A collagen biphase crosslinking material, its preparation method, and an implantable scaffold.

Abstract

This invention provides a collagen biphase crosslinked material, its preparation method, and an implantable scaffold. It relates to compositions of polymeric compounds and also to the field of medical materials technology. This application provides a method for preparing a collagen biphase crosslinked material. During the nucleation phase, a low concentration of hydrogen peroxide is introduced to synergistically promote collagen self-assembly and hydrogen peroxide crosslinking. During the growth phase, a higher concentration of hydrogen peroxide solution is added to allow the covalent crosslinked network to form synchronously with fiber extension. The growth phase self-assembly control process can significantly extend the collagen self-assembly time, resulting in more thorough crosslinking and achieving structure-function synergy at the molecular scale. Finally, hydrogen peroxide decomposes into water and oxygen, leaving no crosslinking agent residue. The prepared collagen biphase crosslinked material exhibits good mechanical properties and resistance to enzymatic degradation, and the complete in vivo degradation time can be controlled between 12 and 24 months.

Description

Technical Field

[0001] This invention relates to compositions of polymeric compounds, and also to the field of medical materials technology, particularly to a collagen biphase crosslinking material, its preparation method, and an implantable scaffold. Background Technology

[0002] Collagen, as a major component of the extracellular matrix, is an ideal material for tissue engineering scaffolds due to its excellent biocompatibility, biodegradability, and biomimetic properties. Currently, most collagen tissue engineering scaffolds are obtained by directly lyophilizing purified collagen solutions. Uncrosslinked pure collagen scaffolds rely solely on non-covalent bonds to maintain the network, resulting in low mechanical strength, uneven pore distribution, and rapid degradation by collagenases in vivo, failing to meet load-bearing or long-term implantation requirements. Some materials crosslink collagen in solution before lyophilization. However, the addition of crosslinking agents in solution leads to disordered collagen molecules, pore collapse, and uneven diffusion of the crosslinking agent, forming a heterogeneous network. Other materials involve immersing lyophilized collagen scaffolds for crosslinking. Due to the poor permeability of the crosslinking agent, a dense crosslinked layer can only be formed on the surface of the material, while the internal fibers remain loose due to the lack of crosslinking, resulting in a "strong surface, weak interior" structural layering that easily swells and collapses in body fluid environments. The fundamental problem with these strategies is that they only crosslink collagen molecules, leading to uneven crosslinking and slow crosslinking rates, resulting in localized collapse. Meanwhile, the residue of cross-linking agents also causes a decrease in the material's tissue affinity, making it prone to adverse reactions.

[0003] Collagen, as a major component of the natural extracellular matrix, possesses a self-assembly capability that forms a crucial foundation for constructing biomimetic materials. Under physiological conditions, collagen molecules can spontaneously aggregate to form an ordered three-dimensional fiber network. This process typically consists of two phases: the nucleation phase, where collagen monomers form initial fiber cores in the liquid phase through intermolecular forces, and the growth phase, which is the key step in the further extension, arrangement, and formation of stable higher-order structures of the fibers. This self-assembly property endows materials with the topological structure of natural tissues, providing an ideal microenvironment for cell adhesion and proliferation. However, existing self-assembly-based collagen scaffolds still have significant limitations. Traditional self-assembly collagen scaffolds rely on physical self-assembly, resulting in inherent defects such as low mechanical strength, poor structural stability, and insufficient resistance to enzymatic degradation, severely restricting their application in load-bearing tissue repair or long-term implantation scenarios. Summary of the Invention

[0004] To address the shortcomings of existing cross-linked materials, such as low mechanical strength, poor structural stability, and insufficient resistance to enzymatic degradation, this application provides the following: First, a method for preparing a collagen biphase cross-linked material. During the nucleation stage, a low concentration of hydrogen peroxide is introduced to facilitate synergistic collagen self-assembly and hydrogen peroxide cross-linking. During the growth stage, a higher concentration of hydrogen peroxide solution is used in a solid state to simultaneously form a covalent cross-linked network and fiber extension, thereby achieving structure-function synergy at the molecular scale. Ultimately, the hydrogen peroxide decomposes into water and oxygen, leaving no cross-linking agent residue. The porosity of the material or product is determined by the amount of hydrogen peroxide added. Second, a collagen biphase cross-linked material with high mechanical properties, resistance to enzymatic degradation, and an ideal pore structure is provided. Third, a collagen-based implant scaffold with high mechanical properties, resistance to enzymatic degradation, and an ideal pore structure is obtained using the collagen biphase cross-linked material of this application.

[0005] Regarding a first aspect of the present invention, this application provides a method for preparing a collagen biphase crosslinked material, comprising the following preparation process:

[0006] Preparation of nucleation stage collagen: Adjust the temperature of the acidic collagen solution to 18-28℃, add hydrogen peroxide solution, adjust the pH value to 6-8, stir for 30-120 min, and obtain solution I with a hydrogen peroxide mass concentration of 0.1-1.8 wt%;

[0007] Preparation of collagen during the growth period: Heat solution I to 30-37℃, adjust the pH to 8-9.5, add hydrogen peroxide solution, stir for 2-6 hours to obtain solution II with a hydrogen peroxide mass concentration of 3-5 wt%;

[0008] Preparation of biphase cross-linked materials: Collagen in solution II was collected, and the collagen concentration was controlled at 5-20 mg / mL. After freeze-drying, the biphase cross-linked materials were obtained.

[0009] In this application, the speed range of rapid stirring is 300-600 rpm, and the speed range of slow stirring is 30-100 rpm.

[0010] At room temperature (18-28℃), collagen self-assembly begins slowly after pH adjustment, which is the nucleation phase. Adding a low concentration of hydrogen peroxide allows the cross-linking reaction to occur simultaneously during the nucleation phase. The cross-linking reaction is slower during the nucleation phase, and steric hindrance affects the rearrangement of collagen molecules during nucleation, slowing down the aggregation of collagen molecules and resulting in more uniform subsequent materials.

[0011] Preparation of biphase cross-linked materials: Centrifuge the growth-stage solution, collect collagen precipitates, wash and control the collagen concentration to 5-20 mg / mL; freeze-dry the collagen to obtain biphase cross-linked materials.

[0012] This invention employs a "two-phase system crosslinking" strategy, precisely embedding crosslinking into different stages of collagen self-assembly. During the nucleation stage, a low-concentration hydrogen peroxide solution is introduced to facilitate crosslinking, allowing collagen self-assembly and hydrogen peroxide crosslinking to proceed synergistically. While maintaining dynamic equilibrium in the liquid phase, the fiber nucleation rate is moderately controlled. This avoids premature gel solidification due to uneven crosslinking caused by the hydrogen peroxide oxidation mechanism, while simultaneously slowing down the self-assembly rate during collagen nucleation through the crosslinking behavior of hydrogen peroxide, providing a longer time window for orderly fiber growth.

[0013] During the growth period, a high concentration of hydrogen peroxide solution is used in the solid state to perform re-crosslinking, so that the covalent crosslinking network is formed synchronously with fiber extension, thereby achieving structure-function synergy at the molecular scale.

[0014] Meanwhile, during subsequent preparation, hydrogen peroxide ultimately decomposes into water and oxygen, leaving no cross-linking agent residue. Simultaneously controlling the concentration of hydrogen peroxide regulates its cross-linking / decomposition rate, allowing the produced oxygen to synchronously control the porosity of the tissue-engineered scaffold.

[0015] After cleaning and purification, the final material is obtained by freeze drying. By controlling the collagen concentration at 5-20 mg / mL, the porosity and pore size of the scaffold material can be optimized.

[0016] Through the aforementioned technical means, not only are the biomimetic advantages of collagen self-assembly (such as highly ordered fiber arrangement and uniform pores) preserved, but the bonding strength and stability of the fiber interface are also enhanced through dynamic cross-linking. Compared with existing technologies, this invention overcomes the unidirectional limitations of "cross-linking interfering with self-assembly" or "self-assembly detaching from cross-linking," ultimately obtaining a collagen-based implant scaffold material with high mechanical properties, resistance to enzymatic degradation, and an ideal pore structure, providing a solution for tissue engineering and regenerative medicine.

[0017] Furthermore, the mass concentration of hydrogen peroxide in solution I is 0.1-1 wt%.

[0018] Furthermore, in the preparation of the nucleation stage collagen, rapid stirring is performed for 30-60 minutes.

[0019] Adjust the pH value to 6-8 to achieve the optimal self-assembly state of collagen and drive collagen into the nucleation stage; stir for 30-60 minutes to introduce shear disturbance to the solution during the nucleation process. When combined with a 0.1-1 wt% hydrogen peroxide solution, it results in a slower rate of collagen nucleation and allows for more uniform and complete nucleation.

[0020] Furthermore, the pH value of the hydrogen peroxide in solution II is 8.5-9.5.

[0021] Furthermore, in the preparation of the collagen during the growth period, stirring is carried out for 3-5 hours.

[0022] The optimal solution temperature for collagen self-assembly is 30-37℃, initiating the growth phase of collagen self-assembly. At this temperature, collagen fibers in the solution begin to grow laterally and extend, becoming thicker and longer. Further adjusting the pH to 8.5-9.5, away from the optimal self-assembly time, slows down the growth rate of collagen fibers during the proliferative phase. At this pH, hydrogen peroxide decomposes simultaneously to produce oxygen, which is beneficial for regulating the scaffold pore structure.

[0023] Adding a high concentration of hydrogen peroxide solution for cross-linking strengthens the cross-linking on the already formed fiber framework, improving material stability. Stirring for 3-5 hours allows for further uniform reinforcement between fibers during the entire process of collagen fiber formation in the growth phase, resulting in a more uniform cross-linked material with better physicochemical properties.

[0024] Furthermore, in the preparation of nucleation-stage collagen, the concentration of the acidic collagen solution is 1-10 mg / mL, the temperature is 2-8℃, and the pH value is 3-5.

[0025] More preferably, the concentration of the acidic collagen solution is 1-5 mg / mL.

[0026] Controlling the acidity and low temperature of the collagen solution ensures uniform collagen dissolution and inhibits self-assembly.

[0027] Furthermore, in the preparation of nucleation-stage collagen, the acidic collagen solution contains sodium chloride, and the concentration of sodium chloride in the acidic collagen solution is 0.5-1.5 w / v.

[0028] Furthermore, in the preparation of nucleation-stage collagen, the acidic collagen solution contains sodium chloride, and the concentration of sodium chloride in the acidic collagen solution is 0.7-1.2 w / v.

[0029] Adding sodium chloride enables salt dissolution, which further disperses collagen molecules in the solution and is beneficial to the degree of cross-linking during the subsequent nucleation stage.

[0030] As an electrolyte, NaCl inhibits excessively rapid aggregation and can partially shield the surface charges of collagen molecules (such as the ionization of carboxyl and amino groups), reducing electrostatic repulsion and allowing collagen molecules to aggregate in a more controllable manner, avoiding uneven fiber formation caused by excessively rapid local nucleation. It also optimizes fiber diameter and arrangement: an appropriate sodium chloride concentration in an acidic collagen solution creates suitable ionic strength, which helps form a uniform microfiber network. This, combined with a moderate hydrogen peroxide concentration, prevents excessively fine fibers (too low a sodium chloride concentration) and excessive fiber aggregation (too high a sodium chloride concentration) in an acidic collagen solution, resulting in unsatisfactory mechanical properties and resistance to enzymatic hydrolysis.

[0031] In a second aspect, this application provides a collagen biphase crosslinking material obtained by the preparation method described in this application.

[0032] In a third aspect, this application provides an implantable stent using the collagen biphasic crosslinking material described in this application.

[0033] The collagen fibers obtained by this invention can be used as injection implantation materials, or freeze-dried and used as tissue engineering scaffolds for applications in burn dressings, tissue hemostasis, soft tissue reconstruction, and other fields.

[0034] Beneficial effects: 1. This application provides a method for preparing a collagen biphase crosslinked material. During the nucleation stage, a low concentration of hydrogen peroxide is introduced to enable collagen self-assembly and hydrogen peroxide crosslinking to proceed synergistically. During the growth stage, a higher concentration of hydrogen peroxide solution is added in the solid state to enable the covalent crosslinked network to form synchronously with fiber extension. The growth stage self-assembly control process can significantly extend the collagen self-assembly time, making the crosslinking more thorough and achieving structure-function synergy at the molecular scale. Finally, hydrogen peroxide decomposes into water and oxygen, leaving no crosslinking agent residue. At the same time, the porosity of the material or product is obtained by adjusting the amount of hydrogen peroxide added. The prepared collagen biphase crosslinked material has good mechanical properties and resistance to enzymatic degradation, and the complete in vivo degradation time can be controlled between 12 and 24 months.

[0035] 2. Further, the concentration, pH value, temperature and stirring time of hydrogen peroxide during the nucleation and growth stages are optimized to further improve the mechanical properties and enzymatic resistance of the prepared collagen biphase crosslinked material.

[0036] 3. Furthermore, this application adds sodium chloride to the acidic collagen solution to achieve salt dissolution, which can further disperse collagen molecules in the solution, improve the degree and uniformity of crosslinking during the subsequent nucleation and / or growth phases, and further enhance the mechanical properties and enzymatic resistance of the prepared collagen biphase system crosslinked material. Attached Figure Description

[0037] Figure 1 These are macroscopic and microscopic morphological structures of the collagen biphase crosslinked materials obtained in Example 3, Comparative Examples 1-3 and Comparative Example 7;

[0038] Figure 2 These are macroscopic and microscopic morphological structures of the collagen biphase crosslinked materials obtained in Example 1, Comparative Example 6, and Comparative Examples 8-9.

[0039] Figure 3 These are macroscopic and microscopic morphological structures of the collagen biphase crosslinked materials obtained in Examples 2, 4-5, and 10.

[0040] Figure 4These are the tensile strength test results of the collagen biphase crosslinked materials obtained in Examples 1-3, Comparative Examples 2-3, and Comparative Examples 5-6;

[0041] Figure 5 The graph shows the tensile strength test results of the collagen biphase crosslinked materials obtained from Comparative Examples 1, 4 and 7-10. Detailed Implementation

[0042] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] Example 1:

[0044] 1) Adjusting the acidic collagen solution: Adjust the pH of the 2 mg / mL acidic collagen solution to 3 and control the solution temperature to 2℃.

[0045] 2) Add sodium chloride: Slowly add sodium chloride crystals to the acidic collagen solution while stirring, ensuring that the added sodium chloride crystals dissolve quickly so that the sodium chloride concentration in the solution system is 0.9 w / v after dissolution.

[0046] 3) Preparation of collagen during the nucleation stage: Place the collagen solution in water and control the solution temperature at 20°C. At the same time, add 30 wt% hydrogen peroxide solution to the solution (in other embodiments, the concentration of hydrogen peroxide solution can be selected from any concentration between 23-50 wt%), so that the final mass concentration of hydrogen peroxide in the solution is 1 wt%. Immediately adjust the pH value of the solution to 6 and stir rapidly for 30 min.

[0047] 4) Preparation of collagen during the growth phase: After stirring, the solution temperature was adjusted to 37℃ and the pH value was adjusted to 8.5. Collagen began to precipitate after slow stirring for 5 minutes. Then, 30 wt% hydrogen peroxide solution was added to the solution to make the final mass concentration of hydrogen peroxide in the solution 5 wt%, and the solution was slowly stirred for 3 hours.

[0048] 5) Preparation of biphase cross-linked materials: After stirring, the collagen precipitates were collected by centrifugation, washed multiple times with 0.9% physiological saline, and the collagen concentration was controlled to be 5 mg / mL before freeze-drying. The precipitates were dispensed into molds (50 mm * 30 mm * 10 mm polytetrafluoroethylene freeze-drying molds) and freeze-dried (freeze-drying conditions: pre-freeze at -20℃ for 24 h, then freeze-dry in a freeze dryer at -40℃, vacuum degree of 0.1-5 Pa, freeze-drying time of 24 h) to obtain collagen biphase cross-linked scaffold materials.

[0049] Example 2:

[0050] 1) Adjusting the acidic collagen solution: Adjust the pH of the 5 mg / mL acidic collagen solution to 5 and control the solution temperature to 8℃.

[0051] Add sodium chloride: Slowly add sodium chloride crystals to the acidic collagen solution while stirring, ensuring that the added sodium chloride crystals dissolve quickly so that the sodium chloride concentration in the solution system is 0.9 w / v after dissolution.

[0052] 3) Preparation of collagen during the nucleation stage: Place the collagen solution in water and control the solution temperature at 20℃. At the same time, add 30 wt% hydrogen peroxide solution to the solution so that the final mass concentration of hydrogen peroxide in the solution is 0.1 wt%. Immediately adjust the pH value of the solution to 8 and stir rapidly for 60 min.

[0053] 4) Preparation of collagen during the growth phase: After stirring, the solution temperature was adjusted to 37℃ and the pH value was adjusted to 9.5. Collagen began to precipitate after slow stirring for 5 minutes. Then, 30 wt% hydrogen peroxide solution was added to the solution to make the final mass concentration of hydrogen peroxide in the solution 3 wt%, and the solution was slowly stirred for 5 hours.

[0054] 5) Preparation of biphase cross-linked materials: After stirring, the collagen precipitates were collected by centrifugation, washed multiple times with 0.9% physiological saline, and the collagen concentration was controlled to be 20 mg / mL before freeze-drying. The precipitates were dispensed into molds and freeze-dried to obtain the collagen biphase cross-linked scaffold material.

[0055] Example 3:

[0056] 1) Adjusting the acidic collagen solution: Adjust the pH of the 10 mg / mL acidic collagen solution to 4 and control the solution temperature to 5℃.

[0057] 2) Add sodium chloride: Slowly add sodium chloride crystals to the acidic collagen solution while stirring, ensuring that the added sodium chloride crystals dissolve quickly so that the sodium chloride concentration in the solution system is 0.9 w / v after dissolution.

[0058] 3) Preparation of collagen during the nucleation stage: Place the collagen solution in water and control the solution temperature at 20℃. At the same time, add 30 wt% hydrogen peroxide solution to the solution so that the final mass concentration of hydrogen peroxide in the solution is 0.5 wt%. Immediately adjust the pH value of the solution to 7 and stir rapidly for 40 min.

[0059] 4) Preparation of collagen during the growth phase: After stirring, the solution temperature was adjusted to 37℃ and the pH value was adjusted to 9. Collagen began to precipitate after slow stirring for 5 minutes. Then, 30 wt% hydrogen peroxide solution was added to the solution to make the final mass concentration of hydrogen peroxide in the solution 4 wt%, and the solution was slowly stirred for 4 hours.

[0060] 5) Preparation of biphase cross-linked materials: After stirring, the collagen precipitates were collected by centrifugation, washed multiple times with 0.9% physiological saline, and the collagen concentration was controlled to be 10 mg / mL before freeze-drying. The precipitates were dispensed into molds and freeze-dried to obtain the collagen biphase cross-linked scaffold material.

[0061] Table 1. List of raw materials, dosages, and process parameters used in Examples 1 to 3

[0062]

[0063] Comparative Example 1 differs from Example 1 in that it does not undergo self-assembly; instead, the scaffold is obtained by cross-linking in a collagen solution with conventional glutaraldehyde followed by freeze-drying. The process is as follows:

[0064] The pH of a 10 mg / mL acidic collagen solution was adjusted to neutral, and a 50 wt% glutaraldehyde solution was added to bring the final glutaraldehyde concentration in the sample solution to 1 wt%. The mixture was stirred for 2 hours to initiate the cross-linking reaction. The cross-linked collagen solution was degassed, pre-frozen, and lyophilized to obtain a cross-linked collagen scaffold. The scaffold was then washed multiple times with 0.9% physiological saline and dried again to obtain the final cross-linked collagen scaffold.

[0065] Comparative Example 2 differs from Example 1 in that the scaffold was obtained by directly freeze-drying the collagen solution and then cross-linking it through immersion. The preparation process is as follows:

[0066] The pH of a 10 mg / mL acidic collagen solution was adjusted to neutral. 15 mL of the acidic collagen solution was placed in a 50 mm * 30 mm * 10 mm polytetrafluoroethylene lyophilization mold, degassed, pre-frozen, and then lyophilized to obtain a cross-linked collagen scaffold. The lyophilized collagen scaffold was then immersed in a 1 wt% glutaraldehyde aqueous solution for 2 hours in a fully submerged state. The scaffold was then removed, rinsed repeatedly with 0.9% physiological saline, and dried again to obtain the final cross-linked collagen scaffold.

[0067] Comparative Example 3 differs from Example 1 in that: after conventional self-assembly, the scaffold is obtained directly by freeze-drying without cross-linking, as follows:

[0068] A 10 mg / mL acidic collagen solution was adjusted to pH 7.4, and sodium chloride was added until its concentration reached 0.9%. The collagen solution was then placed in a constant temperature water bath at 37°C for 3 hours to allow the collagen to self-assemble into a collagen gel scaffold. The obtained gel scaffold was washed multiple times with 0.9% physiological saline and finally lyophilized to obtain the self-assembled collagen scaffold material.

[0069] Comparative Example 4 differs from Example 1 in that the amount of hydrogen peroxide used in step 3) during the nucleation period is higher, and the final hydrogen peroxide concentration is 2 wt%.

[0070] Comparative Example 5 differs from Example 1 in that: in step 4), the amount of hydrogen peroxide used during the growth period is lower, and the final hydrogen peroxide concentration is 2 wt%.

[0071] Comparative Example 6 differs from Example 1 in that sodium chloride was not added to the acidic collagen solution before the nucleation period.

[0072] Comparative Example 7 differs from Example 1 in that the temperature settings for step 3) nucleation period and step 4) growth period are different. The temperature for the nucleation period is set to 8°C, and the temperature for the growth period is set to 20°C.

[0073] Comparative Example 8 differs from Example 1 in that: in step 4), the pH value during the growth period was not adjusted to be far from the optimal self-assembly pH value, and the pH value was set to 7.

[0074] Comparative Example 9 differs from Example 1 in that: in step 3), the stirring time during the nucleation period is insufficient and the stirring is slow, with a slow stirring time of 10 minutes.

[0075] Comparative Example 10 differs from Example 1 in that the collagen concentration controlled before freeze-drying was too high, with a collagen concentration of 30 mg / mL.

[0076] Performance testing

[0077] 1. Macroscopic and microscopic morphology of materials

[0078] The microstructure of the cross-linked materials prepared by the processes in each embodiment and comparative example was observed using an electron microscope (300x magnification). The influence of various process conditions on the sample morphology was analyzed by examining the macroscopic and microscopic pore morphology of each cross-linked material. Figures 1-3 These are macroscopic and microscopic morphological structure diagrams of each embodiment and comparative example.

[0079] As shown in the figures, the macroscopic structures of samples 1-3 all exhibit a uniform and smooth sponge-like appearance, with uniform and fine pores in the sponge cross-linked material. The microscopic morphology of all samples shows uniform and open pores, with no significant difference in pore size. This meets the requirements for tissue engineering cross-linked materials.

[0080] Comparative Example 1 involved cross-linking collagen molecules directly in a collagen solution using glutaraldehyde without collagen self-assembly, followed by freeze-drying. Macroscopic images of the scaffold structure show that the freeze-dried scaffold of Comparative Example 1 exhibits extremely uneven and irregular protrusions. This is because the cross-linking reaction between glutaraldehyde and collagen molecules in solution is rapid and uneven, resulting in uneven protrusions on the collagen scaffold after direct freeze-drying. Furthermore, the microstructure of Comparative Example 1 lacks a porous structure, exhibiting numerous sheet-like structures, which are unsuitable for the cell adhesion and growth required for tissue engineering scaffolds.

[0081] Comparative Example 2 involved lyophilizing a collagen solution and then immersing it in glutaraldehyde solution for cross-linking. The macroscopic structure revealed that this collagen scaffold exhibited an uneven, irregular convex appearance. Simultaneously, its microstructure consisted of numerous collapsed pores with uneven pore sizes.

[0082] Comparative Example 3, obtained by directly freeze-drying collagen after self-assembly, also exhibits an uneven appearance. This is due to the rapid local aggregation caused by the rapid self-assembly of collagen under simulated physiological conditions in vitro. The microstructure of this sample shows a uniform porous structure, but the pores are relatively large.

[0083] The cross-linked material prepared in Comparative Example 4 exhibited an extremely loose, collapsed structure. This was due to the high concentration of hydrogen peroxide used during the nucleation stage, leading to excessive cross-linking in the early stages and inhibiting the self-assembly behavior of collagen during the subsequent growth stage. The subsequent self-assembly process did not occur significantly, resulting in poor mechanical properties of the final cross-linked material. Its microstructure showed an adhesive, finely porous structure.

[0084] Comparative Example 5 had a low concentration of hydrogen peroxide used during the growth period, which resulted in a weak cross-linking effect during the self-assembly process. As a result, the cross-linked material had a loose appearance and the microstructure pores and pore walls were loose. This was due to incomplete cross-linking.

[0085] Comparative Example 6 shows that 0.9% sodium chloride was added to the collagen solution before the nucleation stage. Due to the lack of the dispersing effect of sodium chloride, the self-assembly behavior of collagen was significantly inhibited in the subsequent preparation process, resulting in the subsequent freeze-drying process of the sample being similar to the direct freeze-drying of the collagen solution. The subsequent addition of hydrogen peroxide only had a slight cross-linking effect, and the oxygen produced by the decomposition of hydrogen peroxide caused the scaffold to not exhibit a uniform and continuous porous structure.

[0086] Comparative Example 7 did not regulate the nucleation and growth phases to the optimal self-assembly temperature, resulting in a compact and collapsed scaffold structure. No obvious through-pore structure was observed on the surface, and the pore walls of the microstructure collapsed, which is not conducive to cell adhesion and proliferation during the use of tissue engineering scaffolds.

[0087] Comparative Example 8 failed to adjust the pH of the self-assembly process away from the optimal pH during the growth phase, resulting in an excessively rapid collagen self-assembly rate that did not match the cross-linking rate of hydrogen peroxide. This led to collagen molecules becoming entangled due to the rapid self-assembly, resulting in a cross-linked material with indistinct microstructure pores, consisting entirely of large, closed pores.

[0088] In Comparative Example 9, the stirring speed and time during nucleation were relatively short. Rapid stirring during the nucleation period provides shear disturbance to the solution, slowing down the collagen nucleation process. However, excessively slow stirring speeds lead to accelerated self-assembly during nucleation, which is mismatched with the cross-linking of hydrogen peroxide, ultimately resulting in uneven collagen self-assembly. This manifests as an uneven material surface and excessively coarse and large microstructure pores.

[0089] Comparative Example 10 showed an excessively high collagen concentration. This high concentration caused some of the collagen structures to shrink and collapse, resulting in an uneven scaffold surface and a thick, sheet-like microstructure rather than a porous structure, which is not conducive to cell adhesion and proliferation.

[0090] 2. Mechanical strength

[0091] The obtained crosslinked materials from each embodiment and comparative example were cut into cuboids of the same thickness, measuring 10mm x 40mm. The tensile strength of the samples was measured using a universal tensile testing machine (Shanghai Xiangjie Instruments, electronic universal testing machine; model 830S) at a tensile speed of 30mm / min. The change in force during tensile deformation was recorded. The test results are as follows: Figure 4 , Figure 5 And Table 2.

[0092] Table 2. List of tensile strengths of crosslinked materials obtained in Examples 1-3 and Comparative Examples 1-10

[0093]

[0094] according to Figure 4 and Figure 5 As shown in Table 2, the tensile strength of the samples in Examples 1-3 all exhibited strong breaking strength, with breaking strengths of 2.87 N, 3.03 N, and 2.94 N, respectively. The slight differences in force values ​​among the three groups were caused by the collagen concentration before freeze-drying. The mechanical strength of the three sets of example samples was significantly greater than that of the other comparative examples.

[0095] Comparative Example 1 involved cross-linking collagen directly in a collagen solution using glutaraldehyde without collagen self-assembly, followed by freeze-drying. Because the reaction between glutaraldehyde and collagen molecules was rapid and uneven, the freeze-dried cross-linked material had fewer connections between the pore walls, resulting in very low tensile strength.

[0096] Comparative Example 2 is a collagen solution that has been freeze-dried and then cross-linked by immersion in glutaraldehyde solution. As can be seen from the results in the figure, the tensile strength of Comparative Example 2 is only 0.14 N. This is because the sample is only cross-linked on the surface after immersion in glutaraldehyde solution, and its internal structure is in a non-cross-linked state, resulting in very low mechanical strength.

[0097] Comparative Example 3 was obtained by directly freeze-drying collagen after self-assembly. The uncrosslinked pure crosslinked material has poor mechanical strength, with a breaking strength of only 0.12 N.

[0098] The hydrogen peroxide concentration used in Comparative Example 4 was too high during the nucleation stage, leading to excessive cross-linking in the early stage and inhibiting the self-assembly behavior of collagen in the subsequent growth stage. Therefore, the tensile strength of the sample in Comparative Example 4 was 1.24 N.

[0099] Comparative Example 5 had a low concentration of hydrogen peroxide used during the growth period, resulting in a weaker cross-linking effect during the self-assembly process. As a result, the cross-linked material had a relatively loose appearance, and the tensile breaking strength was measured to be 1.73 N.

[0100] Comparative Example 6 showed that 0.9% sodium chloride was added to the collagen solution before the nucleation stage. Due to the lack of the dispersing effect of sodium chloride, the self-assembly behavior of collagen was significantly inhibited in the subsequent preparation process, resulting in the freeze-drying process of the subsequent sample being similar to the direct freeze-drying of the collagen solution, and the tensile strength obtained was only 1.25 N.

[0101] The nucleation and growth phases of Comparative Example 7 were not regulated to the optimal self-assembly temperature, resulting in the collapse of its support structure. Therefore, the tensile strength of this material is 1.45 N.

[0102] Comparative Example 8 failed to adjust the pH of the self-assembly process away from the optimal pH during the growth phase, resulting in an excessively rapid collagen self-assembly rate that did not match the cross-linking rate of hydrogen peroxide. The final cross-linked material had a tensile strength of 2.09 N.

[0103] Comparative Example 9 involved shorter stirring speeds and times during nucleation. Rapid stirring during the nucleation phase provides shear disturbance to the solution, slowing down the collagen nucleation process. Conversely, excessively slow stirring speeds lead to accelerated self-assembly during nucleation, which is mismatched with the cross-linking effect of hydrogen peroxide, ultimately resulting in uneven collagen self-assembly. The tensile strength of the obtained cross-linked material was measured to be 2.34 N.

[0104] Comparative Example 10 had an excessively high collagen concentration, which caused some of its structure to shrink and collapse. Due to the shrinkage and collapse of the cross-linked material, the support of its pore structure was reduced, and the final tensile strength obtained was 2.48 N.

[0105] 3. Resistance to enzymatic hydrolysis

[0106] Take approximately 0.5 g of the lyophilized sample and immerse it in 10 mL of a 100 U / mL type I collagenase solution (CAS No.: 9001-12-1, collagenase derived from Clostridium histolyticum, enzyme activity ≥125 CDU / mg solid). Incubate the sample in a 37°C water bath for enzymatic digestion. At each digestion time point, remove the sample, rinse the undigested sponge, and re-dry and weigh it. Calculate the digestion rate of the sample based on the mass of the remaining sample.

[0107] The formula for calculating the enzymatic hydrolysis rate is: Enzymatic hydrolysis rate (%) = ((mass of sample to be hydrolyzed - mass of sample after hydrolysis and drying) / mass of sample to be hydrolyzed) * 100

[0108] The enzymatic hydrolysis rates of each group of samples at different times are shown in Table 3:

[0109] Table 3. List of enzymatic hydrolysis rates of crosslinked materials obtained in Examples 1-3 and Comparative Examples 1-10

[0110]

[0111] As can be seen from Table 3, there was no significant difference in the enzymatic hydrolysis rate of the samples in Examples 1-3 at the four time points. With the increase of enzymatic hydrolysis time, the enzymatic hydrolysis rate slowly increased. At 24 h, the enzymatic hydrolysis rate of the three sets of examples was between 50-60%.

[0112] The cross-linking of samples in Comparative Examples 2, 3, 4, and 7 was attributed to several factors, including: direct immersion after freeze-drying resulting in only surface cross-linking; lack of cross-linking during conventional self-assembly; excessive hydrogen peroxide dosage during nucleation inhibiting self-assembly; and failure to assemble at the optimal temperature leading to poor synergistic effects of self-assembly and cross-linking, respectively. The initial enzymatic hydrolysis rate of all four comparative examples was above 20% at 3 hours, and the collagen scaffold material rapidly hydrolyzed with increasing time, ultimately reaching a hydrolysis rate of 80% at 24 hours, indicating poor resistance to enzymatic hydrolysis.

[0113] The three groups of samples (Comparative Examples 1, 5, and 9) had enzymatic hydrolysis rates of over 75% after 24 hours due to the following reasons: inhibition of subsequent self-assembly after solution cross-linking and freeze-drying; uneven self-assembly caused by insufficient secondary hydrogen peroxide dosage during the nucleation period; and excessively rapid self-assembly caused by insufficient stirring during the nucleation period. In addition, rapid collapse occurred during the enzymatic hydrolysis process.

[0114] The samples in Comparative Examples 6 and 8 showed a faster enzymatic hydrolysis rate and weaker enzymatic hydrolysis resistance due to the slow self-assembly caused by the absence of sodium chloride during the self-assembly process and the excessively fast self-assembly rate during the growth period.

[0115] Comparative Example 10 had a slightly higher final enzymatic hydrolysis rate than the Example only because the collagen concentration of the scaffold was too high before freeze-drying.

[0116] 4. Pore size, specific surface area

[0117] Based on the microscopic morphology images of the freeze-dried collagen scaffold samples prepared by each process, the microscopic surface morphology of the samples (N=3) was measured using a scanning electron microscope (magnification of 250). Complete, near-circular pores in the microscopic morphology were used as statistical pore samples. After imaging, the size of 15 different pores in the cross-section was statistically analyzed using Nano Measurer 1.2 software. Five pores were measured for each sample, and the average porosity of the sponge was calculated. The average pore size of each sample was determined. Simultaneously, the freeze-dried samples were taken, and the average specific surface area of ​​the samples was measured. The test results are shown in Table 4.

[0118] Table 4 lists the average porosity and average specific surface area of ​​the crosslinked materials obtained in Examples 1-3 and Comparative Examples 1-10.

[0119]

[0120] As a tissue engineering scaffold material, a pore size between 100-200 μm and a specific surface area greater than 10 m² / g are more suitable for cell growth, nutrient diffusion, and vascularization after implantation. The average pore size and average specific surface area of ​​the scaffold materials in Examples 1-3 are all within the above-mentioned suitable range.

[0121] Due to excessive cross-linking of the solution and excessively high collagen concentration, the freeze-dried scaffold materials of Comparative Examples 1 and 10 lost their normal pore structure and exhibited irregular sheet-like collapse. Therefore, the average porosity of these two groups of samples was extremely high and the specific surface area was very low, making them unsuitable for the application of tissue engineering scaffold materials.

[0122] Comparative Example 6, due to the absence of sodium chloride, inhibited self-assembly behavior, resulting in a freeze-dried scaffold material consisting of a simple collagen solution freeze-dried sponge with numerous fine pore structures. The average pore size was only 86.49 μm, making it unsuitable for cell adhesion and proliferation.

[0123] The average pore size of the remaining seven comparative samples (2 / 3 / 4 / 5 / 7 / 8 / 9) was all above 250 μm, and their average specific surface area was also low, making them unsuitable for tissue engineering scaffold materials to meet the requirements of cell growth, nutrient diffusion, and vascularization.

[0124] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a collagen biphase crosslinked material, characterized in that, The preparation process includes the following: Preparation of nucleation stage collagen: Adjust the temperature of acidic collagen solution to 18-28℃, add hydrogen peroxide solution, adjust the pH value to 6-8, stir for 30-120 min, and obtain solution I with hydrogen peroxide mass concentration of 0.1-1.8 wt%; Preparation of collagen during the growth period: Heat solution I to 30-37℃, adjust the pH to 8-9.5, add hydrogen peroxide solution, stir for 2-6 hours to obtain solution II with a hydrogen peroxide mass concentration of 3-5 wt%; Preparation of biphase cross-linked materials: Collagen in solution II was collected, and the collagen concentration was controlled at 5-20 mg / mL. After freeze-drying, biphase cross-linked materials were obtained.

2. The method for preparing a collagen biphase crosslinked material according to claim 1, characterized in that, The mass concentration of hydrogen peroxide in solution I is 0.1-1 wt%.

3. The method for preparing a collagen biphase crosslinked material according to claim 1 or 2, characterized in that, In the preparation of the nucleation stage collagen, rapid stirring is performed for 30-60 minutes, and the speed range of the rapid stirring is 300-600 rpm.

4. The method for preparing a collagen biphase crosslinked material according to claim 3, characterized in that, The pH value of solution II is 8.5-9.

5.

5. The method for preparing a collagen biphase crosslinked material according to claim 4, characterized in that, In the preparation of the collagen during the growth period, stirring is carried out for 3-5 hours.

6. The method for preparing a collagen biphase crosslinked material according to claim 5, characterized in that, In the preparation of nucleation collagen, the concentration of acidic collagen solution is 1-10 mg / mL, the temperature is 2-8℃, and the pH value is 3-5.

7. A method for preparing a collagen biphase crosslinked material according to any one of claims 1, 2, 4-6, characterized in that, In the preparation of nucleation-stage collagen, the acidic collagen solution contains sodium chloride, and the concentration of sodium chloride in the acidic collagen solution is 0.5-1.5 w / v.

8. The method for preparing a collagen biphase crosslinked material according to claim 7, characterized in that, In the preparation of nucleation collagen, the acidic collagen solution contains sodium chloride, and the concentration of sodium chloride in the acidic collagen solution is 0.7-1.2 w / v.

9. A collagen biphase crosslinking material obtained by the preparation method according to any one of claims 1, 2, 4-6, and 8.

10. An implantable stent comprising the collagen biphasic crosslinked material of claim 9.

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