Collagen-based bone filling material as well as preparation method and application thereof

Abstract

The invention provides a preparation method of a collagen-based bone filling material, which comprises the following steps: S1) heating an acellular matrix biological membrane for thermal crosslinking treatment, and crushing to obtain matrix powder; and S2) mixing the matrix powder with a solvent, performing whipping treatment, then adding inorganic bone particles, performing mixing, and performing freeze drying and thermal crosslinking treatment to obtain the collagen-based bone filling material. Compared with the prior art, the collagen-based bone filling material provided by the invention is composed of the acellular matrix biological membrane and the natural inorganic bone particles, and the acellular matrix biological membrane is subjected to thermal crosslinking treatment, crushing, swelling and whipping treatment to form a stable three-dimensional network structure; the bone filling material prepared by the preparation method disclosed by the invention can obviously prolong the in-vitro degradation time after being physically crosslinked with inorganic bone particles, and the bone filling material has no chemical reagent residue, not only has good biocompatibility, but also has good rebound resilience and plasticity, and is convenient to operate.

Description

Technical Field

[0001] The present invention belongs to the field of material technology, and in particular relates to a collagen-based bone filling material and a preparation method and application thereof. Background Art

[0002] Bone is one of the most important organs in the human body. Clinically, bone defects caused by trauma, tumors, and infections are very common, and millions of patients with bone tissue defects require surgical treatment every year. Although there are currently a variety of treatments for bone defects, they all have their own limitations. Autologous bone transplantation is limited by the limited number of donors; allogeneic bone carries the risk of disease transmission and immune rejection; and various bone repair materials made of metal and polymer materials are mostly implanted as permanent bodies, cannot be degraded, and the results are often unsatisfactory. Therefore, the preparation of ideal artificial bone materials is an urgent need for the clinical treatment of bone defects.

[0003] The ideal artificial bone material should have good osteoconductivity, osteoinductivity, biosafety and clinical operability; at the same time, it should have appropriate porosity to meet the adhesion and proliferation of bone cells to achieve the purpose of bone growth.

[0004] In recent years, a variety of artificial bone repair materials have been introduced to the market and clinically used, primarily including synthetic materials such as hydroxyapatite, hydroxyapatite / collagen complexes, and mineralized collagen. However, pure hydroxyapatite currently suffers from problems such as easy particle scattering and difficulty in handling. Hydroxyapatite / collagen complexes and mineralized collagen often utilize chemical cross-linking processes to enhance structural stability, which can lead to drawbacks such as cytotoxicity (see Chinese patent publication numbers CN106139255, CN104096268, and CN105358189 B).

[0005] During their research, the applicant discovered that physically mixing matrix and bone particles is difficult to form a stable structure. The resulting composite material suffers from bone particle shedding and loosening after absorbing water. Furthermore, the matrix-to-bone particle ratio is a key factor influencing the effectiveness of bone filling materials. Bone particles play a major role in bone defect repair. A low ratio of bone particles can impair osteogenesis in the defect, while a high ratio can compromise the product's plasticity. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a collagen-based bone filling material that adopts physical cross-linking and has high structural stability, as well as a preparation method and application thereof.

[0007] The present invention provides a method for preparing a collagen-based bone filling material, comprising the following steps:

[0008] S1) heating the decellularized matrix biofilm for thermal cross-linking, and then crushing the decellularized matrix biofilm to obtain matrix powder;

[0009] S2) The matrix powder is mixed with a solvent and beaten, and then inorganic bone particles are added and mixed, and the mixture is freeze-dried and thermally cross-linked to obtain a collagen-based bone filling material.

[0010] Preferably, the acellular matrix biofilm is prepared according to the following method:

[0011] A1) After repeated freeze-thaw treatment of peritoneal tissue, fat and excess serosal membrane were removed to obtain a pre-treated membrane;

[0012] A2) treating the pre-treated membrane with a chelating agent solution, a hypertonic alkaline solution, and a hypotonic alkaline solution in sequence, repeating the above treatment steps at least once, and then treating the treated membrane with a hypertonic acidic solution, neutralizing it to neutrality, and freeze-drying it to obtain a decellularized matrix biomembrane;

[0013] And / or, the inorganic bone particles are prepared according to the following method:

[0014] The cancellous bone is cut into thin slices, soaked in purified water to remove blood, dried, and then refluxed with an alkane organic solvent to remove fat components. The slices are then refluxed with an amine organic solvent for deproteinization. The slices are washed to neutrality, dried, and crushed to obtain inorganic bone particles.

[0015] Preferably, in step A1), fat and excess serosal membrane are removed by repeatedly kneading with sodium bicarbonate powder;

[0016] And / or, the chelating agent solution is selected from ethylenediaminetetraacetic acid solution; the molar concentration of the ethylenediaminetetraacetic acid solution is 0.1 to 0.5 mol / L;

[0017] And / or, the hypertonic alkaline solution is selected from a high-concentration sodium hydroxide solution; the mass concentration of the high-concentration sodium hydroxide solution is 2% to 5%;

[0018] And / or, the hypotonic alkaline solution is selected from a low-concentration sodium hydroxide solution; the mass concentration of the low-concentration sodium hydroxide solution is 0.1% to 0.5%;

[0019] And / or, the hypertonic acidic solution is selected from a sodium chloride-hydrochloric acid solution; the mass concentration of hydrochloric acid in the sodium chloride-hydrochloric acid solution is 0.1% to 0.5%; the mass concentration of sodium chloride in the sodium chloride-hydrochloric acid solution is 1% to 2%.

[0020] Preferably, the temperature of treating the membrane after pretreatment with the chelating agent solution in step A2) is 15° C. to 25° C., and the treatment time is 5 to 20 hours;

[0021] And / or, in step A2), the membrane after pretreatment is treated with a hypertonic alkaline solution at a temperature of 15° C. to 25° C. for 1 to 5 hours;

[0022] And / or, in step A2), the pre-treated membrane is treated with a hypotonic alkaline solution at a temperature of 15° C. to 25° C. for 1 to 5 hours;

[0023] and / or, in the process of treating the pre-treated membrane with the hypotonic alkaline solution in step A2), the hypotonic alkaline solution is replaced 1 to 3 times;

[0024] And / or, the number of times the above-mentioned treatment steps are repeated in step A2) is 3 to 5 times;

[0025] And / or, the temperature for treating with the hypertonic acid solution in step A2) is 15° C. to 25° C., the number of times of treating with the hypertonic acid solution is 3 to 5 times, and the time of each treatment with the hypertonic acid solution is 1 to 3 hours.

[0026] Preferably, the temperature of the thermal crosslinking treatment in step S1) is 100° C. to 150° C., and the time of the thermal crosslinking treatment is 24 to 60 hours;

[0027] The temperature of the thermal crosslinking treatment in step S2) is 100° C. to 120° C.; the time of the thermal crosslinking treatment is greater than or equal to 24 hours;

[0028] The thermal cross-linking treatment in step S1) and step S2) is performed under vacuum conditions; the relative vacuum degree of the vacuum conditions is -1 to -0.5 bar.

[0029] Preferably, the particle size of the matrix powder in step S1) is 0.5 to 2 mm;

[0030] and / or, the particle size of the inorganic bone particles is 0.25 to 1 mm;

[0031] And / or, the mass ratio of the matrix powder to the inorganic bone particles is (10:90) to (20:80).

[0032] Preferably, the mass of the matrix powder in step S2) is 5% to 10% of the total mass of the matrix powder and the solvent;

[0033] And / or, the solvent in step S2) is selected from a weak acid; the pH value of the weak acid is 4-6; the weak acid is selected from acetic acid and / or citric acid.

[0034] Preferably, the whipping process has a rotation speed of 6000 to 10000 r / min, and a whipping process time of 15 to 20 min.

[0035] The present invention also provides a collagen-based bone filling material prepared by the above preparation method.

[0036] The present invention also provides a use of the collagen-based bone filling material prepared by the above preparation method in preparing bone defect repair materials.

[0037] The present invention provides a method for preparing a collagen-based bone filling material, comprising the following steps: S1) heating and thermally crosslinking a decellularized matrix biofilm, followed by pulverization to obtain matrix powder; S2) mixing the matrix powder with a solvent and beating the mixture, then adding inorganic bone particles, and freeze-drying and thermally crosslinking the mixture to obtain a collagen-based bone filling material. Compared with the prior art, the collagen-based bone filling material provided by the present invention is composed of a decellularized matrix biofilm and natural inorganic bone particles. The decellularized matrix biofilm is subjected to thermal crosslinking, followed by pulverization, swelling, and beating to form a stable three-dimensional network structure. Physical crosslinking with the inorganic bone particles significantly increases the in vitro degradation time. Furthermore, the bone filling material is free of chemical reagent residues and exhibits excellent biocompatibility, resilience, and plasticity, making it easy to handle. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the X-ray diffraction pattern of the inorganic bone particles obtained in Example 1 of the present invention;

[0039] Figure 2 This is a comparison chart of the viscosity results of the whipped slurry (cross-linked) in Example 1 of the present invention and the whipped slurry (uncross-linked) in Comparative Example 1;

[0040] Figure 3 These are photos of the collagen-based bone filling material sample 1 obtained in Example 1 of the present invention before (a) and after (b) imbibition;

[0041] Figure 4 The scanning electron microscope images of the slurry of the small-particle-size (a) matrix powder and the large-particle-size (b) matrix powder after being beaten in Example 1 of the present invention are shown;

[0042] Figure 5 Graphs showing the results of in vitro degradation tests of collagen-based bone filling materials prepared using large-particle matrix powder and small-particle matrix powder, respectively, in Example 1 of the present invention;

[0043] Figure 6 This is a comparison chart of the viscosity results of slurries with different pH values ​​in Example 1 of the present invention;

[0044] Figure 7 This is a comparison chart of the viscosity results of the slurry under different beating time treatments in Example 1 of the present invention;

[0045] Figure 81 is a scanning electron microscope image of the unwhipped slurry (a) and the whipped slurry (b) in Example 1 of the present invention;

[0046] Figure 9 This is a graph showing the in vitro degradation time of the collagen-based bone filling material prepared from the slurry after being processed for different beating times in Example 1 of the present invention;

[0047] Figure 10 Scanning electron microscope images of the collagen-based bone filling material sample 1(b) obtained in Example 1 of the present invention and the collagen-based bone filling material sample 6(a) obtained in Comparative Example 5;

[0048] Figure 11 The surgical scene for animal experiments of this invention. DETAILED DESCRIPTION

[0049] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] The present invention provides a method for preparing a collagen-based bone filling material, comprising the following steps: S1) heating a decellularized matrix biofilm for thermal cross-linking treatment, and then crushing it to obtain matrix powder; S2) mixing the matrix powder with a solvent and beating it, then adding inorganic bone particles and mixing, and freeze-drying and thermal cross-linking treatment to obtain the collagen-based bone filling material.

[0051] The present invention has no particular limitation on the sources of all raw materials, and they can be commercially available or homemade.

[0052] According to the present invention, the acellular matrix biomembrane is preferably prepared according to the following method: A1) the peritoneal tissue is subjected to repeated freeze-thaw treatment to remove fat and excess serosal membrane to obtain a pre-treated membrane; A2) the pre-treated membrane is treated with a chelating agent solution, a hypertonic alkaline solution, and a hypotonic alkaline solution in sequence, and after repeating the above treatment steps at least once, the treated membrane is further treated with a hypertonic acidic solution, and then neutralized to neutrality, and freeze-dried to obtain the acellular matrix biomembrane.

[0053] In a specific embodiment provided by the present invention, the peritoneal tissue is mammalian peritoneal tissue well known to those skilled in the art, including but not limited to pigs, cows, dogs, sheep, rabbits, mice and the like.

[0054] In a specific embodiment provided by the present invention, fresh peritoneal tissue is subjected to repeated freeze-thaw treatment, and fat and excess serous membrane are removed to obtain a pre-treated membrane; the freezing temperature in the freeze-thaw treatment is preferably -10°C to -30°C, more preferably -15°C to -25°C, and more preferably -20°C; the freezing time is preferably 1 to 5 hours, more preferably 2 to 4 hours, and more preferably 3 hours; the melting in the freeze-thaw treatment is preferably carried out in flowing water at 15°C to 25°C, more preferably in flowing water at 20°C to 25°C, and more preferably in flowing water at 25°C; the number of repeated freeze-thaw treatments is preferably 3 to 5 times, more preferably 4 to 5 times, and more preferably 5 times.

[0055] In a specific embodiment provided by the present invention, after the freeze-thaw treatment, the fat and excess serosal membrane are removed by repeatedly kneading with sodium bicarbonate powder; preferably, after the freeze-thaw treatment, the fat and excess serosal membrane are removed by repeatedly kneading with sodium bicarbonate powder under low temperature conditions; more preferably, after the freeze-thaw treatment, the peritoneal tissue after freeze-thaw treatment is placed on an ice pack with the rough surface facing upward, and sodium bicarbonate powder is sprinkled on the surface of the peritoneal tissue and repeatedly kneaded to remove fat and excess serosal membrane; the amount of the sodium bicarbonate powder used is preferably 0.1 to 1 g / cm 2 , more preferably 0.3 to 0.6 g / cm 2 , and more preferably 0.5g / cm 2 .

[0056] In a specific embodiment provided by the present invention, fat and excess serous membrane are removed, and the membrane is rinsed with running water to obtain a pre-treated membrane; the time for the running water rinse is preferably 8 to 18 hours, more preferably 10 to 15 hours, and even more preferably 12 hours.

[0057] In a specific embodiment provided by the present invention, the pre-treated membrane is treated with a chelating agent solution, a hypertonic alkaline solution and a hypotonic alkaline solution in sequence; the chelating agent solution is preferably an ethylenediaminetetraacetic acid (EDTA) solution; the molar concentration of the EDTA solution is preferably 0.1 to 0.5 mol / L; optionally, the molar concentration of the EDTA solution is 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L or a range between any two of the above values; the hypertonic alkaline solution is preferably It is a high-concentration sodium hydroxide solution; the mass concentration of the high-concentration sodium hydroxide solution is preferably 2% to 5%; optionally, the mass concentration of the high-concentration sodium hydroxide solution is 2%, 3%, 4%, 5% or a range between any two of the above values; the hypotonic alkaline solution is preferably a low-concentration sodium hydroxide solution; the mass concentration of the low-concentration sodium hydroxide solution is preferably 0.1% to 0.5%; optionally, the mass concentration of the low-concentration sodium hydroxide solution is 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or a range between any two of the above values.

[0058] In a specific embodiment provided by the present invention, the temperature for treating the membrane with the chelating agent solution after pretreatment is preferably 15°C to 25°C, more preferably 20°C to 25°C; the time for treating the membrane with the chelating agent solution after pretreatment is preferably 5 to 20 hours; optionally, the time for treating the membrane with the chelating agent solution after pretreatment is 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours or a range between any two of the above values.

[0059] In a specific embodiment provided by the present invention, the temperature for treating the pre-treated membrane with a hypertonic alkaline solution is preferably 15°C to 25°C, more preferably 20°C to 25°C; the time for treating the pre-treated membrane with a hypertonic alkaline solution is preferably 1 to 5 hours; optionally, the time for treating the pre-treated membrane with a hypertonic alkaline solution is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or a range between any two of the above values.

[0060] In a specific embodiment provided by the present invention, the temperature for treating the pre-treated membrane with a hypotonic alkaline solution is preferably 15°C to 25°C, more preferably 20°C to 25°C; the time for treating the pre-treated membrane with a hypotonic alkaline solution is preferably 1 to 5 hours; optionally, the time for treating the pre-treated membrane with a hypotonic alkaline solution is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or a range between any two of the above values.

[0061] In a specific embodiment provided by the present invention, during the treatment of the pre-treated membrane with the hypotonic alkaline solution, the hypotonic alkaline solution is replaced 1 to 3 times, preferably 2 to 3 times.

[0062] In a specific embodiment provided by the present invention, the above-mentioned treatment step is repeated at least once, that is, the above-mentioned steps of treating with a chelating agent solution, a hypertonic alkaline solution and a hypotonic alkaline solution are repeated at least once. Preferably, the above-mentioned treatment steps are repeated 3 to 5 times, and further preferably, the above-mentioned treatment steps are repeated 3 to 4 times.

[0063] In a specific embodiment provided by the present invention, after repeating the above treatment steps at least once, the treated membrane is further treated with a hypertonic acidic solution; the hypertonic acidic solution is preferably a sodium chloride-hydrochloric acid solution; the mass concentration of hydrochloric acid in the sodium chloride-hydrochloric acid solution is preferably 0.1% to 0.5%; optionally, the mass concentration of hydrochloric acid in the sodium chloride-hydrochloric acid solution is 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or a range between any two of the above values; the mass concentration of sodium chloride in the sodium chloride-hydrochloric acid solution is preferably 1% to 2%.

[0064] In a specific embodiment provided by the present invention, the temperature for treatment with a hypertonic acidic solution is preferably 15°C to 25°C, more preferably 20°C to 25°C; the number of treatments with a hypertonic acidic solution is preferably 3 to 5 times, more preferably 3 to 4 times; the time for each treatment with a hypertonic acidic solution is preferably 1 to 3 hours; optionally, the time for each treatment with a hypertonic acidic solution is 1 hour, 2 hours, 3 hours, or a range between any two of the above values.

[0065] In a specific embodiment provided by the present invention, after treatment with a hypertonic acidic solution, the solution is neutralized to neutrality with a sodium bicarbonate solution; the mass concentration of the sodium bicarbonate solution is preferably 0.5% to 5%, more preferably 0.5% to 3%, and even more preferably 1% to 2%.

[0066] In a specific embodiment provided by the present invention, after neutralization to neutrality, it is preferably washed with purified water until the conductivity of the washing liquid drops to 60 μS / cm.

[0067] In a specific embodiment provided by the present invention, after neutralization to neutrality or washing with purified water until the conductivity of the washing liquid drops to 60 μS / cm, freeze-drying is performed to obtain a cell-freeze matrix biofilm; the freeze-drying includes a pretreatment stage, a freezing stage, sublimation drying and desorption drying; the temperature of the pretreatment stage is preferably 0°C to 4°C, more preferably 4°C; the time of the pretreatment stage is preferably 1 to 3 hours; the temperature of the freezing stage is preferably -20°C to -40°C, more preferably -25°C to -35°C, and even more preferably -30°C; the time of the freezing stage is preferably 2 to 4 hours; the temperature of the sublimation drying is preferably 5°C to 10°C; the time of the sublimation drying is preferably 2 to 6 hours; specifically, the sublimation drying includes drying at 5°C for 1 to 3 hours and then drying at 10°C for 1 to 3 hours; the temperature of the desorption drying is preferably 20°C to 25°C; the time of the desorption drying is preferably 16 to 28 hours; specifically, the desorption drying includes drying at 20°C for 15 to 25 hours and then drying at 25°C for 1 to 3 hours.

[0068] In a specific embodiment provided by the present invention, the freeze-drying is specifically 4°C, 1h, -30°C, 3h, 5°C, 3h, 10°C, 2h, 20°C, 18h, 25°C, 3h.

[0069] In a specific embodiment provided by the present invention, the decellularized matrix biofilm is heated for thermal crosslinking treatment; the thermal crosslinking treatment is preferably carried out under vacuum conditions; the relative vacuum degree of the vacuum conditions is preferably -1 to -0.5 bar; the temperature of the thermal crosslinking treatment is preferably 100°C to 150°C; optionally, the temperature of the thermal crosslinking treatment is 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, or a range between any two of the above values; the thermal crosslinking treatment time is preferably 24 to 60 hours; optionally, the thermal crosslinking treatment time is 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, or a range between any two of the above values. Vacuum thermal crosslinking of the decellularized matrix biofilm can increase the crosslinking degree of collagen fibers in the biofilm, and the biofilm matrix powder prepared in this way has a higher viscosity after subsequent beating process, which is more conducive to improving the structural stability of the bone filling material.

[0070] In a specific embodiment provided by the present invention, the decellularized matrix biofilm is heated for thermal cross-linking treatment; the thermal cross-linking treatment is preferably carried out under vacuum conditions; the relative vacuum degree of the vacuum conditions is more preferably -1 bar; the temperature of the thermal cross-linking treatment is preferably 120°C; and the time of the thermal cross-linking treatment is preferably 48 hours.

[0071] In the present invention, unless otherwise specified, relative vacuum refers to the difference between the actual pressure in the vacuum dryer during the thermal crosslinking process and the atmospheric pressure, reflecting the degree of gas rarefaction in the dryer cavity.

[0072] In a specific embodiment provided by the present invention, in order to prevent the biofilm from absorbing moisture, the decellularized matrix biofilm should be crushed by a crusher after the vacuum heat crosslinking is completed, and matrix powders of different particle sizes are prepared by controlling the mesh aperture; the matrix powders of different particle sizes are completely different after subsequent beating. After beating, the slurry prepared by the small-particle matrix powder cannot form interweaving due to the small particle size, resulting in a low crosslinking degree in the later stage, which is not conducive to the structural stability of the bone filling material; while the matrix powder particle size increases, the matrix fibers will be interwoven after beating, and the crosslinking degree of the bone filling material will also increase in the later stage, which is conducive to improving the structural stability of the composite material. However, if the matrix powder particle size is too large, it is difficult to form a uniform slurry, which is not conducive to mixing with bone particles. Therefore, in the present invention, the particle size of the matrix powder is preferably 0.5 to 2 mm; optionally, the particle size of the matrix powder is 0.5 mm, 1 mm, 1.5 mm, 2 mm or a range between any two of the above values.

[0073] In a specific embodiment provided by the present invention, the matrix powder is mixed with a solvent and beaten; the solvent is any solvent well known to those skilled in the art and is not particularly limited. Taking into account the later biocompatibility of the material, the solvent is preferably a weak acid; since lower acidity will cause the bone filling material to exhibit higher cytotoxicity, thereby affecting product safety, the pH value of the weak acid in the present invention is preferably 4 to 6; optionally, the pH value of the weak acid is 4, 5, 6 or a range between any two of the above values; the weak acid can be an inorganic weak acid or an organic weak acid and is not particularly limited. In the present invention, it is preferably an organic weak acid, more preferably acetic acid and / or citric acid; the mass of the matrix powder is preferably 5% of the total mass of the matrix powder and the solvent The weight of the matrix powder is preferably 5%, 6%, 7%, 8%, 9%, 10% of the total weight of the matrix powder and the solvent, or a range between any two of the above values. The speed of the beating process is preferably 6000-10000 r / min. Optionally, the speed of the beating process is 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min, or a range between any two of the above values. The beating time is preferably 15-20 minutes. Optionally, the beating time is 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or a range between any two of the above values. The collagen fibers in the slurry obtained after beating are more fully swollen and interwoven with each other, forming a three-dimensional network structure that can better wrap the bone particles and greatly improve the structural stability of the bone filling material.

[0074] In a specific embodiment provided by the present invention, inorganic bone particles are added after beating; the inorganic bone particles are preferably obtained by removing fat and protein components from cancellous bone, and are more preferably prepared according to the following method: the cancellous bone is cut into slices, soaked in purified water to remove blood, dried, refluxed with an alkane organic solvent to remove fat components, and then refluxed with an amine organic solvent for deproteinization, washed until neutral, dried, and crushed to obtain inorganic bone particles; the thickness of the slices is preferably 0.5-2 cm, more preferably 0.8-1.5 cm, and more preferably 1 cm; the drying temperature after removing blood is preferably 100°C-120°C; the drying time after removing blood is preferably 10-14 h, more preferably 12 h; the alkane organic solvent is any alkane organic solvent well known to those skilled in the art, and is not particularly limited. In the present invention, petroleum ether and / or n-hexane are preferably used; the temperature of the reflux extraction is preferably 80°C-120°C, more preferably 100°C-120°C, and then The temperature of the reflux extraction is preferably 110°C; the number of cycles of the reflux extraction is preferably not less than 24 times; the amine organic solvent is any amine organic solvent well known to those skilled in the art and is not particularly limited. In the present invention, it is preferably one or more of propylamine, ethylenediamine and propylenediamine; the time of the deproteinization treatment is preferably 24 to 72 hours, more preferably 24 to 60 hours, and even more preferably 24 to 48 hours; the drying temperature after washing to neutrality is preferably 100°C to 180°C, more preferably 120°C to 160°C , and more preferably 150°C; the drying time after washing to neutrality is preferably 8 to 15 hours, more preferably 10 to 14 hours, and more preferably 12 hours; the particle size of the inorganic bone particles is preferably 0.25 to 1 mm; the mass ratio of the matrix powder to the inorganic bone particles is preferably (10:90) to (20:80); optionally, the mass ratio of the matrix powder to the inorganic bone particles is 10:90, 12:88, 15:85, 18:82, 20:80 or a range between any two of the above ratios.

[0075] In a specific embodiment provided by the present invention, inorganic bone particles are added and mixed, and then freeze-dried and thermally cross-linked to obtain a collagen-based bone filling material; the freeze-drying includes a pretreatment stage, a freezing stage, sublimation drying and analytical drying; the temperature of the pretreatment stage is preferably 0°C to 4°C, more preferably 4°C; the time of the pretreatment stage is preferably 1 to 3 hours; the temperature of the freezing stage is preferably -20°C to -40°C, more preferably -25°C to -35°C, and more preferably -30°C; the time of the freezing stage is preferably 3 to 6 hours; the temperature of the sublimation drying is preferably 5°C to 10°C; the time of the sublimation drying is preferably 2 to 6 hours; specifically, the sublimation drying includes first drying at 5°C for 1 to 3 hours, and then drying at 1 The thermal crosslinking treatment is preferably carried out under vacuum conditions; the relative vacuum degree of the vacuum conditions is preferably -1 to -0.5 bar, more preferably -1 bar; the temperature of the thermal crosslinking treatment is preferably 100°C to 120°C, more preferably 105°C to 115°C, and more preferably 110°C; the time of the thermal crosslinking treatment is preferably greater than or equal to 24h, more preferably 24 to 90h, more preferably 48 to 84h, more preferably 60 to 78h, and most preferably 72h.

[0076] In a specific embodiment provided by the present invention, the freeze-drying is specifically 4°C, 2h, -30°C, 5h, 5°C, 3h, 10°C, 2h, 20°C, 28h, 25°C, 2h.

[0077] The collagen-based bone filling material provided by the present invention is composed of acellular matrix biofilm and natural inorganic bone particles. The acellular matrix biofilm is subjected to thermal cross-linking treatment and then to crushing, swelling, and beating treatment to form a stable three-dimensional network structure. After physical cross-linking with the inorganic bone particles, the in vitro degradation time can be significantly improved. In addition, the bone filling material has no chemical reagent residue, not only has good biocompatibility, but also has good resilience and plasticity, and is easy to operate.

[0078] The present invention also provides a collagen-based bone filling material prepared by the above preparation method.

[0079] In a specific embodiment provided by the present invention, the collagen-based bone filling material is preferably composed of an acellular matrix biofilm and inorganic bone particles, and more preferably, the inorganic bone particles are interwoven with fibers of the acellular matrix biofilm. The main component of the acellular matrix biofilm is type I collagen, which promotes cell adhesion and proliferation, stimulates tissue growth, and provides nutritional support for bone regeneration in the defect area. The inorganic bone particles are granular inorganic salt materials purified from bovine bones. They are similar to the minerals in human bones and have a macroscopic and microscopic porous structure, which promotes new bone formation in the defect area.

[0080] In a specific embodiment provided by the present invention, the collagen-based bone filling material includes a three-dimensional network structure formed by acellular matrix biofilm and inorganic bone particles; the inorganic bone particles are distributed inside and on the surface of the three-dimensional network structure.

[0081] In a specific embodiment provided by the present invention, the collagen-based bone filling material has a porous structure, and the inorganic bone particles are evenly distributed inside and on the surface of the collagen-based bone filling material.

[0082] The present invention also provides a use of the collagen-based bone filling material in preparing a material for repairing bone defects.

[0083] To further illustrate the present invention, a collagen-based bone filling material, a preparation method thereof, and applications thereof provided by the present invention are described in detail below with reference to examples.

[0084] The reagents used in the following examples are all commercially available.

[0085] Example 1

[0086] 1.1 Preparation of decellularized matrix powder

[0087] Pretreatment: Fresh porcine peritoneal tissue was frozen at -20°C for 3 hours, then thawed in running water at 25°C. This process was repeated five times. The frozen-thawed peritoneum was placed on an ice pack with the rough surface facing up. Sodium bicarbonate powder was sprinkled on the peritoneal surface at a ratio of 0.5g of sodium bicarbonate per square centimeter. The tissue was rubbed repeatedly to remove surface fat and excess serosal membrane. The tissue was then rinsed with running water for 12 hours.

[0088] After pretreatment, the membrane was transferred to a 0.5 mol / L EDTA solution and shaken at 25°C for 15 hours. The membrane was then transferred to a 2% sodium hydroxide solution and shaken at room temperature for 2 hours. The membrane was then transferred to a 0.5% sodium hydroxide solution and shaken for 3 hours, with the sodium hydroxide solution being replaced every hour. The pretreatment steps were repeated three times (i.e., the steps of transferring to a 0.5 mol / L EDTA solution, a 2% sodium hydroxide solution, and a 0.5% sodium hydroxide solution were repeated in sequence). The membrane was then treated in a 0.5% hydrochloric acid-1% sodium chloride solution three times for 2 hours each. The membrane was then neutralized with a 1% sodium bicarbonate solution to a pH of 7.0. The membrane was then washed with purified water until the conductivity of the washings dropped to 60 μS / cm. The membrane was then removed and freeze-dried in a freeze dryer. The freeze-drying conditions were: 4°C for 1 hour, -30°C for 3 hours, 5°C for 3 hours, 10°C for 2 hours, 20°C for 18 hours, and 25°C for 3 hours. The freeze-dried membrane was then placed in a vacuum drying oven, the relative vacuum degree was set to -1 bar, and thermally cross-linked at 120°C for 48 hours, and finally the acellular matrix biomembrane 1 was obtained.

[0089] The decellularized matrix biofilm 1 was crushed by a crusher to obtain 1-2 mm matrix powder.

[0090] 1.2 Preparation of inorganic bone particles

[0091] Bovine cancellous bone was sliced ​​into approximately 1 cm thick slices, repeatedly rinsed with purified water to remove blood, and then dried at 100°C for 12 hours. The completely dried cancellous bone was placed in a Soxhlet extractor and the fat component was extracted with petroleum ether at reflux at 110°C, following 24 cycles of extraction to obtain defatted bone. The defatted bone was then deproteinized in a propylene diamine solution for 24 hours. After deproteinization, the bone tissue was removed and repeatedly rinsed with purified water until neutral, followed by drying at 150°C for 12 hours to obtain dried deproteinized bone. The dried bone tissue was then pulverized and sieved to obtain inorganic bovine cancellous bone particles of 0.25 mm to 1 mm in size.

[0092] The fat and protein content of the obtained inorganic bone particles were tested: 2 g of inorganic bone particles were taken and tested according to the first method, Soxhlet extraction method, in accordance with GB5009.6 2016 National Food Safety Standard - Determination of Fat in Foods, and the fat content test result was 0.85%; another 2 g of inorganic bone particles were taken and tested according to the second method, forin-phenol method (Lowry method), in accordance with Part IV 0731 of the Pharmacopoeia of the People's Republic of China, and the protein content test result was 0.06%.

[0093] The inorganic bone particles obtained by the above preparation method were subjected to X-ray diffraction analysis, and the X-ray diffraction pattern thereof was as follows: Figure 1As shown in the figure, the diffraction peak characteristics of the XRD curve are consistent with the ICDD PDF card No. 09-0432, proving that the main component of the inorganic bone particles is hydroxyapatite.

[0094] 1.3 Prepare acetic acid solution with a pH value of 4, add the matrix powder at a concentration of 5% to fully soak it, and use a high-speed disperser to beat the slurry at a speed of 6000r / min for 20min. Then use a rotational viscometer to measure its viscosity. The results are as follows: Figure 2 As shown. Inorganic bone particles were added in a mass ratio of matrix powder to inorganic bone particles of 20:80, and the materials were stirred for 2.5 hours using a dual planetary mixer to ensure thorough mixing. After stirring, the materials were placed in the corresponding molds and freeze-dried under the following conditions: 4°C for 2 hours, -30°C for 5 hours, 5°C for 3 hours, 10°C for 2 hours, 20°C for 28 hours, and 25°C for 2 hours. The freeze-dried samples were transferred to a vacuum drying oven for vacuum thermal crosslinking, with the specific temperature set at 110°C, relative vacuum at -1 bar, and time set for 72 hours, to obtain collagen-based bone filling material sample 1.

[0095] The collagen-based bone filling material sample 1 was immersed in the methylene blue solution for 2 minutes, and the photos of the collagen-based bone filling material sample 1 before and after absorption of the solution were obtained as shown in the figure. Figure 3 As shown, a is the collagen-based bone filling material sample 1 before liquid absorption, and b is the collagen-based bone filling material sample 1 after liquid absorption.

[0096] Determination of Decellularized Matrix Particle Size: To prevent moisture absorption by the biofilm, the decellularized matrix biofilm should be pulverized using a pulverizer immediately after vacuum thermal crosslinking. By controlling the mesh size, matrix powders of varying particle sizes were prepared: 0.05-0.2 mm, 0.5-1 mm, and 1-2 mm. Aside from the different particle sizes obtained by controlling the mesh size, the remaining steps for preparing the collagen-based bone filler material were identical to those described in 1.1-1.3. Studies have shown that the blending of matrix powders of varying particle sizes with acetic acid solution produces distinct states after whipping. After whipping, the slurry prepared with small-particle powders exhibits a reduced interweaving of the matrix particles due to their smaller particle size, resulting in a lower degree of crosslinking and poor structural stability. However, as the particle size increases, the matrix fibers intertwine after whipping, leading to a higher degree of crosslinking in the bone filler material. The crosslinking degrees of collagen-based bone fillers prepared with matrix powders of varying particle sizes, crosslinked under the same vacuum thermal crosslinking conditions, are shown in Table 1.

[0097] Cross-linking degree determination method:

[0098] 1. Sample testing:

[0099] 1.1 Sample preparation and testing:

[0100] Accurately weigh 11 mg of sample into a 50 mL stoppered test tube. Add 1 mL each of 4% NaHCO₃ solution and 0.5% TNBC solution. Heat in a 40°C water bath for 2 h. Add 3 mL of 6 mol / L hydrochloric acid solution and hydrolyze in a boiling water bath for 1 h to obtain a hydrolyzed collagen solution. Cool the resulting solution to room temperature, dilute with 5 mL of water, and extract with ether three times, 20 mL each time. Discard the ether layer. Pipette 5 mL of the aqueous phase solution into a test tube and heat in a boiling water bath for 15 min. Cool to room temperature, dilute with 15 mL of purified water, mix, and measure absorbance at 346 nm.

[0101] 1.2 Preparation and testing of blank samples:

[0102] Accurately weigh 11 mg of the freeze-dried sample (without vacuum thermal crosslinking) and place it in a 50 mL stoppered test tube. Add 1 mL of 4% NaHCO3 solution and heat it in a water bath at 40°C for 2 h. Then add 3 mL of 6 mol / L hydrochloric acid solution and 1 mL of 0.5% TNBC solution in sequence and mix well. Place it in a boiling water bath for hydrolysis for 1 h to obtain a hydrolyzed collagen solution. The other steps are the same as above.

[0103] 2. Calculation of cross-linking degree:

[0104] The calculation formula for the number of ε-amino groups in the lysine side chain in the sample (t) is:

[0105]

[0106] Where: A: absorbance value

[0107] 8: Dilution multiple

[0108] 5: 5 mL of solution after digestion

[0109] b: optical path

[0110] 14600: Molar absorption coefficient of trinitrobenzene derivatives (mL / (mmol·cm))

[0111] M: sample weight (mg)

[0112]

[0113] Table 1 Cross-linking degree of collagen-based bone filling materials prepared from matrix particles of different particle sizes

[0114] Matrix powder particle size (mm) Cross-linking degree of collagen bone filling material (%) 0.05～0.2 5.08 0.5～1 21.56 1～2 32.86

[0115] From the data in Table 1, we can see that the cross-linking degree of collagen-based bone filling materials increases with the increase of the particle size of the matrix powder used. The reason is that the three-dimensional network structures formed by the matrix powders with different particle sizes after mixing with the acetic acid solution are different. Figure 4 As shown, Figure 4 These are SEM images of slurries obtained after beating matrix powder of different particle sizes (a: 0.05-0.2 mm, b: 1-2 mm). It can be seen that larger matrix powder particles create a more solid and compact three-dimensional network structure. Matrix powder with smaller particle sizes has larger pores after beating, which is clearly not conducive to encapsulating bone particles.

[0116] The in vitro degradation of collagen-based bone filling materials prepared from matrix powders of different particle sizes was tested: Tris-HCl-CaCl2 solution was used as the buffer system, collagenase was added, and 100U / mL enzymatic hydrolysate was prepared. The samples were placed in 15mL centrifuge tubes, 5mL of enzymatic hydrolysate was added to each tube, and the samples were immersed in the enzymatic hydrolysate for 1 minute. After that, they were taken out and weighed and recorded as m0. Then, the centrifuge tubes were placed in a (37±1)℃ water bath. The block samples in the centrifuge tubes were taken out at 4h, 8h, 12h, 16h, 20h, and 24h, and the weights were recorded. The in vitro degradation diagrams of collagen-based bone filling materials prepared from matrix powders of different particle sizes were obtained as shown in the figure below. Figure 5 As shown (the particle size of the matrix powder of large particles is 1 to 2 mm, and the particle size of the matrix powder of small particles is 0.05 to 0.2 mm), Figure 5 The in vitro degradation test results show that the collagen bone filling material prepared with large-particle matrix powder takes longer to degrade, so the present invention selects matrix powder with a particle size of 1 to 2 mm as the raw material for preparing the collagen-based bone filling material.

[0117] Solvent determination: Solvents need to be added when the matrix powder and bone particles are mixed. Considering the later biocompatibility of the material, the solvents that can be selected include water, weak acid, phosphate buffer, physiological saline, etc.

[0118] The present invention takes organic weak acid as an example, uses acetic acid to prepare acid solutions with pH values ​​of 2, 3, and 4 respectively, and then adds matrix powder at a ratio of 5% by mass concentration of matrix powder. After stirring and soaking completely, it is beaten using a high-speed disperser. The viscosity of the slurry with different pH values ​​is as follows: Figure 6 As shown in the figure, it can be seen that the lower the pH value, the greater the degree of swelling of the matrix powder and the greater the viscosity of the slurry. Figure 6 It can be seen that although a lower pH value can make the matrix slurry have a higher viscosity after being whipped, the lower pH value can cause the collagen-based bone filling material to exhibit higher cytotoxicity, affecting product safety. The cytotoxicity (MTT colorimetric method) test results of collagen-based bone filling materials prepared with slurries of different pH values ​​are shown in Table 2. The data in Table 2 show that when the pH value of the slurry is lower than 3, the collagen-based bone filling material prepared therefrom is potentially cytotoxic (GB / T16886.5: Potential cytotoxicity exists when the cell viability is lower than 70%). Therefore, the present invention selects acetic acid solution with a pH value of 4 as the solvent.

[0119] Table 2 Average OD values ​​and survival rates of cytotoxicity assay

[0120] Group <![CDATA[OD 570 ]]> Survival rate (%) Samples with a pH of 2 0.458±0.002 58.2 Samples with a pH of 3 0.531±0.002 67.5 Samples with a pH of 4 0.624±0.001 79.3 Negative control 0.754±0.001 96 Medium control group 0.787±0.001 / Positive control 0.026±0.013 3

[0121] Whipping process: The applicant found that if the matrix slurry is simply stirred, the viscosity of the matrix slurry is poor, it cannot be tightly combined with the bone particles, and the material structure is easy to loosen. Therefore, a special treatment method is used to treat the matrix slurry, that is, a high-speed disperser is used to whip the matrix slurry after soaking at a speed of 6000-10000 r / min. It can be seen that the viscosity of the matrix slurry after whipping increases significantly, and the whipping time also has a great influence on the viscosity of the slurry. The viscosity of the slurry under the speed of 6000 r / min and different whipping times is as follows: Figure 7 As shown, it can be seen that the viscosity of the slurry increases with the extension of the beating time. Figure 8 The scanning electron microscope images of the unbeaten pulp and the pulp after beating at 6000r / min for 20min, where a is the unbeaten pulp and b is the beaten pulp. Figure 8 As can be seen, compared to unwhipped slurry, the collagen fibers in the whipped slurry swell more fully, interweaving the fibers together. The resulting three-dimensional network structure better encapsulates the bone particles, significantly improving the structural stability of the bone filling material. To ensure ease of subsequent mixing with bone particles, the present invention limits the whipping time to 15-20 minutes.

[0122] The slurry was beaten at a speed of 6000 r / min for 0 min, 10 min, 15 min, 20 min, and 25 min, and then mixed with inorganic bone particles according to the method in step 1.3 to prepare collagen-based bone filling materials. The in vitro degradability was determined as follows: Tris-HCl-CaCl2 solution was used as the buffer system, collagenase was added, 100 U / mL enzymatic hydrolysate was prepared, and the sample was placed in a 15 mL centrifuge tube, 5 mL enzymatic hydrolysate was added to each tube, and the sample was immersed in the enzymatic hydrolysate for 1 min, then taken out and weighed and recorded as m0. After that, the centrifuge tube was placed in a (37±1)°C water bath, and the block sample in the centrifuge tube was taken out at 4h, 8h, 12h, 16h, 20h, and 24h for weighing and recording the weight. The results are as follows: Figure 9 As shown, Figure 9 The in vitro degradation time of collagen-based bone filler prepared from slurries treated with different beating times is shown in Figure 2. The degradation time of the collagen-based bone filler increases with increasing beating time. Samples treated for 20 and 25 minutes have the same final degradation time, with both samples completely disintegrating within 24 hours. This demonstrates that the beating process employed in the present invention significantly increases the structural stability of the bone filler and prolongs its in vitro degradation time.

[0123] Example 2

[0124] The mass ratio of matrix powder to inorganic bone particles was changed to 10:90, and other conditions were the same as those in Example 1 to prepare collagen-based bone filling material sample 2.

[0125] Comparative Example 1

[0126] Preparation of acellular matrix powder

[0127] Pretreatment: Fresh porcine peritoneal tissue was frozen at -20°C for 3 hours, then thawed in running water at 25°C. This process was repeated five times. The frozen-thawed peritoneum was placed on an ice pack with the rough surface facing up. Sodium bicarbonate powder was sprinkled on the peritoneal surface at a ratio of 0.5g of sodium bicarbonate per square centimeter. The tissue was rubbed repeatedly to remove surface fat and excess serosal membrane. The tissue was then rinsed with running water for 12 hours.

[0128] After pretreatment, the membrane was transferred to a 0.5 mol / L EDTA solution and shaken at 25°C for 15 hours. The membrane was then transferred to a 2% sodium hydroxide solution and shaken at room temperature for 2 hours. The membrane was then transferred to a 0.5% sodium hydroxide solution and shaken for 3 hours, with the sodium hydroxide solution being replaced every hour. The above pretreatment steps were repeated three times (i.e., the steps of transferring to a 0.5 mol / L EDTA solution, a 2% sodium hydroxide solution, and a 0.5% sodium hydroxide solution were repeated in sequence). The membrane was then placed in a 0.5% hydrochloric acid-1% sodium chloride solution for treatment three times, each for 2 hours. The membrane was then neutralized with a 1% sodium bicarbonate solution to a pH of 7.0. The membrane was then washed with purified water until the conductivity of the washing solution dropped to 60 μS / cm. The membrane was removed and freeze-dried in a freeze dryer to obtain acellular matrix biomembrane 2.

[0129] The decellularized matrix biofilm 2 was crushed by a crusher to obtain 1-2 mm matrix powder.

[0130] Prepare acetic acid solution with a pH value of 4, add the matrix powder at a concentration of 5% to fully soak it, and then use a high-speed disperser to beat the slurry at a speed of 6000r / min for 20 minutes. Then use a rotational viscometer to measure its viscosity. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the viscosity of the matrix slurry prepared by crushing the biofilm after thermal cross-linking treatment is significantly higher than that of the matrix slurry prepared by crushing the biofilm without thermal cross-linking treatment.

[0131] Comparative Example 2

[0132] The mass ratio of matrix powder to inorganic bone particles was changed to 50:50, and other conditions were the same as those in Example 1 to prepare collagen-based bone filling material sample 3.

[0133] Comparative Example 3

[0134] The mass ratio of matrix powder to inorganic bone particles was changed to 40:60, and other conditions were the same as those in Example 1 to prepare collagen-based bone filling material sample 4.

[0135] Comparative Example 4

[0136] The mass ratio of matrix powder to inorganic bone particles was changed to 30:70, and other conditions were the same as those in Example 1 to prepare collagen-based bone filling material sample 5.

[0137] Comparative Example 5

[0138] Commercially available type I collagen (CAS: 9007-34-5) was dissolved in an acetic acid solution with a pH of 4 to a collagen concentration of 5%. After thorough infiltration, the mixture was blended using a high-speed disperser at 6000 rpm for 20 minutes. The mass ratio of collagen to inorganic bone particles (prepared in Example 1) was set at 20:80. The specific procedures were the same as in Example 1 to prepare collagen-based bone filling material sample 6.

[0139] The collagen-based bone filling material sample 1 obtained in Example 1 and the collagen-based bone filling material sample 6 obtained in Comparative Example 5 were analyzed using a scanning electron microscope, and the scanning electron microscope images thereof were as follows: Figure 10 As shown, a is collagen-based bone filling material sample 6, and b is collagen-based bone filling material sample 1. Figure 10 As can be seen, the microstructure of bone filling material sample 6, prepared using commercially available collagen (CAS: 9007-34-5), showed flaky collagen with large pores, resulting in poor resistance to enzymatic degradation and a short in vitro degradation time. In contrast, bone filling material sample 1, prepared using acellular matrix powder in Example 1, had a mesh-like collagen interior that tightly wrapped around the bone particles, resulting in better structural stability, strong resistance to enzymatic degradation, and a long in vitro degradation time.

[0140] Animal experimental research

[0141] Animal trials were conducted to study the effectiveness of bone filling materials with different proportions, and the optimal ratio of matrix powder to bone particles was determined based on the results of the animal trials.

[0142] Animal test model: Healthy adult New Zealand rabbits were used as test animals. Circular bone defects with a diameter of 6 mm and a depth of 2 mm were prepared in the edentulous area of ​​the left and right mandibles. Bone filling material was implanted in the defects, and then covered with absorbable biofilm as a physical barrier. The wounds were sutured (with surgical photos attached). Figure 11 ).

[0143] Evaluation criteria: All animals were euthanized after one month of feeding and sample collection. Micro-CT analysis was performed, and the results were shown in Table 3. New bone formation was analyzed. The optimal ratio was determined based on the test data.

[0144] BV / TV: bone volume / total tissue volume, which can directly reflect the changes in bone mass;

[0145] Tb.Th: average thickness of trabecular bone;

[0146] Tb.N: trabecular number, the number of intersections between bone tissue and non-bone tissue within a given length;

[0147] Tb.Sp: average trabecular spacing, the average width of the medullary cavity between trabeculae. When osteoporosis occurs, the value of Tb.Sp increases.

[0148] Table 3 Micro-CT detection data

[0149]

[0150] Note: n=6, *: P<0.05.

[0151] The data in Table 3 show that the BV / TV values ​​of sample 1 and sample 2 are significantly higher than those of the other sample groups, while the Tb.Sp values ​​are significantly lower than those of the other sample groups, indicating that the materials prepared according to the mass ratio of matrix powder to bone particles selected in the present invention can more effectively promote new bone formation compared with other samples.

[0152] Conclusion: Samples 1 and 2 have good plasticity and resilience, and can be tailored to repair bone defects of different shapes. They also have good bone repair effects. After implantation into the human body, they can act as new bone scaffolds to conduct bone and contribute to the formation and growth of new bones.

[0153] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a collagen-based bone filling material, characterized in that: The following steps are involved: S1) heating the decellularized matrix biofilm for thermal cross-linking, and then crushing the decellularized matrix biofilm to obtain matrix powder; S2) The matrix powder is mixed with a solvent and beaten, and then inorganic bone particles are added and mixed, and the mixture is freeze-dried and thermally cross-linked to obtain a collagen-based bone filling material.

2. The preparation method according to claim 1, characterized in that The acellular matrix biofilm is prepared according to the following method: A1) After repeated freeze-thaw treatment of peritoneal tissue, fat and excess serosal membrane were removed to obtain a pre-treated membrane; A2) treating the pre-treated membrane with a chelating agent solution, a hypertonic alkaline solution, and a hypotonic alkaline solution in sequence, repeating the above treatment steps at least once, and then treating the treated membrane with a hypertonic acidic solution, neutralizing it to neutrality, and freeze-drying it to obtain a decellularized matrix biomembrane; And / or, the inorganic bone particles are prepared according to the following method: The cancellous bone is cut into thin slices, soaked in purified water to remove blood, dried, and then refluxed with an alkane organic solvent to remove fat components. The slices are then refluxed with an amine organic solvent for deproteinization. The slices are washed to neutrality, dried, and crushed to obtain inorganic bone particles.

3. The preparation method according to claim 2, characterized in that In step A1), fat and excess serosal membrane are removed by repeatedly kneading with sodium bicarbonate powder; And / or, the chelating agent solution is selected from ethylenediaminetetraacetic acid solution; the molar concentration of the ethylenediaminetetraacetic acid solution is 0.1 to 0.5 mol / L; And / or, the hypertonic alkaline solution is selected from a high-concentration sodium hydroxide solution; the mass concentration of the high-concentration sodium hydroxide solution is 2% to 5%; and / or, the hypotonic alkaline solution is selected from a low concentration sodium hydroxide solution; The mass concentration of the low-concentration sodium hydroxide solution is 0.1% to 0.5%; and / or, the hypertonic acidic solution is selected from sodium chloride-hydrochloric acid solution; The mass concentration of hydrochloric acid in the sodium chloride-hydrochloric acid solution is 0.1% to 0.5%; the mass concentration of sodium chloride in the sodium chloride-hydrochloric acid solution is 1% to 2%.

4. The preparation method according to claim 2, characterized in that The temperature of treating the membrane after pretreatment with the chelating agent solution in step A2) is 15°C to 25°C, and the treatment time is 5 to 20 hours; And / or, in step A2), the membrane after pretreatment is treated with a hypertonic alkaline solution at a temperature of 15° C. to 25° C. for 1 to 5 hours; And / or, in step A2), the pre-treated membrane is treated with a hypotonic alkaline solution at a temperature of 15° C. to 25° C. for 1 to 5 hours; and / or, in the process of treating the pre-treated membrane with the hypotonic alkaline solution in step A2), the hypotonic alkaline solution is replaced 1 to 3 times; And / or, the number of times the above-mentioned treatment steps are repeated in step A2) is 3 to 5 times; And / or, the temperature for treating with the hypertonic acid solution in step A2) is 15° C. to 25° C., the number of times of treating with the hypertonic acid solution is 3 to 5 times, and the time of each treatment with the hypertonic acid solution is 1 to 3 hours.

5. The preparation method according to claim 1, characterized in that The temperature of the thermal crosslinking treatment in step S1) is 100°C to 150°C, and the time of the thermal crosslinking treatment is 24 to 60 hours; The temperature of the thermal crosslinking treatment in step S2) is 100° C. to 120° C.; the time of the thermal crosslinking treatment is greater than or equal to 24 hours; The thermal cross-linking treatment in step S1) and step S2) is performed under vacuum conditions; the relative vacuum degree of the vacuum conditions is -1 to -0.5 bar.

6. The preparation method according to claim 1, characterized in that The particle size of the matrix powder in step S1) is 0.5 to 2 mm; and / or, the particle size of the inorganic bone particles is 0.25 to 1 mm; And / or, the mass ratio of the matrix powder to the inorganic bone particles is (10:90) to (20:80).

7. The preparation method according to claim 1, characterized in that The mass of the matrix powder in step S2) is 5% to 10% of the total mass of the matrix powder and the solvent; And / or, the solvent in step S2) is selected from a weak acid; the pH value of the weak acid is 4-6; the weak acid is selected from acetic acid and / or citric acid.

8. The preparation method according to claim 1, characterized in that The whipping process has a rotation speed of 6000 to 10000 r / min, and a whipping process time of 15 to 20 minutes.

9. The collagen-based bone filling material prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the collagen-based bone filling material prepared by the preparation method according to any one of claims 1 to 8 in the preparation of bone defect repair materials.

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