Bone repair material with mineralized collagen layered structure and preparation method thereof

Abstract

The invention relates to a bone repair material with a mineralized collagen layered structure and a preparation method thereof, and the preparation method comprises the following steps: regulating and controlling related parameters of mineralized collagen suspensions required for forming each mineralized collagen layer in the mineralized collagen layered structure, and regulating and controlling technological parameters of freeze-drying treatment; the pore sizes from the bottom mineralized collagen layer to the top mineralized collagen layer are gradually increased in sequence; a mineralized collagen suspension corresponding to an upper mineralized collagen layer suitable for forming two adjacent mineralized collagen layers covers the surface of a lower mineralized collagen layer which is formed through freeze drying treatment, and permeates into a pore channel which is formed through freeze drying treatment in the lower mineralized collagen layer; and a wet interface interpenetration area is formed, and a continuous compact interface is formed after pressure applying and freeze drying treatment. The mineralized collagen material with obvious hierarchical differentiation and controllable gradient can be constructed, interlayer continuity and mechanical strengthening are further achieved, and the overall compression performance, the peel strength and the fatigue stability of the material are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bone repair materials, in particular to a bone repair material with a layered structure of mineralized collagen and a preparation method thereof. BACKGROUND

[0002] Natural bone is a hierarchical composite of "organic matrix (type I collagen) + inorganic mineral (hydroxyapatite, HA)", with mineralization inside / outside collagen, nanocrystal orientation and pore hierarchy at the microscale, and density and mechanical gradient of cortical bone-cancellous bone at the macroscale. This hierarchical and gradient structure determines the load-bearing, conduction and regeneration capacity of bone, so the biomimetic materials in tissue engineering need to consider the mineralization mechanism, hierarchical pores, interlayer continuity and interface mechanical stability. Mineralized collagen (MC) bone repair material is a biomaterial that simulates the composition and structure of natural bone tissue for bone defect repair. It is mainly composed of collagen and minerals, where collagen provides the organic framework; minerals are usually hydroxyapatite, etc., which give the material certain hardness and strength to support the growth of bone tissue and bear mechanical load. This material has good biological activity and can form a close bond with the surrounding tissue, promoting the formation and growth of new bone, and has a wide application prospect in the field of bone repair. With the development of bone repair materials, it has become possible to prepare bone repair materials with various complex hierarchical structures and functions. Especially in the field of bone tissue engineering, it is necessary to control the pore structure and porosity of bone repair material scaffolds to promote cell growth and autologous bone tissue regeneration. In the past decade, a large number of studies have shown that "mineralized collagen" is superior to pure collagen or single HA in terms of osteogenic-related cell adhesion, differentiation and osteogenic induction, but it is limited to complex defect repair scenarios due to the lack of overall mechanical and structural controllability.

[0003] The main approaches to biomimetic construction of "mineralized collagen" include: ① Polymer-induced liquid precursor (PILP) and other approaches to realize intra-fiber mineralization in (collagen-like) collagen, improve the biomimetic degree and mechanics of collagen-HA composite (including doping such as Sr to tune the lattice and biological response); ② Complex with degradable polymers (such as polylactic acid (PLA), polycaprolactone (PCL)) to improve designability and formability; ③ Use low-temperature solidification / freeze-drying to obtain high-porosity three-dimensional scaffolds. However, most of the work still focuses on "single pore structure and one-time forming", which has limited structure and performance space, making it difficult to reproduce the layered / gradient characteristics of real bone.

[0004] To access the true transition between bone-soft tissue or cortical-cancellous bone, the "gradient / multilayer" scaffold design has emerged in recent years: through the continuous or segmented changes in composition, pore size, and mechanics in space, the cell migration, angiogenesis, and interface integration are improved; the preparation methods involve freeze casting / directional solidification, layer-by-layer self-assembly (LbL), multi-channel / microfluidic, and 3D printing. However, how to obtain a stable interlayer interface in the "water-rich collagen / mineralized collagen system" and achieve repeatable and scalable manufacturing is still a difficulty for many reviews and original studies.

[0005] In terms of freeze-drying / freeze casting forming, the process can control the pore orientation and size through temperature gradient and ice crystal templating, which is a common method for building porous scaffolds; however, if "one-time forming" is used, only single-layer, uniform or gradually changing but not layered pore structures can be obtained; if "multiple pouring and stacking are directly followed by unified freeze-drying", the interlayer is mostly physically attached, affected by shrinkage, interfacial moisture migration and recrystallization, and is prone to delamination or mechanical weak zone. The review on freeze casting and freeze drying also points out that multilayer / gradient design should consider "stable pore structure within the layer" and "continuity between layers", otherwise the overall mechanical and fatigue resistance cannot meet the requirements of load-bearing parts.

[0006] From the published prior art, there are technical solutions for "multilayer collagen / HA scaffolds". For example, the prior art discloses a multilayer collagen (containing HA or different types of collagen / GAG) freeze-dried layered structure, the basic steps of which are: preparing a first suspension and freeze-drying into a first layer → (if necessary) rehydrating the freeze-dried layer → pouring the next layer of suspension → freeze-drying again, and repeating to obtain a multilayer structure. However, this technical idea emphasizes "rehydration / pouring / re-freezing" layer-by-layer stacking, which can obtain layered bodies of different formulations, but does not involve active engineering means for interlayer interface strength (such as interpenetration reconstruction and compaction densification), and the interlayer bonding mainly depends on the physical lapping and dry consolidation after rehydration.

[0007] On the other hand, there are a lot of experiences in the field of polymer and composite scaffolds for interface enhancement, such as improving interfacial peel and shear resistance through interfacial interdiffusion, thermal / pressure densification or introducing transition phase; related reviews and experimental studies point out that the processing window (temperature / pressure / time) and the formation of interfacial diffusion / interpenetrating network are the key control quantities for determining the interlayer strength. However, it is still lacking of a systematic freeze-drying-re-wetting-compaction-re-freezing solution to transfer these controllable interface engineering means to the "mineralized collagen interface in wet state" which is water-rich, heat-sensitive and contains inorganic particles.

[0008] Based on the above analysis, there are the following technical gaps or defects in the prior art: 1. Mineralized collagen-based bone repair materials have recognized advantages in osteogenic induction and structural simulation, but the single structure caused by one-time forming limits the application of load-bearing and complex defect repair. 2. Existing "multi-layer / gradient" strategies focus on formula or aperture differences, and the interface between layers is often physically attached or rehydrated and bonded, lacking the interface engineering process of "wetting interpenetration + cold compaction + independent freeze-drying". 3. Although there are proposals for "multi-layer freeze-drying stacking" in existing technologies, there is a lack of parameterized control and scalable manufacturing path for "wet interface reconstruction - compaction - shaping", making it difficult to stably obtain a unified body with high interface strength and mechanical gradient. 4. Consistent reviews point out that to achieve a biomimetic level close to cortical bone-cancellous bone, it is necessary to have a coordinated design of controllable intra-layer pore structure + continuous dense interface between layers + replicable process.

[0009] The above existing technical gaps or defects are the core technical problems that the technical scheme of the present application aims to solve. SUMMARY

[0010] The technical scheme of the present application relates to the field of biological medical materials and tissue engineering scaffold preparation, and particularly relates to a multi-layer mineralized collagen bone repair material prepared by layer-by-layer independent freeze-drying forming, interlayer solution wetting interpenetration, and interface hammering / cold compaction densification technology. The material has controllable layered structure, porosity gradient and mineralization gradient, can realize stable interlayer bonding, and can simulate the hierarchical structure characteristics of natural bone tissue.

[0011] The technical scheme of the present application fills the technical gap that existing mineralized collagen materials can only achieve single structure or simple laminated structure, provides a multi-layer mineralized collagen material preparation method with controllable interface, designable structure, and industrialized repeated construction, has significant novelty and creativity, and is suitable for three types of bone repair medical devices.

[0012] The bone repair material with mineralized collagen layered structure provided by the technical scheme has the following preparation method: more than one portion of mineralized collagen suspension is prepared respectively, each portion of the mineralized collagen suspension is subjected to freeze-drying treatment in sequence to form each layer of the mineralized collagen layer in which the mineralized collagen layered structure is stacked in the thickness direction, at least one of the related parameters of each portion of the mineralized collagen suspension and the process parameters of the freeze-drying treatment is adjusted to make the pore size of the mineralized collagen layer at the bottom to the mineralized collagen layer at the top of the formed mineralized collagen layered structure gradually increase, and a continuous dense interface is formed between two adjacent layers of the mineralized collagen layer by the following method: the mineralized collagen suspension suitable for forming the mineralized collagen layer at the upper layer of the two adjacent layers of the mineralized collagen layer is covered on the surface of the mineralized collagen layer at the lower layer which has been formed by the freeze-drying treatment, and is left to stand for a preset time to make it penetrate into the pore formed by the freeze-drying treatment in the mineralized collagen layer at the lower layer, and a wet interface interpenetration zone is formed, pressure is applied to the wet interface interpenetration zone, and the freeze-drying treatment is performed on the mineralized collagen suspension covered on the surface of the mineralized collagen layer at the lower layer and the wet interface interpenetration zone after the pressure is applied to form the continuous dense interface.

[0013] Optionally, the related parameters of the mineralized collagen suspension include at least one of the collagen concentration, the inorganic phase ratio, the viscosity, the solid content, the target pH value and the adjustment rate thereof, and the process parameters of the freeze-drying treatment include at least one of the pre-freezing temperature, the cooling rate of the freezing stage, and the vacuum degree, the heating rate and the sublimation rate of the vacuum sublimation drying stage.

[0014] Optionally, the adjustment of the related parameters of each portion of the mineralized collagen suspension includes: the collagen concentration, the inorganic phase ratio, the viscosity, the solid content, the target pH value and the adjustment rate thereof of the mineralized collagen suspension suitable for forming the mineralized collagen layer at the bottom to the mineralized collagen layer at the top gradually decrease, and the adjustment of the process parameters of the freeze-drying treatment includes: the cooling rate, the vacuum degree, the heating rate and the sublimation rate of the freeze-drying treatment suitable for forming the mineralized collagen layer at the bottom to the mineralized collagen layer at the top gradually decrease, and the pre-freezing temperature gradually increases.

[0015] Optionally, the gradual decrease of the target pH value and the adjustment rate thereof of the mineralized collagen suspension suitable for forming the mineralized collagen layer at the bottom to the mineralized collagen layer at the top includes: an alkaline solution is added into each portion of the mineralized collagen suspension suitable for forming the mineralized collagen layer at the bottom to the mineralized collagen layer at the top in a gradually decreasing adjustment rate from the same initial pH value to a gradually decreasing target pH value.

[0016] Optionally, the alkaline solution is one of a sodium hydroxide solution, a potassium hydroxide solution, an ammonia water solution, a sodium carbonate solution and a sodium bicarbonate solution.

[0017] Optionally, the preparing of the plurality of mineralized collagen suspensions respectively comprises: mixing a collagen solution, a mineral solution and a phosphoric acid solution to prepare the plurality of mineralized collagen suspensions.

[0018] Optionally, the preparing of the plurality of mineralized collagen suspensions respectively comprises: mixing a collagen solution and a nano-hydroxyapatite powder to prepare the plurality of mineralized collagen suspensions.

[0019] Optionally, the preparing of the plurality of mineralized collagen suspensions respectively comprises: sequentially adding a sodium dihydrogen phosphate solution and a calcium chloride solution into a collagen solution to prepare the plurality of mineralized collagen suspensions.

[0020] Optionally, the plurality of mineralized collagen suspensions are obtained by dividing a mineralized collagen suspension prepared by the same preparation process of the mineralized collagen suspension into a plurality of portions, or each of the plurality of mineralized collagen suspensions is prepared by the same preparation process of the mineralized collagen suspension.

[0021] Optionally, the method further comprises: before the freeze-drying treatment, concentrating each of the plurality of mineralized collagen suspensions with the adjusted parameters (including pH value); and the concentrating comprises at least one of dialysis by loading the mineralized collagen suspension into a dialysis bag, suction filtration by using a Buchner funnel, and pressure removal of water.

[0022] Optionally, the target pH value of the mineralized collagen suspension and the decreasing rate of the target pH value gradually decrease from the mineralized collagen layer adapted to form the bottom layer to the mineralized collagen layer adapted to form the top layer.

[0023] Optionally, the pressure applied to the wet interfacial interpenetration zone of the mineralized collagen layer closer to the bottom layer is greater than the pressure applied to the wet interfacial interpenetration zone of the mineralized collagen layer farther from the bottom layer.

[0024] Optionally, the pressure is applied to the wet interfacial interpenetration zone by hammering or cold pressing.

[0025] Optionally, the preset time of the mineralized collagen suspension of the mineralized collagen layer located in the lower layer is greater than or equal to the preset time of the mineralized collagen suspension of the mineralized collagen layer located in the upper layer.

[0026] Optionally, the mineralized collagen layered structure comprises 2-10 layers of mineralized collagen layers.

[0027] Optionally, the mineralized collagen layered structure comprises a first mineralized collagen layer and a second mineralized collagen layer stacked in sequence, and is prepared by: placing a prepared first portion of mineralized collagen suspension into a mold, and forming the first mineralized collagen layer by a first freeze-drying process; adding a prepared second portion of mineralized collagen suspension to a surface of the first mineralized collagen layer, and allowing it to infiltrate into pores formed by the first freeze-drying process in the first mineralized collagen layer for a first preset time to form a first wet interfacial interpenetration zone; applying a first pressure to the first wet interfacial interpenetration zone, and performing a second freeze-drying process on the first wet interfacial interpenetration zone after the first pressure is applied and the second portion of mineralized collagen suspension covering the surface of the first mineralized collagen layer to form a first continuous dense interface and the second mineralized collagen layer; the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value and its adjustment rate of the first portion of mineralized collagen suspension are greater than or equal to the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value and its adjustment rate of the second portion of mineralized collagen suspension, respectively; the cooling rate, vacuum degree, warming rate and sublimation rate of the first freeze-drying process are greater than or equal to the cooling rate, vacuum degree, warming rate and sublimation rate of the second freeze-drying process, respectively, and the pre-freezing temperature of the first freeze-drying process is lower than or equal to the pre-freezing temperature of the second freeze-drying process.

[0028] Optionally, the mineralized collagen layered structure further comprises a third mineralized collagen layer stacked on the second mineralized collagen layer, and the preparation of the mineralized collagen layered structure further comprises: adding a prepared third portion of mineralized collagen suspension to the surface of the second mineralized collagen layer, and allowing it to infiltrate into the pores formed in the second mineralized collagen layer by the second freeze-drying process for a second predetermined time to form a second wet interfacial interpenetration zone; applying a second pressure to the second wet interfacial interpenetration zone, and performing a third freeze-drying process on the second wet interfacial interpenetration zone after the application of the second pressure and the third portion of mineralized collagen suspension covering the surface of the second mineralized collagen layer to form a second continuous dense interface and the third mineralized collagen layer; the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value and its adjustment rate of the second portion of mineralized collagen suspension are greater than or equal to the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value and its adjustment rate of the third portion of mineralized collagen suspension, respectively; the cooling rate, vacuum degree, warming rate and sublimation rate of the second freeze-drying process are greater than or equal to the cooling rate, vacuum degree, warming rate and sublimation rate of the third freeze-drying process, respectively, and the pre-freezing temperature of the second freeze-drying process is lower than or equal to the pre-freezing temperature of the third freeze-drying process; the first pressure is greater than the second pressure; the first predetermined time is greater than or equal to the second predetermined time; the difference between the target pH value of the second portion of mineralized collagen suspension and the target pH value of the third portion of mineralized collagen suspension is less than the difference between the target pH value of the first portion of mineralized collagen suspension and the target pH value of the second portion of mineralized collagen suspension.

[0029] Optionally, the first portion of the mineralized collagen suspension has a collagen concentration of 1.0-1.2 w / v %, an inorganic phase ratio of 65-70 %, a viscosity of 800-1000 mPa·s, a solid content of 13-16 %, a target pH value of 8-9, and a target pH value adjustment rate of 10-20 ml / min; the second portion of the mineralized collagen suspension has a collagen concentration of 0.8-1.0 w / v %, an inorganic phase ratio of 60-65 %, a viscosity of 600-800 mPa·s, a solid content of 10-13 %, a target pH value of 7-8, and a target pH value adjustment rate of 5-15 ml / min; the third portion of the mineralized collagen suspension has a collagen concentration of 0.5-0.8 w / v %, an inorganic phase ratio of 55-60 %, a viscosity of 400-600 mPa·s, a solid content of 7-10 %, a target pH value of 6.5-7.5, and a target pH value adjustment rate of 3-10 ml / min; the first freeze-drying treatment has a pre-freezing temperature of -50℃ to -40℃, a cooling rate of 1.0-2.0℃ / min, a vacuum degree of 10-15 Pa, a heating rate of 0.15-0.30℃ / min, and a sublimation rate of 1.5-3.0 % / h; the second freeze-drying treatment has a pre-freezing temperature of -40℃ to -30℃, a cooling rate of 0.5-1.0℃ / min, a vacuum degree of 15-20 Pa, a heating rate of 0.10-0.15℃ / min, and a sublimation rate of 1.0-1.5 % / h; the third freeze-drying treatment has a pre-freezing temperature of -30℃ to -20℃, a cooling rate of 0.1-0.5℃ / min, a vacuum degree of 20-25 Pa, a heating rate of 0.05-0.10℃ / min, and a sublimation rate of 0.5-1.0 % / h; the first pressure is 0.03-0.05 MPa, the second pressure is 0.02-0.03 MPa; the first preset time is 45-60 seconds, and the second preset time is 60-80 seconds; the first mineralized collagen layer has a pore size of 20-80 μm, the second mineralized collagen layer has a pore size of 80-150 μm, and the third mineralized collagen layer has a pore size of 150-300 μm.

[0030] To solve the above technical problems, the technical scheme of the present application further provides a bone repair material with a mineralized collagen layered structure formed by the preparation method of the bone repair material with a mineralized collagen layered structure.

[0031] Compared with the prior art, the technical scheme of the present application has at least the following advantages: This invention proposes a mineralized collagen bone repair material with a layered structure and its preparation method, aiming to solve the technical bottlenecks of existing mineralized collagen raw materials, such as simple structure, insufficient interlayer bonding, and inability to achieve mechanical and pore gradient design. Although existing technologies have proposed the concept of multi-layer freeze-drying or segmented casting, they generally rely on "physical bonding after rehydration" or "gradual structure during a single freeze-drying process," lacking technical means to engineer the interface of wet mineralized collagen, resulting in significant deficiencies in the material's load-bearing capacity, structural integrity, and biomimetic accuracy. This invention, through an innovative process of "independent freeze-drying layer by layer—interface wetting and interpenetration—hammering / cold pressing densification—secondary shaping," achieves a mineralized collagen composite material that can simulate the hierarchical structure of natural cortical bone and cancellous bone and possesses high interfacial strength.

[0032] The core technical solution of this invention includes: first, preparing a mineralized collagen suspension with controllable mineralization; by adjusting the collagen concentration, inorganic phase ratio, viscosity, and solid content, enabling the collagen system of different layers to have designable pore characteristics and mineralization distribution; second, freeze-drying the first layer (bottom layer) solution to obtain an initial support layer with an independent structure; then, adding a second layer of mineralized collagen solution to the surface of this layer, allowing the solution to partially penetrate into the freeze-dried pores, forming a wet interfacial interpenetration zone, creating conditions for interfacial structure reconstruction; then applying appropriate hammering or cold pressing to this wet interfacial interpenetration zone, causing local entanglement of fibers, rearrangement of inorganic particles, and pore collapse to form a dense interface; subsequently, performing a second freeze-drying to solidify the interpenetration-compaction-formed interfacial structure and obtain a second layer of independent pore structure. Repeating the above steps can construct a stable layered structure of 2 to 10 layers.

[0033] Compared with existing technologies, the innovation of this invention is mainly reflected in three aspects: First, it adopts wet interfacial interpenetration technology, which enables the new solution and the underlying structure to interlock at the molecular and fiber scales, unlike the "simple rehydration contact" in the disclosed technology, fundamentally improving the interfacial bonding mode; Second, it introduces wet hammer pressing / cold pressing process to form a dense transition layer in the interpenetration interface, which significantly improves the interlayer bonding strength and achieves a continuous rather than sectional transition. This is an interface engineering method that has not been established in existing mineralized collagen multilayer materials; Third, through the cyclic system of "independent freeze-drying - interface engineering - independent re-freeze-drying", the porosity, mineralization and mechanical properties of each layer can be individually controlled, realizing a layered biomimetic structure close to natural bone tissue, rather than a structure that cannot be layered by one freeze-drying or whose interface cannot be stabilized by traditional lamination.

[0034] Therefore, this invention not only constructs a mineralized adhesive raw material with clear layer distinctions and controllable gradients, but also achieves interlayer continuity and mechanical strengthening, significantly improving the overall compressive strength, peel strength, and fatigue stability of the material. Simultaneously, the pore size gradient of the multilayer structure promotes cell migration from the dense layer to the loose layer, enhancing angiogenesis and osteogenic properties. The preparation method provided by this invention has clearly defined parameters, high reproducibility, and is easy to industrialize. It is applicable to three types of bone repair implants, including cranial repair blocks, bone defect filling materials, maxillofacial repair materials, and alveolar bone augmentation materials, and has significant application value. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the overall structure of the bone repair material with a mineralized collagen layered structure according to Example 1 of the present invention. Figure 2 This is a schematic diagram of the preparation process of a bone repair material with a mineralized collagen layered structure according to Example 1 of the present invention; Figure 3 This is a schematic diagram of the second mineralized collagen suspension being added to the surface of the first mineralized collagen layer and forming a wet interfacial interpenetration zone in Example 1 of the present invention. Figure 4 This is a schematic diagram of the hammer pressing / cold pressing treatment of the wet interface interpenetration zone in Example 1 of the present invention; Figure 5 This is a schematic diagram of the overall structure of the bone repair material with a mineralized collagen layered structure, as described in Example 2 of this invention. Figure 6 This is an electron micrograph of the micropore structure of the bone repair material with a three-layer mineralized collagen layer structure according to Embodiment 2 of the present invention. Figure 7 This is a physical image of the bone repair material with a three-layer mineralized collagen layer structure according to Embodiment 2 of the present invention. Detailed Implementation

[0036] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0037] The specific implementation steps of the method for preparing bone repair materials with a layered structure of mineralized collagen according to the technical solution of the present invention include the following: Step S1: Prepare N portions of mineralized collagen suspension, including: the first portion of mineralized collagen suspension, the second portion of mineralized collagen suspension, ..., the Nth portion of mineralized collagen suspension, where N≥2 (i.e., the number of portions of mineralized collagen suspension prepared is more than one).

[0038] In this embodiment of the invention, multiple portions of mineralized collagen suspension are prepared separately and used to form each layer of mineralized collagen in a subsequent bone repair material with a layered mineralized collagen structure (formed by freeze-drying). For example, the first portion of mineralized collagen suspension is freeze-dried to form the first mineralized collagen layer, the second portion is freeze-dried to form the second mineralized collagen layer, and so on, until the Nth portion of mineralized collagen suspension is freeze-dried to form the Nth mineralized collagen layer. The mineralized collagen layers are stacked in the thickness direction to collectively constitute the layered mineralized collagen structure. In actual implementation, the layered mineralized collagen structure generally contains 2 to 10 layers of mineralized collagen. Considering both process complexity and meeting actual material performance requirements, 3 to 5 layers of mineralized collagen are preferred.

[0039] The specific preparation process of the mineralized collagen suspension can be achieved through various technical means. For example, it can be prepared by mixing a collagen solution, a mineral solution (e.g., a solution containing at least one mineral selected from CaCl2, MgCl2, and ZnCl2), and a phosphoric acid solution; or by physically mixing a collagen solution with nano-hydroxyapatite powder; or by sequentially adding a sodium dihydrogen phosphate solution and a calcium chloride solution to the collagen solution. In specific embodiments, one of these technical means can be selected to prepare the mineralized collagen suspension.

[0040] Specifically, the preparation of N parts of mineralized collagen suspension by mixing collagen solution, mineral solution, and phosphoric acid solution can be implemented in the following way: Method (1): Weigh type I lyophilized collagen powder and add it to 0.01~0.02mol / L acetic acid solution to dissolve it completely. Adjust the collagen mass concentration to 0.3~1.5% (w / v) to form a collagen solution. Prepare 0.5 mol / L CaCl2 solution and 0.3 mol / L Na2HPO4 solution, and add them to the collagen solution in sequence according to the calcium-to-phosphorus ratio Ca / P = 1.67. Stir magnetically for 30 minutes at 4℃ to obtain a uniform mineralized collagen suspension.

[0041] Method (2): Dissolve 1.2g of type I collagen in 2L of 0.02% acetic acid solution to form a collagen solution. Seal the solution with plastic wrap and stir overnight with a magnetic stirrer to ensure complete dissolution of the collagen. Mix the weighed CaCl2 and MgCl2 / ZnCl2 powders in a ratio of 2:1 to 3:1 and stir evenly in 1500ml of water to obtain CaCl2 and MgCl2 / ZnCl2 solutions. Slowly add the CaCl2 and MgCl2 / ZnCl2 solutions to the collagen solution at a rate of 5ml / min and stir for at least 2 hours to ensure that the minerals are fully mixed with the collagen. Dissolve 53g of phosphoric acid in 1500ml of deionized water and stir evenly to obtain a phosphoric acid solution. Slowly add the phosphoric acid solution to the collagen solution at a rate of 5ml / min and stir for at least 2 hours to ensure that the minerals in the mineralized collagen suspension are completely deposited.

[0042] The preparation of N parts of mineralized collagen suspension by physically mixing collagen solution and nano-hydroxyapatite powder can be carried out in the following way: Dissolve 1.2g of recombinant collagen in 2L of 0.02% acetic acid solution to form a collagen solution, seal with plastic wrap, and stir overnight with a magnetic stirrer; place the collagen solution in a magnetic stirrer, slowly add 0.4g of nano-hydroxyapatite powder, and stir evenly to form a mineralized collagen suspension.

[0043] The preparation of N mineralized collagen suspensions by sequentially adding sodium dihydrogen phosphate solution and calcium chloride solution to collagen solution can be implemented as follows: Take 2g of type I collagen and dissolve it in 0.5mol / L acetic acid solution to prepare a collagen solution with a mass concentration of 2mg / mL; dissolve sodium dihydrogen phosphate and calcium chloride in deionized water to prepare 500ml of sodium dihydrogen phosphate solution with a concentration of 0.05mol / L and calcium chloride solution with a concentration of 0.075mol / L respectively, simulating the calcium and phosphorus ion concentrations in human body fluids; place the collagen solution in a magnetic stirrer and slowly add the corresponding volume of sodium dihydrogen phosphate solution at a constant temperature of 25℃ at a dropping rate of 1ml / min, stirring until homogeneous, and the collagen molecules are initially dispersed; add calcium chloride solution at a rate of 1mL / min; the collagen fibers self-assemble to form a three-dimensional network structure, and under the guidance of the collagen fibers, calcium and phosphorus ions are loosely and orderly deposited on the surface and inside of the collagen fibers to form a mineralized collagen suspension.

[0044] In practice, the formation of N portions of mineralized collagen suspension can be achieved by dividing the mineralized collagen suspension prepared using the same mineralized collagen suspension preparation process into N portions, or by forming each portion of mineralized collagen suspension separately using the same mineralized collagen suspension preparation process.

[0045] Step S2: Adjust the relevant parameters of each mineralized collagen suspension, including at least one of collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value and its adjustment rate.

[0046] In this embodiment of the invention, to simulate the natural cortical-cancellous bone hierarchical structure, the pore size of the mineralized collagen layer from the bottom layer to the top layer in the formed mineralized collagen layered structure increases sequentially. This is achieved by adjusting the relevant parameters of each mineralized collagen suspension (including at least one of collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate). Specifically, adjusting the relevant parameters of each mineralized collagen suspension includes: to make it suitable for forming the bottom layer to the top layer of mineralized collagen, the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the corresponding mineralized collagen suspension decrease sequentially.

[0047] Specifically, the solid content of the first mineralized collagen suspension can be greater than that of the second mineralized collagen suspension (or the solute concentration of the first mineralized collagen suspension is greater than that of the second mineralized collagen suspension), the solid content of the second mineralized collagen suspension is greater than that of the third mineralized collagen suspension (or the solute concentration of the second mineralized collagen suspension is greater than that of the third mineralized collagen suspension), and so on, decreasing sequentially until the solid content of the (N-1)th mineralized collagen suspension is greater than that of the Nth mineralized collagen suspension (or the solute concentration of the (N-1)th mineralized collagen suspension is greater than that of the N-1th mineralized collagen suspension).

[0048] The differences in solid content (solute concentration) among the various mineralized collagen suspensions play a crucial role in regulating pore structure during the subsequent freeze-drying process: if the mineralized collagen suspension has a relatively higher solid content and solute concentration, it can inhibit the growth rate of ice crystals, reduce the size of ice crystals, and thus form a relatively finer and denser pore structure; if the mineralized collagen suspension has a relatively lower solid content and solute concentration, it indicates that it contains more free water, which will produce larger ice crystals at the same cooling rate. After sublimation (the sublimation stage of freeze-drying), it will form larger pores, which is beneficial for bone cell ingrowth and angiogenesis.

[0049] Besides the difference in solid content, the volume fraction of the pore-forming agent in the first mineralized collagen suspension can also be less than that in the second mineralized collagen suspension, the volume fraction of the pore-forming agent in the second mineralized collagen suspension can be less than that in the third mineralized collagen suspension, and so on, increasing sequentially until the volume fraction of the pore-forming agent in the (N-1)th mineralized collagen suspension is less than that in the Nth mineralized collagen suspension. Obviously, the larger the volume fraction of the pore-forming agent, the larger the pore size will be after subsequent freeze-drying. Furthermore, the viscosity of the first mineralized collagen suspension can be made greater than that of the second mineralized collagen suspension, the viscosity of the second mineralized collagen suspension can be greater than that of the third mineralized collagen suspension, and so on, decreasing sequentially until the viscosity of the (N-1)th mineralized collagen suspension is greater than that of the Nth mineralized collagen suspension. Since higher viscosity means greater resistance to the migration of solvent molecules and ions in the solution, this will inhibit the nucleation rate of ice crystals and slow down the growth rate of ice crystals during subsequent freeze-drying, resulting in smaller ice crystal sizes and thus forming relatively smaller pores. In contrast, low-viscosity solutions allow for easier migration of solute and solvent molecules, resulting in higher nucleation rates and the formation of relatively larger pores.

[0050] The collagen concentration of the first mineralized collagen suspension can be greater than that of the second mineralized collagen suspension, the second mineralized collagen suspension can be greater than that of the third mineralized collagen suspension, and so on, decreasing sequentially until the collagen concentration of the (N-1)th mineralized collagen suspension is greater than that of the Nth mineralized collagen suspension. Since a higher collagen concentration results in a more viscous solution, greater resistance to ice crystal growth, and smaller ice crystals, a relatively smaller pore size is formed. In contrast, a low collagen concentration solution is relatively thin, with less resistance to ice crystal growth, resulting in larger and more interconnected ice crystals, and a relatively larger pore size is formed.

[0051] Furthermore, the inorganic phase ratio of the first mineralized collagen suspension can be greater than that of the second mineralized collagen suspension, the inorganic phase ratio of the second mineralized collagen suspension can be greater than that of the third mineralized collagen suspension, and so on, decreasing sequentially until the inorganic phase ratio of the (N-1)th mineralized collagen suspension is greater than that of the Nth mineralized collagen suspension. Since a larger inorganic phase ratio results in greater steric hindrance within the solution and greater resistance to ice crystal growth, continuous growth is impossible, resulting in relatively smaller pore sizes. In contrast, solutions with a low inorganic phase ratio have relatively smaller steric hindrance, less resistance to ice crystal growth, and easier growth and interconnection, leading to relatively larger pore sizes.

[0052] In this embodiment of the invention, for each prepared mineralized collagen solution, an alkaline solution can be added at a predetermined adjustment rate to regulate and induce collagen fiber formation, thereby stabilizing the liquid crystal tissue into a dense fibrous matrix. By controlling the adjustment of the target pH value and the adjustment rate of each mineralized collagen suspension, a gradual change in density is formed from the bottom layer to the top layer of the mineralized collagen layered structure. This allows for the controlled assembly of mineralized collagen into a bone repair material with a gradually dense layered structure using a simple and effective process, enabling the bone repair material to simultaneously possess excellent properties such as uniformity, stable mechanical properties, and good bone regeneration effect.

[0053] Specifically, the target pH value and adjustment rate of the mineralized collagen suspensions suitable for forming the bottom layer (first mineralized collagen layer) to the top layer (Nth mineralized collagen layer) can be sequentially decreased. This allows the pore size of the subsequently formed first, second, ..., up to the Nth mineralized collagen layer to increase sequentially (with sequentially decreasing intralayer density). In practice, an alkaline solution is added to each portion of the prepared mineralized collagen suspension suitable for forming the bottom layer to the top layer at a sequentially decreasing adjustment rate, adjusting the initial pH value to a sequentially decreasing target pH value. For example, the pH value of the first portion of mineralized collagen suspension can be adjusted to a first target pH value using an alkaline solution at a first predetermined adjustment rate, and the pH value of the second portion of mineralized collagen suspension can be adjusted to a second target pH value using a second predetermined adjustment rate, where the second target pH value is less than the first target pH value, and the second predetermined adjustment rate is less than the first predetermined adjustment rate; and so on, until the pH value of the Nth portion of mineralized collagen suspension is adjusted to the Nth target pH value using an alkaline solution at an Nth predetermined adjustment rate, where the Nth predetermined adjustment rate is less than the (N-1)th predetermined adjustment rate, and the Nth target pH value is less than the (N-1)th target pH value. In this embodiment of the invention, the Nth target pH value generally needs to be at least greater than or equal to 6.5, which can be considered as the minimum critical range of target pH values ​​for each portion of mineralized collagen suspension to ensure the feasibility of the technical solution. In actual implementation, if the Nth target pH value is set to be greater than or equal to 7, the implementation effect is better. The alkaline solution can be one of the following: sodium hydroxide solution, potassium hydroxide solution, ammonia solution, sodium carbonate solution, sodium bicarbonate solution, etc.

[0054] In this embodiment of the invention, the target pH value and its adjustment rate of the mineralized collagen suspension, from the bottom layer to the top layer of the mineralized collagen layer, can be gradually reduced. Taking N=3 as an example: if the first target pH value is 8.5, the second target pH value is 7.5, and the third target pH value is 7, the decrease in the second target pH value relative to the first target pH value is 1, and the decrease in the third target pH value relative to the second target pH value is 0.5. Thus, the decrease in the target pH value is gradually reduced. In addition to reducing the decrease in the target pH value, the predetermined adjustment rate of the pH value of each mineralized collagen solution from the bottom layer to the top layer of the mineralized collagen layered structure can also be gradually reduced. Again, taking N=3 as an example: if the adjustment rate of the first target pH value is 20 ml / min, the adjustment rate of the second target pH value is 10 ml / min, and the adjustment rate of the third target pH value is 5 ml / min. The rate of decrease in the second target pH adjustment relative to the first target pH adjustment rate is 10 ml / min, and the rate of decrease in the third target pH adjustment rate relative to the second target pH adjustment rate is 5 ml / min. This demonstrates that the rate of decrease in the target pH adjustment rate gradually decreases. By progressively reducing the rate of decrease in the target pH values ​​of the first to Nth portions of mineralized collagen solution, coupled with a progressively decreasing rate of decrease in the predetermined adjustment rate, the slower pH change makes it relatively difficult for collagen to rapidly form a dense fibrous structure. This prevents the dense accumulation of a large number of hydroxyapatite particles between collagen fibers, thus gradually reducing the intralayer density (increasing the pore size). Therefore, by controlling the predetermined adjustment rate of the target pH value, a layered structure with progressively decreasing density can be formed more effectively.

[0055] In this invention, the principle of controlled assembly of mineralized collagen through pH adjustment is as follows: In neutral or slightly acidic environments, collagen molecules carry a large number of positive charges, resulting in electrostatic repulsion and weak intermolecular interactions. As the pH increases, the concentration of hydroxide ions in the solution increases, causing some acidic groups on the collagen molecules to dissociate, reducing the positive charge on the collagen molecules. This promotes the collagen molecules to approach each other and arrange themselves in an orderly manner, thereby forming collagen fibers. Simultaneously, under alkaline conditions, collagen molecules contain a suitable amount of negatively charged groups and Ca2+. 2+ Zn 2+ Mg 2+ and PO4 3- Interactions; the electrochemical properties of collagen molecules can regulate the behavior of inorganic ions in the matrix, and the carboxyl groups provide nucleation sites for hydroxyapatite, while the orderly arrangement of collagen fibers provides nucleation space for hydroxyapatite crystallization.

[0056] It should be noted that in actual implementation, steps S1 and S2 can be combined, meaning that the corresponding parameters are adjusted simultaneously with the preparation of each mineralized collagen suspension, ensuring that the prepared mineralized collagen suspension has all parameters adjusted. The following example illustrates this by adjusting the target pH value while preparing the mineralized collagen suspension: In the process of preparing the Nth mineralized collagen suspension by sequentially adding sodium dihydrogen phosphate solution and calcium chloride solution to the collagen solution, 2g can be taken first... Type I collagen was dissolved in 0.5 mol / L acetic acid solution to prepare a collagen solution with a mass concentration of 2 mg / mL. Sodium dihydrogen phosphate and calcium chloride were dissolved in deionized water to prepare 500 mL solutions of 0.05 mol / L sodium dihydrogen phosphate and 0.075 mol / L calcium chloride, respectively, to simulate the calcium and phosphorus ion concentrations in human body fluids. The collagen solution was placed in a magnetic stirring apparatus, and at a constant temperature of 25°C, the corresponding volume of sodium dihydrogen phosphate solution was slowly added dropwise at a dropping rate of 1 mL / min. After stirring evenly, the pH of the mineralized collagen suspension is approximately 4.5, and the collagen molecules are initially dispersed. Calcium chloride solution is added dropwise at a rate of 1 mL / min, while the pH of the system is adjusted with 5 mol / L sodium hydroxide solution at a rate of 5 mL / min, gradually increasing from 4.5 to 7 ± 0.2. Collagen fibers self-assemble to form a three-dimensional network structure. Simultaneously, under the dual guidance of pH changes and collagen fibers, calcium and phosphorus ions are loosely and orderly deposited on the surface and inside of the collagen fibers, forming the Nth mineralized collagen suspension with complete pH adjustment.

[0057] Step S3: Place the first mineralized collagen suspension with the relevant parameters adjusted at the bottom of the mold, and form the first mineralized collagen layer through the first freeze-drying process.

[0058] Those skilled in the art know that freeze drying mainly includes two stages: a freezing stage (pre-freezing stage) and a vacuum sublimation drying stage (preserving its morphology by slowly raising the temperature). Therefore, the two stages are sequential and belong to a continuous process of "pre-freezing + vacuum sublimation drying".

[0059] The embodiments of the present invention provide the following two specific implementation processes for the first freeze-drying process: One specific implementation process of the first freeze-drying treatment: After the first mineralized collagen suspension with the relevant parameters adjusted is placed at the bottom of the mold, it is pre-frozen at a predetermined freezing temperature (e.g., -50℃ to -40℃) for a predetermined time (e.g., 2 to 3 hours); the freeze-drying program is started: the freezing stage temperature rises from the predetermined freezing temperature (-50℃ to -40℃) to the initial temperature suitable for vacuum sublimation drying (e.g., -25℃) and is maintained for a certain time (e.g., 1 hour) to prepare for the subsequent vacuum sublimation drying stage and prevent the risk of runaway internal thermal stress and interfacial stress in ice crystals that may be caused by direct sublimation at a lower temperature; the vacuum sublimation drying stage includes two stages: the initial drying stage and the final drying stage. In the initial drying stage, the chamber pressure is 10 to 15 Pa and the plate temperature rises from -25℃ to -10℃; in the final drying stage, the temperature rises to 5℃ and is maintained for 6 hours to form the first mineralized collagen layer in the mineralized collagen layered structure, thereby obtaining the initial support layer of the independent structure.

[0060] The second specific implementation process of the first freeze-drying treatment: After the first batch of mineralized collagen suspension with the relevant parameters adjusted is placed at the bottom of the mold, it is first pre-frozen at a first temperature (e.g., -50℃ to -40℃) without vacuum for a predetermined time (e.g., 2 to 3 hours) to quickly lock in high ice core density and inhibit ice crystal growth. Then, the temperature is continuously reduced through a gradient cooling program at a first cooling rate (e.g., 1.0 to 2.0℃ / min) to reduce the freezing temperature from the first temperature to a second temperature (cooling range of 15℃ to 25℃) to complete the freeze-locking, locking the water into a solid ice crystal framework to prevent structural collapse during subsequent vacuuming. After freezing, the pressure reduction operation (vacuuming) performed in the vacuum sublimation drying stage reaches the first target vacuum level (e.g., 10 to 15 Pa). Under this vacuum level, the temperature is gradually increased to a third temperature (e.g., -10 to -5℃) at a first heating rate (e.g., 0.15 to 0.30℃ / min), controlled at a first sublimation rate (e.g., 1.5 to 3.0℃). Finally, the temperature is raised to the fourth temperature and dried for a preset time (e.g., 6 hours). Through "isothermal pre-freezing → programmed cooling → vacuum sublimation drying", the first mineralized collagen layer in the mineralized collagen layered structure can be formed, thereby obtaining the initial support layer of the independent structure (the first mineralized collagen layer is independently freeze-dried).

[0061] The thickness of the first mineralized collagen layer is about 1.5 to 2.0 mm, and the pore size is about 20 to 80 μm.

[0062] Step S4: The second mineralized collagen suspension, after the relevant parameters have been adjusted, is added to the surface of the first mineralized collagen layer and left to stand for a first preset time to allow it to penetrate into the pores formed by the first freeze-drying treatment in the first mineralized collagen layer, forming the first wet interface interpenetration zone.

[0063] In practice, the second mineralized collagen suspension can be uniformly added to the surface of the first mineralized collagen layer by dripping, at a rate of 0.20~0.25 mL / cm². 2 The first preset time is 45 to 60 seconds.

[0064] Step S5: Apply a first pressure to the first wet interface interpenetration zone.

[0065] In specific implementation, the first pressure can be applied to the wet interfacial interpenetration zone by hammer pressing or cold pressing. The first pressure is 0.03~0.05 MPa, and the application time is 20~25 seconds. After the second mineralized collagen suspension penetrates into the first mineralized collagen layer, hammer pressing or cold pressing is applied to the first wet interfacial interpenetration zone to form a dense transition layer at the interface. The pores at the interface are compressed, the fibers are further entangled, and the inorganic particles are rearranged, thereby significantly enhancing the interlayer (between the first and second mineralized collagen layers) bonding strength.

[0066] In this embodiment of the invention, by introducing a wet hammer pressing / cold pressing process, a dense transition layer is formed in the first wet interface interpenetration zone, which significantly improves the interlayer bonding strength and achieves a continuous rather than sectional transition.

[0067] Step S6: Perform a second freeze-drying treatment on the first wet interpenetrating zone of the interface after the first pressure is applied and the second mineralized collagen suspension covering the surface of the first mineralized collagen layer to form a first continuous dense interface and the second mineralized collagen layer.

[0068] In this embodiment of the invention, to simulate the natural cortical bone-cancellous bone hierarchical structure, the pore size of the mineralized collagen layer from the bottom layer to the top layer in the formed mineralized collagen layered structure increases sequentially. Besides adjusting the relevant parameters of the mineralized collagen suspension in step S2, this can also be achieved by controlling the process parameters of the freeze-drying process (including at least one of the pre-freezing temperature and cooling rate in the freezing stage, and the vacuum degree, heating rate, and sublimation rate in the vacuum sublimation drying stage). Specifically, the control of the freeze-drying process parameters includes: to make the formation of the bottom layer of mineralized collagen layer to the top layer of mineralized collagen layer, the corresponding freeze-drying cooling rate, vacuum degree, heating rate, and sublimation rate decrease sequentially, while the pre-freezing temperature increases sequentially.

[0069] Besides the individual parameters of the mineralized collagen suspension affecting subsequent pore size, differences in the corresponding freeze-drying process parameters can also influence pore size. Among the most important freeze-drying process parameters are freezing temperature (pre-freezing temperature) and freezing rate (cooling rate). Higher freezing temperatures and slower freezing processes favor the formation of larger, more orderly arranged ice crystals. These large ice crystals, after sublimation, leave behind larger pores. Conversely, slower freezing temperatures lead to the formation of numerous small ice crystals, which, after sublimation, also form relatively small pores. Temperature gradients directly affect the nucleation rate and growth direction of ice crystals. Regions with slower cooling rates form coarse ice crystals, resulting in large pores after sublimation; regions with faster cooling rates form fine ice crystals, resulting in small pores after sublimation. The control of parameters such as vacuum degree, heating rate, and sublimation rate during the vacuum sublimation drying stage also has a certain impact on the pore size of the formed material. In contrast, higher vacuum levels, heating rates, and sublimation rates primarily serve to accelerate drying, form fine porous structures, and maintain a smooth surface. Conversely, lower vacuum levels primarily serve to maintain structural stability, inhibit pore wall collapse, and ensure the formation of large, interconnected pores.

[0070] By introducing a spatial temperature gradient and time control program during the freeze-drying process, and adjusting the pre-freezing temperature, vacuum degree and heating rate, the ice crystal growth rate of each mineralized collagen layer in the layered structure of mineralized collagen is significantly different. Thus, after freeze-drying, the difference in solvent sublimation rate is used to form a stable and controllable translayer pore size gradient.

[0071] Therefore, in step S6, the process parameters of the freeze-drying process can be adjusted so that the cooling rate, vacuum degree, heating rate and sublimation rate of the first freeze-drying process are greater than or equal to the cooling rate, vacuum degree, heating rate and sublimation rate of the second freeze-drying process, and the pre-freezing temperature of the first freeze-drying process is lower than or equal to the pre-freezing temperature of the second freeze-drying process.

[0072] The second freeze-drying process in step S6 is basically the same as the first freeze-drying process in step S3, with the main differences being in the process parameters and the target material: compared to the first freeze-drying process, the second freeze-drying process has a higher pre-freezing temperature and lower cooling rate, vacuum degree, heating rate, and sublimation rate; regarding the target material, the first freeze-drying process is carried out on the first mineralized collagen suspension, while the second freeze-drying process, in addition to targeting the second mineralized collagen suspension, also includes the first wet interfacial interpenetration zone after the first pressure is applied. In actual implementation, the pre-freezing temperature of the second freeze-drying process is -40℃ to -30℃, the cooling rate is 0.5 to 1.0℃ / min, the vacuum degree is 15 to 20 Pa, the heating rate is 0.10 to 0.15℃ / min, and the sublimation rate is 1.0 to 1.5% / h. The corresponding implementation process is as follows: For the first wet interpenetrating zone after the first pressure is applied and the second mineralized collagen suspension covering the surface of the first mineralized collagen layer, pre-freeze for a predetermined time (e.g., 1-2 hours) at a predetermined freezing temperature (e.g., -40℃ to -30℃); start the freeze-drying process: the freezing stage temperature rises from the predetermined freezing temperature (-40℃ to -30℃) to an initial temperature suitable for vacuum sublimation drying (e.g., -25℃) and is maintained for a certain time (e.g., 1 hour) to prepare for the subsequent vacuum sublimation drying stage and prevent the risk of runaway internal thermal stress and interfacial stress in ice crystals that may be caused by direct sublimation at a lower temperature; vacuum sublimation drying stage: it includes two stages: initial drying stage and final drying stage. In the initial drying stage, the chamber pressure is 15-20 Pa and the plate temperature rises from -25℃ to -15℃; in the final drying stage, the temperature rises to 5℃ and is maintained for 6 hours to form the second mineralized collagen layer in the mineralized collagen layered structure.

[0073] It should be noted that in actual implementation, if the pre-freezing temperature is close to the initial temperature suitable for vacuum sublimation drying, the vacuum sublimation drying stage can be started directly from the pre-freezing temperature (equivalent to skipping the preparatory work before the vacuum sublimation drying stage).

[0074] For details on the implementation of the second freeze-drying process, please refer to the first freeze-drying process involved in step S3 (the relevant process parameters of the two are different), which will not be repeated here.

[0075] The second mineralized collagen layer obtained after the second freeze-drying process has a thickness of approximately 1.0–1.5 mm and a pore size of approximately 80–150 μm.

[0076] Step S7: The third mineralized collagen suspension, after the relevant parameters have been adjusted, is added to the surface of the second mineralized collagen layer and left to stand for a second preset time to allow it to penetrate into the pores formed by the second freeze-drying treatment within the second mineralized collagen layer, forming the second wet interface interpenetration zone.

[0077] Step S7 is similar to step S4. The third mineralized collagen suspension can also be uniformly added to the surface of the second mineralized collagen layer by dripping. The difference is that the amount of the third mineralized collagen suspension added is relatively larger, and the standing time can be longer. Specifically, the amount of the third mineralized collagen suspension added can be 0.25~0.30 mL / cm³. 2 The second preset time is 60 to 80 seconds.

[0078] Step S8: Apply a second pressure to the second wet interface interpenetration zone.

[0079] The implementation process of step S8 is similar to that of step S5. Specifically, the second pressure can be applied to the wet interfacial interpenetration zone by hammer pressing or cold pressing. The second pressure can be 0.02~0.03 MPa, and the application time is 15~20 seconds. After the third mineralized collagen suspension penetrates the second mineralized collagen layer, hammer pressing or cold pressing is applied to the second wet interfacial interpenetration zone to form a dense transition layer at the interface. This process compresses the pores at the interface, further entangles the fibers, and rearranges the inorganic particles, thereby significantly enhancing the interlayer (between the second and third mineralized collagen layers) bonding strength. By introducing a wet hammer pressing / cold pressing process, a dense transition layer is formed in the second wet interfacial interpenetration zone, significantly improving the interlayer bonding strength and achieving a continuous rather than discontinuous transition.

[0080] It should be noted that, in this embodiment of the invention, the pressure applied to the wet interfacial interpenetration zone of the mineralized collagen layer closer to the bottom layer is greater than the pressure applied to the wet interfacial interpenetration zone of the mineralized collagen layer farther from the bottom layer. This is because, in order to obtain a denser intralayer structure, it is necessary to apply greater pressure to the mineralized collagen layer closer to the bottom layer, while the mineralized collagen layer farther from the bottom layer has larger intralayer pores, and therefore the applied pressure should be smaller. Therefore, the second pressure in step S8 is less than the first pressure in step S5, and the application time of the second pressure is also less than or equal to the application time of the first pressure.

[0081] Step S9: Perform a third freeze-drying treatment on the second wet interpenetrating zone after the second pressure is applied and the third mineralized collagen suspension covering the surface of the second mineralized collagen layer to form a second continuous dense interface and the third mineralized collagen layer.

[0082] The third freeze-drying process in step S9 is basically the same as the second freeze-drying process in step S6. The main difference lies in the various process parameters. Compared with the second freeze-drying process, the third freeze-drying process has a higher pre-freezing temperature and lower cooling rate, vacuum degree, heating rate, and sublimation rate. In actual implementation, the pre-freezing temperature of the third freeze-drying process is -30℃ to -20℃, the cooling rate is 0.1 to 0.5℃ / min, the vacuum degree is 20 to 25 Pa, the heating rate is 0.05 to 0.10℃ / min, and the sublimation rate is 0.5 to 1.0% / h. The corresponding implementation process is as follows: For the second wet interpenetrating zone after the application of the second pressure and the third mineralized collagen suspension covering the surface of the second mineralized collagen layer, pre-freeze for a predetermined time (e.g., 1-2 hours) at a predetermined freezing temperature (e.g., -30℃ to -20℃); Vacuum sublimation drying stage: This includes two stages: an initial drying stage and a final drying stage. In the initial drying stage, the chamber pressure is 20-25 Pa, and the plate temperature is directly raised from the predetermined freezing temperature (e.g., -25℃) to -20℃; In the final drying stage, the temperature is raised to 5℃ and maintained for 6 hours, forming the third mineralized collagen layer in the mineralized collagen layered structure. It should be noted that if the pre-freezing temperature of the third freeze-drying treatment is already close to the initial temperature suitable for vacuum sublimation drying, the vacuum sublimation drying stage can be started directly (equivalent to skipping the preparatory work before the vacuum sublimation drying stage).

[0083] For details on the specific implementation process of the third freeze-drying process, please refer to the second freeze-drying process involved in step S6 (the relevant process parameters of the two are different), which will not be repeated here.

[0084] The thickness of the third mineralized collagen layer obtained after the third freeze-drying process is approximately 1.5–2.0 mm, and the pore size is approximately 150–300 μm.

[0085] Step S9 and subsequent steps can be repeated in a cycle similar to steps 3 to 6 or steps 7 to 9, thereby continuously forming the mineralized collagen layers that are stacked sequentially in the thickness direction in the layered structure of mineralized collagen, until the formation of the Nth mineralized collagen layer is completed, and finally a bone repair material with a layered structure of N mineralized collagen layers is formed.

[0086] Furthermore, in this embodiment of the invention, before freeze-drying, each mineralized collagen suspension with adjusted parameters (including pH value) can be concentrated. The concentration process includes at least one of the following: dialysis of the adjusted mineralized collagen suspension in a dialysis bag, filtration using a Buchner funnel, and removal of water by pressure. Through concentration operations such as dialysis and filtration, small molecule impurities in the solution can be removed, solvent content reduced, and the concentration and purity of the mineralized collagen increased.

[0087] Specifically, the first portion of mineralized collagen suspension, after pH adjustment, can be subjected to a settling and concentration process to solidify the first portion of mineralized collagen solution. The solidified first portion of mineralized collagen suspension is then placed at the bottom of a mold, and freeze-dried to form the first mineralized collagen layer (or "bottom layer") of the layered mineralized collagen structure. In practice, the settling process involves placing the first portion of mineralized collagen suspension, after parameter adjustment (including pH adjustment), at a specific temperature (e.g., 37°C) for a preset time. The purpose of this settling process is to allow the suspension particles to precipitate. This is typically performed before the concentration process. After both the settling and concentration processes are completed, the solidified first portion of mineralized collagen suspension is placed at the bottom of the mold. Alternatively, the settling and concentration processes can be performed inside the mold. In this case, the first portion of mineralized collagen suspension, after parameter adjustment, can be placed directly at the bottom of the mold and set at a specific temperature (e.g., 37°C) for a preset time. Then, a pressure-based concentration process can be used.

[0088] It should be noted that the settling process for the first mineralized collagen suspension differs from the settling process for the other mineralized collagen suspensions. The duration and function of the two processes differ; the former requires a longer settling time (usually over one hour) to solidify the first mineralized collagen suspension, while the latter requires a shorter settling time (generally around tens of seconds) to allow the (non-solidified) mineralized collagen suspension to penetrate into the pores formed by freeze-drying within the underlying mineralized collagen layer, creating a corresponding wet interfacial interpenetration zone. Furthermore, the pressurized concentration of the first mineralized collagen suspension is significantly different from the pressurization process applied to the other mineralized collagen suspensions to form a denser transition layer at the interface. It is particularly important to emphasize that the pressurization operation of the second to Nth portions of mineralized collagen suspension in the embodiments of the present invention is a wet pressurization process. This is significantly different from the interlayer bonding effect achieved by applying pressure to multi-layered pre-formed mineralized collagen laminates or multi-layered mineralized collagen laminates in a solidified state in the prior art. The interlayer bonding formed by the latter method is mostly physical adhesion, which is prone to delamination or mechanically weak zones due to drying shrinkage, interfacial moisture migration and recrystallization. In contrast, the former method (the technical solution of the present invention) can stably obtain high interfacial strength and mechanical gradient. The difference in function means that the latter cannot provide technical inspiration for the former.

[0089] For those skilled in the art, forming a multilayer mineralized collagen layer as a whole through cross-linking or freeze-drying is a common technique due to its relatively high process efficiency, and it is frequently used in actual production. However, the "layer-by-layer freeze-drying" method usually increases the complexity of the process, and the bonding strength of the interlayer interfaces and the overall mechanical properties of the material are relatively poor. Therefore, those skilled in the art rarely consider using the "layer-by-layer freeze-drying" method when implementing materials with multilayer mineralized collagen layer structures. This can be considered a kind of technical bias among those skilled in the art. The technical solution provided by the embodiments of the present invention, based on "layer-by-layer freeze-drying," further combines wet interface interpenetration technology and wet hammer pressing / cold pressing processes to form an interface engineering process of "wetting interpenetration + cold pressing densification + independent re-freeze-drying." This can fundamentally improve the interface bonding mode, enhance the bonding strength of the interlayer interfaces and the overall mechanical properties of the material, and make the technical solution practically applicable, thereby overcoming the aforementioned technical bias of those skilled in the art. Therefore, the technical solution of the present invention has outstanding substantive features and significant progress.

[0090] The parameter ranges in the above steps of the embodiments of the present invention are merely illustrative examples. Those skilled in the art can easily understand that, in actual implementation, the relevant parameters of mineralized collagen used in each step and the relevant process parameters of freeze-drying treatment can be adjusted based on the actual situation according to the change of the number N of mineralized collagen layers in the layered structure. They should not be regarded as limitations on the embodiments of the present invention.

[0091] Based on the above embodiments of the present invention, two examples are given below: Example 1 (Method for forming a layered structure of mineralized collagen in two mineralized collagen layers) In Example 1, the mineralized collagen layered structure consists of two mineralized collagen layers, as follows: Figure 1 As shown, the mineralized collagen layered structure includes a first mineralized collagen layer 1, a second mineralized collagen layer 2, and a first continuous dense interface 12 located between the first mineralized collagen layer 1 and the second mineralized collagen layer 2, stacked sequentially. Further combined with... Figure 2 It is prepared in the following manner: Step S11, place the first prepared mineralized collagen suspension into a mold (the mold is in...) Figure 2 (not shown in the image), and the first mineralized collagen layer 1 is formed by a first freeze-drying process (as shown in the image). Figure 2As shown in (a)); it should be noted that step S11 is the process of injecting the first mineralized collagen suspension into the mold and forming the first porous structure (first mineralized collagen layer) through an independent freeze-drying process. This mainly includes steps such as suspension injection, pre-freezing, and sublimation drying, ultimately obtaining the first layer of material (first mineralized collagen layer) with the designed pore size. The "independently formed first mineralized collagen layer" is the basis of the technical solution process of this invention.

[0092] Step S12: The prepared second portion of mineralized collagen suspension 2' is added to the surface of the first mineralized collagen layer 1, and left to stand for a first preset time to allow it to penetrate into the pores formed by the first freeze-drying treatment within the first mineralized collagen layer 1, forming a first wet interface interpenetration zone 12a (e.g., Figure 2 (as shown in (b)). Further integration is possible Figure 3 , Figure 3 The process is shown whereby the second mineralized collagen suspension 2' is dropped onto the dry surface of the first mineralized collagen layer 1 along the dropping direction 31, and then seeps into the pores of the first mineralized collagen layer 1 to form the first wet interface interpenetration zone 12a. Figure 3 The figure illustrates the formation of the liquid infiltration channel (infiltration channel direction 32), the first wet interface interpenetration region 12a, and the initial entanglement 33 of collagen fibers at the interface. This figure demonstrates the "interface interpenetration reconstruction mechanism" that distinguishes the technical solution of this invention from the prior art.

[0093] Step S13, apply a first pressure P (e.g., ...) to the first wet interface interpenetration zone 12a. Figure 2 (as shown in (c)); can be further combined Figure 4 This involves applying hammering / cold pressing treatment to the first wet interface interpenetration zone 12a. Figure 4 This illustrates the process by which, after the second mineralized collagen suspension 2' permeates into the pores of the first mineralized collagen layer 1 formed by the first mineralized collagen suspension through a first freeze-drying process, hammer pressure or cold pressure (pressure application direction 42) is applied to the first wet interfacial interpenetration zone 12a under the combined action of the upper pressure plate 40 and the lower pressure plate 41, causing the interface to form a denser transition layer. The pores at the interface are compressed, the fibers further entangle, and the inorganic particles rearrange, thereby significantly enhancing the interlayer bonding strength. Figure 4 One of the key innovations of this invention is interface reconstruction and compaction.

[0094] Step S14: Perform a second freeze-drying treatment on the first wet interfacial interpenetration zone 12b after applying the first pressure P and the second mineralized collagen suspension 2' covering the surface of the first mineralized collagen layer 1, to form a first continuous dense interface 12 and the second mineralized collagen layer 2 (e.g., ...). Figure 2 (as shown in d); In this example, the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the first mineralized collagen suspension are greater than or equal to the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the second mineralized collagen suspension; the cooling rate, vacuum degree, heating rate, and sublimation rate of the first freeze-drying treatment are greater than or equal to the cooling rate, vacuum degree, heating rate, and sublimation rate of the second freeze-drying treatment; and the pre-freezing temperature of the first freeze-drying treatment is lower than or equal to the pre-freezing temperature of the second freeze-drying treatment.

[0095] For specific implementation details of this example, please refer to the corresponding content described in the above embodiments of the present invention, which will not be repeated here.

[0096] Example 2 (Method for forming a three-layer mineralized collagen structure) like Figure 5 As shown, based on Example 1 above, Example 2 can further form a mineralized collagen layered structure with three mineralized collagen layers. That is, the mineralized collagen layered structure of Example 2 can further include a third mineralized collagen layer 3 stacked on the second mineralized collagen layer 2 and a first continuous dense interface 23 located between the second mineralized collagen layer 2 and the third mineralized collagen layer 3. The preparation method is as follows: Steps S21 to S24 correspond to steps S11 to S14 in Example 1, respectively, and will not be repeated here.

[0097] In this example, step S25 is further included: adding the prepared third portion of mineralized collagen suspension to the surface of the second mineralized collagen layer 2, and allowing it to stand for a second preset time to allow it to penetrate into the pores formed by the second freeze-drying treatment within the second mineralized collagen layer 2, forming a second wet interface interpenetration zone; applying a second pressure to the second wet interface interpenetration zone, and performing a third freeze-drying treatment on the second wet interface interpenetration zone after applying the second pressure and the third portion of mineralized collagen suspension covering the surface of the second mineralized collagen layer, forming a second continuous dense interface 23 and the third mineralized collagen layer 3; the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the second portion of mineralized collagen suspension are respectively greater than or equal to the third portion of mineralized collagen suspension. The collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the mineralized collagen suspension; the cooling rate, vacuum degree, heating rate, and sublimation rate of the second freeze-drying treatment are greater than or equal to the cooling rate, vacuum degree, heating rate, and sublimation rate of the third freeze-drying treatment, respectively; the pre-freezing temperature of the second freeze-drying treatment is lower than or equal to the pre-freezing temperature of the third freeze-drying treatment; the first pressure is greater than the second pressure; the first preset time is greater than or equal to the second preset time; the difference between the target pH value of the second mineralized collagen suspension and the target pH value of the third mineralized collagen suspension is less than the difference between the target pH value of the first mineralized collagen suspension and the target pH value of the second mineralized collagen suspension.

[0098] In actual implementation of Examples 1 or 2 above, the first mineralized collagen suspension has a collagen concentration of 1.0~1.2 w / v%, an inorganic phase ratio of 65~70%, a viscosity of 800~1000 mPa·s, a solid content of 13~16%, a target pH value of 8~9, and a target pH adjustment rate of 10~20 ml / min; the second mineralized collagen suspension has a collagen concentration of 0.8~1.0 w / v%, an inorganic phase ratio of 60~65%, a viscosity of 600~800 mPa·s, a solid content of 10~13%, a target pH value of 7~8, and a target pH adjustment rate of 5~15 ml / min; the third mineralized collagen suspension has a collagen concentration of 0.5~0.8 w / v%, an inorganic phase ratio of 55~60%, and a viscosity of 400~600 ml / min. The freeze-drying process has the following parameters: mPa·s, solid content 7-10%, target pH 6.5-7.5, and target pH adjustment rate 3-10 ml / min; the first freeze-drying process has a pre-freezing temperature of -50℃ to -40℃, a cooling rate of 1.0-2.0℃ / min, a vacuum of 10-15 Pa, a heating rate of 0.15-0.30℃ / min, and a sublimation rate of 1.5-3.0% / h; the second freeze-drying process has a pre-freezing temperature of -40℃ to -30℃, a cooling rate of 0.5-1.0℃ / min, a vacuum of 15-20 Pa, a heating rate of 0.10-0.15℃ / min, and a sublimation rate of 1.0-1.5% / h; the third freeze-drying process has a pre-freezing temperature of -30℃ to -20℃, a cooling rate of 0.1-0.5℃ / min, and a vacuum of 20-25 Pa. The temperature ranges as follows: Pa, heating rate of 0.05~0.10 ℃ / min, sublimation rate of 0.5~1.0 % / h; first pressure of 0.03~0.05 MPa, second pressure of 0.02~0.03 MPa; first preset time of 45~60 seconds, second preset time of 60~80 seconds; pore size of the first mineralized collagen layer of 20~80 μm, pore size of the second mineralized collagen layer of 80~150 μm, and pore size of the third mineralized collagen layer of 150~300 μm.

[0099] Related to Examples 1 and 2 above Figure 1 , Figure 5 The overall structure of the mineralized collagen layered bone repair material obtained by the present invention is shown, including at least two layers of dry mineralized collagen porous structure. The porosity, collagen concentration and mineralization degree of each layer are different, forming a clear layered or gradient structure. Figure 5 The image shows the structural relationship of the first mineralized collagen layer 1, the second mineralized collagen layer 2, and the third mineralized collagen layer 3 arranged in sequence. The layers are connected by continuous dense interfaces rather than by simple physical bonding.

[0100] The actual implementation of the technical solution of the present invention is described below with specific examples: Example 1: Preparation method of double-layer mineralized collagen layered bone repair material This embodiment illustrates the preparation method of the present invention, and can be repeated by those skilled in the art. The parameters described in the embodiment are specific values ​​within an optional range and do not constitute the unique limitation of the present invention.

[0101] Step S101: Preparation of the first mineralized collagen suspension Weigh type I lyophilized collagen powder and dissolve it completely in 0.01 mol / L acetic acid solution, adjusting the collagen concentration to 1.0% (w / v). Prepare 0.5 mol / L CaCl2 solution and 0.3 mol / L Na2HPO4 solution, and add them sequentially to the collagen solution at a Ca / P molar ratio of 1.67. Stir magnetically at 4℃ for 30 minutes to obtain a homogeneous mineralized collagen suspension (first mineralized collagen suspension).

[0102] Step S102: Freeze-drying the first mineralized collagen layer The above-mentioned mineralized collagen suspension was poured into a mold with dimensions of 40 mm × 40 mm × 2 mm and pre-frozen at -40°C for 2 hours. Then, the freeze-drying process was initiated: during the freezing stage, the temperature was increased from -40°C to -25°C and maintained for 1 hour; during the initial drying stage, the chamber pressure was controlled at 20 Pa, and the plate temperature was increased from -25°C to -10°C; during the final drying stage, the temperature was increased to 5°C and maintained for 6 hours. After freeze-drying, a first layer of dried mineralized collagen porous material with a thickness of approximately 1.5–2.0 mm (first layer of mineralized collagen) was obtained.

[0103] Step S103: Addition and interface wetting of the second mineralized collagen suspension Prepare a second mineralized collagen suspension with a collagen concentration adjusted to 0.5% (w / v). The remaining preparation method is the same as that for the first mineralized collagen suspension. Lay the first layer of dried material (the first mineralized collagen layer) flat and evenly drop the second mineralized collagen suspension onto its surface at a rate of 0.20 mL / cm². 2 Then let it stand for 60 seconds to allow part of the suspension to seep into the pores of the first layer of mineralized collagen, forming a wet interpenetrating interface (the first wet interpenetrating interface zone).

[0104] Step S104: Hammer pressing / cold pressing densification of the wet interosmotic interface (first wet interosmotic zone) The material forming the wet interpenetrating interface (first wet interpenetrating interface zone) is placed between flat mold plates and a cold pressing pressure of 0.05 MPa is applied for 20 seconds, causing the pores at the interface to collapse locally, and the collagen fibers to interlock and strengthen, forming a dense interlayer structure (first continuous dense interface).

[0105] Step S105: Independent freeze-drying of the second mineralized collagen layer The cold-pressed material was then placed back into a freeze dryer for an independent freeze-drying process: pre-freezing temperature -35°C for 1 hour; initial drying stage chamber pressure of 15 Pa, plate temperature increased from -35°C to -15°C for 2 hours; final drying stage temperature increased to 5°C and maintained for 6 hours. After freeze-drying, a second layer of mineralized collagen dry structure (second mineralized collagen layer) was obtained, with an overall material thickness of approximately 3.0–3.5 mm.

[0106] The final bilayer mineralized adhesive raw material has a layered structure with obvious differences in pore size between the upper and lower layers. The upper layer has larger pores and the lower layer has smaller pores. The interlayer interface is a continuous and dense transition zone with a thickness of about 50 to 150 μm and no delamination phenomenon.

[0107] Example 2: Preparation method of mineralized adhesive raw material for a three-layer biomimetic cortical bone-transition layer-cancellous bone structure This embodiment illustrates the specific application of the present invention in the construction of multi-layer structures, and the parameters are specific examples under optional conditions.

[0108] Step S201: Preparation of the corresponding mineralized collagen suspension (first mineralized collagen suspension) for the first layer (dense cortical bone biomimetic layer) mineralized collagen layer. Weigh type I lyophilized collagen powder and dissolve it in 0.01 mol / L acetic acid solution to adjust the collagen concentration to 1.2% (w / v). Prepare 0.5 mol / L CaCl2 solution and 0.3 mol / L Na2HPO4 solution, and add the collagen solution at a Ca / P molar ratio of 1.67. Stir at 4℃ for 30 minutes to obtain a mineralized collagen suspension (first mineralized collagen suspension). This suspension has a high viscosity (900 mPa·s) and can form a dense microporous structure.

[0109] Step S202: Freeze-drying the first mineralized collagen layer The suspension was injected into a mold measuring 40 mm × 40 mm × 2 mm and pre-frozen at -40°C for 2 hours. The freeze-drying process was then initiated: during the freezing stage, the temperature was increased from -40°C to -25°C and maintained for 1 hour; during the initial drying stage, the chamber pressure was 20 Pa, and the plate temperature was increased from -25°C to -10°C; during the final drying stage, the temperature was increased to 5°C and maintained for 6 hours. The resulting first mineralized collagen layer was approximately 1.5–2.0 mm thick with a pore size of approximately 20–80 μm.

[0110] Step S203: Preparation and interface wetting of the corresponding mineralized collagen suspension (second mineralized collagen suspension) for the second mineralized collagen layer (transition layer). A second mineralized collagen suspension was prepared, with the collagen concentration adjusted to 0.8% (w / v) and the suspension viscosity set at 700 mPa·s. The remaining mineralization steps were the same as those for the first mineralized collagen suspension. The second mineralized collagen suspension was then pumped at 0.25 mL / cm³. 2 The solution is added evenly to the surface of the first layer and left to stand for 45 seconds, allowing part of the solution to penetrate into the pores of the first layer to form a wet interosmotic interface (the first wet interosmotic zone).

[0111] Step S204: Interface cold pressing and second-layer freeze drying The moistened material was placed between mold plates and subjected to a cold pressing pressure of 0.04 MPa for 20 seconds to form a dense transition layer structure at the interface. A second freeze-drying process was then performed: pre-freezing temperature –35°C for 1 hour; initial drying stage with a chamber pressure of 25 Pa and a temperature increase from –35°C to –15°C; final drying stage with a temperature increase to 5°C and maintained for 6 hours. The resulting second mineralized collagen layer had a thickness of approximately 1.0–1.5 mm and a pore size of approximately 80–150 μm.

[0112] Step S205: Injection and interface construction of the corresponding mineralized collagen suspension (third mineralized collagen suspension) in the third mineralized collagen layer (cancellous bone biomimetic layer). Prepare a third mineralized collagen suspension, adjusting the collagen concentration to 0.5% (w / v) and the suspension viscosity to 500 mPa·s, and stir thoroughly. Then, pump the suspension at a flow rate of 0.30 mL / cm³. 2 The amount of slurry added is dropped onto the surface of the second mineralized collagen layer and left to stand for 60 seconds, allowing some of the slurry to penetrate into the pores of the second mineralized collagen layer.

[0113] Step S206: Hammer pressing and freeze-drying of the second wet interpenetrating interface (second wet interpenetrating zone) A pressure of 0.03 MPa was applied to the wetting interface for 15 seconds to form a dense interfacial region (the second continuous dense interface). A third independent freeze-drying process was then performed: pre-freezing temperature –35°C for 1 hour; initial drying stage chamber pressure of 30 Pa, with the plate temperature increased from –35°C to –20°C; final drying stage temperature increased to 5°C and held for 6 hours. The resulting third mineralized collagen layer had a thickness of approximately 1.5–2.0 mm and a pore size of approximately 150–300 μm.

[0114] The resulting three-layer mineralized adhesive raw material exhibits a distinct hierarchical structure: the first layer has the smallest pores and the highest mechanical strength; the second layer is a transition layer with pore size and mechanical properties intermediate between the upper and lower layers; and the third layer is a macroporous layer, which facilitates cell penetration and angiogenesis. All three layers form continuous, dense interpenetrating zones without delamination. The overall thickness of the material is approximately 4–5 mm.

[0115] Please refer to the micropore structure of the bone repair material with a mineralized collagen layered structure formed in this embodiment. Figure 6 . Figure 6 The left image shows the microstructure of the top layer, while the right image shows the microstructure of the interlayer interface. It can be seen that the pore structure of the interlayer interface is relatively chaotic, forming a continuous and dense interface, thus achieving interlayer continuity and mechanical strengthening. Figure 7 This is a photograph of the three-layered mineralized collagen bone repair material of Embodiment 2 of the present invention. Figure 7 The figure illustrates the final three-layer mineralized adhesive raw material formed by the "independent freeze-drying + interpenetration + cold pressing + independent re-freeze-drying" process of Embodiment 2 of the present invention. The different layers in the figure have different pore sizes and mineralization characteristics; the interlayer regions are continuous and dense transition zones; and there is essentially no interface separation. This figure visually demonstrates the structural characteristics of the final product of the technical solution of the present invention.

[0116] Example 3: Preparation method of a five-layer gradient mineralized collagen layered bone repair material This embodiment illustrates the application of the present invention in the construction of multi-layer gradient structures, and is suitable for bone defect repair materials that require high overall mechanical properties and good tissue permeability.

[0117] Step S301: Preparation of mineralized collagen suspensions of different layers Five groups of mineralized collagen suspensions with collagen mass concentrations of 1.2%, 1.0%, 0.8%, 0.6%, and 0.4% (w / v) were prepared according to the required five-layer structure. All suspensions used type I collagen as the matrix, dissolved in 0.01 mol / L acetic acid solution, and CaCl2 and Na2HPO4 solutions were added at a Ca / P molar ratio of 1.67. The mixture was stirred at 4℃ for 30 minutes to obtain a homogeneous suspension. The higher concentration was used to construct the dense layer (relatively dense mineralized collagen layer) in the mineralized collagen layered structure, while the lower concentration was used to construct the loose layer (relatively loose mineralized collagen layer) in the mineralized collagen layered structure.

[0118] Step S302: Freeze-drying of the first mineralized collagen layer (the densest layer) A 1.2% (w / v) mineralized collagen suspension (the first mineralized collagen suspension) was injected into a mold measuring 40 mm × 40 mm × 2 mm and pre-frozen at –40°C for 2 hours. The freeze-drying process was then initiated: the freezing stage temperature was increased from –40°C to –25°C and held for 1 hour; the initial drying stage pressure was 20 Pa, and the plate temperature was increased from –25°C to –10°C; the final drying stage temperature was increased to 5°C and held for 6 hours. The resulting first mineralized collagen layer was approximately 1.5 mm thick with a pore size of approximately 20–60 μm.

[0119] Step S303: Addition, interface wetting, and freeze-drying of the second mineralized collagen suspension The 1.0% (w / v) mineralized collagen suspension (second mineralized collagen suspension) was prepared at 0.20 mL / cm³. 2 The additive was dropped onto the surface of the first mineralized collagen layer and allowed to stand for 60 seconds. Then, a cold press of 0.05 MPa was applied for 20 seconds to form a dense interpenetrating structure at the interface. A second freeze-drying process was performed (pre-freezing at –35℃ for 1 hour, initial drying conditions were 15 Pa, –35℃ → –15℃, final drying conditions were 5℃ for 6 hours) to obtain the second mineralized collagen layer, approximately 1.2 mm thick with a pore size of approximately 60–100 μm.

[0120] Step S304: Construction of the third mineralized collagen layer The 0.8% (w / v) suspension (third mineralized collagen suspension) was prepared at 0.25 mL / cm³. 2 The additive amount was added dropwise to the second mineralized collagen layer and allowed to stand for 45 seconds. A pressure of 0.04 MPa was applied to the interface for 15 seconds. Subsequently, a third freeze-drying was performed (using the same process as the second freeze-drying). The resulting third mineralized collagen layer was approximately 1.0 mm thick with a pore size of approximately 100–150 μm, serving as a transition layer.

[0121] It should be noted that, compared to the second mineralized collagen layer, the concentration of the mineralized collagen suspension used to form the third mineralized collagen layer is lower, which is equivalent to more water. As a result, more water will penetrate into the layers below. Therefore, although the third mineralized collagen layer uses a larger amount of mineralized collagen suspension, has a shorter settling time, and is subjected to less pressure, the overall thickness of the third mineralized collagen layer is smaller than that of the second mineralized collagen layer.

[0122] Step S305: Addition, compaction and freeze-drying of the fourth mineralized collagen suspension The 0.6% (w / v) suspension (fourth mineralized collagen suspension) was prepared at 0.30 mL / cm³. 2 The solution was dropped onto the third mineralized collagen layer, allowed to stand for 60 seconds, and then subjected to cold pressure of 0.03 MPa for 15 seconds. A fourth freeze-drying process was then performed (pre-freezing at –35℃, initial drying at 15 Pa, and final drying at 5℃). The resulting fourth layer was approximately 1.0 mm thick with a pore size of approximately 150–220 μm.

[0123] Step S306: Construction of the fifth mineralized collagen layer (the most porous layer) The 0.4% (w / v) mineralized collagen suspension (the fifth mineralized collagen suspension) was prepared at 0.35 mL / cm³. 2 The slurry was added to the fourth mineralized collagen layer and allowed to stand for 60 seconds to allow it to fully penetrate the interface. A hammer pressure of 0.02 MPa was then applied for 10 seconds to form the corresponding dense wet interface layer. A fifth independent freeze-drying process was then performed, following the same steps as described above. The resulting fifth mineralized collagen layer was approximately 1.2 mm thick with a pore size of approximately 200–350 μm.

[0124] The final material exhibits a distinct five-layer gradient structure: the first mineralized collagen layer is the densest, with the smallest pore size and highest load-bearing capacity; the pore size gradually increases between the second and third mineralized collagen layers, forming a structural transition zone; the fourth and fifth mineralized collagen layers have the largest porosity, which is conducive to cell migration and angiogenesis. The interfaces between each layer are continuous, dense, and interpenetrating structures, approximately 50–200 μm thick, without delamination or cracks. The overall thickness of the material is approximately 6 mm.

[0125] The five-layer mineralized adhesive raw material prepared in this embodiment exhibits a high overall compression modulus, while also possessing excellent bio-permeability and gradient biomimetic characteristics, making it suitable for repairing bone defects in load-bearing areas.

[0126] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0127] The following are the experimental data for each of the above embodiments: Table 1: Comparison of Data from Various Embodiments of the Invention

[0128] 1. Comparison of mechanical properties Compressive strength test: By measuring the strength of bone repair materials from different embodiments under compressive load, the mechanical stability of each embodiment in different layers of mineralized collagen raw materials can be compared. Generally speaking, the more layers and the denser the structure of the mineralized collagen, the higher its compressive strength.

[0129] 2. Comparison of mineralization degree TGA analysis: The proportion of inorganic and organic matter in the mineralized adhesive raw materials in different embodiments was analyzed using TGA to detect the degree of mineralization.

[0130] The decreasing degree of mineralization from bottom to top indicates that more layers and the control of parameters such as pH adjustment and inorganic phase ratio can effectively control the mineralization process and form a higher quality mineralized structure.

[0131] 3. Porosity and pore size distribution Scanning electron microscopy (SEM) image analysis: The pore structure of the materials in different embodiments was observed using SEM, including the distribution, size, and uniformity of pores. Ideal bone repair materials should have suitable porosity to promote cell growth and bone regeneration.

[0132] 4. Comparison of horizontal interface clipping performance Special fixtures are required to fix one layer and push another until the interface slips or breaks. The transverse interfacial shear force of each layer in different embodiments is tested. Generally, the higher the concentration and the denser the structure of the mineralized collagen, the higher its transverse interfacial shear strength.

[0133] The multi-layered structure has a more uniform porosity and pore size distribution, and is closer to the natural bone structure, resulting in better bioactivity and bone cell adhesion.

[0134] The technical solutions of this invention can be extended to other mineralized collagen-related applications, such as customized material design for different bone defect environments, fracture repair, maxillofacial bone defect repair, cranioplasty, and plastic and cosmetic surgery. They can also be combined with 3D printing technology to construct scaffolds with biomimetic structures, and to develop novel medical devices with dynamic mineralization regulation.

[0135] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for preparing a bone repair material with a layered structure of mineralized collagen, characterized in that, include: Prepare one or more portions of mineralized collagen suspension, and freeze-dry each portion of mineralized collagen suspension in sequence to form the mineralized collagen layered structure with each layer of mineralized collagen stacked in the thickness direction. By adjusting the relevant parameters of each mineralized collagen suspension and controlling at least one of the process parameters of the freeze-drying process, the pore size of the bottom mineralized collagen layer to the top mineralized collagen layer in the formed mineralized collagen layered structure increases sequentially. A continuous and dense interface is formed between two adjacent mineralized collagen layers by the following method: A mineralized collagen suspension suitable for forming the upper mineralized collagen layer of the two adjacent mineralized collagen layers is applied to the surface of the lower mineralized collagen layer, which has been formed by freeze-drying, and left to stand for a preset time to allow it to penetrate into the pores formed by freeze-drying in the lower mineralized collagen layer, forming a wet interface interpenetration zone; pressure is applied to the wet interface interpenetration zone, and the mineralized collagen suspension covering the surface of the lower mineralized collagen layer and the wet interface interpenetration zone after applying pressure are freeze-dried to form the continuous and dense interface.

2. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 1, characterized in that, The relevant parameters of the mineralized collagen suspension include at least one of collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value and its adjustment rate; the process parameters of the freeze-drying treatment include at least one of the pre-freezing temperature and cooling rate of the freezing stage, and the vacuum degree, heating rate and sublimation rate of the vacuum sublimation drying stage.

3. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 2, characterized in that, The adjustment of relevant parameters for each mineralized collagen suspension includes: from the bottom layer to the top layer of mineralized collagen, the collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the mineralized collagen suspension decrease sequentially; the control of process parameters for the freeze-drying process includes: from the bottom layer to the top layer of mineralized collagen, the cooling rate, vacuum degree, heating rate, and sublimation rate of the freeze-drying process decrease sequentially, and the pre-freezing temperature increases sequentially.

4. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 3, characterized in that, The method of adjusting the target pH value and its adjustment rate of the mineralized collagen suspensions from the bottom layer to the top layer to form a mineralized collagen layer in a sequentially decreasing manner includes: adding an alkaline solution to each portion of the prepared mineralized collagen suspension from the bottom layer to the top layer at a sequentially decreasing adjustment rate, thereby adjusting the pH value from the same initial value to a sequentially decreasing target pH value.

5. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 3 or 4, characterized in that, The target pH value and its adjustment rate of the mineralized collagen suspension, which are suitable for forming the bottom layer of mineralized collagen to the top layer, are gradually reduced.

6. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 1, characterized in that, The pressure exerted on the wet interfacial interpenetration zone of the mineralized collagen layer closer to the bottom layer is greater than the pressure exerted on the wet interfacial interpenetration zone of the mineralized collagen layer farther away from the bottom layer.

7. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 1, characterized in that, Pressure is applied to the wet interosmotic zone by hammer pressing or cold pressing.

8. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 1, characterized in that, The preset time for the mineralized collagen suspension in the lower mineralized collagen layer to stand is greater than or equal to the preset time for the mineralized collagen suspension in the upper mineralized collagen layer to stand.

9. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 1, characterized in that, The mineralized collagen layered structure contains 2 to 10 layers of mineralized collagen.

10. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 1, characterized in that, The mineralized collagen layered structure comprises a first mineralized collagen layer and a second mineralized collagen layer stacked sequentially, and is prepared in the following manner: The first mineralized collagen suspension was placed in a mold and the first mineralized collagen layer was formed by the first freeze-drying process. The second mineralized collagen suspension is added to the surface of the first mineralized collagen layer and left to stand for a first preset time to allow it to penetrate into the pores formed by the first freeze-drying treatment in the first mineralized collagen layer, forming a first wet interface interpenetration zone. A first pressure is applied to the first wet interface interpenetration zone, and a second freeze-drying treatment is performed on the first wet interface interpenetration zone after the first pressure is applied and the second mineralized collagen suspension covering the surface of the first mineralized collagen layer to form a first continuous dense interface and the second mineralized collagen layer. The collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the first mineralized collagen suspension are greater than or equal to those of the second mineralized collagen suspension; the cooling rate, vacuum degree, heating rate, and sublimation rate of the first freeze-drying treatment are greater than or equal to those of the second freeze-drying treatment; and the pre-freezing temperature of the first freeze-drying treatment is lower than or equal to the pre-freezing temperature of the second freeze-drying treatment.

11. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 10, characterized in that, The mineralized collagen layered structure further includes a third mineralized collagen layer stacked on top of the second mineralized collagen layer, and the preparation of the mineralized collagen layered structure further includes: The third portion of the prepared mineralized collagen suspension is added to the surface of the second mineralized collagen layer and left to stand for a second preset time to allow it to penetrate into the pores formed by the second freeze-drying treatment in the second mineralized collagen layer, forming a second wet interface interpenetration zone. A second pressure is applied to the second wet interface interpenetration zone, and a third freeze-drying treatment is performed on the second wet interface interpenetration zone after the second pressure is applied and the third mineralized collagen suspension covering the surface of the second mineralized collagen layer to form a second continuous dense interface and the third mineralized collagen layer. The collagen concentration, inorganic phase ratio, viscosity, solid content, target pH value, and adjustment rate of the second mineralized collagen suspension are greater than or equal to those of the third mineralized collagen suspension; the cooling rate, vacuum degree, heating rate, and sublimation rate of the second freeze-drying treatment are greater than or equal to those of the third freeze-drying treatment; the pre-freezing temperature of the second freeze-drying treatment is lower than or equal to the pre-freezing temperature of the third freeze-drying treatment; the first pressure is greater than the second pressure; the first preset time is greater than or equal to the second preset time; the difference between the target pH value of the second mineralized collagen suspension and the target pH value of the third mineralized collagen suspension is less than the difference between the target pH value of the first mineralized collagen suspension and the target pH value of the second mineralized collagen suspension.

12. The method for preparing bone repair material with a layered structure of mineralized collagen according to claim 11, characterized in that, The first mineralized collagen suspension has a collagen concentration of 1.0–1.2 w / v%, an inorganic phase ratio of 65–70%, a viscosity of 800–1000 mPa·s, a solid content of 13–16%, a target pH of 8–9, and a target pH adjustment rate of 10–20 ml / min; the second mineralized collagen suspension has a collagen concentration of 0.8–1.0 w / v%, an inorganic phase ratio of 60–65%, a viscosity of 600–800 mPa·s, a solid content of 10–13%, a target pH of 7–8, and a target pH adjustment rate of 5–15 ml / min; the third mineralized collagen suspension has a collagen concentration of 0.5–0.8 w / v%, an inorganic phase ratio of 55–60%, and a viscosity of 400–600 mPa·s. The freeze-drying process has the following parameters: mPa·s, solid content 7-10%, target pH 6.5-7.5, and target pH adjustment rate 3-10 ml / min; the first freeze-drying process has a pre-freezing temperature of -50℃ to -40℃, a cooling rate of 1.0-2.0℃ / min, a vacuum of 10-15 Pa, a heating rate of 0.15-0.30℃ / min, and a sublimation rate of 1.5-3.0% / h; the second freeze-drying process has a pre-freezing temperature of -40℃ to -30℃, a cooling rate of 0.5-1.0℃ / min, a vacuum of 15-20 Pa, a heating rate of 0.10-0.15℃ / min, and a sublimation rate of 1.0-1.5% / h; the third freeze-drying process has a pre-freezing temperature of -30℃ to -20℃, a cooling rate of 0.1-0.5℃ / min, and a vacuum of 20-25 Pa. The temperature ranges as follows: Pa, heating rate of 0.05~0.10 ℃ / min, sublimation rate of 0.5~1.0 % / h; first pressure of 0.03~0.05 MPa, second pressure of 0.02~0.03 MPa; first preset time of 45~60 seconds, second preset time of 60~80 seconds; pore size of the first mineralized collagen layer of 20~80 μm, pore size of the second mineralized collagen layer of 80~150 μm, and pore size of the third mineralized collagen layer of 150~300 μm.

13. A bone repair material with a mineralized collagen layered structure prepared by the method for preparing a bone repair material with a mineralized collagen layered structure as described in any one of claims 1 to 12.

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