Collagen-based repair membrane material for oral cavity and preparation method of collagen-based repair membrane material

Abstract

The invention relates to the technical field of biomedical materials, in particular to a collagen-based repair membrane material for an oral cavity and a preparation method of the collagen-based repair membrane material. The invention relates to an anti-aging collagen gel, which specifically comprises the following components: collagen type I, bioactive glass nanoparticles, gamma-aminopropyltriethoxysilane, a cross-linking system, polyaspartic acid, CaCl2, K2HPO4 and a Sr-Zn solution. According to the preparation method, in-situ mineralization of calcium and phosphorus is guided by polyaspartic acid to form a nanocrystalline template with orientation compatibility, and in combination with chemical coupling and continuous ion release of silanized bioactive glass, time sequence synergy of mineral deposition and ion supply is achieved; a structure gradient with a compact cavity surface and a porous bone side is constructed by utilizing unidirectional freezing, and the structure gradient is endowed with two-way functions of seepage prevention and cell guidance; the wet strength is improved and swelling is controlled by combining alcohol-containing mild crosslinking; furthermore, a Sr-Zn solution is used for activating a single side of the bone, so that a directional microenvironment is formed, mineralization and maturation of an interface are promoted, collagen degradation is delayed, and a multi-module synergistic oral cavity repairing function is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a collagen-based repair membrane material for oral cavity and a preparation method thereof. BACKGROUND

[0002] As the primary interface between the human body and the external environment, the oral cavity is in a complex and dynamic microecological environment for a long time. Its health status not only depends on the host's own immune defense and tissue repair capacity, but also is continuously influenced by multiple factors such as oral microbial community, physical and chemical stimulation, and biomechanical load. When pathogenic microorganisms form a highly structured biofilm on the surface of the tooth or soft tissue, their metabolic products can cause enamel demineralization, and then cause dental caries. If the plaque biofilm extends to the subgingival area, it may activate the immune inflammatory response of the periodontal tissue, cause alveolar bone resorption and periodontal attachment loss, and eventually lead to tooth loosening or even shedding. In the field of oral bone tissue regeneration, how to effectively guide and accelerate the repair of the defect area has always been a core problem faced by the clinic, and the repair membrane used in guided bone regeneration technology plays a key role in this process. The repair membranes currently widely used in clinical and research, whether based on natural collagen, synthetic polymers or composite systems, have increasingly highlighted the functional limitations in the complex and dynamic environment of the oral cavity. Some materials exhibit obvious swelling phenomenon and mechanical property attenuation under wet conditions, and are difficult to maintain a stable barrier space throughout the healing period. Some other materials are biologically inert and cannot actively regulate the local microenvironment, and can only act as passive physical barriers. In addition, most membrane materials lack functional considerations in structural design and cannot simultaneously achieve the dual requirements of soft tissue isolation and bone tissue guidance. The homogenization of the form seriously limits its repair efficiency. In terms of active component loading, whether it is the simple mixing of inorganic fillers or the physical adsorption of biological factors, there are generally technical bottlenecks such as uncontrollable active release, weak interface combination, initial burst release or easy inactivation, which seriously restrict the application prospect of functional tissue regeneration. SUMMARY

[0003] In view of the defects of the prior art, the purpose of the present application is to provide a collagen-based oral repair membrane material and a preparation method thereof. In order to solve the problems of weak mechanical properties, easy falling off of inorganic phase, poor remineralization endurance and lack of cavity surface barrier-defect surface guided partition of the existing oral repair membrane, the present application is based on the collagen limited in-situ mineralization induced by polyaspartic acid to form an in-situ mineralization template compatible with collagen, to realize rapid remineralization and morphological stability; the bioactive glass nanoparticles are surface modified by gamma-aminopropyl triethoxysilane and chemically coupled with collagen in an EDC / NHS system to construct a continuous interface network, prevent wet state pulverization and falling off and enhance load transmission; the unidirectional freezing structure is used to build a gradient structure of dense cavity surface and porous defect surface to realize the anti-infiltration and anti-disturbance of the same piece material and tissue guidance; the mild crosslinking with alcohol and glycine quenching are used to reduce swelling and regulate degradability and compatibility; the isosmotic near-neutral Sr-Zn single surface activation is used to make the ions release locally, to cooperatively promote remineralization, interface integration and enzyme degradation regulation with the template and interface; to realize multi-level cooperation, significantly improve the wet state stability, anti-disturbance and directional regeneration performance.

[0004] The technical effects of the present application are realized by the following technical scheme: a collagen-based oral repair membrane material, the raw material composition of which comprises the following components: type I collagen, bioactive glass nanoparticles, gamma-aminopropyl triethoxysilane, a crosslinking system, polyaspartic acid, CaCl2, K2HPO4 and a Sr-Zn solution.

[0005] Preferably, the bioactive glass nanoparticles are silicate system; Preferably, the crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide at a molar ratio of 3-5:1; Preferably, the Sr-Zn solution is prepared by using HEPES buffer, NaCl, SrCl2.6H2O, ZnCl2 and deionized water at a dosage 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 application is to provide a preparation method of a collagen-based oral repair membrane material, which specifically comprises the following steps: S101: type I collagen is added into 0.02M acetic acid to prepare an acidic solution with a concentration of 0.5-1wt%, and is swelled at 4℃ for 12-24h, and is stirred and defoamed before use; S102: NaOH is slowly added dropwise to the acidic solution in step S101 to adjust the pH of the system to 6.8-7.4, then 4wt% polyaspartic acid aqueous solution is slowly added dropwise and stirred to disperse uniformly, then acetic acid and NaOH are added dropwise to control the pH at 7.1-7.3 to obtain a pre-neutralization system; S103: Under the condition of constant temperature of 25-30℃, 0.5M CaCl2 solution and 0.3M K2HPO4 solution were added into the pre-neutralized system in step S102 at a speed of 0.2-0.6mL / min, and the pH of the system was controlled to 7.6-8 by NaOH. After the addition was completed, the temperature was raised to 37℃, and the stirring was continued for 2-4h to obtain a mineralized collagen sol; S104: The bioactive glass nanoparticles, ethanol and deionized water were mixed in proportion, and ultrasonic treatment was carried out for 10-15min to disperse uniformly. Then, γ-aminopropyl triethoxysilane was added, and the pH was adjusted to 4.5-5 by glacial acetic acid. Subsequently, stirring treatment was carried out at 400-500rpm for 45-60min at 60℃. After cooling and centrifugation, the silane was removed by ethanol resuspension and washing. Finally, vacuum drying was carried out at 40℃ for 6-8h, and the product was ground to 200 mesh to obtain silanized BG nanoparticles; S105: The silanized BG nanoparticles in step S104 were added into part of the mineralized collagen sol in step S103 at a mass-volume ratio of 1:4, and pre-pasting was carried out by stirring at 1000rpm for 2-3min. Then, the remaining mineralized collagen sol in step S103 was added, and the product was obtained by sectional shearing to obtain a composite collagen system; S106: The inner wall of the mold was coated with a thin layer of polyvinyl alcohol, and the lower cold plate was pre-cooled to -40℃. Then, the mold was filled with a wet thickness of 1-2mm. After air exhaust by light vibration for 10s, unidirectional freezing was carried out, and then the product was dried in stages. The temperature was raised from -20℃ to 0℃ at a speed of 0.2℃ / min, the cavity pressure was 20Pa, and the drying time was 6-8h. Then, the temperature was raised from 0℃ to 20℃ at a speed of 0.2℃ / min, the cavity pressure was 15Pa, and the drying time was 4-6h to obtain a mold blank; S107: The cross-linking system was dissolved in an 80wt% ethanol solution to obtain a cross-linking solution with a total concentration of 30mM. The mold blank in step S106 was placed into the cross-linking solution, and the liquid film ratio was 20mL:1cm 2 . After light shaking at 60rpm for 4h, the product was quenched in 10mM glycine aqueous solution for 10min, and then the product was washed in stages. After the surface water was absorbed, the product was placed on a polytetrafluoroethylene plate and stood for 30-60min to obtain a cross-linked mold blank; S108: The porous surface of the cross-linked mold blank in step S107 was placed downward in a Sr-Zn solution, and the liquid film ratio was 10mL:1cm 2 . After light shaking at 60rpm for 6-12h at 37℃, the product was quickly washed twice with PBS buffer. Then, the product was dried at 20Pa by raising the temperature from -20℃ to 20℃ at a speed of 0.5℃ / min for 4-6h to obtain a collagen-based repair membrane material; Preferably, in step S102, the addition amount of the polyaspartic acid aqueous solution is 0.1% of the volume of the acidic solution; Preferably, in step S103, the volume ratio of CaCl2 solution to K2HPO4 solution is 1:1, and the total addition volume is 3-6% of the volume of the acidic solution. Preferably, in step S104, the ratio of the amount of the bioactive glass nanoparticles, ethanol and deionized water is 1g: 15-20mL: 1mL; Preferably, in step S104, the amount of the γ-aminopropyl triethoxysilane added is 2-3% of the mass of the bioactive glass nanoparticles; Preferably, in step S104, the ratio of the amount of the silanized BG nanoparticles and the total mineralized collagen sol is 1g: 140-160mL; Preferably, in step S105, the segmental shearing parameters are: 800-1200rpm stirring for 3min, 2000-2500rpm stirring for 2min, and ice bath temperature control throughout the shearing process; Preferably, in step S106, the unidirectional freezing is specifically performed as follows: programmed stepwise temperature reduction, the lower cold plate temperature set point is sequentially -20℃ constant temperature for 5-10min, -40℃ constant temperature for 5-10min and -60℃, the top of the sample is maintained at 0-5℃ throughout, and the freezing is performed until complete hardening; Preferably, in step S107, the hierarchical washing parameters are: 50wt% ethanol repeated washing for 2 times, and then deionized water washing until the washing liquid conductivity is <50μS / cm.

[0006] The beneficial effects of the present application are as follows: Compared with the prior art, the application constructs a collagen-based repair membrane which can work stably in a wet oral environment through a continuous integration process of in-situ confined mineralization, interface chemical coupling, structure gradient forming, mild crosslinking and ion microenvironment activation. Specifically, the application first guides calcium and phosphorus to nucleate and grow inside and outside the collagen confinement with polyaspartic acid, forming an in-situ mineralized template of calcium phosphate nanocrystals compatible with the orientation of collagen, providing crystal nuclei for remineralization and matrix deposition, while avoiding the coarse deposition of inorganic phases on the outer surface of collagen, thus giving consideration to tissue adhesion and mechanical continuity from the early stage of material construction. Subsequently, bioactive glass nanoparticles (BG) modified by silanization are compounded into the mineralized collagen sol, and a stable network mainly composed of amide bonds is constructed in an alcohol-containing medium through mild crosslinking, so that the glass particles are upgraded from physical mixing to chemical bonding, reducing the risk of migration and shedding of the glass particles in a wet environment; at the same time, the active ions released are limited to diffuse in the collagen network, which is easy to form a functional layer along the thickness direction of the membrane, and cooperates with the calcium phosphate template formed in the early stage to realize the time sequence connection of mineral deposition and ion supply. Through unidirectional freezing and freeze-drying, a structure gradient with a dense oral cavity side (cavity surface) and a porous tissue defect side (bone / tooth side) is spontaneously generated in the thickness direction: the dense side provides a barrier against permeation and shear disturbance on the oral cavity side, and the porous side provides a channel for cell migration, nutrient exchange and new matrix entry on the defect side; at the same time, the phase distribution effect induced by the directional growth of ice crystals further promotes the functional division of inorganic phase distribution and pore structure, and alcohol-containing crosslinking can reduce the swelling and deformation of the material in a wet state, thereby achieving a balance between wet mechanical strength, sutureability and controllable degradation; the residual intermediates after crosslinking are removed by timely quenching and elution, improving biocompatibility. Finally, the bone / tooth side is activated by an isotonic and nearly neutral Sr-Zn solution to form a narrow multi-ion microenvironment: Sr tends to promote the mineralization maturity and interface integration of hard tissues, and Zn tends to provide mild antibacterial and matrix metalloproteinase regulation, thereby delaying the rapid degradation of collagen and stabilizing the early barrier function; this activation is superimposed on the aforementioned chemical coupling and structure gradient, so that the ion effect is concentrated on the interface and direction that needs it, avoiding indiscriminate release of the whole phase. In summary, the rapid nucleation of the calcium phosphate in-situ mineralized template, the sustained ion supply and load transfer of the silanized bioactive glass, the functional gradient formed by unidirectional freezing, the wet stability given by alcohol-containing mild crosslinking, and the localized microenvironment constructed by Sr-Zn single-sided activation, together form a multi-level coherent regeneration process. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 is a biocompatibility type I extraction test result graph of the collagen-based repair membrane material of examples 1 to 3 and comparative examples 1 to 4 of the application; Figure 2 is a biocompatibility type II extraction test result graph of the collagen-based repair membrane material of example 1 and comparative example 4 of the application; Figure 3 is a graph of the cumulative permeation amount curve test results of the dense side of the collagen-based repair membrane material of the present application Example 1 and Comparative Examples 1-4; Figure 4 is a graph of the cumulative permeation amount curve test results of the porous side of the collagen-based repair membrane material of the present application Example 1 and Comparative Examples 1-4; Figure 5 is a graph of the surface metallographic weight gain area density test results of the collagen-based repair membrane material of the present application Example 1 and Comparative Examples 1-4; Figure 6 is a graph of the Ca removal amount normalization test results of the collagen-based repair membrane material of the present application Example 1 and Comparative Examples 1-4; Figure 7 is a graph of the surface microhardness recovery rate SMHR test results of the collagen-based repair membrane material of the present application Example 1 and Comparative Examples 1-4. DETAILED DESCRIPTION

[0008] The technical solutions of the present application will be described in detail below with reference to the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments; unless otherwise specified, the raw materials involved in the present application are purchased through conventional commercial channels; the experimental methods without specific conditions are conventional methods and conventional conditions familiar to the field, or according to the conditions recommended by the instrument manufacturer.

[0009] Example 1: An oral collagen-based repair membrane material, the raw material composition of which comprises the following components: type I collagen, bioactive glass nanoparticles, γ-aminopropyl triethoxysilane, a crosslinking system, polyaspartic acid, CaCl2, K2HPO4, and a Sr-Zn solution.

[0010] 1. The crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide at a molar ratio of 4:1; 2. The Sr-Zn solution is prepared from HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2, and deionized water at a dosage ratio of 10 mmol: 15 mmol: 1 mmol: 0.1 mmol: 1000 mL, and the pH is adjusted to 7.4 with NaOH and HCl; 3. The preparation of the oral collagen-based repair membrane material comprises the following steps: S101: 8g of type I collagen is added to 1000mL of 0.02M acetic acid to prepare an acidic solution with a concentration of 0.8wt%, and then it is swelled at 4℃ for 20h, and after stirring and defoaming, it is ready for use; S102: Slowly add NaOH to the acid solution in step S101 to adjust the pH to 7, then slowly add 1 mL of 4wt% polyaspartic acid aqueous solution and stir to disperse uniformly, then add acetic acid and NaOH to control the pH to 7.2, and obtain a pre-neutralized system; S103: Under constant temperature conditions at 28°C, 25 mL of 0.5M CaCl2 solution and 25 mL of 0.3M K2HPO4 solution are added to the pre-neutralized system in step S102 at a speed of 0.5 mL / min, NaOH is used to control the pH of the system to 7.8, and after the addition is completed, the temperature is raised to 37°C for 3h of aging and stirring, and a mineralized collagen sol is obtained; S104: Mix 7g of bioactive glass nanoparticles, 120mL of ethanol and 7mL of deionized water in proportion, ultrasonic treatment for 14min to disperse uniformly, add 0.2g of γ-aminopropyl triethoxysilane, adjust the pH to 4.8 with glacial acetic acid, then stir at 450rpm at 60°C for 55min, cool and centrifuge, wash with ethanol to remove the silane, vacuum dry at 40°C for 7h, grind to 200 mesh, and obtain silanized BG nanoparticles; S105: Add 7g of silanized BG nanoparticles in step S104 to 28mL of mineralized collagen sol in step S103, pre-paste at 1000rpm for 2.5min, then add the remaining mineralized collagen sol in step S103, and segment the shear, stir at 1000rpm for 3min, and stir at 2300rpm for 2min, the whole process is controlled by ice bath, and a composite collagen system is obtained; S106: Coat the inner wall of the mold with a thin layer of polyvinyl alcohol, pre-cool the lower cold plate to -40°C, and pour the wet thickness of 1.6mm; exhaust for 10s, freeze in one direction, and program the temperature of the lower cold plate to -20°C for 8min, -40°C for 8min and -60°C, respectively, and maintain the top of the sample at 4°C throughout the process; freeze until completely hardened; then dry in stages, increase the temperature from -20°C to 0°C at a rate of 0.2°C / min, the cavity pressure is 20Pa, dry for 7h, the temperature is 23°C, the cavity pressure is 15Pa, dry for 5h, and obtain a mold; S107: Add the crosslinking system to an 80wt% ethanol solution, dissolve uniformly, and obtain a total concentration of 30mM crosslinking solution; place the 80x120mm mold in step S106 into the crosslinking solution, the liquid film ratio is 20mL:1cm 2 , crosslink at 60rpm for 4h, quench with 10mM glycine aqueous solution for 10min, grade wash, repeat 2 times with 50wt% ethanol, then wash with deionized water until the conductivity of the washing solution is <50μS / cm; absorb the surface water, lay on a polytetrafluoroethylene plate and stand for 50min, and obtain a crosslinked mold; S108: Place the porous side of the crosslinked mold in step S107 facing down in the Sr-Zn solution with a liquid film ratio of 10 mL: 1 cm 2 ; After 10 h of light shaking at 37 °C and 60 rpm, rinse twice with PBS buffer; then, dry for 5 h by increasing the temperature from -20 °C to 20 °C at a rate of 0.5 °C / min under 20 Pa 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 comprises the following components: type I collagen, bioactive glass nanoparticles, γ-aminopropyl triethoxysilane, a crosslinking 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 at a molar ratio of 5:1; 2. The Sr-Zn solution is prepared from HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2, and deionized water at a use ratio of 10 mmol: 15 mmol: 1 mmol: 0.1 mmol: 1000 mL, and the pH is adjusted to 7.4 with NaOH and HCl; 3. The preparation of the collagen-based repair membrane material for oral use comprises the following steps: S101: 10 g of type I collagen is added to 1000 mL of 0.02 M acetic acid to prepare an acidic solution with a concentration of 1 wt%, and is swelled at 4 °C for 24 h. After defoaming by stirring, it is ready for use; S102: Slowly add NaOH to the acidic solution in step S101 to adjust the pH to 7.4, then slowly add 1 mL of 4 wt% polyaspartic acid aqueous solution and stir to disperse uniformly. Add acetic acid and NaOH to control the pH to 7.1 to obtain a pre-neutralization system; S103: Under constant temperature conditions at 25 °C, 30 mL of 0.5 M CaCl2 solution and 30 mL of 0.3 M K2HPO4 solution are added to the pre-neutralization system in step S102 at a rate of 0.6 mL / min, and the pH of the system is controlled to 8 with NaOH. After the addition is completed, the temperature is increased to 37 °C and the stirring is continued for 4 h to obtain a mineralized collagen sol; S104: Mix 7 g of bioactive glass nanoparticles, 140 mL of ethanol, and 7 mL of deionized water in proportion, ultrasonic treatment for 15 min to disperse uniformly, add 0.21 g of γ-aminopropyl triethoxysilane, adjust the pH to 4.5 with glacial acetic acid, then stir at 500 rpm at 60 °C for 60 min, cool and centrifuge, resuspend and wash with ethanol to remove the silane, vacuum dry at 40 °C for 8 h, and grind to 200 mesh to obtain silanized BG nanoparticles; S105: 6.63 g of silanized BG nanoparticles in step S104 were added to 26.5 mL of the mineralized collagen sol in step S103 and pre-gelatinized by stirring at 1000 rpm for 3 min, and then added to the remaining mineralized collagen sol in step S103, and sheared in stages, stirred at 1200 rpm for 3 min, and stirred at 2500 rpm for 2 min, with ice bath temperature control throughout the shearing process, to obtain a composite collagen system; S106: The inner wall of the mold was coated with a thin layer of polyvinyl alcohol, and the lower cold plate was pre-cooled to -40°C, and the wet thickness was 2 mm; air was removed by gentle shaking for 10 s, and unidirectional freezing was performed with programmed stepwise temperature reduction, with the lower cold plate temperature set point being -20°C for 10 min, -40°C for 10 min, and -60°C, and the top of the sample being maintained at 0°C throughout, and frozen to complete hardening; then dried in stages, with the temperature being raised from -20°C to 0°C at a rate of 0.2°C / min, the cavity pressure being 20 Pa, and the drying time being 8 h at 0°C, and the cavity pressure being 15 Pa, and the drying time being 6 h at 25°C, to obtain a mold blank; S107: The crosslinking system was added to an 80 wt% ethanol solution and dissolved uniformly to obtain a crosslinking solution with a total concentration of 30 mM; an 80x120 mm mold blank in step S106 was placed in the crosslinking solution, with a liquid film ratio of 20 mL:1 cm 2 , and crosslinked at 60 rpm for 4 h, quenched with 10 mM glycine aqueous solution for 10 min, washed in stages, washed with 50 wt% ethanol twice, and then washed with deionized water until the wash water conductivity was <50 μS / cm; the surface water was absorbed and the mold blank was placed on a polytetrafluoroethylene plate and left to stand for 60 min to obtain a crosslinked mold blank; S108: The porous surface of the crosslinked mold blank in step S107 was placed in the Sr-Zn solution with a liquid film ratio of 10 mL:1 cm 2 ; after shaking at 37°C and 60 rpm for 12 h, the mold blank was quickly rinsed twice with PBS buffer; then dried at 20 Pa by raising the temperature from -20°C to 20°C at a rate of 0.5°C / min for 6 h to obtain a collagen-based repair membrane material.

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

[0014] 1. The crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide at a molar ratio of 3:1; 2. The Sr-Zn solution is prepared by HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2 and deionized water in the proportion of 10 mmol: 15 mmol: 1 mmol: 0.1 mmol: 1000 mL, and the pH is adjusted to 7.4 with NaOH and HCl; 3. The preparation of the oral collagen-based repair membrane material comprises the following steps: S101: 5g of type I collagen is added to 1000mL of 0.02M acetic acid to prepare an acidic solution with a concentration of 0.5wt%, and is swelled at 4℃ for 12h. After stirring and defoaming, it is ready for use; S102: Slowly add NaOH to the acidic solution in step S101 to adjust the pH of the system to 6.8, then slowly add 1mL of 4wt% polyaspartic acid aqueous solution and stir to disperse uniformly. Then add acetic acid and NaOH to control the pH to 7.3 to obtain a pre-neutralization system; S103: Under constant temperature conditions at 30℃, 15mL of 0.5M CaCl2 solution and 15mL of 0.3M K2HPO4 solution are added to the pre-neutralization system in step S102 at a rate of 0.2mL / min, and the pH of the system is controlled by NaOH to 7.6. After the addition is completed, the temperature is raised to 37℃ and the stirring is continued for 2h to obtain a mineralized collagen sol; S104: 8g of bioactive glass nanoparticles, 120mL of ethanol and 8mL of deionized water are mixed in proportion, ultrasonic treated for 10min to disperse uniformly, 0.16g of γ-aminopropyl triethoxysilane is added, the pH is adjusted to 5 with glacial acetic acid, then stirred at 400rpm for 45min at 60℃, cooled and centrifuged, washed with ethanol to remove silane, vacuum dried at 40℃ for 6h, ground to 200 mesh to obtain silanized BG nanoparticles; S105: 7.35g of silanized BG nanoparticles in step S104 is added to 29.4mL of mineralized collagen sol in step S103 and stirred at 1000rpm for 2min to pre-paste, then the remaining mineralized collagen sol in step S103 is added, and the shearing is divided into two stages, 800rpm for 3min and 2000rpm for 2min, and the whole shearing process is controlled by ice bath to obtain a composite collagen system; S106: The inner wall of the mold is coated with a thin layer of polyvinyl alcohol, the lower cold plate is pre-cooled to -40℃, and the wet thickness is 1mm; air is exhausted by light vibration for 10s, unidirectional freezing is carried out, and the temperature of the lower cold plate is set to -20℃ for 5min, -40℃ for 5min and -60℃, respectively, and the top of the sample is maintained at 5℃ throughout the process; freeze until completely hardened; then dry in stages, increase the temperature from -20℃ to 0℃ at a rate of 0.2℃ / min, the cavity pressure is 20Pa, dry for 6h, the temperature is 20℃, the cavity pressure is 15Pa, dry for 4h to obtain a mold; S107: The cross-linking system was added into 80wt% ethanol solution and dissolved uniformly to obtain a cross-linking solution with a total concentration of 30mM; the 80x120mm mold blank of step S106 was put into the cross-linking solution, and the liquid film ratio was 20mL:1cm 2 , and the cross-linking was performed at 60rpm for 4h, the quenching was performed with 10mM glycine aqueous solution for 10min, the washing was performed in stages, the 50wt% ethanol was repeated for 2 times, and then the deionized water was washed until the washing liquid conductivity was <50μS / cm; the surface water was absorbed, and the cross-linked mold blank was laid on a polytetrafluoroethylene plate and stood for 30min to obtain a cross-linked mold blank; S108: The porous surface of the cross-linked mold blank of step S107 was placed downward in the Sr-Zn solution, and the liquid film ratio was 10mL:1cm 2 ; after being shaken at 60rpm for 6h at 37℃, the PBS buffer was used for rapid washing for 2 times; then the temperature was raised from-20℃ to 20℃ at a speed of 0.5℃ / min under 20Pa, and the drying was performed for 4h to obtain a collagen-based repair film material.

[0015] Comparative Example 1: The raw materials and process of Comparative Example 1 were basically the same as those of Example 1, and the main difference was that the bioactive glass nanoparticles in Comparative Example 1 were not subjected to silanization treatment, that is, the addition of γ-aminopropyl triethoxysilane in step S104 was cancelled, and the remaining operations in step S104 were performed as usual; the remaining steps and parameters were consistent with those of Example 1.

[0016] Comparative Example 2: The raw materials and process of Comparative Example 2 were basically the same as those of Example 1, and the main difference was that the unidirectional freezing in Comparative Example 2 was directly replaced by rapid freezing, that is, the whole casted slurry was isothermally frozen in a-80℃ environment, and the temperature difference of the lower cold plate and the programmed stepwise cooling were not set; the remaining steps and parameters were consistent with those of Example 1.

[0017] Comparative Example 3: The raw materials and process of Comparative Example 3 were basically the same as those of Example 1, and the main difference was that the polyaspartic acid in step S102 was deleted in Comparative Example 3, and the CaCl2 / K2HPO4 was not added in step S103; instead, the same amount of nano-hydroxyapatite as the theoretical calcium phosphate mineral was directly added and mechanically dispersed uniformly in the acid solution after neutralization in step S102; the remaining steps and parameters were consistent with those of Example 1.

[0018] Comparative Example 4: The raw materials and process of Comparative Example 4 were basically the same as those of Example 1, and the main difference was that the Sr-Zn single surface activation treatment in step S108 was not performed in Comparative Example 4, but was directly completed according to the drying program after being washed twice with PBS under the same conditions; the remaining steps and parameters were consistent with those of Example 1.

[0019] Performance test: Biocompatibility test: Human keratinocyte HaCaT (corresponding to the cavity side) and human gingival fibroblast HGF (corresponding to the bone / tooth side) were selected, and the sample was the collagen-based repair membrane material (membrane) of Examples 1-3 and Comparative Examples 1-4. The extraction solution was prepared according to the membrane area / volume ratio of 6 cm 2 / mL, and extracted at 37°C and 60 rpm for 24 hours. Two types of extraction systems were set up: the culture medium extraction solution (corresponding to the complete culture medium of each cell) as the first type of extraction system; to reflect the mechanism of single-sided ion activation, another bone / tooth side directional conditioning extraction was set up. The membrane was placed in a Transwell to simulate an asymmetric release environment, and only the bone / tooth side was in contact with the lower cavity medium (the cavity side was isolated), and the conditioned medium in the lower cavity was collected after 24 hours of incubation at 37°C as the second type of extraction solution (only for Example 1 and Comparative Example 4 to reflect the difference in Sr / Zn single-sided activation). HaCaT and HGF were inoculated in 96-well plates, and after the cells reached 70%-80% confluence, the medium was discarded, and 100% extraction solution was used for treatment at 100 µL / well. The negative control was fresh culture medium, and the positive control was culture medium containing 10% DMSO. Incubation was carried out at 37°C and 5% CO2 for 48 hours. CCK-8 working solution was added according to the instructions (10 µL per well), incubated for 2 hours, and OD 450 was measured. Survival rate (%) = (OD sample-OD positive control) / (OD negative control-OD positive control) × 100%. Each group of conditions was set up with 3 parallel holes, and 3 batches of independent repeats were carried out. The results are shown in Figure 1 and Figure 2 .

[0020] Based on Figure 1 and Figure 2The collagen-based repair membrane prepared in the embodiment of the present application effectively reduces the exposure of soluble stimulants and free particles based on the synergistic effect of multiple levels, and exhibits good biocompatibility within 48 h. In Comparative Example 1, after removing γ-aminopropyl triethoxysilane (APTES), BG only exists in physical mixing, and particle migration / micro-aggregation and interface micro-separation are more likely to occur in the wet state. The trace amount of suspended inorganic debris and faster early ion burst generated during the extraction process, together with the slight pH / osmotic pressure drift, make the cell membrane surface be subjected to higher instantaneous ion intensity and hard particle contact stimulation, and compared with Example 1, the metabolic activity is obviously decreased, and the survival rate is obviously low. In Comparative Example 2, the layered structure of the cavity surface densification / bone side porosity is lost, the double-sided pores of the membrane body are open, and the specific surface area is increased, which is theoretically more likely to form a faster early extraction and diffusion path. Under the same extraction conditions, this may lead to a higher instantaneous concentration of soluble components and ions in the extraction liquid, thereby exhibiting a slight decrease in compatibility compared with Example 1. In Comparative Example 3, without polyaspartic acid induction and Ca / P drop, the nano-hydroxyapatite exists in the form of micro-aggregated particles, and the orientation matching and internal / external fiber positioning are insufficient, which makes more surface-exposed inorganic particle interfaces and local rough deposition appear in the collagen network, and mechanical fine particle contact and local Ca / P microenvironment fluctuation are easy to occur during extraction; at the same time, the in-situ mineralization template is missing, and the time sequence relay of BG ion is weakened, and the overall environment is less friendly to cells, and the survival rate is obviously decreased. In Comparative Example 4, the first type of extraction liquid is very similar to Example 1; this may be because this extraction method does not distinguish between the two sides of the membrane body, and the single-sided localized advantage of Sr / Zn is diluted under the homogenization condition of large volume and double-sided soaking, and Example 1 and Comparative Example 4 are consistent in APTES coupling + structure gradient + crosslinking elution, so HaCaT / HGF remains high survival rate, and the difference is not significant. In the second type of extraction liquid, the survival rate of HGF in Example 1 is higher than that in Comparative Example 4, while HaCaT has little difference, which may be due to the fact that only the bone / dental side is in contact with the culture medium under this condition, and Example 1 will release a low dose of Sr (with very low Zn) locally, forming a mild and near-neutral localized ion field, which supports the metabolism and spreading of fibroblast / osteogenic-related cells and gently regulates the activity of MMP; Comparative Example 4 lacks this favorable microenvironment on the bone side, and HGF is more sensitive to the difference, so the compatibility gap at 48 h is reflected; HaCaT is not sensitive to this directional condition itself (its corresponding cavity surface is not directly in contact with the extraction liquid), so the difference between the two groups is small.

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

[0022] Table 1. Test results of examples and comparative examples

[0023] Based on the result analysis of Table 1, the collagen-based repair membrane material prepared in the examples of the present application has excellent wet tensile strength and wet elongation at break, and at the same time has a lower water absorption rate, showing excellent wet mechanical properties. In Comparative Example 1, the BG is only physically mixed, and the interfacial shear strength between the particles and collagen / mineralized collagen under wet conditions decreases significantly; the load transmission path is interrupted, which further leads to a decrease in strength; at the same time, it is more prone to produce micro-agglomeration and particle detachment during hydration / stirring / formation, which becomes a brittle cracking point, and the elongation decreases. In addition, the lack of hydrophobic effect of silane-amide bond increases the interfacial water absorption channel, which leads to an increase in overall water absorption, further diluting the network and reducing the strength. Comparative Example 2 has no structure gradient of dense cavity surface and porous bone side: the isotropic pore structure increases the specific surface area and water penetration path of the membrane body, and the water absorption increases; there is no layer / fiber arrangement formed by the orientation of ice crystals, and the crack is difficult to deflect and consume energy between layers, which shows a small decrease in strength and elongation at the same time; the lack of dense cavity surface leads to more liquid penetration and swelling under wet conditions, and the network is passively loosened, which further affects the mechanics. Comparative Example 3 lacks the internal / external fiber orientation and orientation matching induced by PASP, and the nano-hydroxyapatite exists in the form of exogenous agglomerate particles, which is difficult to form a continuous composite phase with collagen. The agglomerate becomes a multi-order micro-defect under wet conditions, and stress concentration causes a decrease in strength and elongation at the same time. In addition, the exogenous particles are unevenly filled and the interface is not tightly bonded, and the capillary and interfacial water phase are more likely to enter, which increases the water absorption rate and reduces the strength in the opposite direction. In Comparative Example 4, Sr / Zn activation belongs to interface level regulation, which does not change the crosslinking degree and pore structure of the bulk phase, and the in-situ mineralization, APTES coupling, one-way freezing and alcohol-containing crosslinking are all retained, so the strength and elongation at break of the matrix are close to those of Example 1.

[0024] Barrier function verification test: The collagen-based repair membrane material pieces (effective area about 3.14 cm 2 , diameter 20 mm round piece) of Example 1 and Comparative Examples 1-4 were pre-wetted for 10 min and loaded into a double-chamber diffusion cell. Two assembly directions were set: the cavity surface (dense surface) towards the donor side; the bone / tooth side (porous surface) towards the donor side; the receptor side PBS 37°C, 60 rpm stirring; the donor side was added with a tracer dye solution (such as methylene blue 0.05 mg / mL), and 1 mL of the receptor side sample was taken at 10, 20, 30, 60, 90, 120 min and replaced with an equal volume of buffer. The absorbance at the maximum absorption wavelength of methylene blue was determined by ultraviolet-visible spectrophotometry (UV-Vis), and the cumulative permeation amount per unit membrane area Q(t) / A was calculated; then the same batch of membrane pieces were turned over and the test was repeated (new samples that have not been tested before), and the negative reference was an open channel without membrane. The cumulative permeation amount / time curve test results are shown in Figure 1.Figure 3 (dense surface) and Figure 4 (porous surface) as shown.

[0025] Based on Figure 3 and Figure 4 Result analysis, the collagen-based repair membrane material prepared in the embodiment of the application can significantly improve the tortuosity of the pore channel, reduce the effective pore size and the connectivity rate based on the cavity surface dense layer formed by one-way freezing, and inhibit the interface cracking caused by wet swelling by combining the interface chemical coupling effect of the silane coupling agent. Specifically, the permeation is reduced, and the difference between the front and back surfaces is obvious. Comparative Example 1 lacks chemical coupling, and there is a microcrack / weakly bonded area between the BG and the collagen interface, which is more prone to form a seepage bypass under a wet state; a shorter lag phase and a higher slope appear earlier; the porous surface is more permeable in the first place, and the local large pores or channels caused by the superimposed particle micro-migration / agglomeration cause the cumulative permeation to increase significantly; although the dense layer still exists, the interface integrity is poor, and the cavity surface advantage is weakened. The no-cavity surface dense-bone side porous gradient structure of Comparative Example 2 presents an approximately symmetrical connected pore, and the turning surface curve is basically coincident, and the cumulative permeation is relatively high as a whole; this may be due to the fact that isotropic rapid freezing usually forms shorter and less tortuous seepage paths, and the specific surface area and swelling are increased; although the structure is not layered, the interface microcrack and particle migration are less due to the retention of coupling and in-situ mineralization template, and the performance is still better than that of Comparative Example 1. Comparative Example 3 is prone to micro-agglomeration / surface exposure due to the external addition of nano-hydroxyapatite, and micro-defects and rough channels are formed in the process of crosslinking / drying / humidification; the continuity of the dense layer is discounted, and the slope and cumulative permeation increase; the uneven distribution of the external added particles and the non-adhesion of the interface make the probability of large pores and semi-penetrating pores higher, and the permeation increases significantly; the one-way freezing is retained, and there is still a difference between the two surfaces, but the difference is reduced. Comparative Example 4 has a basic structure similar to that of Example 1, and its performance is also close to that of Example 1; this may be because the short-term liquid permeation is mainly determined by the pore structure and the interface integrity; the absence of Sr / Zn does not significantly change these physical channels.

[0026] Mineralization test: collagen-based repair membrane material film pieces (effective area about 3.14 cm 2 , diameter 20 mm round piece) of Example 1 and Comparative Examples 1-4 were activated by PBS for 10 min, and the bone / tooth side (porous surface) was exposed to the clamp to contact SBF (37°C, pH7.4) with the cavity surface isolated from the solution; the volume of SBF was prepared according to 10 mL / cm 2 , and the film was shaken at 60 rpm; the sampling time points were set to 6, 12, 24 and 48 h, and each film was replaced with SBF to avoid cross-influence; the dry constant weight of the film was recorded before the test; the PBS was taken out and washed twice, and then vacuum freeze-dried to constant weight to record the mass m t ; the SBF sample solutions at each time point were saved synchronously, and the Ca 2 +(o-CPC method) Colorimetric determination of Ca concentration in initial and final SBF, surface mineralization weight gain surface density (mg / cm 2 )=(dry constant weight of membrane after test-initial dry constant weight of membrane) / effective area of membrane; Ca removal amount normalization (mg / cm 2 )=(initial Ca concentration in SBF-end point Ca concentration in SBF) x SBF volume / effective area of membrane, test results are shown in Figure 5 and Figure 6 .

[0027] Remineralization test: polished tooth slice (5x5x2mm) was prepared, artificial lesion (enamel 24h) was obtained by acid etching with lactic acid buffer (pH4.5, 37°C), after washing with water and drying, initial micro Vickers hardness was measured (load 200g, 10s, 5 points average); the same tooth slice reserved healthy self-control window as healthy control; the membrane bone / tooth side was attached to the tooth slice surface, after fixed with elastic ring, it was placed in the remineralization liquid (PBS buffer, 2mM Ca 2+ , 1.2mM PO4 2- , pH7.2, 37°C, 10mL / slice) with 60rpm light shaking, sampling at 1, 3 and 7 days, daily liquid change during the period, after light washing, natural drying, re-measuring hardness under the original load, calculating surface micro hardness recovery rate SMHR (%)=(re-measured hardness-initial hardness) / (healthy hardness-initial hardness) x 100%, results are shown in Figure 7 .

[0028] Based on Figure 5 , Figure 6 and Figure 7The collagen-based repair membrane material prepared in the embodiment of the present application shows excellent deposition efficiency, and a mild Sr-rich ion field is formed on the bone side surface, which can promote the growth and maturation of initial crystal nucleus; and the deposition on the interface of the tooth piece is made more dense and better adhered during remineralization. In Comparative Example 1, the physical mixing of BG leads to micro-agglomeration in a wet state and interface debonding, forming a weak substrate and micro-seam, resulting in incoherent deposition, low weight gain; the ion non-local burst release causes excessively high local supersaturation, low calcium utilization efficiency, and low fixed amount; and finally, the mineralized layer is loose and contains weak layers with particles, and the hardness recovery is slow. In Comparative Example 2, the lack of a dense partition structure on the bone side porous / cavity surface leads to a thin boundary layer on the bone side, enhanced convection, and difficulty in maintaining local supersaturation, resulting in low deposition efficiency and low calcium retention rate; the ions repeatedly migrate in the system but are not fixed enough, the bone side guidance is weak, the interface deposition is discontinuous, and the formation of the dense layer adhering to the tooth piece surface is delayed. In Comparative Example 3, the nano-hydroxyapatite exists in the form of exogenous micro-agglomerates, and lacks fiber internal positioning, resulting in isolated heterogeneous nucleation points, fragmented deposition layer and insufficient weight gain; the effective nucleation area is low and there is no local ion supply, and the calcium fixation efficiency is poor; finally, a porous mineral layer containing inclusions and weak adhesion is formed on the interface of the tooth piece, and the hardness recovery is slow. In Comparative Example 4, the interface activation of Sr is missing, and the early crystal nucleation and densification is blocked, resulting in low deposition amount and calcium fixation rate within 24 hours; the perfect bulk phase structure gradually approaches the embodiment 1 in the SBF under the unlimited calcium source; the tooth piece remineralization depends on early interface integration, and the single-sided activation of Sr is beneficial to early integration and crystal maturation, and the performance is still slightly lower.

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

Claims

1. A collagen-based repair membrane material for oral use, characterized by, The raw material composition comprises the following components: collagen type I, bioactive glass nanoparticles, gamma-aminopropyl triethoxysilane, a crosslinking system, polyaspartic acid, CaCl2, K2HPO4 and a Sr-Zn solution.

2. The collagen-based repair membrane material for oral use according to claim 1, characterized in that, The crosslinking system is a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide at a molar ratio of 3-5:

1.

3. The collagen-based repair membrane material for oral use according to claim 2, characterized in that, The Sr-Zn solution is prepared from HEPES buffer, NaCl, SrCl2·6H2O, ZnCl2 and deionized water at a dosage ratio of 10 mmol:15 mmol:1 mmol:0.1 mmol:1000 mL, and the pH is adjusted to 7.4 with NaOH and HCl.

4. A method for producing the collagen-based repair membrane material for oral cavity according to any one of claims 1 to 3, characterized by, Specifically comprising the following steps: S101: Collagen type I is added to acetic acid to prepare an acidic solution, which is left to swell, and then stirred to remove bubbles for standby; S102: In the acidic solution in step S101, the pH of the system is adjusted by slowly adding NaOH, then the polyaspartic acid aqueous solution is slowly added and stirred to disperse uniformly, then acetic acid and NaOH are added to control the pH, and a pre-neutralization system is obtained; S103: Under constant temperature conditions, the pre-neutralization system in step S102 is slowly added with CaCl2 solution and K2HPO4 solution at the same time, the pH of the system is controlled by NaOH, and after the addition is completed, the temperature is raised for curing and stirring to obtain a mineralized collagen sol; S104: Bioactive glass nanoparticles, ethanol and deionized water are mixed in proportion, ultrasonic treatment is performed to disperse uniformly, gamma-aminopropyl triethoxysilane is added, glacial acetic acid is used to adjust the pH, then the temperature is raised for stirring treatment, cooling and centrifugation, ethanol is used for resuspension and washing to remove silane, vacuum drying, grinding, to obtain silanized BG nanoparticles; S105: The silanized BG nanoparticles in step S104 are added to part of the mineralized collagen sol in step S103 for pre-gelatinization, and then the remaining mineralized collagen sol in step S103 is added, and the mixture is cut into sections to obtain a composite collagen system; S106: A thin layer of polyvinyl alcohol is coated on the inner wall of the mold, the lower cold plate is pre-cooled, casting is performed, the mold is gently shaken to exhaust air, unidirectional freezing is performed, and then the mold is dried in stages to obtain a mold blank; S107: The crosslinking system is added to an 80wt% ethanol solution to dissolve uniformly to obtain a crosslinking solution; the mold blank in step S106 is placed into the crosslinking solution, gently shaken for crosslinking, quenched with glycine aqueous solution, washed in stages, surface water is absorbed, and then the crosslinked mold blank is obtained by laying on a polytetrafluoroethylene plate for standing; S108: The porous surface of the crosslinked mold blank in step S107 is placed downward in the Sr-Zn solution, gently shaken, and then quickly rinsed with PBS buffer; and then dried to obtain a collagen-based repair membrane material.

5. A method for producing the collagen-based repair membrane material for oral cavity according to claim 4, characterized by, In step S102, the addition amount of the polyaspartic acid aqueous solution is 0.1% of the volume of the acidic solution.

6. A method for producing the collagen-based repair membrane material for oral cavity according to claim 4, characterized by, In step S103, the volume ratio of the CaCl2 solution and the K2HPO4 solution is 1:1; The total addition volume is 3-6% of the volume of the acidic solution.

7. A method for producing the collagen-based repair membrane material for oral cavity according to claim 4, characterized by, In step S104, the ratio of the amount of the bioactive glass nanoparticles, ethanol and deionized water is 1g:15-20mL:1mL; the amount of the added γ-aminopropyl triethoxysilane is 2-3% of the mass of the bioactive glass nanoparticles; and the ratio of the amount of the silanized BG nanoparticles and the total mineralized collagen sol is 1g:140-160mL.

8. A method for producing the collagen-based repair membrane material for oral cavity according to claim 4, characterized by, In step S105, the segmental shearing parameters are as follows: 800-1200rpm stirring for 3min, 2000-2500rpm stirring for 2min, and ice bath temperature control throughout the shearing process.

9. A method for producing the collagen-based repair membrane material for oral cavity according to claim 4, characterized by, In step S106, the one-way freezing is specifically operated as follows: programmed stepwise temperature reduction, the lower cold plate temperature set point is sequentially-20℃ constant temperature for 5-10min, -40℃ constant temperature for 5-10min and -60℃, the top of the sample is maintained at 0-5℃ throughout the freezing process, and the freezing is performed until complete hardening.

10. A method for producing the collagen-based repair membrane material for oral cavity according to claim 4, characterized by, In step S107, the hierarchical washing parameters are as follows: 50wt% ethanol repeated washing for 2 times, and then deionized water washing until the washing liquid conductivity is <50μS / cm.

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