A collagen-based bone filling material and a preparation method and application thereof

Abstract

The application provides a preparation method of a collagen-based bone filling material, comprising the following steps: S1) heating and performing heat crosslinking treatment on a decellularized matrix biomembrane, and then crushing the decellularized matrix biomembrane to obtain a matrix powder; S2) mixing the matrix powder with a solvent to perform whipping treatment, then adding inorganic bone particles to mix, and then performing freeze-drying and heat crosslinking treatment to obtain the collagen-based bone filling material. Compared with the prior art, the collagen-based bone filling material provided by the application is composed of a decellularized matrix biomembrane and natural inorganic bone particles, wherein the decellularized matrix biomembrane is subjected to heat crosslinking treatment, and then is subjected to crushing, swelling and whipping treatment, so that a stable three-dimensional network structure is formed; after physical crosslinking of the inorganic bone particles, the in-vitro degradation time is significantly prolonged; the bone filling material has no chemical reagent residue, has good biocompatibility, good resilience and plasticity, and is convenient to operate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of materials, and particularly relates to a collagen-based bone filling material and a preparation method and application thereof. BACKGROUND

[0002] Bone is one of the most important organs of the human body, and bone defects caused by trauma, tumors and infections are very common in clinical practice. At present, although there are various treatment methods for bone defects, each has its own limitations. Autologous bone transplantation is limited by the limited donor; allogeneic bone has the risk of disease transmission and immune rejection; various bone repair materials made of metal and high polymer materials are mostly implanted as permanent bodies and cannot be degraded, and the effect is often unsatisfactory. Therefore, it is an urgent need in clinical treatment of bone defects to prepare an ideal artificial bone material.

[0003] An ideal artificial bone material should have good bone conduction, bone induction, biological safety and clinical operability; at the same time, it should have a suitable 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 marketed and used in clinical practice, mainly including hydroxyapatite, hydroxyapatite / collagen composite, mineralized collagen and other artificial synthetic materials. However, the current pure hydroxyapatite has the problems of easy particle shedding and difficult operation; the hydroxyapatite / collagen composite and the mineralized collagen use chemical cross-linking process to enhance the structural stability, and have the disadvantages of cytotoxicity (Chinese patents with publication numbers CN106139255, CN104096268, CN105358189B, etc.).

[0005] The applicant found in the research that the matrix and bone particles are difficult to form a stable structure by physical mixing, and the composite material formed has the phenomena of easy shedding of bone particles and easy loosening of the structure after water absorption; and the compounding ratio of the matrix and the bone particles is also a key factor affecting the effectiveness of the bone filling material. In the process of bone defect repair, bone particles play a major role, and a too low ratio of bone particles may affect the osteogenesis effect at the bone defect site, and a too high ratio may affect the plasticity of the product. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide a collagen-based bone filling material with high structural stability by physical cross-linking and a preparation method and application thereof.

[0007] The present application provides a preparation method of a collagen-based bone filling material, comprising the following steps:

[0008] S1) heating and cross-linking a decellularized matrix biomembrane, crushing the cross-linked biomembrane to obtain a matrix powder;

[0009] S2) mixing the matrix powder with solvent and whipping, then adding inorganic bone particles, after freeze-drying and thermal cross-linking, obtaining collagen-based bone filling material.

[0010] Preferably, the decellularized matrix biological membrane is prepared by the following method:

[0011] A1) treating the peritoneal tissue by repeated freeze-thawing, removing fat and excess serosa, obtaining a pretreated membrane;

[0012] A2) treating the pretreated membrane with chelating agent solution, high osmotic alkaline solution and low osmotic alkaline solution in turn, repeating the above steps at least once, then treating the membrane with high osmotic acid solution, and then neutralizing to neutral, freeze-drying, obtaining a decellularized matrix biological membrane;

[0013] and / or, the inorganic bone particles are prepared by the following method:

[0014] After cutting the cancellous bone into thin slices, soaking in purified water to remove blood, drying, refluxing with alkane organic solvent to remove fat components, and then deproteinizing with amine organic solvent, washing to neutral, drying, and crushing, obtaining inorganic bone particles.

[0015] Preferably, in step A1), the fat and excess serosa are removed by rubbing 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 0.1-0.5 mol / L;

[0017] and / or, the high osmotic alkaline solution is selected from high concentration sodium hydroxide solution; the mass concentration of the high concentration sodium hydroxide solution is 2%-5%;

[0018] and / or, the low osmotic alkaline solution is selected from low concentration sodium hydroxide solution; the mass concentration of the low concentration sodium hydroxide solution is 0.1%-0.5%;

[0019] and / or, the high osmotic acid 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%-0.5%; the mass concentration of sodium chloride in the sodium chloride-hydrochloric acid solution is 1%-2%.

[0020] Preferably, in step A2), the temperature for treating the pretreated membrane with chelating agent solution is 15-25°C, and the treatment time is 5-20 h;

[0021] and / or, in step A2), the temperature for treating the pretreated membrane with high osmotic alkaline solution is 15-25°C, and the treatment time is 1-5 h;

[0022] and / or, the temperature for treating the pretreated membrane with the low-osmotic alkaline solution in step A2) is 15-25℃, and the treating time is 1-5h;

[0023] and / or, the low-osmotic alkaline solution is replaced 1-3 times during the treating process of the pretreated membrane with the low-osmotic alkaline solution in step A2);

[0024] and / or, the number of repeating the above treating step in step A2) is 3-5 times;

[0025] and / or, the temperature for treating the pretreated membrane with the high-osmotic acid solution in step A2) is 15-25℃, and the number of treating the pretreated membrane with the high-osmotic acid solution is 3-5 times, and the treating time for each treating with the high-osmotic acid solution is 1-3h.

[0026] Preferably, the temperature for the heat cross-linking treatment in step S1) is 100-150℃, and the heat cross-linking treatment time is 24-60h;

[0027] the temperature for the heat cross-linking treatment in step S2) is 100-120℃, and the heat cross-linking treatment time is greater than or equal to 24h;

[0028] the heat cross-linking treatment in step S1) and step S2) is carried out under vacuum condition, and the relative vacuum degree of the vacuum condition is -1 to -0.5bar.

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

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

[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%-10% of the total mass of the matrix powder and the solvent;

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

[0034] Preferably, the rotating speed for the whipping treatment is 6000-10000r / min, and the whipping treatment time is 15-20min.

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

[0036] The application further provides application of the collagen-based bone filling material prepared by the preparation method in preparation of bone defect repair materials.

[0037] The application provides a preparation method of a collagen-based bone filling material, comprising the following steps: S1) performing heat crosslinking treatment on a decellularized matrix biomembrane, and then crushing the decellularized matrix biomembrane to obtain matrix powder; S2) mixing the matrix powder with a solvent to perform whipping treatment, and then mixing inorganic bone particles, and then performing freeze-drying and heat crosslinking treatment to obtain the collagen-based bone filling material. Compared with the prior art, the collagen-based bone filling material provided by the application is composed of a decellularized matrix biomembrane and natural inorganic bone particles, wherein the decellularized matrix biomembrane is subjected to heat crosslinking treatment, and then is subjected to crushing, swelling and whipping treatment to form a stable three-dimensional network structure, and then is subjected to physical crosslinking with the inorganic bone particles to significantly prolong the in-vitro degradation time, and the bone filling material has no chemical reagent residue, has good biocompatibility, and has good resilience and plasticity, and is convenient to operate. BRIEF DESCRIPTION OF DRAWINGS

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

[0039] Figure 2 It is a viscosity comparison chart of the slurry (after crosslinking) after whipping treatment in Example 1 of the application and the slurry (without crosslinking) after whipping treatment in Comparative Example 1;

[0040] Figure 3 It is a photo of a sample 1 of the collagen-based bone filling material obtained in Example 1 of the application before liquid absorption (a) and after liquid absorption (b);

[0041] Figure 4 It is a scanning electron microscope (SEM) image of the slurry after whipping treatment of the small-particle-size (a) matrix powder and the large-particle-size (b) matrix powder in Example 1 of the application;

[0042] Figure 5 It is a graph of in-vitro degradation test results of the collagen-based bone filling materials prepared by using the large-particle-size matrix powder and the small-particle-size matrix powder respectively in Example 1 of the application;

[0043] Figure 6 It is a viscosity comparison chart of the slurries with different pH values in Example 1 of the application;

[0044] Figure 7 It is a viscosity comparison chart of the slurries treated for different whipping times in Example 1 of the application;

[0045] Figure 8 It is a scanning electron microscope (SEM) image of the slurry (a) without whipping treatment and the slurry (b) after whipping treatment in Example 1 of the application;

[0046] Figure 9 Figure 1 shows the in vitro degradation time graph of the collagen-based bone filling material prepared from the slurry treated with different whipping times in Example 1 of the present application;

[0047] Figure 10 Figure 5 shows the scanning electron microscope graph of the collagen-based bone filling material sample 1(b) obtained in Example 1 of the present application and the collagen-based bone filling material sample 6(a) obtained in Comparative Example 5.

[0048] Figure 11 Figure 6 shows the surgical scene for the animal experiment of the present application. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0050] The present application provides a preparation method of a collagen-based bone filling material, comprising the following steps: S1) heating and performing heat crosslinking treatment on a decellularized matrix biomembrane, and then crushing to obtain a matrix powder; S2) mixing the matrix powder with a solvent to perform whipping treatment, and then adding inorganic bone particles to mix, and then performing freeze-drying and heat crosslinking treatment to obtain the collagen-based bone filling material.

[0051] In the present application, there is no special limitation on the source of all raw materials, which can be commercially available or self-made.

[0052] According to the present application, the decellularized matrix biomembrane is preferably prepared according to the following method: A1) repeatedly performing freeze-thaw treatment on peritoneal tissue, and then removing fat and excess serosa to obtain a pretreated membrane; A2) sequentially treating the pretreated membrane with a chelating agent solution, a high-osmotic alkaline solution and a low-osmotic alkaline solution, repeating the above treatment steps at least once, then treating the treated membrane with a high-osmotic acid solution, and then neutralizing to neutral, and then freeze-drying to obtain the decellularized matrix biomembrane.

[0053] In a specific embodiment provided in the present application, the peritoneal tissue is the peritoneal tissue of mammals familiar to those skilled in the art, including but not limited to pigs, cows, dogs, sheep, rabbits and mice, etc.

[0054] In one specific embodiment provided by the present application, the fresh peritoneal tissue is treated by repeated freeze-thawing, and then the fat and excess serosa are removed to obtain the pretreated membrane; the freezing temperature in the freeze-thawing is preferably -10℃ to -30℃, more preferably -15℃ to -25℃, and even more preferably -20℃; the freezing time is preferably 1 to 5 hours, more preferably 2 to 4 hours, and even more preferably 3 hours; the thawing is preferably performed in flowing water at 15℃ to 25℃, more preferably at 20℃ to 25℃, and even more preferably at 25℃; and the number of times of the repeated freeze-thawing is preferably 3 to 5, more preferably 4 to 5, and even more preferably 5.

[0055] In one specific embodiment provided by the present application, after the freeze-thawing, the fat and excess serosa are removed by repeatedly rubbing with sodium bicarbonate powder; preferably, after the freeze-thawing, the fat and excess serosa are removed by repeatedly rubbing with sodium bicarbonate powder under low temperature; more preferably, after the freeze-thawing, the freeze-thawed peritoneal tissue is placed on an ice bag with the rough surface facing upward, and sodium bicarbonate powder is sprinkled on the surface of the peritoneal tissue to remove the fat and excess serosa by repeatedly rubbing; and the amount of the sodium bicarbonate powder is preferably 0.1 to 1 g / cm 2 , more preferably 0.3 to 0.6 g / cm 2 , and even more preferably 0.5 g / cm 2 .

[0056] In one specific embodiment provided by the present application, after the fat and excess serosa are removed, the membrane is washed with flowing water to obtain the pretreated membrane; and the washing time is preferably 8 to 18 hours, more preferably 10 to 15 hours, and even more preferably 12 hours.

[0057] In one specific embodiment provided by the present application, the pretreated membrane is sequentially treated with a chelating agent solution, a high-ionic-strength alkaline solution and a low-ionic-strength alkaline solution; the chelating agent solution is preferably an ethylenediaminetetraacetic acid (EDTA) solution; the molar concentration of the EDTA solution is preferably 0.1-0.5 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 high-ionic-strength alkaline solution is preferably a high-concentration sodium hydroxide solution; the mass concentration of the high-concentration sodium hydroxide solution is preferably 2%-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 low-ionic-strength alkaline solution is preferably a low-concentration sodium hydroxide solution; the mass concentration of the low-concentration sodium hydroxide solution is preferably 0.1%-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 one specific embodiment provided by the present application, the temperature at which the pretreated membrane is treated with the chelating agent solution is preferably 15-25°C, more preferably 20-25°C; the time for which the pretreated membrane is treated with the chelating agent solution is preferably 5-20 h; optionally, the time for which the pretreated membrane is treated with the chelating agent solution is 5 h, 8 h, 10 h, 12 h, 15 h, 18 h, 20 h or a range between any two of the above values.

[0059] In one specific embodiment provided by the present application, the temperature at which the pretreated membrane is treated with the high-ionic-strength alkaline solution is preferably 15-25°C, more preferably 20-25°C; the time for which the pretreated membrane is treated with the high-ionic-strength alkaline solution is preferably 1-5 h; optionally, the time for which the pretreated membrane is treated with the high-ionic-strength alkaline solution is 1 h, 2 h, 3 h, 4 h, 5 h or a range between any two of the above values.

[0060] In one specific embodiment provided by the present application, the temperature at which the pretreated membrane is treated with the low-ionic-strength alkaline solution is preferably 15-25°C, more preferably 20-25°C; the time for which the pretreated membrane is treated with the low-ionic-strength alkaline solution is preferably 1-5 h; optionally, the time for which the pretreated membrane is treated with the low-ionic-strength alkaline solution is 1 h, 2 h, 3 h, 4 h, 5 h or a range between any two of the above values.

[0061] In one specific embodiment provided by the present application, the low-ionic-strength alkaline solution is replaced 1-3 times during the treatment of the pretreated membrane with the low-ionic-strength alkaline solution, preferably 2-3 times.

[0062] In one specific embodiment provided by the present application, the above-mentioned treatment steps are repeated at least once, i.e. the above-mentioned treatment steps using the chelating agent solution, the high-osmotic alkaline solution and the low-osmotic alkaline solution are repeated at least once. Preferably, the above-mentioned treatment steps are repeated 3 to 5 times, and more preferably, the above-mentioned treatment steps are repeated 3 to 4 times.

[0063] In one specific embodiment provided by the present application, after the above-mentioned treatment steps are repeated at least once, the treated membrane is further treated with a high-osmotic acid solution. The high-osmotic acid 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-mentioned values. The mass concentration of sodium chloride in the sodium chloride-hydrochloric acid solution is preferably 1% to 2%.

[0064] In one specific embodiment provided by the present application, the temperature for treating with the high-osmotic acid solution is preferably 15°C to 25°C, and more preferably 20°C to 25°C. The number of times for treating with the high-osmotic acid solution is preferably 3 to 5 times, and more preferably 3 to 4 times. The time for treating with the high-osmotic acid solution each time is preferably 1 to 3 hours. Optionally, the time for treating with the high-osmotic acid solution each time is 1 hour, 2 hours, 3 hours or a range between any two of the above-mentioned values.

[0065] In one specific embodiment provided by the present application, after treating with the high-osmotic acid solution, the treated membrane is neutralized to neutral 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 one specific embodiment provided by the present application, after being neutralized to neutral, the treated membrane is preferably washed with purified water until the conductivity of the washing solution is reduced to 60 μS / cm.

[0067] In a specific embodiment provided by the application, after neutralization to neutral or washing with purified water until the conductivity of the washing solution is reduced to 60 μS / cm, the acellular matrix biomembrane is obtained by freeze-drying; the freeze-drying comprises a pretreatment stage, a freezing stage, sublimation drying and desorption drying; the temperature of the pretreatment stage is preferably 0-4°C, more preferably 4°C; the time of the pretreatment stage is preferably 1-3 h; the temperature of the freezing stage is preferably -20 to -40°C, more preferably -25 to -35°C, and more preferably -30°C; the time of the freezing stage is preferably 2-4 h; the temperature of the sublimation drying is preferably 5-10°C; the time of the sublimation drying is preferably 2-6 h; specifically, the sublimation drying comprises first drying at 5°C for 1-3 h, and then drying at 10°C for 1-3 h; the temperature of the desorption drying is preferably 20-25°C; the time of the desorption drying is preferably 16-28 h; specifically, the desorption drying comprises first drying at 20°C for 15-25 h, and then drying at 25°C for 1-3 h.

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

[0069] In a specific embodiment provided by the application, the acellular matrix biomembrane is heated for heat crosslinking treatment; the heat crosslinking treatment is preferably performed under vacuum conditions; the relative vacuum degree of the vacuum conditions is preferably -1 to -0.5 bar; the temperature of the heat crosslinking treatment is preferably 100-150°C; optionally, the temperature of the heat 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 time of the heat crosslinking treatment is preferably 24-60 h; optionally, the time of the heat crosslinking treatment is 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, 60 h, or a range between any two of the above values. Vacuum heat crosslinking treatment of the acellular matrix biomembrane can increase the crosslinking degree of collagen fibers in the biomembrane, so that the viscosity of the slurry obtained after the subsequent whipping process of the biomembrane matrix powder is greater, which is more conducive to improving the structural stability of the bone filling material.

[0070] In a specific embodiment provided by the application, the acellular matrix biomembrane is heated for heat crosslinking treatment; the heat crosslinking treatment is preferably performed under vacuum conditions; the relative vacuum degree of the vacuum conditions is more preferably -1 bar; the temperature of the heat crosslinking treatment is preferably 120°C; the time of the heat crosslinking treatment is preferably 48 h.

[0071] In the present application, relative vacuum degree refers to the difference between the actual pressure in the vacuum drying machine during the heat cross-linking process and the atmospheric pressure, reflecting the degree of gas thinning in the drying machine cavity, unless otherwise specified.

[0072] In a specific embodiment provided by the present application, to prevent the biological membrane from absorbing moisture, the decellularized matrix biological membrane should be crushed by a crusher immediately after the vacuum heat cross-linking is completed, and different particle sizes of matrix powder are prepared by controlling the mesh size. Different particle sizes of matrix powder have completely different states after subsequent whipping. The slurry prepared by small particle size matrix powder cannot form interweaving between the matrix powder particles after whipping, resulting in low cross-linking degree in the later stage, which is not conducive to the structural stability of the bone filling material. The matrix fiber will interweave together after whipping when the particle size of the matrix powder increases, and the cross-linking degree of the bone filling material in the later stage will also increase, which is conducive to improving the structural stability of the composite material. However, if the particle size of the matrix powder is too large, it is not easy to form a uniform slurry, which is not conducive to the mixing with bone particles. Therefore, the particle size of the matrix powder is preferably 0.5-2 mm in the present application. Alternatively, 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 in the present application, the matrix powder is mixed with a solvent for whipping treatment; the solvent can be any solvent known to those skilled in the art, and there is no special limitation, and the solvent is preferably a weak acid in consideration of the biocompatibility of the material in the later stage; since lower acidity will lead to higher cytotoxicity of the bone filling material, thereby affecting the safety of the product, therefore in the present application, the pH value of the weak acid is preferably 4-6; 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 inorganic or organic, and there is no special limitation, and the present application preferably is an organic weak acid, more preferably acetic acid and / or citric acid; the mass of the matrix powder is preferably 5%-10% of the total mass of the matrix powder and the solvent; optionally, the mass of the matrix powder is 5%, 6%, 7%, 8%, 9%, 10% or a range between any two of the above values of the total mass of the matrix powder and the solvent; the rotation speed of the whipping treatment is preferably 6000-10000r / min; optionally, the rotation speed of the whipping treatment is 6000r / min, 7000r / min, 8000r / min, 9000r / min, 10000r / min or a range between any two of the above values; the whipping treatment time is preferably 15-20min; optionally, the whipping time is 15min, 16min, 17min, 18min, 19min, 20min or a range between any two of the above values. After whipping, the collagen fibers inside the slurry swell more fully, the fibers interweave together, and the three-dimensional network structure formed can have better wrapping degree for bone particles, and can greatly improve the structural stability of the bone filling material.

[0074] In a specific embodiment provided in the present application, the inorganic bone particles are mixed after the whipping process; the inorganic bone particles are preferably obtained by removing fat components and protein components from cancellous bone, and more preferably prepared by the following method: the cancellous bone is cut into thin slices, blood is removed by soaking in purified water, dried, and then fat components are removed by reflux extraction with an alkane organic solvent, and then deproteinization treatment is performed by reflux with an amine organic solvent, washed to neutral, dried, crushed, and inorganic bone particles are obtained; the thickness of the thin 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 100-120°C; the drying time after removing blood is preferably 10-14 h, and more preferably 12 h; the alkane organic solvent can be any alkane organic solvent known to those skilled in the art without special limitation, and in the present application, petroleum ether and / or n-hexane are preferred; the reflux extraction temperature is preferably 80-120°C, more preferably 100-120°C, and more preferably 110°C; the reflux extraction cycle number is preferably not less than 24 times; the amine organic solvent can be any amine organic solvent known to those skilled in the art without special limitation, and in the present application, one or more of propylamine, ethylenediamine, and propylenediamine are preferred; the deproteinization treatment time is preferably 24-72 h, more preferably 24-60 h, and more preferably 24-48 h; the drying temperature after washing to neutral is preferably 100-180°C, more preferably 120-160°C, and more preferably 150°C; the drying time after washing to neutral is preferably 8-15 h, more preferably 10-14 h, and more preferably 12 h; the particle size of the inorganic bone particles is preferably 0.25-1 mm; the mass ratio of the matrix powder to the inorganic bone particles is preferably (10:90)-(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 application, the inorganic bone particles are added and mixed, and then subjected to freeze-drying and thermal crosslinking treatment to obtain the collagen-based bone filling material; the freeze-drying comprises a pretreatment stage, a freezing stage, sublimation drying and desorption drying; the temperature of the pretreatment stage is preferably 0-4°C, more preferably 4°C; the time of the pretreatment stage is preferably 1-3h; the temperature of the freezing stage is preferably -20- -40°C, more preferably -25- -35°C, and more preferably -30°C; the time of the freezing stage is preferably 3-6h; the temperature of the sublimation drying is preferably 5-10°C; the time of the sublimation drying is preferably 2-6h; specifically, the sublimation drying comprises first drying at 5°C for 1-3h, and then drying at 10°C for 1-3h; the temperature of the desorption drying is preferably 20-25°C; the time of the desorption drying is preferably 21-33h; specifically, the desorption drying comprises first drying at 20°C for 20-30h, and then drying at 25°C for 1-3h; the thermal crosslinking treatment is preferably carried out under vacuum conditions; the relative vacuum degree of the vacuum conditions is preferably -1--0.5bar, more preferably -1bar; the temperature of the thermal crosslinking treatment is preferably 100-120°C, more preferably 105-115°C, and more preferably 110°C; the time of the thermal crosslinking treatment is preferably greater than or equal to 24h, more preferably 24-90h, more preferably 48-84h, more preferably 60-78h, and most preferably 72h.

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

[0077] The collagen-based bone filling material provided by the present application is composed of a decellularized matrix biomembrane and natural inorganic bone particles, wherein the decellularized matrix biomembrane is subjected to thermal crosslinking treatment, and then subjected to crushing, swelling and whipping treatment to form a stable three-dimensional network structure, and then is subjected to physical crosslinking with the inorganic bone particles to significantly increase the in-vitro degradation time, and the bone filling material has no chemical reagent residue, has good biocompatibility, good resilience and plasticity, and is convenient to operate.

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

[0079] In a specific embodiment provided by the present application, the collagen-based bone filling material is preferably composed of a decellularized matrix biomembrane and inorganic bone particles, and more preferably formed by fibers of the decellularized matrix biomembrane interweaving and wrapping the inorganic bone particles. The main component of the decellularized matrix biomembrane is collagen type I, which has the effects of promoting cell adhesion and proliferation and stimulating tissue growth, and provides nutritional support for bone regeneration in the defect area. The inorganic bone particles are granular inorganic salt materials prepared by purifying bovine bone, which are similar to the minerals in human bone and have macroscopic and microscopic porous structures, thus being helpful to new bone formation in the defect area.

[0080] In a specific embodiment provided by the present application, the collagen-based bone filling material comprises a three-dimensional network structure formed by a decellularized matrix biomembrane and inorganic bone particles, and the inorganic bone particles are distributed in the interior and on the surface of the three-dimensional network structure.

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

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

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

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

[0085] Example 1

[0086] 1.1 Preparation of decellularized matrix powder

[0087] Pre-treatment: fresh porcine peritoneal tissue was frozen at -20℃ for 3h, and then thawed in flowing water at 25℃, and the freezing and thawing was repeated for 5 times. The thawed peritoneal tissue was placed on an ice bag with the rough surface facing upward, and 0.5g of sodium bicarbonate was added per square centimeter to the surface of the peritoneal tissue, and the sodium bicarbonate powder was rubbed repeatedly to remove the fat and excess serous membrane on the surface, and then the tissue was washed with flowing water for 12h.

[0088] After the pretreatment, the membrane was transferred into a 0.5 mol / L EDTA solution, and shaken for 15 h at 25℃, then transferred into a 2% sodium hydroxide solution, and shaken for 2 h at room temperature, and then transferred into a 0.5% sodium hydroxide solution, and shaken for 3 h, during which the sodium hydroxide solution was replaced every 1 h. The above-mentioned pretreatment steps were repeated 3 times (i.e., the operations of transferring into the 0.5 mol / L EDTA solution, 2% sodium hydroxide solution, and 0.5% sodium hydroxide solution were repeated in turn). Then the membrane was transferred into a 0.5% hydrochloric acid-1% sodium chloride solution for 3 times, each for 2 h, and then neutralized to a pH value of 7.0 using a 1% sodium bicarbonate solution, and washed with purified water until the conductivity of the washing solution decreased to 60 μS / cm, and then the membrane was taken out and freeze-dried in a freeze dryer under the following conditions: 4℃ for 1 h, -30℃ for 3 h, 5℃ for 3 h, 10℃ for 2 h, 20℃ for 18 h, and 25℃ for 3 h. The freeze-dried membrane was then placed in a vacuum drying oven, and set to a relative vacuum degree of -1 bar, and heat-crosslinked at 120℃ for 48 h, and finally a decellularized matrix biomembrane 1 was obtained.

[0089] The decellularized matrix biomembrane 1 was crushed by a crusher to obtain a matrix powder with a size of 1-2 mm.

[0090] 1.2 Preparation of inorganic bone particles

[0091] The cancellous bone of a cow was cut into slices with a thickness of about 1 cm, and repeatedly soaked and washed with purified water to remove blood, and then dried at 100℃ for 12 h. The completely dried cancellous bone was placed in a Soxhlet extractor, and the fat component was extracted by reflux extraction with petroleum ether at an extraction temperature of 110℃ for 24 cycles to obtain defatted bone. The defatted bone was placed in a propylene diamine solution for 24 h for deproteinization treatment. After the deproteinization was completed, the bone tissue was taken out, repeatedly washed with purified water until neutral, and then dried at 150℃ for 12 h to obtain dried deproteinized bone. The dried bone tissue was crushed and sieved to obtain inorganic bone particles of the cancellous bone of the cow with a size of 0.25-1 mm.

[0092] The obtained inorganic bone particles were detected for fat content and protein content: 2 g of the inorganic bone particles were detected according to GB5009.6 2016 National Food Safety Standard-Determination of Fat in Foods-First Method Soxhlet Extraction Method, and the fat content detection result was 0.85%; and another 2 g of the inorganic bone particles were detected according to the Protein Content Determination Method Second Method Folin Phenol Method (Lowry Method) in the Fourth Volume of the Pharmacopoeia of the People's Republic of China 0731, and the protein content detection result was 0.06%.

[0093] The inorganic bone particles obtained according to the above preparation method were subjected to X-ray diffraction analysis, and the X-ray diffraction spectrum thereof is shown in FIG. 1. Figure 1As shown in the figure, the diffraction peak characteristics of the XRD curve conform to ICDD PDF card No. 09-0432, proving that the main component of inorganic bone particles is hydroxyapatite.

[0094] 1.3 Prepare an acetic acid solution with a pH of 4. Add the matrix powder at a ratio of 5% by mass, ensuring thorough wetting. Then, use a high-speed disperser to agitate the slurry at 6000 r / min for 20 min. Finally, measure the viscosity using a rotational viscometer. The results are as follows: Figure 2 As shown. Inorganic bone particles were added according to a mass ratio of matrix powder:inorganic bone particles = 20:80. The material was stirred using a dual planetary mixer for 2.5 hours to ensure thorough and uniform mixing. After stirring, the material was placed into the corresponding molds and freeze-dried under the following conditions: 4℃ for 2 hours, -30℃ for 5 hours, 5℃ for 3 hours, 10℃ for 2 hours, 20℃ for 28 hours, and 25℃ for 2 hours. The freeze-dried samples were then transferred to a vacuum drying oven for vacuum thermal cross-linking. The specific temperature settings were 110℃, relative vacuum of -1 bar, and time of 72 hours, yielding collagen-based bone filling material sample 1.

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

[0096] Determination of decellularized matrix powder 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 sieve aperture, matrix powders of different particle sizes were prepared, specifically 0.05–0.2 mm, 0.5–1 mm, and 1–2 mm. Apart from the different particle sizes obtained by controlling the sieve aperture, the other steps in preparing collagen-based bone filler materials are the same as described in sections 1.1–1.3 above. The study found that the states of matrix powders of different particle sizes after mixing with acetic acid solution and undergoing whipping treatment were completely different. The slurry prepared from small-particle-size matrix powder, due to its small particle size, could not form interweaving between matrix powder particles after whipping, resulting in a lower degree of crosslinking in the later stages, which was detrimental to the structural stability of the bone filler material. Conversely, with larger matrix powder particle sizes, the matrix fibers would interweave after whipping, and the degree of crosslinking of the bone filler material would increase accordingly. The degree of crosslinking of collagen-based bone filler materials prepared from matrix powders of different particle sizes under the same conditions (vacuum thermal crosslinking) is shown in Table 1.

[0097] Method for determining the degree of crosslinking:

[0098] 1. Sample testing:

[0099] 1.1 Sample preparation and testing:

[0100] Precisely weigh 11 mg of sample into a 50 mL test tube with a stopper, add 1 mL of 4% NaHCO3 solution and 1 mL of 0.5% TNBC solution, heat in a water bath at 40°C for 2 h, then 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. After cooling to room temperature, add 5 mL of water to dilute, extract with diethyl ether 3 times, 20 mL each time, and discard the diethyl ether layer. Take 5 mL of aqueous solution and place it in a test tube in a boiling water bath for 15 min, then cool to room temperature, add 15 mL of purified water to dilute, mix well, and measure the absorbance at a wavelength of 346 nm.

[0101] 1.2 Preparation and detection of blank sample:

[0102] Precisely weigh 11 mg of sample (not vacuum heat crosslinked) into a 50 mL test tube with a stopper, add 1 mL of 4% NaHCO3 solution, heat 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, mix well, and hydrolyze in a boiling water bath for 1 h to obtain a hydrolyzed collagen solution. The other steps are the same as above.

[0103] 2. Calculation of crosslinking degree:

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

[0105]

[0106] A: absorbance value

[0107] 8: dilution factor

[0108] 5: 5 mL solution after digestion

[0109] b: optical path

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

[0111] M: sample weight (mg)

[0112]

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

[0114] Matrix powder particle size (mm) Collagen bone filler cross-linking degree (%) 0.05～0.2 5.08 0.5～1 21.56 1～2 32.86

[0115] From the data in Table 1, it can be seen that the crosslinking degree of the collagen-based bone filling material increases with the increase of the particle size of the matrix powder used. The reason is that the three-dimensional network structures formed after the mixing of matrix powders of different particle sizes with acetic acid solution and whipping treatment are different, as shown in Figure 4 the figure.Figure 4 The SEM images of the slurry obtained after whipping treatment of the matrix powder with different particle sizes (the particle size of a is 0.05-0.2 mm, and the particle size of b is 1-2 mm) can be seen that the larger the particle size of the matrix powder, the more three-dimensional network structure formed is stereoscopic and compact. The matrix powder with small particle size has larger holes after whipping, and obviously this structure is not conducive to the wrapping of bone particles.

[0116] The in-vitro degradation of the collagen-based bone filling material prepared from the matrix powder with different particle sizes was tested: a Tris-HCl-CaCl2 solution was used as a buffer system, collagenase was added, and 100 U / mL enzyme solution was configured, the sample was placed in a 15 mL centrifuge tube, 5 mL enzyme solution was added to each tube, the sample was immersed in the enzyme solution for 1 min, then taken out and weighed and recorded as m0, then the centrifuge tube was placed in a (37±1) ℃ water bath, and the block sample in the centrifuge tube was taken out at 4 h, 8 h, 12 h, 16 h, 20 h and 24 h for weighing, and the weight was recorded, and the in-vitro degradation of the collagen-based bone filling material prepared from the matrix powder with different particle sizes was obtained as shown in Figure 5 , wherein the particle size of the large-particle matrix powder is 1-2 mm, and the particle size of the small-particle matrix powder is 0.05-0.2 mm, Figure 5 The in-vitro degradation test results show that the collagen-based bone filling material prepared from the matrix powder with large particle size has a longer degradation time, and therefore the matrix powder with a particle size of 1-2 mm is selected as the raw material for preparing the collagen-based bone filling material.

[0117] Solvent determination: a solvent needs to be added when the matrix powder is mixed with bone particles, and considering the biocompatibility of the material in the later stage, the solvent can be water, weak acid, phosphate buffer, physiological saline and the like.

[0118] Taking organic weak acid as an example, acetic acid is used to configure acid solutions with pH values of 2, 3 and 4, respectively, then the matrix powder is added according to a proportion of 5% of the mass concentration of the matrix powder, and after complete stirring and immersion, whipping is performed using a high-speed dispersion machine. The viscosities of the slurries with different pH values are shown in Figure 6 It can be seen that the lower the pH value, the greater the swelling degree of the matrix powder, and the greater the viscosity of the slurry. It can be known from Figure 6 that although a lower pH value can make the matrix slurry have a higher viscosity after whipping, a lower pH value can cause the collagen-based bone filling material to have higher cytotoxicity, affecting the safety of the product. The cytotoxicity (MTT colorimetric method) test results of the collagen-based bone filling material prepared from the slurries with different pH values are shown in Table 2, and it can be known from the data in Table 2 that when the pH value of the slurry is lower than 3, the collagen-based bone filling material prepared therefrom has potential cytotoxicity (when the cell survival rate is lower than 70%, there is potential cytotoxicity), and therefore the acetic acid solution with a pH value of 4 is selected as the solvent.

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

[0120] Group OD 570 ]] Survival rate (%) Sample with pH value of 2 0.458±0.002 58.2 Sample with pH value of 3 0.531±0.002 67.5 Sample with pH value 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 simply using stirring method, the substrate slurry has poor viscosity and cannot be tightly combined with bone particles, and the material structure is easy to be loose. Therefore, the substrate slurry is treated by a special treatment method, that is, the substrate slurry after soaking is whipped by a high-speed dispersion machine at a speed of 6000-10000 r / min. It can be seen that the viscosity of the substrate slurry after whipping increases obviously, and the whipping time also has a great influence on the viscosity of the slurry. The viscosity of the slurry treated at a speed of 6000 r / min for different whipping times is shown in Table 1. Figure 7 As can be seen from Table 1, the viscosity of the slurry increases with the extension of the whipping time. Figure 8 The scanning electron micrographs of the un-whipped slurry and the slurry treated by whipping at a speed of 6000 r / min for 20 min are shown in Figure 1, wherein a is the un-whipped slurry, and b is the slurry after whipping treatment. Figure 8 As can be seen from Figure 1, compared with the un-whipped slurry, the collagen fibers in the slurry after whipping treatment swell more fully, and the fibers interweave together to form a three-dimensional network structure which can better wrap the bone particles and greatly improve the structural stability of the bone filling material. Considering the operability of subsequent mixing with bone particles, the whipping treatment time is controlled to 15-20 min in the present application.

[0122] The slurry is whipped at a speed of 6000 r / min for 0 min, 10 min, 15 min, 20 min and 25 min respectively, and then mixed with inorganic bone particles according to the method in step 1.3 to prepare a collagen-based bone filling material. The in-vitro degradation of the collagen-based bone filling material is measured according to the following method: taking Tris-HCl-CaCl2 solution as the buffer system, adding collagenase to prepare 100 U / mL enzyme solution, taking the sample into a 15 mL centrifuge tube, adding 5 mL enzyme solution into each tube, taking out the sample after immersing in the enzyme solution for 1 min, weighing and recording as m0, then placing the centrifuge tube in a (37±1)℃ water bath, taking out the block sample in the centrifuge tube at 4h, 8h, 12h, 16h, 20h and 24h for weighing, and recording the weight. The results are shown in Figure 2. Figure 9 As can be seen from Figure 2, the degradation time of the collagen-based bone filling material increases with the extension of the whipping time. The samples treated by whipping for 20 min and 25 min have the same final degradation time, both of which are completely scattered at 24h. Figure 9 Figure 3 is a graph of the in-vitro degradation time of the collagen-based bone filling material prepared from the slurry treated by different whipping times. As can be seen from Figure 3, the degradation time of the collagen-based bone filling material increases with the extension of the whipping time. The samples treated by whipping for 20 min and 25 min have the same final degradation time, both of which are completely scattered at 24h. It can be seen that the whipping process adopted in the present application can significantly increase the structural stability of the bone filling material and prolong the in-vitro degradation time thereof.

[0123] Example 2

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

[0125] Comparative Example 1

[0126] Preparation of the decellularized matrix powder

[0127] Pre-treatment: fresh porcine peritoneal tissue was frozen at -20°C for 3 h, and then thawed in flowing water at 25°C for 12 h. The thawed peritoneal tissue was placed on an ice bag with the rough surface facing up, and 0.5 g of sodium bicarbonate powder was added per square centimeter and rubbed repeatedly to remove the fat and excess serous membrane on the surface.

[0128] After the pre-treatment, the membrane was transferred into a 0.5 mol / L EDTA solution, shaken for 15 h at 25°C, and then transferred into a 2% sodium hydroxide solution, shaken for 2 h at room temperature. The membrane was then transferred into a 0.5% sodium hydroxide solution and shaken for 3 h, with the sodium hydroxide solution being replaced every 1 h. The above-mentioned pre-treatment steps were repeated 3 times (i.e., the operations of transferring into 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 3 times, each for 2 h, and then neutralized to a pH value of 7.0 using a 1% sodium bicarbonate solution. The membrane was washed with purified water until the conductivity of the washing solution decreased to 60 μS / cm, and then freeze-dried in a freeze dryer to obtain a decellularized matrix biological membrane 2.

[0129] The decellularized matrix biological membrane 2 was crushed using a crusher to obtain a matrix powder with a size of 1-2 mm.

[0130] An acetic acid solution with a pH value of 4 was prepared, and the matrix powder was added at a mass concentration of 5% to allow it to be fully infiltrated. A high-speed dispersing machine was used to whip the slurry at a speed of 6000 r / min for 20 min, and then a rotary viscometer was used to measure the viscosity of the slurry, with the results shown in Table 1. Figure 2 As can be seen from Table 1, the viscosity of the matrix slurry prepared by crushing the biological membrane after the heat crosslinking treatment was significantly higher than that of the matrix slurry prepared by crushing the biological membrane without the heat crosslinking treatment. Figure 2

[0131] Comparative Example 2

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

[0133] Comparative Example 3 ​

[0134] By changing the mass ratio of matrix powder to inorganic bone particles to 40:60, and keeping other conditions the same as in Example 1, collagen-based bone filling material sample 4 was prepared.

[0135] Comparative Example 4

[0136] By changing the mass ratio of matrix powder to inorganic bone particles to 30:70, and keeping other conditions the same as in Example 1, collagen-based bone filling material sample 5 was prepared.

[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, resulting in a collagen mass concentration of 5%. After thorough soaking, the slurry was whipped at 6000 rpm for 20 minutes using a high-speed disperser. The mass ratio of collagen to inorganic bone particles (prepared in Example 1) was set to 20:80, and the specific operation was the same as in Example 1, resulting in 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 scanning electron microscopy, and their scanning electron micrographs are shown below. Figure 10 As shown, sample a is collagen-based bone filling material sample 6, and sample b is collagen-based bone filling material sample 1. Figure 10 It can be seen that the bone filler sample 6, prepared with commercially available collagen (CAS: 9007-34-5), exhibits a microstructure in which the internal collagen is sheet-like with large pores, resulting in poor resistance to enzymatic degradation and a short in vitro degradation time. In contrast, the bone filler sample 1, prepared using decellularized matrix powder in Example 1, has an internal collagen network that tightly encapsulates bone particles, resulting in better structural stability, strong resistance to enzymatic degradation, and a longer in vitro degradation time.

[0140] Animal experimental research

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

[0142] Animal experimental model: Healthy adult New Zealand rabbits were used as experimental animals. Circular bone defects with a diameter of 6 mm and a depth of 2 mm were created in the edentulous areas of the left and right mandibles. Bone filling material was implanted into the defects, followed by covering with an absorbable biofilm as a physical barrier, and the wounds were sutured. (Surgical photos attached) Figure 11 ).

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

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

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

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

[0147] Tb.Sp: mean trabecular spacing, the average width of the marrow cavity between the trabecular bone, which increases when osteoporosis occurs.

[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 other sample groups, and the Tb.Sp values are significantly smaller than those of other sample groups, indicating that the material prepared according to the mass ratio of the matrix powder and bone particles selected in the application can more effectively promote new bone formation compared to other samples.

[0152] Conclusion: Sample 1 and sample 2 have good plasticity and resilience, and can be suitable for repairing bone defects of different shapes by cutting; at the same time, they have good bone repair effect, and can play a bone conduction role as a new bone scaffold after being implanted into the human body, which is helpful for new bone formation and growth.

[0153] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A method for preparing a collagen-based bone filling material, characterized by, The method comprises the following steps: S1) heating and cross-linking the decellularized matrix biological membrane, and then crushing the membrane to obtain a matrix powder; the particle size of the matrix powder is 0.5-2 mm; S2) mixing the matrix powder with a solvent, whipping the mixture, adding natural inorganic bone particles, and then performing freeze-drying and heat cross-linking to obtain a collagen-based bone filling material; In step S2), the mass of the matrix powder is 5-10% of the total mass of the matrix powder and the solvent; The solvent in step S2) is selected from weak acids; the pH value of the weak acid is 4-6; The whipping speed is 6000-10000 r / min; the whipping time is 15-20 min; The particle size of the natural inorganic bone particles is 0.25-1 mm; The mass ratio of the matrix powder to the natural inorganic bone particles is (10:90)-(20:80).

2. The production method according to claim 1, characterized by, The decellularized matrix biological membrane is prepared by the following method: A1) repeatedly freezing and thawing peritoneal tissue, removing fat and excess serosa to obtain a pretreated membrane; A2) sequentially treating the pretreated membrane with a chelating agent solution, a high-osmotic alkaline solution and a low-osmotic alkaline solution, repeating the above steps at least once, then treating the membrane with a high-osmotic acid solution, neutralizing to neutral, and freeze-drying to obtain a decellularized matrix biological membrane; And / or, the inorganic bone particles are prepared by the following method: Cutting cancellous bone into thin slices, soaking in purified water to remove blood, drying, refluxing with an alkane organic solvent to remove fat components, and then performing deproteinization treatment with an amine organic solvent, washing to neutral, drying, and crushing to obtain inorganic bone particles.

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

4. The production method according to claim 2, characterized by, In step A2), the temperature for treating the pretreated membrane with the chelating agent solution is 15-25°C, and the treatment time is 5-20 h; And / or, in step A2), the temperature for treating the pretreated membrane with the high-osmotic alkaline solution is 15-25°C, and the treatment time is 1-5 h; And / or, in step A2), the temperature for treating the pretreated membrane with the low-osmotic alkaline solution is 15-25°C, and the treatment time is 1-5 h; And / or, in step A2), the low-osmotic alkaline solution is replaced 1-3 times during the treatment of the pretreated membrane with the low-osmotic alkaline solution. And / or, the number of times of repeating the above-mentioned processing steps in the step A2) is 3-5 times; And / or, the temperature of the treatment with the hypertonic acid solution in the step A2) is 15-25℃, the number of times of the treatment with the hypertonic acid solution is 3-5 times, and the time of each treatment with the hypertonic acid solution is 1-3 h.

5. The preparation method according to claim 1, characterized in that, The temperature of the heat cross-linking treatment in the step S1) is 100-150℃, and the time of the heat cross-linking treatment is 24-60 h. The temperature of the heat cross-linking treatment in the step S2) is 100-120℃, and the time of the heat cross-linking treatment is greater than or equal to 24 h. The heat cross-linking treatment in the step S1) and the step S2) is carried out under vacuum condition, and the relative vacuum degree of the vacuum condition is -1--0.5 bar.

6. The method of claim 1, wherein, The particle size of the matrix powder in the step S1) is 1-2 mm.

7. The preparation method according to claim 1, characterized in that, The mass of the matrix powder in the step S2) is 5%-8% of the total mass of the matrix powder and the solvent. And / or, the pH value of the weak acid is 4-5, and the weak acid is selected from acetic acid and / or citric acid.

8. The method of claim 1, wherein, The rotating speed of the whipping treatment is 6000-8000 r / min, and the time of the whipping treatment is 18-20 min.

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

10. The use of the collagen-based bone filling material prepared by the preparation method of any one of claims 1-8 in the preparation of bone defect repairing materials.

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