Platelet-like gel repairing preparation, preparation method thereof and application of platelet-like gel repairing preparation in promoting wound healing

Abstract

The invention provides a platelet-like gel repair preparation, a preparation method thereof and application of the platelet-like gel repair preparation in promoting wound healing. The platelet-like gel repair preparation comprises the following growth factors: PDGF-BB, EGF, FGF-2, SDF-1alpha, VEGF and IGF-1. The platelet-like gel repair preparation has an excellent effect of promoting wound repair, and provides a new choice for clinical wound treatment.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a platelet-imitation gel repair preparation and a preparation method thereof and an application of the preparation method for promoting wound healing. Background Art

[0002] Platelets play an important role in physiological wound healing, angiogenesis, and coagulation. In response to trauma, platelets are activated and migrate to the site of trauma. Platelet surface glycoprotein GP IIb / IIIa binds to fibrinogen in plasma, causing platelets to further aggregate. Fibrinogen forms fibrin under the action of thrombin to stop bleeding from the wound. Then the substances in the lysosomes play an antibacterial role. Finally, a variety of active substances in alpha granules are released, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), stromal cell-derived factor-1α (SDF-1α), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1), etc., so that platelets can promote wound healing, epidermal, vascular, and nerve regeneration [Platelet Derivatives and the Immunomodulation of Wound Healing], and are biologically active mediators shared by other cells (endothelial cells, macrophages, neutrophils, and fibroblasts) involved in the regeneration cascade. The action of thrombin and fibrin causes platelets, white blood cells, etc. to form a gel at the wound site, which stops bleeding and exerts an antibacterial effect.

[0003] Platelet gel (PG) plays an important role in treating chronic wounds that are difficult to heal, accelerating wound healing of diabetic foot and venous ulcers, and promoting cartilage formation in osteoarthritis. However, the production of platelet gel requires extracting platelets from the patient's own blood and then processing them in vitro, which is not only time-consuming and labor-intensive, but also causes trauma to the patient. Summary of the invention

[0004] The purpose of the present invention is to provide a platelet-like gel repair preparation for promoting wound healing.

[0005] The platelet-imitation gel repair preparation of the present invention comprises growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF and IGF-1; the weight ratio of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF and IGF-1 is (10-1000): (10-200): (1-100): (0.5-100): (0.01-50): (0.1-50).

[0006] Preferably, the preparation is a solution, wherein the final concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are (10-1000) ug / ml, (10-200) ug / ml, (1-100) ug / ml, (0.5-100) ug / ml, (0.01-50) ug / ml, and (0.1-50) ug / ml, respectively.

[0007] Further preferably, the concentrations of the PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / ml, 200ug / ml, 1ug / ml, 100ug / ml, 0.01ug / ml, and 0.1ug / ml, respectively; or, the concentrations of the PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 1000ug / ml, 10ug / ml, 100ug / ml, 0.5ug / ml, 50ug / ml, and 50ug / ml, respectively.

[0008] More preferably, the preparation further comprises fibrinogen and thrombin, and the final concentrations of fibrinogen and thrombin are (8-12) mg / ml and (3-7) mg / ml. Particularly preferably, in the preparation, the final concentrations of fibrinogen and thrombin are 10 mg / ml and 5 mg / ml.

[0009] Preferably, the preparation is a hydrogel, which is a hydrogel made of carbomer and triethanolamine as a matrix and added with a growth factor solution; the final content of the growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are (10-1000) ug / g, (10-200) ug / g, (1-100) ug / g, (0.5-100) ug / g, (0.01-50) ug / g, and (0.1-50) ug / g, respectively.

[0010] Further preferably, the weight ratio of carbomer to triethanolamine is 2:2.7; or, the final contents of the growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / g, 200ug / g, 5ug / g, 1ug / g, 0.1ug / g, and 0.1ug / g, respectively.

[0011] The aforementioned solution or growth factor solution uses physiological saline as a solvent.

[0012] The present invention also provides a method for preparing the aforementioned platelet-mimicking gel repair preparation for promoting wound healing, the steps of which are as follows: dissolving growth factors in water, adding fibrinogen and thrombin, and mixing to form a solution; or mixing the growth factor solution with a hydrogel matrix to form a hydrogel.

[0013] The present invention also provides the use of the platelet-imitation gel repair preparation in a medicine for promoting wound healing.

[0014] The growth factors of the present invention are defined as follows:

[0015] PDGF-BB: human platelet-derived growth factor-BB;

[0016] EGF: epidermal growth factor;

[0017] FGF-2: basic fibroblast growth factor;

[0018] SDF-1α: stromal cell-derived factor-1α;

[0019] VEGF: vascular endothelial growth factor;

[0020] IGF-1: insulin-like growth factor 1.

[0021] Fibrinogen: It is a normal component in human blood. It is a glycoprotein (22v2) synthesized and secreted by liver cells. It is an important protein involved in the coagulation and hemostasis process.

[0022] Thrombin: It is a white to off-white non-crystalline substance, available as freeze-dried powder.

[0023] The preparation method of the platelet-imitation gel repair preparation of the present invention is simple, does not need to be extracted from platelets, has an excellent effect of promoting wound healing, and has a good prospect for clinical application.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Wound repair effect diagram.

[0027] Figure 2 Wound repair effect diagram. DETAILED DESCRIPTION

[0028] The various experimental raw materials and reagents used below are all commercially available, among which human fibrinogen was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; thrombin lyophilized powder was purchased from Zhejiang Hangkang Pharmaceutical Co., Ltd.

[0029] Example 1 Platelet-like gel repair preparation of the present invention

[0030] Take physiological saline to prepare PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents into a solution, and add thrombin to the prepared liquid to obtain. The final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / ml, 200ug / ml, 1ug / ml, 100ug / ml, 0.01ug / ml, and 0.1ug / ml, respectively, and the final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively.

[0031] The repair preparation of the present invention is a preparation used after temporary configuration, and can be prepared into two raw material packages before use: raw material package 1 contains a solution of growth factors and fibrinogen, and raw material package 2 is thrombin, and the two can be mixed when used.

[0032] Example 2 Platelet-like gel repair preparation of the present invention

[0033] Add 2g of carbomer to 80ml of distilled water, stir well to fully swell, add 2.7g of triethanolamine while stirring to make a hydrogel matrix. Dissolve PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 in 2ml of normal saline, add to the gel matrix while stirring, add distilled water to 100g, stir well, and the final content of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 in the final product is 10ug / g, 200ug / g, 5ug / g, 100ug / g, 0.01ug / g, and 0.1ug / g, respectively.

[0034] Example 3 Platelet-like gel repair preparation of the present invention

[0035] Take physiological saline to prepare PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents into a solution, and add thrombin to the prepared liquid to obtain. The final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are: 1000ug / ml, 10ug / ml, 100ug / ml, 0.5ug / ml, 50ug / ml, and 50ug / ml, and the final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively.

[0036] The beneficial effects of the present invention are described below by way of test examples:

[0037] Experimental Example 1 Repairing Effect on Animal Skin Wounds

[0038] 1. Effects of different types of growth factors on repair effects

[0039] 1. Experimental methods

[0040] 1. Product preparation

[0041] Product 1: Take physiological saline to prepare PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents into a solution, and add thrombin to the prepared liquid. The final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / ml, 200ug / ml, 1ug / ml, 100ug / ml, 0.01ug / ml, and 0.1ug / ml, respectively, and the final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively. This product should be used on the affected area immediately after preparation.

[0042] Product 2:

[0043] Add 2g of carbomer to 80ml of distilled water, stir well to fully swell, add 2.7g of triethanolamine under stirring to make a hydrogel matrix. Dissolve PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 in 2ml of normal saline, add to the gel matrix under stirring, add distilled water to 100g, stir well, and the final content of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / g, 200ug / g, 5ug / g, 1ug / g, 0.1ug / g, and 0.1ug / g, respectively.

[0044] Product 3:

[0045] The preparation method of this product is the same as that of product 2, except that the types of raw materials are reduced and only three cell growth factors are used, with final concentrations of EGF, SDF-1α, and VEGF of 200ug / g, 1ug / g, and 0.1ug / g, respectively.

[0046] 2. Experimental Grouping

[0047] SPF-grade 2.5-3kg New Zealand rabbits were used as experimental animals and divided into product group 1 (using product 1), product group 2 (using product 2), product group 3 (using product 3) and a control group (not using any drugs).

[0048] 3. Modeling and drug administration method

[0049] Rabbits were anesthetized by injecting 0.5% sodium pentobarbital at a dose of 6 ml / kg through the ear vein, and then 4*4 cm of skin was excised from the back of the rabbits. Products 1, 2, and 3 were applied to the skin wounds, respectively, once a day. The growth factor component in product group 1 was mixed with fibrinogen and thrombin before use and then applied to the wounds. The healing of the rabbit skin was observed every 3-5 days.

[0050] (II) Experimental results

[0051] 55 days after skin excision, the wounds of the four groups of animals healed as Figure 1 As shown. It can be seen that the wound surface of the experimental animals in the control group has huge fibrous connective tissue (indicated by the red arrow), the skin structure of the experimental animals of product 3 is intact but not covered with much hair, the skin structure of the experimental animals using products 1 and 2 is intact and the wound is covered with hair, the effect of product 1 is better than that of product 2, the wound skin of the experimental animals of product 1 heals and the hair almost covers the entire wound surface. The medicine of the present invention has a good skin repair effect, and the wound repair effect of product 1 is better than that of product 2.

[0052] The results show that the platelet-like gel repair preparation (Product 1 and Product 2) prepared by using the growth factor solution of the present invention (including PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1) with fibrinogen and thrombin or with a hydrogel matrix can repair the wound surface better, while the repair effect is poor when using other growth solutions.

[0053] 2. Effects of different concentrations of growth factors on repair effects

[0054] 1. Experimental methods

[0055] 1. Product preparation

[0056] Product 1: Take physiological saline to prepare PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents into a solution, and add thrombin to the prepared liquid. The final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / ml, 200ug / ml, 1ug / ml, 100ug / ml, 0.01ug / ml, and 0.1ug / ml, respectively, and the final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively. This product should be used on the affected area immediately after preparation.

[0057] Product 4: The preparation and use of this product are the same as those of Product 1, except that the dosage and proportion of the raw materials are adjusted so that the final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are: 1000ug / ml, 10ug / ml, 100ug / ml, 0.5ug / ml, 50ug / ml, and the final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively. This product should be used on the affected area immediately after preparation.

[0058] 2. Experimental Grouping

[0059] SPF-grade 2.5-3kg New Zealand rabbits were used as experimental animals and divided into product group 1 (using product 1) and product group 4 (using product 4).

[0060] 3. Modeling and drug administration method

[0061] Rabbits were anesthetized by injecting 0.5% sodium pentobarbital at a dose of 6 ml / kg through the ear vein, and then 4*4 cm of skin was excised from the back of the rabbits. The growth factor components in products 1 and 4 were mixed with fibrinogen and thrombin before use and applied to the wound.

[0062] (II) Experimental results

[0063] The healing of the rabbit skin was observed every 3-5 days. Figure 2 As shown:

[0064] After using the drug in product group 1, it was found that the wound surface was eventually covered by the skin, but in the later stage, the skin still had contracture and scars were visible.

[0065] After using the drug in product group 4, it was found that the skin matured within 10 days, was basically healed within 20 days, hair could grow on the wound surface within 30 days, and there were no obvious scars.

[0066] The results show that the platelet-like gel repair preparations (Product 1 and Product 4) made from the two growth factor solutions of the present invention (including PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1) and fibrinogen and thrombin can effectively repair the wound surface, among which the repair effect of Product 4 is particularly excellent, significantly superior to that of Product 1.

[0067] In summary, the experiment confirms that the platelet-like gel repair preparation made from the specific growth factor solution of the present invention has a very good effect in promoting wound repair, and provides a new option for clinical wound repair.

Claims

1. A platelet-like gel repair preparation, characterized in that: The preparation includes growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF and IGF-1; the weight ratio of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF and IGF-1 is (10-1000): (10-200): (1-100): (0.5-100): (0.01-50): (0.1-50).

2. The preparation according to claim 1, characterized in that: The preparation is a solution, wherein the final concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are (10-1000) ug / ml, (10-200) ug / ml, (1-100) ug / ml, (0.5-100) ug / ml, (0.01-50) ug / ml, and (0.1-50) ug / ml, respectively.

3. The preparation according to claim 2, characterized in that: The concentrations of the PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / ml, 200ug / ml, 1ug / ml, 100ug / ml, 0.01ug / ml, and 0.1ug / ml, respectively; or, the concentrations of the PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 1000ug / ml, 10ug / ml, 100ug / ml, 0.5ug / ml, 50ug / ml, and 50ug / ml, respectively.

4. The preparation according to claim 2, characterized in that: The preparation also includes fibrinogen and thrombin, and the final concentrations of fibrinogen and thrombin are (8-12) mg / ml and (3-7) mg / ml.

5. The preparation according to claim 4, characterized in that: In the preparation, the final concentrations of fibrinogen and thrombin were 10 mg / ml and 5 mg / ml.

6. The preparation according to claim 1, characterized in that: The preparation is a hydrogel, which is prepared by adding a growth factor solution into a hydrogel prepared by taking a hydrogel prepared by carbomer and triethanolamine as a matrix; the final contents of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF and IGF-1 in the growth factor solution are (10-1000) ug / g, (10-200) ug / g, (1-100) ug / g, (0.5-100) ug / g, (0.01-50) ug / g and (0.1-50) ug / g respectively.

7. The preparation according to claim 8, characterized in that: The weight ratio of the carbomer to triethanolamine is 2:2.7; or, the final contents of the PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / g, 200ug / g, 5ug / g, 1ug / g, 0.1ug / g, and 0.1ug / g, respectively.

8. The preparation according to any one of claims 2 to 7, characterized in that: The solution or growth factor solution uses physiological saline as a solvent.

9. A method for preparing the preparation according to any one of claims 1 to 8, characterized in that: The steps are as follows: dissolving growth factors and fibrinogen in water, adding thrombin, and mixing to form a solution; or mixing the growth factor solution with a hydrogel matrix to form a hydrogel.

10. Use of the preparation according to any one of claims 1 to 8 in the preparation of a medicament for promoting wound healing.

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