Efficient mineralized hydrogel for bone repair as well as preparation method and application of efficient mineralized hydrogel

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to efficient mineralized hydrogel for bone repair as well as a preparation method and application of the efficient mineralized hydrogel. According to the hydrogel for promoting hard bone repair provided by the invention, hydroxybutyl chitosan (HBC) and deferoxamine (DFO) are introduced into a fibrinogen-thrombin / bioglass system, so that the in-vitro mineralization ability of the material is remarkably improved. HBC provides a good adhesion scaffold for osteoblasts and promotes formation of a mineralized matrix; dFO is chelated with iron ions, a hypoxia environment is simulated, expression of osteogenesis related genes is activated, and deposition of mineralized matrixes is further promoted. And more and denser inorganic mineral substances are deposited on the surface of the material through the synergistic effect of the two.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a high-efficiency mineralized hydrogel for bone repair, and a preparation method and application thereof. Background Art

[0002] In bone repair applications, the material should not be toxic to surrounding tissues or induce adverse immune responses; it should have sufficient mechanical strength to support bone tissue regeneration, be able to respond to external stimuli (such as temperature, pH value, etc.) to control its behavior and function in the body; and be able to carry and release bioactive components, such as drugs, proteins, or other growth factors, to promote tissue repair. Traditional hydrogel materials can often only meet some of these requirements, such as biocompatibility or stimulus responsiveness, but may lack in mechanical properties and functionality.

[0003] Compared with cartilage repair, hard bone repair occupies an extremely important position in bone tissue engineering and regenerative medicine. The reason is that the structure and function of hard bones are crucial. They not only support the mechanical needs of the body, but also protect important internal organs. The repair of hard bones (such as long bones, spine, hip bones, etc.) is often more complicated, especially in cases of bone loss, poor fracture healing, bone defects or lesions. The clinical demand for efficient bone repair materials is very urgent. The repair of hard bones depends on the biomineralization process, which forms a hard mineral matrix through the deposition of calcium salts (such as hydroxyapatite) to ensure the hardness and strength of bone tissue. Bone repair materials must not only be able to induce the proliferation and differentiation of bone cells, but also must promote mineralization to achieve the regeneration of functional bones.

[0004] Fibrinogen-thrombin system and bioglass materials are widely used in bone repair and can provide certain biocompatibility and mineralization promotion effects. However, the existing systems are still insufficient in promoting mineralized matrix deposition and osteoblast proliferation. Summary of the invention

[0005] The first aspect of the present invention aims to provide a hydrogel.

[0006] The purpose of the second aspect of the present invention is to provide a method for preparing the hydrogel described in the first aspect of the present invention.

[0007] The purpose of the third aspect of the present invention is to provide an application of the hydrogel described in the first aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a product.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0010] A first aspect of the present invention provides a hydrogel, wherein the hydrogel comprises a thermosensitive hydrogel matrix, an angiogenesis-promoting component, and bioglass.

[0011] Preferably, the thermosensitive hydrogel matrix comprises at least one of hydroxybutyl chitosan (HBC), polyethylene glycol-lactide block copolymer, poloxamer, and polyethylene glycol-polylactic acid block copolymer.

[0012] In some embodiments of the present invention, the thermosensitive hydrogel matrix is ​​hydroxybutyl chitosan.

[0013] Preferably, the angiogenesis-promoting components include deferoxamine (DFO), fibrinogen, and thrombin.

[0014] Preferably, the mass ratio of the thermosensitive hydrogel matrix to fibrinogen, bioglass and deferoxamine in the hydrogel is (20-50):(5-15):(5-15):1.

[0015] Preferably, the ratio of the enzyme activity of thrombin to deferoxamine: mass is (5-15) IU: 1 mg.

[0016] In some embodiments of the present invention, the ratio of the thermosensitive hydrogel matrix, fibrinogen, bioglass, deferoxamine, and thrombin in the hydrogel is (40-60) mg: (10-20) mg: (10-20) mg: 1.6 mg: (10-30) IU. More preferably, it is (45-55) mg: (10-20) mg: (10-20) mg: 1.6 mg: (15-25) IU.

[0017] Preferably, the gel temperature of the thermosensitive hydrogel matrix is ​​10°C to 40°C.

[0018] Further preferably, the gel temperature of the thermosensitive hydrogel matrix is ​​16°C to 38°C.

[0019] More preferably, the gel temperature of the thermosensitive hydrogel matrix is ​​22°C to 37°C.

[0020] Preferably, the gel time of the thermosensitive hydrogel matrix is ​​0.01 min to 60 min.

[0021] The second aspect of the present invention provides a method for preparing the hydrogel according to the first aspect of the present invention, comprising the following steps:

[0022] Component A: Mix fibrinogen with bioglass;

[0023] Component B: mixing the thermosensitive hydrogel matrix, thrombin and deferoxamine;

[0024] Mix components A and B to obtain.

[0025] The third aspect of the present invention provides use of the hydrogel described in the first aspect of the present invention in the preparation of a product for promoting bone tissue repair.

[0026] Preferably, the bone tissue repair is hard bone repair. The hard bone refers to a mineralized connective tissue composed of a large amount of calcium and phosphorus. Its structure is relatively dense and consists of osteocytes, bone matrix and periosteum.

[0027] Preferably, the product includes at least one of biomedical materials and drugs.

[0028] A fourth aspect of the present invention provides a product, wherein the product comprises the hydrogel described in the first aspect of the present invention.

[0029] Preferably, the product comprises pharmaceutically acceptable excipients.

[0030] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, preservatives, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0031] Preferably, the dosage form of the drug includes but is not limited to an injectable dosage form or a dressing.

[0032] Further preferably, the injectable dosage forms include but are not limited to injection solutions, injection solutions, intravenous injection solutions, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0033] Preferably, the product is administered to a mammal.

[0034] Preferably, the mammal comprises a human.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides a hydrogel that promotes bone repair. By introducing hydroxybutyl chitosan (HBC) and deferoxamine (DFO) into a fibrinogen-thrombin / bioglass system, the in vitro mineralization ability of the material is significantly improved. HBC provides a good adhesion scaffold for osteoblasts and promotes the formation of mineralized matrix; DFO activates the expression of osteogenesis-related genes by chelating iron ions and simulating a hypoxic environment, further promoting the deposition of mineralized matrix. The synergistic effect of the two allows more and denser inorganic minerals to be deposited on the surface of the material.

[0037] The combination of HBC and DFO enables the material to exhibit a faster and more efficient mineralization process in in vitro experiments. Compared with single components or traditional mineralized materials, the composite material with the addition of HBC and DFO exhibits more calcium ion deposition and a denser mineralized matrix in the mineralization experiment, which is clearly confirmed in the osteoblast mineralization experiment, greatly improving the application prospects of the material. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the in vitro mineralization result of the hydrogel of the present invention.

[0039] Figure 2 This is the qualitative result of Alizarin red staining of the hydrogel of the present invention.

[0040] Figure 3 This is the quantitative analysis result of Alizarin red staining of the hydrogel of the present invention.

[0041] Figure 4 This is the qualitative result of ALP staining of the hydrogel of the present invention.

[0042] Figure 5 This is the quantitative analysis result of ALP staining of the hydrogel of the present invention. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0044] Example 1 Preparation method of DFO@FT / BG / HBC hydrogel

[0045] Ultrapure water was used to prepare solutions of the following concentrations: 10 mg / mL fibrinogen solution (F), 50 IU / mL thrombin solution (T), 100 mg / mL hydroxybutyl chitosan solution (HBC), and 6 μmol / mL deferoxamine hydrochloride solution (DFO).

[0046] 1.6 mL of fibrinogen solution (F) was mixed evenly with 15 mg of bioglass (BG) to obtain component A; 0.4 mL of thrombin solution, 0.5 mL of hydroxybutyl chitosan solution (HBC) and 0.5 mL of deferoxamine hydrochloride solution (DFO) were mixed to obtain component B.

[0047] The A and B components were fully mixed to obtain DFO@FT / BG / HBC hydrogel.

[0048] Comparative Example 1: Preparation method of FT / BG hydrogel

[0049] Ultrapure water was used to prepare the following solutions: 10 mg / mL fibrinogen solution (F), 50 IU / mL thrombin solution (T).

[0050] 1.6 mL of fibrinogen solution (F) was mixed evenly with 15 mg of bioglass (BG) to obtain component A; 0.4 mL of thrombin solution and 1.0 mL of ultrapure water were mixed to obtain component B.

[0051] The A and B components were fully mixed to obtain FT / BG hydrogel.

[0052] Comparative Example 2: Preparation method of FT / BG / HBC hydrogel

[0053] Ultrapure water was used to prepare solutions of the following concentrations: 10 mg / mL fibrinogen solution (F), 50 IU / mL thrombin solution (T), and 100 mg / mL hydroxybutyl chitosan solution (HBC).

[0054] 1.6 mL of fibrinogen solution (F) was mixed evenly with 15 mg of bioglass (BG) to obtain component A; 0.4 mL of thrombin solution was mixed with 1.0 mL of hydroxybutyl chitosan solution (HBC) to obtain component B.

[0055] The A and B components were fully mixed to obtain FT / BG / HBC hydrogel.

[0056] Comparative Example 3DFO@FT / BG hydrogel preparation method

[0057] Ultrapure water was used to prepare the following solutions: 10 mg / mL fibrinogen solution (F), 50 IU / mL thrombin solution (T), and 6 μmol / mL deferoxamine hydrochloride solution (DFO).

[0058] 1.6 ml of fibrinogen solution (F) was mixed evenly with 15 mg of bioglass (BG) to obtain component A; 0.4 ml of thrombin solution was mixed with 1.0 ml of deferoxamine hydrochloride solution (DFO) to obtain component B.

[0059] The AB components were fully mixed to obtain DFO@FT / BG hydrogel.

[0060] Effect Example 1 In vitro mineralization results

[0061] 2 mL of each group of composite hydrogels (10 mm × 6 mm) were placed in a 15 mL centrifuge tube containing 10 mL of simulated body fluid, and then the centrifuge tube was incubated in a shaker at 37°C and 60 rpm / min, and the simulated body fluid (Yuanye Bio, R24029) was replaced every 2 days. After 7 days of incubation, the simulated body fluid was removed, and the residual simulated body fluid on the surface of the hydrogel was washed with deionized water. After freeze-drying, the mineralized nodules were observed under an electron microscope.

[0062] The results are as follows Figure 1 As shown. In the in vitro mineralization experiment, the surface of the double network hydrogel (DFO@FT / BG / HBC) prepared in Example 1 was able to deposit more inorganic salt components, showing a more adequate mineralization ability. This enhanced mineralization property directly proves the positive effect of the material on osteoblast activity, helps to induce new bone formation, and especially exhibits excellent osteoinduction properties during bone repair and regeneration. This result fully demonstrates the innovative advantages of the present invention in promoting bone tissue regeneration. Effect Example 2 Osteoblast Mineralization Results

[0063] 1) Alizarin red staining

[0064] The same concentration of material extract (100 mg / mL extraction concentration, complete medium extraction) was added to osteoblasts and cultured until the mineralization induction period, and then the cells were fixed with 4wt% paraformaldehyde for 10 minutes and rinsed with PBS. Then 2wt% Alizarin Red S staining solution (pH 4.1-4.3) was added and incubated at 37°C for 30 minutes. After staining, the cells were repeatedly washed with distilled water until the background was free of staining, and the results were observed under a microscope and statistically analyzed.

[0065] The results are as follows Figure 2 , 3 As shown, the hydrogel of Example 1 had the best mineralization result.

[0066] 2) ALP staining

[0067] After adding the same concentration of material extract (100 mg / mL extraction concentration, complete medium extraction) to osteoblasts and culturing until the induction period, fix with 4wt% paraformaldehyde and rinse with PBS. Then add ALP staining solution and incubate at room temperature for 15 minutes until a blue deposit is formed, indicating ALP activity. After washing, observe the stained area under a microscope.

[0068] Osteoblasts have alkaline phosphatase on their cell membranes, and alkaline phosphatase staining can be used to determine the presence and activity of osteoblasts. Figure 4 ,5 As shown, the hydrogel of Example 1 has a stronger promoting effect on osteoblasts.

Claims

1. A hydrogel, characterized in that: The hydrogel comprises a thermosensitive hydrogel matrix, an angiogenesis-promoting component, and bioglass.

2. The hydrogel according to claim 1, characterized in that: The thermosensitive hydrogel matrix comprises at least one of hydroxybutyl chitosan, polyethylene glycol glycolide lactide block copolymer, poloxamer, and polyethylene glycol polylactic acid block copolymer; Preferably, the thermosensitive hydrogel matrix is ​​hydroxybutyl chitosan.

3. The hydrogel according to claim 2, characterized in that: The pro-angiogenic components include deferoxamine, fibrinogen, and thrombin.

4. The hydrogel according to claim 3, characterized in that: The mass ratio of the thermosensitive hydrogel matrix to fibrinogen, bioglass and deferoxamine in the hydrogel is (20-50):(5-15):(5-15):

1.

5. The hydrogel according to claim 4, characterized in that: The ratio of the enzyme activity of thrombin to deferoxamine to mass is (5-15) IU: 1 mg.

6. The hydrogel according to claim 1, characterized in that: The gel temperature of the hydrogel is 10°C to 40°C.

7. The method for preparing the hydrogel according to claims 1 to 6, comprising the following steps: Component A: Mix fibrinogen with bioglass; Component B: mixing the thermosensitive hydrogel matrix, thrombin and deferoxamine; Mix components A and B to obtain.

8. Use of the hydrogel according to claims 1 to 6 in the preparation of products promoting bone tissue repair; Preferably, the bone tissue repair is hard bone repair.

9. The use according to claim 8, characterized in that: The product includes at least one of biomedical materials and drugs.

10. A product, characterized in that: The product comprises the hydrogel according to claims 1 to 6; Preferably, the product comprises pharmaceutically acceptable excipients.

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