Preparation method and application of gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane

Abstract

The invention discloses a preparation method and application of a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, and the preparation method comprises the following steps: (1) respectively dissolving gelatin, sodium alginate and mesoporous bioactive glass in pure water, mixing, and degassing in vacuum; and (2) carrying out membrane casting molding on the degassed homogeneous mixed solution, and sequentially carrying out condensation, ion and chemical crosslinking, cleaning and freeze drying to obtain the regenerated membrane. The prepared regeneration membrane has a compact surface layer and can effectively inhibit penetration of fibrous connective tissues; meanwhile, proper roughness is achieved, and cell adhesion and bone surface osteogenesis are facilitated. The membrane has good mechanical performance, biocompatibility, barrier performance, controllable degradation rate and excellent osteogenesis performance, and can effectively promote bone regeneration and defect repair while obstructing soft tissue growth. In addition, the medicine is wide in raw material source, simple in preparation process, mild in condition and low in cost, and has a remarkable clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of oral medical materials technology, specifically relating to a method for preparing and applying a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane. Background Technology

[0002] Guided bone regeneration (GBR) is a surgical technique that uses physical barriers to regulate cell migration behavior and promote targeted bone repair. Its core principle is based on the difference in migration rates of different cell types in the defect area: fibroblasts and epithelial cells migrate faster, while osteoblasts migrate slower. By implanting a barrier membrane in the bone defect area, soft tissue cells can be effectively prevented from invading, providing osteoblasts with preferential space for proliferation, thereby achieving bone regeneration and repair.

[0003] Currently, GBR membranes can be divided into two categories: non-absorbable membranes and absorbable membranes. While non-absorbable membranes (such as polytetrafluoroethylene and titanium-based membranes) possess good mechanical strength and space-holding capacity, their materials are typically too rigid and lack flexibility, making it difficult to fit tightly to bone defects. They also require secondary surgery for removal, which can easily lead to complications such as soft tissue dehiscence and infection, increasing patient suffering and limiting their clinical application. Absorbable membranes are composed of synthetic polymers (such as polycaprolactone PCL and polylactic acid), natural polymers (such as collagen, chitosan, and silk fibroin), and biodegradable metals (such as magnesium and zinc). Compared to non-absorbable membranes, absorbable membranes do not require secondary surgery, are easy to operate, and have good biocompatibility. However, existing materials, such as the Bio-Gide membrane, currently the gold standard for GBR in clinical practice, suffer from drawbacks such as high cost, complex manufacturing processes, insufficient mechanical strength, excessively rapid degradation rates (degradation time of 2-4 weeks), poor osteogenic properties, and the risk of disease transmission. These drawbacks make it difficult to stably maintain the space required for bone regeneration and meet large-scale clinical needs. Therefore, developing novel composite membrane materials that combine excellent mechanical strength, biocompatibility, controllable degradation, and active osteogenic induction capabilities has become a research hotspot in the field of GBR membranes.

[0004] Currently, membrane fabrication processes mainly include solution casting, electrospinning, and 3D printing. Solution casting is a simple process suitable for large-scale production, where a polymer solution is poured into a mold, and the target product is obtained after the solvent evaporates. In contrast, while electrospinning and 3D printing are widely used in membrane fabrication, they have significant disadvantages. For example, patent CN119681268A discloses a method for preparing a 3D-printed porous biodegradable metal GBR membrane, which promotes bone repair through personalized design and porous structure; patent CN119185645A discloses a biodegradable zinc-based GBR membrane and its preparation method and application, which uses 3D printing technology to prepare a biodegradable zinc-based GBR membrane, involving complex mixing, extrusion, and printing steps; patent CN115518206A discloses a self-mineralizing GBR membrane and its preparation method, which also prepares a self-mineralizing GBR membrane by 3D printing a metal scaffold and combining it with a collagen membrane. It is evident that current 3D printing preparation of GBR membranes typically relies on special skeleton components or complex processes, resulting in cumbersome preparation and high costs, which hinders the promotion of absorbable GBR membranes.

[0005] Meanwhile, in the field of bioresorbable barrier membranes, research has largely focused on electrospinning technology, concentrating on optimizing membrane material properties, such as mechanical properties, osteogenic properties, and antibacterial properties, as exemplified by patents CN111494720A and CN109224134A. However, electrospinning requires tens of hours to produce a single membrane, has low yield, and involves expensive and bulky equipment, consuming significant human and material resources, making it difficult to meet the demands of large-scale, rapid production.

[0006] Based on this, the present invention proposes a simple and low-cost preparation method to solve the above problems. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention utilizes a combination of materials to form a biodegradable composite membrane, leveraging the advantages of each material while overcoming the limitations of polymer-based membranes in certain applications. The purpose of this invention is to provide a gelatin-sodium alginate mesoporous bioactive glass composite guided bone tissue regeneration membrane, its preparation method, and its applications. This regeneration membrane possesses advantages such as good mechanical properties, excellent barrier function, high bioactivity, and controllable degradation rate.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, as follows:

[0010] S1. Slurry preparation:

[0011] Sodium alginate was dissolved in pure water and mechanically stirred until completely dissolved to obtain a sodium alginate solution.

[0012] Gelatin solution is prepared by dissolving gelatin in pure water;

[0013] To prepare mesoporous bioactive glass, the mesoporous bioactive glass was dissolved in pure water to form a dispersion solution.

[0014] S2, Degassing: Sodium alginate solution, gelatin solution and mesoporous bioactive glass dispersion solution are mixed evenly to obtain a mixture, and then the mixture is subjected to vacuum degassing treatment;

[0015] S3. Mold setting: Add the degassed mixture into the mold, and after it is evenly cast, place it in a 4℃ refrigerator for 2 hours.

[0016] S4. Crosslinking: Add appropriate amounts of metal chloride and crosslinking agent solution to the mixture in the mold in sequence to carry out ionic and chemical crosslinking to obtain a composite membrane. After the reaction is completed, remove the composite membrane from the mold.

[0017] S5. Cleaning and freeze-drying: After rinsing the composite membrane, soak it in pure water for 12-24 hours to remove the residual crosslinking agent in the composite membrane; finally, freeze it at -80℃ for 2 hours, and then put it into a freeze dryer and freeze-dry it at -40℃ for 24 hours to obtain the bone tissue regeneration guiding membrane.

[0018] Preferably, in step S1, the mesoporous bioactive glass is prepared as follows:

[0019] Dodecylamine was added to a mixed solution of ethanol and ultrapure deionized water, and heated and stirred in a water bath at 40°C to obtain the first mixed solution.

[0020] Add hexadecyltrimethylammonium bromide to the first mixed solution, maintain stirring, and add tetraethyl orthosilicate, triethyl phosphate and calcium nitrate tetrahydrate solution dropwise every 30 min, and continue stirring to obtain the second mixed solution;

[0021] The second mixed solution is allowed to age until a white precipitate forms.

[0022] The collected sediment was washed and then dried.

[0023] The dried precipitate was sintered at 650–700℃ to obtain mesoporous bioactive glass particles.

[0024] More preferably, the drying temperature is 60–75°C.

[0025] Preferably, in step S1, the sodium alginate solution has a mass concentration of 5% and the gelatin solution has a mass concentration of 10%.

[0026] Preferably, in step S2, the mass ratio of sodium alginate to gelatin in the mixture is 1:1, and the mass of mesoporous bioactive glass accounts for ≤0.3% of the total mass of sodium alginate and gelatin.

[0027] Preferably, the mesoporous bioactive glass particles have a particle size of 0.5–0.6 μm and a pore size of 2–10 nm.

[0028] Preferably, in step S4, the metal chloride includes one or more of calcium chloride, copper chloride, strontium chloride, and barium chloride, and the crosslinking agent includes one or more of glutaraldehyde, genipin, glutamin transferase, carbodiimide / succinimide, and oxidized amylose.

[0029] In a second aspect, the present invention provides a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, comprising gelatin, sodium alginate and mesoporous bioactive glass, wherein the mass of the mesoporous bioactive glass accounts for 5 to 10% of the total mass of gelatin and sodium alginate.

[0030] Preferably, the mass ratio of gelatin to sodium alginate is (1-1.5):(1-1.5).

[0031] Preferably, the thickness of the regenerated membrane is 60–100 μm.

[0032] In a third aspect, this invention proposes the application of a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, which is used in dental implantation and maxillofacial surgery. Research and exploration have shown that when the mass percentages of natural polymer materials and mesoporous bioactive glass in the mixed solution described in this application are controlled within the aforementioned range, a relatively ideal film-forming effect can be achieved, with good membrane mechanical properties, barrier function, and a suitable degradation rate.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention comprises gelatin, sodium alginate, and mesoporous bioactive glass. Gelatin and sodium alginate possess excellent biocompatibility and biodegradability, facilitating cell adhesion and proliferation. The mesoporous bioactive glass has the function of locally releasing calcium and phosphorus ions, which can activate osteogenic-related signaling pathways, promote osteoblast differentiation, and enhance osteogenic induction. The preparation method provided by this invention allows for the preparation of a guided bone tissue regeneration membrane with a dense surface, thereby inhibiting fibrous connective tissue penetration. Appropriate roughness facilitates cell adhesion, thus promoting bone surface osteoogenesis. The regeneration membrane prepared using this method possesses good mechanical properties, biocompatibility, barrier properties, a suitable degradation rate, and superior osteogenic properties. It can better promote bone tissue regeneration and bone defect repair while blocking fibrous tissue ingrowth. Simultaneously, the preparation time of the regeneration membrane is significantly shortened, and the operation method is simple, greatly saving production costs and enabling large-scale production of guided bone tissue regeneration membranes, showing potential clinical application prospects. Attached Figure Description

[0035] Figures 1-4 The figures shown are morphological characterization images of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3, wherein... Figure 1 In the image, 'a' represents SEM. Figure 2 In the image, b and c are energy spectrum scans, respectively. Figure 3 In the middle, d represents EDS analysis. Figure 4 The image in the middle, e, is a 3D atomic force microscope image. Figures 2-4 Middle order and Figure 1 Same order;

[0036] Figure 5 The figures show the mechanical property test results of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3, where a is a tensile process diagram, and be and b are respectively: b stress-strain curves of different samples; c tensile strength; d elastic modulus; e elongation at break;

[0037] Figure 6 The results of the in vitro degradation test of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3 are shown. In the figure, a is the sample mass change curve from week 0 to week 9, b is the mass change of each group of composite membranes at week 9, and c is the actual image of each membrane degrading in SBF at week 10.

[0038] Figure 7 The figures show the apparent hydrophilicity test results of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3, where a is the average contact angle measurement data and b is a photograph of a water droplet in contact with the membrane material for 5 seconds.

[0039] Figure 8The results shown are the CCK-8 values ​​of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3 at 1, 3, and 5 days.

[0040] Figure 9 The results of live and dead staining of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3 are shown on the 3rd day.

[0041] Figure 10 The figures show the cell adhesion and quantitative results of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3 on day 3, where a and c represent cell adhesion, and b and d represent the quantitative results of cell adhesion.

[0042] Figure 11 The figures show the barrier performance test results of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3. In the figure, b is a schematic diagram of the barrier function evaluation by cell migration experiment; a is the corresponding optical image of the lower chamber of the culture plate of the control group and various membranes after 4 days of culture; c is the cell counting results of the lower chamber of all groups; d is the representative fluorescence image of L929 cells after 4 days of culture on various membrane surfaces.

[0043] Figure 12 The image shown is an Alizarin Red (ARS) staining image of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3 on day 21; (Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) Detailed Implementation

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention and some comparative examples. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] This invention proposes a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane. The main components include gelatin, sodium alginate, and mesoporous bioactive glass, wherein the mesoporous bioactive glass accounts for 5-10% of the total mass of gelatin and sodium alginate. Gelatin and sodium alginate exhibit good biocompatibility and biodegradability, which are beneficial for cell adhesion and proliferation. The mesoporous bioactive glass has the effect of locally releasing calcium and phosphorus ions, promoting bone tissue regeneration.

[0051] Preferably, the thickness of the regeneration membrane is 60–100 μm to ensure the mechanical strength of the membrane to meet the needs of bone tissue regeneration.

[0052] Preferably, the mass ratio of gelatin to sodium alginate is (1-1.5):(1-1.5), and more preferably, the mass ratio of gelatin to sodium alginate is 1:1. A 1:1 ratio of gelatin to sodium alginate exhibits excellent emulsion stability, as they form an irreversible gel. The mesoporous bioactive glass contained within the bone tissue regeneration membrane effectively improves the hydrophilicity and mechanical strength of the polymer-based membrane material, and significantly enhances the tensile strength of the bone tissue regeneration membrane.

[0053] The method for preparing the mesoporous bioactive glass of the present invention is as follows:

[0054] Dodecylamine was added to a mixed solution of ethanol and ultrapure deionized water, and heated and stirred in a water bath at 40°C for 10 minutes to obtain the first mixed solution.

[0055] Add hexadecyltrimethylammonium bromide to the first mixed solution and stir for 30 min. While maintaining the stirring conditions, add tetraethyl orthosilicate, triethyl phosphate and calcium nitrate tetrahydrate solution dropwise every 30 min. Continue stirring for 6 h to obtain the second mixed solution.

[0056] The second mixed solution should be aged for ≥3 hours to allow a white precipitate to form.

[0057] The collected precipitate was washed with deionized water and ethanol and then dried in an oven at 60-75°C for, for example, 24 hours.

[0058] The dried precipitate is sintered at 650–700°C to remove organic components and water, yielding mesoporous bioactive glass particles. Preferably, the mesoporous bioactive glass particles have a particle size of 0.5–0.6 μm and a pore size of 2–10 nm.

[0059] The mesoporous bioactive glass of this invention is dispersed in a gelatin-sodium alginate system, effectively solving the problem of insufficient mechanical strength in membrane materials. Membrane materials containing mesoporous bioactive glass exhibit good biocompatibility and bioactivity, promoting osteoblast proliferation and differentiation. The gelatin-sodium alginate and mesoporous bioactive glass membrane material, prepared in a 1:1 ratio, forms a membrane material whose degradation rate matches the regeneration rate of oral bone tissue. The degradation time of the membrane material is no less than 10 weeks, allowing it to remain in the patient's oral cavity for a relatively long time. Its barrier properties effectively prevent soft tissue from invading the bone defect area.

[0060] This invention also proposes a method for preparing a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, as follows:

[0061] S1. Slurry preparation:

[0062] Sodium alginate was dissolved in pure water and mechanically stirred until completely dissolved to obtain a sodium alginate solution.

[0063] Gelatin solution is prepared by dissolving gelatin in pure water;

[0064] To prepare mesoporous bioactive glass, the mesoporous bioactive glass was dissolved in pure water to form a dispersion solution.

[0065] S2, Degassing: Sodium alginate solution, gelatin solution and mesoporous bioactive glass dispersion solution are mixed evenly to obtain a mixture, and then the mixture is subjected to vacuum degassing treatment;

[0066] S3. Mold shaping: Add the degassed mixture into the polytetrafluoroethylene sheet mold, and after the casting is uniform, place it in 4℃ for 2 hours.

[0067] S4. Crosslinking: Add appropriate amounts of metal chloride and crosslinking agent solution to the mold in sequence to carry out ionic and chemical crosslinking to obtain a composite membrane. After the reaction is completed, remove the composite membrane from the mold.

[0068] S5. Cleaning and freeze-drying: First, rinse the composite membrane with running pure water for 10 seconds, repeat three times, then soak it in pure water for 12-24 hours to remove the residual crosslinking agent in the composite membrane; finally, freeze it at -80℃ for 2 hours, and then put it into a freeze dryer and freeze-dry it at -40℃ for 24 hours to obtain the bone tissue regeneration guiding membrane.

[0069] The freeze-dried guiding bone tissue regeneration membrane is processed into guiding bone tissue regeneration membrane products through cutting, packaging, and sterilization.

[0070] Preferably, in step S1, the sodium alginate solution has a mass concentration of 5% and the gelatin solution has a mass concentration of 10%.

[0071] Preferably, in step S2, the mass ratio of sodium alginate to gelatin in the mixture is 1:1, and the mass of mesoporous bioactive glass accounts for ≤0.3% of the total mass of sodium alginate and gelatin.

[0072] In step S3, the material is refrigerated at 4°C for 2 hours to promote gel formation, ensuring that the material structure remains unchanged during subsequent cross-linking. Otherwise, the addition of glutaraldehyde and calcium chloride solution in step S4 would cause pitting, affecting the material structure. After refrigeration, the material in the mold initially solidifies and takes shape.

[0073] Preferably, in step S4, the metal chloride includes one or more of calcium chloride, copper chloride, strontium chloride, and barium chloride, and the crosslinking agent includes one or more of glutaraldehyde, genipin, glutamin transferase, carbodiimide / succinimide, and oxidized amylose.

[0074] In step S4, specifically, a calcium chloride solution with a mass concentration of 10% is added to the mold, immersing the material in the calcium chloride solution for 10 minutes. Then, excess calcium chloride is removed by rinsing with distilled water. Finally, a glutaraldehyde solution with a mass concentration of 1.0% is added, and the reaction is carried out for 30 minutes to obtain the composite membrane.

[0075] In step S5, the first freezing at -80°C for 2 hours is to freeze the composite film in a refrigerator until it meets the requirements for being placed in a freeze dryer. The subsequent second freezing is to freeze-dry the composite film in the freeze dryer.

[0076] The technical solution of the present invention will be described in detail below with specific embodiments.

[0077] Example 1

[0078] In this embodiment, the mass concentration of mesoporous bioactive glass (mBG) in the mixture is 0.25%, the mass concentration of sodium alginate (SA) in the sodium alginate solution is 2.5%, and the mass concentration of gelatin (Gel) in the gelatin solution is 2.5%. The specific preparation is as follows:

[0079] Sodium alginate was dissolved in pure water at 60°C and mechanically stirred until completely dissolved to obtain a sodium alginate solution. Gelatin was dissolved in pure water at 60°C to obtain a gelatin solution. Mesoporous bioactive glass was dissolved in pure water at 60°C to prepare a homogeneous solution.

[0080] The sodium alginate solution, gelatin solution, and mesoporous bioactive glass solution were mixed evenly to obtain a mixture. In the mixture, the mass of the mesoporous bioactive glass accounted for 5% of the total mass of sodium alginate and gelatin. Then, the mixture was subjected to vacuum degassing treatment.

[0081] Add the degassed mixture into a polytetrafluoroethylene sheet mold, and after it is evenly cast, refrigerate it at 4°C for 2 hours.

[0082] A suitable amount of calcium chloride and glutaraldehyde solution were added sequentially to the mixture in the mold to carry out ionic and chemical cross-linking to obtain a composite membrane. After the reaction was completed, the composite membrane was removed from the mold.

[0083] First, rinse the composite membrane with running pure water for 10 seconds, repeat three times, then soak it in pure water for 15 hours to remove the residual crosslinking agent in the composite membrane; finally, freeze it at -80℃ for 2 hours, and then put it into a freeze dryer and freeze dry it at -40℃ for 24 hours to obtain the bone tissue regeneration guiding membrane (denoted as mBG-0.25).

[0084] Example 2

[0085] Compared with Example 1, the mass concentration of mesoporous bioactive glass in the mixture of this example is 0.5%, and the mass of mesoporous bioactive glass accounts for 10% of the total mass of sodium alginate and gelatin. The remaining steps are the same as in Example 1 to obtain a bone tissue regeneration guiding membrane (denoted as mBG-0.5).

[0086] Comparative Example 1

[0087] Compared with Example 1, no mesoporous bioactive glass was added in this comparative example, and the remaining steps were the same as in Example 1 to obtain the membrane material (denoted as mBG-0).

[0088] Comparative Example 2

[0089] Compared with Example 1, the mass concentration of mesoporous bioactive glass in this comparative example mixture is 1.0%, and the mass of mesoporous bioactive glass accounts for 20% of the total mass of sodium alginate and gelatin. The remaining steps are the same as in Example 1, and the membrane material (denoted as mBG-1) is obtained.

[0090] Comparative Example 3

[0091] Compared with Example 1, the mass concentration of the mesoporous bioactive glass in this comparative example is 1.5%, and the mass of the mesoporous bioactive glass accounts for 30% of the total mass of sodium alginate and gelatin. The remaining steps are the same as in Example 1, and the membrane material (denoted as mBG-1.5) is obtained.

[0092] Comparative Example 4

[0093] Compared with Example 1, the mass concentration of mesoporous bioactive glass in this comparative example mixture is 2.0%, and the mass of mesoporous bioactive glass accounts for 40% of the total mass of sodium alginate and gelatin. The remaining steps are the same as in Example 1, and the membrane material (denoted as mBG-2) is obtained.

[0094] The composite membrane materials obtained in Examples 1-2 and Comparative Examples 1-3 were subjected to physicochemical and biological performance tests, as follows:

[0095] I. Comparative Analysis of Morphological Structure

[0096] Examples 1-2 and Comparative Examples 1-3 employed solution casting technology, i.e., the process of this invention, to prepare composite films with and without mBG doping, achieving standardized production. Scanning electron microscopy (SEM) was used to evaluate the surface morphology of the freeze-dried composite films; silicon content was determined by SEM-EDS to verify the amount of mBG added, and surface elemental distribution mapping analysis was performed; finally, atomic force microscopy (AFM) was used to detect the film surface roughness.

[0097] Electron micrographs, surface elemental analysis, and roughness results of the membrane material are as follows: Figures 1-4 As shown in the figure. SEM results show that at low concentrations (0.25%-0.5%), mBG is uniformly dispersed in the polymer matrix with a smooth and dense surface; at high concentrations (1% and above), mBG particles show obvious agglomeration and clumping. EDS detected peaks for Si, Ca, and P elements. The intensity of the corresponding characteristic element Si significantly increased with increasing mBG content, indicating successful introduction of mBG. Mapping results further confirm this. AFM results show that different mBG contents result in different surface roughness of the material. When the mBG content is 1.0% and above, the surface roughness is more pronounced. The membrane material prepared by this invention has a moderate surface roughness; when mBG is excessive, the membrane material surface becomes too rough.

[0098] II. Comprehensive Performance Comparison Analysis

[0099] Mechanical property testing: The films obtained in Examples 1-2 and Comparative Examples 1-3 were cut into strips of 6x1.5cm each, and their tensile properties were tested using a universal tensile testing machine. The tensile speed was set to 5mm / min. The ultimate tensile strength, elongation at break, and Young's modulus were calculated based on the stress-strain curves. Figure 5 As shown, the ultimate tensile strength and Young's modulus exhibit similar trends: with increasing mBG content, the ultimate tensile strength initially increases and then gradually decreases. Specifically, the UTS of mBG-0.25 (0.40±0.10 MPa) and mBG-0.5 (0.35±0.10 MPa) is greater than that of mBG-0 (0.20±0.06 MPa), while there is no significant difference between mBG-1 (0.30±0.04 MPa), mBG-1.5 (0.26±0.03), and mBG-0. Similarly, the addition of mBG (below 1.5 wt%) does not affect the elongation at break of the membrane; except for the mBG-1.5 group, there are no significant differences among mBG-0.25, mBG-0.5, mBG-1, and mBG-0. Regarding Young's modulus, only the mBG-0.25 (1.47±0.06MPa) and mBG-0.5 (1.23±0.24MPa) films have significantly higher modulus than the mBG-0 film (0.55±0.10MPa). This shows that neither adding nor adding excessive amounts of mesoporous bioactive glass is conducive to improving the mechanical strength of the obtained composite film. The membrane material prepared by adding an appropriate amount of mesoporous bioactive glass in this invention has better mechanical properties.

[0100] Degradation performance evaluation: The composite membranes from Examples 1-2 and Comparative Examples 1-3 were immersed in SBF to evaluate their degradation behavior. The long-term stability of different composite membranes was assessed through degradation tests, such as… Figure 6 As shown, the dissolution rate of the mBG group was slightly faster than that of the control group. The addition of mBG accelerated the degradation process of the composite membrane, and this process became even faster with increasing mBG content. The lower degradation rate of the mBG-0 composite membrane may be due to the fact that the degradation of SA and Gel is mainly caused by hydrolysis, while the cross-linking reaction of glutaraldehyde can prevent the hydrolysis of SA and Gel, thereby further stabilizing the composite membrane and slowing down the degradation behavior. By day 70, the mass of mBG-1 and mBG-1.5 had decreased by nearly half of their initial weight, and the membrane morphology integrity was compromised due to the gradual release of mBG. Figure 6 As shown, the membrane materials of Examples 1-2 remained stable after immersion in simulated body fluid (SBF) for 80 days.

[0101] Surface hydrophilicity testing: The membrane materials prepared in Examples 1-2 and Comparative Examples 1-3 were characterized by water contact angle (WCA) analysis at room temperature using a water contact angle meter. Figure 7As shown, the introduction of mBG can significantly reduce the water contact angle of the gelatin-sodium alginate composite membrane. The higher the mBG content, the smaller the water contact angle. This is mainly because mBG is a hydrophilic material, and as the concentration increases, it increases the roughness of the composite membrane surface, making the membrane surface more hydrophilic.

[0102] III. Comparative Analysis of In Vitro Biocompatibility, Barrier Performance, and Osteogenesis Promotion Capacity

[0103] Cell compatibility: Cell compatibility was tested on the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3. Rat bone marrow mesenchymal stem cells (rBMSCs) and L929 fibroblasts were cultured on the respective membranes for 1, 3, and 5 days, and cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) method. Furthermore, considering that the composite membranes need to directly contact epithelial and connective tissues in clinical applications, the effect of each membrane group on the viability of L929 fibroblasts was further evaluated using a live / dead cell staining experiment. Simultaneously, the attachment morphology of rBMSCs and L929 cells on the surface of each membrane group was observed using scanning electron microscopy (SEM), and the amount of cell adhesion was quantitatively analyzed using a cell counting chamber.

[0104] like Figure 8 As shown, compared with the control (TCP) group, the CCK-8 assay results further demonstrated that cells in all groups continued to proliferate over time, exhibiting good proliferative activity on the composite membrane, indicating that the material supports cell growth. It was also found that the viability of L929 cells increased significantly over time compared to rBMSCs, because fibroblasts generally grow faster than osteoblast-like cells. Live / dead cell staining 3 days after inoculation confirmed this (see...). Figure 9 In Examples 1-2 and Comparative Examples 1-3, L929 cells on the membrane material surface exhibited high-density green fluorescence (live cells), while only sporadic red fluorescence (dead cells) was observed in random fields of view, further indicating that the composite membranes of different groups did not cause adverse damage to the cells.

[0105] Cell adhesion experiments confirmed (see...) Figure 10 rBMSCs and L929 fibroblasts performed better on rough surfaces, exhibiting superior adhesion and proliferation capabilities. SEM analysis confirmed that L929 cells displayed excellent cell attachment morphology on various membrane groups, indicating that the materials promoted cell adhesion and growth. Higher concentrations of mBG resulted in greater cell extension and anchorage on the membrane surface, clearly demonstrating that L929 fibroblasts preferred rough surfaces due to their larger surface area, which promotes cell adhesion. In rBMSCs, increasing surface roughness on nearly smooth surfaces promoted cell adhesion and diffusion.

[0106] Barrier performance testing: To evaluate the barrier membrane's ability to block fibroblasts, 12 mm diameter membrane sheets were adhered to Transwell chambers with gelatin and secured with stainless steel rings to prevent floating. L929 cell suspension (100 μL / sample) was evenly seeded onto the membrane surface. After 2 hours, 400 μL of serum-free culture medium was added to the upper chamber and 1000 μL of serum-containing culture medium was added to the lower chamber. After 4 days of culture, the cells were fixed with 4% PFA and stained with DAPI. Cell growth on the membrane surface was observed using a fluorescence microscope, and cell penetration at the bottom of the pores was detected using an optical microscope to verify the barrier efficacy.

[0107] Figure 11 In group a, the Control group is the one above the transwel chamber where no material has been placed; all other conditions are the same as the group where material has been placed. Figure 11 Numerous L929 cells were observed growing on the membrane surface in well c, while almost no cells were observed at the bottom of the well plate. Light microscopic observation of the lower layer cells in different groups demonstrated the material's excellent barrier properties, with almost no fibroblasts penetrating the GBR membrane. This result indicates that the addition of mBG allows the composite membrane to effectively prevent fibroblast infiltration, thereby successfully isolating bone defects from the upper fibrous and connective tissues.

[0108] Osteogenic differentiation capacity: To further investigate the in vitro osteoblast differentiation capacity of the membrane materials prepared in Examples 1-2 and Comparative Examples 1-3, the osteogenic properties of the prepared membranes were evaluated by observing the staining of Alizarin Red S. Third- to fifth-generation rBMSCs were introduced at a rate of 4 × 10⁻⁶ cells per membrane. 4 Samples were seeded at a density of 1000 individuals on a membrane and cultured in osteogenic medium. On day 21, the samples were washed three times with PBS, fixed in 4% paraformaldehyde for 30 minutes, then immersed in 1% ARS solution for 10 minutes, thoroughly washed with PBS, and observed under an optical microscope.

[0109] like Figure 12 As shown, 21 days after osteogenic induction, the mBG-0.5 and mBG-1 groups exhibited stronger ARS staining compared to the mBG-0 group, while the mBG-0 group showed the lightest staining. The ARS results may also be attributed to the presence of mBG in the membrane. These results further demonstrate that mBG micro / nanoparticles can effectively promote osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), thereby promoting bone tissue repair in the treatment of bone defects.

[0110] Based on the above test results, it can be seen that the overall performance of the membrane materials prepared in Examples 1-2 of the present invention is better than that of the membrane materials prepared in Comparative Examples 1-3.

[0111] The embodiments provided by the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention, and the descriptions of the embodiments above are only for the purpose of helping to understand the core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, characterized in that, as follows: S1. Slurry preparation: Sodium alginate was dissolved in pure water and mechanically stirred until completely dissolved to obtain a sodium alginate solution. Gelatin solution is prepared by dissolving gelatin in pure water; To prepare mesoporous bioactive glass, the mesoporous bioactive glass was dissolved in pure water to form a dispersion solution. S2, Degassing: Sodium alginate solution, gelatin solution and mesoporous bioactive glass dispersion solution are mixed evenly to obtain a mixture, and then the mixture is subjected to vacuum degassing treatment; S3. Mold setting: Add the degassed mixture into the mold, and after it is evenly cast, place it in a 4℃ refrigerator for 2 hours. S4. Crosslinking: Metal chloride and crosslinking agent solutions are added to the mixture in the mold to carry out ionic and chemical crosslinking to obtain a composite membrane. After the reaction is completed, the composite membrane is removed from the mold. S5. Cleaning and freeze-drying: After rinsing the composite membrane, soak it in pure water for 12-24 hours to remove the residual crosslinking agent in the composite membrane; finally, freeze it at -80℃ for 2 hours, and then put it into a freeze dryer and freeze-dry it at -40℃ for 24 hours to obtain the bone tissue regeneration guiding membrane.

2. The preparation method according to claim 1, characterized in that, In step S1, the mesoporous bioactive glass is prepared as follows: Dodecylamine was added to a mixed solution of ethanol and ultrapure deionized water, and heated and stirred in a water bath at 40°C to obtain the first mixed solution. Add hexadecyltrimethylammonium bromide to the first mixed solution, maintain stirring, and add tetraethyl orthosilicate, triethyl phosphate and calcium nitrate tetrahydrate solution dropwise every 30 min, and continue stirring to obtain the second mixed solution; The second mixed solution is allowed to age until a white precipitate forms. The collected sediment was washed and then dried. The dried precipitate was sintered at 650–700℃ to obtain mesoporous bioactive glass particles.

3. The preparation method according to claim 2, characterized in that, The mesoporous bioactive glass particles have a particle size of 0.5–0.6 μm and a pore size of 2–10 nm.

4. The preparation method according to claim 1, characterized in that, In step S1, the sodium alginate solution has a mass concentration of 5%, and the gelatin solution has a mass concentration of 10%.

5. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of sodium alginate to gelatin in the mixture is 1:1, and the mass of mesoporous bioactive glass accounts for ≤0.3% of the total mass of sodium alginate and gelatin.

6. The preparation method according to claim 1, characterized in that, In step S4, the metal chloride includes one or more of calcium chloride, copper chloride, strontium chloride, and barium chloride, and the crosslinking agent solution includes one or more of glutaraldehyde, genipin, glutaminase, carbodiimide / succinimide, and oxidized amylose.

7. A gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, characterized in that, The preparation method according to any one of claims 1-6 comprises gelatin, sodium alginate and mesoporous bioactive glass, wherein the mass of the mesoporous bioactive glass accounts for 5-10% of the total mass of gelatin and sodium alginate.

8. The regenerated membrane according to claim 7, characterized in that, The mass ratio of gelatin to sodium alginate is (1-1.5):(1-1.5).

9. The regenerated membrane according to claim 7, characterized in that, The thickness of the regenerated membrane is 60–100 μm.

10. The application of a gelatin / sodium alginate / mesoporous bioactive glass composite guided bone tissue regeneration membrane, characterized in that, The regenerative membrane according to any one of claims 7-9 is used in the fields of oral implantology and maxillofacial surgery.

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