber porosity to prevent pore blockage and mechanical property mismatch. Within this thickness range, the functional group density of the coating is maximized, significantly improving adhesion. If the coating is too thin, it may not completely cover the fiber surface, leaving uncovered areas and resulting in lower-than-expected adhesion; if the coating is too thick, it may block pores, reducing fiber surface permeability and bioactivity, or even detaching from the fiber due to differences in mechanical properties, thus weakening the PRP's function. Therefore, a dopamine coating thickness of 10 nm to 50 nm represents the optimal range balancing coverage, functionality, and porosity preservation, effectively enhancing the interfacial adhesion between the nanofiber material and the PRP.

[0011] Preferably, the mass ratio of the PRP to the dopamine-modified 3D nanofiber sponge is 1:5~50.

[0012] Preferably, the buffer solution is a Tris-HCl buffer solution, and more preferably, the pH of the Tris-HCl buffer solution is 8.5.

[0013] The pH buffering range of the Tris-HCl buffer solution is 7.0–9.2, with a preferred pH of 8.5. This is because a pH of 8.5 provides a stable, weakly alkaline environment that promotes the self-polymerization of dopamine into polydopamine. Theoretically, other buffer systems can stably maintain a pH around 8.5 (such as carbonate buffers and borax buffers), but the buffering capacity of carbonate buffers is significantly affected by CO2 in the air. Borate buffers may have borate ions that coordinate with the catechol groups of dopamine, potentially interfering with coating formation and properties. Phosphate buffers have weaker buffering capacity above pH 8.0 and are less stable than Tris-HCl. Therefore, the Tris-HCl buffer solution is preferred.

[0014] The preparation of dopamine solution must be strictly carried out in accordance with the procedure, that is, the dopamine powder is directly dissolved in the pre-prepared pH 8.5 Tris-HCl buffer solution and used immediately.

[0015] Preferably, the temperature for soaking the dopamine-modified 3D nanofiber sponge in PRP is 36℃~38℃, and the soaking time is 1h~4h.

[0016] At physiological temperatures, this method promotes platelet activity and the release of growth factors while avoiding high temperatures that could cause protein denaturation or loss of activity in the PRP. This temperature and time range facilitates the uniform penetration and binding of PRP components to the dopamine coating and nanofiber surface, forming a stable composite layer. This improves the interfacial bonding between the nanofiber material and the PRP and reduces the problem of uneven PRP loading.

[0017] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge, wherein the preparation steps of the 3D nanofiber sponge are as follows: The polymer material is dissolved in the corresponding solvent to obtain the electrospinning solution, wherein the mass-volume ratio of the polymer material to the solvent is 1g:10-20mL; Preparing electrospinning solutions by limiting the mass-to-volume ratio of polymeric materials to solvent to 1g:10-20mL creates a huge specific surface area. After dopamine modification, a large number of active functional groups (catechols, amino groups) are exposed on the surface. This provides numerous binding sites for proteins, growth factors, and platelets in PRP, greatly enhancing the overall interfacial binding force and making PRP less prone to detachment. The formation of an optimized hierarchical porous structure ensures that the PRP solution can easily, quickly, and uniformly fill the entire interior of the sponge through gravity, capillary action, and external pressure. This fundamentally solves the problem of uneven loading caused by dense material structures, resulting in PRP only adhering to the surface and the interior remaining "dry."

[0018] Two-dimensional nanofiber membranes were prepared by electrospinning the electrospinning solution. After reacting a two-dimensional nanofiber membrane with a crosslinking agent, the membrane is cleaned and dried to obtain a polymer nanofiber membrane. The polymer nanofiber membrane was destroyed and homogenized to obtain a uniform nanofiber suspension. After the nanofiber suspension is shaped, it is cooled and freeze-dried to obtain a 3D nanofiber sponge.

[0019] Preferably, the polymer material is polyvinyl alcohol, polylactic acid, or chitosan, the crosslinking agent is glutaraldehyde, N,N'-methylenebisacrylamide, or phthalate, and the solvent is water, ethanol, or chloroform.

[0020] This invention provides a method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge.

[0021] This invention provides the application of PRP-loaded dopamine-modified 3D nanofiber sponge in the repair of chronic, refractory wounds.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: The PRP-loaded dopamine-modified 3D nanofiber sponge prepared in this invention utilizes a biomimetic design strategy, inspired by the adhesion mechanism of marine mussels. A polydopamine (PDA) surface modification method is employed. The 3D nanofiber sponge is immersed in a dopamine solution of 5 mg / mL to 50 mg / mL for 2 to 12 hours, allowing dopamine to polymerize on the surface of the 3D nanofiber sponge, forming a uniform and complete polydopamine coating with strong adhesion and biocompatibility, and a thickness of 10 nm to 50 nm (this polydopamine coating is a bioactive interface layer). The dopamine-modified 3D nanofiber sponge is then immersed in PRP. The active functional groups on the PDA surface covering the 3D nanofiber sponge combine with the growth factors in the PRP, forming a stable PDA-PRP composite functional layer. This solves the problem of uneven loading between the nanofiber material and PRP, improves the loading efficiency and interfacial bonding strength of PRP, enhances the surface activity of the 3D nanofiber sponge, and achieves controllable slow release of growth factors, extending the slow release period of the growth factors.

[0023] This is because the PDA surface contains a large number of active functional groups such as catechol and amino groups. These groups can form various stable chemical bonds and non-covalent interactions with the growth factors in PRP. On the one hand, covalent bonds such as Schiff base reactions and Michael addition reactions can be used to firmly anchor the growth factors to the PDA coating surface, preventing them from rapidly detaching. On the other hand, non-covalent bonds such as hydrogen bonds and hydrophobic interactions can further strengthen the bonding effect. By stably fixing the growth factors in the composite layer, the characteristic of easy free diffusion of growth factors is broken, causing them to slowly detach from the binding site and be released, thus prolonging the release period.

[0024] Furthermore, the PDA polymerization on the surface of the 3D nanofiber sponge forms a uniform and dense coating. This coating itself acts as a physical barrier, preventing growth factors from rapidly penetrating and diffusing into the surrounding environment. The 3D nanofiber sponge itself has a porous structure, and the PDA-PRP composite layer adheres to the inner walls of the sponge's pores and the fiber surface. When growth factors want to be released from the composite layer, they need to slowly permeate through the pores of the sponge and the gaps between the PDA coating, thus slowing down the release rate. At the same time, the activated PRP forms a fibrin gel, which intertwines with the network structure of the nanofiber sponge to form a "double network" barrier, further hindering the rapid diffusion of growth factors.

[0025] Various proteolytic enzymes exist in living organisms, and unprotected growth factors are easily degraded by these enzymes, losing their activity and shortening their actual release period. The PDA-PRP composite functional layer forms a protective shell; the PDA coating encapsulates the growth factors, reducing their contact with proteolytic enzymes, lowering the probability of degradation, and indirectly extending their effective release period. Furthermore, PDA has good biodegradability and degrades slowly under physiological conditions. As the PDA coating gradually degrades, its binding sites with the growth factors are gradually exposed, releasing the growth factors. This degradation rate matches the release rate of the growth factors, avoiding burst release and achieving controlled, sustained release, rather than releasing all growth factors at once.

[0026] When traditional nanofiber materials and PRP are unevenly loaded, an excess of growth factors in some areas can lead to burst release, while a deficiency in others prevents them from exerting their effects. However, the PDA coating allows PRP to adhere evenly to the surface and pores of the 3D nanofiber sponge, ensuring a uniform distribution of growth factors throughout the composite layer. This uniformly distributed growth factor releases synchronously and slowly from all sites, avoiding rapid diffusion caused by excessively high local concentrations and guaranteeing the stability of the overall release process, thereby extending the sustained-release period.

[0027] The interconnected porous network of the 3D high-porosity sponge prepared in this invention allows PRP solution to freely permeate through capillary forces and gravity, ensuring full contact with PRP from the material surface to the core region, thereby achieving uniform loading in three-dimensional space. Traditional 2D nanofiber membranes, with their limited surface area, restrict the number of available binding sites. The high porosity of the 3D high-porosity sponge creates an extremely large specific surface area. After dopamine modification, this vast surface area can accommodate a large number of active functional groups. Upon contact with PRP, these functional groups can bind to the biomacromolecules in PRP through multiple chemical bonds, significantly enhancing the overall interfacial bonding force and preventing easy detachment of PRP. This results in the 3D nanofiber sponge possessing high porosity and suitable mechanical properties. These suitable mechanical properties resist external forces during loading, maintaining the open state of its porous structure and providing stable channels and space for the uniform permeation and distribution of PRP. Good toughness allows the sponge to withstand certain deformations without breaking, ensuring the integrity of the loaded substrate. This means that the formed uniform loading and strong bonding interface can be maintained in subsequent applications. The sponge, with its flexibility and elasticity, can adapt to the contours of the wound, forming a close fit. This close contact ensures that the active ingredients of PRP released from the material can act evenly on the entire wound surface. In contrast to traditional 2D nanofiber membranes, where PRP can only adhere to the surface and cannot effectively penetrate the material's interior, resulting in low loading capacity and extremely uneven distribution, 3D high-porosity sponges offer high loading capacity and extremely uniform distribution. Attached Figure Description

[0028] Figure 1These are SEM images comparing PRP-loaded nanofiber sponges before and after dopamine modification; (a) is the SEM image of the nanofiber sponge before dopamine modification; (b) is the SEM image of the PRP-loaded nanofiber sponge at 5 μm after dopamine modification; and (c) is the SEM image of the PRP-loaded nanofiber sponge at 10 μm after dopamine modification.

[0029] Figure 2 The FTIR spectra are those of dopamine (DA), polyvinyl alcohol (PVA), platelet-rich plasma (PRP), PVA / DA (Comparative Example 1), and dopamine-modified PRP-loaded 3D nanofiber sponge (Example 1).

[0030] Figure 3 These are contact angle diagrams of 3D nanofiber sponges modified with different concentrations of dopamine, where (a) is the contact angle diagram of the 3D nanofiber sponge modified with a dopamine solution of 0.5 mg / mL; (b) is the contact angle diagram of the 3D nanofiber sponge modified with a dopamine solution of 2.5 mg / mL; and (c) is the contact angle diagram of the 3D nanofiber sponge modified with a dopamine solution of 5 mg / mL.

[0031] Figure 4 The figures show the mechanical properties of 3D nanofiber sponges modified with different concentrations of dopamine. (a) shows the compressive strength of the 3D nanofiber sponge modified with 0.5 mg / mL dopamine solution under different strains; (b) shows the compressive strength of the 3D nanofiber sponge modified with 2.5 mg / mL dopamine solution under different strains; (c) shows the compressive strength of the 3D nanofiber sponge modified with 5 mg / mL dopamine solution under different strains; and (d) shows the compressive strength of the 3D nanofiber sponges modified with 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL dopamine solutions.

[0032] Figure 5 These are blood compatibility images of 3D nanofiber sponges modified with different concentrations of dopamine. (a) is a digital photograph of the 3D nanofiber sponges after hemolysis after modification with dopamine solutions of concentrations of 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL; (b) is a hemolysis rate image of the 3D nanofiber sponges after modification with dopamine solutions of concentrations of 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL.

[0033] Figure 6The images show the whole blood coagulation time of 3D nanofiber sponges modified with different concentrations of dopamine. (a) shows the whole blood coagulation samples of 3D nanofiber sponges modified with dopamine solutions at concentrations of 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL; (b) shows the whole blood coagulation test results of 3D nanofiber sponges modified with dopamine solutions at concentrations of 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL.

[0034] Figure 7 The results show the in vitro coagulation index of 3D nanofiber sponges modified with different concentrations of dopamine. (a) is a sample image of 3D nanofiber sponges modified with dopamine solutions at concentrations of 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL; (b) is a test image of the in vitro coagulation index of 3D nanofiber sponges modified with dopamine solutions at concentrations of 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL.

[0035] Figure 8 The graphs show the bonding strength of 3D nanofiber sponges modified with different concentrations of dopamine. (a) is the stress-displacement curve of the 3D nanofiber sponges modified with 0.5 mg / mL, 2.5 mg / mL and 5 mg / mL dopamine solutions, and (b) is the maximum stress histogram of the 3D nanofiber sponges modified with 0.5 mg / mL, 2.5 mg / mL and 5 mg / mL dopamine solutions. Detailed Implementation

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0037] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in Examples 1 to 23, preferred embodiments are described in this invention to avoid redundancy. However, this invention is not limited to these, but can be implemented in other ways within the scope of the technical solutions defined in the appended claims. All raw materials, reagents, instruments, and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0038] The following detailed description, in conjunction with embodiments of the present invention and accompanying drawings, provides a clear and complete illustration of the technical solutions in these embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] The following examples all use this spinning solution to prepare two-dimensional nanofiber membranes by electrospinning. The steps are as follows: The electrospinning solution is loaded into the syringe of the electrospinning equipment, a high-voltage power supply and a receiving device are connected, and spinning is carried out at room temperature. The nanofibers are collected by aluminum foil attached to the roller to obtain a uniform two-dimensional nanofiber membrane.

[0040] Example 1 A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0041] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0042] Example 2 A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0043] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving in Tris-HCl buffer solution at pH 8.5) for 6 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 25 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0044] Example 3 A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0045] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving in Tris-HCl buffer solution at pH 8.5) for 2 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 10 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0046] Example 4 The only difference between Example 4 and Example 1 is the concentration of the dopamine solution.

[0047] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 h to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0048] The prepared 3D nanofiber sponge was immersed in a 5 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0049] Example 5 The only difference between Example 5 and Example 1 is the concentration of the dopamine solution.

[0050] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0051] The prepared 3D nanofiber sponge was immersed in 50 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0052] Example 6 The only difference between Example 6 and Example 1 is that the soaking time of the PDA-modified sponge in PRP is different.

[0053] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0054] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 1 h to load PRP onto the 3D nanofiber sponge.

[0055] Example 7 The only difference between Example 7 and Example 1 is that the soaking time of the dopamine-modified 3D nanofiber sponge in PRP is different.

[0056] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0057] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 4 h to load PRP onto the 3D nanofiber sponge.

[0058] Example 8 The only difference between Example 8 and Example 1 is the quality of the dopamine-modified 3D nanofiber sponge.

[0059] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0060] The prepared 3D nanofiber sponge was immersed in a 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 30 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0061] Example 9 The only difference between Example 9 and Example 1 is the quality of the dopamine-modified 3D nanofiber sponge.

[0062] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 h to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0063] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 150 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0064] Example 10 The only difference between Example 10 and Example 1 is that the loading method of PRP and dopamine-modified 3D nanofiber sponge is changed from immersion method to surface adsorption method.

[0065] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 h to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0066] The prepared 3D nanofiber sponge was immersed in a 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized on the surface of the 3D nanofiber sponge to form a uniform dopamine coating with a thickness of 50 nm. 3 mL of PRP was loaded onto the surface of the 15 mg dopamine-modified 3D nanofiber sponge by surface adsorption at 37 °C for 2 h, thus loading PRP onto the 3D nanofiber sponge.

[0067] Example 11 The only difference between Example 11 and Example 1 is that the raw material of the 3D nanofiber sponge is replaced by polylactic acid instead of polyvinyl alcohol, and the corresponding solvent is replaced by dichloromethane instead of deionized water.

[0068] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: Polylactic acid powder (10% by mass) was added to dichloromethane and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol (PVA) spinning solution. The PVA spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was crosslinked with glutaraldehyde to obtain a water-insoluble PVA nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The PVA nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0069] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of PDA-modified sponge was immersed in 3 ml of PRP for 2 h to load PRP onto the 3D nanofiber sponge.

[0070] Example 12 The only difference between Example 12 and Example 1 is that the raw material of the 3D nanofiber sponge is replaced with chitosan instead of polyvinyl alcohol.

[0071] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) chitosan powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol (PVA) spinning solution. The PVA spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble PVA nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The PVA nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0072] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0073] Example 13 The only difference between Example 13 and Example 1 is the crosslinking agent.

[0074] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 h to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was crosslinked with N,N'-methylenebisacrylamide to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure.

[0075] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of PDA-modified sponge was immersed in 3 ml of PRP for 2 h to load PRP onto the 3D nanofiber sponge.

[0076] Example 14 The only difference between Example 14 and Example 1 is the crosslinking agent.

[0077] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 10% polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 h to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with diester to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure.

[0078] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0079] Example 15 The only difference between Example 15 and Example 1 is that the mass-volume ratio of the polymer material to the solvent is different.

[0080] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 15% polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0081] The prepared 3D nanofiber sponge was immersed in a 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 ml of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0082] Example 16 The only difference between Example 16 and Example 1 is that the mass-volume ratio of the polymer material to the solvent is different.

[0083] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 20% polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0084] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0085] Example 17 The only difference between Example 17 and Example 1 is that the mass-volume ratio of the polymer material to the solvent is different.

[0086] A method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge includes the following steps: 15% polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a uniform two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0087] The prepared 3D nanofiber sponge was immersed in 25 mg / mL dopamine solution (dopamine was prepared by dissolving dopamine in Tris-HCl buffer solution at pH 8.5) for 12 h. Dopamine polymerized to form a uniform dopamine coating with a thickness of 50 nm on the surface of the 3D nanofiber sponge. Then, 15 mg of dopamine-modified 3D nanofiber sponge was immersed in 3 mL of PRP at 37 °C for 2 h to load PRP onto the 3D nanofiber sponge.

[0088] Example 18 The only difference between Example 18 and Example 1 is that the pH of the Tris-HCl buffer is 7.0.

[0089] Example 19 The only difference between Example 19 and Example 1 is that the pH of the Tris-HCl buffer is 9.2.

[0090] Example 20 The only difference between Example 20 and Example 1 is that Tris-HCl buffer solution is replaced with borax buffer solution.

[0091] Example 21 The only difference between Example 21 and Example 1 is that Tris-HCl buffer solution is replaced with carbonate buffer solution.

[0092] Example 22 The only difference between Example 22 and Example 1 is that the temperature of soaking the dopamine-modified 3D nanofiber sponge in PRP is replaced with 36°C and the soaking time is replaced with 1 hour.

[0093] Example 23 The only difference between Example 23 and Example 1 is that the temperature of soaking the dopamine-modified 3D nanofiber sponge in PRP is replaced with 38°C and the soaking time is replaced with 4 hours.

[0094] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the 3D nanofiber sponge was directly applied without dopamine modification.

[0095] 10% (w / w) polyvinyl alcohol powder was added to deionized water and heated and stirred for 12 hours to obtain a transparent polyvinyl alcohol spinning solution. The polyvinyl alcohol spinning solution was then electrospun to obtain a two-dimensional nanofiber membrane. The nanofiber membrane was cross-linked with glutaraldehyde to obtain a water-insoluble polyvinyl alcohol nanofiber membrane. Unvolatile substances in the nanofiber membrane were removed by vacuum drying in an oven. The polyvinyl alcohol nanofiber membrane was then broken up and homogenized to obtain a uniform nanofiber suspension. This suspension was poured into a specific mold and frozen using a low-temperature cold source. After complete freezing, it was freeze-dried using a vacuum freeze dryer to obtain a nanofiber sponge with a three-dimensional interconnected porous structure, i.e., a 3D nanofiber sponge.

[0096] Then, 15 mg of the prepared 3D nanofiber sponge was soaked in 3 mL of PRP at 37°C for 2 h, so that PRP was loaded onto the 3D nanofiber sponge-2.

[0097] PRP-loaded dopamine-modified 3D nanofiber sponges were prepared using Examples 1 to 23 above. The PRP-loaded dopamine-modified 3D nanofiber sponge prepared in Example 1 was selected for efficacy verification.

[0098] Experimental verification (a) Structural confirmation (1) Morphological characteristics Figure 1 These are scanning electron microscope (SEM) comparison images of PRP-loaded nanofiber sponges before and after dopamine modification. Figure 1 Image (a) is a SEM image of the nanofiber sponge before dopamine modification (Comparative Example 1); [The image is presented in the original text but is not translated here.] Figure 1 As shown in (a), the internal structure of the 3D nanofiber sponge is clear and uniformly distributed, and a porous structure can be observed. Figure 1 (b) and Figure 1 (c) in the image are SEM images of PRP-loaded nanofiber sponges after dopamine modification, obtained through... Figure 1 As shown in (b), a large number of platelets adhere to the fiber surface, with slightly reduced pore size, but still maintaining strong connectivity. Through Figure 1 As shown in (c), platelets are activated and extend pseudopodia, adhering tightly to the fiber surface. Figure 1 (b) and (c) in the middle Figure 1 The comparison in (a) showed that the dopamine-modified nanofiber sponge had good platelet adhesion.

[0099] (2) Functional group representation Figure 2The FTIR spectra of dopamine (DA), polyvinyl alcohol (PVA), platelet-rich plasma (PRP), PVA / DA (Comparative Example 1), and dopamine-modified PRP-loaded 3D nanofiber sponge (Example 1) were obtained by... Figure 2 This confirms the successful modification of 3D nanofiber sponge with DA and its loading with PRP. At 1610 cm⁻¹ -1 and 1520cm -1 The absorption peaks appear on both sides, which are attributed to the stretching vibration of the aromatic ring C=C, at 1250 cm⁻¹. -1 ~1350cm -1 The presence of an absorption peak in the region, attributed to the CN stretching vibration of the aromatic amine, confirms that dopamine has been successfully polymerized and modified onto the PVA fiber surface. At 1630 cm⁻¹... -1 ~1660cm -1 The absorption peak at 1520 cm⁻¹ is due to the stretching vibration of the protein's C=O structure. -1 ~1550cm -1 The peaks appearing are CN stretching vibration peaks, which are characteristic functional groups of proteins. Their presence proves that PRP has been successfully loaded.

[0100] (II) Performance Verification (3) Hydrophilicity test Figure 3 These are contact angle diagrams of 3D nanofiber sponges modified with different concentrations of dopamine, among which... Figure 3 (a) in the figure is the contact angle diagram of the 3D nanofiber sponge after modification with a dopamine solution of concentration of 5 mg / mL (DA-0.5); Figure 3 (b) in the figure is the contact angle diagram of the 3D nanofiber sponge after modification with a dopamine solution of concentration of 25 mg / mL (DA-2.5); Figure 3 (c) shows the contact angle diagram of the 3D nanofiber sponge modified with a 50 mg / mL (DA-5) dopamine solution. At the same concentration, Figure 3 (a) Figure 3 (b) and Figure 3 In (c), the contact angle decreases significantly over time; at the same time interval, Figure 3 (a) Figure 3 (b) and Figure 3 The contact angle of (c) gradually decreases. This verifies that it has good hydrophilicity.

[0101] (4) Mechanical property testing The mechanical properties of 3D nanofiber sponges modified with different concentrations of dopamine were tested using a universal testing machine. The test results are as follows: Figure 4 As shown, where Figure 4(a) in the figure shows the compressive strength of the 3D nanofiber sponge modified with a 5 mg / mL (DA-0.5) dopamine solution under different strains. Figure 4 (b) in the figure shows the compressive strength of the 3D nanofiber sponge modified with a 25 mg / mL (DA-2.5) dopamine solution under different strains. Figure 4 (c) in the figure shows the compressive strength of the 3D nanofiber sponge modified with 50 mg / mL (DA-5) dopamine solution under different strains. Figure 4 The compressive strength diagrams of 3D nanofiber sponges modified with dopamine solutions at concentrations of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 50 mg / mL (DA-5) are shown. Figure 4 As shown in (a), although the 3D nanofiber sponge modified with 5 mg / mL dopamine has a certain elasticity, its fatigue resistance is generally poor; through Figure 4 As shown in (b), the 3D nanofiber sponge modified with 25 mg / mL dopamine exhibits excellent elastic recovery and fatigue resistance; through Figure 4 As shown in (c), although the 3D nanofiber sponge modified with 50 mg / mL dopamine has a certain compressive strength, its elasticity and fatigue resistance are reduced; through Figure 4 As shown in (d), under the same strain, the compressive strength of 3D nanofiber sponges modified with different concentrations of dopamine varies, with the 25 mg / mL dopamine-modified 3D nanofiber sponge exhibiting the best mechanical properties. This demonstrates that the 3D nanofiber sponge modified with an appropriate concentration of dopamine possesses good elasticity, compressive strength, and fatigue resistance. It also confirms that the growth factors are stably fixed in the composite layer, breaking the characteristic of easy free diffusion of growth factors, causing them to slowly detach from their binding sites and be released, thus prolonging the release cycle.

[0102] (5) Biocompatibility evaluation The biocompatibility of 3D nanofiber sponges modified with different concentrations of dopamine was evaluated using in vitro hemolysis experiments. Figure 5 (a) shows digital photographs of 3D nanofiber sponges after hemolysis following modification with dopamine solutions at concentrations of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 50 mg / mL (DA-5); Figure 5 (b) shows the hemolysis rate of 3D nanofiber sponges modified with dopamine solutions at concentrations of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 50 mg / mL (DA-5). Figure 5As shown in (a), after co-culturing erythrocytes with extracts of dopamine-modified 3D nanofiber sponges at different concentrations (5 mg / mL, 25 mg / mL, 50 mg / mL), the supernatants of each experimental group (125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) were similar in color to the negative control group (PBS), appearing clear or light red, with no obvious hemolysis observed to the naked eye; while the positive control group (Triton) appeared dark red, indicating complete hemolysis of erythrocytes. This indicates that the dopamine-modified 3D nanofiber sponges did not induce a significant hemolytic reaction under different concentrations of extract; through Figure 5 As shown in (b), the hemolysis rate of dopamine-modified 3D nanofiber sponges at different concentrations of extract was below the international blood compatibility safety threshold of 5% at all extract concentrations. Furthermore, the hemolysis rate showed a slight upward trend with increasing extract concentration. Among all tested concentrations, the 25 mg / mL dopamine-modified 3D nanofiber sponge exhibited the lowest hemolysis rate, demonstrating the best blood compatibility. This proves that the dopamine-modified 3D nanofiber sponge has good blood compatibility and is safe to use.

[0103] (6) Evaluation of in vitro coagulation performance Figure 6 The coagulation ability of 3D nanofiber sponges modified with different concentrations of dopamine was evaluated using whole blood coagulation time. Figure 6 (a) shows whole blood coagulation samples of 3D nanofiber sponges modified with dopamine solutions at concentrations of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 5 mg / mL (DA-50). Figure 6 (b) shows the whole blood coagulation test results of 3D nanofiber sponges modified with dopamine solutions at concentrations of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 5 mg / mL (DA-50). Figure 6 As shown in (a), after 3D nanofiber sponges modified with dopamine at different concentrations (5 mg / mL, 25 mg / mL, 50 mg / mL) came into contact with whole blood, their coagulation state was different from that of the blank control group (Blank), forming a dense blood clot, indicating that the 3D nanofiber sponge can promote the blood coagulation process. Figure 6 As shown in (b), the whole blood clotting time in the dopamine-modified groups was shorter than that in the blank control group. This indicates that the dopamine-modified 3D nanofiber sponge can effectively promote whole blood clotting and has good procoagulant properties.

[0104] (7) In vitro coagulation index assessment Figure 7 These are the in vitro coagulation index results of 3D nanofiber sponges modified with different concentrations of dopamine, among which... Figure 7(a) Sample images of 3D nanofiber sponges modified with dopamine solutions at concentrations of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 5 mg / mL (DA-50); Figure 7 (b) shows the in vitro coagulation index of 3D nanofiber sponges and gelatin sponges modified with dopamine solutions at concentrations of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 5 mg / mL (DA-50). Figure 7 As shown in (a), the blood in the blank group remained liquid and incompletely coagulated. A small amount of blood clots appeared in the gelatin sponge group. However, the blood in the 3D nanofiber sponges modified with different concentrations (5 mg / mL, 25 mg / mL, 50 mg / mL) of dopamine was coagulated, and the amount of uncoagulated blood in the glass dish was significantly reduced. Figure 7 As shown in (b), the in vitro coagulation index (BCI) of each group decreased over time, indicating an increase in blood coagulation. The BCI values ​​of the dopamine-modified 3D nanofiber sponge groups were significantly lower than those of the gelatin group at all time points, indicating that the dopamine-modified 3D nanofiber sponge has good procoagulant properties. The use of the in vitro coagulation index to assess the coagulation ability of the 3D nanofiber sponge after contact with blood confirmed that it effectively accelerates blood coagulation.

[0105] (8) Test of interfacial bonding strength of 3D nanofiber sponge Figure 8 These are bonding strength diagrams for 3D nanofiber sponges modified with different concentrations of dopamine. Figure 8 In Figure (a), the stress-displacement curves of 3D nanofiber sponges modified with dopamine solutions of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 5 mg / mL (DA-50) are shown. Figure 8 (b) in the figure shows the maximum stress histograms of the 3D nanofiber sponges modified with dopamine solutions of 5 mg / mL (DA-0.5), 25 mg / mL (DA-2.5), and 5 mg / mL (DA-50). Figure 8 As shown in (a), different concentrations of dopamine modification have a significant impact on the interfacial adhesion strength of 3D nanofiber sponges. Figure 8 As shown in (b), the interfacial binding strength gradually increases with the increase of dopamine concentration, proving that dopamine modification can effectively enhance the binding strength of 3D nanofiber sponge. It also confirms that the growth factor coated by PDA coating reduces the contact between the growth factor and proteolytic enzyme, reduces the degradation probability, indirectly prolongs the effective release cycle of the growth factor, and achieves controlled slow release.

[0106] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a PRP-loaded dopamine-modified 3D nanofiber sponge, characterized by, Includes the following steps: Preparation of 3D nanofiber sponges; Dopamine was dissolved in a buffer solution with a pH of 7.0 to 9.2 to prepare a dopamine solution with a concentration of 5 mg / mL to 50 mg / mL. The 3D nanofiber sponge was then immersed in the dopamine solution for 2 to 12 hours to allow the dopamine to form a uniform coating on the surface of the 3D nanofiber sponge, thus obtaining a dopamine-modified 3D nanofiber sponge. The thickness of the dopamine coating on the surface of the dopamine-modified 3D nanofiber sponge was 10 nm to 50 nm. PRP was loaded into dopamine-modified 3D nanofiber sponge by immersion or surface adsorption to obtain PRP-loaded dopamine-modified 3D nanofiber sponge.

2. The method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge according to claim 1, characterized in that, The mass ratio of PRP to dopamine-modified 3D nanofiber sponge is 1:5~50.

3. The method of claim 1, wherein the PRP loaded dopamine modified 3D nanofibrous sponges are prepared by the steps of: The buffer solution is Tris-HCl buffer solution.

4. The method for preparing PRP-loaded dopamine-modified 3D nanofiber sponge according to claim 1, characterized in that, The temperature for soaking dopamine-modified 3D nanofiber sponges in PRP is 36℃~38℃, and the soaking time is 1h~4h.

5. The method of claim 1, wherein the PRP loaded dopamine modified 3D nanofibrous sponges are prepared by the steps of: The preparation steps of the 3D nanofiber sponge are as follows: The polymer material is dissolved in a solvent to obtain an electrospinning solution, wherein the mass-volume ratio of the polymer material to the solvent is 1g:10-20mL. Two-dimensional nanofiber membranes were prepared by electrospinning solution using an electrospinning method. After reacting a two-dimensional nanofiber membrane with a crosslinking agent, the membrane is cleaned and dried to obtain a polymer nanofiber membrane. The polymer nanofiber membrane was destroyed and homogenized to obtain a uniform nanofiber suspension. After the nanofiber suspension is shaped, it is cooled and freeze-dried to obtain a 3D nanofiber sponge.

6. The method of claim 5, wherein the PRP loaded dopamine modified 3D nanofibrous sponges are prepared by, The polymer material is polyvinyl alcohol, polylactic acid, or chitosan; the crosslinking agent is glutaraldehyde, N,N'-methylenebisacrylamide, or phthalate; and the solvent is water, ethanol, or chloroform.

7. The PRP-loaded dopamine-modified 3D nanofiber sponge is prepared by the method according to any one of claims 1 to 6.

8. The application of the PRP-loaded dopamine-modified 3D nanofiber sponge according to claim 7 in the repair of chronic, refractory wounds.

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