A method of preparing and use of an injectable high-strength bone tissue adhesive

Abstract

The application discloses a preparation method and application of an injectable high-strength bone tissue adhesive, and belongs to the technical field of biomaterials. The hydrogel tissue adhesive comprises micron or nanometer organic particles carrying amino groups, water-soluble polymer which can be chemically crosslinked with the amino groups, and micron or nanometer inorganic particles which can promote osteogenesis; under the action of water, the hydrogel tissue adhesive can form a continuous and porous particle network, and the chemical reaction between the particles and the polymer can provide good adhesive performance, and the adhesive strength can reach 5Mpa. In addition, the non-covalent interaction between the particles can make the material better stay in the defect site, and realize the filling of the bone defect. The inorganic component in the material component of the application can release ions which can promote osteogenesis, and promote the repair of the bone defect. In addition, the hydrogel tissue adhesive can also be used as a bone wound hemostatic material and a cell scaffold material, and has a wide application prospect in the field of bone repair.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and particularly relates to a method for preparing and using an injectable high-strength bone tissue adhesive. Background Technology

[0002] Fracture treatment usually requires surgical refixation of the fracture site to achieve bone regeneration and ultimately fracture healing. Traditional fracture fixation methods include the use of metal plates, pins, screws, etc. However, the materials implanted by traditional methods are usually non-degradable and require a second surgery for removal. There are also risks of infection of surrounding damaged tissues and osteoporosis at the wound site caused by stress shielding, which can eventually lead to refracture. In addition, traditional methods cannot effectively fix comminuted fractures or periarticular fractures. Polymer-based tissue adhesives are considered an effective solution for fracture fixation. Biodegradable polymer adhesives do not require a second surgery to remove the fixation device and reduce the risk of infection and other complications. Bone adhesives can effectively fix small bone fragments such as comminuted fractures. However, the current tissue adhesive design based on in-situ chemical cross-linking reaction still faces great challenges in achieving effective bone tissue adhesion in vivo; (1) the fracture site is usually in a liquid environment of continuous bleeding and tissue fluid, so liquid molecules will hinder the effective contact between the adhesive and the tissue, making it difficult for the adhesive to adhere effectively; (2) bone tissue itself has high strength and is usually in a continuous stress environment, so the bonding interface and materials are prone to yielding and failure. In summary, developing biomaterials with high tissue adhesion strength, tailored to the physiological characteristics of bone tissue, is an urgent problem to be solved in the development of bone tissue bonding materials.

[0003] Currently, commonly used bone adhesives in clinical practice can be divided into two categories: natural material bone adhesives and synthetic material bone adhesives. Natural polymers, derived from nature, have been widely explored for medical applications, such as bone adhesives, sealants, and hemostatic agents. Compared to synthetic adhesives, adhesives composed of natural polymers generally exhibit excellent biocompatibility, degradability, and non-immunogenicity. However, they have lower mechanical strength (e.g., fibrin glue, bio-glue, gelatin-based materials). Synthetic polymers are man-made polymers prepared through chemical reactions. Compared to natural polymers, they generally exhibit higher mechanical strength, allowing them to withstand greater loads. However, their biocompatibility is usually inferior to that of natural polymers, and they often generate heat during polymerization, which may negatively impact surrounding tissues. Poor degradability is also a problem with synthetic adhesives. Common synthetic bone adhesives include polyurethane, polycyanoacrylate, and polymethyl methacrylate.

[0004] Furthermore, all of the above-mentioned bone adhesive precursors are in a liquid state and are easily flowable, making it difficult for the material to remain at the desired site. In practical applications, the moist environment of wounds or defects can also dilute the adhesive precursors, resulting in poor adhesion. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention aims to provide a method for preparing and using an injectable, malleable, high-strength bone tissue adhesive based on a simple mixture of micro / nano particles and polymers. The initial state of the bone adhesive is an injectable, self-healing gel that ensures filling of bone defects under physiological conditions. When the gel comes into contact with tissue, adhesion to the defective tissue is achieved through a covalent chemical reaction between the polymer, particles, and tissue. Furthermore, the inorganic components possess excellent osteogenic activity, effectively promoting the regeneration of bone defects and achieving physical adhesion of fracture defects while simultaneously promoting bone tissue healing.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a hydrogel tissue adhesive composed of organic / inorganic particles and polymer chains. The hydrogel tissue adhesive includes micron- or nano-sized organic particles carrying amino groups, a water-soluble polymer that undergoes a chemical cross-linking reaction with the amino groups, and micron- or nano-sized inorganic particles with osteogenic properties. The mass ratio of organic particles, inorganic particles, and water-soluble polymer is 1-20:1:0.1-5. The volume percentage of particles in the hydrogel tissue adhesive is 10-80%. The organic / inorganic particles are formed by mixing organic and inorganic particles, or by organic particles encapsulating inorganic particles to form a core-shell structured composite particle. Under the action of water, the hydrogel tissue adhesive can form a continuous, porous particle network through covalent interactions and / or reversible non-covalent interactions between particles, endowing the hydrogel tissue adhesive with injectability, shapeability, and self-healing properties.

[0008] Based on the above technical solutions, further, reversible non-covalent interactions include electrostatic adsorption, hydrophilic-hydrophobic interactions, and hydrogen bonding.

[0009] Based on the above technical solution, the particle size is further specified as 1 nm to 50 μm; and the particle potential under neutral conditions is -30 mV to +30 mV.

[0010] Based on the above technical solution, the volume percentage of particles in the hydrogel tissue adhesive is further 20-60%.

[0011] Based on the above technical solution, the composition of the micron or nano-sized organic particles carrying amino groups is one or more of gelatin, chitosan, polylysine, polyethyleneimine, albumin, protamine, fibroin, silk fibroin, and silkworm epidermal protein.

[0012] Based on the above technical solution, further, the molecular weight of the components of the micron or nano-sized organic particles carrying amino groups is 0.5-100 kDa.

[0013] Based on the above technical solution, further, the micron or nano-sized inorganic particles with osteogenic promoting effect account for ≥10 wt% of the dry weight percentage of the hydrogel tissue adhesive; the inorganic particles are calcium phosphate, calcium carbonate, calcium silicate, or bioglass inorganic non-metallic material particles; the morphology is one or a combination of two or more of the following: needle-like, fibrous, granular, and blocky.

[0014] Based on the above technical solution, the inorganic particles are further selected from one or more of the following: silica particles, tricalcium phosphate, calcium phosphate, calcium carbonate, nano-clay particles, calcium silicate, and bioglass particles.

[0015] Based on the above technical solution, the inorganic particles are further described as hydroxyapatite.

[0016] Based on the above technical solution, the inorganic particles are further modified with amino groups on their surface.

[0017] Based on the above technical solution, the process of modifying the surface of inorganic particles with amino groups is as follows: inorganic particles are added to n-hexane to obtain a uniform dispersion of 1-500 mg / mL, and then 3-aminopropyltriethoxysilane that reacts with the hydroxyl groups on the surface of inorganic particles is added. The mixture is stirred at 50-80℃ for 4-12 h, and then freeze-dried to obtain aminated inorganic particles.

[0018] Based on the above technical solution, further, the active groups of the water-soluble polymer that undergoes chemical cross-linking reaction with the amino groups include, but are not limited to, the following groups: succinimide, aldehyde, maleimide, epoxy, and carboxyl.

[0019] Based on the above technical solution, the backbone of the water-soluble polymer that undergoes chemical cross-linking reaction with the amino groups is one or more of polyethylene glycol, polyacrylic acid, block polyether F-127, block polyether F-108, sodium alginate, hyaluronic acid, chondroitin sulfate, dextran, cellulose, and agarose.

[0020] Based on the above technical solution, the water-soluble polymer is further defined as linear, multi-arm, or hyperbranched, with a molecular weight of 0.5-100 kDa. The water-soluble polymer accounts for 10-50 wt% of the dry weight of the hydrogel tissue adhesive, and the molar ratio of amino groups to reactive groups in the polymer chain is 0.1-100.

[0021] The present invention also provides a method for preparing the above-mentioned hydrogel tissue adhesive, comprising the following two methods:

[0022] Method 1: Includes the following steps:

[0023] (1) Preparation of micron or nano-sized organic particles carrying amino groups:

[0024] a) When the particle size is greater than 5 μm, the preparation process is as follows: Dissolve the raw material in water at 20℃~60℃ to obtain an aqueous solution with a concentration of 1~100 mg / mL, add a crosslinking agent, stir and crosslink at room temperature for 1~20 h, remove the remaining crosslinking agent, and prepare microsphere particles by spray drying.

[0025] b) When the particle size is 1nm-5μm and the raw materials are gelatin, polylysine, polyethyleneimine, albumin, and protamine, the raw materials are dissolved in water at 20℃-60℃ to obtain an aqueous solution with a concentration of 1-100mg / mL; the pH of the aqueous solution is adjusted to 9-14, and 1-10 times the volume of the aqueous solution of a polar organic solvent is added dropwise to the above aqueous solution. After stirring for 5-60min, a crosslinking agent is added, and the mixture is stirred at room temperature for 1-20h for crosslinking. The remaining crosslinking agent is removed, and after washing, a micro / nano particle dispersion is obtained. After freeze-drying, particle powder is obtained.

[0026] c) When the particle size is 1nm-5μm and the raw material is chitosan, the preparation process is the same as step b), the only difference being that the pH of the aqueous solution is adjusted to 3-5.

[0027] d) When the particle size is 1nm-5μm and the raw material is silk fibroin, firstly, natural silkworm cocoons are degummed to obtain silk fibroin fibers, which are then dissolved in an aqueous solution of lithium bromide or an aqueous solution of calcium chloride / ethanol to obtain a regenerated silk fibroin solution with a concentration of 0.01-0.1g / mL. The solution is then freeze-dried under vacuum to obtain soluble freeze-dried silk fibroin, and then the particles are synthesized according to step b).

[0028] (2) Preparation of hydrogel tissue binder:

[0029] The micron or nano-sized organic particles carrying amino groups prepared in step (1), the water-soluble polymer that undergoes a chemical cross-linking reaction with the amino groups, and the micron or nano-sized inorganic particles that promote osteogenic activity are mixed evenly to obtain the final product.

[0030] Method 2: Includes the following steps:

[0031] 1) Preparation of core-shell structured composite particles:

[0032] Inorganic particles serving as the core layer are ultrasonically dispersed in an aqueous solution containing dissolved organic polymers for the outer shell layer. The concentration of the outer shell material is 1–100 mg / mL, and the concentration ratio of the core material to the outer shell material is 1:2–1:50. The pH is adjusted to 9–14. A polar organic solvent is added dropwise to the above aqueous solution, with the volume of the added polar organic solvent being 1–10 times the volume of the aqueous solution. After stabilizing for 10–30 min, a crosslinking agent is added, and the crosslinking reaction is carried out at room temperature for 1–12 h to obtain a suspension of micro / nano particles with a core-shell structure. The remaining crosslinking agent is removed, and after washing, a core-shell particle dispersion is obtained. After drying, a composite particle powder with a core-shell structure is obtained.

[0033] 2) Preparation of hydrogel tissue binder:

[0034] The core-shell composite particles prepared in step 1) are mixed evenly with a water-soluble polymer that has undergone a chemical cross-linking reaction with amino groups to obtain the final product.

[0035] Based on the above technical solution, further, the polar organic solvent is one or a mixture of two or more of methanol, ethanol, isopropanol, butanol, acetone, acetonitrile, and tetrahydrofuran; the crosslinking agent is one or a combination of two or more of carbodiimide / N-hydroxysuccinimide, glyceraldehyde, glutaraldehyde, formaldehyde, paraformaldehyde, succinaldehyde, and genipin.

[0036] Another aspect of the present invention provides a method for using the above-mentioned hydrogel tissue adhesive, comprising the following steps: mixing the hydrogel tissue adhesive and water evenly, repeatedly blowing and agitating the mixture 5 to 25 times through a Luer adapter syringe to obtain an injectable gel, and then squeezing it out.

[0037] Based on the above technical solution, the mass ratio of hydrogel tissue adhesive to water is further 0.1 to 1:1.

[0038] In another aspect, the present invention provides the application of the above-mentioned hydrogel tissue adhesive as a bone tissue adhesive. The hydrogel tissue adhesive is mixed with water to obtain an injectable gel, which is then squeezed out to the site of bone fragments or around an implanted device. After the reaction and curing, it forms an adhesion to the bone or a seal for bleeding from the wound. The curing time is 1 to 600 seconds, and the adhesion strength is 10 kPa to 5 MPa.

[0039] In another aspect, the present invention provides the application of the above-mentioned hydrogel tissue adhesive as a drug carrier or cell carrier, wherein the drug or cells, the hydrogel tissue adhesive and water are mixed evenly and extruded to the wound site, thereby achieving the effect of drug release.

[0040] Based on the above technical solution, the drug components are further defined as one or a combination of two or more of the following: vitamins, amino acids, mineral elements, microecological regulators, growth factors, small molecule drugs, protein macromolecule drugs, nucleic acid drugs, antibiotics, hormones, anesthetics, antiviral drugs, antibacterial drugs, anticancer drugs, and immunomodulatory drugs, which are applied to the repair and filling of trauma or defects in bone tissue, cartilage tissue, muscles, and blood vessels.

[0041] The present invention has the following beneficial effects:

[0042] 1. Fractures, as one of the most common traumatic injuries, endanger the health of tens of millions of people worldwide. Severe comminuted fractures significantly increase surgical difficulty, healing time, and the incidence of surgical failure. Traditional fixation methods often use metal plates, pins, and screws to stabilize the fracture. However, these non-degradable devices require removal through a secondary surgery. Furthermore, metal fixation devices can increase the risk of wound infection, tissue damage, and refracture. In particular, traditional fixation is ineffective in treating comminuted fractures or periarticular fractures, and bone fragments are generally not preserved during surgery. The injectable, biodegradable bone adhesive proposed in this invention has a larger contact area with bone tissue, thereby reducing the risk of secondary injury due to stress concentration; it can also achieve the bonding and fixation of bone fragments, and the osteogenic components in the adhesive can promote new bone formation. Simultaneously, it can also be used as a hemostatic material for bone wounds and a cell scaffold material.

[0043] 2. The present invention is a bone filler material with adhesive properties composed of micro / nano particles and polymers. The particles and polymers can undergo covalent reactions. When not covalently cross-linked, the material has shear thinning and self-healing characteristics due to the non-covalent interactions between the particles. This can ensure the smooth extrusion of the material. This characteristic makes it have broad application prospects in the fields of bone adhesive materials, hemostatic materials and cell scaffolds.

[0044] 3. The bone bonding material prepared by this invention possesses excellent properties such as injectability, self-healing, and plasticity. Furthermore, it can achieve high mechanical strength and structural stability through covalent cross-linking between particles and polymers, forming a hydrogel. By controlling the mass fraction of the gel, the storage modulus can be adjusted between 10 and 500 kPa, far exceeding that of traditional particulate gels formed through physical interactions, while ensuring the structural integrity and high mechanical strength of the gel within the body. When used for bone wound hemostasis, bone filling, and bone tissue bonding, the micro- and nano-particles in the material components of this invention allow the material to better adhere to the defect site. Traditional polymer-based adhesives, due to their flow characteristics and humid environments, tend to flow easily on tissue surfaces and cannot stably form at the desired location, leading to reduced filling effect or adhesive failure. In this invention, the particulate components exhibit non-covalent interactions, making the hydrogel not a readily flowing liquid before polymerization but a colloid with shear-thinning properties. After the material is extruded to the desired location, the covalent reaction between the material components allows the material to stabilize at the defect site and form a strong adhesive interface. Currently, the adhesive strength of clinically bonded fibrin glue is around 17 kPa; the adhesive strength of polyethylene glycol adhesive Coseal is around 30 kPa, while the adhesive strength of this invention can reach 200 kPa, which is far higher than that of currently commercially available fibrin glue and polyethylene glycol adhesive.

[0045] 4. The material of this invention exhibits excellent osteogenic effects when used as a bone adhesive. Firstly, the inorganic components in the material release ions that promote osteogenic growth. Furthermore, the material possesses a porous microstructure, providing space for cell growth. In practical applications, the shear-thinning property of the colloidal gel ensures that the material can smoothly fill the gaps in bone defects or bone fragments. Once the material reaches the designated location, the chemical reaction between the polymer and the particles promptly enhances the material's mechanical strength. Simultaneously, a chemical reaction occurs between the polymer and the tissue, resulting in a firm bond between the material and bone tissue, thus achieving bone adhesion. After bone adhesion is achieved, compared to traditional polymer adhesives, this hydrogel-based bone adhesive material, composed of biodegradable polymers (such as collagen or polyethylene glycol) and biodegradable inorganic materials (bioglass or calcium phosphate), can be gradually degraded and absorbed by the body when integrating fragmented bone particles. Surrounding new cells and tissues will grow into the hydrogel scaffold matrix and eventually be completely degraded and absorbed, replaced by new bone tissue. Attached Figure Description

[0046] Figure 1 This is a transmission electron microscope image of the albumin nanoparticles in Example 1.

[0047] Figure 2 This is a schematic diagram of the preparation of colloidal gels using a Luer adapter syringe;

[0048] Figure 3 This is a shear-thinning diagram of albumin colloidal gel in Example 1.

[0049] Figure 4 This is the transmission electron microscopy morphology of hydroxyapatite in Example 8.

[0050] Figure 5 This is the transmission electron microscopy morphology of albumin-hydroxyapatite composite particles in Example 8.

[0051] Figure 6 Example 8 shows the shear thinning of albumin-hydroxyapatite composite particles.

[0052] Figure 7 This is the stress-strain curve of albumin particles after cross-linking in Example 13.

[0053] Figure 8 This is a comparison diagram of the filling effect of albumin hydroxyapatite composite particles and CA glue in Example 17.

[0054] Figure 9 This is a comparison chart of the stability of the particulate adhesive and Bio-glue in a humid environment in Example 18.

[0055] Figure 10 This is a comparison chart of the hemocoagulant effects of albumin-hydroxyapatite composite particles and bone wax in Example 19.

[0056] Figure 11 This is a staining image of the albumin-hydroxyapatite composite cell scaffold in Example 20.

[0057] Figure 12 This is a staining image of live and dead cells in albumin-hydroxyapatite complex particles from Example 22.

[0058] Figure 13 This is a staining image of alkaline phosphatase (ALP) and alizarin red S (ARS), an indicator of late osteogenic differentiation, in the in vitro osteogenic experiment of Example 24. Detailed Implementation

[0059] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used can be purchased from biological or chemical companies.

[0060] Example 1

[0061] 1. Preparation of albumin nanoparticles

[0062] At room temperature, 5g of albumin was dissolved in 100mL of deionized water, and the pH was adjusted to 10. 200mL of acetone was added over 30 minutes, and the mixture was stirred rapidly to allow the protein molecules to slowly dehydrate and curl into nanospheres. Then, 100μL of the cross-linking agent glutaraldehyde was added, and the mixture was stirred for 12 hours. After cross-linking was complete, 1g of glycine was added to remove any remaining cross-linking agent. The mixture was then centrifuged three times at 10000rpm, washed three times, and freeze-dried to obtain albumin nanoparticle powder. A portion of the nanoparticles was used to prepare a suspension, and its morphology was observed using a transmission electron microscope. The results are as follows: Figure 1 The hydrated particle size and zeta potential were determined by nanolaser particle size and zeta potential meter, and the results are shown in Table 1.

[0063] Table 1. Hydration particle size and zeta potential of albumin nanoparticles

[0064] Particle size 491±5nm Potential -20.4±2.1mV

[0065] 2. Albumin colloidal gel

[0066] Albumin particle powder aqueous solutions of 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, and 1 g / mL were repeatedly blown 10 times using a Luer adapter syringe to obtain injectable albumin particle gels. The storage modulus G' of the particle gels was obtained using a rotational rheometer in time-scan mode. The self-healing efficiency was obtained by comparing the storage modulus of the particle gels after oscillatory shearing with that before shearing (strain 0.1–1000%). The results are shown in Table 2. The shear-thinning properties are as follows: Figure 3 As shown, the frequency is 1 Hz and the strain is 0.5%. At this point, the albumin particle gel has a certain self-healing efficiency and shear-thinning mechanical characteristics, and is injectable and printable.

[0067] Table 2 Storage modulus and self-healing rate of albumin colloidal gel

[0068] 0.2g / mL 0.3g / mL 0.5g / mL 1g / mL Energy storage modulus (kPa) 1.079 7.14 47.84 110.762 Self-repair rate (%) 98.21019 86.5004 48.3381 9.81583

[0069] Example 2 (Different Materials)

[0070] At room temperature, 5g of gelatin (molecular weight 100kDa), polylysine (molecular weight 20kDa), protamine (molecular weight 620.943Da), chitosan (molecular weight 60kDa), silk fibroin (molecular weight 250kDa after degumming), and silkworm epidermal protein (all materials purchased from Sigma) were dissolved in 100mL of deionized water, and the pH was adjusted to 10 (the pH of chitosan was adjusted to 3). 200mL of acetone was added over 30 minutes, and the mixture was stirred rapidly to allow the molecules to slowly dehydrate and curl into nanoscale spheres. Then, 100μL of the cross-linking agent glutaraldehyde was added, and the mixture was stirred for 12 hours. After cross-linking, 1g of glycine was added to remove any remaining cross-linking agent. The mixture was then centrifuged three times at 10000rpm, washed three times, and freeze-dried to obtain nanoparticle powder. A portion of the nanoparticle suspension was taken and its hydrated particle size and zeta potential were measured using a nanolaser particle size analyzer and a zeta potential analyzer. The results are shown in Table 3.

[0071] Table 3 Hydration particle size and zeta potential of nanoparticles prepared from different materials

[0072] gelatin Chitosan Polylysine Protamine Silk fibroin Epidermal protein Particle size (nm) 236.6±8.9 333±43 763.6±18.2 146.9±13.9 291±5 812±38 Potential (mV) 3.7±0.6 3.3±0.4 16.9±0.3 6.7±1.08 -17.9±2.1 -19.5±2.1

[0073] As shown in Table 3, the nanoparticles prepared from different materials have different sizes. This is because different materials have different tolerances to the same solvent conditions (including type and volume ratio). Materials that are easy to dehydrate will form smaller nanoparticles, and vice versa.

[0074] 0.3g of the above nanoparticle powder was repeatedly blown 10 times with 1mL of deionized water through a Luer adapter syringe to obtain an injectable particulate gel. The storage modulus G' of the particulate gel was obtained using the time-scan mode of a rotational rheometer.

[0075] Table 4 Storage modulus and self-healing rate of particulate gels

[0076] gelatin Chitosan Polylysine Protamine Silk fibroin Epidermal protein Energy storage modulus (kPa) 21.3 3.7 6.25 1.2 13.5 19.5 Self-repair rate (%) 96.51 84.55 67.33 78.15 85.42 72.12

[0077] Example 3 (Different Solvents)

[0078] At room temperature, 5g of albumin was dissolved in 100mL of deionized water, and the pH was adjusted to 10. Within 30 minutes, 200mL each of methanol, ethanol, isopropanol, acetonitrile, and tetrahydrofuran were added, and the mixture was stirred rapidly to allow the protein molecules to slowly dehydrate and curl into nanospheres. Then, 100μL of the cross-linking agent glutaraldehyde was added, and the mixture was stirred for 12 hours. After cross-linking was complete, 1g of glycine was added to remove any remaining cross-linking agent. The mixture was then centrifuged three times at 10000rpm, washed three times, and freeze-dried to obtain albumin nanoparticle powder. A portion of the nanoparticle suspension was taken and its hydration particle size and zeta potential were measured using a nanolaser particle size analyzer and a zeta potential analyzer. The results are shown in Table 5.

[0079] Table 5. Hydration particle size and zeta potential of albumin nanoparticles

[0080] methanol ethanol Isopropanol Acetonitrile Tetrahydrofuran Particle size (nm) 456.4 256.8 462.2 893.4 399.2 Potential (mV) -19.9 -10.9 -18.6 -13.8 -21.6

[0081] 0.3 g of the above-mentioned granular powder was repeatedly blown 10 times through a Luer adapter syringe with 1 mL of deionized water to obtain injectable albumin granular gel. The storage modulus G' of the granular gel was obtained using a rotational rheometer in time-scan mode.

[0082] Table 6 Storage modulus and self-repair rate of albumin particle gel

[0083] methanol ethanol Isopropanol Acetonitrile Tetrahydrofuran Energy storage modulus (kPa) 5.4 6.9 4.1 3.5 7.6 Self-repair (%) 94.48 95.67 93.64 98.30 95.72

[0084] Example 4 (Different crosslinking agents)

[0085] At room temperature, 5g of albumin was dissolved in 100mL of deionized water, and the pH was adjusted to 10. 200mL of acetone was added over 30 minutes, and the mixture was stirred rapidly to allow the protein molecules to slowly dehydrate and coil into nanospheres. Subsequently, the following cross-linking agents were added: carbodiimide / N-hydroxysuccinimide (EDC / NHS), glyceraldehyde, succinaldehyde, and genipin, and the mixture was stirred for 12 hours. After cross-linking was complete, 1g of glycine was added to remove any remaining cross-linking agents. The mixture was then centrifuged three times at 10000rpm and freeze-dried to obtain albumin nanoparticle powder. A portion of the nanoparticle suspension was used to determine its hydration particle size and zeta potential using a nanolaser particle size analyzer and a zeta potential analyzer. The results are shown in Table 7.

[0086] Table 7 Hydration particle size and zeta potential of albumin nanoparticles

[0087] EDC / NHS Glyceraldehyde Butylene dialdehyde Kinnipin Particle size (nm) 463.8 511.4 539.5 397.3 Potential (mV) -13.5 -21.6 -27.9 -21.1

[0088] 0.3 g of the above-mentioned granular powder was repeatedly blown 10 times through a Luer adapter syringe with 1 mL of deionized water to obtain injectable albumin granular gel. The storage modulus G' of the granular gel was obtained using a rotational rheometer in time-scan mode.

[0089] Table 8 Storage modulus and self-repair rate of albumin particle gel

[0090] EDC / NHS Glyceraldehyde Butylene dialdehyde Kinnipin Energy storage modulus (kPa) 8.6 7.5 4.9 8.1 Self-repair (%) 97.10 95.67 95.84 96.81

[0091] Example 5 Different Sizes

[0092] First, a 5 wt% albumin aqueous solution was rapidly added to a 10 wt% glutaraldehyde solution (volume ratio 1:1) to form a dispersion of albumin particles. Then, particles were prepared by spray drying using a 0.75 mm nozzle. The precursor solution was prepared in… With the assistance of a pressure nozzle, the feed flow rate is 15 mL / min and the air flow rate is 140 m / min. 3 / h, atomization pressures of 0.06, 0.04, and 0.01 MPa produce albumin particles with particle sizes of 5μm, 20μm, and 50μm.

[0093] 0.3 g of the above-mentioned granular powder was repeatedly blown 10 times with 1 mL of deionized water through a Luer adapter syringe to obtain injectable albumin granular gel. The storage modulus G' of the granular gel was obtained using the time-scan mode of a rotational rheometer. The self-healing efficiency was obtained by comparing the storage modulus of the granular gel after oscillatory shearing with that before shearing (strain 0.1–1000%).

[0094] Table 9. Storage modulus and self-repair rate of albumin particles of different sizes.

[0095] 5μm 20μm 50μm Energy storage modulus (kPa) 2.69 1.6 0.85 Self-repair rate (%) 82 73 65

[0096] As shown in Table 9, compared with nanoscale particles, micron-scale colloidal gels have lower mechanical strength when not covalently crosslinked, but still have shear thinning and self-healing properties.

[0097] Example 6 (Albumin-Inorganic Mixed Particles)

[0098] 0.3 g of albumin granule powder, 1 mL of deionized water, and 50 mg, 100 mg, and 500 mg of hydroxyapatite nanoparticles (purchased from Maclean, particle size 60-80 nm) were repeatedly blown 10 times using a Luer adapter syringe to obtain injectable particulate gels. The storage modulus G' of the particulate gels was obtained using a rotational rheometer in time-scan mode. The self-healing efficiency was obtained by comparing the storage modulus of the particulate gels after oscillatory shearing with that before shearing (strain 0.1-1000%).

[0099] Table 10 Storage modulus and self-healing rate of particulate gels with different amounts of hydroxyapatite

[0100] 50mg 100mg 500mg Energy storage modulus (kPa) 10.23 50.26 100.37 Self-repair rate (%) 87.56 78.56 84.12

[0101] 0.3g of albumin particle powder, 1mL of deionized water, and 100mg each of silica nanoparticles, calcium silicate nanoparticles, tricalcium phosphate, calcium carbonate, nanoclay particles, and bioglass nanoparticles (all materials purchased from Maclean, particle size 60-80nm) were repeatedly blown 10 times using a Luer adapter syringe to obtain an injectable albumin particle gel. The storage modulus of the particle gel was obtained using a rotational rheometer in time-scan mode.

[0102] Table 11 Storage modulus and self-healing rate of particulate gels with different types of nanoparticles

[0103] silicon dioxide Calcium silicate Tricalcium phosphate Calcium carbonate Nano clay bioglass Energy storage modulus (kPa) 32.5 29.3 24.12 20.62 29.5 27.9 Self-repair rate (%) 72.67 64.22 69.45 58.91 87.48 74.047

[0104] 0.3g of albumin granule powder, 1mL of deionized water, and 100mg of needle-like hydroxyapatite, fibrous hydroxyapatite, and blocky calcined bone (all materials purchased from McLean) were repeatedly blown 10 times using a Luer adapter syringe to obtain an injectable albumin granule gel. The storage modulus G' of the granule gel was obtained using a rotational rheometer in time-scan mode.

[0105] Table 12 Storage modulus and self-healing efficiency of particulate gels with different shapes of hydroxyapatite added.

[0106]

[0107]

[0108] Example 7 HA Amino Modification

[0109] 100 mg / ml hydroxyapatite (HA, purchased from Maclean) particles were added to 100 mL of n-hexane to obtain a homogeneous emulsion. 5 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred at 60 °C for 8 h. After lyophilization, aminated hydroxyapatite HA-NH2 was obtained. The hydration particle size and zeta potential of the nanoparticle suspension were measured using a nanolaser particle size analyzer and a zeta potential analyzer. The results are shown in Table 13.

[0110] Table 13 Hydrated particle size and zeta potential of aminated hydroxyapatite

[0111] Particle size 261±5nm Potential +10.3±1.4mV

[0112] 0.3 g of albumin granule powder, 1 mL of deionized water, and 100 mg of HA-NH2 were repeatedly agitated 10 times using a Luer adapter syringe to obtain an injectable granular gel. The storage modulus G' of the granular gel was obtained using a rotational rheometer in time-scan mode. The self-healing efficiency was obtained by comparing the storage modulus of the granular gel after oscillatory shearing with that before shearing (strain 0.1–1000%).

[0113] Table 14 Storage modulus and self-healing rate of particulate gels with added aminated hydroxyapatite

[0114] 100mg Energy storage modulus (kPa) 103.42 Self-repair rate (%) 98.56

[0115] Example 8 (Albumin-hydroxyapatite composite nanoparticles)

[0116] 1. Preparation of hydroxyapatite nanoparticles

[0117] Hydroxyapatite nanoparticles were prepared by liquid-phase chemical precipitation. 100 ml of 75 mM H₂PO₄ was added dropwise to 100 ml of 125 mM Ca(OH)₂, and the mixture was stirred continuously at room temperature for 14-16 hours. The pH of the solution was then adjusted to 7, and the nanoparticles were obtained after centrifugation and washing three times. A portion of the nanoparticle suspension was subjected to transmission electron microscopy (TEM). The size and morphology of the particles are shown in the figure. Figure 4 As shown.

[0118] 2. Preparation of albumin-hydroxyapatite composite nanoparticles (albumin@HA NP)

[0119] First, hydroxyapatite was ultrasonically dispersed in 100 ml of albumin aqueous solution, with an albumin concentration of 50 mg / ml; the concentration ratio of hydroxyapatite to albumin was 1:8. The pH of the aqueous solution was adjusted to 10, and 200 mL of acetone was added over 30 min. After stabilization for 15 min, 100 μL of glutaraldehyde was added to obtain a covalently cross-linkable particle suspension. After cross-linking, 1 g of glycine was added to remove the remaining cross-linking agent. Then, the suspension was centrifuged and washed three times at 10,000 rpm, and freeze-dried to obtain core-shell structured albumin nanoparticle powder. A portion of the nanoparticle suspension was used to determine its hydration particle size and zeta potential using a nanolaser particle size analyzer and a zeta potential analyzer.

[0120] Table 15 Hydration particle size and zeta potential of core-shell structured albumin nanoparticles

[0121] Particle size 484.2nm Potential -21.35mV

[0122] Transmission electron microscopy (TEM) was performed on a suspension of core-shell albumin nanoparticles, and the size and morphology of the particles were as follows: Figure 5 As shown, hydroxyapatite appears as needles, and the size of the hydroxyapatite / albumin core-shell particles is about 500 nm. Furthermore, through transmission electron microscopy, it can be clearly observed that the dark black hydroxyapatite is encased inside the light gray albumin particles, forming a core-shell structure.

[0123] 3. Preparation of albumin@HA NP colloidal gel

[0124] 0.3 g of albumin@HA particle powder was repeatedly blown 10 times with 1 mL of deionized water using a Luer adapter syringe to obtain injectable albumin@HA particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time-scan mode. The self-healing efficiency was obtained by comparing the storage modulus of the particle gel after oscillatory shearing with that before shearing (strain 0.1–1000%). The results are shown in Table 2. The shear-thinning properties are as follows: Figure 6 As shown in the figure. At this point, the albumin particle gel has a certain self-healing efficiency and shear-thinning mechanical properties, and is injectable and printable.

[0125] Table 16 Storage modulus and self-repair rate of albumin@HA particle gel

[0126] 0.3g Energy storage modulus 23.1 kPa Self-repair rate (%) 89％

[0127] Example 9

[0128] The hydroxyapatite from Example 8 was ultrasonically dispersed in 100 ml of albumin aqueous solution with an albumin concentration of 50 mg / ml; the concentration ratio of hydroxyapatite to albumin was 1:8. The pH of the aqueous solution was adjusted to 1-11, and 200 mL of acetone was added within 30 min. After stabilizing for 15 min, 100 μL of glutaraldehyde was added to obtain a suspension of covalently crosslinkable particles. The crosslinking reaction was carried out at room temperature for 12 h. After crosslinking was completed, 1 g of glycine was added to remove the remaining crosslinking agent. Then, the particles were centrifuged and washed three times at 10000 rpm, and freeze-dried to obtain nanoparticle powder. A portion of the nanoparticle suspension was used to determine its hydrated particle size and zeta potential using a nanolaser particle size analyzer and a zeta potential analyzer.

[0129] Table 17 Hydration particle size and zeta potential of albumin nanoparticles

[0130]

[0131]

[0132] As shown in Table 17, composite protein particles can be prepared in the pH range of 6 to 11. When the solution pH is 1 or 2, no nanoparticles are obtained after multiple high-speed centrifugations of the product solution, indicating that this pH condition is insufficient to form stable nanospheres. Fine control of solution pH is crucial for the synthesis of such composite protein nanomaterials.

[0133] Example 10

[0134] The hydroxyapatite from Example 8 was ultrasonically dispersed in 100 ml of albumin aqueous solution with an albumin concentration of 50 mg / ml; the concentration ratio of hydroxyapatite to albumin was 1:8. The pH of the aqueous solution was adjusted to 10, and acetone was added within 30 min. After stabilizing for 15 min, 100 μL of glutaraldehyde was added to obtain a suspension of covalently crosslinkable particles. The volume of the polar organic solvent acetone added was 0.5, 1, 2, 4, 8, 10, and 12 times the volume of the aqueous solution. The crosslinking reaction was carried out at room temperature for 12 h. After crosslinking was completed, 1 g of glycine was added to remove the remaining crosslinking agent. Then, the mixture was centrifuged and washed three times at 10,000 rpm, and freeze-dried to obtain nanoparticle powder. A portion of the nanoparticle suspension was examined using a nano-laser particle size analyzer to determine whether particles were generated.

[0135] Table 18. Particle generation in the prepared nanoparticle suspension.

[0136] Acetone ratio Are there any particles? 0.5 no 1 yes 2 yes 4 yes 8 yes 10 yes 12 Particle aggregation and precipitation

[0137] As shown in Table 18, the volume ratio of organic solvent to water can be 1 to 10 to obtain nanoparticle powder.

[0138] Example 11

[0139] The hydroxyapatite from Example 8 was ultrasonically dispersed in 100 ml of albumin aqueous solution with an albumin concentration of 50 mg / ml. The concentration ratio of hydroxyapatite to albumin was 1:1, 1:2, 1:4, 1:8, and 1:16. The pH of the aqueous solution was adjusted to 10. 200 mL of acetone was added over 30 minutes, and after stabilization for 15 minutes, 100 μL of glutaraldehyde was added to obtain a suspension of covalently cross-linkable particles. The cross-linking reaction was carried out at room temperature for 12 hours. After cross-linking, 1 g of glycine was added to remove the remaining cross-linking agent. The mixture was then centrifuged three times at 10,000 rpm and freeze-dried to obtain nanoparticle powder. A portion of the nanoparticle suspension was examined using a nano-laser particle size analyzer to determine if particles were formed.

[0140] Table 19. Particle generation in the prepared nanoparticle suspension

[0141] Hydroxyapatite / Albumin Are there any particles? 1\1 no 1\2 no 1\4 no 1\8 yes 1\16 yes

[0142] As shown in Table 19, the mass ratio of hydroxyapatite to albumin is 1 / 8 to 1 / 16, which can yield nanoparticle powder.

[0143] Example 12 (Different Kernels)

[0144] First, silica nanoparticles, calcium silicate nanoparticles, tricalcium phosphate, calcium carbonate, nanoclay particles, and bioglass nanoparticles were ultrasonically dispersed in 100 ml of albumin aqueous solution, with an albumin concentration of 50 mg / ml. The concentration ratio of the above materials to albumin was 1:8. The pH of the aqueous solution was adjusted to 10, and 200 mL of acetone was added within 30 min. After stabilization for 15 min, 100 μL of glutaraldehyde was added to obtain a covalently cross-linkable particle suspension. The cross-linking reaction was carried out at room temperature for 12 h. After cross-linking was completed, 1 g of glycine was added to remove the remaining cross-linking agent. Then, the particles were centrifuged and washed three times at 10,000 rpm, and freeze-dried to obtain albumin nanoparticle powder. A portion of the nanoparticle suspension was taken and its hydrated particle size and zeta potential were measured using a nanolaser particle size analyzer and a zeta potential analyzer.

[0145] Table 20 Hydration particle size and zeta potential of albumin nanoparticles

[0146] silicon dioxide Calcium silicate Tricalcium phosphate Calcium carbonate Nano clay bioglass Particle size (nm) 598.6 497.3 491.8 557.9 893.4 462.2 Potential (mV) -11.9 -23.5 -21.6 -17.9 -13.8 -18.6

[0147] Comparative Example 1

[0148] Albumin powder and gelatin powder were dissolved in water to obtain solutions with a concentration of 200 mg / mL. At room temperature, 200 mg / mL albumin was a liquid, while gelatin formed a gel. The storage modulus G' of the particulate gel was obtained using a rotational rheometer in time-scan mode, and the results are shown in Table 21. At the same concentration, the modulus of albumin particles was higher than that of albumin solution.

[0149] Table 21 Storage modulus of albumin and gelatin

[0150] albumin solution Gelatin solution Energy storage modulus (kPa) 0.035 12.24

[0151] Example 13 (Nanoparticle Crosslinking)

[0152] 1. 0.3 g of albumin particle powder and 1 mL of deionized water were mixed with 10 mg (10 mg / mL), 100 mg (100 mg / mL), and 500 mg (500 mg / mL) of 8-arm PEG-NHS (molecular weight 5000), respectively. The mixture was then repeatedly blown and agitated 10 times using a Luer adapter syringe to obtain cross-linked albumin particle gels. The storage modulus G' of the particle gels was obtained using a rotational rheometer in time-scan mode.

[0153] After obtaining albumin nanoparticle gel according to the above formula, the gel was immersed in PBS at 37°C and its swelling value was measured. The results are shown in Table 22.

[0154] Albumin nanoparticle gel was prepared according to the above formula, and cylindrical gels were obtained using a mold with a diameter of 5 mm and a height of 10 mm. The mechanical properties of the gel were determined using a universal testing machine. The compression speed was 1 mm / min. The stress-strain curve is shown below. Figure 7-1 As shown.

[0155] Table 22 Storage modulus and swelling percentage of albumin nanoparticle gel

[0156] Amount of PEG 10mg / mL 100mg / mL 500mg / mL Energy storage modulus (kPa) 45.71 50.696 168.32 Swelling value (%) 250.45 160.23 150.56

[0157] 2. Albumin / hydroxyapatite (0.3g albumin, 100mg HA) mixed granular powder and 1mL deionized water were mixed with 10mg (10mg / mL), 100mg (100mg / mL), and 500mg (500mg / mL) 8-arm PEG-NHS, respectively. The mixture was then repeatedly blown and agitated 10 times using a Luer adapter syringe to obtain cross-linked albumin granular gel. The storage modulus G' of the granular gel was obtained using a rotational rheometer in time-scan mode.

[0158] After obtaining albumin-hydroxyapatite nanoparticle gel according to the above formula, the gel was immersed in PBS at 37°C to determine its swelling value. The results are shown in Table 23.

[0159] Albumin / hydroxyapatite nanoparticle gel was prepared according to the above formula, and cylindrical gels were obtained using a mold with a diameter of 5 mm and a height of 10 mm. The mechanical properties of the gel were determined using a universal testing machine. The compression speed was 1 mm / min. The stress-strain curve is shown below. Figure 7-2 As shown.

[0160] Table 23 Storage modulus and swelling percentage of albumin particle gel

[0161]

[0162]

[0163] 3. Albumin / HA-NH2 (0.3g albumin and 100mg HA-NH2) mixed granular powder, 1mL deionized water, and 10mg (10mg / mL), 100mg (100mg / mL), and 500mg (500mg / mL) 8-arm PEG-NHS were mixed and repeatedly blown 10 times using a Luer adapter syringe to obtain cross-linked albumin granular gel. The storage modulus G' of the granular gel was obtained using the time-scan mode of a rotational rheometer.

[0164] After obtaining albumin-hydroxyapatite nanoparticle gel according to the above formula, the gel was immersed in PBS at 37°C and its swelling value was measured. The results are shown in Table 24.

[0165] Albumin-hydroxyapatite nanoparticle gel was prepared according to the above formula, and cylindrical gels were obtained using a mold with a diameter of 5 mm and a height of 10 mm. The mechanical properties of the gel were determined using a universal testing machine. The compression speed was 1 mm / min. The stress-strain curve is shown below. Figure 7-3 As shown.

[0166] Table 24 Storage modulus and swelling percentage of albumin-hydroxyapatite nanoparticle gel

[0167] 10mg / mL 100mg / mL 500mg / mL Energy storage modulus (kPa) 152.89 224.46 418.41 Swelling (%) 184.32 153.47 131.26

[0168] 4. Mix 0.3g of albumin@HA particle powder and 1mL of deionized water with 10mg (10mg / mL), 100mg (100mg / mL), and 500mg (500mg / mL) 8-arm PEG-NHS respectively. The mixture is then repeatedly blown and agitated 10 times using a Luer adapter syringe to obtain cross-linked albumin particle gels. The storage modulus G' of the particle gels is obtained using a rotational rheometer in time-scan mode.

[0169] After obtaining albumin-hydroxyapatite nanoparticle gel according to the above formula, the gel was immersed in PBS at 37°C and its swelling value was measured. The results are shown in Table 25.

[0170] Albumin-hydroxyapatite nanoparticle gel was prepared according to the above formula, and cylindrical gels were obtained using a mold with a diameter of 5 mm and a height of 10 mm. The mechanical properties of the gel were determined using a universal testing machine. The compression speed was 1 mm / min. Figure 7-4 As shown.

[0171] Table 25 Storage modulus and swelling percentage of albumin-hydroxyapatite nanoparticle gel

[0172] 10mg / mL 100mg / mL 500mg / mL Energy storage modulus (kPa) 63.19 180.34 230.52 Swelling (%) 174.94 163.8 152.64

[0173] Comparative Example 2

[0174] 0.3 g of albumin powder, 1 mL of deionized water, and 10 mg (10 mg / mL), 100 mg (100 mg / mL), and 500 mg (500 mg / mL) of 8-arm PEG-NHS with a molecular weight of 5000 were mixed and repeatedly blown 10 times using a Luer adapter syringe to obtain cross-linked albumin gel. The storage modulus G' of the particulate gel was obtained using a rotational rheometer in time-scan mode.

[0175] Table 26 Storage modulus of gels prepared from albumin powder

[0176] 10mg / mL 100mg / mL 500mg / mL Energy storage modulus (kPa) 24.35 50.24 134.12

[0177] Compared with Table 26, it can be seen that the storage modulus of cross-linked albumin nanoparticles is higher than that of uncross-linked albumin hydrogel at the same concentration.

[0178] Example 14 (Different Crosslinking Agents)

[0179] 0.3 g of albumin particle powder and 1 mL of deionized water were mixed with 100 mg of linear, 4-arm, 8-arm PEG-NHS (molecular weight 5000) and hyperbranched PEG-NHS, respectively. The mixture was then repeatedly blown and agitated 10 times using a Luer adapter syringe to obtain cross-linked albumin particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time-scan mode.

[0180] Table 27 Storage modulus of cross-linked albumin gel

[0181] PEG type Straight-chain PEG-NHS 4-arm PEG-NHS 8-arm PEG-NHS Hyperbranched PEG-NHS Energy storage modulus (kPa) 29.52 33.32 50.696 83.14

[0182] 0.3 g of albumin particle powder and 1 mL of deionized water were mixed with 100 mg of 8-arm PEG-NHS with molecular weights of 5000, 10000, and 20000, respectively. The mixture was then repeatedly blown and agitated 10 times using a Luer adapter syringe to obtain cross-linked albumin particle gels. The storage modulus G' of the particle gels was obtained using a rotational rheometer in time-scan mode.

[0183] Table 28 Storage modulus of cross-linked albumin gel

[0184] PEG molecular weight 5000 10000 20000 Energy storage modulus (kPa) 50.696 33.60 11.61

[0185] 0.3 g of albumin particle powder and 1 mL of deionized water were mixed with 100 mg of PF127-NHS (Shenzhen Meiluo Technology Co., Ltd.), PF108-NHS (Shenzhen Meiluo Technology Co., Ltd.), sodium alginate-NHS (Qiyue Biotechnology), hyaluronic acid-NHS (Shaanxi Xingbei Aike Biotechnology), chondroitin sulfate-NHS (Ruixi Biotechnology), dextran-NHS (Ruixi Biotechnology), cellulose-NHS (Qiyue Biotechnology), polyacrylic acid-NHS (Qiyue Biotechnology), and agarose-NHS (Beike Nanotechnology). The mixture was then repeatedly blown and agitated 10 times using a Luer adapter syringe to obtain a cross-linked albumin particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time-scan mode.

[0186] Table 29 Storage modulus of cross-linked albumin gel

[0187]

[0188]

[0189] Example 15 (Different Groups)

[0190] 0.3 g of albumin particle powder and 1 mL of deionized water were mixed with 100 mg of maleimide polyethylene glycol (molecular weight 5000), epoxy polyethylene glycol, aldehyde polyethylene glycol, and carboxyl-terminated polyethylene glycol (molecular weight 5000), respectively. The mixture was then repeatedly blown and agitated 10 times using a Luer adapter syringe to obtain a cross-linked albumin particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time-scan mode.

[0191] Table 30 Storage modulus of cross-linked albumin gel

[0192] Group type maleimide Epoxy Aldehyde group carboxyl Energy storage modulus (kPa) 39.8 15.26 31.10 18.79

[0193] Example 16 (Adhesive Application)

[0194] Using the nanoparticle hydrogel from Example 8.3, 0.3g of albumin@HA+PEG-NHS was mixed with 1mL of deionized water and 10mg (10mg / mL), 100mg (100mg / mL), and 500mg (500mg / mL) of 8-arm PEG-NHS. The mixture was then repeatedly blown and bonded onto the surfaces of two bone fragments (5.0cm × 2.0cm rectangles) using a Luer adapter syringe, with the overlapping area being a 1.5cm × 2.0cm rectangle. The overlapping sample was sheared and peeled using a tensile tester with a 500N force sensor (peeling rate: 10mm / min), and the stress-strain curves during the peeling process were obtained. The point of maximum stress on the curve was used to define the bonding strength. The curing time of the corresponding components was also measured: timing began immediately after the material was extruded, and the material was considered fully cured when the bone fragments could no longer bond together. As can be seen from Table 31, the bonding strength of the nanoparticle hydrogel of the present invention is higher than that of pure polymer systems or nanoparticle systems alone, and also higher than that of commercially available protein adhesive products.

[0195] Table 31 Adhesion strength and curing time of different nanoparticle hydrogels

[0196] 10mg / mL 100mg / mL 500mg / mL Bond strength (kPa) 100.25±23.73 180.13±27.88 5300.52±96.89 Curing time (s) 268.39±12 124.92±7.88 52.12±14.8

[0197] Comparative Example 3

[0198] 1. Mix 0.3g of albumin granule powder and 1mL of deionized water evenly, and then quickly overlap and bond them to the surfaces of two bone fragments (5.0cm × 2.0cm rectangles), with the overlapping area being a (1.5cm × 2.0cm rectangle). Use a tensile tester with a 500N force sensor to shear and peel the overlapping sample (peeling rate: 10mm / min), and obtain the stress-strain curve of the peeling process. The point of maximum stress on the curve is used to define the bonding strength.

[0199] 2. Mix 0.3g of albumin granule powder, 100mg of HA, and 1mL of deionized water evenly, and then quickly overlap and bond them to the surfaces of two bone fragments (5.0cm × 2.0cm rectangles), with the overlapping area being a (1.5cm × 2.0cm rectangle). Use a tensile tester with a 500N force sensor to shear and peel the overlapped sample (peeling rate: 10mm / min), obtaining the stress-strain curve during the peeling process. The point of maximum stress on the curve is used to define the bonding strength.

[0200] 3. Apply Bio-glue adhesive (purchased from Cryolife) to the surfaces of two bone fragments (5.0cm × 2.0cm rectangles), with an overlap area of ​​1.5cm × 2.0cm. Use a tensile tester with a 500N force sensor to perform shear peeling on the overlapped sample (peeling rate: 10mm / min), obtaining the stress-strain curve during the peeling process. The point of maximum stress on the curve is used to define the adhesive strength.

[0201] 4. Fiber adhesive (Yueling brand) is used to bond two bone fragments (5.0cm × 2.0cm rectangles) together, with the overlapping area being a 1.5cm × 2.0cm rectangle. A tensile tester with a 500N force sensor is used to shear and peel the overlapping sample (peeling rate: 10mm / min), obtaining the stress-strain curve during the peeling process. The point of maximum stress on the curve is used to define the adhesive strength.

[0202] Table 32 Adhesive strength of different types of nanoparticle hydrogels

[0203] Example Adhesion strength Example 16 180.13 kPa Comparative Example 3.1 115.11 kPa Comparative Example 3.2 14.63 kPa Comparative Example 3.3 17.56 kPa Comparative Example 3.4 14.6 kPa

[0204] Example 17 (Application of Bone Filling)

[0205] Using the nanoparticle hydrogel in Example 8.3, 0.3g albumin@HA + 100mg PEG-NHS: 0.3g albumin@HA particle powder, 1mL deionized water and 100mg (100mg / mL) 8-arm PEG-NHS were mixed and repeatedly blown through a Luer adapter syringe and then quickly squeezed into the bone defect site to fill the bone defect.

[0206] Comparative Example 4

[0207] Using medical CA glue (brand: Baiyun) as a control, it was squeezed into the bone defect area to fill the bone defect.

[0208] The results are as follows Figure 8 As shown, from Figure 8 As can be seen, the nanoparticle hydrogel of this invention has a better filling effect compared to medical CA glue.

[0209] Example 18 (Liquid Environment)

[0210] Using the nanoparticle hydrogel from Example 8.3, 0.3g of BSA albumin@HA+PEG-NHS: 0.3g of BSA albumin@HA particle powder and 1mL of deionized water were mixed with 100mg (100mg / mL) of 8-arm PEG-NHS. After repeated blowing and agitation using a Luer adapter syringe, the mixture was overlapped and bonded to the surface of two bone fragments (5.0cm × 2.0cm rectangles) soaked in PBS. Figure 9The overlapping area was a rectangle measuring 1.5cm × 2.0cm. A tensile tester with a 500N force sensor was used to shear and peel the overlapping sample (peeling rate: 10mm / min), obtaining the stress-strain curve during the peeling process. The point of maximum stress on the curve was used to define the adhesive strength.

[0211] Comparative Example 5

[0212] Bio-glue (purchased from Cryolife) was used to bond two bone fragments (5.0cm × 2.0cm rectangles) to the surface of the bone fragments soaked in PBS. Figure 9 The overlapping area was a rectangle measuring 1.5cm × 2.0cm. A tensile tester with a 500N force sensor was used to shear and peel the overlapping sample (peeling rate: 10mm / min), obtaining the stress-strain curve during the peeling process. The point of maximum stress on the curve was used to define the adhesive strength. As can be seen from the figure, the colloidal adhesive of this invention can exist stably in a liquid environment, while Bio-glue adhesive diffuses in the liquid, leading to a decrease in adhesive strength.

[0213] Table 33 Adhesive strength in humid environments

[0214] Example Adhesion strength Example 18 200.54 kPa Comparative Example 5 11 kPa

[0215] Example 19 (Application in Bone Hemostasis)

[0216] Using the nanoparticle hydrogel from Example 8.3, 0.3g albumin@HA + 100mg PEG-NHS: 0.3g albumin@HA particle powder, 1mL deionized water, and 100mg (100mg / mL) 8-arm PEG-NHS were mixed and repeatedly extruded using a Luer adapter syringe to obtain the hydrogel. The coagulation properties of the bone adhesive were investigated through an in vitro coagulation experiment. The specific implementation steps are as follows: First, 50μL of fresh blood was dropped onto the sample surface after the nanoparticle hydrogel had gelled. Next, the sample was incubated in an oven at 37℃ for 2-10min, then the sample surface was rinsed with PBS solution, and the absorbance of hemoglobin in the solution was measured using a UV-Vis spectrophotometer at a wavelength of 540nm.

[0217] The in vitro coagulation test was performed using Johnson & Johnson bone wax. The specific steps were as follows: First, 50 μL of fresh blood was dropped onto the sample surface after bone wax treatment. Then, the sample was incubated in an oven at 37°C for 2-10 minutes. Next, the sample surface was rinsed with PBS solution. The supernatant was then measured using a UV-Vis spectrophotometer at a wavelength of 540 nm to measure the absorbance of hemoglobin in the solution.

[0218] The results are as follows Figure 10As shown in the figure, the nanoparticle hydrogel of the present invention has a better effect on promoting blood clotting than bone wax.

[0219] Example 20 (Cellular Scaffold)

[0220] Taking the particulate gel prepared by 0.3g albumin@HA+100mg PEG-NHS as an example, MC3T3-E1 cells were mixed with the hydrogel (cell concentration was 5.0×10⁻⁶). 3 Cells / mL) were extruded to obtain a cell-carrying colloidal gel scaffold with stable mechanical properties; after culturing at 37°C and 5% carbon dioxide for 72 h, the cell viability encapsulated in the hydrogel was evaluated by fluorescence live / dead assay, where green fluorescence represents live cells and red fluorescence represents dead cells.

[0221] The results are as follows Figure 11 As shown in the figure, the scaffold structure is clear and complete, and the survival rate of live cells is high. The material of this invention can be used as a cell scaffold.

[0222] Example 21 (Drug Delivery Application)

[0223] Using a particulate gel prepared from 0.3g albumin@HA+100mg PEG-NHS as an example, the drug release behavior was studied. First, the gel was mixed with arginine, vitamin D, and the natural active factor VEGF (Glucose-containing mRNA). Then, the gel was placed in PBS at 37℃ and shaken on a shaker to simulate the in vivo environment. At predetermined time points, the absorbance of the supernatant was measured using a UV-Vis spectrophotometer, and compared with a standard curve to determine the release amount at each time point.

[0224] Table 34 Cumulative Release Amounts of Various Drugs at Different Time Points

[0225] 1d 4d 7d 14d 28d Arginine 10.18％ 16.84％ 25.04％ 33.48％ 48.42％ Vitamin D 13.99％ 34.20％ 51.26％ 64.95％ 78.95％ GLuc mRNA 10.44％ 25.82％ 34.87％ 73.87％ 80.06％ VEGF 16.80％ 27.36％ 49.68％ 59.43％ 63.20％

[0226] The above results indicate that albumin colloidal gel can serve as a drug carrier, ensuring long-term drug release.

[0227] Example 22 (Cell Experiment)

[0228] Using a particulate gel prepared from 0.3g albumin@HA+100mg PEG-NHS as an example, the biocompatibility of the material was investigated through in vitro cell culture experiments. The specific implementation steps are as follows: After extruding, curing, and lyophilizing the adhesive, the gel was immersed in culture medium (concentration 0.2g / mL) for 24 hours for later use; bone marrow mesenchymal stem cells (MC3T3-E1 pre-osteoblast cell line) were digested with trypsin and seeded on the adhesive surface. The culture medium was aspirated and replaced with gel extract, and the cells were cultured at 37℃ and 5% carbon dioxide for 72 hours. The effect of the hydrogel on cell viability was evaluated using a fluorescence live / dead assay, where green fluorescence represents live cells and red fluorescence represents dead cells.

[0229] The results are as follows Figure 12 As shown, the results indicate that the adhesive has good biocompatibility.

[0230] Example 23 (Subcutaneous Implantation Experiment)

[0231] The particulate gel prepared using 0.3g albumin@HA+100mg PEG-NHS was injected subcutaneously into both sides of mice using a 22G needle, with a total of 100μL of gel material. After feeding for 1, 4, 8 and 12 weeks, the mice were sacrificed, and the implanted material was removed and weighed to evaluate the in vivo degradation performance of the material.

[0232] 100 mg of CA gel was injected subcutaneously into both sides of mice using a 22G needle, resulting in a total of 100 μL of gel material. Mice were euthanized after 1, 4, 8, and 12 weeks of feeding, and the implanted material was removed and weighed for comparison of degradation performance.

[0233] Table 35 Degradation rate of particulate gel materials at different time points

[0234] time Albumin degradation rate (%) CA degradation rate (%) 1 week 13.88 1.54 4 weeks 20.26 3.21 8 weeks 44.25 8.85 12 weeks 53.03 13.48

[0235] The above results indicate that the composite protein particle gel of the present invention can be stably filled subcutaneously in mice for 12 weeks.

[0236] Example 24 (In vitro osteogenic experiment)

[0237] A particulate gel was prepared using 0.3 g albumin@HA + 100 mg PEG-NHS. Tissue adhesive or CA gel was injected into the bottom of a 24-well plate, completely cured, and then sterilized. The plate was then washed three times with PBS. MC-3T3-E1 cells (5 × 10⁻⁶) were then added. 4 Cells were seeded per well in DMEM containing 2.5% FBS for 7 and 14 days. Cell mineralization and ALP secretion were observed under a light microscope using Alizarin Red staining kit and ALP staining kit, respectively.

[0238] Compared to CA glue and the blank group, the tissue adhesive of the present invention showed more blue precipitate and more red calcium nodules. Figure 13 This indicates that the tissue adhesive of the present invention has a superior osteogenic effect.

Claims

1. A hydrogel tissue adhesive composed of organic / inorganic particles and polymer chains, characterized in that, The hydrogel tissue adhesive comprises micron- or nano-sized organic particles carrying amino groups, a water-soluble polymer that undergoes a chemical cross-linking reaction with the amino groups, and micron- or nano-sized inorganic particles that promote osteogenic growth; the mass ratio of organic particles, inorganic particles, and water-soluble polymer is 1~20:1:0.1~5; the volume percentage of particles in the hydrogel tissue adhesive is 10~80%; the organic / inorganic particles are formed by mixing organic and inorganic particles, or by organic particles encapsulating inorganic particles to form core-shell structured composite particles. Under the action of water, the hydrogel tissue adhesive can form a continuous, porous particle network through covalent interactions and / or reversible non-covalent interactions between particles, giving the hydrogel tissue adhesive injectability, shapeability, and self-healing properties. The particle size ranges from 1 nm to 50 μm; the particle potential under neutral conditions ranges from -30 mV to +30 mV. The component of the micron or nano-sized organic particles carrying amino groups is albumin; the molecular weight of the component of the micron or nano-sized organic particles carrying amino groups is 0.5-100 kDa. The micron or nano-sized inorganic particles with osteogenic properties account for ≥10 wt% of the dry weight of the hydrogel tissue adhesive; the inorganic particles are calcium phosphate, calcium carbonate, calcium silicate, or bioglass-like inorganic non-metallic material particles; the morphology is one or a combination of two or more of the following: needle-like, fibrous, granular, and blocky. The active groups of the water-soluble polymer that undergoes chemical cross-linking with amino groups include the following groups: succinimide, aldehyde, maleimide, epoxy, and carboxyl; the backbone of the water-soluble polymer that undergoes chemical cross-linking with amino groups is one or a combination of two or more of polyethylene glycol, polyacrylic acid, block polyether F-127, block polyether F-108, sodium alginate, hyaluronic acid, chondroitin sulfate, dextran, cellulose, and agarose. The inorganic particles are modified with amino groups on their surface. The process of modifying the surface of the inorganic particles with amino groups is as follows: the inorganic particles are added to n-hexane to obtain a uniform dispersion of 1~500 mg / mL, and then 3-aminopropyltriethoxysilane that reacts with the hydroxyl groups on the surface of the inorganic particles is added. The mixture is stirred at 50~80℃ for 4~12 h, and then freeze-dried to obtain the aminated inorganic particles.

2. The hydrogel tissue adhesive according to claim 1, characterized in that, The water-soluble polymer has a linear, multi-arm, or hyperbranched shape, a molecular weight of 0.5-100 kDa, and accounts for 10-50 wt% of the dry weight of the hydrogel tissue adhesive. Furthermore, the molar ratio of amino groups to reactive groups in the polymer chain is 0.1-100.

3. The method for preparing the hydrogel tissue adhesive according to any one of claims 1-2, characterized in that, This includes the following two methods: Method 1: Includes the following steps: (1) Preparation of micron or nano-sized organic particles carrying amino groups: a) When the size of the prepared particles is greater than 5 μm, the preparation process is as follows: Dissolve the raw material in water at 20℃~60℃ to obtain an aqueous solution with a concentration of 1~100 mg / mL, add a crosslinking agent, stir and crosslink at room temperature for 1~20 h, remove the remaining crosslinking agent, and prepare microsphere particles by spray drying. b) When the particle size is 1 nm When the raw material is albumin, the raw material is dissolved in water at 20℃~60℃ to obtain an aqueous solution with a concentration of 1~100 mg / mL. The pH of the aqueous solution is adjusted to 9~14, and 1~10 times the volume of the aqueous solution of a polar organic solvent is added dropwise to the above aqueous solution. After stirring for 5~60 min, a crosslinking agent is added, and the mixture is stirred at room temperature for 1~20 h for crosslinking. The remaining crosslinking agent is removed, and after washing, a micro / nano particle dispersion is obtained. After freeze-drying, a particle powder is obtained. (2) Preparation of hydrogel tissue adhesive: The micron or nano-sized organic particles carrying amino groups prepared in step (1), the water-soluble polymer that undergoes a chemical cross-linking reaction with the amino groups, and the micron or nano-sized inorganic particles that promote osteogenic activity are mixed evenly to obtain the product. Method 2: Includes the following steps: 1) Preparation of core-shell structured composite particles: Inorganic particles serving as the core layer are ultrasonically dispersed in an aqueous solution containing dissolved organic polymers for the outer shell layer. The concentration of the outer shell material is 1–100 mg / mL, and the concentration ratio of the core material to the outer shell material is 1:2–1:

50. The pH is adjusted to 9–14. A polar organic solvent is added dropwise to the above aqueous solution, with the volume of the added polar organic solvent being 1–10 times the volume of the aqueous solution. After stabilizing for 10–30 min, a crosslinking agent is added, and the crosslinking reaction is carried out at room temperature for 1–12 h to obtain a suspension of micro / nano particles with a core-shell structure. The remaining crosslinking agent is removed, and after washing, a core-shell particle dispersion is obtained. After drying, a composite particle powder with a core-shell structure is obtained. 2) Preparation of hydrogel tissue binder: The core-shell composite particles prepared in step 1) are mixed evenly with a water-soluble polymer that has undergone a chemical cross-linking reaction with amino groups to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The polar organic solvent is one or a mixture of two or more of methanol, ethanol, isopropanol, butanol, acetone, acetonitrile, and tetrahydrofuran; the crosslinking agent is carbodiimide / N One or more of the following: hydroxysuccinimide, glyceraldehyde, glutaraldehyde, formaldehyde, paraformaldehyde, succinaldehyde, and genipin.

5. The method of using the hydrogel tissue adhesive according to any one of claims 1-2, characterized in that, The process includes the following steps: mixing the hydrogel tissue adhesive according to any one of claims 1-2 with water until homogeneous, repeatedly blowing and agitating it 5-25 times with a Luer adapter syringe to obtain an injectable gel, which is then squeezed out.

6. The use of the hydrogel tissue adhesive according to any one of claims 1-2 as a bone tissue adhesive.

7. The use of the hydrogel tissue adhesive according to any one of claims 1-2 as a drug carrier or cell carrier.

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