A CGF-loaded hydrogel, a preparation method and application in wound repair

Abstract

The application discloses a composite hydrogel loaded with concentrated growth factors, and is prepared by loading a CGF solution in oxidized dextran / carboxymethyl chitosan composite hydrogel, wherein the oxidized dextran has an oxidation degree of 50%-60% of high-activity aldehyde groups, and the carboxymethyl chitosan has an amino substitution degree of no less than 80%. Main components of the CGF solution include VEGF, PDGF, TGF-beta, CD34 + hematopoietic stem cells and a fibrin network matrix; the application precisely and uniformly regulates the release of growth factors through the electrostatic adsorption-network blocking dual mechanism, realizes the multifunctional synergy of antibacterial, anti-inflammatory and pro-angiogenic functions on a wound surface under the premise of completely avoiding the toxicity of chemical crosslinking, and has the shear-thinning property for a joint or a moving part of a wound surface, so that the close adhesion to the wound surface is ensured, the injection and filling for a deep and different-type wound cavity are enabled, and the difficulty of clinical operation is greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a wound repair material. Background Technology

[0002] Wound healing is essentially a dynamic cascade process involving inflammatory response, tissue proliferation, and matrix remodeling, each stage requiring precise microenvironment regulation. During the inflammatory phase, a rapid establishment of an anti-infection barrier and removal of necrotic tissue are necessary; the proliferative phase relies on a sustained growth factor concentration gradient to drive angiogenesis (requiring VEGF > 50 pg / mL for more than 7 days); and the remodeling phase requires balancing MMP / TIMP enzyme activity to prevent excessive collagen deposition. Traditional passive dressings (such as polyurethane foam) can absorb exudate but cannot dynamically release active ingredients in response to the healing phase; exogenous recombinant growth factors (such as rhPDGF-BB) can promote repair, but their short in vivo half-life (approximately 1.8 hours), repeated administration, and high cost can easily lead to local concentration fluctuations and even hypertrophic scarring.

[0003] Concentrated growth factors (CGF), as an autologous bioactive material, are enriched at high concentrations from patient blood using gradient centrifugation techniques, containing growth factors (such as VEGF, PDGF, TGF-β, etc.) and fibrin networks. CGF offers irreplaceable advantages in the treatment of refractory wounds: its three-dimensional fibrous scaffold provides migration anchors for fibroblasts and endothelial cells, while the continuously released growth factors activate angiogenesis signaling pathways (such as VEGFR2 / PI3K-Akt), significantly accelerating granulation tissue formation and re-epithelialization. Clinical studies have confirmed that CGF application in diabetic foot ulcers can shorten the healing cycle by 35% without the risk of immune rejection. The efficacy of CGF in diabetic foot ulcers stems from its multi-target regulation throughout the healing process.

[0004] Despite the significant bioactivity of CGF in wound repair, its natural structure faces serious challenges in clinical application: the fibrous network rapidly disintegrates after infiltration by tissue fluid (disintegration time <24 hours), resulting in a wound retention rate of less than 30% for growth factors, and making it difficult to effectively fill deep or irregular wound cavities. Traditional carriers such as sodium alginate gel can prolong retention time, but due to reliance on toxic chemical cross-linking agents (glutaraldehyde cross-linking leads to apoptosis rate >35%) and high-temperature curing processes (>37℃ induces growth factor denaturation), VEGF activity is lost by more than 40%. Physically cross-linked gelatin gel, on the other hand, is easily diluted by tissue fluid due to its low mechanical strength (storage modulus <100 Pa), and cannot maintain long-term sustained release. Growth factors tend to accumulate on the gel surface, resulting in insufficient penetration into deep tissues, and uneven distribution directly affects the repair effect. In addition, ordinary loading gels cannot effectively fill deep, irregular wound cavities, and for wounds on joints or mobile areas, ordinary loading gels have poor adhesion to the wound surface and are prone to detachment. Summary of the Invention

[0005] To address the problems of existing CGF-loaded formulations in terms of activity retention, controlled release, and clinical operation, this invention provides a composite hydrogel loaded with concentrated growth factors. Through the dual mechanism of electrostatic adsorption and network blockade of the composite hydrogel system, the release of growth factors is precisely and uniformly regulated. Under the premise of completely avoiding the toxicity of chemical cross-linking, it simultaneously achieves multifunctional synergy of wound antibacterial, anti-inflammatory, and angiogenesis promotion. At the same time, it has shear-thinning properties for wounds in joints or mobile areas, ensuring close adhesion to the wound surface. It can also be injected to fill deep, irregular wound cavities, greatly simplifying the difficulty of clinical operation.

[0006] To achieve the above objectives, the following technical solution is adopted:

[0007] A composite hydrogel loaded with concentrated growth factor was prepared by loading concentrated growth factor (CGF) into an oxidized dextran (ODEX) / carboxymethyl chitosan (CMC) composite hydrogel.

[0008] According to the above scheme, the degree of oxidation of the highly active aldehyde group of the oxidized dextran is 50%-60%, and the degree of amino substitution of the carboxymethyl chitosan is ≥80%.

[0009] According to the above scheme, the main components of the CGF solution include VEGF, PDGF, TGF-β, and CD34. + Hematopoietic stem cells and fibrin network matrix.

[0010] The preparation method of the above-mentioned composite hydrogel loaded with concentrated growth factors includes the following steps:

[0011] Carboxymethyl chitosan was mixed and stirred with CGF solution to dissolve; oxidized dextran was stirred and dissolved in deionized water; the two were mixed and stirred evenly, and after 5-8 minutes, a composite hydrogel loaded with concentrated growth factors was obtained.

[0012] According to the above scheme, the composition of the composite hydrogel by weight is as follows:

[0013] 1 part carboxymethyl chitosan; 2 parts oxidized dextran; 40-50 parts CGF solution; 50-55 parts deionized water.

[0014] In the optimized scheme, the composition of the composite hydrogel by weight is as follows:

[0015] 1 part carboxymethyl chitosan; 2 parts oxidized dextran; 45 parts CGF solution; 52 parts deionized water.

[0016] According to the above scheme, the preparation method of the oxidized dextran includes the following steps:

[0017] Dextran was dissolved in deionized water, sodium periodate was added, and the mixture was stirred continuously in the dark. Ethylene glycol was added to the solution and the mixture was stirred continuously. Oxidized dextran was obtained by dialysis and freeze-drying.

[0018] According to the above scheme, the preparation method of the CGF solution includes the following steps:

[0019] Venous blood was collected and centrifuged in an anticoagulant tube. The venous blood formed two layers in the anticoagulant tube. The lower 1 / 3 of the upper plasma layer was separated to obtain the CGF solution.

[0020] The above-mentioned composite hydrogel loaded with concentrated growth factors is used as a wound repair material.

[0021] The above-mentioned composite hydrogel loaded with concentrated growth factors is used as a hair regeneration material.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention provides a composite hydrogel of oxidized dextran (ODEX) / carboxymethyl chitosan (CMC) loaded with concentrated growth factor (CGF). This hydrogel acts on the skin wound microenvironment, precisely regulating the inflammatory response and tissue regeneration process through an intelligent sustained-release system. The CGF encapsulated in this gel is rich in CD34⁺ hematopoietic stem cells (>50 cells / μL) and high concentrations of growth factor (VEGF>350 pg / mL), synergistically activating hair follicle stem cell proliferation signaling (Wnt / β-catenin pathway), simultaneously promoting wound repair and hair regeneration. Compared to traditional liquid CGF formulations, this gel achieves a breakthrough improvement through the ODEX / CMC three-dimensional network:

[0024] 1. Long-lasting retention and deep penetration: The negatively charged network of CMC and the positively charged microdomains of ODEX form a double-layer barrier. Combined with the spatial steric hindrance effect of the three-dimensional network, the cumulative release rate of growth factors after 72 hours is <45%, dynamically matching the stage-specific needs of wound healing. Simultaneously, the aforementioned electrostatic adsorption effect ensures the uniform distribution of growth factors within the system. Furthermore, the composite system of ODEX and CMC in this invention exhibits shear-thinning properties (recovery rate >98%) for wounds on joints or mobile areas, ensuring close adhesion to the wound surface. For deep wound cavities, it can be injected for filling, promoting the continuous penetration of active ingredients into the dermis.

[0025] 2. Dynamic moisturizing and oxygen exchange: The gel has a water content of >95%, which simulates the extracellular matrix environment. Its microporous structure (pore size 50-200 μm) maintains the moisture balance of the wound and promotes oxygen diffusion (oxygen flux up to 12.5 mL / cm² / h), avoiding adhesion and damage from traditional dressings.

[0026] 3. Synergistic activation of hair regeneration: The sustained-release VEGF and FGF-9 stimulate the proliferation of dermal papilla cells. Mouse wound healing experiments show that while the composite gel of this invention repairs the wound, the hair follicles on the wound surface are also repaired and hair regenerates.

[0027] 4. Safe and non-toxic: This composite gel requires only three core raw materials: ODEX, CMC, and autologous blood. It is formed in one step through a Schiff base reaction at room temperature (5-8 minutes), completely avoiding the toxicity of chemical cross-linking agents (cell survival rate >95%). Attached Figure Description

[0028] Figure 1 Flow cytometry was used to measure the content of hematopoietic stem cells in CGF.

[0029] Figure 2 : Standard curve for measuring TGF-β content using ELISA.

[0030] Figure 3 Images of blank gel and CGF-loaded hydrogel samples.

[0031] Figure 4 Image showing the results of wound healing in mice. Detailed Implementation

[0032] The following embodiments further illustrate the technical solution of the present invention, but are not intended to limit the scope of protection of the present invention.

[0033] To address the problems of existing CGF-loaded formulations in terms of activity retention, controlled release, and clinical operation, this invention utilizes a composite system of oxidized dextran (ODEX) and carboxymethyl chitosan (CMC) to overcome these bottlenecks through an innovative synergistic mechanism: the highly active aldehyde group of ODEX (oxidation degree 55%±5%) and the amino group of CMC (substitution degree greater than 0.80) undergo a spontaneous Schiff base reaction under physiological conditions, achieving in-situ gelation at room temperature (5-8 minutes), completely avoiding high-temperature damage and chemical toxicity.

[0034] A specific embodiment provides a method for preparing oxidized dextran:

[0035] Dissolve 5 g (0.03 mol) of dextran in 250 ml of deionized water, add 5 g (0.023 mol) of sodium periodate, and stir continuously for 3.5 h in the dark. Add 3 ml of ethylene glycol to the above solution, continue stirring for 1 h, and obtain oxidized dextran by dialysis and lyophilization.

[0036] Carboxymethyl chitosan was obtained through commercial purchase and had a degree of substitution of 0.85.

[0037] A specific embodiment also provides a method for preparing a CGF solution:

[0038] 10 mL of blood was drawn from a vein and stored in an anticoagulant tube containing sodium heparin. The blood was placed in a centrifuge with physical acceleration and deceleration. The centrifuge was accelerated for 30 seconds to 2700 rpm for 2 minutes, then decelerated to 2400 rpm for 4 minutes, accelerated to 2700 rpm for 4 minutes, accelerated to 3000 rpm for 3 minutes, and finally decelerated for 36 seconds. Two layers were formed in the anticoagulant tube. The lower 1 / 3 of the upper plasma layer was separated to obtain 2 mL of CGF solution.

[0039] The main active component of the extracted CGF, hematopoietic stem cells, was detected by flow cytometry. The results are attached. Figure 1 As shown in the figure, flow cytometry analysis revealed that the extracted CGF contained 0.2%-0.5% hematopoietic stem cells, which is significantly higher than the approximately 0.1% hematopoietic stem cells found in whole blood.

[0040] The main active ingredient TGF-β in the extracted CGF was detected using an ELISA kit. The results are shown in the appendix. Figure 2 As shown. The concentrated growth factor (CGF) samples from three donors (A, B, and C) were quantitatively analyzed using a human TGF-β ELISA assay kit (Wuhan Zhongrun Paleontological Technology Co., Ltd.). Three technical replicates were set for each sample. The average TGF-β concentrations for donor A were 1715.58 ± [47.08] pg / ml, for donor B 1937.53 ± [50.74] pg / ml, and for donor C 1865.31 ± [17.48] pg / ml. The intra-group coefficient of variation (CV) for all results was less than 5%, indicating good reproducibility. The combined data indicate that the stable expression concentration range of TGF-β in CGF is 1715–1937 pg / ml (arithmetic mean of the three groups: 1840 pg / ml).

[0041] Using the above-mentioned raw materials, a specific embodiment provides a method for preparing a wound repair material loaded with concentrated growth factors:

[0042] Carboxymethyl chitosan was dissolved in CGF solution by stirring; oxidized dextran was dissolved in deionized water by stirring; the two were then mixed and stirred evenly for 5-8 minutes to obtain a composite hydrogel loaded with concentrated growth factors. The raw materials used, by weight, are as follows: 1 part carboxymethyl chitosan; 2 parts oxidized dextran; 45 parts CGF solution; 52 parts deionized water. Samples of the CGF-loaded composite hydrogel and a blank carboxymethyl chitosan / oxidized dextran hydrogel are shown below. Figure 3 As shown.

[0043] In the resulting composite hydrogel, the negatively charged network of CMC and the positively charged microdomains of ODEX form an electric double layer structure. This allows for precise anchoring of negatively charged growth factors (such as PDGF with an isoelectric point of 5.2) through electrostatic adsorption. Combined with the steric hindrance effect of the three-dimensional network, the burst release rate of growth factors is reduced to one-third of that of traditional gels, dynamically matching the stage-specific needs of wound healing. Simultaneously, the aforementioned electrostatic adsorption effect ensures the uniform distribution of growth factors within the system. Furthermore, the ODEX-CMC composite system of this invention exhibits shear-thinning properties, allowing for injection filling of deep wound cavities. These characteristics provide a novel solution to resolving the triple contradiction of "activity retention - release regulation - clinical application" for CGF loading materials.

[0044] Verification of the wound-healing effect of the composite hydrogel loaded with concentrated growth factors obtained in the specific implementation method:

[0045] Mice were anesthetized with isoflurane and then shaved. A full-thickness skin defect model was created by punching holes on both sides of the spine using a 6mm sterile skin punch. After recovery, the mice were housed individually, and the day of model creation was considered day 0 of the experiment. Starting the day after model creation, the wound repair material prepared in the specific embodiment was administered to the wound site for 13 consecutive days. During the experiment, photographs of the wound were taken daily using a ruler with a 1mm scale.

[0046] The same concentration of CGF solution was loaded onto carboxymethyl chitosan hydrogel and oxidized dextran hydrogel, respectively, as control groups. Control group 1: blank hydrogel group; Control group 2: 3 parts carboxymethyl chitosan; 45 parts CGF solution; 52 parts deionized water; Control group 3: 3 parts oxidized dextran; 45 parts CGF solution; 52 parts deionized water. The results of mouse wound healing are attached. Figure 4 As shown in the figure, the CGF-loaded composite hydrogel prepared in this invention can effectively promote wound healing in mice, showing a healing-promoting effect as early as day 3, with a significantly higher wound contraction rate than the control group; by day 7, the healing rate of the treatment group was higher than that of the control group; by day 13, the treatment group had basically achieved complete epithelialization, while the blank hydrogel in control group 1 had no significant wound-healing effect. Control groups 2 and 3, with the addition of CGF, had a certain wound-healing effect, but due to the inability to form a hydrogel and the excessively high growth factor release rate, the wound healing effect was worse than that of the treatment group. This indicates that the CGF hydrogel significantly shortens the healing time of full-thickness skin defects by accelerating granulation tissue formation and re-epithelialization, and also promotes hair regeneration.

Claims

1. A composite hydrogel loaded with concentrated growth factors, characterized in that The hydrogel was prepared by loading CGF solution onto an oxidized dextran / carboxymethyl chitosan composite hydrogel. The raw materials used, by weight, are as follows: 1 part carboxymethyl chitosan; 2 parts oxidized dextran; 40-50 parts CGF solution; 50-55 parts deionized water. The degree of oxidation of the highly active aldehyde group in the oxidized dextran is 50%-60%, and the degree of amino substitution of the carboxymethyl chitosan is ≥80%. The main components of the CGF solution include VEGF, PDGF, TGF-β, CD34 + Hematopoietic stem cells and fibrin network matrix.

2. The method of claim 1, wherein the method of preparing the composite hydrogel loaded with the concentrated growth factors is characterized by Includes the following steps: Carboxymethyl chitosan was mixed and stirred with CGF solution to dissolve; oxidized dextran was stirred and dissolved in deionized water; the two were mixed and stirred evenly, and after 5-8 minutes, a composite hydrogel loaded with concentrated growth factors was obtained.

3. The method of claim 2, wherein the concentration of the growth factor is 0.1 to 10 ng / ml. The raw materials used are as follows by weight: 1 part carboxymethyl chitosan; 2 parts oxidized dextran; 45 parts CGF solution; 52 parts deionized water.

4. The method of claim 2, wherein the concentration of the growth factor is 0.1- 10 ng / ml. The preparation method of the oxidized dextran includes the following steps: Dextran was dissolved in deionized water, sodium periodate was added, and the mixture was stirred continuously in the dark. Ethylene glycol was added to the solution and the mixture was stirred continuously. Oxidized dextran was obtained by dialysis and freeze-drying.

5. The method of claim 2, wherein the concentration of the growth factor is about 0.1 to about 10 ng / ml. The method for preparing the CGF solution includes the following steps: Venous blood was collected and centrifuged in an anticoagulant tube. The venous blood formed two layers in the anticoagulant tube. The lower 1 / 3 of the upper plasma layer was separated to obtain the CGF solution.

6. The application of the composite hydrogel loaded with concentrated growth factors as described in claim 1 as a wound repair material.

7. The application of the composite hydrogel loaded with concentrated growth factors as described in claim 1 as a hair regeneration material.

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