Collagen biphasic system cross-linking material, preparation method thereof and implant stent

Abstract

The invention provides a collagen biphasic system cross-linking material, a preparation method thereof and an implantation stent, relates to a high-molecular compound composition, relates to a high-molecular compound composition, and also relates to the technical field of medical materials. According to the preparation method of the collagen two-phase system cross-linking material, in the nucleation period, low-concentration hydrogen peroxide is introduced, so that collagen self-assembly and hydrogen peroxide cross-linking are carried out synergistically; in the growing period, a hydrogen peroxide solution with relatively high concentration is added, so that a covalent cross-linked network and fiber extension are synchronously formed, and a self-assembly regulation process in the growing period can greatly prolong the collagen self-assembly time, so that cross-linking is more thorough, and structure-function synergy on a molecular scale is realized; finally, hydrogen peroxide is decomposed into water and oxygen, no crosslinking agent is left, the prepared collagen biphasic system crosslinking material has good mechanical performance and enzymolysis resistance, and the in-vivo complete degradation time can be controlled within 12-24 months.

Description

Technical Field

[0001] The present invention relates to a composition of polymer compounds and also to the technical field of medical materials, in particular to a collagen two-phase system cross-linked material and a preparation method and an implantation stent thereof. Background Art

[0002] Collagen, 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, collagen tissue engineering scaffolds are primarily obtained by direct freeze-drying of purified collagen solutions. Uncross-linked pure collagen scaffolds rely solely on non-covalent bonds to maintain their network, resulting in low mechanical strength, uneven pore distribution, and rapid degradation by collagenase in the body, making them incapable of bearing loads or long-term implantation. Some materials cross-link the collagen in solution before freeze-drying. The addition of cross-linking agents in solution can lead to disordered collagen molecules, pore collapse, and uneven diffusion of the cross-linker, resulting in a heterogeneous network. Other materials also cross-link the freeze-dried collagen scaffolds by soaking them in water. Due to the poor permeability of the cross-linker, a dense cross-linked layer forms only on the surface of the material, leaving the internal fibers loose due to the lack of cross-linking. This results in a stratified structure that is "strong on the surface, weak on the inside," and susceptible to swelling and collapse in body fluids. The fundamental problem with these strategies is that they only cross-link the collagen molecules, leading to uneven cross-linking and problems such as slow cross-linking and localized collapse. At the same time, the residue of cross-linking agent also causes the tissue affinity of the material to deteriorate, which is prone to adverse reactions.

[0003] As the main component of the natural extracellular matrix, collagen's self-assembly ability is an important basis for the construction of biomimetic materials. Under physiological conditions, collagen molecules can form an ordered three-dimensional fiber network through spontaneous aggregation. This process is usually divided into two stages: "nucleation phase" and "growth phase": the nucleation phase is the process in which collagen monomers form the initial fiber core through intermolecular forces in the liquid phase, while the growth phase is the key step for the fibers to further extend, arrange and form a stable high-level structure. This self-assembly property gives the material a topological structure similar to natural tissue, providing an ideal microenvironment for cell adhesion and proliferation. However, the existing technology of collagen scaffolds based on self-assembly still has significant limitations. The physical self-assembly of traditional self-assembling collagen scaffolds has inherent defects such as low mechanical strength, poor structural stability, and insufficient resistance to enzymatic hydrolysis, which seriously restricts its application in load-bearing tissue repair or long-term implantation scenarios. Summary of the Invention

[0004] In order to solve the defects of low mechanical strength, poor structural stability and insufficient resistance to enzymatic hydrolysis of cross-linked materials in the prior art, the first aspect of the present application is to provide a preparation method of a collagen two-phase system cross-linked material. During the nucleation period, by introducing a low concentration of hydrogen peroxide, collagen self-assembly and hydrogen peroxide cross-linking are carried out synergistically; during the growth period, a higher concentration of hydrogen peroxide solution is used in the solid phase state to make the covalent cross-linked network and fiber extension form synchronously, thereby realizing structure-function synergy on the molecular scale; finally, hydrogen peroxide decomposes into water and oxygen without any cross-linking agent residue, and the porosity of the material or product is obtained by the amount of hydrogen peroxide added; secondly, a collagen two-phase system cross-linked material with high mechanical properties, resistance to enzymatic hydrolysis and ideal pore structure is provided; thirdly, a collagen-based implant scaffold with high mechanical properties, resistance to enzymatic hydrolysis and ideal pore structure is obtained by using the collagen two-phase system cross-linked material of the present application.

[0005] In the first aspect of the present invention, the present application provides a method for preparing a collagen two-phase system cross-linked material, comprising the following preparation process:

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

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

[0008] Preparation of a biphasic system cross-linked material: Collect collagen in solution II, control the collagen concentration to 5-20 mg / mL, and obtain a biphasic system cross-linked material after freeze-drying.

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

[0010] At room temperature (18-28°C), after adjusting the pH, collagen self-assembly begins slowly, marking the nucleation phase. Adding low concentrations of hydrogen peroxide for crosslinking allows the crosslinking reaction to proceed simultaneously with the nucleation phase. The crosslinking reaction is slower during the nucleation phase, and steric hindrance affects the rearrangement of collagen molecules during this phase, slowing their aggregation and ensuring a more uniform material.

[0011] Preparation of a biphasic system cross-linked material: centrifuging the growth phase solution, collecting the collagen precipitates, and controlling the collagen concentration to 5-20 mg / mL after washing; and freeze-drying the collagen to obtain a biphasic system cross-linked material.

[0012] This invention employs a "two-phase crosslinking" strategy, precisely embedding crosslinking into different stages of collagen self-assembly. During the nucleation phase, crosslinking is carried out by introducing a low-concentration hydrogen peroxide solution, 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 regulated, preventing uneven crosslinking and premature curing of the collagen caused by hydrogen peroxide's oxidation mechanism. Furthermore, the crosslinking behavior of hydrogen peroxide slows the self-assembly rate during the collagen nucleation process, providing a longer time window for orderly fiber growth.

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

[0014] During the subsequent preparation process, hydrogen peroxide ultimately decomposes into water and oxygen, leaving no crosslinker residue. Controlling the hydrogen peroxide concentration simultaneously regulates its crosslinking / decomposition rate, allowing the generated oxygen to simultaneously control the porosity of the tissue engineering scaffold.

[0015] After washing and purification, the final material is freeze-dried to obtain the final material. The collagen concentration is controlled at 5-20 mg / mL, which can optimize the porosity and pore size of the scaffold material.

[0016] This technological approach not only preserves the biomimetic advantages of collagen self-assembly (such as highly ordered fiber arrangement and uniform porosity), but also enhances the bonding strength and stability of the fiber interfaces through dynamic crosslinking. Compared to existing technologies, this invention overcomes the unidirectional limitations of "crosslinking interfering with self-assembly" or "self-assembly decoupled from crosslinking," ultimately resulting in a collagen-based implantable 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 the solution I is 0.1-1 wt%.

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

[0019] The pH value is adjusted to 6-8 to achieve the optimal self-assembly state of collagen and drive the collagen into the nucleation phase; the stirring time is 30-60 minutes, and the solution is given shear disturbance during the nucleation process. When combined with a 0.1-1 wt% hydrogen peroxide solution, the collagen nucleation phase speed is slowed down, and more uniform and sufficient nucleation can be achieved.

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

[0021] Furthermore, during the preparation of the growth phase collagen, stirring is performed for 3-5 hours.

[0022] The solution temperature was set to 30-37°C, the optimal temperature for collagen self-assembly, and the growth phase of collagen self-assembly began. At this point, collagen fibers in the solution began to grow and extend laterally, thickening and lengthening. Furthermore, the pH was adjusted to 8.5-9.5, moving away from the optimal collagen self-assembly time, slowing the growth of collagen fibers during the growth phase. This pH value allows hydrogen peroxide to decompose and generate oxygen, which is beneficial for regulating the pore structure of the scaffold.

[0023] A higher concentration of hydrogen peroxide solution is added for cross-linking, which strengthens the cross-linking on the formed fiber framework and improves the stability of the material. Stirring for 3-5 hours is to allow collagen to further evenly reinforce the fibers during the entire process of fiber generation during the growth period. The final cross-linked material is more uniform and has better physical and chemical properties.

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

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

[0026] Control the acidity and low temperature of the collagen solution to ensure uniform collagen dissolution and inhibit 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 to achieve salt dissolution can further disperse the collagen molecules in the solution, which is beneficial to the degree of cross-linking in the subsequent nucleation stage.

[0030] NaCl acts as an electrolyte to inhibit excessive aggregation, partially shield the surface charges of collagen molecules (such as the ionization of carboxyl and amino groups), reduce electrostatic repulsion, and enable collagen molecules to aggregate in a more controllable manner, thus avoiding uneven fibers caused by local excessive nucleation. Optimize fiber diameter and arrangement: Appropriate sodium chloride concentration in the acidic collagen solution forms a suitable ionic strength that helps to form a uniform microfiber network, which works in conjunction with a moderate hydrogen peroxide concentration to avoid excessively thin fibers due to too low a sodium chloride concentration in the acidic collagen solution, and excessive fiber aggregation due to too low a sodium chloride concentration in the acidic collagen solution, resulting in unsatisfactory mechanical properties and enzymatic resistance.

[0031] Regarding the second aspect of the present invention, the present application provides a collagen two-phase system cross-linked material obtained by the preparation method described in the present application.

[0032] Regarding the third aspect of the present invention, the present application provides an implantable stent using the collagen two-phase system cross-linked material described in the present application.

[0033] The collagen fibers obtained by the present invention can be used as injection implant materials, or as tissue engineering scaffolds after freeze-drying, and can be used in the fields of burn dressings, tissue hemostasis, soft tissue reconstruction, etc.

[0034] Beneficial effects: 1. The present application provides a method for preparing a collagen two-phase system cross-linked material. During the nucleation period, a low concentration of hydrogen peroxide is introduced to enable collagen self-assembly and hydrogen peroxide cross-linking to proceed synergistically; during the growth period, a higher concentration of hydrogen peroxide solution is added in the solid phase to enable the covalent cross-linked network and fiber extension to be formed synchronously. The growth period self-assembly regulation process can greatly extend the collagen self-assembly time, make the cross-linking more thorough, and achieve structure-function synergy on the molecular scale; finally, hydrogen peroxide decomposes into water and oxygen without any cross-linking agent residue. At the same time, the porosity of the material or product is obtained by the amount of hydrogen peroxide added. The prepared collagen two-phase system cross-linked material has good mechanical properties and enzymatic resistance, and the complete degradation time in vivo can be controlled between 12-24 months.

[0035] 2. Furthermore, the concentration, pH value, temperature and stirring time of hydrogen peroxide during the nucleation and growth phases are optimized to further improve the mechanical properties and enzymatic resistance of the prepared collagen two-phase system cross-linked material.

[0036] 3. Furthermore, the present application adds sodium chloride to the acidic collagen solution to achieve salt dissolution, which can further disperse the collagen molecules in the solution, improve the degree of cross-linking and cross-linking uniformity in the subsequent nucleation period and / or growth period, and further improve the prepared collagen two-phase system cross-linked material to have good mechanical properties and resistance to enzymatic hydrolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The macroscopic and microscopic morphological structures of the collagen two-phase system cross-linked materials obtained in Example 3, Comparative Examples 1-3 and Comparative Example 7;

[0038] Figure 2 The macroscopic and microscopic morphological structures of the collagen dual-phase cross-linked materials obtained in Example 1, Comparative Example 6, and Comparative Examples 8-9 are shown;

[0039] Figure 3 The macroscopic and microscopic morphological structures of the collagen dual-phase cross-linked materials obtained in Example 2, Comparative Examples 4-5 and Comparative Example 10;

[0040] Figure 4Graph showing the tensile strength test results of the collagen two-phase system cross-linked materials obtained in Examples 1-3, Comparative Examples 2-3, and Comparative Examples 5-6;

[0041] Figure 5 Graph showing the tensile strength test results of the collagen two-phase system cross-linked material obtained from Comparative Examples 1, 4, and Comparative Examples 7-10. DETAILED DESCRIPTION

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

[0043] Example 1:

[0044] 1) Regulating the acidic collagen solution: Adjust the pH of the 2 mg / mL acidic collagen solution to 3 and maintain the solution temperature at 2°C.

[0045] 2) Adding 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 of the dissolved solution system is 0.9 w / v%.

[0046] 3) Preparation of Collagen in the Nucleation Stage: The collagen solution was placed in water at a temperature of 20°C. A 30 wt% hydrogen peroxide solution (in other embodiments, the concentration of the hydrogen peroxide solution can be selected from 23-50 wt%) was added to the solution to a final concentration of 1 wt% hydrogen peroxide. The pH of the solution was immediately adjusted to 6 and rapidly stirred for 30 minutes.

[0047] 4) Preparation of Growth-Stage Collagen: After stirring, adjust the solution temperature to 37°C and the pH to 8.5. Stir slowly for 5 minutes until collagen begins to precipitate. Then, add 30 wt% hydrogen peroxide solution to a final concentration of 5 wt% hydrogen peroxide, and stir slowly for 3 hours.

[0048] 5) Preparation of biphasic cross-linked materials: After stirring, the collagen precipitates were collected by centrifugation and washed multiple times with 0.9% saline. The collagen concentration before lyophilization was controlled to 5 mg / mL. The precipitates were then divided into molds (50 mm x 30 mm x 10 mm polytetrafluoroethylene freeze-drying molds) and lyophilized (lyophilization conditions: pre-freeze at -20°C for 24 hours, then freeze-dry in a freeze-dryer at -40°C, vacuum of 0.1-5 Pa, and lyophilization for 24 hours) to obtain a biphasic collagen cross-linked scaffold material.

[0049] Example 2:

[0050] 1) Regulating the acidic collagen solution: Adjust the pH of the 5 mg / mL acidic collagen solution to 5 and maintain the solution temperature at 8°C.

[0051] Adding 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 of the solution system after dissolution is 0.9 w / v%.

[0052] 3) Preparation of Collagen during the Nucleation Phase: The collagen solution was placed in water at a temperature of 20°C. A 30 wt% hydrogen peroxide solution was added to the solution to a final concentration of 0.1 wt%. The pH of the solution was immediately adjusted to 8 and the solution was rapidly stirred for 60 min.

[0053] 4) Preparation of Growth-Stage Collagen: After stirring, adjust the solution temperature to 37°C and the pH to 9.5. Stir slowly for 5 minutes until collagen begins to precipitate. Then, add 30 wt% hydrogen peroxide solution to a final concentration of 3 wt% hydrogen peroxide, and stir slowly for 5 hours.

[0054] 5) Preparation of a biphasic cross-linked material: After stirring, the collagen precipitates were collected by centrifugation and washed multiple times with 0.9% saline. The collagen concentration was controlled to 20 mg / mL before lyophilization. The precipitates were then dispensed into molds and lyophilized to obtain a biphasic collagen cross-linked scaffold material.

[0055] Example 3:

[0056] 1) Regulating the acidic collagen solution: Adjust the pH of the 10 mg / mL acidic collagen solution to 4 and maintain the solution temperature at 5°C.

[0057] 2) Adding 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 of the dissolved solution system is 0.9 w / v%.

[0058] 3) Preparation of Collagen during the Nucleation Phase: The collagen solution was placed in water at a temperature of 20°C. A 30 wt% hydrogen peroxide solution was added to the solution to a final concentration of 0.5 wt%. The pH of the solution was immediately adjusted to 7 and the mixture was rapidly stirred for 40 min.

[0059] 4) Preparation of Growth-Stage Collagen: After stirring, adjust the solution temperature to 37°C and the pH to 9. Stir slowly for 5 minutes until collagen begins to precipitate. Then, add 30 wt% hydrogen peroxide solution to a final concentration of 4 wt% hydrogen peroxide, and stir slowly for 4 hours.

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

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

[0062]

[0063] Comparative Example 1 differs from Example 1 in that: self-assembly is not performed, and the scaffold is directly cross-linked in a collagen solution using conventional glutaraldehyde and then freeze-dried to obtain the scaffold. The process is as follows:

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

[0065] Comparative Example 2 differs from Example 1 in that the collagen solution is directly freeze-dried and then the scaffold is soaked and cross-linked to obtain the scaffold. The preparation process is as follows:

[0066] Adjust the pH of a 10 mg / mL acidic collagen solution to neutral. Place 15 mL of the acidic collagen solution in the aforementioned 50 mm x 30 mm x 10 mm polytetrafluoroethylene freeze-drying mold, degas, pre-freeze, and freeze-dry to obtain a cross-linked collagen scaffold. Soak the freeze-dried collagen scaffold in a 1 wt% aqueous solution of glutaraldehyde for 2 hours, completely submerged. Remove the scaffold, rinse it multiple times with 0.9% saline, and then dry it again to obtain the final cross-linked collagen scaffold.

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

[0068] A 10 mg / mL acidic collagen solution was adjusted to a pH of 7.4, and sodium chloride was added to a concentration of 0.9% after dissolution. The collagen solution was 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 resulting gel scaffold was washed multiple times with 0.9% saline and finally freeze-dried 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) the nucleation period is relatively high, and the final hydrogen peroxide concentration is 2 wt %.

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

[0071] Comparative Example 6 differs from Example 1 in that no sodium chloride is added to the acidic collagen solution before the nucleation phase.

[0072] Comparative Example 7 differs from Example 1 in that the temperature settings in step 3) the nucleation period and step 4) the growth period are different: the temperature in the nucleation period is set to 8° C., and the temperature in 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 away from the optimal self-assembly pH value, but 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 speed is slow, and the stirring is slow for 10 minutes.

[0075] Comparative Example 10 differs from Example 1 in that the collagen concentration before freeze-drying was too high, namely, 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 of each embodiment and comparative example was observed by electron microscopy (magnification 300 times). The influence of each process condition on the sample morphology was analyzed based on the macroscopic and microscopic pore morphology of each cross-linked material. Figure 1-Figure 3 The macroscopic and microscopic structure diagrams of various embodiments and comparative examples are shown.

[0079] The results in the figures show that the macrostructures of the samples from Examples 1-3 all exhibit a uniform and smooth sponge-like appearance, with uniform and fine pores in the cross-linked sponge materials. The microstructures all exhibit uniform and open pores with no significant variation in pore size, demonstrating their suitability for cross-linked materials in tissue engineering.

[0080] Comparative Example 1 bypasses collagen self-assembly and instead directly cross-links the collagen solution with glutaraldehyde before freeze-drying. Macroscopic images of the scaffold structure reveal that the freeze-dried collagen scaffold in Comparative Example 1 exhibits highly uneven, irregular protrusions. This is due to the rapid and uneven cross-linking reaction between glutaraldehyde and collagen molecules in the solution, resulting in uneven protrusions in the collagen scaffold after direct freeze-drying. Furthermore, the microstructure of Comparative Example 1 lacks pores and instead exhibits a largely flaky structure, making it unsuitable for the cell adhesion and growth required for tissue engineering scaffolds.

[0081] Comparative Example 2 involved freeze-drying the collagen solution and then soaking it in a glutaraldehyde solution for cross-linking. The macroscopic appearance of the collagen scaffold showed uneven, irregular protrusions. Meanwhile, its microstructure showed a large number of collapsed pores with uneven pore sizes.

[0082] Comparative Example 3, obtained by direct freeze-drying after collagen self-assembly, also exhibited an uneven appearance. This was due to rapid localized aggregation caused by the rapid self-assembly of collagen under simulated physiological conditions in vitro. The microstructure of this sample exhibited a uniform pore structure, but the pores were relatively large.

[0083] The cross-linked material prepared in Comparative Example 4 exhibited a very loose, collapsed structure. This was due to the high hydrogen peroxide concentration used during the nucleation phase, which resulted in excessive cross-linking in the early stages and suppressed collagen self-assembly during the subsequent growth phase. Subsequent self-assembly was not significantly initiated, resulting in poor mechanical properties of the resulting cross-linked material. Its microstructure exhibited a cohesive, fine pore structure.

[0084] In Comparative Example 5, the hydrogen peroxide concentration used during the growth period was relatively low, resulting in a weak cross-linking effect during the self-assembly process during the growth period. Therefore, the cross-linked material had a loose appearance, and the pores and pore walls of the microstructure were loose, which was caused by incomplete cross-linking.

[0085] In Comparative Example 6, 0.9% sodium chloride was added to the collagen solution before the nucleation phase. Due to the lack of sodium chloride's dispersing effect, the collagen's self-assembly behavior was significantly inhibited during the subsequent preparation process, resulting in the freeze-drying process of the subsequent sample being similar to the collagen solution being freeze-dried directly. The subsequent addition of hydrogen peroxide resulted in only a slight cross-linking effect, and the oxygen generated by the decomposition of hydrogen peroxide prevented the scaffold from exhibiting a uniform, continuous pore structure.

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

[0087] In Comparative Example 8, the pH for self-assembly was not adjusted away from the optimal pH during the growth phase. This resulted in excessively rapid collagen self-assembly, which was mismatched with the cross-linking speed of hydrogen peroxide. This rapid self-assembly led to entanglement between collagen molecules, resulting in a cross-linked material with a microstructure characterized by relatively large closed pores and lack of apparent porosity.

[0088] In Comparative Example 9, the stirring speed and duration during the nucleation process were relatively short. Rapid stirring during the nucleation phase creates shear disturbances in the solution, slowing collagen nucleation. However, too slow a stirring speed accelerates the self-assembly process during the nucleation phase, which is inconsistent with the cross-linking effect of hydrogen peroxide and ultimately leads to uneven collagen self-assembly. This results in an uneven surface and overly coarse and large pores in the microstructure.

[0089] In Comparative Example 10, the controlled collagen concentration is too high. The excessively high collagen concentration causes part of the structure to shrink and collapse, the scaffold surface is uneven, and the microstructure is thick sheet-like rather than porous, which is not conducive to cell adhesion and proliferation.

[0090] 2. Mechanical strength

[0091] The cross-linked materials obtained in each embodiment and comparative example were cut into rectangular blocks of 10 mm*40 mm with the same thickness. The tensile strength of the samples was measured using a universal tensile testing machine (Shanghai Xiangjie Instrument, electronic universal material testing machine; model 830S) (tensile speed: 30 mm / min). The changes in the force value of the samples during the tensile deformation process were recorded. The test results are shown in FIG. Figure 4 、 Figure 5 And Table 2.

[0092] Table 2. Tensile strength of cross-linked materials obtained in Examples 1-3 and Comparative Examples 1-10

[0093]

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

[0095] Comparative Example 1 is to crosslink the collagen solution directly with glutaraldehyde before freeze-drying without using collagen self-assembly. Since glutaraldehyde reacts rapidly and unevenly with collagen molecules, the cross-linked material obtained by freeze-drying has few links between the pore walls, resulting in a very low tensile strength of the material.

[0096] Comparative Example 2, a collagen solution freeze-dried and then immersed in a glutaraldehyde solution for cross-linking, shows a fracture strength of only 0.14 N. This is because the sample's internal structure remains uncross-linked, resulting in very low mechanical strength.

[0097] Comparative Example 3 is obtained by directly freeze-drying the collagen after self-assembly. The pure cross-linked material without cross-linking has poor mechanical strength, with a breaking strength of only 0.12 N.

[0098] The high hydrogen peroxide concentration used in the nucleation phase of Comparative Example 4 resulted in excessive cross-linking in the early stages, which inhibited the self-assembly of collagen in the subsequent growth phase. Therefore, the tensile strength of the sample in Comparative Example 4 was 1.24 N.

[0099] In Comparative Example 5, since the hydrogen peroxide concentration used during the growth period was relatively low, the cross-linking effect during the self-assembly process during the growth period was relatively weak, and the cross-linked material had a relatively loose appearance. The tensile strength at break was measured to be 1.73N.

[0100] In Comparative Example 6, 0.9% sodium chloride was added to the collagen solution before the nucleation phase. Due to the lack of sodium chloride's dispersing effect, the collagen's self-assembly behavior was significantly inhibited during the subsequent preparation process. This resulted in the freeze-drying process of the subsequent sample being similar to the direct freeze-drying of the collagen solution, resulting in a tensile strength of only 1.25 N.

[0101] In Comparative Example 7, the nucleation and growth phases were not adjusted to the optimal self-assembly temperature, resulting in a compact and collapsed scaffold structure. Therefore, the tensile strength of the material was 1.45 N.

[0102] In Comparative Example 8, the pH of the self-assembly process was not adjusted away from the optimal pH during the growth phase, resulting in excessively fast collagen self-assembly during the growth phase, which did not match the cross-linking speed of hydrogen peroxide. The resulting cross-linked material had a breaking strength of 2.09 N.

[0103] In Comparative Example 9, the stirring speed and duration during the nucleation process were relatively short. Rapid stirring during the nucleation phase creates shear disturbance in the solution, slowing collagen nucleation. However, excessively slow stirring speeds can accelerate self-assembly during the nucleation phase, mismatching the crosslinking effect of hydrogen peroxide and ultimately leading to uneven collagen self-assembly. The tensile strength of the resulting crosslinked material was measured to be 2.34 N.

[0104] In Comparative Example 10, the controlled collagen concentration was too high. The excessively high collagen concentration caused part of its structure to shrink and collapse. Due to the shrinkage and collapse of the cross-linked material, the support of its void structure was reduced. The final tensile strength measured was 2.48 N.

[0105] 3. Enzyme resistance

[0106] Submerge approximately 0.5 g of freeze-dried sample in 10 mL of 100 U / mL collagenase type I (CAS No. 9001-12-1, derived from Clostridium histolyticum, activity ≥125 CDU / mg solid) in a 37°C water bath for enzymatic digestion. Remove the sample at each digestion time point, rinse the undigested sponge, re-dry, and re-weigh it. Calculate the digestion efficiency based on the remaining sample mass.

[0107] The formula for calculating the enzymatic hydrolysis rate is: Enzymatic hydrolysis rate (%) = ((mass of sample to be hydrolyzed - mass of sample after enzymatic 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. Enzyme hydrolysis rates of cross-linked materials obtained in Examples 1-3 and Comparative Examples 1-10

[0110]

[0111] As can be seen from Table 3, there is no significant difference in the enzymatic hydrolysis rate of the samples of Examples 1-3 at the four time points. As the enzymatic hydrolysis time increases, the enzymatic hydrolysis rate slowly increases. At 24 h, the enzymatic hydrolysis rates of the three groups of Examples are between 50-60%.

[0112] Comparative Examples 2, 3, 4, and 7 exhibited poor crosslinking due to the following reasons: freeze-dried samples were crosslinked only on the surface after direct immersion; no crosslinking occurred during conventional self-assembly; excessive hydrogen peroxide dosage during the nucleation phase inhibited self-assembly; and self-assembly was not performed at the optimal temperature, resulting in poor synergistic crosslinking. The initial enzymatic degradation rates for all four comparative examples were above 20% within 3 hours. Over time, the collagen scaffolds rapidly degraded, reaching a final degradation rate of 80% at 24 hours, indicating poor resistance to enzymatic degradation.

[0113] In comparative examples 1, 5, and 9, the three groups of samples had enzymatic hydrolysis rates of more than 75% within 24 h, and rapid collapse occurred during the enzymatic hydrolysis process, respectively, due to the inhibition of subsequent self-assembly after solution cross-linking and freeze-drying, the low amount of secondary hydrogen peroxide used during the nucleation period resulting in uneven self-assembly, and insufficient stirring during the nucleation period resulting in excessive self-assembly.

[0114] In the two groups of samples of Comparative Example 6 / 8, the enzymatic hydrolysis rate increased rapidly with time, and the enzymatic hydrolysis resistance was weak because no sodium chloride was added during the self-assembly process, resulting in too slow self-assembly and too fast self-assembly during the growth period.

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

[0116] 4. Pore size, specific surface area

[0117] Based on the microscopic morphology of freeze-dried collagen scaffold samples prepared by each process, scanning electron microscopy (SEM) at a magnification of 250 was used to measure the surface morphology of three samples (N = 3). Complete, circular pores within the microscopic morphology were used as statistical pore samples. After photographing, the sizes of 15 different pores in the cross-section were counted using NanoMeasurer 1.2 software. Five pores were measured for each sample, and the average pore size of the sponge was calculated. The average pore size of each sample was determined. Simultaneously, 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. List of average pores and average specific surface areas of cross-linked 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 ranges.

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

[0122] In Comparative Example 6, since no sodium chloride was added, the self-assembly behavior was inhibited, and the freeze-dried scaffold material obtained was a simple collagen solution freeze-dried sponge with a large number of fine pore structures. The average pore size was only 86.49 μm, which was not suitable for cell adhesion and proliferation.

[0123] The average pore sizes of the remaining seven groups of samples in comparative examples 2 / 3 / 4 / 5 / 7 / 8 / 9 were all above 250 μm, and the average specific surface areas were all low, which were not suitable for the requirements of tissue engineering scaffold materials for cell growth, nutrient diffusion and vascularization.

[0124] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing a collagen two-phase system cross-linked material, characterized in that: The preparation process includes the following: Preparation of collagen during the nucleation phase: Adjust the temperature of the acidic collagen solution to 18-28°C, add hydrogen peroxide solution, adjust the pH to 6-8, and stir for 30-120 minutes to obtain solution I with a hydrogen peroxide concentration of 0.1-1.8 wt%; Preparation of collagen during growth phase: Heat solution I to 30-37°C, adjust the pH to 8-9.5, add hydrogen peroxide solution, and stir for 2-6 hours to obtain solution II with a hydrogen peroxide concentration of 3-5 wt%; Preparation of a biphasic system cross-linked material: Collect collagen in solution II, control the collagen concentration to 5-20 mg / mL, and obtain a biphasic system cross-linked material after freeze-drying.

2. The method for preparing a collagen two-phase system cross-linked material according to claim 1, characterized in that: The mass concentration of hydrogen peroxide in the solution I is 0.1-1 wt%.

3. The method for preparing a collagen two-phase system cross-linked material according to claim 1 or 2, characterized in that: During the preparation of the nucleation stage collagen, rapid stirring is performed for 30-60 minutes.

4. The method for preparing a collagen two-phase system cross-linked material according to claim 3, characterized in that: The pH value of the solution II is 8.5-9.

5.

5. The method for preparing a collagen two-phase system cross-linked material according to claim 4, characterized in that: During the preparation of the growth phase collagen, stirring is performed for 3-5 hours.

6. The method for preparing a collagen two-phase system cross-linked material according to claim 5, characterized in that: In the preparation of collagen in the nucleation stage, the concentration of the acidic collagen solution is 1-10 mg / mL, the temperature is 2-8°C, and the pH value is 3-5.

7. The method for preparing a collagen two-phase system cross-linked 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 two-phase system cross-linked material according to claim 7, 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 0.7-1.2 w / v%.

9. A collagen two-phase system cross-linked 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 two-phase system cross-linked material according to claim 9.

Images