A dual cross-linked hydrogel for synergizing platelet-rich plasma and releasing growth factors, and a preparation method and application thereof

Abstract

The application discloses a kind of synergic platelet-rich plasma and sustained-release growth factor double crosslinked hydrogel and its preparation method and application, belong to the technical field of biomedical materials.The hydrogel is prepared by oxidized modified polysaccharide, sulfated modified polysaccharide and platelet-rich plasma, the aldehyde group of oxidized modified polysaccharide is formed with the amino group of protein, polypeptide component in platelet-rich plasma Schiff base dynamic crosslinking, the fibrin network formed after platelet-rich plasma is activated by activator can be jointly constituted with the above crosslinking double crosslinking network, and the sulfate group of sulfated modified polysaccharide provides affinity binding site for platelet-rich plasma derived growth factor by electrostatic interaction and hydrogen bond.The hydrogel has good structural stability, and can reduce the early burst release of growth factor, realizes pH-responsive regulation and sustained delivery of growth factor in acidic environment, can provide favorable microenvironment for cell behavior and tissue angiogenesis, and is suitable for preparing medical dressings for acute and chronic wound repair.

Description

Technical Field

[0001] This application relates to a double cross-linked hydrogel that synergistically releases platelet-rich plasma (PRP) and growth factors, its preparation method, and its application, belonging to the field of biomedical materials technology. Background Technology

[0002] The skin is the largest organ in the human body and a vital barrier for maintaining fluid balance and protecting against external stimuli. Cuts, burns, mechanical injuries, and diabetes-related chronic wounds can all disrupt the integrity of skin tissue and impair barrier function, thereby reducing patients' quality of life. The skin wound healing process is complex, typically involving consecutive stages such as hemostasis, inflammation, proliferation, tissue remodeling, and scar formation. For chronic wounds, persistent inflammation, increased oxidative stress, insufficient angiogenesis, and abnormal extracellular matrix deposition can further delay healing.

[0003] Growth factors are crucial regulators of endogenous repair mechanisms. Platelet-rich plasma (PRP), derived from autologous or allogeneic blood, contains high concentrations of platelets and various growth factors, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), after centrifugation and enrichment. These active factors promote collagen synthesis, angiogenesis, mesenchymal cell proliferation, extracellular matrix formation, and tissue repair and regeneration. Due to its low immunogenicity and relatively convenient preparation, PRP has attracted widespread attention in wound repair and tissue regeneration.

[0004] However, direct use of PRP still has significant limitations. The growth factors in PRP are easily released and inactivated rapidly within a short time, resulting in a short therapeutic window, insufficient bioavailability, and a rapid decrease in local effective concentration. This necessitates frequent dressing changes during treatment to maintain the repair effect, thus increasing patient pain and medical costs. To address these issues, researchers have developed various wound dressings in recent years, including porous foams, electrospun nanofibers, freeze-dried sponges, biocompatible films, and hydrogels. Among these, hydrogels, due to their high water content, porous structure, and unique swelling capacity, are suitable for wound coverage and delivery of active factors. Furthermore, hydrogels offer advantages such as minimally invasive drug delivery, adaptation to irregular wounds, in-situ gelation, and good adhesion to the wound surface.

[0005] However, existing hydrogels mostly rely on physical embedding of PRP to retain active factors. The carrier lacks sufficient binding sites for high affinity interactions with growth factors, potentially leading to burst release and ineffective release. Furthermore, the local pH of the wound can change with inflammation and the healing process, making it difficult for carriers with fixed release rates to regulate growth factor delivery according to microenvironmental changes. On the other hand, some hydrogel systems require the addition of small-molecule cross-linking agents, initiators, or redox systems, which may introduce potential toxic side effects and affect biocompatibility. Therefore, developing a hydrogel with relatively simple components, capable of in-situ gelation, synergistic with PRP, and possessing both affinity-based sustained release and microenvironment-responsive release capabilities is of great significance for prolonging the duration of growth factor action, protecting its bioactivity, matching the wound healing stage, and promoting wound healing to address the aforementioned clinical problems. Summary of the Invention

[0006] To address the issues of insufficient growth factor loading capacity, easy burst release, and potential biocompatibility risks in partially cross-linked systems in platelet-rich plasma (PRP) hydrogels, this application provides a dual-crosslinked hydrogel technology that synergistically enhances PRP and sustains growth factor release. This technology utilizes a mixture of oxidized and sulfated modified polysaccharides and PRP as raw materials. First, the aldehyde groups of the oxidized modified polysaccharides form a Schiff base dynamic crosslinking network with proteins and peptides containing primary amino groups in the PRP. Then, a calcium-containing activator activates fibrinogen in the PRP to form a fibrin network. These two components interpenetrate to form a dual-crosslinked structure. The sulfated modified polysaccharides interact with growth factors via electrostatic and hydrogen bonding through their sulfate groups. This structure achieves physical encapsulation, affinity binding, and pH-responsive dynamic release without the need for small organic molecule crosslinking agents or free radical initiators, thus enabling stable immobilization, sustained release, and wound-microenvironment-responsive delivery of PRP-derived growth factors.

[0007] The technical solution adopted in this application is as follows: According to a first aspect of this application, a dual crosslinked hydrogel that synergistically releases platelet-rich plasma and growth factors is provided, comprising: Double cross-linked network matrix, platelet-rich plasma; The dual cross-linked network matrix is ​​formed by the interpenetration of a polysaccharide cross-linked network and a fibrin network. The polysaccharide cross-linking network is formed by cross-linking oxidized modified polysaccharides, sulfated modified polysaccharides, and proteins and peptides containing primary amino groups in platelet-rich plasma; The fibrin network is formed by the activation of fibrin in platelet-rich plasma by an activator. The platelet-rich plasma is loaded into the double cross-linked network matrix through physical encapsulation and chemical cross-linking.

[0008] Optionally, in the synergistic platelet-rich plasma and sustained-release growth factor double crosslinked hydrogel, the mass concentration of oxidized modified polysaccharide is 0.5%-10% (w / v). The mass concentration of sulfated modified polysaccharides is 0.5%-10% (w / v); The volume fraction of platelet-rich plasma is 5-30% (v / v); Optionally, the polysaccharides in the oxidized modified polysaccharide and the sulfated modified polysaccharide are independently selected from at least one of xyloglucan, hyaluronic acid, sodium alginate, chitosan, carboxymethyl chitosan, chondroitin sulfate, heparin, heparan sulfate, dextran, and carboxymethyl cellulose. The two modified polysaccharides may be derived from the same polysaccharide or from different polysaccharides.

[0009] Optionally, the polysaccharides in the oxidized modified polysaccharide and the sulfated modified polysaccharide are both xylo-glucan.

[0010] According to a second aspect of this application, a method for preparing a double crosslinked hydrogel that synergistically releases platelet-rich plasma and growth factors is provided, comprising: We provide oxidized modified polysaccharides and sulfated modified polysaccharides. Provide platelet-rich plasma; An activator is added to a solution containing the oxidized modified polysaccharide, the sulfated modified polysaccharide, and the platelet-rich plasma, and a reaction is carried out to obtain the bicrosslinked hydrogel that synergistically enriches platelet-rich plasma and releases growth factors.

[0011] Optionally, the mass concentration of the oxidized polysaccharide in the solution is 0.5%-10% (w / v). The mass concentration of sulfated modified polysaccharides is 0.5%-10% (w / v); The volume fraction of platelet-rich plasma is 5-30% (v / v).

[0012] Optionally, the activator is selected from physiologically compatible solutions containing calcium ions.

[0013] Optionally, the activator is selected from at least one of calcium chloride solution, calcium gluconate solution, or calcium chloride-thrombin mixed solution.

[0014] Optionally, the volume ratio of the activator to platelet-rich plasma in the solution is approximately 1:5 to 1:20.

[0015] Optionally, the activator contains Ca. 2+ The concentration is 0.5-5 mM.

[0016] Optionally, the solvent in the solution containing the oxidized modified polysaccharide, the sulfated modified polysaccharide, and the platelet-rich plasma is selected from at least one of phosphate buffer, deionized water, Green's solution, and glycerol.

[0017] Optionally, the method for preparing the oxidized modified polysaccharide includes: Under light-protected conditions and with continuous stirring, the oxidizing agent is slowly added to an aqueous solution containing polysaccharides to carry out an oxidation reaction. The solution obtained from the oxidation reaction is then dialyzed and freeze-dried to obtain the oxidized modified polysaccharide.

[0018] Optionally, the oxidizing agent is selected from an aqueous solution of sodium periodate.

[0019] Optionally, the mass ratio of the oxidizing agent to the polysaccharide is 0.05-1.0:1.

[0020] Optionally, the conditions for the oxidation reaction include: a reaction temperature of 15-35°C, a reaction time of 2-24 hours, and the addition of a terminator to terminate the reaction.

[0021] Optionally, the terminating agent is selected from at least one of ethylene glycol, propylene glycol, and glycerin; Optionally, the dialysis conditions in the preparation method of oxidatively modified polysaccharides include: a molecular weight cutoff of 8000-14000 Da and a dialysis time of 3-5 days.

[0022] Optionally, the method for preparing the sulfated modified polysaccharide includes: Under light-protected conditions and with continuous stirring, a sulfation reagent is added to an organic solution containing polysaccharides and a sulfation reaction is carried out. The solution obtained from the sulfation reaction is diluted with water and then dialyzed and freeze-dried to obtain the sulfated modified polysaccharide.

[0023] Optionally, the sulfation reagent is selected from an organic solution of a sulfur trioxide-pyridine complex.

[0024] Optionally, the mass ratio of the sulfating agent to the polysaccharide is 1-10:1; Optionally, the organic solvent in the polysaccharide-containing organic solution is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and formamide.

[0025] Optionally, the mass-volume concentration of the polysaccharide in the organic solution containing the polysaccharide is 0.1%-5% (w / v). Optionally, the conditions for the sulfation reaction include: a reaction temperature of 40-80°C, a reaction time of 4-24 h, and the addition of a neutralizing agent after the reaction; Optionally, the neutralizing agent is selected from at least one of sodium hydroxide solution, sodium carbonate solution, and sodium bicarbonate solution; Optionally, the dialysis conditions in the preparation method of sulfated modified polysaccharides include: a molecular weight cutoff of 8000-14000 Da and a dialysis time of 3-5 days.

[0026] Optionally, the polysaccharide is selected from at least one of xyloglucan, hyaluronic acid, sodium alginate, chitosan, carboxymethyl chitosan, chondroitin sulfate, heparin, heparan sulfate, dextran, and carboxymethyl cellulose. The two modified polysaccharides may be derived from the same polysaccharide or from different polysaccharides; preferably, both modified polysaccharides are derived from xyloglucan.

[0027] Optionally, the method for preparing the platelet-rich plasma includes: Provide anticoagulated whole blood to the target population; The anticoagulated whole blood was centrifuged for the first time, and the upper plasma layer was collected while avoiding the aspiration of the red blood cell layer and the white membrane layer. Add a separating gel to the upper plasma layer and perform a second centrifugation to place red blood cells and white blood cells below the separating gel or at the separating gel interface. Collect the platelet-rich plasma above the separating gel, discard 60% to 90% of the supernatant plasma, and resuspend the precipitated platelets in the remaining plasma to obtain the platelet-rich plasma. Optionally, the platelet concentration in the platelet-rich plasma is 2-8 times that of the whole blood platelet concentration.

[0028] Optionally, the platelet-rich plasma contains 5 × 10⁻⁶ whole blood platelets. 8 ~3×10 9 per mL.

[0029] According to a third aspect of this application, a dual cross-linked hydrogel of synergistic platelet-rich plasma and sustained-release growth factor is provided, and the application of the dual cross-linked hydrogel of synergistic platelet-rich plasma and sustained-release growth factor prepared according to the aforementioned preparation method in the preparation of medical wound dressings is described.

[0030] Optionally, the wound dressing is selected from at least one of chronic wound dressings such as mechanical injury wound dressings, burn wound dressings, and diabetic wound dressings.

[0031] The beneficial effects of this application include: (1) This application provides a dual crosslinking method in which an initial dynamic crosslinking network is formed by mixing oxidized modified polysaccharide, sulfated modified polysaccharide and PRP, and then a fibrin network is formed by a physiologically compatible calcium-containing activator. No heating is required, nor is it necessary to introduce organic small molecule crosslinking agents, free radical initiators or redox systems. Both modified polysaccharides can be derived from natural polysaccharides, and the raw material composition is relatively simple. The hydrogel preparation conditions are mild, the operation is convenient and reproducible, and it has good cell, blood and tissue compatibility.

[0032] (2) In this application, the sulfate group can be used as a binding site for interaction with growth factors such as PDGF, VEGF, and EGF. Combined with the three-dimensional porous structure of the hydrogel, it can achieve the fixation and sustained release of growth factors, reduce ineffective release before treatment, and help maintain the activity of growth factors and prolong their action time.

[0033] (3) The hydrogel of this application is injectable, self-healing, antioxidant and hemostatic. It can adapt to irregular wounds and maintain a moist microenvironment. The dynamic Schiff base network can accelerate dissociation under acidic conditions and regulate the release of PRP-derived growth factors. The sulfate affinity binding and three-dimensional network physical embedding can reduce early burst release and prolong release time, thereby synergistically promoting cell survival, tissue repair and closure of diabetic chronic wounds. Attached Figure Description

[0034] Figure 1 shows the chemical structure characterization of the hydrogel and related materials obtained in Example 3 of this application, wherein Figure 1(A) shows XG, OXG, SXG, PRP and OSP@Ca 2+ The Fourier transform infrared spectra of XG, OXG, and SXG are shown in Figure 1(B). Figure 1(C) shows the proton NMR spectra of PRP, OXG, SXG, and OSP@Ca. 2+ The full X-ray photoelectron spectra of the OXG, SXG, and OSP@Ca are shown in Figure 1(D), which are the C1s high-resolution spectra of OXG, SXG, and OSP@Ca. 2+ C1s high-resolution spectrum and OSP@Ca 2+ The N 1s high-resolution spectrum; Figure 2 shows the gelation, gelation time, and microstructure of the hydrogel of this application. Figure 2(A) shows the pre-gelation mixture, OSP, and OSP@Ca. 2+ PRP and PRP@Ca 2+ An inverted image of the actual product, Figure 2 (B) shows the OP. 10 OP 20 OP 30 OS2P 20 OS4P 20 OS6P 20 OS4P 20 @Ca2+ and PRP@Ca 2+ The statistical results of gelation time are shown in Figure 2(C) for PRP@Ca 2+ OSP and OSP@Ca 2+ Scanning electron microscope images of the freeze-dried samples, Figure 2(D) shows the statistical results of the average pore size of the corresponding samples; Figure 3 shows the different OSP systems in this application in Ca 2+ Rheological property characterization diagrams before and after activation, where Figure 3(A) shows the OSP. 20 and OSP 20 @Ca 2+ The shear-thinning curve and injection photos of OSP are shown in Figure 3(B). 10 OSP 10 @Ca 2+ OSP 20 and OSP 20 @Ca 2+ The time-scan results are shown in Figure 3(C), which shows the strain scan results for each group mentioned above, and Figure 3(D) shows the OSP results. 20 @Ca 2+ Modulus recovery results under alternating low and high strain conditions; Figure 4 shows the total protein and growth factor release results of different systems in this application, where Figure 4(A) shows the PRP@Ca 2+ OP@Ca 2+ OS2P@Ca 2+ and OS4P@Ca 2+ Total protein, Figure 4(B) shows PRP@Ca 2+ OP@Ca 2+ OS2P@Ca 2+ and OS4P@Ca 2+ EGF, Figure 4(C) shows PRP@Ca 2+ OP@Ca 2+ OS2P@Ca 2+ and OS4P@Ca 2+ PDGF and Figure 4(D) are PRP@Ca 2+ OP@Ca 2+ OS2P@Ca 2+ and OS4P@Ca 2+ The cumulative release curve of VEGF, Figure 4(E) shows the OS4P@Ca 2+ Total protein at pH 8.5, pH 7.4, and pH 5.5, Figure 4(F) shows OS4P@Ca 2+ EGF at pH 8.5, pH 7.4 and pH 5.5, Figure 4(G) shows OS4P@Ca 2+PDGF at pH 8.5, pH 7.4, and pH 5.5 and Figure 4(H) for OS4P@Ca 2+ Cumulative VEGF release curves at pH 8.5, pH 7.4, and pH 5.5; Figure 5 shows the biocompatibility characterization of the hydrogel of this application, where Figure 5(A) is the control group and PRP@Ca 2+ Group, OP@Ca 2+ Groups, OSP groups, and OSP@Ca 2+ The results of live / dead cell staining of L929 cells on days 1, 3, and 5 after treatment are shown in Figure 5(B). 2+ Photographs of the implantation site and H&E staining results on days 1, 7, 20, and 32 after subcutaneous injection. Figure 5(C) shows the results of Triton X-100, PBS, and PRP@Ca. 2+ OP, OSP and OSP@Ca 2+ Hemolysis rate and supernatant images for each group; Figure 6 shows the hemostatic performance characterization of the hydrogel of this application, where Figure 6(A) shows the control group, OSP group, and PRP@Ca group. 2+ Groups and OSP@Ca 2+ Typical photographs of the liver hemostasis process at 20 s, 40 s, 60 s, and 80 s are shown in Figure 6(B). Figure 6(C) shows the blood loss statistics for each group. 2+ Groups and OSP@Ca 2+ Coagulation index results of the group at different time points; Figure 7 shows the effect of the hydrogel of this application on chronic wound repair in diabetic mice, where Figure 7(A) shows the control group, the commercial EGF group, and the PRP@Ca group. 2+ Group, OP@Ca 2+ Groups and OSP@Ca 2+ Typical photos of wounds on days 0, 3, 7, 14 and 21 of each group, and schematic diagrams of wound contour changes at different time points in each group. Figure 7(B) shows the statistical curves of wound area changes in each group. Detailed Implementation

[0035] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0036] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased through commercial channels.

[0037] Unless otherwise specified, all test methods are standard and all instrument settings are those recommended by the manufacturer.

[0038] According to one embodiment of this application, a dual cross-linked hydrogel that synergistically releases platelet-rich plasma and growth factors comprises: Double cross-linked network matrix, platelet-rich plasma; The dual cross-linked network matrix is ​​formed by the interpenetration of a polysaccharide cross-linked network and a fibrin network. The polysaccharide cross-linking network is formed by cross-linking oxidized modified polysaccharides, sulfated modified polysaccharides, and proteins and peptides containing primary amino groups in platelet-rich plasma; The fibrin network is formed by the activation of fibrin in platelet-rich plasma by an activator. The platelet-rich plasma is loaded into the double cross-linked network matrix through physical encapsulation and chemical cross-linking.

[0039] In one embodiment, the mass concentration of the oxidized polysaccharide in the synergistic platelet-rich plasma and sustained-release growth factor double crosslinked hydrogel is 0.5%-10% (w / v). The mass concentration of sulfated modified polysaccharides is 0.5%-10% (w / v); The volume fraction of platelet-rich plasma is 5-30% (v / v); In one embodiment, the polysaccharide is selected from at least one of xyloglucan, hyaluronic acid, sodium alginate, chitosan, carboxymethyl chitosan, chondroitin sulfate, heparin, heparan sulfate, dextran, and carboxymethyl cellulose. The two modified polysaccharides may be derived from the same polysaccharide or from different polysaccharides; preferably, both modified polysaccharides are derived from xyloglucan.

[0040] The bicrosslinked hydrogel of this application, which synergistically utilizes PRP to release growth factors, is formed by crosslinking two modified polysaccharides and PRP. The two modified polysaccharides are polysaccharides containing hydroxyl groups that can be modified, which are then oxidized and sulfated, respectively, to produce polysaccharides containing aldehyde and sulfate groups. The polysaccharides (e.g., xyloglucan) can be directly oxidized to generate oxidized modified polysaccharides (e.g., oxidized xyloglucan), creating aldehyde crosslinking sites. Schiff base bonds are constructed by crosslinking the amino groups on the PRP with the aldehyde groups of the oxidized modified polysaccharide chains. Simultaneously, the oxidized modified polysaccharide, sulfated modified polysaccharide (e.g., sulfated xyloglucan), and PRP are crosslinked through hydrogen bonding and electrostatic forces. After PRP is activated by an activator (e.g., CaCl2) solution, fibrinogen forms a fibrin network. This constitutes a bicrosslinked hydrogel containing a dynamic covalent network, endowing the hydrogel with self-healing function and allowing for injection. Under acidic conditions, the Schiff base bond dissociation rate is increased, promoting growth factor release, while the sulfate affinity binding and three-dimensional network embedding inhibit early burst release and prolong the release cycle. The purpose of this application is to provide the application of a dual crosslinked hydrogel that synergistically releases PRP and growth factors in the preparation of medical wound dressings; the applications include dressings for chronic wounds such as mechanically injured wounds, burn wounds, and diabetic wounds.

[0041] According to one embodiment of this application, a method for preparing a bicrosslinked hydrogel that synergistically releases platelet-rich plasma and growth factors includes: We provide oxidized modified polysaccharides and sulfated modified polysaccharides. Provide platelet-rich plasma; An activator is added to a solution containing the oxidized modified polysaccharide, the sulfated modified polysaccharide, and the platelet-rich plasma, and a reaction is carried out to obtain the bicrosslinked hydrogel that synergistically enriches platelet-rich plasma and releases growth factors.

[0042] In one embodiment, the mass concentration of the oxidized polysaccharide in the solution is 0.5%-10% (w / v). The mass concentration of sulfated modified polysaccharides is 0.5%-10% (w / v); The volume fraction of platelet-rich plasma is 5-30% (v / v).

[0043] In one embodiment, the physiologically compatible solution containing calcium ions is preferably a calcium chloride solution, a calcium gluconate solution, or a calcium chloride-thrombin mixed solution. In one embodiment, the volume ratio of the activator to platelet-rich plasma in the solution is approximately 1:5 to 1:20, or the activator contains Ca... 2+ The concentration is 0.5-5 mM.

[0044] In one embodiment, the solvent in the solution containing the oxidized modified polysaccharide, the sulfated modified polysaccharide, and the platelet-rich plasma is selected from at least one of phosphate buffer, deionized water, Green's solution, and glycerol.

[0045] In one embodiment, the method for preparing the oxidized modified polysaccharide includes: Under light-protected conditions and with continuous stirring, the oxidizing agent is slowly added to an aqueous solution containing polysaccharides to carry out an oxidation reaction. The solution obtained from the oxidation reaction is then dialyzed and freeze-dried to obtain the oxidized modified polysaccharide.

[0046] In one embodiment, the oxidizing agent is selected from an aqueous solution of sodium periodate.

[0047] In one embodiment, the mass ratio of the oxidizing agent to the polysaccharide is 0.05-1.0:1.

[0048] In one embodiment, the conditions for the oxidation reaction include: a reaction temperature of 15-35°C, a reaction time of 2-24 hours, and the addition of a terminator to terminate the reaction.

[0049] In one embodiment, the terminating agent is selected from at least one of ethylene glycol, propylene glycol, and glycerin.

[0050] In one embodiment, the dialysis conditions in the preparation method of the oxidized modified polysaccharide include: a molecular weight cutoff of 8000-14000 Da and a dialysis time of 3-5 days.

[0051] In one embodiment, the sulfated modified polysaccharide comprises: Under light-protected conditions and with continuous stirring, a sulfation reagent is added to an organic solution containing polysaccharides and a sulfation reaction is carried out. The solution obtained from the sulfation reaction is diluted with water and then dialyzed and freeze-dried to obtain the sulfated modified polysaccharide.

[0052] In one embodiment, the sulfation reagent is selected from an organic solution of a sulfur trioxide-pyridine complex.

[0053] In one embodiment, the mass ratio of the sulfation reagent to the polysaccharide is 1-10:1.

[0054] In one embodiment, the sulfation reaction conditions include: a reaction temperature of 40-80°C, a reaction time of 4-24 h, and the addition of a neutralizing agent after the reaction.

[0055] In one embodiment, the neutralizing agent is selected from at least one of sodium hydroxide solution, sodium carbonate solution, and sodium bicarbonate solution.

[0056] In one embodiment, the dialysis conditions in the preparation method of sulfated modified polysaccharide include: a molecular weight cutoff of 8000-14000 Da and a dialysis time of 3-5 days.

[0057] In one embodiment, the polysaccharide is selected from at least one of xyloglucan, hyaluronic acid, sodium alginate, chitosan, carboxymethyl chitosan, chondroitin sulfate, heparin, heparan sulfate, dextran, and carboxymethyl cellulose. The two modified polysaccharides may be derived from the same polysaccharide or from different polysaccharides; preferably, both modified polysaccharides are derived from xyloglucan.

[0058] In one embodiment, the polysaccharide is obtained from the corresponding natural plant through extraction and freeze-drying.

[0059] In one embodiment, the method for preparing the platelet-rich plasma includes: Provide anticoagulated whole blood to the target population; The anticoagulated whole blood was centrifuged for the first time, and the upper plasma layer was collected while avoiding the aspiration of the red blood cell layer and the white membrane layer. Add a separating gel to the upper plasma layer and perform a second centrifugation to position red blood cells and white blood cells below or at the separating gel interface. Collect the platelet-rich plasma above the separating gel, discard 60% to 90% of the supernatant plasma, and resuspend the precipitated platelets in the remaining plasma to obtain the platelet-rich plasma.

[0060] In one embodiment, the platelet concentration in the platelet-rich plasma is 2-8 times the whole blood platelet concentration, or the whole blood platelet count is 5 × 10⁻⁶. 8 ~3×10 9 per mL.

[0061] In one embodiment, the conditions for the two centrifugations can be selected as needed, with the first centrifugation aimed at removing most of the red blood cells (exemplarily, conditions of 4 °C, 2000 rpm, 20 min) and the second centrifugation aimed at removing white blood cells (exemplarily, conditions of 4 °C, 3500 rpm, 20 min).

[0062] This application utilizes a double-crosslinked hydrogel that synergistically enriches platelet-rich plasma and releases growth factors. This hydrogel has a high water content and abundant three-dimensional porous structure, which can provide favorable conditions for cell infiltration, proliferation and differentiation, as well as the germination of blood vessels in granulation tissue.

[0063] According to one embodiment of this application, the aforementioned synergistic platelet-rich plasma and sustained-release growth factor dual cross-linked hydrogel, and the synergistic platelet-rich plasma and sustained-release growth factor dual cross-linked hydrogel prepared according to the aforementioned preparation method, are applied in the preparation of medical wound dressings.

[0064] In one embodiment, the wound dressing is selected from at least one of chronic wound dressings such as mechanical injury wound dressings, burn wound dressings, and diabetic wound dressings.

[0065] The dual-crosslinked hydrogel of this application, which synergistically encapsulates PRP and its active ingredients through a three-dimensional network, also immobilizes growth factors through affinity binding sites provided by sulfate groups, thereby reducing early burst release and prolonging the duration of action of growth factors. The dynamic Schiff base network can reversibly dissociate with local pH changes, promoting the release of total protein and growth factors such as EGF, PDGF, and VEGF during wound healing; Ca... 2+ The activated fibrin network further enhances the hydrogel structure and improves hemostatic properties. This hydrogel is simple to prepare, highly reproducible, and exhibits good cell, blood, and tissue compatibility, making it suitable for treating mechanically induced wounds, burn wounds, and chronic wounds such as those caused by diabetes.

[0066] Example 1: Preparation of Modified Polysaccharides (1) Preparation of xylo-glucan (XG) 10 g of tamarind powder was dissolved in 1000 mL of deionized water, and the solution was gradually heated to 40 °C and stirred continuously for 24 h to obtain a homogeneous solution. Then, the solution was centrifuged at 8000 r for 10 min to remove insoluble impurities. The supernatant was retained after removing the impurities. The supernatant was frozen at -80 °C and then freeze-dried. The spongy solid obtained after freeze-drying was xyloglucan, named XG.

[0067] (2) Preparation of oxidized xylo-glucan (OXG) 1 g of XG prepared in step (1) was completely dissolved in 100 mL of deionized water. 10 mL of 2% sodium periodate was slowly added dropwise under light-protected conditions and stirred continuously. After reacting at room temperature for 4 h, 1 mL of ethylene glycol was slowly added dropwise and stirred continuously for 2 h to terminate the reaction and obtain the reaction solution. Then the reaction solution was transferred into a dialysis bag (molecular weight cutoff MWCO = 8000 Da) and dialyzed in deionized water for 3 days. After dialysis, the solution was placed in a -80℃ freezer and then freeze-dried in a freeze dryer. After freeze-drying, oxidized xylo-glucan was obtained and named OXG.

[0068] (3) Preparation of sulfated xylo-glucan (SXG) 6 g of sulfur trioxide-pyridine complex was diluted in 10 mL of DMSO to prepare a sulfation reagent; 1 g of XG prepared in step (1) was dissolved in 100 mL of DMSO and stirred at room temperature in the dark for 4 h, then the sulfation reagent was added, and then stirred at 60 ℃ for 10 h to obtain a reaction solution; the reaction solution was diluted with deionized water and neutralized with 2.5 mM NaOH to pH=7, then the reaction solution was transferred to a dialysis bag (molecular weight cutoff MWCO=8000 Da), dialyzed in deionized water for 3 days, and after dialysis, it was frozen in a -80℃ freezer and then freeze-dried in a freeze dryer to finally obtain SXG.

[0069] Example 2: Preparation of PRP Whole blood was collected from the patient via abdominal aortic puncture and placed in an EDTA centrifuge tube. The whole blood was centrifuged twice using a high-speed centrifuge to enrich platelets and reduce residual red blood cells and white blood cells. First, the whole blood was centrifuged at 2000 rpm for 20 min at 4 °C to separate most red blood cells. After centrifugation, the supernatant plasma was collected, avoiding the aspiration of the red blood cell layer and the white blood cell layer. Then, a separating gel was added to the supernatant serum, and the mixture was centrifuged at 3500 rpm for 20 min at 4 °C to create a stratified structure. The separating gel and its interface effectively blocked red blood cells and white blood cells, which were mainly located below the separating gel or at the separating gel interface. The platelet-rich plasma above the separating gel was collected. After discarding approximately three-quarters of the supernatant plasma layer, the precipitated platelets were resuspended in the remaining plasma to obtain PRP (platelet-rich plasma), with a platelet concentration of 1 × 10⁻⁶. 9 The number of cells / mL is approximately four times the concentration of whole blood platelets.

[0070] Example 3: OXG / SXG / PRP hydrogel (OS4P) 20 @Ca 2+ Preparation of ) 0.05 g of OXG prepared in Example 1, 0.04 g of SXG prepared in Example 1, and 0.2 mL of PRP prepared in Example 2 were added to PBS and brought to a final volume of 1 mL to prepare a pregelation mixture. The OXG concentration was 5% (w / v), the SXG concentration was 4% (w / v), and the PRP concentration was 20% (v / v). The mixture was magnetically stirred for 10 min until homogeneous. Then, 0.02 mL of a 10% (w / v) CaCl2 solution was added in one go, and the mixture was gently stirred for another 30 s. The mixture was allowed to stand until gelation, yielding a hydrogel named OS4P. 20 @Ca 2+ Hydrogel, abbreviated as OSP@Ca in the attached diagram. 2+ .

[0071] Comparative Example 1 OP@Ca 2+ Preparation of hydrogels 0.05 g of OXG prepared in Example 1 and 0.2 mL of PRP prepared in Example 2 were added to PBS and brought to a final volume of 1 mL to prepare a pregelation mixture. The OXG concentration was 5% (w / v), and the PRP concentration was 20% (v / v). The mixture was magnetically stirred for 10 min until homogeneous. Then, 0.02 mL of a 10% (w / v) CaCl2 solution was added in one go, and the mixture was gently stirred for another 30 s. The mixture was allowed to stand until gelation occurred, yielding OP@Ca 2+ Hydrogel.

[0072] Comparative Example 2: PRP@Ca 2+ Gel preparation 0.2 mL of the PRP prepared in Example 2 was added to PBS and the volume was adjusted to 1 mL to prepare a pregelation mixture. The volume fraction of PRP was 20% (v / v). The mixture was magnetically stirred for 10 min until homogeneous. Then, 0.02 mL of a 10% (w / v) CaCl2 solution was added in one go, and the mixture was gently stirred for another 30 s. The mixture was allowed to stand until gelation occurred, yielding PRP@Ca 2 + gel.

[0073] Comparative Example 3 OS2P 20 @Ca 2+ Preparation of hydrogels 0.05 g of OXG prepared in Example 1, 0.02 g of SXG prepared in Example 1, and 0.2 mL of PRP prepared in Example 2 were added to PBS and diluted to a final volume of 1 mL to prepare a pregelation mixture. The OXG concentration was 5% (w / v), the SXG concentration was 2% (w / v), and the PRP concentration was 20% (v / v). The mixture was magnetically stirred for 10 min until homogeneous. Then, 0.02 mL of a 10% (w / v) CaCl2 solution was added in one go, and the mixture was gently stirred for another 30 s. The mixture was allowed to stand until gelation occurred, yielding OS2P20@Ca 2+ Hydrogel.

[0074] Comparative Example 4: OP systems with different PRP volume fractions (OP) 10 OP 20 and OP 30 Preparation of ) 0.05 g of OXG prepared in Example 1 and 0.1 mL, 0.2 mL, or 0.3 mL of PRP prepared in Example 2 were added to PBS, and the volumes were adjusted to 1 mL to make the OXG concentration 5% (w / v) and the PRP volume fractions 10%, 20%, and 30% (v / v), respectively. Each mixture was magnetically stirred for 10 min until homogeneous and then allowed to stand. These mixtures were named OP. 10 OP 20 and OP 30 The corresponding Ca needs to be prepared. 2+ To activate the system, add 0.01 mL, 0.02 mL, or 0.03 mL of a 10% (w / v) CaCl2 solution, respectively, and continue to stir gently for 30 s and let stand.

[0075] Comparative Example 5: OSP systems with different SXG mass concentrations (OS2P) 20 OS4P20 and OS6P 20 Preparation of ) 0.05 g of OXG prepared in Example 1, 0.02 g, 0.04 g, or 0.06 g of SXG prepared in Example 1, and 0.2 mL of PRP prepared in Example 2 were added to PBS and brought to a final volume of 1 mL, so that the mass-volume concentration of OXG was 5% (w / v), the mass-volume concentration of SXG was 2%, 4%, and 6% (w / v), and the volume fraction of PRP was 20% (v / v). Each mixture was magnetically stirred for 10 min until homogeneous and then allowed to stand; these mixtures were named OS2P. 20 OS4P 20 and OS6P 20 The corresponding Ca needs to be prepared. 2+ When activating the system, add 0.02 mL of CaCl2 solution with a mass-volume concentration of 10% (w / v), continue to stir gently for 30 s and let stand.

[0076] Comparative Example 6: OSP systems with different PRP volume fractions (OS4P) 10 and OS4P 20 Preparation of ) 0.05 g of OXG prepared in Example 1, 0.04 g of SXG prepared in Example 1, and 0.1 mL or 0.2 mL of PRP prepared in Example 2 were added to PBS and brought to a final volume of 1 mL, so that the mass-volume concentration of OXG was 5% (w / v), the mass-volume concentration of SXG was 4% (w / v), and the volume fraction of PRP was 10% and 20% (v / v), respectively. Each mixture was magnetically stirred for 10 min until homogeneous and then allowed to stand; these mixtures were named OS4P. 10 and OS4P 20 ; It is necessary to prepare Ca 2+ To activate the system, add 0.01 mL or 0.02 mL of a 10% (w / v) CaCl2 solution, stir gently for 30 s, and let stand.

[0077] For ease of explanation, OSP in the attached diagram represents a pre-crosslinked system formed by OXG, SXG, and PRP. OSP@Ca 2+ This indicates that the system is further processed by Ca 2+ The double cross-linked hydrogel formed after activation; OP and PRP "@Ca 2+ "The markings have the same meaning. Unless otherwise specified in the attached figures, OSP and OSP@Ca..." 2+ They refer to OS4P respectively 20 and OS4P 20 @Ca 2+ .

[0078] Test Example 1: Characterization of Chemical Structure, Gel Formation, and Microstructure XG, OXG, SXG, PRP, and OSP@Ca obtained in Example 3 2+ Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and X-ray photoelectron spectroscopy were performed to characterize the signal, and the results are shown in Figure 1. Figure 1(A) shows that OXG exhibits absorption signals related to carbonyl groups, SXG exhibits characteristic absorptions related to sulfate groups, PRP shows amide-related absorptions, and OSP@Ca... 2+ The retention of characteristic signals from both the modified polysaccharide and PRP indicates that all components coexist within the hydrogel. Figure 1(B) shows the chemical shift changes after XG oxidation and sulfation modification, demonstrating that the polysaccharide chemical environment was regulated. Figure 1(C) shows the OSP@Ca 2+ The signals of C, O, N and S elements are present simultaneously; in the high-resolution C 1s and N 1s spectra of Figure 1 (D), signals of CC, CO, C=O, C=N, -NH- / CO-NH and positively charged nitrogen can be observed, which supports the formation of Schiff base dynamic crosslinking between OXG aldehyde group and PRP amino-containing component, and proves that SXG sulfate group is introduced into the system.

[0079] The inverted gelation state, gelation time, and lyophilized morphology of different systems were observed, and the results are shown in Figure 2. In Figure 2(A), the pre-gelation mixture can flow in the inverted state; forming an OSP pre-crosslinked system or further adding Ca 2+ Afterwards, the sample can remain inverted and non-flowing. PRP retains its flowability when present alone, and after being treated with Ca... 2+ After activation, PRP@Ca is formed. 2+ Gel. Figure 2(B) shows that the gel time of the OP system decreases with increasing PRP volume fraction; the gel time of the OSP system is affected by SXG concentration, with OS4P... 20 It can form an initial gel relatively quickly. Ca 2+ Upon activation, it further forms a fibrin network, thereby constructing a double cross-linked structure.

[0080] Figure 2 (C) shows PRP@Ca 2+ OSP and OSP@Ca 2+ Both have porous structures. Compared to OSP, OSP@Ca 2+ The pore structure is more compact and the average pore size is reduced; the pore size statistics in Figure 2(D) are consistent with the scanning electron microscopy observations, indicating that Ca 2+ The activated fibrin network can regulate the internal structure of the hydrogel, providing space for water retention, cell adhesion, and substance exchange.

[0081] Test Example 2: Rheological Test Testing OSP systems with different PRP volume fractions and their Ca2+ Rheological properties before and after activation. Tests were conducted using a rotational rheometer (TA Instruments DHR-2, USA). The effects of OSP and OSP@Ca were compared. 2+ Perform for 0.1-100 s - ¹Shear rate scan; for OSP 10 OSP 10 @Ca 2+ OSP and OSP@Ca 2+ A 120 s time scan was performed at 1% strain and 10 rad / s; a 0.1%–10000% strain scan was performed at 10 rad / s; and the OSP@Ca 2+ Alternating time scans of 1% low strain and 800% high strain were performed, each strain stage lasting 60 s, for a total of 5 cycles. The results are shown in Figure 3. In Figure 3(A), OSP and OSP@Ca 2+ The viscosity of all samples decreased with increasing shear rate, and the samples could be extruded through a needle, demonstrating their shear-thinning and injectable properties; Ca 2+ The viscosity of the system increased after activation. Figure 3(B) shows that the storage modulus G′ of each group was higher than the loss modulus G″ and remained relatively stable during the test, indicating the formation of a stable gel network, in which OSP@Ca 2+ It exhibits a high modulus. Figure 3(C) shows that in the low strain region, G′ is higher than G″, and the network gradually breaks down with increasing strain; Ca2+ activation can increase the system modulus and enhance structural stability. Figure 3(d) shows OSP@Ca 2+ The significant decrease in G′ under high strain, followed by repeated recovery after the return to low strain, indicates that the hydrogel has self-healing ability.

[0082] Test Example 3: Protein and Growth Factor Release Experiment Test PRP@Ca 2+ OP@Ca 2+ OS2P@Ca 2+ and OS4P@Ca 2+ The total protein and growth factor release performance was evaluated, and the OSP@Ca expression was assessed. 2+ Release behavior under different pH conditions.

[0083] 200 μL of each sample group was added to 300 μL of PBS in 48-well culture plates and incubated at 37°C. The release solution was collected at 1 h, 3 h, 6 h, 12 h, 18 h, 24 h, 48 h, 120 h, and 168 h after incubation, and an equal volume of fresh PBS was added. Total protein content was determined using a BCA assay kit, and EGF, PDGF, and VEGF concentrations were determined using an ELISA kit. OSP@Ca 2+Release was measured using the same method after placing the samples in buffer solutions at pH 8.5, pH 7.4, and pH 5.5, respectively. The results are shown in Figure 4. Figures 4(A) to 4(D) show the release of PRP@Ca 2+ and OP@Ca 2+ Early release is faster, while OS2P@Ca containing SXG 2+ and OSP@Ca 2+ The release was more gradual and lasted longer; increasing SXG content further reduced early burst release, indicating that sulfate affinity binding and three-dimensional network embedding together contribute to the immobilization and sustained release of total protein, EGF, PDGF, and VEGF from PRP. Figures 4(E) to 4(H) show that OSP@Ca 2+ The cumulative release was highest at pH 5.5, followed by pH 7.4, and lowest at pH 8.5, indicating that an acidic environment can promote the dissociation of the dynamic bonds of Schiff bases, thereby achieving pH-responsive release regulation.

[0084] Test Example 4: Biocompatibility Test The cell, blood, and tissue compatibility of the hydrogel was evaluated using L929 cell live / dead staining, hemolysis assay, and subcutaneous implantation assay in mice.

[0085] PRP@Ca was prepared under aseptic conditions. 2+ OP@Ca 2+ OSP and OSP@Ca 2+ Extracts were prepared, with complete culture medium serving as a control. L929 fibroblasts were seeded into culture plates, and after cell adhesion, the respective group's extracts were added for culture. Live / dead cell staining was performed on days 1, 3, and 5 using Calcein-AM and propidium iodide, and observed under a fluorescence microscope. The results are shown in Figure 5(A). All groups showed predominantly green live cells, with the cell number increasing with prolonged culture time. OSP@Ca 2+ The group showed high cell density on days 3 and 5, indicating that the hydrogel has good cell compatibility and is conducive to cell survival and proliferation.

[0086] OSP@Ca 2+ The hydrogel was injected subcutaneously into the back of C57 mice to evaluate its tissue compatibility and in vivo degradation. After anesthetizing the mice, 0.2 mL of sterile hydrogel was injected subcutaneously into the back using a 20 G needle. The implantation site was observed and samples were collected for H&E staining on days 1, 7, 20, and 32. The results are shown in Figure 5(B). No obvious redness or purulent swelling was observed at the implantation site, and the hydrogel gradually degraded over time. Normal local reactions were observed on day 1, inflammatory cells decreased on day 7, the hydrogel volume significantly decreased on day 20 with tissue ingrowth, and the hydrogel was essentially degraded with intact surrounding tissue structure on day 32, indicating good tissue compatibility and degradability.

[0087] Fresh rat red blood cells were used to evaluate blood compatibility. Washed red blood cells were prepared into a 5% suspension with physiological saline and then subjected to PRP@Ca... 2+ OP, OSP and OSP@Ca 2+ Co-incubation was performed; PBS was used as a negative control, and Triton X-100 as a positive control. After incubation at 37°C for 1 h, centrifugation was performed, and the absorbance of the supernatant at 540 nm was measured. The hemolysis rate was calculated as follows: (A... 样品 -A 阴性 ) / (A 阳性 -A 阴性 The result is calculated as PRP@Ca × 100%. The result is shown in Figure 5(C). 2+ OP, OSP and OSP@Ca 2+ The hemolysis rate in all groups was less than 4%, with OSP@Ca being the most effective. 2+ The lowest values ​​in group A indicate that the materials in each group have good blood compatibility.

[0088] Test Example 5: Liver Hemostasis Performance Test Testing OSP, PRP@Ca 2+ and OSP@C a2+ Its hemostatic properties.

[0089] A standardized mouse model of liver hemorrhage was used to evaluate hemostasis. Four-week-old C57BL / 6 mice weighing 18-22 g were anesthetized, and the liver was exposed via a midline abdominal incision after aspiration of surrounding tissue fluid. A 3 mm long wound was made on the liver surface using a scalpel, and 100 μL of OSP and PRP@Ca were immediately applied. 2+ Or OSP@Ca 2+ The control group received no material treatment. The hemostasis process was filmed at 20, 40, 60, and 80 seconds, and blood loss was calculated by measuring the mass change of the filter paper after absorbing blood. The procoagulant ability of the materials was evaluated using the in vitro coagulation index method. Gauze, OSP, and PRP@Ca were used in this study. 2+ and OSP@Ca 2+ The absorbance of the uncoagulated red blood cell release fluid was measured at 30, 60, 90, 120, 150 and 180 min after contact with anticoagulated whole blood, and the coagulation index was calculated.

[0090] The results are shown in Figure 6. In Figure 6(A), OSP@Ca 2+ Bleeding decreased rapidly after treatment and a stable covering layer formed within the observation period; OSP and PRP@Ca 2+ Different degrees of blood seepage were still visible in the group. Figure 6(B) shows OSP@Ca 2+ The group with the lowest blood loss was significantly lower than the control group, OSP group, and PRP@Ca.2+ Group. Figure 6(C) shows OSP@Ca 2+ The coagulation index of the group was low at all time points, indicating that it can promote blood coagulation. These results suggest that the initial polysaccharide network and Ca... 2+ The activated fibrin network synergistically enhances the rapid hemostatic ability of the hydrogel.

[0091] Test Example 6: Chronic Wound Test in Diabetic Mice Evaluation of OSP@Ca 2+ OP@Ca 2+ PRP@Ca 2+ And the repair effect of commercial epidermal growth factor (EGF) preparations on chronic wounds in diabetic mice.

[0092] Male C57BL / 6 mice, weighing 18-22 g and 6-8 weeks old, were used. After a 12-hour fast, streptozotocin was administered daily at 50 mg / kg for 5 consecutive days to establish a diabetic model. Circular full-thickness skin wounds (8 mm in diameter) were created on the backs of the diabetic mice, and 100 μL of PRP@Ca was administered to each wound. 2+ OP@Ca 2+ Or OSP@Ca 2+ Treatment: The commercial EGF group received topical administration at an equivalent dose; the control group received 100 μL of normal saline topically. All wounds were covered with sterile transparent dressings. Wounds were photographed and recorded on days 0, 3, 7, 14, and 21, and changes in wound area were calculated. Results are shown in Figure 7. Figure 7(A) shows that the wound area decreased over time in all treatment groups, with OSP@Ca... 2+ The group showed the fastest shrinkage, with the wound essentially closed by day 21; the statistical results in Figure 7(B) further show that OSP@Ca 2+ The residual wound area was the lowest in the group, which was superior to the control group, the commercial EGF group, and the PRP@Ca group. 2+ Group and OP@Ca 2+ Group. The above results indicate that OSP@Ca 2+ By prolonging the duration of action of PRP-derived growth factors, providing pH-responsive release, and improving local hemostasis and cell compatibility, it can promote the repair of chronic diabetic wounds.

[0093] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A bicrosslinked hydrogel that synergistically releases platelet-rich plasma and growth factors, characterized in that, include: Double cross-linked network matrix, platelet-rich plasma; The dual cross-linked network matrix is ​​formed by the interpenetration of a polysaccharide cross-linked network and a fibrin network. The polysaccharide cross-linking network is formed by cross-linking oxidized modified polysaccharides, sulfated modified polysaccharides, and proteins and peptides containing primary amino groups in platelet-rich plasma; The fibrin network is formed by the activation of fibrin in platelet-rich plasma by an activator. The platelet-rich plasma is loaded into the double cross-linked network matrix through physical encapsulation and chemical cross-linking.

2. The double cross-linked hydrogel with synergistic platelet-rich plasma and sustained-release growth factor according to claim 1, characterized in that, In the bicrosslinked hydrogel that synergistically enriches platelet-rich plasma and releases growth factors, the mass concentration of oxidized modified polysaccharide is 0.5%-10% w / v. The mass concentration of sulfated modified polysaccharides is 0.5%-10% w / v; The volume fraction of platelet-rich plasma is 5-30% v / v.

3. The double cross-linked hydrogel with synergistic platelet-rich plasma and sustained-release growth factor according to claim 1, characterized in that, The polysaccharides in the oxidized modified polysaccharides and sulfated modified polysaccharides are independently selected from at least one of xyloglucan, hyaluronic acid, sodium alginate, chitosan, carboxymethyl chitosan, chondroitin sulfate, heparin, heparan sulfate, dextran, and carboxymethyl cellulose. Preferably, the polysaccharides in the oxidized modified polysaccharide and the sulfated modified polysaccharide are both xyloglucans.

4. A method for preparing a bi-crosslinked hydrogel that synergistically releases platelet-rich plasma and growth factors, characterized in that, include: We provide oxidized modified polysaccharides and sulfated modified polysaccharides. Provide platelet-rich plasma; An activator is added to a solution containing the oxidized modified polysaccharide, the sulfated modified polysaccharide, and the platelet-rich plasma, and a reaction is carried out to obtain the bicrosslinked hydrogel that synergistically enriches platelet-rich plasma and releases growth factors.

5. The preparation method according to claim 4, characterized in that, The mass concentration of the oxidized polysaccharide in the solution is 0.5%-10% w / v; The mass concentration of sulfated modified polysaccharides is 0.5%-10% w / v; The volume fraction of platelet-rich plasma is 5-30% v / v.

6. The preparation method according to claim 4, characterized in that, The activator is selected from physiologically compatible solutions containing calcium ions; Preferably, the activator is selected from at least one of calcium chloride solution, calcium gluconate solution, or calcium chloride-thrombin mixed solution; Preferably, the volume ratio of the activator to platelet-rich plasma in the solution is approximately 1:5 to 1:20; Preferably, the activator contains Ca 2+ The concentration is 0.5-5 mM. Preferably, the solvent in the solution is selected from at least one of phosphate buffer, deionized water, Green's solution, and glycerol.

7. The preparation method according to claim 4, characterized in that, The method for preparing the oxidized modified polysaccharide includes: Under light-protected conditions and with continuous stirring, the oxidizing agent was slowly added to an aqueous solution containing polysaccharides and an oxidation reaction was carried out. The solution obtained from the oxidation reaction was dialyzed and freeze-dried to obtain the oxidized modified polysaccharide. Preferably, the oxidizing agent is selected from an aqueous solution of sodium periodate; Preferably, the mass ratio of the oxidizing agent to the polysaccharide is 0.05-1.0:1; Preferably, the conditions for the oxidation reaction include: a reaction temperature of 15-35°C, a reaction time of 2-24 hours, and the addition of a terminator to terminate the reaction. Preferably, the terminating agent is selected from at least one of ethylene glycol, propylene glycol, and glycerin; Preferably, the dialysis conditions in the preparation method of oxidatively modified polysaccharides include: a molecular weight cutoff of 8000-14000 Da and a dialysis time of 3-5 days; Preferably, the method for preparing the sulfated modified polysaccharide includes: Under light-protected conditions and with continuous stirring, a sulfation reagent is added to an organic solution containing polysaccharides and a sulfation reaction is carried out. The solution obtained from the sulfation reaction is diluted with water and then dialyzed and freeze-dried to obtain the sulfated modified polysaccharide. Preferably, the sulfation reagent is selected from an organic solution of a sulfur trioxide-pyridine complex; Preferably, the mass ratio of the sulfation reagent to the polysaccharide is 1-10:1; Preferably, the organic solvent in the polysaccharide-containing organic solution is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and formamide; Preferably, the mass-volume concentration of the polysaccharide in the organic solution containing the polysaccharide is 0.1%-5% (w / v). Preferably, the conditions for the sulfation reaction include: a reaction temperature of 40-80°C, a reaction time of 4-24 h, and the addition of a neutralizing agent after the reaction; Preferably, the neutralizing agent is selected from at least one of sodium hydroxide solution, sodium carbonate solution, and sodium bicarbonate solution; Preferably, the dialysis conditions in the preparation method of sulfated modified polysaccharides include: a molecular weight cutoff of 8000-14000 Da and a dialysis time of 3-5 days.

8. The preparation method according to claim 4, characterized in that, The method for preparing platelet-rich plasma includes: Provide anticoagulated whole blood to the target population; The anticoagulated whole blood was centrifuged for the first time, and the upper plasma layer was collected while avoiding the aspiration of the red blood cell layer and the white membrane layer. Add a separating gel to the upper plasma layer and perform a second centrifugation to place red blood cells and white blood cells below the separating gel or at the separating gel interface. Collect the platelet-rich plasma above the separating gel, discard 60% to 90% of the supernatant plasma, and resuspend the precipitated platelets in the remaining plasma to obtain the platelet-rich plasma. Preferably, the platelet concentration in the platelet-rich plasma is 2-8 times that of whole blood platelets; Preferably, the platelet-rich plasma contains 5 × 10⁻⁶ whole blood platelets. 8 ~3×10 9 per mL.

9. The application of the synergistic platelet-rich plasma and sustained-release growth factor dual crosslinked hydrogel according to any one of claims 1 to 3, or the synergistic platelet-rich plasma and sustained-release growth factor dual crosslinked hydrogel prepared by the preparation method according to any one of claims 3 to 8, in the preparation of medical wound dressings.

10. The application according to claim 9, characterized in that, The wound dressing is selected from at least one of the following: dressings for mechanical injury wounds, dressings for burn wounds, dressings for diabetic wounds, and other chronic wound dressings.

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