Oral collagen-based repair membrane material and preparation method thereof

Abstract

The present application relates to the technical field of biological medicine materials, and particularly relates to a collagen-based repair membrane material for oral cavity and a preparation method thereof. Specifically, the present application comprises the following components: collagen type I, bioactive glass nanoparticles, gamma-aminopropyl triethoxysilane, a crosslinking system, polyaspartic acid, CaCl2, K2HPO4 and Sr-Zn solution. In the present application, polyaspartic acid is used to guide in-situ mineralization of calcium and phosphorus to form an oriented compatible nanocrystalline template, chemical coupling and continuous ion release of silanized bioactive glass are combined to realize time sequence cooperation of mineral deposition and ion supply; a unidirectional freezing structure is used to build a structure gradient with a dense cavity surface and a porous bone side, so as to give the structure the functions of anti-permeation and cell guidance; mild crosslinking containing alcohol is combined to improve the wet strength and control swelling; further, the bone side is activated by Sr-Zn solution to form a directional microenvironment, so as to promote interface mineralization maturity and delay collagen degradation, and realize the oral cavity repair function of multi-module cooperation.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a collagen-based repair membrane material for oral use and its preparation method. Background Technology

[0002] As the primary interface between the human body and the external environment, the oral cavity exists within a complex and dynamic microecological environment. Its health depends not only on the host's own immune defense and tissue repair capabilities but also on the continuous interaction of multiple factors, including the oral microbiome, physicochemical stimuli, and biomechanical load. When pathogenic microorganisms form highly structured biofilms on the surface of teeth or soft tissues, their metabolic products can lead to enamel demineralization, resulting in tooth decay. If the plaque biofilm extends into the subgingival region, it may activate the immune inflammatory response of periodontal tissues, causing alveolar bone resorption and loss of periodontal attachment, ultimately leading to tooth loosening or even loss. In the field of oral bone regeneration, effectively guiding and accelerating the repair of damaged areas has always been a core clinical challenge, and the repair membranes used in guided bone regeneration technology play a crucial role in this process. Currently, repair membranes widely used in clinical practice and research, whether based on natural collagen, synthetic polymers, or composite systems, are increasingly showing functional limitations in the complex and dynamic environment of the oral cavity. Some materials exhibit significant swelling and mechanical property degradation under humid conditions, making it difficult to maintain a stable barrier space throughout the healing cycle. Other materials, due to their biological inertness, cannot actively regulate the local microenvironment and can only serve as passive physical membranes. Furthermore, most membrane materials lack functional considerations in their structural design, failing to simultaneously meet the dual requirements of soft tissue isolation and bone tissue guidance; their homogeneous morphology severely limits their repair efficacy. Regarding the loading of active components, both simple mixing of inorganic fillers and physical adsorption of biological factors generally face technical bottlenecks such as uncontrollable release of active ingredients, weak interfacial binding, initial burst release, or easy inactivation, severely restricting their application prospects in functional tissue regeneration. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide an oral collagen-based repair membrane material and its preparation method. To address the shortcomings of existing oral repair membranes, such as weak mechanical properties, easy detachment of the inorganic phase, poor remineralization sustainability, and lack of cavity surface barrier-defect surface guiding zones, this invention utilizes polyaspartic acid-induced collagen-confined in-situ mineralization to form a collagen-compatible in-situ mineralization template, achieving rapid remineralization and morphological stability. Bioactive glass nanoparticles, after surface modification with γ-aminopropyltriethoxysilane, are chemically coupled with collagen in an EDC / NHS system to construct a continuous interfacial network, preventing wet-state powdering and detachment and enhancing load transfer. Unidirectional freezing is employed to construct a gradient structure of dense cavity surface and porous defect surface, achieving impermeability, disturbance resistance, and tissue guidance within the same membrane. Mild alcohol-containing crosslinking and glycine quenching reduce swelling and regulate degradability and compatibility. Ion localization and release are achieved through isotonic near-neutral Sr-Zn unilateral activation, synergistically promoting remineralization, interfacial integration, and enzymatic degradation regulation. This multi-level synergy significantly improves wet-state stability, disturbance resistance, and directional regeneration performance.

[0004] The technical effects described in this invention are achieved through the following technical solution: a collagen-based repair membrane material for oral use, the raw material composition of which includes the following components: type I collagen, bioactive glass nanoparticles, γ-aminopropyltriethoxysilane, cross-linking system, polyaspartic acid, CaCl2, K2HPO4 and Sr-Zn solution.

[0005] Preferably, the bioactive glass nanoparticles are silicate systems; Preferably, the crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 3 to 5:1; Preferably, the Sr-Zn solution is prepared by using HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2 and deionized water in a volume ratio of 10mmol:15mmol:1mmol:0.1mmol:1000mL, and adjusting the pH to 7.4 with NaOH and HCl. Preferably, another aspect of the present invention provides a method for preparing an oral collagen-based repair membrane material, specifically comprising the following steps: S101: Add type I collagen to 0.02M acetic acid to prepare an acidic solution of 0.5-1wt%, let it stand at 4℃ for 12-24h to swell, stir to remove foam and then use it for later use; S102: Slowly add NaOH to the acidic solution in step S101 to adjust the pH of the system to 6.8-7.4, then slowly add 4wt% polyaspartic acid aqueous solution and stir to disperse evenly. Then add acetic acid and NaOH to control the pH at 7.1-7.3 to obtain a pre-neutralized system. S103: Under constant temperature conditions of 25-30℃, add 0.5M CaCl2 solution and 0.3M K2HPO4 solution dropwise at a rate of 0.2-0.6 mL / min to the pre-neutralization system in step S102. Control the pH of the system to 7.6-8 with NaOH. After the addition is complete, heat to 37℃ and stir for 2-4 hours to obtain mineralized collagen sol. S104: Bioactive glass nanoparticles, ethanol, and deionized water are mixed in a certain proportion and ultrasonically treated for 10-15 min to disperse them evenly. γ-aminopropyltriethoxysilane is added, and the pH is adjusted to 4.5-5 with glacial acetic acid. Then, the mixture is stirred at 400-500 rpm at 60℃ for 45-60 min, cooled, centrifuged, resuspended in ethanol, washed to remove silane, vacuum dried at 40℃ for 6-8 h, and ground through a 200-mesh sieve to obtain silanized BG nanoparticles. S105: Add the silanized BG nanoparticles from step S104 to a portion of the mineralized collagen sol from step S103 at a mass-to-volume ratio of 1:4 and stir at 1000 rpm for 2-3 minutes to pre-gelatinize. Then add the remaining mineralized collagen sol from step S103 and shear in segments to obtain a composite collagen system. S106: Coat the inner wall of the mold with a thin layer of polyvinyl alcohol, pre-cool it to -40℃ with the lower cold plate, and pour the wet thickness of 1-2mm; gently vibrate for 10s to degas, freeze in one direction, and then dry in stages. Heat from -20℃ to 0℃ at a rate of 0.2℃ / min, with a cavity pressure of 20Pa, and dry for 6-8 hours; then dry at 20-25℃ with a cavity pressure of 15Pa for 4-6 hours to obtain the mold blank. S107: Add the crosslinking system to an 80wt% ethanol solution and dissolve it evenly to obtain a crosslinking solution with a total concentration of 30mM; immerse the mold blank from step S106 into the crosslinking solution with a liquid-to-film ratio of 20mL:1cm. 2 Crosslinking by gently shaking at 60 rpm for 4 hours, quenching with 10 mM glycine aqueous solution for 10 minutes, washing in stages, absorbing surface water, spreading on a polytetrafluoroethylene plate and standing for 30-60 minutes to obtain a crosslinked mold blank. S108: Place the cross-linked preform from step S107 with its porous surface facing down in the Sr-Zn solution, with a liquid-to-film ratio of 10 mL: 1 cm⁻¹. 2 After shaking gently at 60 rpm for 6–12 h at 37 °C, the material was rinsed twice with PBS buffer. Then, the temperature was increased from -20 °C to 20 °C at a rate of 0.5 °C / min under 20 Pa, and the material was dried for 4–6 h to obtain the collagen-based repair membrane material. Preferably, in step S102, the amount of the polyaspartic acid aqueous solution added is 0.1% of the volume of the acidic solution; Preferably, in step S103, the volume ratio of the CaCl2 solution to the K2HPO4 solution is 1:1; and the total added volume is 3-6% of the volume of the acidic solution. Preferably, in step S104, the ratio of the bioactive glass nanoparticles, ethanol, and deionized water is 1g:15-20mL:1mL. Preferably, in step S104, the amount of γ-aminopropyltriethoxysilane added is 2-3% of the mass of the bioactive glass nanoparticles; Preferably, in step S104, the ratio of the amount of silanized BG nanoparticles to the total mineralized collagen sol is 1g:140-160mL. Preferably, in step S105, the segmented shearing parameters are: stirring at 800-1200 rpm for 3 minutes, stirring at 2000-2500 rpm for 2 minutes, and temperature controlled by an ice bath throughout the shearing process; Preferably, in step S106, the unidirectional freezing operation is as follows: programmed step cooling, with the temperature set points of the lower cold plate being -20℃ for 5-10 minutes, -40℃ for 5-10 minutes, and -60℃ respectively, and the top of the sample being maintained at 0-5℃ throughout the process, until it is completely hardened. Preferably, in step S107, the graded washing parameters are: repeated washing twice with 50wt% ethanol, followed by washing with deionized water until the conductivity of the washing solution is <50μS / cm.

[0006] The beneficial effects of this invention are as follows: Compared to existing technologies, this invention constructs a collagen-based repair membrane that can function stably in a moist oral environment through a continuous integrated process of in-situ confined mineralization, interfacial chemical coupling, structural gradient shaping, mild cross-linking, and ionic microenvironment activation. Specifically, this invention first uses polyaspartic acid to guide the nucleation and growth of calcium phosphate within and outside the collagen confinement area, forming an in-situ mineralization template of calcium phosphate nanocrystals compatible with collagen orientation. This provides nuclei for remineralization and matrix deposition, while avoiding coarse deposition of inorganic phases on the collagen surface. From the early stages of material construction, it balances tissue adhesion and mechanical continuity. Subsequently, silanized bioactive glass nanoparticles (BG) were incorporated into mineralized collagen sol and a stable network dominated by amide bonds was constructed through mild cross-linking in an alcohol-containing medium. This upgraded the physical mixing of glass particles to chemical bonding, reducing the risk of migration and detachment in a humid environment. At the same time, the released active ions were confined to the collagen network and diffused locally, which facilitated the formation of functional layers along the film thickness direction. In conjunction with the previously formed calcium phosphate template, the time sequence of mineral deposition and ion replenishment was achieved. Through unidirectional freezing and freeze-drying, a structural gradient is spontaneously generated in the thickness direction: the oral cavity side (cavity surface) is dense, and the tissue defect side (bone / tooth side) is porous. The dense surface provides a barrier against seepage and shear disturbance on the oral cavity side, while the porous surface provides channels for cell migration, nutrient exchange, and the entry of new matrix towards the defect side. At the same time, the phase distribution effect induced by the directional growth of ice crystals further promotes the functional partitioning of inorganic phase distribution and pore structure. Alcohol crosslinking can reduce the swelling and deformation of the material in the wet state, thereby achieving a balance between wet mechanical strength, sutureability, and controllable degradation. Residual intermediates after crosslinking are removed by timely quenching and elution, improving biocompatibility. Finally, isotonic, near-neutral Sr-Zn solution was used for unilateral activation of the bone / dental side, forming a narrow-domain multi-ion microenvironment: Sr tends to promote hard tissue-related mineralization maturation and interfacial integration, while Zn tends to provide mild antibacterial and matrix metalloproteinase regulation, thereby delaying excessive collagen degradation and stabilizing early barrier function. This activation, combined with the aforementioned chemical coupling and structural gradient, concentrates the ionic effect on the desired interface and direction, avoiding indiscriminate release of the entire phase. In summary, the rapid nucleation of the calcium phosphate in-situ mineralization template, the continuous ion supply and load transfer of the silanized bioactive glass, the functional gradient formed by unidirectional freezing, the wet-state stability conferred by mild alcohol-containing crosslinking, and the localized microenvironment constructed by Sr-Zn unilateral activation collectively form a multi-level coherent regeneration process. Attached Figure Description

[0007] Figure 1 These are the results of a class I extraction test on the biocompatibility of the collagen-based repair membrane materials of Examples 1-3 and Comparative Examples 1-4 of this invention; Figure 2 This is a graph showing the biocompatibility of the collagen-based repair membrane materials of Example 1 and Comparative Example 4 of this invention, obtained from a type II extraction test. Figure 3 This is a graph showing the cumulative permeation curve test results of the collagen-based repair membrane materials of Example 1 and Comparative Examples 1-4 of the present invention. Figure 4 This is a graph showing the cumulative permeation curve test results of the porous surface of the collagen-based repair membrane material in Example 1 and Comparative Examples 1-4 of the present invention; Figure 5 These are the surface mineralization weight gain areal density test results of the collagen-based repair membrane materials of Embodiment 1 and Comparative Examples 1-4 of the present invention; Figure 6 This is a graph showing the normalized test results of Ca removal of collagen-based repair membrane materials in Example 1 and Comparative Examples 1-4 of the present invention; Figure 7 The graph shows the surface microhardness recovery rate (SMHR) test results of the collagen-based repair membrane materials of Embodiment 1 and Comparative Examples 1-4 of this invention. Detailed Implementation

[0008] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0009] Example 1: A collagen-based repair membrane material for oral use, the raw material composition of which includes the following components: type I collagen, bioactive glass nanoparticles, γ-aminopropyltriethoxysilane, cross-linking system, polyaspartic acid, CaCl2, K2HPO4 and Sr-Zn solution.

[0010] 1. The crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 4:1; 2. The Sr-Zn solution was prepared by using HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2 and deionized water in a volume ratio of 10 mmol:15 mmol:1 mmol:0.1 mmol:1000 mL, and adjusting the pH to 7.4 with NaOH and HCl. 3. The preparation of the oral collagen-based repair membrane material includes the following steps: S101: Add 8g of type I collagen to 1000mL of 0.02M acetic acid to prepare an acidic solution of 0.8wt%, let it stand at 4℃ for 20h to swell, stir to remove foam and then set aside; S102: Slowly add NaOH to the acidic solution in step S101 to adjust the pH of the system to 7, then slowly add 1 mL of 4 wt% polyaspartic acid aqueous solution and stir to disperse evenly. Then add acetic acid and NaOH to control the pH at 7.2 to obtain a pre-neutralized system. S103: Under constant temperature of 28℃, 25 mL of 0.5 M CaCl2 solution and 25 mL of 0.3 M K2HPO4 solution were added dropwise at a rate of 0.5 mL / min to the pre-neutralization system in step S102. The pH of the system was controlled to 7.8 with NaOH. After the addition was completed, the temperature was raised to 37℃ and stirred for 3 hours to obtain mineralized collagen sol. S104: 7g of bioactive glass nanoparticles, 120mL of ethanol and 7mL of deionized water were mixed in proportion, and ultrasonically treated for 14min to disperse evenly. 0.2g of γ-aminopropyltriethoxysilane was added, and the pH was adjusted to 4.8 with glacial acetic acid. Then, the mixture was stirred at 450rpm at 60℃ for 55min, cooled and centrifuged, resuspended in ethanol and washed to remove silane, vacuum dried at 40℃ for 7h, and ground through a 200-mesh sieve to obtain silanized BG nanoparticles. S105: Add 7g of silanized BG nanoparticles from step S104 to 28mL of mineralized collagen sol from step S103 and stir at 1000rpm for 2.5min to pregelatinize. Then add the remaining mineralized collagen sol from step S103, shear in segments, stir at 1000rpm for 3min, stir at 2300rpm for 2min, and control the temperature in an ice bath throughout the shearing process to obtain a composite collagen system. S106: Coat the inner wall of the mold with a thin layer of polyvinyl alcohol, pre-cool to -40℃ with the lower cold plate, and pour with a wet thickness of 1.6mm; gently vibrate for 10s to degas, freeze unidirectionally, and perform programmed step cooling. The temperature set points of the lower cold plate are -20℃ for 8min, -40℃ for 8min, and -60℃, respectively. The top of the sample is maintained at 4℃ throughout the process, and the sample is frozen until it is completely hardened. Then, dry in stages: heat from -20℃ to 0℃ at a rate of 0.2℃ / min, with a cavity pressure of 20Pa, for 7h, and then dry at 23℃ with a cavity pressure of 15Pa for 5h to obtain the mold blank. S107: Add the crosslinking system to an 80wt% ethanol solution and dissolve thoroughly to obtain a 30mM crosslinking solution; immerse the 80×120mm mold blank from step S106 into the crosslinking solution at a liquid-to-film ratio of 20mL:1cm. 2 Crosslinking was performed by gently shaking at 60 rpm for 4 h, followed by quenching with 10 mM glycine aqueous solution for 10 min, followed by graded washing, repeated washing twice with 50 wt% ethanol, and then washing with deionized water until the conductivity of the washing solution was <50 μS / cm; the surface water was dried, and the mixture was laid flat on a polytetrafluoroethylene plate and allowed to stand for 50 min to obtain the crosslinked preform. S108: Place the cross-linked preform from step S107 with its porous surface facing down in the Sr-Zn solution, with a liquid-to-film ratio of 10 mL: 1 cm⁻¹. 2 After shaking gently at 60 rpm for 10 h at 37℃, the material was rinsed twice with PBS buffer. Then, the temperature was increased from -20℃ to 20℃ at a rate of 0.5℃ / min under 20 Pa, and the material was dried for 5 h to obtain the collagen-based repair membrane material.

[0011] Example 2: A collagen-based repair membrane material for oral use, the raw material composition of which includes the following components: type I collagen, bioactive glass nanoparticles, γ-aminopropyltriethoxysilane, cross-linking system, polyaspartic acid, CaCl2, K2HPO4 and Sr-Zn solution.

[0012] 1. The crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 5:1; 2. The Sr-Zn solution was prepared by using HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2 and deionized water in a volume ratio of 10 mmol:15 mmol:1 mmol:0.1 mmol:1000 mL, and adjusting the pH to 7.4 with NaOH and HCl. 3. The preparation of the oral collagen-based repair membrane material includes the following steps: S101: Add 10g of type I collagen to 1000mL of 0.02M acetic acid to prepare a 1wt% acidic solution, let it stand at 4℃ for 24h to swell, stir to remove foam and then use it for later use; S102: Slowly add NaOH to the acidic solution in step S101 to adjust the pH of the system to 7.4, then slowly add 1 mL of 4 wt% polyaspartic acid aqueous solution and stir to disperse evenly. Then add acetic acid and NaOH to control the pH at 7.1 to obtain a pre-neutralized system. S103: Under constant temperature of 25℃, 30 mL of 0.5 M CaCl2 solution and 30 mL of 0.3 M K2HPO4 solution were added dropwise at a rate of 0.6 mL / min to the pre-neutralization system in step S102. The pH of the system was controlled to 8 with NaOH. After the addition was completed, the temperature was raised to 37℃ and stirred for 4 h to obtain mineralized collagen sol. S104: 7g of bioactive glass nanoparticles, 140mL of ethanol and 7mL of deionized water were mixed in proportion, and ultrasonically treated for 15min to disperse evenly. 0.21g of γ-aminopropyltriethoxysilane was added, and the pH was adjusted to 4.5 with glacial acetic acid. Then, the mixture was stirred at 500rpm at 60℃ for 60min, cooled and centrifuged, resuspended in ethanol and washed to remove silane, vacuum dried at 40℃ for 8h, and ground through a 200-mesh sieve to obtain silanized BG nanoparticles. S105: Add 6.63g of silanized BG nanoparticles from step S104 to 26.5mL of mineralized collagen sol from step S103 and stir at 1000rpm for 3min for pregelatinization. Then add the remaining mineralized collagen sol from step S103, perform segmented shearing, stir at 1200rpm for 3min and at 2500rpm for 2min, and control the temperature in an ice bath throughout the shearing process to obtain a composite collagen system. S106: Coat the inner wall of the mold with a thin layer of polyvinyl alcohol, pre-cool to -40℃ with the lower cold plate, and pour with a wet thickness of 2mm; gently vibrate for 10s to vent, freeze unidirectionally, and perform programmed step cooling. The temperature set points of the lower cold plate are -20℃ for 10min, -40℃ for 10min, and -60℃, respectively. The top of the sample is kept at 0℃ throughout the process and frozen until completely hardened. Then, dry in stages, heating from -20℃ to 0℃ at a rate of 0.2℃ / min, with a cavity pressure of 20Pa, for 8 hours, and then drying at 25℃ with a cavity pressure of 15Pa for 6 hours to obtain the mold blank. S107: Add the crosslinking system to an 80wt% ethanol solution and dissolve thoroughly to obtain a 30mM crosslinking solution; immerse the 80×120mm mold blank from step S106 into the crosslinking solution at a liquid-to-film ratio of 20mL:1cm. 2 Crosslinking was performed by gently shaking at 60 rpm for 4 h, followed by quenching with 10 mM glycine aqueous solution for 10 min, followed by graded washing, repeated washing twice with 50 wt% ethanol, and then washing with deionized water until the conductivity of the washing solution was <50 μS / cm; the surface water was dried, and the mixture was laid flat on a polytetrafluoroethylene plate and allowed to stand for 60 min to obtain the crosslinked preform. S108: Place the cross-linked preform from step S107 with its porous surface facing down in the Sr-Zn solution, with a liquid-to-film ratio of 10 mL: 1 cm⁻¹. 2 After shaking gently at 60 rpm for 12 hours at 37°C, the material was rinsed twice with PBS buffer. Then, the temperature was increased from -20°C to 20°C at a rate of 0.5°C / min under 20 Pa, and the material was dried for 6 hours to obtain the collagen-based repair membrane material.

[0013] Example 3: A collagen-based repair membrane material for oral use, the raw material composition of which includes the following components: type I collagen, bioactive glass nanoparticles, γ-aminopropyltriethoxysilane, cross-linking system, polyaspartic acid, CaCl2, K2HPO4 and Sr-Zn solution.

[0014] 1. The crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 3:1; 2. The Sr-Zn solution was prepared by using HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2 and deionized water in a volume ratio of 10 mmol:15 mmol:1 mmol:0.1 mmol:1000 mL, and adjusting the pH to 7.4 with NaOH and HCl. 3. The preparation of the oral collagen-based repair membrane material includes the following steps: S101: Add 5g of type I collagen to 1000mL of 0.02M acetic acid to prepare a 0.5wt% acidic solution, let it stand at 4℃ for 12h to swell, stir to remove foam and then set aside. S102: Slowly add NaOH to the acidic solution in step S101 to adjust the pH of the system to 6.8, then slowly add 1 mL of 4 wt% polyaspartic acid aqueous solution and stir to disperse evenly. Then add acetic acid and NaOH to control the pH at 7.3 to obtain a pre-neutralized system. S103: Under constant temperature of 30℃, 15 mL of 0.5 M CaCl2 solution and 15 mL of 0.3 M K2HPO4 solution were added dropwise at a rate of 0.2 mL / min to the pre-neutralization system in step S102. The pH of the system was controlled to 7.6 with NaOH. After the addition was completed, the temperature was raised to 37℃ and stirred for 2 hours to obtain mineralized collagen sol. S104: Mix 8g of bioactive glass nanoparticles, 120mL of ethanol and 8mL of deionized water in a certain proportion, sonicate for 10min to disperse evenly, add 0.16g of γ-aminopropyltriethoxysilane, adjust the pH to 5 with glacial acetic acid, then stir at 400rpm for 45min at 60℃, cool and centrifuge, resuspend in ethanol and wash to remove silane, vacuum dry at 40℃ for 6h, grind through 200 mesh to obtain silanized BG nanoparticles; S105: Add 7.35g of silanized BG nanoparticles from step S104 to 29.4mL of mineralized collagen sol from step S103 and stir at 1000rpm for 2min for pregelatinization. Then add the remaining mineralized collagen sol from step S103, perform segmented shearing, stir at 800rpm for 3min and at 2000rpm for 2min, and control the temperature in an ice bath throughout the shearing process to obtain a composite collagen system. S106: Coat the inner wall of the mold with a thin layer of polyvinyl alcohol, pre-cool to -40℃ with the lower cold plate, and pour with a wet thickness of 1mm; gently vibrate for 10s to vent, freeze unidirectionally, and perform programmed step cooling. The temperature set points of the lower cold plate are -20℃ for 5min, -40℃ for 5min, and -60℃, respectively. The top of the sample is kept at 5℃ throughout the process and frozen until completely hardened. Then, dry in stages, heating from -20℃ to 0℃ at a rate of 0.2℃ / min, with a cavity pressure of 20Pa, for 6h, and then drying at 20℃ with a cavity pressure of 15Pa for 4h to obtain the mold blank. S107: Add the crosslinking system to an 80wt% ethanol solution and dissolve thoroughly to obtain a 30mM crosslinking solution; immerse the 80×120mm mold blank from step S106 into the crosslinking solution at a liquid-to-film ratio of 20mL:1cm. 2 Crosslinking was performed by gently shaking at 60 rpm for 4 h, followed by quenching with 10 mM glycine aqueous solution for 10 min, followed by graded washing, repeated washing twice with 50 wt% ethanol, and then washing with deionized water until the conductivity of the washing solution was <50 μS / cm; the surface water was dried, and the mixture was laid flat on a polytetrafluoroethylene plate and allowed to stand for 30 min to obtain the crosslinked preform. S108: Place the cross-linked preform from step S107 with its porous surface facing down in the Sr-Zn solution, with a liquid-to-film ratio of 10 mL: 1 cm⁻¹. 2 After shaking gently at 60 rpm for 6 hours at 37℃, the material was rinsed twice with PBS buffer. Then, the temperature was increased from -20℃ to 20℃ at a rate of 0.5℃ / min under 20 Pa, and the material was dried for 4 hours to obtain the collagen-based repair membrane material.

[0015] Comparative Example 1: The raw materials and processes of Comparative Example 1 are basically the same as those of Example 1. The main difference is that the bioactive glass nanoparticles in Comparative Example 1 are not silanized, that is, the addition of γ-aminopropyltriethoxysilane in step S104 is cancelled, while the remaining operations in step S104 are performed as before; the remaining steps and parameters are consistent with those of Example 1.

[0016] Comparative Example 2: The raw materials and processes of Comparative Example 2 are basically the same as those of Example 1. The main difference is that in Comparative Example 2, unidirectional freezing is directly replaced by rapid freezing, that is, the entire slurry after pouring is placed in an environment of -80℃ for isothermal freezing, without setting the temperature difference of the lower cooling plate and the programmed step cooling; the remaining steps and parameters are consistent with those of Example 1.

[0017] Comparative Example 3: The raw materials and processes of Comparative Example 3 are basically the same as those of Example 1. The main difference is that in Comparative Example 3, polyaspartic acid is removed from step S102, and step S103 does not involve the time-drop mineralization of CaCl2 / K2HPO4, etc. Instead, after neutralization in the acidic solution in step S102, nano-hydroxyapatite of the same mass as the calcium phosphate mineral theoretically generated in Example 1 is directly added and mechanically dispersed evenly. The remaining steps and parameters are consistent with those of Example 1.

[0018] Comparative Example 4: The raw materials and processes of Comparative Example 4 are basically the same as those of Example 1. The main difference is that in Comparative Example 4, step S108 does not involve Sr-Zn single-sided activation treatment. Instead, it is completed by quickly rinsing twice with PBS under the same conditions and then directly following the drying procedure. The remaining steps and parameters are consistent with those of Example 1.

[0019] Performance testing: Biocompatibility testing: Human keratinocytes (HaCaT) (corresponding to the cavity surface) and human gingival fibroblasts (HGF) (corresponding to the bone / tooth side) were selected. The samples were collagen-based repair membrane materials (sheets) from Examples 1-3 and Comparative Examples 1-4, with a membrane area / volume ratio of 6 cm². 2 The extraction solution was prepared at 37°C and 60 rpm with gentle shaking for 24 h. Two extraction systems were set up: the culture medium extract (corresponding to the complete culture medium of each cell) was used as the first extraction system; to reflect the mechanism of single-sided ion activation, a bone / tooth side directional conditional extraction was set up. The membrane was placed in the Transwell insert to simulate the asymmetric release environment, with only the bone / tooth side facing the lower cavity culture medium (cavity surface isolated). The lower cavity conditional culture medium was collected after incubation at 37°C for 24 h as the second extraction solution (only for Example 1 and Comparative Example 4, to reflect the difference in single-sided activation of Sr / Zn); HaCaT and HGF were seeded into 96-well plates, and after culture to 70%-80% confluence, the medium was discarded, and each was treated with 100 µL of 100% extraction solution per well; the negative control was fresh culture medium, and the positive control was culture medium containing 10% DMSO. Incubate at 37°C with 5% CO2 for 48 hours; add CCK-8 working solution (10 µL per well) according to the instructions, incubate for 2 hours, and then measure the OD. 450 Survival rate (%) = (OD sample - OD positive control) / (OD negative control - OD positive control) × 100%; Three parallel wells were set up for each condition, and three batches were independently replicated. The results are as follows: Figure 1 and Figure 2 As shown.

[0020] based on Figure 1 and Figure 2Results analysis showed that the collagen-based repair membrane prepared in the embodiments of the present invention, based on a multi-layered synergistic effect, effectively reduced the exposure of soluble irritants and free particles, exhibiting good biocompatibility within 48 hours. In Comparative Example 1, after removing γ-aminopropyltriethoxysilane (APTES), BG existed only in a physically mixed state, making it more prone to particle migration / micro-aggregation and interfacial micro-detachment under wet conditions. The trace amounts of suspended inorganic fragments and rapid early ion bursts generated during extraction, coupled with slight pH / osmotic pressure shifts, resulted in higher instantaneous ionic intensity and contact stimulation of hard particles on the cell membrane surface, leading to a significant decline in metabolic activity and a significantly lower survival rate compared to Example 1. In Comparative Example 2, after losing the dense luminal / porous layered structure on the bone side, the membrane's two-sided pores opened, and the specific surface area increased, theoretically facilitating the formation of faster early extraction and diffusion pathways. Under the same extraction conditions, this may lead to a higher instantaneous concentration of early soluble components and ions in the extract, resulting in a slight decrease in biocompatibility compared to Example 1. Comparative Example 3, without polyaspartic acid induction and Ca / P addition, contained nano-hydroxyapatite with externally incorporated micro-aggregates, resulting in insufficient orientation matching and intracellular / extracellular localization. This led to more exposed inorganic particle interfaces and localized rough deposits within the collagen network, making it prone to mechanical fine particle contact and local Ca / P microenvironment fluctuations during extraction. Simultaneously, the lack of an in-situ mineralization template weakened the sequential relay of BG ion propagation, creating a less cell-friendly environment and significantly reducing cell viability. Comparative Example 4 showed little difference from Example 1 in the first type of extraction solution. This may be because this extraction method does not distinguish between the two sides of the membrane, and the single-sided localization advantage of Sr / Zn is diluted under homogenization conditions of large volume and double-sided immersion. Furthermore, Example 1 and Comparative Example 4 were consistent in terms of APTES coupling, structural gradient, and cross-linking elution, thus both HaCaT / HGF maintained high viability with no significant difference. The survival rate of HGF in the second type of extract was higher in Example 1 than in Comparative Example 4, while the difference for HaCaT was not significant. This may be because under these conditions, only the bone / dental side is in contact with the culture medium. Example 1 releases a low dose of Sr (with very low Zn) locally, forming a mild, near-neutral localized ion field that supports the metabolism and spread of fibroblast / osteoblast-related cells and mildly regulates MMP activity. Comparative Example 4 lacks this bone-side-favorable microenvironment, and HGF is more sensitive to the difference, hence the compatibility difference at 48 h. HaCaT is not sensitive to this orientation condition itself (its corresponding luminal surface is not in direct contact with the extract), so the difference between the two groups is small.

[0021] Wet mechanical tests: The collagen-based repair membrane materials (membranes) of Examples 1-3 and Comparative Examples 1-4, after being soaked in PBS at 37°C for 30 min, were tested for wet tensile strength and wet elongation at break according to the method standard of GB / T 16578.2-2009. At the same time, the wet swelling rate before and after moisture absorption was measured. The test results are shown in Table 1 below.

[0022] Table 1. Test Results of Examples and Comparative Examples

[0023] Based on the results in Table 1, the collagen-based repair membrane material prepared in this embodiment of the invention exhibits excellent wet tensile strength and wet elongation at break, while also having a low swelling rate, demonstrating superior wet mechanical properties. In Comparative Example 1, BG was only physically mixed, resulting in a significant decrease in the interfacial shear strength between the particles and collagen / mineralized collagen in the wet state; the load transfer pathway was interrupted, leading to a decrease in strength; simultaneously, micro-agglomeration and particle loosening were more likely to occur during hydration / stirring / forming, becoming brittle initiation points and reducing elongation. Furthermore, the lack of silane-amide bond dehydration effect increased the interfacial water absorption channels, leading to an increase in the overall swelling rate, further diluting the network and reducing strength. Comparative Example 2: Dense on the surface and porous on the bone side without cavities: The isotropic porous structure increases the specific surface area and water permeation pathway of the membrane, leading to increased water absorption; the absence of ice crystal-oriented layered / fiber arrangement makes it difficult for cracks to deflect and dissipate energy between layers, resulting in a simultaneous slight decrease in strength and elongation; the lack of dense cavities makes it easier for liquid to permeate and swell in the wet state, passively loosening the network and further affecting the geology. Comparative Example 3: Lacking PASP-induced internal / external fiber positioning and orientation matching, the nano-hydroxyapatite exists in the form of exogenous aggregated particles, making it difficult to form a continuous composite phase with collagen. The aggregates become multi-order-of-magnitude micro-defects in the wet state, and stress concentration causes a simultaneous decrease in strength and elongation. In addition, the uneven filling of exogenous particles and the lack of interface tightness make it easier for capillary and interfacial aqueous phases to enter, increasing the swelling rate and conversely reducing the strength. In Comparative Example 4, Sr / Zn activation is an interface-level regulation that does not change the bulk crosslinking degree and pore structure. Confined in-situ mineralization, APTES coupling, unidirectional freezing, and alcohol-containing crosslinking are all retained. Therefore, the matrix strength and elongation at break are close to those of Example 1.

[0024] Barrier function verification test: Collagen-based repair membrane material sheets (effective area approximately 3.14 cm²) from Example 1 and Comparative Examples 1-4 were used. 2 After pre-wetting for 10 min, 20 mm diameter discs were placed into a dual-chamber diffusion cell. Two assembly orientations were set: the cavity surface (dense surface) facing the donor side; the bone / tooth side (porous surface) facing the donor side; and the recipient side was stirred with PBS at 37°C and 60 rpm. A tracer dye solution (e.g., methylene blue 0.05 mg / mL) was added to the donor side. At 10, 20, 30, 60, 90, and 120 min, 1 mL of the recipient side sample was taken and replenished with an equal volume of buffer. The absorbance at the maximum absorption wavelength of methylene blue was measured using UV-Vis spectrophotometry, and the cumulative permeate volume Q(t) / A per unit membrane area was calculated. Subsequently, the same batch of membranes was flipped over and the test was repeated (new samples that had not been tested before). The negative baseline was a channel without membrane openings. The cumulative permeate volume / time curve test results are shown below. Figure 3 (dense surface) and Figure 4 As shown in the diagram (porous surface).

[0025] based on Figure 3 and Figure 4 Analysis of the results shows that the collagen-based repair membrane material prepared in this embodiment of the invention, based on the cavity-surface dense layer formed by unidirectional freezing, can significantly improve the tortuosity of the pores, reduce the effective pore size and connectivity, and simultaneously, combined with the interfacial chemical coupling effect of the silane coupling agent, inhibit the interfacial cracking caused by wet swelling, specifically manifested as reduced permeability, with significant differences between the front and back sides. Comparative Example 1 lacks chemical coupling, and the BG-collagen interface has micro-cracks / weak binding zones, making it easier to form permeation bypasses in the wet state; it exhibits a shorter initiation hysteresis and a higher slope in the early stages; the porous surface is already more permeable, and the cumulative permeability is significantly increased due to the addition of localized macropores or channels caused by particle micromigration / aggregation. Although the dense layer still exists, its interfacial integrity deteriorates, and the cavity-surface advantage is weakened. Comparative Example 2, with its non-cavitary, dense-bonded porous gradient structure, exhibits approximately symmetrical interconnected pores, with the flip-over curves largely overlapping, and a generally high cumulative permeability. This may be due to the shorter, less tortuous flow paths typically formed by isotropic rapid freezing, which also increases specific surface area and swelling capacity. Despite lacking structural layering, its performance is still superior to Comparative Example 1 due to the retention of coupling and in-situ mineralization templates, resulting in fewer interfacial micro-cracks and particle migration. Comparative Example 3, due to the easy micro-agglomeration / surface exposure of the exfoliated nano-hydroxyapatite, forms micro-defects and rough channels during cross-linking / drying / wetting processes. The continuity of the dense layer is compromised, leading to an increase in slope and cumulative permeability. Uneven distribution of exfoliated particles and poor interfacial adhesion result in a higher probability of macropores and semi-through pores, significantly increasing permeability. While unidirectional freezing is retained, there is still a difference between the two sides, but the gap is narrowing. Comparative Example 4 has an overall structure basically consistent with Example 1, and its performance is also quite close to Example 1. This may be because short-term liquid permeability is mainly determined by pore structure and interfacial integrity; the absence of Sr / Zn does not significantly alter these physical channels.

[0026] Mineralization test: Collagen-based repair membrane material sheets (effective area approximately 3.14 cm²) from Example 1 and Comparative Examples 1-4 2 20mm diameter discs were pre-wetted with PBS for 10 min for activation. The discs were then placed in a single-sided exposed fixture with the bone / tooth side (porous side) facing down in contact with SBF (37°C, pH 7.4), while the cavity side remained isolated from the solution. The SBF volume was 10 mL / cm³. 2 Prepare the solution and gently shake at 60 rpm; set sampling time points of 6, 12, 24, and 48 hours, and replace SBF independently for each membrane to avoid cross-influence; record the membrane's dry weight before the experiment begins; at the designated time, remove the membrane, wash it twice with PBS, and freeze-dry it under vacuum until constant weight, then record the mass m. t Simultaneously save SBF samples at each time point, using Ca... 2 +(o-CPC method) Colorimetric determination of Ca concentration in SBF at the initial and endpoint points, and calculation of surface mineralization gain density (mg / cm³). 2 = (Post-experiment membrane dry constant weight - Initial membrane dry constant weight) / Effective membrane area; Normalized Ca removal (mg / cm³) 2 The formula is: (Initial Ca concentration in SBF - Final Ca concentration in SBF) × SBF volume / Effective membrane area. The test results are as follows: Figure 5 and Figure 6 As shown.

[0027] Remineralization test: Polished dental slides (5×5×2mm) were prepared and artificial lesions (enamel 24h) were obtained by acid etching with lactate buffer (pH 4.5, 37°C). After washing and drying, the initial Vickers microhardness was measured (load 200g, 10s, average of 5 points). A healthy self-control window reserved on the same dental slide was used as a healthy control. The bone / tooth side of the membrane was attached to the surface of the dental slide, fixed with an elastic ring, and placed in remineralization solution (PBS buffer, 2mM Ca). 2+ 1.2mM PO4 2- The solution was gently shaken at 60 rpm in a solution containing 10 mL / tablet (pH 7.2, 37°C). Samples were taken on days 1, 3, and 7, with the solution changed daily. After each sample was removed, it was gently washed, allowed to air dry, and then the hardness was retested under the original load. The surface microhardness recovery rate (SMHR) was calculated as follows: SMHR(%) = (retested hardness - initial hardness) / (healthy hardness - initial hardness) × 100%. The results are shown below. Figure 7 As shown.

[0028] based on Figure 5 , Figure 6 and Figure 7Analysis of the results shows that the collagen-based repair membrane material prepared in the embodiments of the present invention exhibits excellent deposition efficiency. A mild Sr-enriched ion field is formed on the bone-side surface, which can promote the growth and maturation of early crystal nuclei. During remineralization, the deposition on the dental radiograph interface becomes denser and has better adhesion. Comparative Example 1, due to the physical mixing of BG, resulted in micro-agglomeration and interface debonding in the wet state, forming a weak substrate and micro-cracks, leading to discontinuous deposition and low weight gain. The unlocalized release of ions caused excessively high local supersaturation, low calcium utilization efficiency, and low fixation amount. The final mineralized layer was loose and contained a weak layer with particles, resulting in slow hardness recovery. Comparative Example 2, due to the lack of a porous / cavity-dense partitioned structure on the bone side, resulted in a thin bone-side boundary layer, enhanced convection, difficulty in maintaining local supersaturation, and low deposition efficiency and calcium retention rate. Ions migrated back and forth in the system but were not sufficiently fixed, resulting in weak bone-side guidance and discontinuous interface deposition, which led to slow formation of a dense, adherent layer on the dental radiograph surface. Comparative Example 3, due to the presence of exogenous micro-aggregates of nano-hydroxyapatite and the lack of intrafiber localization, resulted in isolated heterogeneous nucleation sites, fragmented deposition layers, and insufficient weight gain. The effective nucleation area was low, and there was no localized ion supply, leading to poor calcium fixation efficiency. Ultimately, a porous, inclusion-containing, weakly adherent mineral layer was formed at the dental slice interface, with slow hardness recovery. Comparative Example 4, due to the lack of Sr interface activation, hindered early crystal nucleation and densification, resulting in lower deposition amount and calcium fixation rate within 24 hours. Its intact bulk structure, under the unlimited calcium source of SBF, gradually led to deposition amounts approaching those of Example 1. Dental slice remineralization depends on early interface integration; Sr's one-sided activation facilitates early integration and crystal maturation. While initially lagging significantly, it partially caught up later due to bulk advantages, but ultimately its performance remained slightly lower.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A collagen-based repair membrane material for oral use, characterized in that, Its raw material composition includes the following components: type I collagen, bioactive glass nanoparticles, γ-aminopropyltriethoxysilane, cross-linking system, polyaspartic acid, CaCl2, K2HPO4 and Sr-Zn solution; The crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 3 to 5:1; The Sr-Zn solution was prepared by using HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2 and deionized water in a volume ratio of 10 mmol:15 mmol:1 mmol:0.1 mmol:1000 mL, and adjusting the pH to 7.4 with NaOH and HCl. The preparation of the oral collagen-based repair membrane material includes the following steps: S101: Add type I collagen to acetic acid to prepare an acidic solution, let it stand to swell, stir to defoam, and then set aside for use; S102: Slowly add NaOH to the acidic solution in step S101 to adjust the pH of the system, then slowly add polyaspartic acid aqueous solution and stir to disperse evenly, then add acetic acid and NaOH to control the pH to obtain a pre-neutralized system; S103: Under constant temperature conditions, CaCl2 solution and K2HPO4 solution are added dropwise to the pre-neutralization system in step S102 at equal intervals. NaOH is used to control the pH of the system. After the addition is completed, the temperature is raised and stirred to obtain mineralized collagen sol. S104: Bioactive glass nanoparticles, ethanol and deionized water are mixed in proportion, ultrasonically dispersed, γ-aminopropyltriethoxysilane is added, pH is adjusted with glacial acetic acid, then heated and stirred, cooled and centrifuged, resuspended in ethanol and washed to remove silane, vacuum dried, and ground to obtain silanized BG nanoparticles. S105: Add the silanized BG nanoparticles from step S104 to a portion of the mineralized collagen sol from step S103 and stir to pre-gelatinize. Then add the remaining mineralized collagen sol from step S103 and shear in segments to obtain a composite collagen system. S106: Coat the inner wall of the mold with a thin layer of polyvinyl alcohol, pre-cool it with the lower cold plate, pour it, gently vibrate to vent the air, freeze it in one direction, and then dry it in stages to obtain the mold blank. S107: Add the crosslinking system to an 80wt% ethanol solution and dissolve it evenly to obtain a crosslinking solution; put the mold blank from step S106 into the crosslinking solution, gently shake to crosslink, quench with glycine aqueous solution, wash in stages, absorb the surface water, lay it flat on a polytetrafluoroethylene plate and let it stand to obtain a crosslinked mold blank. S108: Place the cross-linked preform from step S107 with its porous side down in the Sr-Zn solution, shake gently, and rinse quickly with PBS buffer; then dry to obtain the collagen-based repair membrane material. In step S104, the ratio of the bioactive glass nanoparticles, ethanol, and deionized water is 1g:15-20mL:1mL; the amount of γ-aminopropyltriethoxysilane added is 2-3% of the mass of the bioactive glass nanoparticles; and the ratio of the amount of silanized BG nanoparticles to the total mineralized collagen sol is 1g:140-160mL. In step S105, the segmented shearing parameters are: stirring at 800-1200 rpm for 3 minutes, stirring at 2000-2500 rpm for 2 minutes, and temperature controlled by an ice bath throughout the shearing process; In step S106, the specific operation of the unidirectional freezing is as follows: programmed step cooling, with the temperature set points of the lower cold plate being -20℃ for 5 to 10 minutes, -40℃ for 5 to 10 minutes, and -60℃ in sequence, while the top of the sample is maintained at 0 to 5℃ throughout the process, until it is completely hardened.

2. The oral collagen-based repair membrane material according to claim 1, characterized in that, In step S102, the amount of the polyaspartic acid aqueous solution added is 0.1% of the volume of the acidic solution.

3. The oral collagen-based repair membrane material according to claim 2, characterized in that, In step S103, the volume ratio of the CaCl2 solution to the K2HPO4 solution is 1:

1. The total volume added is 3 to 6% of the volume of the acidic solution.

4. The oral collagen-based repair membrane material according to claim 3, characterized in that, In step S107, the graded washing parameters are as follows: wash twice with 50wt% ethanol, and then wash with deionized water until the conductivity of the washing solution is <50μS / cm.

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