A double-layer artificial periosteum for guided bone tissue regeneration and a preparation method thereof

Abstract

This invention belongs to the field of artificial biomimetic materials technology, specifically relating to a double-layer artificial bone membrane for guided bone tissue regeneration and its preparation method. The double-layer artificial bone membrane provided by this invention is composed of a dense layer and a porous layer. The dense layer is prepared by photocrosslinking with PBS buffer containing the following mass / volume fractions of raw materials: 1-40% methacryloyl gelatin with a grafting degree of 60%-90%, and 0.1-10% photoinitiator. The porous layer is prepared by photocrosslinking with PBS buffer containing the following mass / volume fractions of raw materials: 1-40% methacryloyl gelatin with a grafting degree of 60%-90%, 0.1-10% photoinitiator, and 5-20 mg / ml nano-sized hydroxyapatite. This invention, by optimizing the material composition and preparation process of the double-layer artificial bone membrane, obtains a double-layer artificial bone membrane with excellent repair properties for bone defects, as well as good swelling coefficient, mechanical strength, and degradation rate, showing great application potential.

Description

Technical Field

[0001] This invention belongs to the field of artificial biomimetic materials technology, specifically relating to a double-layer artificial bone membrane for guided bone tissue regeneration and its preparation method. Background Technology

[0002] Bone defects caused by trauma, tumors, inflammation, and developmental abnormalities are common clinical problems that endanger patients' health and quality of life. Bone defects in the maxillofacial region often lead to tooth loosening or even loss, and limit the implementation of related treatments such as implant restoration and orthodontics. The repair and reconstruction of bone defects is a significant challenge frequently faced by orthodontists and dentists. Guided bone regeneration (GBR) is a commonly used clinical method for bone defect repair. Its principle is to use a barrier membrane to prevent rapidly growing epithelial cells and fibroblasts from ingrowing into the bone defect area, thus preventing them from interfering with bone repair. This creates and maintains a growth space for osteogenic cells and blood vessels, protects blood clots and other bone repair materials within the defect, and promotes the growth, proliferation, differentiation, and migration of osteogenic cells, thereby accelerating new bone formation. The barrier membrane plays a crucial role in GBR, and its performance largely determines the success or failure of the procedure. An ideal barrier membrane should possess good biocompatibility, cellular barrier function, suitable physicochemical and mechanical properties, biodegradability, and osteogenic promotion effects.

[0003] Clinically used GBR membranes can be divided into two categories: non-absorbable membranes and absorbable membranes. Non-absorbable membranes often require a second surgery for removal, and the removal time is difficult to control, which can easily affect the treatment effect. Traditional absorbable membranes, including natural collagen, chitosan, and synthetic polyester materials, have good biocompatibility and biodegradability, but their physical properties are often poor, their mechanical properties are insufficient, their degradation rate is too fast, and their swelling ratio is too high. These issues can easily allow rapidly growing fibroblasts to invade the bone defect area, thereby hindering bone tissue repair and failing to fully meet clinical needs. Currently, the GBR membranes widely used in clinical practice are mainly developed by Geithner AG of Switzerland. It is an absorbable barrier membrane with a bilayer structure of varying density, composed of porcine skin type I and type II collagen, without cross-linking treatment. As a pure collagen membrane, It also suffers from drawbacks such as a high swelling ratio, excessively rapid degradation, and poor mechanical properties, requiring further improvement and optimization. The properties of adhesive raw materials can be improved through chemical cross-linking, but chemical cross-linking agents, such as glutaraldehyde, have problems such as cytotoxicity or hindering bone tissue regeneration.

[0004] Methacrylic anhydride (MA) can undergo amidation reactions with active amino groups in materials such as collagen, gelatin, and chitosan, thereby acquiring methacryl groups and forming gelatin methacryloyl (GelMA). Adding different types of photoinitiators can endow the materials with the ability to crosslink and cure under visible or ultraviolet light irradiation. The photocrosslinking process is simple and has low requirements for conditions, requiring only room temperature, a neutral pH range, and aqueous solutions. Furthermore, the degree of crosslinking can be controlled by adjusting the range and duration of light irradiation. These materials retain the biological activities of natural materials, such as promoting cell adhesion and osteogenic activity, while also possessing highly tunable physicochemical properties, enabling the creation of heterogeneous bilayer structures for GBR barrier membranes and the improvement of their physicochemical properties. Based on this idea, Chinese invention patent "CN114558172B A Double-Layer Bionic Artificial Osteome and Its Preparation Method and Application" provides a double-layer bionic artificial osteomembrane, which includes a bionic germinal layer and a bionic fiber layer. The bionic germinal layer is made of a hydrogel prepared from methacrylamide gelatin and nano-hydroxyapatite. The bionic fiber layer is made of a hydrogel prepared from methacrylamide gelatin and N-acryloyl-2-glycine.

[0005] Although methacrylamide gelatin has been used in the preparation of bilayer artificial bone membranes, the following problems still exist in its application: the biocompatibility, mechanical properties, and porosity of methacrylic anhydride vary significantly with factors such as the grafting rate of methacrylic anhydride, the light exposure during the preparation process, and the selection and dosage of composite materials. In GBR membranes, high requirements are placed on the biocompatibility, mechanical properties, and porosity of both the dense and porous layers. Therefore, how to adjust the properties of methacrylamide gelatin-related composite materials to meet the performance requirements of the GBR membrane bilayer structure remains a problem to be solved in this field. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides a double-layer artificial periosteum for guided bone tissue regeneration and its preparation method. The aim is to optimize the composition of the two layers of materials of the artificial periosteum to obtain an artificial periosteum with good physicochemical and mechanical properties, good biocompatibility, biosafety and bone defect repair capabilities.

[0007] A double-layered artificial periosteum for guided bone regeneration, which is composed of a dense layer and a porous layer;

[0008] The dense layer is prepared by photocrosslinking with PBS buffer containing the following raw materials in the specified mass-volume fractions:

[0009] 1-40% methacryloyl gelatin with a grafting degree of 60%-90%

[0010] Photoinitiator 0.1-10%;

[0011] The loose layer was prepared by photocrosslinking with PBS buffer containing the following raw materials in the following mass-volume fractions:

[0012] 1-40% methacryloyl gelatin with a grafting degree of 60%-90%

[0013] Photoinitiator 0.1-10%,

[0014] Nano-sized hydroxyapatite 5-20 mg / ml.

[0015] Preferably, the dense layer is prepared by photocrosslinking with PBS buffer containing the following raw materials in the specified mass-volume fractions:

[0016] 10% methacryloyl gelatin with a grafting degree of 60%-90%

[0017] Photoinitiator 0.5%.

[0018] Preferably, the grafting degree of the methacrylamide gelatin used to prepare the dense layer is 90%.

[0019] Preferably, the loose layer is prepared by photocrosslinking with PBS buffer containing the following raw materials in the specified mass-volume fractions:

[0020] 10% methacryloyl gelatin with a grafting degree of 60%-90%

[0021] Photoinitiator 0.5%,

[0022] Nano-sized hydroxyapatite 10-20 mg / ml.

[0023] Preferably, the grafting degree of the methacryloyl gelatin used to prepare the porous layer is 60%.

[0024] Preferably, the amount of nano-sized hydroxyapatite used in the raw materials for preparing the porous layer is 10-20 mg / ml.

[0025] Preferably, the amount of nano-sized hydroxyapatite used in the raw materials for preparing the porous layer is 10 mg / ml.

[0026] Preferably, the photocrosslinking time of the dense layer and / or the loose layer is 10-30 seconds.

[0027] Preferably, the photocrosslinking time of the dense layer is 30 seconds;

[0028] And / or, the time for photocrosslinking of the loose layer is 10s.

[0029] The present invention also provides a method for preparing the above-mentioned double-layer artificial periosteum, comprising the following steps:

[0030] Step 1: Add the raw materials and PBS solution used to prepare the dense layer into the mold and perform photocrosslinking;

[0031] Step 2: Add the raw materials for preparing the loose layer and the PBS solution to the mold in which the dense layer has been formed, and perform photocrosslinking;

[0032] Step 3: Vacuum freeze-drying to obtain the product.

[0033] This invention optimizes the composition of the two layers of artificial bone membrane. In summary, the nano-hydroxyapatite-methacryl gelatin lyophilized hydrogel bilayer barrier membrane constructed in this study has good physicochemical characterization and mechanical properties, and has good in vivo and in vitro biocompatibility, biosafety performance and bone defect repair ability. From the perspective of tissue engineering, it provides a new direction for GBR barrier membrane to repair defects and provides a new option for the repair of skull, maxillofacial bones, vertebral bones and other bones.

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0036] Figure 1 The 1H NMR spectra of gelatin and methacrylonitrile gelatin with different grafting degrees;

[0037] Figure 2 This is the statistical result of the grafting degree integral of methacryloyl gelatin;

[0038] Figure 3 The FTIR infrared spectra of gelatin and methacrylonitrile gelatin are shown.

[0039] Figure 4 FTIR infrared spectra of hydroxyapatite and nano-hydroxyapatite methacryloyl gelatin;

[0040] Figure 5 This is the general appearance of the freeze-dried hydrogel;

[0041] Figure 6 Micropore size and average pore size of gelatin and methacrylonitrile gelatin lyophilized hydrogels;

[0042] Figure 7Micropore size and average pore size of nano-hydroxyapatite-methacryl gelatin freeze-dried hydrogel.

[0043] Figure 8 The stress-strain curves are for gelatin and each group of modified gelatin.

[0044] Figure 9 A general image of gelatin and methacrylonitrile gelatin after swelling for 24 hours to reach swelling equilibrium;

[0045] Figure 10 The swelling properties of gelatin and methacrylonitrile gelatin;

[0046] Figure 11 The swelling properties of nano-hydroxyapatite-methacryl gelatin;

[0047] Figure 12 The in vitro degradation rates of gelatin and methacrylonitrile gelatin;

[0048] Figure 13 The in vitro degradation rate of nano-hydroxyapatite-methacryloyl gelatin;

[0049] Figure 14 Approximate photographs of each group of materials after sampling and freeze-drying;

[0050] Figure 15 The percentage of the remaining mass of gelatin and each group of modified gelatin after 14 days of in vivo degradation.

[0051] Figure 16 HE staining results of rat visceral tissue sections from each experimental group and the blank control group;

[0052] Figure 17 The wetting depth of L929 on the cross sections of four groups of methacryloyl gelatin lyophilized hydrogels;

[0053] Figure 18 Statistics on the wetting depth of L929 on the cross sections of four groups of methacryloyl gelatin lyophilized hydrogels;

[0054] Figure 19 ALP staining results on the surface of nano-hydroxyapatite-methacryl gelatin lyophilized hydrogel;

[0055] Figure 20 The results show the quantitative activity of ALP on the surface of nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogel.

[0056] Figure 21 The images show the Micro CT 3D reconstruction images of five groups of samples and the corresponding cross-sectional images of the defects.

[0057] Figure 22Analysis results of BV / TV and BMD data after MicroCT reconstruction for five groups of samples;

[0058] Figure 23 HE staining results for five groups of samples;

[0059] Figure 24 Masson staining results for five groups of samples;

[0060] Figure 25 The results of OCN immunohistochemical staining and statistical analysis of five groups of samples;

[0061] Figure 26 Col I immunohistochemical staining and statistical results for five groups of samples;

[0062] Figure 27 The results of CD31 immunohistochemical staining and statistical analysis of five groups of samples are presented. Detailed Implementation

[0063] In the following examples and experimental cases, reagents and materials not specifically described are all commercially available products.

[0064] Some of the experimental reagents and materials are as follows:

[0065] Gelatin, AR, Beijing, Solarbio;

[0066] Methacrylic anhydride, AR, USA, Sigma-Aldrich;

[0067] 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (photoinitiator 2959), AR, USA, Sigma-Aldrich;

[0068] PBS buffer, AR, USA, Hyclone;

[0069] Nano-sized hydroxyapatite, AR, USA, Sigma-Aldrich;

[0070] Type I collagenase, USP grade, Germany, BioFroxx.

[0071] Example 1: Double-layer artificial periosteum

[0072] This embodiment provides a double-layered artificial periosteum that can be used in guided bone tissue regeneration, and its preparation method is as follows:

[0073] 1. Preparation of Methacrylgelatin

[0074] Methacrylonitrile gelatin was prepared by grafting gelatin with methacrylic anhydride. 10g of gelatin was dissolved in 10ml of PBS buffer in a 60°C constant-temperature water bath with magnetic stirring. Then, 10ml and 30ml of methacrylic anhydride were slowly added dropwise to the gelatin solution, respectively. The temperature of the water bath was adjusted to 50°C, and the reaction was carried out for 3 hours with magnetic stirring. Subsequently, 200ml of PBS buffer preheated to 50°C was added to terminate the methacrylic anhydride-mediated grafting reaction. All the liquid was transferred to a 12-14kDa dialysis bag and dialyzed in ultrapure water at 37°C for 7 days, changing the ultrapure water every 6 hours to remove unreacted methacrylic anhydride monomers. After 7 days of dialysis, the remaining liquid in the dialysis bag was transferred to a container, frozen at -20°C for 24 hours, and then freeze-dried in a vacuum freeze dryer for 72 hours to obtain two types of methacrylic anhydride gelatin with different grafting degrees.

[0075] The grafting degree of methacrylamide gelatin was determined using the following method: ¹H NMR spectroscopy (NMR) was employed. 1 H-NuclearMagnetic Resonance, 1 The degree of grafting, or 1H-NMR, is determined by the substitution of free amino groups with methacryloyl groups in gelatin. Five mg of pure gelatin and five mg of two different types of methacryloyl gelatin with varying grafting degrees were weighed and completely dissolved in 500 μl of deuterium water (D₂O) at 50°C. The solutions were transferred to NMR tubes and, after cooling, detected using a Bruker AV II-400MHz NMR spectrometer (Bruker AV, Switzerland) to obtain the corresponding 1H NMR spectra. The formula for calculating the grafting degree of methacryloyl gelatin is:

[0076]

[0077] According to the test results: the grafting degree of methacryl gelatin prepared by adding 10 ml of methacrylic anhydride was 60%; the grafting degree of methacryl gelatin prepared by adding 30 ml of methacrylic anhydride was 90%.

[0078] 2. Preparation method of double-layer artificial periosteum

[0079] Methacrylamide gelatin with a grafting degree of 90% and a photocrosslinking time of 30 seconds was selected as the dense layer, and nano-hydroxyapatite-methacrylamide gelatin with a concentration of 10 mg / ml hydroxyapatite was selected as the loose layer. A 96-well plate was used as the preparation mold, and the specific preparation method is as follows:

[0080] Take 90% grafted methacryloyl gelatin and add it to PBS buffer containing 0.5% photoinitiator 2959 at a 10% (w / v) weight fraction (10g methacryloyl gelatin dissolved in 100ml PBS buffer). Stir magnetically in a 65℃ constant temperature water bath for 1 hour until completely dissolved. Pour into a mold and crosslink and cure under 450nm ultraviolet light for 30 seconds in a dark room to form a dense layer.

[0081] Methacrylamide gelatin with a grafting degree of 60% was added at 10% by volume to PBS buffer containing 0.5% by volume of photoinitiator 2959. Nano-sized hydroxyapatite powder (Sigma-Aldrich, 677418) was then added at a concentration of 10 mg / ml. The mixture was magnetically stirred in a constant temperature water bath at 65°C for 1 hour until completely homogeneous, protected from light. The liquid was then poured into a mold and cross-linked and cured under 450 nm UV light for 10 seconds in a dark room to form a loose layer. The volume ratio of the liquid added to the mold when preparing the dense and loose layers was 1:1.

[0082] After being frozen at -20 degrees Celsius for 24 hours, a freeze dryer vacuum freeze-drysed a double-layered film with a diameter of 5 mm and a total thickness of 1 mm to obtain the film.

[0083] Example 2: Artificial Periosteum

[0084] The artificial bone membrane provided in this embodiment is prepared in the same way as in Example 1. The difference is that the amount of nano-hydroxyapatite powder added during the preparation of the porous layer is adjusted to 5 mg / ml and 20 mg / ml, respectively, to prepare two kinds of double-layer artificial bone membranes with different contents of nano-hydroxyapatite.

[0085] Comparative Example 1: Modified gelatin lyophilized hydrogel used in the comparative experiment

[0086] 1. Pure gelatin freeze-dried hydrogel

[0087] After dissolving pure gelatin in water, it was frozen at -20°C for 24 hours and then freeze-dried in a vacuum freeze dryer for 48 hours to obtain gelatin freeze-dried hydrogel, which was named the Gelatin group.

[0088] 2. Methacrylamide gelatin lyophilized hydrogel

[0089] Two types of methacrylamide gelatin with different grafting degrees prepared in Example 1 were added to PBS buffer containing 0.5% photoinitiator 2959 at a mass volume fraction of 10%. The mixture was magnetically stirred in a constant temperature water bath at 65°C for 1 hour until completely dissolved. The solution was then poured into a mold and crosslinked and cured by irradiation with 450nm ultraviolet light for 10 seconds and 30 seconds, respectively, in a dark room. After the hydrogel cooled, it was placed in a freezer at -20°C for 24 hours and then freeze-dried in a vacuum freeze dryer for 48 hours to obtain four types of methacrylamide gelatin freeze-dried hydrogels with different grafting degrees and crosslinking times, which were named GM60-10s, GM60-30s, GM90-10s, and GM90-30s, respectively.

[0090] 3. Preparation of methacryloyl gelatin and nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogel

[0091] Methacrylamide gelatin with a grafting degree of 60% was added to PBS buffer containing 0.5% photoinitiator 2959 at a mass volume fraction of 10%. Then, nano-sized hydroxyapatite powder (Sigma Aldrich, 677418) was added at concentrations of 5 mg / ml, 10 mg / ml, and 20 mg / ml, respectively. The mixture was magnetically stirred in a constant temperature water bath at 65°C for 1 hour under light-protected conditions until completely homogeneous. The liquid was poured into a mold and cross-linked and cured under 450 nm ultraviolet light for 10 seconds in a dark room. The lyophilization process was as described above, resulting in three types of methacrylamide gelatin with different hydroxyapatite contents, named 5HA, 10HA, and 20HA, respectively. The methacrylamide gelatin group with a grafting degree of 60% without any added hydroxyapatite was named the 0HA group (the 0HA group is also the GM60-10s group).

[0092] Comparative Example 2: Single-layer membrane material

[0093] Using the same mold as in Example 1, GM90-30s and 10HA from Comparative Example 1 were respectively made into single-layer film materials with a thickness of 1 mm.

[0094] The beneficial effects of the present invention will be further illustrated by the following experiments.

[0095] Characterization of modified gelatin lyophilized hydrogel in Experiment Example 1

[0096] This experiment characterizes the physicochemical properties of various modified gelatin freeze-dried hydrogels prepared in Comparative Example 1.

[0097] I. Experimental Methods

[0098] 1. Infrared spectral analysis of modified gelatin lyophilized hydrogels

[0099] Gelatin, hydroxyapatite, various groups of methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogel were ground into powders, and the characteristic groups in each group of materials were detected using a Fourier transform infrared spectrometer (Nicolet is10, Thermo Scientific, MA, USA).

[0100] 2. Microstructure observation of modified gelatin lyophilized hydrogel

[0101] The microstructure of each group of methacryloyl gelatin and nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogels was observed using scanning electron microscopy (Inspect F50, FEI, Hillsboro, OR, US). Gelatin and each modified gelatin group were prepared into lyophilized hydrogels with a diameter of 8 mm and a height of 10 mm. Each group of materials was transversely cut with a scalpel perpendicular to its long axis to expose its internal structure. The cross-sectional surfaces were sputtered with gold and then scanned using electron microscopy. At least 50 pores in each modified gelatin group were selected, and their pore size was measured using ImageJ software.

[0102] 3. Mechanical property testing of modified gelatin lyophilized hydrogel

[0103] The compressibility of gelatin, various groups of methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogels was tested using an Instron 5967 electronic universal testing machine (Instron 5967). Gelatin and the modified gelatin groups were prepared into cylindrical lyophilized hydrogels with a diameter of 5 mm and a height of 2 mm. At room temperature, the sample surface was vertically compressed at a constant force of 1 N / s using a 10 cm diameter compression probe until the sample height was compressed to 80% of its initial height. The stress-strain curves were recorded and plotted.

[0104] 4. Swelling property test of modified gelatin lyophilized hydrogel

[0105] The swelling properties of gelatin, each group of methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin were determined using a gravimetric method. Gelatin and each group of modified gelatin were prepared into lyophilized hydrogels with a diameter of 8 mm and a height of 10 mm. Their initial mass was measured as W0. Each group of materials was immersed in PBS buffer at pH 7.4 and placed in a 37°C incubator. After 6, 12, and 24 hours, the materials were removed, surface moisture was absorbed using qualitative filter paper, and their weight was recorded as W0. x The formula for calculating the swelling coefficient at each time point is as follows:

[0106]

[0107] In the formula, W x The mass of the material at each measurement time point is defined, where W0 is the initial mass of the material, and three parallel samples are set up for each group.

[0108] 5. In vitro degradation performance testing of modified gelatin lyophilized hydrogels

[0109] The in vitro degradation properties of gelatin, various groups of methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin were detected using an enzymatic method. Type I collagenase was dissolved in PBS buffer at a concentration of 5 mg / ml to obtain a collagenase solution. Gelatin and the modified gelatin groups were prepared into lyophilized hydrogels with a diameter of 8 mm and a height of 10 mm. Each group of materials was first immersed in PBS buffer at pH 7.4 for 24 hours to reach swelling equilibrium. After removing surface moisture, the initial weight was measured and recorded as W0. Subsequently, each group of materials was immersed in the collagenase solution and placed in a 37°C incubator. The collagenase solution was changed daily. Within the first 24 hours, materials were removed at 6, 9, 12, and 24 hours, and then daily thereafter. After blotting the surface moisture with qualitative filter paper, the weight was measured and recorded as W0. x The material degradation coefficient at each time point is calculated using the following formula:

[0110]

[0111] In the formula, W x The remaining mass of the material at each measurement time point is given, and W0 is the initial mass of the material after reaching swelling equilibrium. Three parallel samples are set up for each group.

[0112] 6. In vivo degradation properties and biotoxicity of modified gelatin lyophilized hydrogels

[0113] The in vivo degradation properties of gelatin, methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin were investigated using a subcutaneous implantation model in SD rats. Eight-week-old healthy SD rats (SPF grade, weighing 230-250g) purchased from Chengdu Dashuo Experimental Animal Co., Ltd. were randomly divided into nine groups of three rats each. Gelatin and modified gelatin were prepared into lyophilized hydrogels with a diameter of 10mm and a thickness of 2mm. Their initial mass was measured and recorded as W0. Nine replicates were prepared for each group. Surgical procedure: The rats were fasted for 12 hours before surgery. The SD rats were weighed and intraperitoneally anesthetized with 0.33ml of 10% chloral hydrate solution per 100g. After anesthesia took effect, the skin on the rat's back was prepared and disinfected with povidone-iodine. A 1.5cm incision was made along the midline near the neck on the back. The subcutaneous fascia was bluntly dissected, and three subcutaneous cysts were created at the upper left, lower left, and right sides of the incision. The implant was placed into each of these three cysts around the incision, ensuring no contact between the materials. The incision was then tightly sutured with 4-0 sutures. Postoperatively, erythromycin ointment was applied to the incision site for routine infection prevention. 200,000 units of potassium penicillin solution were injected intramuscularly into the rat's thigh. The rats were placed on an animal heating blanket until they recovered, and then separated into different cages for feeding. The rats' recovery and feeding were continuously monitored postoperatively. Two weeks postoperatively, the rats were euthanized using an overdose anesthesia method. The skin at the implantation site on the back was harvested, and the surrounding soft tissue was separated to obtain the remaining material. After freeze-drying, the mass was measured and recorded as W. x .

[0114]

[0115] At the same time as collecting the materials, samples of the heart, liver, spleen, lungs, and kidneys of each group of rats were collected for biotoxicity testing.

[0116] 7. Sample dehydration and embedding / sectioning

[0117] The fixed visceral samples were sequentially immersed in graded ethanol for 40 minutes each for dehydration, then immersed in xylene for clearing treatment for 30 minutes, and then immersed in paraffin (I, II, III) for paraffin permeation treatment. After paraffin embedding, sections were prepared along the cross-section of each organ with a section thickness of 5 μm.

[0118] 8. HE staining

[0119] After drying the slides in a 60℃ constant temperature oven for 2 hours, the slides were dewaxed by immersing them in xylene (I,II) for 10 minutes each. Then, they were successively immersed in 100% ethanol (I,II), 95% ethanol (I,II), 85% ethanol, 75% ethanol, and distilled water for 5 minutes each. The slides were then washed with distilled water 3 times for 1 minute each time. After staining with hematoxylin for 2 minutes, the slides were rinsed 3 times for 1 minute each time. After staining with eosin for 1 minute, the slides were washed 3 times for 1 minute each time. The slides were then treated with 75% ethanol, 85% ethanol, 95% ethanol (I,II), and 100% ethanol (I,II) for 5 minutes each time in sequence. After mounting with neutral resin, the slides were air-dried and observed and photographed under a microscope.

[0120] 9. Statistical Analysis

[0121] At least three parallel samples were set for each group. The results were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPadprism 8. The t-test was used to compare data between two groups, and one-way ANOVA was used to compare data between multiple groups. A p < 0.05 was recorded as a significant difference.

[0122] II. Experimental Results

[0123] 1. 1H NMR spectrum of gelatin and modified gelatin

[0124] The 1H NMR spectra of gelatin and two types of methacrylamide gelatin with different MA grafting degrees are shown below. Figure 1 As shown in the figure, in the grafting reaction, methacrylic anhydride reacts with the free amino groups in gelatin. The spectra at δ = 5.3 ppm and δ = 5.5 ppm correspond to the methacrylic group -2HC=C(CH3)-, and the peak at δ = 7.3 ppm represents the unreacted free amino groups in gelatin. The figure shows that the two methacryloyl gelatin groups with different grafting degrees show obvious peaks at δ = 5.3 ppm and δ = 5.5 ppm compared to the pure gelatin group, indicating that the methacrylic anhydride has successfully reacted with the free amino groups in gelatin to generate methacrylic groups. The significant decrease or disappearance of the peak of methacryloyl gelatin at δ = 2.8 ppm compared to the pure gelatin group is also related to the modification with methacrylic acid.

[0125] The grafting degree of the obtained methacryloyl gelatin can be adjusted by changing the amount of MA added in the grafting reaction. The grafting degree is calculated by integrating and summing the waveforms at δ = 5.3 ppm and δ = 5.5 ppm, and then comparing the sum with the integral of the waveform at δ = 7.3 ppm. The statistical results are shown below. Figure 2 As shown, the grafting degree of the methacryl gelatin increases with the amount of methacrylic anhydride added in the grafting reaction. The grafting degrees of the methacryl gelatin corresponding to the addition of 10 ml and 30 ml of methacrylic anhydride are 60% and 90%, respectively. Therefore, the two modified gelatins are named GelMA60 and GelMA90, respectively.

[0126] 2. Infrared spectra of gelatin and modified gelatin

[0127] The Fourier transform infrared (FTIR) spectroscopy results for gelatin, hydroxyapatite, various groups of methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin are as follows: Figure 3 , Figure 4 As shown. Figure 3 Medium-grafted gelatin and methacryloyl gelatin with different grafting degrees and crosslinking times all exhibited vibrational peaks of characteristic groups. (1240 cm⁻¹) -1 The peak at 1541 cm represents the tensile vibration of CN. -1 The peak at 1640 cm⁻¹ represents an NH bond. -1 The peak represents the tensile vibration of the C=O double bond, 2934 cm⁻¹ -1 The peak at 3303 cm⁻¹ represents the vibration of the CH bond. -1 Another NH bond stretching vibration of the grafted acyl group can also be seen at this location.

[0128] Figure 4 Hydroxyapatite nanopowder at 1061 cm⁻¹ -1 A characteristic peak appeared at this point; compared with the 0HA group which did not contain hydroxyapatite, the nano-hydroxyapatite-methacryloyl gelatin in all groups was at 1061 cm⁻¹. -1 The appearance of peaks at the point indicates that nano-hydroxyapatite has been successfully blended into each group of freeze-dried hydrogels, and the higher the concentration of hydroxyapatite, the more obvious the peaks.

[0129] 3. Microstructural observation of gelatin and modified gelatin

[0130] Freeze-dried hydrogels were prepared using 48-well plates as molds. After freezing at -20°C followed by vacuum freeze-drying, the water within the hydrogels sublimated directly upon forming ice crystals. The remaining hydrogel components formed microporous structures of varying sizes, with a general appearance as follows: Figure 5 As shown.

[0131] Electron microscopy revealed that the lyophilized hydrogels possessed a microporous scaffold structure. The statistical results of the microstructure and average pore size of gelatin, various groups of methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogels are shown below. Figure 6 , Figure 7As shown, gelatin has the largest average pore size, approximately 383 μm. After reacting the free amino groups of gelatin with methacrylic anhydride and crosslinking under ultraviolet light, the average pore size of the modified gelatin gradually decreases, and the morphology becomes more uniform and regular. The average pore size decreases with increasing grafting degree, and also gradually decreases with prolonged photocrosslinking time. Statistical results show that the average pore size of the methacryloyl gelatin lyophilized hydrogels in each group gradually decreases from 342 μm in the GM60-10s group, 244 μm in the GM60-30S group, and 187 μm in the GM90-10s group, down to 93 μm in the GM90-30s group.

[0132] When the grafting degree is maintained at 60% and the photocrosslinking time is 10 seconds, the effect of different hydroxyapatite concentrations on the average pore size of methacrylamide gelatin is as follows: Figure 7 As shown, with the increase of hydroxyapatite concentration, there is no significant difference in the average pore size of each group, which remains at 340-360 μm, indicating that the concentration of hydroxyapatite has no effect on the average pore size of the modified gelatin.

[0133] 4. Mechanical properties of gelatin and modified gelatin

[0134] The barrier membrane possesses certain compressive mechanical properties, ensuring the material maintains its morphological stability and integrity during practical applications, thereby guaranteeing its barrier and bone-promoting functions. This is an important mechanical parameter. This experiment tested the compressive properties of gelatin, various groups of methacryloyl gelatin with different grafting degrees and photocrosslinking times, and various groups of lyophilized hydrogels of nano-hydroxyapatite-methacryloyl gelatin with different concentrations. Corresponding stress-strain curves were plotted, such as... Figure 8 As shown, compared with the pure gelatin group, the compressive strength of all groups of methacryloyl gelatin freeze-dried hydrogels was improved. With the increase of grafting degree, the content of methacrylic double bonds increased, and the compressive strength of the freeze-dried hydrogel increased. With the increase of photocrosslinking time, the number of copolymerized methacryloyl gelatin molecular chains increased, and its compressive strength also increased, with the GM90-30s group showing the strongest compressive strength. Compared with the GM60-10s methacryloyl gelatin (0HA group) without hydroxyapatite, the compressive strength of all groups of nano-hydroxyapatite-methacryloyl gelatin was also enhanced. With the increase of the concentration of added hydroxyapatite, its compressive strength increased, with the 20HA group showing the strongest compressive strength, which was already stronger than the GM90-30s group. This indicates that adding hydroxyapatite to the GM60-10s group can significantly enhance the compressive strength of the freeze-dried hydrogel.

[0135] 5. Swelling properties of gelatin and modified gelatin

[0136] For GBR barrier membranes, an excessively high swelling rate will cause their volume to increase rapidly after implantation, compressing surrounding soft tissues and blood supply, and also affecting their mechanical properties. Therefore, a suitable GBR barrier membrane should have a low swelling rate. The swelling properties of gelatin, methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin after soaking in PBS buffer for 6 hours, 12 hours, and 24 hours are shown below. Figure 10 , 11 As shown in the figure. Compared with the other groups of methacryloyl gelatin, the gelatin group had a significantly higher swelling rate at all three time points, and the increase in swelling rate was significantly faster than that of the other groups of methacryloyl gelatin as the swelling time increased. After reaching swelling equilibrium at 24 hours, the gelatin group had a significantly larger volume than the methacryloyl gelatin group and no longer formed a solid shape, with an appearance as shown in the figure. Figure 9 As shown, it does not possess the basic properties of a barrier membrane.

[0137] Figure 10 As the grafting degree increases, the swelling rate of methacryloyl gelatin gradually decreases. As the photocrosslinking time gradually increases, the swelling rate of methacryloyl gelatin also gradually decreases. Moreover, the increase in swelling rate gradually decreases with the increase in swelling time. That is, when the grafting degree is 90% and the photocrosslinking time is 30 seconds, methacryloyl gelatin has the lowest swelling rate.

[0138] Figure 11 The results showed that, with the same grafting degree of 60% and the same photocrosslinking time of 10 seconds, the swelling rate of nano-hydroxyapatite-methacryloyl gelatin with different hydroxyapatite concentrations did not show significant statistical differences at various time points, indicating that the hydroxyapatite concentration does not affect the swelling rate of the modified gelatin.

[0139] 6. In vitro degradation properties of gelatin and modified gelatin

[0140] The degradation of gelatin, methacryloyl gelatin, and nano-hydroxyapatite-methacryloyl gelatin after immersion in collagenase I solution is as follows: Figure 12 , 13As shown, with increasing soaking time, the remaining mass of each group of materials gradually decreased. Gelatin had the shortest degradation time, rapidly degrading from the initial contact with collagenase and completely degrading after 24 hours of enzymatic hydrolysis. For methacryloyl gelatin, the GM60-10s and GM60-30s groups were completely degraded on days 6 and 7, respectively, while the GM90-10s and GM90-30s groups were completely degraded on day 9. There was no statistically significant difference in the in vitro enzymatic hydrolysis amount of methacryloyl gelatin between the two groups with the same grafting degree but different photocrosslinking times at each time point. However, for methacryloyl gelatin with different grafting degrees, there were statistically significant differences in the degradation ratio at each in vitro enzymatic hydrolysis time point (p<0.01). This indicates that as the grafting degree increases, the in vitro degradation rate of methacryloyl gelatin slows down, while the photocrosslinking time has little effect on the in vitro enzymatic hydrolysis rate.

[0141] Figure 13 When the grafting degree was 60% and the photocrosslinking time was 10 seconds, the in vitro complete degradation time of methacryloyl gelatin with nano-hydroxyapatite was extended to day 9, and the degradation rate was significantly slower than that of the 0HA group without hydroxyapatite. However, there was no statistically significant difference in the degradation rate of the 5HA, 10HA and 20HA groups with added hydroxyapatite at different time points, indicating that the amount of hydroxyapatite added does not affect the degradation rate of methacryloyl gelatin.

[0142] 7. In vivo degradation properties of gelatin and modified gelatin

[0143] Gelatin, methacryloyl gelatin of various groups, and nano-hydroxyapatite-methacryloyl gelatin were implanted subcutaneously in rats for 14 days. The implants were then removed, surrounding soft tissue was removed, and the materials were freeze-dried before calculating the percentage of remaining mass. The materials were encapsulated within fibrous capsules subcutaneously, with a small amount of fibrous tissue and blood vessels ingrowth into the surface. After freeze-drying, the materials appeared approximately as follows: Figure 14 As shown.

[0144] The remaining mass percentage results for each group of materials are as follows: Figure 15 As shown, the gelatin group completely degraded 14 days after subcutaneous implantation in rats, with no material residue. This is consistent with previous literature descriptions of gelatin's instability and easy degradation at body temperature. However, the methacryloyl gelatin group and the nano-hydroxyapatite-methacryloyl gelatin group had a significant amount of material residue. Except for the gelatin group, although the remaining mass of the other groups did not differ much and remained above 90%, there was still a certain trend: with the increase of grafting degree and photocrosslinking time, the remaining mass increased and the in vivo degradation rate slowed down. With the increase of hydroxyapatite concentration, the remaining mass increased and the in vivo degradation rate slowed down.

[0145] 8. Biotoxicity testing of gelatin and modified gelatin

[0146] Figure 16The image shows the HE staining results of rat heart, liver, spleen, lung, and kidney sections from the gelatin group, methacryloyl gelatin group, nano-hydroxyapatite-methacryloyl gelatin group, and blank control group. Compared with the blank control group, parallel-arranged cardiomyocytes were visible in the heart sections of each experimental group. The cells were short columnar and connected by intercalated disc structures. Polyhedral stem cells with large, centrally located nuclei were visible in the liver sections of each experimental group, along with radially distributed hepatic lobule structures. Germinal centers and purplish-red red pulp and blue white pulp structures were visible in the spleen sections of each experimental group. Alveolar structures composed of a single layer of cells were visible in the lung sections of each experimental group. Normal glomerular structures were visible in the kidney sections of each experimental group. This indicates that the materials in each group showed no tissue toxicity and possessed a certain degree of safety.

[0147] The above experiments demonstrate that methacrylamide gelatin and nano-hydroxyapatite-methacrylamide gelatin possess excellent physicochemical properties. Their swelling coefficient, mechanical strength, and degradation rate are all superior to ordinary gelatin, and they are non-toxic. Therefore, methacrylamide gelatin and nano-hydroxyapatite-methacrylamide gelatin have the potential to prepare artificial bone membranes.

[0148] Experimental Example 2: Barrier Effects and Bone Function Promotion of Methacrylamide Gelatin and Nano-hydroxyapatite-Methacrylamide Gelatin

[0149] This experiment investigated the barrier function of methacryloyl gelatin prepared in Comparative Example 1 and the osteogenic function of nano-hydroxyapatite-methacryloyl gelatin.

[0150] I. Experimental Methods

[0151] The following are some of the reagents and materials used in this experiment:

[0152]

[0153] 1. Detection of the barrier function of methacrylamide gelatin lyophilized hydrogel

[0154] Using a 48-well plate as a mold, lyophilized hydrogels of methacrylamide gelatin were prepared into 8 mm diameter and 5 mm high. After sterilization by UV light for 30 minutes, the hydrogels were placed in 48-well plates and immersed in PBS buffer containing 10% penicillin-streptomycin solution for 24 hours to obtain sterile lyophilized hydrogels of methacrylamide gelatin. Each group of lyophilized hydrogels was then immersed in culture medium for 24 hours to fully wet the medium. L929 was seeded on the surface of the material at a density of 1*104 / well. After adhering to the wells for 1 hour in a 5% CO2 incubator at 37°C, 250 μL of fresh culture medium was added to each well, and the medium was changed every two days. Three days after seeding the cells, the culture medium was aspirated. After washing with PBS, the cells in the plate were fixed under 4% paraformaldehyde treatment. Then, 0.5% Triton X-100 was added to the well plate to permeate the cells for 15 minutes. After rinsing with PBS, the cells were blocked with 10% bovine serum albumin for 30 minutes. The cells were rinsed three times with PBS buffer for 5 minutes each time. Rhodamine-labeled phalloidin was added at a 1:200 dilution and stained for 30 minutes in the dark. After removing the staining agent, an anti-fluorescence attenuation agent containing DAPI was added. The lyophilized hydrogel was cut vertically along its diameter to expose its internal longitudinal section. Cell morphology and invasion depth were observed and photographed using a laser confocal microscope with excitation wavelengths of 488 nm and 562 nm. Invasion depth was measured at three locations in each group and the results were statistically analyzed.

[0155] 2. Osteogenic Properties Testing of Nano-hydroxyapatite-methacryloyl gelatin Lyophilized Hydrogel

[0156] Using 48-well plates as molds, nano-hydroxyapatite-methacrylamide gelatin was prepared into lyophilized hydrogels with a diameter of 8 mm and a height of 5 mm. After sterilization by UV irradiation for 30 minutes, the gels were immersed in PBS buffer containing 10% penicillin-streptomycin solution for 24 hours to obtain sterile lyophilized hydrogels, which were then immersed in α-MEM medium for 24 hours. The acquisition and purification of primary rat BMSCs followed the same steps as described above. BMSCs were seeded at a density of 4*10⁴ / well on the material surface and cultured in a 5% CO₂ incubator at 37°C for 1 hour until adherence. Then, 250 μL of fresh medium was added, and the medium was changed every two days. By day 3, the cell density had reached approximately 80% of the material surface area. The medium was removed, and a prepared osteogenic induction solution containing ascorbic acid, sodium β-glycerophosphate pentahydrate, and dexamethasone was added. The cells were cultured under light-protected conditions for 7 days, with the osteogenic induction solution changed every two days.

[0157] (1) ALP staining:

[0158] Seven days after adding osteogenic induction solution, the induction solution was aspirated, and the cells were washed three times with PBS buffer for 5 minutes each time. The cells were then fixed in 4% paraformaldehyde for 20 minutes. Using the BCIP / NBT alkaline phosphatase assay kit, an appropriate amount of staining agent was added, and the cells were stained at 37°C for 2 hours in the dark. The staining agent was aspirated, and the ALP staining results were observed by taking pictures using a stereomicroscope.

[0159] (2) Quantitative detection of ALP:

[0160] Seven days after adding osteogenic induction solution, the induction solution was aspirated, and the cells were washed with PBS buffer. BMSCs cells were then lysed on ice for 25 minutes using cell lysis buffer without phosphatase inhibitors. After repeated pipetting, the lysate was aspirated and centrifuged at 12,000 rpm for 15 minutes in a centrifuge pre-cooled to 4°C. The supernatant was collected to obtain the total protein solution. The protein concentration of each group was detected using a BCA kit, and the absorbance was measured at 562 nm using a microplate reader. Subsequently, a preliminary ALP quantification experiment was performed. The obtained total protein solution was diluted 5-fold, 10-fold, and 50-fold, respectively. After reacting with an alkaline phosphatase kit at 37°C in the dark for 30 minutes, the absorbance was measured at 405 nm using a microplate reader. After selecting an appropriate dilution factor, the total protein solution of each group was diluted accordingly, added to the detection kit, and reacted at 37°C in the dark for 30 minutes. The absorbance was then measured at 405 nm using a microplate reader.

[0161] 3. Statistical Analysis

[0162] At least three parallel samples were set for each group. The results were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPadprism 8. The t-test was used to compare data between two groups, and one-way ANOVA was used to compare data between multiple groups. A p < 0.05 was recorded as a significant difference.

[0163] II. Experimental Results

[0164] 1. Barrier properties of methacryloyl gelatin lyophilized hydrogel

[0165] The infiltration of L929 cells into the cross-sections of methacryloyl gelatin lyophilized hydrogels in each group, as captured by laser confocal microscopy, is shown below. Figure 17As shown in the image, the top of the image shows the upper surface of the lyophilized hydrogel cross-section, with the depth of cell infiltration increasing closer to the bottom of the image. Comparing four groups of methacryloyl gelatin lyophilized hydrogels with different grafting degrees and photocrosslinking times, it can be seen that L929 has the greatest infiltration depth in the GM60-10s group with the largest pore size, with an average infiltration depth of 300.0 μm. The cells are also the most extended, exhibiting short spindle-shaped or triangular forms, occasionally with pseudopodia. The distance between adjacent cells is the greatest, meaning the cell density is the lowest within the same infiltration depth range. As the pore size gradually decreases, the infiltration depth of L929 gradually decreases in the corresponding GM60-30s and GM90-10s groups, to 240.6 μm and 100.6 μm, respectively. The degree of cell extension gradually decreases, and the cells become more aggregated. Within the same infiltration depth range, the cell density gradually increases. The shallowest infiltration depth is found in the GM90-30s group with the smallest pore size, with an average infiltration depth of only 45.3 μm. The cells are in close contact and overlap, with a relatively flattened morphology.

[0166] The statistical results of the wetting depth of L929 in four material sections are as follows: Figure 18 As shown, as the grafting degree increased from 60% to 90%, the wetting depth of L929 within the lyophilized hydrogel gradually decreased. Similarly, as the photocrosslinking time increased from 10 seconds to 30 seconds, the wetting depth of L929 within the lyophilized hydrogel also gradually decreased, with statistically significant differences between groups. This trend is consistent with the trend in the internal pore size of the four groups of methacryloyl gelatin: higher grafting degree, longer photocrosslinking time, and smaller pore size result in shallower wetting depth and better barrier function.

[0167] 2. Osteogenic effect of nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogel

[0168] (1) ALP staining: ALP staining of BMSCs on the surface of nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogels with different hydroxyapatite concentrations after 7 days of osteogenic induction is shown in the figure. Figure 19 As shown, with the increase of hydroxyapatite concentration, the freeze-dried hydrogel gradually changes from translucent to milky white and opaque, and the ALP staining depth gradually increases. The staining depth is most obvious when the hydroxyapatite concentration is 10 mg / ml. When the hydroxyapatite concentration increases to 20 mg / ml, the ALP staining depth decreases slightly.

[0169] (2) ALP Quantitative Detection: ALP quantitative results of BMSCs on the surface of nano-hydroxyapatite-methacryloyl gelatin lyophilized hydrogels with different hydroxyapatite concentrations after 7 days of osteogenic formation are as follows: Figure 20As shown, the trend is consistent with the ALP staining trend. With the increase of hydroxyapatite concentration, ALP activity first increases, reaching its strongest osteogenic effect on BMSCs in the 10HA group. Subsequently, as the hydroxyapatite concentration increases to 20 mg / ml, ALP activity decreases slightly. All groups with added hydroxyapatite showed statistically significant differences in ALP activity compared to the 0HA group without added hydroxyapatite.

[0170] The experimental data from this example show that the GM90-30s group of methacryloyl gelatin exhibits the best barrier function and can be preferentially used as a dense layer to isolate connective tissue cells and fibroblasts; the 10HA group of nano-hydroxyapatite-methacryloyl gelatin exhibits the best osteogenic function and can be preferentially used as a porous layer to promote osteogenic repair. Using these two materials to fabricate the bilayer artificial periosteum of this application is a preferred choice.

[0171] Experimental Example 3: Repair of Limiting Bone Defects in Rat Skulls with Nano-hydroxyapatite-methacryloyl gelatin Bilayer Film

[0172] This experimental example examines the double-layer artificial periosteum of Example 1 (denoted as the 10HA-GM90-30s group), the GM90-30s membrane group prepared in Comparative Example 2, the 10HA membrane group, and the commercial membrane. The repair effect of the barrier membrane (positive control group) on the limit bone defects of the rat skull.

[0173] I. Experimental Methods

[0174] The following are some of the reagents and materials used in this experiment:

[0175]

[0176] 1. Grouping of experimental animals

[0177] This experiment was approved by the Animal Ethics Committee of West China Hospital of Stomatology, Sichuan University. Thirty SPF-grade 8-week-old male SD rats (Chengdu, Dashuo), weighing 250-300g, were purchased. The experimental environment and facilities met the relevant standards of "Experimental Animal Environment and Facilities" (GB14925-2001), and animal husbandry and experimental procedures complied with relevant regulations. Based on experimental requirements, the rats were randomly divided into 5 groups of 6 rats each, as follows:

[0178] 1) 10HA-GM90-30s nano-hydroxyapatite-methacryloyl gelatin bilayer membrane assembly (using the bilayer artificial bone membrane prepared in Example 1);

[0179] 2) GM90-30s methacryloyl gelatin monolayer membrane group (using the monolayer membrane prepared in Comparative Example 2);

[0180] 3) 10HA nano-hydroxyapatite-methacryloyl gelatin monolayer membrane (using the monolayer membrane prepared in Comparative Example 2);

[0181] 4) Positive control group;

[0182] 5) Blank control group.

[0183] 2. Establishment and tissue sampling of a rat model of extreme skull defects

[0184] 10% chloral hydrate anesthetic was injected at a dose of 0.33 ml / 100 g. After the rats were fully anesthetized, the surgical area was shaved and prepared. The rats were then fixed in place, and the surgical area was disinfected with povidone-iodine. Local anesthesia with articaine containing adrenaline was injected into the surgical area to reduce bleeding. An incision was made along the midline of the rat's head from the midpoint between the eyes to the midpoint between the ears using a scalpel. The subcutaneous fascia and periosteum of the skull were bluntly dissected using ophthalmic scissors to expose the skull, avoiding the sutures. A 5 mm trephine was used to create a 5 mm ring defect on one side of the skull at low speed. The area was then rinsed with saline to cool the rat. The modified gelatin barrier membranes of each group were then applied. A barrier membrane was placed over the defect, while no material was placed in the blank control group. The skin layer of the wound was sutured using 4-0 sutures with needles. Erythromycin ointment was evenly applied to the wound to prevent infection. On the day of surgery and two days after surgery, 200,000 units of penicillin solution were injected intramuscularly into the leg. After surgery, the rats were placed on a warm blanket until they were fully recovered and then separated into different cages for feeding. The rats' eating habits, wound healing, and weight were continuously observed after surgery. Eight weeks after surgery, the rats were sacrificed using an overdose anesthesia method. The rat skull was separated and excised, trimmed, and then soaked in 4% paraformaldehyde for 48 hours for fixation. Subsequently, it was transferred to 0.5% paraformaldehyde for preservation.

[0185] 3. Micro CT analysis

[0186] The fixed rat skull samples were placed in a 34mm MicroCT scanning tube for scanning. The scanning voltage was set to 40kV, the current to 250mA, and the scanning accuracy to 10μm. The samples were reconstructed and analyzed using Scanco software to obtain the bone mineral density (BMD) and bone volume / total volume (BV / TV) of the defect area.

[0187] 4. Sample decalcification, dehydration, and embedding / sectioning

[0188] The fixed samples were immersed in 10% EDTA solution and placed on a shaker for decalcification. The decalcification solution was changed daily. Decalcification was completed when a 1ml needle could penetrate the sample without resistance. After rinsing the samples with distilled water, they were successively immersed in 70%, 80%, 90%, and 100% ethanol for 40 minutes each for dehydration. Then, they were immersed in xylene for 30 minutes for clearing treatment, followed by immersion in paraffin (I, II, III) for paraffin permeation treatment. After paraffin embedding, sections were made along the longitudinal section at the defect site, with a section thickness of 5μm.

[0189] 5. HE staining

[0190] The slides were dried in a 60°C oven for 2 hours, then dewaxed by immersing them in xylene (I, II) for 10 minutes each. They were then immersed in a series of alcohols (100% (I, II), 95%, 85%, 75%) and distilled water for 5 minutes each. The slides were then rinsed with distilled water three times for 1 minute each time to hydrate them. After hematoxylin staining for 2 minutes, the slides were rinsed with double-distilled water three times for 1 minute each time. After eosin staining for 1 minute, the slides were rinsed with double-distilled water three times for 1 minute each time. Finally, the slides were immersed in a series of alcohols of different concentrations for 5 minutes each. After mounting with neutral resin, the slides were air-dried and observed and photographed under a microscope.

[0191] 6. Masson staining

[0192] The slides were dried in a 60°C oven for 2 hours. Cells were cleared with xylene (I, II) for 10 minutes, then immersed in 100% ethanol (I, II), 95% ethanol, 85% ethanol, 75% ethanol, and distilled water for 5 minutes each. The slides were stained with iron hematoxylin for 5 minutes, differentiated with acidic ethanol differentiation solution for 10 seconds, rinsed with double-distilled water, blued with Masson's blue solution for 5 minutes, rinsed with double-distilled water for 1 minute, stained with fuchsin for 5 minutes, rinsed with weak acid working solution for 1 minute, rinsed with phosphomolybdic acid solution for 1 minute, rinsed with weak acid working solution for 1 minute, stained with aniline blue for 1 minute, rinsed with weak acid working solution for 1 minute, dehydrated with 95% ethanol and anhydrous ethanol for 5 minutes, cleared with xylene (I, II) for 10 minutes, mounted with neutral resin, air-dried, and observed and photographed under a microscope.

[0193] 7. Immunohistochemistry (IHC) staining

[0194] The dewaxing and hydration steps are as shown in 1.8. The slides were soaked in preheated blocking and permeabilizing buffer (Triton X-100) for 30 minutes, rinsed with PBS 3 times for 3 minutes each time, and then the antigenicity was restored by heat retrieval using antigen retrieval buffer. Serum was added and the slides were blocked at 37°C for 30 minutes. The slides were then soaked in primary antibody solution and incubated at 4°C overnight. After warming, the primary antibody solution was washed off with PBS 3 times for 3 minutes each time. The slides were then soaked in secondary antibody solution and treated at 37°C for 1 hour. After washing with PBS, the slides were developed with DAB chromogenic reagent for 10 minutes. Distilled water was added to stop the development. The slides were then mounted with neutral resin and observed and photographed under a microscope.

[0195] 8. Statistical Analysis

[0196] At least three parallel samples were set for each group. The results were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPadprism 8. A t-test was performed between two groups, and a one-way ANOVA was performed among multiple groups. A p < 0.05 was recorded as a significant difference.

[0197] II. Experimental Results

[0198] 1. Micro CT Analysis

[0199] Five sets of samples were subjected to micro-CT scanning, resulting in 3D reconstructed images and corresponding longitudinal section DICOM images of the defects, as shown below. Figure 21 As shown, in the blank control group, almost no new bone formation was observed at the defect site 8 weeks post-surgery, indicating that the rats' own repair capabilities alone could not repair a 5mm skull defect. In contrast, the 10HA-GM90-30s double-layer membrane group showed significant new bone formation at the defect site 8 weeks post-surgery, almost completely repairing the defect. Its repair effect was comparable to... The positive control group showed almost no difference, but Dicom cross-sectional images showed that the morphology and thickness of the new bone at the defect site were not similar. The positive control group was uniform. New bone formation was also observed at the defect site 8 weeks postoperatively in the GM90-30s dense monolayer membrane group and the 10HA loose monolayer membrane group, but the amount of new bone formation was significantly less than that in the double membrane group. Dicom cross-sectional images showed that the new bone formation in the monolayer membrane group was thinner and could not completely fill the defect site.

[0200] Micro CT data analysis results as follows Figure 22 As shown, the bilayer membrane group had the highest values ​​for both BV / TV and BMD, which were significantly different from the blank control group (p<0.01). There were no statistically significant differences between the positive and control groups. The BV / TV and BMD values ​​in the GM90-30s dense monolayer membrane group were both greater than those in the 10HA loose monolayer membrane group. Although the BV / TV and BMD values ​​in both monolayer membrane groups were higher than those in the blank control group, they were significantly lower than those in the bilayer membrane group. Positive control group (p<0.05). Micro-CT analysis showed that the bilayer GBR barrier membrane prepared using 10 mg / ml hydroxyapatite nano-hydroxyapatite-methacryloyl gelatin as the loose layer and GM90-30s methacryloyl gelatin as the dense layer... Barrier membranes have similar defect repair capabilities.

[0201] 2. HE staining results

[0202] The results of HE staining of the five groups of samples are as follows Figure 23 As shown in the figure. Similar to the results of MicroCT reconstruction, no new bone formation was observed in the defect area in the blank control group, which was mainly covered by soft tissues such as fibrous tissue. New bone formation was observed in the defect area of ​​the nano-hydroxyapatite-methacryloyl gelatin double barrier membrane group, connecting the fracture ends. The new bone was dense and thick, and stained the most deeply. New bone formation was also observed in the defect areas of the GM90-30s dense single-layer membrane group and the 10HA loose single-layer membrane group, but the amount of new bone was small and could not completely cover the defect area, which was still covered by soft tissue. The positive control group also showed significant new bone formation, completely covering the defect area with uniform bone quality. No obvious inflammatory response was observed in any group, indicating good tissue compatibility of the barrier membrane in each group.

[0203] 3. Masson staining results

[0204] Masson staining results of five groups of samples are as follows Figure 24 As shown, type I collagen in bone can be stained red; the deeper the red, the more mature the bone. New bone appears as alternating red and blue. The 10HA-GM90-30s double-layer membrane and... In the positive control group, there was a large amount of bone formation. The bone surface in the defect area was relatively mature, and the red staining was deep. The new bone completely covered the defect area. The GM90-30s and 10HA single-layer membrane groups also had new bone formation, but the new bone stained lightly red. The bone maturity was worse than that of the bilateral membrane and the positive control group. The new bone only covered part of the defect area. In the blank control group, no new bone formation was observed at the defect site. Only a large amount of soft tissue covered the defect area.

[0205] 4.2.3 IHC staining results

[0206] Immunohistochemical results of five groups of samples are as follows Figure 25 , 26 As shown in Figure 27, three groups of indicators were subjected to immunohistochemical staining: OCN osteogenic specific indicator, Col I mid-osteogenic indicator, and CD31 angiogenesis indicator. A brown staining result indicates that the indicator is positive. Figure 25 The results of OCN staining showed that, except for the blank control group, all groups had positive staining at the site of new bone formation, with the 10HA-GM90-30s double-layer membrane group and The positive control group showed the deepest staining. Statistical results showed that the 10HA-GM90-30s bilayer membrane group exhibited the strongest positive staining, with a statistically significant difference compared to the blank control group. There was no statistically significant difference between them.

[0207] Figure 26 The Col I staining results show that positive staining was observed at the new bone formation sites at the defect margins in all groups, but the 10HA-GM90-30s double-layer membrane group and... The positive control group showed the deepest staining, while the blank control group showed the lightest staining, indicating that the 10HA-GM90-30s bilayer membrane group and... The positive control group showed the best osteogenic activity, and the statistical results also showed the same trend.

[0208] Figure 27 The CD31 staining results show that angiogenesis was present at the defect sites in all groups, except... In the positive control group, intraosseous vessels were observed, while in the other groups, neovascularization was mostly located within soft tissue. Although neovascularization was observed in the 10HA-GM90-30s double-layer membrane group, it was not significantly different from that in the GM90-30s double-layer membrane group. The positive control group showed less angiogenesis in the soft tissue, which may be related to the fact that new bone had basically formed and covered the defect area when the two groups were sampled, so the blood supply demand was less. However, the immunohistochemical results showed that the staining depth was still the deepest in the two groups, and the difference was not statistically significant.

[0209] The repair effect of extreme defects can be scored according to the following criteria: ① 4 points: The new bone bridge completely spans the maximum diameter of the defect; ② 3 points: The new bone bridge only covers a part of the maximum diameter of the defect and does not completely cover the defect; ③ 2 points: New bone is generated only at the edge of the extreme defect; ④ 1 point: There are only scattered punctate new bone at the defect site; ⑤ 0 points: No new bone formation.

[0210] The reconstruction and analysis results of MicroCT showed that the nano-hydroxyapatite-methacryl gelatin bilayer membrane had the highest BV / TV and BMD values, and... There was no statistically significant difference compared to the positive control group. Furthermore, the repair effect of the five groups of extreme defects could be scored based on the three-dimensional reconstructed images. The blank control group only showed a small amount of new bone at the defect edge and sporadic punctate new bone in the center of the defect, indicating that a 5 mm defect indeed cannot be repaired by the body itself; its score was between 1 and 2 points. In contrast, the 10HA-GM90-30s double-layer membrane group and... In the first group, the defect was almost completely repaired by new bone formation, which covered the maximum diameter of the defect, scoring 4 points. In the GM90-30s and 10HA single-layer membrane groups, although new bone formation occurred, the area of ​​new bone was smaller and could not completely cover the maximum diameter of the defect, scoring 3 points. The results indicate that the double-layer membrane group had the best repair effect compared to the single-layer membrane group, and its effect was comparable to... The positive control group was comparable.

[0211] As can be seen from the above embodiments and experimental examples, the present invention, by optimizing the material composition and preparation process of the double-layer artificial bone membrane, has obtained a double-layer artificial bone membrane that has excellent repair effect on bone defects, and at the same time has good swelling coefficient, mechanical strength and degradation rate, and has good application prospects.

Claims

1. A double-layered artificial periosteum for guided bone tissue regeneration, characterized in that: It is composed of a dense layer and a loose layer; The dense layer is prepared by photocrosslinking with PBS buffer containing the following raw materials in the specified mass-volume fractions: 10% methacryloyl gelatin with a grafting degree of 90% Photoinitiator 0.5%; The dense layer undergoes photocrosslinking for 30 seconds; The loose layer was prepared by photocrosslinking with PBS buffer containing the following raw materials in the following mass-volume fractions: 10% methacryloyl gelatin with a grafting degree of 60% Photoinitiator 0.5%, Nano-sized hydroxyapatite 10 mg / ml; The time for photocrosslinking of the loose layer is 10 seconds.

2. The method for preparing the double-layer artificial periosteum for guided bone tissue regeneration as described in claim 1, characterized in that, Includes the following steps: Step 1: Add the raw materials for preparing the dense layer and PBS buffer into the mold and perform photocrosslinking; Step 2: Add the raw materials for preparing the loose layer and PBS buffer to the mold that has formed the dense layer, and perform photocrosslinking; Step 3: Vacuum freeze-drying to obtain the product.

Images