Preparation method and application of functional gradient layered bone-imitating GBR membrane

Abstract

The invention relates to a preparation method and application of a functional gradient layered bone-imitating GBR (Guarantee Boron Reactor) membrane, the GBR membrane is a novel GBR membrane obtained by taking decellularized fish scales as a base material, constructing an EGCG-fish scale complex through EGCG cross-linking modification and then combining biomimetic mineralization, and the GBR membrane has certain mechanical properties and can maintain a space required by bone regeneration; meanwhile, mineralized hydroxyapatite wraps fish scale collagen, so that the degradation speed of fish scales is reduced, the action time is prolonged, and sufficient time is provided for bone repair; in addition, the mineralized fish scales are similar to natural cortical bones in structure and composition, a cell growth microenvironment is simulated, good biocompatibility is achieved, adhesion and differentiation of osteoblasts are promoted, and the healing speed is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, particularly relates to bone regeneration materials, and specifically relates to a preparation method and application of a functionally graded layered bone-like GBR membrane. Background Art

[0002] Bone defects can be caused by various reasons, including trauma, tumors, congenital deformities, and various diseases. Widely used guided bone regeneration (GBR) membrane materials, such as collagen, polytetrafluoroethylene, and other synthetic polymers, still have the following problems when solving critical bone defects: ① insufficient mechanical strength and poor space maintenance ability; ② easy to degrade and unstable degradation, resulting in the destruction of its barrier function; ③ no obvious promotion effect on the healing of hard and soft tissues. Therefore, it is necessary to construct a GBR membrane with excellent biocompatibility, mechanical properties, degradation properties, and osteoinductive activity.

[0003] Simulating the bone structure is widely regarded as a bone defect repair method with great potential. Natural bone tissue has a highly ordered multi-layered structure, and its main components include type I collagen and apatite crystals. Its multi-level structure exhibits outstanding mechanical and physical properties. Although researchers are committed to imitating the bone structure, for example, Liang Chen (The effect of fish scale collagen membrane on the adhesion, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells [D]. Sichuan: Sichuan University, 2021). This study prepared a decellularized fish scale collagen membrane (FS) from grass carp scales, and after characterizing its physicochemical properties, in vitro cell experiments were carried out. The results showed that FS had good thermal stability and cell compatibility, could promote cell proliferation, and was outstanding in the late stage of osteogenic differentiation, and had potential application prospects in the field of bone tissue regeneration. However, so far, an innovative repair material with a certain mechanical mechanical support to maintain the osteogenic space and a bundle-like bone hierarchical structure has not been successfully developed.

[0004] Therefore, it is of great significance to develop a guided bone regeneration membrane with a layered simulated bone structure with good mechanical properties, degradation properties, and osteogenic activity. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of a functionally graded layered bone-like GBR membrane, and to develop an innovative repair material with a bone hierarchical structure, which has good mechanical properties, degradation properties, and osteogenic activity.

[0006] The present invention realizes the above purpose through the following technical solutions: The present invention provides a preparation method of a functionally graded layered bone-like GBR membrane derived from the fish scale structure. The decellularized fish scales obtained by decellularizing fresh fish scales are sequentially subjected to cross-linking treatment with an EGCG solution and modification treatment with a biomimetic mineralization solution to obtain the GBR membrane.

[0007] As a further optimized solution of the above invention, it includes the following steps: (1) Select fresh fish scales, perform decellularization treatment to obtain decellularized fish scales FS, and place the decellularized fish scales FS into an EGCG solution at room temperature for cross-linking reaction to obtain a modified decellularized fish scale EGCG-FS material; (2) Place the modified decellularized fish scale EGCG-FS material into a biomimetic mineralization solution, mineralize it in a constant temperature shaker for 5 days, rinse it, and then store it by freeze-drying to obtain biomimetic mineralized decellularized grass carp scales EGCG-FS5d, that is, the GBR membrane.

[0008] As a further optimized solution of the above invention, the decellularization treatment of the fresh fish scales is: using a combined physical-chemical enzyme decellularization method to remove the original cell components in the grass carp scales to obtain a decellularized grass carp scale material.

[0009] As a further optimized solution of the above invention, the decellularization treatment of the fresh fish scales specifically includes the following steps: (1) Immerse the fresh fish scales in an aqueous solution containing NaOH and H2O2 for decolorization and degreasing, and then undergo at least 4 freeze-thaw cycles; (2) After the freeze-thaw cycle ends, use Triton X-100 solution, PBS solution, and sodium dodecyl sulfate solution to perform at least 2 chemical decellularization treatments on the fish scales; (3) After the chemical decellularization treatment, immerse the fish scales in a solution containing deoxyribonuclease and ribonuclease, and shake them in a shaker at 37°C for 24 hours.

[0010] As a further optimized solution of the above invention, the parameters of the freeze-thaw cycle are: -80°C, freeze for 4 hours, and thaw at 37°C for 30 minutes.

[0011] As a further optimized solution of the above invention, the chemical decellularization treatment is specifically: place the fish scales in a Triton X-100 solution and stir, then immerse the fish scales in a PBS solution for ultrasonic treatment, then immerse the fish scales in a sodium dodecyl sulfate solution and stir, and then immerse them in a PBS solution again for ultrasonic treatment.

[0012] As a further optimized solution of the above invention, the pH value of the EGCG solution is 8.0 - 10.0, and the concentration is 1 - 10 mg / mL.

[0013] As a further optimized solution of the above invention, the raw materials of the biomimetic mineralization solution are 1 - 5 mM soluble calcium salt, 1 - 10 mM soluble phosphate, 100 - 200 mM soluble sodium salt, and 120 - 360 μg / mL water-soluble polymer.

[0014] As a further optimized solution of the above invention, the parameters for the mineralization modification treatment of the bionic mineralization solution are: mineralization in a constant temperature shaker at 37°C and 100 rpm for 5 - 10 days.

[0015] The present invention also provides an application of the functional gradient layered bone-like GBR membrane prepared by the above preparation method in the preparation of bone repair materials, osteogenic materials or bone materials.

[0016] The beneficial effects of the present invention are as follows: The present invention uses natural source fish scales and prepares a GBR membrane with a higher degree of mimicking the natural bone tissue structure by the modification method combining EGCG and bionic mineralization, reproducing the surface chemical properties and structural characteristics at the nanoscale similar to natural bone. The material prepared by this method has good biocompatibility, mechanical strength and appropriate degradation rate, and can provide a microenvironment similar to natural bone for cell growth, thereby promoting the adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Description of the Drawings

[0017] Figure 1 Evaluation before and after decolorization and decellularization of grass carp scales prepared in the experiment: A: Gross view before and after decolorization and decellularization of scales. B: HE staining images before and after decolorization and decellularization of scales. C: DAPI staining image after decolorization and decellularization of scales; Figure 2 For the structure and morphology of decolorized and decellularized fish scales (FS). A: Scanning electron microscope image of the outer surface microstructure of FS. B: Scanning electron microscope image of the inner surface microstructure of FS. C: Scanning electron microscope image of the cross-section of FS. D: Transmission electron microscope image of the cross-section of FS; Figure 3 For the chemical composition characterization of decolorized and decellularized fish scales (FS). A: Mapping diagram of the cross-section of FS. B: Element content and Ca / P ratio in the three layers of FS. C: XRD of the outer surface of FS. D: Infrared of the inner and outer surfaces of FS. E: Thermogravimetry of FS; Figure 4 For EGCG-modified decolorized and decellularized fish scales. A: Infrared spectra before and after EGCG modification. B: Contact angles before and after EGCG modification. C: Zeta potential of the solid surface after EGCG modification; Figure 5 . Scanning and transmission electron microscope images of the mineralized samples and the femurs of SD rats; the inset in the upper right corner is the SAED result; Figure 6Evaluation of the physical and mechanical properties of the GBR membrane prepared for this invention. A: Infrared spectroscopy analysis of FS, EGCG-FS, FS5d, and EGCG-FS5d. B-C: Thermodynamic degradation curves of FS, EGCG-FS, FS5d, and EGCG-FS5d. D: X-ray diffraction analysis of FS, EGCG-FS, FS5d, and EGCG-FS5d. E: Stress-strain curves of FS, EGCG-FS, FS5d, and EGCG-FS5d. F: Tensile elastic modulus of FS, EGCG-FS, FS5d, and EGCG-FS5d. G: Flexural elastic modulus of FS, EGCG-FS, FS5d, and EGCG-FS5d. H: Flexural strength of FS, EGCG-FS, FS5d, and EGCG-FS5d. I: Microhardness of FS, EGCG-FS, FS5d, and EGCG-FS5d. J: Water contact angle of FS, EGCG-FS, FS5d, and EGCG-FS5d. K: In vitro degradation of FS, EGCG-FS, FS5d, and EGCG-FS5d. (*P<0.05, **P<0.01, ***P<0.001); Figure 7 Element content and Ca / P ratio of the three lower layers under the cross-section of the GBR membrane constructed by EGCG-binding biomimetic mineralization modification prepared for this invention; Figure 8 In vitro cell evaluation of the GBR membrane prepared for this invention. A: Biocompatibility detection of RBMSCs in each group at different time points (the blank control group is denoted as BLANK). B: Adhesion of RBMSCs in each group. C: Influence of materials in each group on the morphology of RBMSCs. (*P<0.05, **P<0.01, ***P<0.001); Figure 9 Evaluation of the in vitro osteogenic effect of the GBR membrane prepared for this invention. A: ALP staining of RBMSCs in each group at 4 and 7 days of osteogenic induction and differentiation. B: Results of ALP activity determination of RBMSCs in each group at 4 and 7 days of osteogenic induction and differentiation. C: OCN immunofluorescence images of RBMSCs in each group at 21 days of osteogenic induction and differentiation. D: OCN expression of RBMSCs in each group at 21 days of osteogenic induction and differentiation. E-F: Expression of COL-1 and Runx-2 genes in each group at 7 days of osteogenic induction and differentiation. G-H: Expression of OCN and OPN genes in each group at 7 days of osteogenic induction and differentiation. (*P<0.05, **P<0.01, ***P<0.001); Figure 10In vivo bone tissue regeneration evaluation of the GBR membrane prepared for this invention. (A) Construction of a skull defect model in SD rats; (B) Quantitative analysis of bone volume / total tissue volume (BV / TV); (C) Quantitative analysis of bone mineral density (BMD). (*P<0.05, **P<0.01, ***P<0.001). Detailed implementation manners

[0018] The following further describes the present application in conjunction with the attached drawings. It is necessary to point out here that the following detailed implementation manners are only used for further explanation of the present application and cannot be understood as a limitation on the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0019] 1. Description Epigallocatechin gallate, EGCG, pH = 8.0 - 10.0, concentration 1 - 10 mg / mL.

[0020] Bionic mineralization solution: 1 - 5 mM soluble calcium salt, 1 - 10 mM soluble phosphate, 100 - 200 mM soluble sodium salt, and 120 - 360 μg / mL water-soluble polymer, where the water-soluble polymer can be selected from polyaspartic acid (P-Asp) or polyacrylic acid (PAA).

[0021] The methods used in this invention are all conventional methods known to those skilled in the art unless otherwise specified. The reagents and other materials used are all commercially available products unless otherwise specified. The instruments used are all conventional instruments known to those skilled in the art unless otherwise specified.

[0022] 2. Preparation of decellularized grass carp scales (FS), modified decellularized grass carp scales (EGCG-FS), and bionic mineralized decellularized grass carp scales before and after EGCG crosslinking (FS5d, EGCG-FS5d) (1) Use tweezers to pluck fresh grass carp scales and wash them thoroughly; (2) Immerse the fish scales in step 1 in an aqueous solution containing 1% NaOH and 3% H2O2 for 12 h for decolorization and degreasing. Then place them in a refrigerator at -80 °C for 4 hours, and subsequently put them into a water bath at 37 °C to thaw for about 30 minutes. Such a process is called one freeze-thaw cycle and needs to be repeated 4 times. After the last freeze-thaw cycle, place the fish scales in a 1% (vol / vol) Triton X-100 solution and stir on a magnetic stirrer at room temperature for 24 hours. Subsequently, immerse them in PBS solution and perform ultrasonic treatment 3 times, 10 minutes each time. Then, immerse the fish scales in a 1% sodium dodecylsulfate (SDS) solution and stir on a magnetic stirrer at room temperature for 24 hours, and immerse them in PBS solution again for ultrasonic treatment 3 times, 10 minutes each time. Such a process is called one chemical decellularization, and this process needs to be repeated 2 times. After that, take out the fish scales, immerse them in a solution containing 150 U / ml deoxyribonuclease (Danes) and 100 μg / ml ribonuclease (Rnase), and shake in a shaker at 37 °C for 24 hours.

[0023] (3) Thoroughly rinse the fish scale material obtained in step 2, freeze-dry and store it to obtain decellularized grass carp fish scales FS ( Figure 1 ); (4) At room temperature, place the material obtained in step 2 into a 2 mg / ml EGCG solution (pH = 9.0) for crosslinking for 24 h. Thoroughly rinse the obtained fish scale material, freeze-dry and store it to obtain EGCG-modified decellularized grass carp fish scales (EGCG-FS) ( Figure 4 ); (5) Put the materials obtained in steps 2 and 4 into a mineralization solution (the raw materials of the mineralization solution are: 1.67 mM CaCl2, 9.5 mM Na2HPO4, 150 mM NaCl and 240 μg / mL P-Asp), and mineralize in a constant temperature shaker at 37 °C and 100 rpm for 5 days. Thoroughly rinse the obtained materials, freeze-dry and store them to obtain biomimetic mineralized decellularized grass carp fish scales before and after EGCG crosslinking (FS5d, EGCG-FS5d) ( Figure 5 ).

[0024] 3. Characterization, effects and properties of FS, EGCG-FS, EGCG-FS5d materials 3.1 Characterization of FS materials The FS material obtained in 2.1-(3) removed the original cellular components on the premise that the structure was not damaged ( Figure 1 ).

[0025] SEM shows that the outer surface of the fish scale is composed of ridges and radial grooves, and is divided into three regions: the base region, the top region, and the side region ( Figure 2 A). The inner surface of fish scales is composed of dense collagen fibers, and the collagen fibers are arranged in an orderly staggered manner ( Figure 2 B). From the cross-section, the fish scale structure is mainly composed of three layers: the limiting layer (LL), the outer elastic layer (EE) and the inner elastic layer (IE) ( Figure 2 C). Transmission electron microscopy shows that LL in fish scales is composed of randomly distributed mineralized collagen fibers ( Figure 2 D). In the inner and outer elastin layers, adjacent collagen fiber layers are arranged in parallel to form a plywood-like structure. In addition, in the elastin layer, a clear mineralized boundary between the EE layer and the IE layer can be seen ( Figure 2 D).

[0026] The cross-sectional mapping diagram shows that fish scales mainly contain oxygen, phosphorus, nitrogen, carbon and calcium, and the calcium and phosphorus content gradually decreases from LL to IE ( Figure 3 A). Quantitative analysis further elucidated the composition of the different layers, where the weight percentages of calcium in LL, EE, and IE were 6.12%, 5.32%, and 0.02%, respectively ( Figure 3 B). XRD of the outer surface of fish scales shows obvious peaks at 2θ=25.8 (002), 2θ=31.7 (211), 2θ=39.23 (321), 2θ=49.66 and 2θ=53.1 (004), which match the standard peaks of hydroxyapatite (Ca10(PO4)6(OH)2) ( Figure 3 C). The strong and broad peak at 1024 cm-1 on the outer surface of the fish scale is the stretching vibration of PO43-, and the peaks at 869 cm-1 and 1400 cm-1 correspond to the in-plane bending vibration peaks of CO32-. At the same time, the peak at 1652 cm-1 is the bending vibration peak of -OH, and 579 and 607 cm-1 are the characteristic peaks of the bending mode of PO43; the inner surface has a peak at 1653 cm-1 for the vibration peak of amide I with C=O, 1558 cm-1 for the bending vibration peak of amide II with NH, 1249 cm-1 for the characteristic peak of amide III, and 1451 cm-1 and 1379 cm-1 are caused by the bending vibration of the CH bond. In addition, the peaks at about 3300 cm-1 corresponding to the amide A peak (NH stretching combined with hydrogen bonding) and 2920 cm-1 corresponding to the amide B peak (CH asymmetric stretching) ( Figure 3D). Therefore, it can be determined that the protein is an organic component in fish scales. Thermogravimetric analysis shows that the water content of fish scales is 10.67%, the organic components account for 56.71%, and the inorganic components account for 32.62%. In the degradation curve, three significant peaks can be observed. The peak at 80 - 100 °C is attributed to the decomposition of water molecules in the fish scale matrix, the peak in the range of 330 - 400 °C is due to the degradation of collagen organic matter, and the small peak at 400 - 500 °C is caused by the CO2 released from the thermal decomposition of proteins at high temperatures ( Figure 3 E).

[0027] 3.2 Effect of EGCG modification in EGCG-FS For the EGCG-FS material obtained in 2.1-(4), compared with FS, the absorption peak intensities of EGCG-FS are enhanced at the five peaks of amide A, B, I, II, and III. In addition, a slight red shift is observed in the amide A and B peaks, while a slight blue shift is observed in the amide I, II, and III peaks ( Figure 4 A). It shows that there is a hydrogen bond interaction between EGCG and collagen. EGCG-FS exhibits higher hydrophilicity than FS, 72.96 ± 1.13 vs 60.14 ± 1.82 ( Figure 4 B). The zeta potential of EGCG-FS decreases from -7.91 ± 0.19 mV to -22.78 ± 0.23 mV ( Figure 4 C).

[0028] 3.3 Effect and difference of mineralization in FS5d and EGCG-FS5d For the FS5d and EGCG-FS5d materials obtained in 2.1-(5), from the SEM images, it can be seen that the hydroxyapatite (HA) crystals on the inner surface of FS5d show a random and disordered growth state on the collagen fibers, and the degree of mineralization is relatively low. Exposed collagen fibers can still be observed ( Figure 5 ). On the inner surface of EGCG-FS5d, the HA crystals grow orderly along the c-axis of the collagen fibers, and the collagen fibers are completely mineralized, and no exposed collagen fibers can be observed ( Figure 5 ). From the TEM images, obvious mineral depositions can be seen in FS5d and EGCG-FS5d ( Figure 5 A). Among them, the mineralization of FS5d is uneven, and mineralization does not occur in some areas, and the HA is arranged disorderly ( Figure 5 A). In EGCG-FS5d, the mineralization process shows the characteristics of uniformity. The HA crystals are distributed in the void areas between the collagen fibers and their vicinity, and are arranged along the c-axis direction of the collagen fibers ( Figure 5A). The results of selected area electron diffraction (SAED) experiments on the selected area showed that the mineral phase on the collagen fibers was HA. In EGCG-5d, the (002) diffraction arc presented an obvious symmetric arc, indicating a clear direction of HA crystal arrangement. In FS5d, a complete ring feature was presented ( Figure 5 A). EGCG-FS5d was similar to the mineralized collagen fibers in natural bone. The collagen fibers showed an ordered arrangement, and the HA crystals were distributed along the long axis direction of the collagen fibers ( Figure 5 A). EGCG-FS5d reproduced the composite structure of hydroxyapatite in the collagen fibers of natural bone, achieving a high degree of biomimesis, which was beneficial to the regeneration and repair of bone tissue.

[0029] Quantitative analysis further clarified the composition of different layers of the GBR membrane constructed by EGCG-binding biomimetic mineralization. The weight percentages of calcium in LL, EE, and IE were 12.44%, 5.34%, and 6.60% respectively( Figure 7 ), and the results showed that the functional gradient characteristics still existed even after 5 days of mineralization cycle.

[0030] 3.4 Evaluation of the physical and mechanical properties of FS, EGCG-FS, and EGCG-FS5d materials ATR-FTIR was used to monitor the formation of mineral crystals during the fiber mineralization process. After the mineralization treatment, significant absorption peaks appeared on the inner surface of FS at 1020 cm−1, 560 cm−1, and 602 cm−1. These peaks corresponded to the bending vibration modes υ3PO4, υ4PO4, and υ2PO4 of the phosphate group (O-P-O) in the hydroxyapatite (HA) crystal ( Figure 6 A). Compared with FS5d, the absorption peaks of EGCG-FS5d at the corresponding wavelengths were more significant. The mineral content and thermodynamic degradation mode of the mineralized fish scales were detected by thermogravimetry. The mineral contents of FS5d and EGCG-FS5d were 38.07 and 45.40 respectively( Figure 6 B-C). The X-ray diffraction (XRD) patterns of FS5d and EGCG-FS5d matched well with the standard peaks of hydroxyapatite (Ca10 (PO4)6 (OH)2), confirming that the mineral components on the inner surface of the mineralized fish scales were all hydroxyapatite, and the peak value of EGCG-FS5d was higher than that of FS5d ( Figure 6 D). Therefore, the degree of mineralization of FS5d was lower than that of EGCG-FS5d.

[0031] The elastic modulus of EGCG-FS5d was up to 405.45±59.93 MPa. Although the elastic moduli of EGCG-FS (219.70±31.06 MPa) and FS5d (227.49±27.10 MPa) were greater than that of FS (168.96±48.03 MPa), there was no statistical difference (P>0.05) ( Figure 6 E-F). Compared with FS, EGCG-FS, and FS5d, EGCG-FS5d had good flexural strength (15.19±2.94 MPa) and flexural elastic modulus (1.10±0.25 GPa) ( Figure 6 G-H). The microhardness value of EGCG-FS5d (29.46±1.36) was higher than that of FS, EGCG-FS, and FS5d ( Figure 6 I).

[0032] The contact angles measured by FS were 77.44±2.18, those by EGCG-FS were 59.45±6.03, and those by FS5d were 43.61±6.18. The droplets on the surface of the EGCG-FS5d group disappeared instantly only within a few seconds after contact and could not be measured ( Figure 6 J). This indicated that the hydrophilic effect of EGCG-FS5d was excellent. The in vitro degradation rate showed that the weights of each group (FS, EGCG-FS, FS5d, and EGCG-FS5d) decreased during the whole process, but the degradation rate of the FS group was the largest from the beginning to the end. In contrast, compared with the FS group, the weight loss of the EGCG-FS, FS5d groups, and EGCG-FS5d group was significantly reduced ( Figure 6 K). Among them, the EGCG-FS5d group had the least weight loss at each time point. The above results showed that cross-linking modification of EGCG biomimetic mineralization could effectively improve the mechanical properties, hydrophilic properties of the material, and slow down the degradation rate.

[0033] 3.5 Effects of FS, EGCG-FS, and EGCG-FS5d materials on the growth and behavior of RBMSC cells Rat bone marrow mesenchymal stem cells (RBMSCs) were seeded on FS, EGCG-FS, and EGCG-FS5d materials, and the group cultured on the bottom of the well plate was the Blank group. On the 1st, 3rd, 5th, and 7th days, as the culture time extended, the absorbance of each group gradually increased. Moreover, there was no statistical difference between FS, EGCG-FS, EGCG-FS5d and the Blank group (P>0.05), indicating that FS, EGCG-FS, and EGCG-FS5d had good biocompatibility ( Figure 8 A).

[0034] Using the same culture method, the adhesion of RBMSCs on each group of samples was observed by scanning electron microscopy after 24 h of culture. In each group, RBMSCs were fully spread out and extended pseudopodia. In the Blank group, RBMSCs were randomly oriented, while in FS, EGCG-FS, and EGCG-FS5d, RBMSCs grew orderly along the collagen fibers of the fish scales ( Figure 8 B). The cell morphology was observed by optical microscopy at 1, 3, 5, and 7 days of culture. As the culture time extended, the cell number gradually increased, reaching about 70-80% on the 7th day. And in FS, EGCG-FS, and EGCG-FS5d, the cells were spindle-shaped or long spindle-shaped and grew in a certain direction ( Figure 8 C). In EGCG-FS5d, it was visible that the cells extended plasmodesmata, which was considered the state of cell differentiation. The experiment showed that the material modified by EGCG combined with biomimetic mineralization not only had good biocompatibility, but also could regulate the cell growth direction and promote cell differentiation.

[0035] 3.6 Osteogenic differentiation ability of FS, EGCG-FS, and EGCG-FS5d materials in vitro Rat bone marrow mesenchymal stem cells (RBMSCs) were seeded on FS, EGCG-FS, and EGCG-FS5d materials, and the group cultured on the bottom of the well plate was used as the Blank group. The osteogenic potential of EGCG-FS5d was evaluated by measuring the alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression level. At 4 and 7 days of culture, the ALP staining results showed that obvious dark blue granular deposits were present on the membranes of each group, and the staining in the EGCG-FS5d group was the most obvious ( Figure 9 A). Since the samples were brown themselves, it might affect the observation of the ALP staining results. In this study, the ALP staining results needed to be further corroborated by the ALP activity detection results. The quantitative detection of ALP activity showed that as the culture time extended, the enzyme activity of each group also gradually increased. And compared with other groups, the enzyme activity of EGCG-FS5d was the highest ( Figure 9 B). After 21 days of culture, the OCN staining results showed obvious green fluorescence signals in each group, indicating positive cyanate staining. Among them, the green fluorescence signal in EGCG-FS5d was the strongest, suggesting the strongest expression of OCN in the cells ( Figure 9 C). The Elisa results of OCN also showed the same trend, that is, the expression level of OCN in EGCG-FS5d was significantly higher than that of other groups ( Figure 9 D). Detection of osteogenesis-related genes in vitro ( RUNX2 , Col-1 , OCN , OPN), indicating that the expression levels of osteogenesis-related genes in EGCG-FS5d were significantly enhanced compared with those in other groups ( Figure 9 E-H). The above results show that the material combined with EGCG and biomimetic mineralization has good ability to induce osteogenic differentiation of rBMSCs cells.

[0036] 3.7 Guided bone tissue regeneration effect of EGCG-FS5d material and commercial GBR membrane (Heal-All) in vivo EGCG-FS5d and Heal-All materials were covered on the critical bone defects of the skulls of SD rats ( Figure 10 A), and the bone defect without any treatment was used as the control group. The bone tissue regeneration effect was observed at 4 weeks and 8 weeks. The three-dimensional reconstruction images of the skull defects of SD rats at 4 weeks and 8 weeks after surgery showed that the amount of bone defect regeneration in each group gradually increased with time. Especially for EGCG-FS5d, it showed better bone defect repair effect than the commercial Heal-ALL membrane at the 4th week and the 8th week ( Figure 10 A). Quantitative analysis of the Micro-CT scan results was performed to obtain bone volume / bone tissue volume (BV / TV) and bone mineral density (BMD). At the 4th week and the 8th week after implantation, compared with the blank control group and the Heal-ALL group, the BMD value of the EGCG-FS5d group increased significantly; in addition, the BV / TV value of the EGCG-FS5d group was significantly higher than that of the blank control group and the Heal-All group ( Figure 10 B-C). The above results show that the material constructed by combining EGCG and biomimetic mineralization can significantly guide bone tissue regeneration in vivo.

[0037] 4. Conclusion In summary, the present invention uses natural grass carp scales and prepares a GBR membrane with a high degree of mimicking the natural bone tissue structure by a modification method of combining EGCG and biomimetic mineralization, reproducing the surface chemical properties and structural characteristics at the nanoscale similar to natural bone. This material has good biocompatibility, mechanical strength and appropriate degradation rate, and can provide a microenvironment similar to natural bone for cell growth, thereby promoting the adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and is very suitable for preparing bone repair materials, osteogenic materials or bone materials.

[0038] The above embodiments only represent several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A preparation method of a functionally graded layered bone-like GBR membrane, characterized in that, The decellularized fish scales obtained by decellularizing fresh fish scales are sequentially subjected to crosslinking treatment with an EGCG solution and modification treatment with a biomimetic mineralization solution to obtain the GBR membrane.

2. The preparation method of a functionally graded layered bone-mimicking GBR membrane according to claim 1, wherein It includes the following steps: (1) Select fresh fish scales and perform decellularization treatment to obtain decellularized fish scales FS. At room temperature, place the decellularized fish scales FS into an EGCG solution for crosslinking reaction to obtain a modified decellularized fish scale EGCG-FS material; (2) Put the modified decellularized fish scale EGCG-FS material into a biomimetic mineralization solution and mineralize it in a constant temperature shaker for 5 days. After rinsing, store it by freeze-drying to obtain biomimetic mineralized decellularized grass carp fish scales EGCG-FS5d, that is, the GBR membrane.

3. The preparation method of a functionally graded layered bone-like GBR membrane according to claim 1, characterized in that, The decellularization treatment of the fresh fish scales is: using a physical, chemical and enzymatic combined decellularization method to remove the original cell components in the fish scales to obtain a decellularized fish scale material.

4. The preparation method of a functionally graded layered bone-mimicking GBR membrane according to claim 3, characterized in that, The decellularization treatment of the fresh fish scales specifically includes the following steps: (1) Immerse the fresh fish scales in an aqueous solution containing NaOH and H2O2 for decolorization and defatting, and then go through at least 4 freeze-thaw cycles; (2) After the freeze-thaw cycle ends, use Triton X-100 solution, PBS solution and sodium dodecyl sulfate solution to perform at least 2 chemical decellularization treatments on the fish scales; (3) After chemical decellularization treatment, immerse the fish scales in a solution containing deoxyribonuclease and ribonuclease, and shake them in a shaker at 37°C for 24 h.

5. The preparation method of a functionally graded layered bone-mimicking GBR membrane according to claim 4, wherein The parameters of the freeze-thaw cycle are: -80°C, freeze for 4 h, and thaw at 37°C for 30 min.

6. The preparation method of a functionally graded layered bone-mimicking GBR membrane according to claim 4, characterized in that, The chemical decellularization treatment is specifically: place the fish scales in a Triton X-100 solution and stir, then immerse the fish scales in a PBS solution for ultrasonic treatment, then immerse the fish scales in a sodium dodecyl sulfate solution and stir, and then immerse them in a PBS solution again for ultrasonic treatment.

7. The preparation method of a functionally gradient layered bone-mimicking GBR membrane according to claim 1, characterized in that The pH value of the EGCG solution is 8.0 - 10.0, and the concentration is 1 - 10 mg / mL.

8. The preparation method of a functionally graded layered bone-mimicking GBR membrane according to claim 1, characterized in that, The raw materials of the biomimetic mineralization solution are 1 - 5 mM soluble calcium salt, 1 - 10 mM soluble phosphate, 100 - 200 mM soluble sodium salt and 120 - 360 μg / mL water-soluble polymer.

9. The preparation method of a functionally graded layered bone-mimicking GBR membrane according to claim 8, characterized in that, The parameters for the mineralization modification treatment of the biomimetic mineralization solution are: mineralize in a constant temperature shaker at 37°C and 100 rpm for 5 - 10 days.

10. Application of the functional gradient layer-like bone-mimicking GBR membrane prepared by the preparation method according to any one of claims 1 - 9 in the preparation of bone repair materials, osteogenic materials or bone materials.

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