Guided bone regeneration membrane, its preparation method and its application

Abstract

The present invention provides a guided bone regeneration membrane, a preparation method thereof, and an application thereof. The guided bone regeneration membrane includes a substrate layer, the substrate layer containing at least 99% by weight of zinc; and at least one self-assembled mineralized collagen layer, the self-assembled mineralized collagen layer being disposed on the substrate layer and containing collagen fibers and apatite crystals bonded to the collagen fibers. The guided bone regeneration membrane of the present invention not only functions as a barrier, but also has good biocompatibility, degradability, osteoinductivity, and uniform slow release of zinc, solving the problem that the treatment application effect of the existing guided bone regeneration membrane is limited by the single material structure.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and in particular to a guided bone regeneration membrane, a preparation method thereof and an application thereof. Background Art

[0002] Guided Bone Regeneration Membrane (GBR) technology is a treatment method that uses a barrier membrane to achieve bone tissue regeneration. Among them, the GBR membrane, as the key to GBR technology, can seal the bone defect area, prevent non-osteoblasts from growing in, and create a good environment for bone tissue regeneration. The ideal GBR membrane should have good biocompatibility, a suitable degradation rate, and sufficient support to seal the defect.

[0003] Currently, GBR membranes on the market can be divided into absorbable membranes and non-absorbable membranes. Among them, the mainstream absorbable collagen membranes have insufficient mechanical properties and uncontrollable degradation rates; while non-absorbable membranes require secondary surgery to remove, which can easily cause wound exposure and infection. In addition, existing GBR membrane materials often lack bone inductivity and require bone filling materials, and cannot have both degradability and mechanical support properties.

[0004] The optimization and modification of existing GBR membranes focus on: component optimization such as loading bioactive factors and stem cells, structural optimization such as surface modification, etc. Degradable metal zinc has attracted extensive attention due to its good degradation performance and biocompatibility. However, since the release of excessive zinc ions can cause cytotoxicity and inhibit osteogenesis, finding a suitable method to slowly release zinc ions and promote osteogenesis is very important for guiding bone regeneration.

[0005] However, the prior art still lacks a biocompatible GBR membrane that can both slowly release zinc ions and guide bone regeneration. Summary of the invention

[0006] In view of the shortcomings of the existing GBR membrane, the problem to be solved by the present invention is to prepare a new double-layer GBR membrane - covalent self-assembled collagen zinc membrane, which combines zinc and hierarchical functional mineralized collagen (Hierarchical Mineralized Collagen / Zn via Surface Covalent Modification and Self-Assembly, ss-HMC / Zn) through covalent surface modification and self-assembly, so that it not only plays a barrier function, but also has good biocompatibility, degradability, bone induction, and uniform sustained release of zinc, thereby solving the problem that the existing GBR membrane has a single material structure that limits its therapeutic application effect.

[0007] In order to solve the above technical problems, this application adopts the following technical solutions:

[0008] 1. A guided bone regeneration membrane, comprising: a substrate layer, the substrate layer comprising at least 99 wt% zinc; and at least one self-assembled mineralized collagen layer, the self-assembled mineralized collagen layer being disposed on the substrate layer and comprising collagen fibers and apatite crystals adhered to the collagen fibers.

[0009] Optionally, the mineralized collagen layer is formed by first forming a porous collagen scaffold layer on the substrate layer and then catalyzing the formation of a mineralization solution within the pores of the collagen scaffold layer with polyacrylic acid, wherein the composition of the mineralization solution is:

[0010]

[0011] Optionally, relative to the total amount of the mineralization solution, the concentration of polyacrylic acid is 0.20 - 0.30 mM.

[0012] Optionally, the substrate layer is a surface-pretreated zinc film, wherein the surface pretreatment is selected from any one of acid etching pretreatment in a phosphoric acid solution with a pH value of 2 - 3 and mechanical grinding pretreatment with sandpapers of various different particle sizes.

[0013] Optionally, relative to the total thickness of the guided bone regeneration membrane, the thickness of the mineralized collagen layer accounts for 74 - 80%, wherein in the mineralized collagen, the content of the collagen fibers is 80 wt% - 85 wt%, and the content of the apatite crystals is 15 wt% - 20 wt%.

[0014] Optionally, the guided bone regeneration membrane has a yield strength of 45 - 80 MPa, an ultimate tensile strength of 55 - 80 MPa, and a water contact angle of 30 - 53°.

[0015] 2. A method for preparing a guided bone regeneration membrane, comprising the following steps:

[0016] S1 - Provide a substrate layer, the substrate layer comprising at least 99 wt% zinc;

[0017] S2 - Apply a collagen solution on the substrate layer to self-assemble the collagen into a collagen fiber layer and dry and shape it to form a porous scaffold layer; and

[0018] S3 - Add a mineralization solution into the pores of the porous scaffold layer, perform mineralization treatment, and catalyze the formation of apatite crystals from the mineralization solution within the pores of the collagen scaffold layer, thereby obtaining the guided bone regeneration membrane.

[0019] Optionally, before step S2, the substrate layer is surface-pretreated, and then the collagen solution is applied to the pretreated substrate layer, wherein the surface pretreatment comprises at least one of the following:

[0020] C) Acid-etching pretreatment of the substrate layer in a mixed solution of zinc nitrate and phosphoric acid with a pH value of 2 - 3, or

[0021] D) Mechanically grinding the substrate layer successively with silicon carbide sandpapers of 600, 800, 1200, 1500, and 2000 meshes.

[0022] Optionally, step S2 includes dropping a type I procollagen solution with a concentration of 0.5 - 2.0 mg / ml onto the substrate layer at a rate of 0.5 to 2 ml / min, self-assembling for 24 - 48 hours, and then freeze-drying for 12 - 24 hours to form a dry shape.

[0023] Optionally, the method further includes ultrasonically cleaning the surface-treated substrate with distilled water, acetone, and absolute ethanol at a temperature of 40 - 60 °C for 1 - 2 minutes after surface pretreatment.

[0024] Optionally, step S3 includes adding 1 - 3 ml of the mineralization solution to each of the pores, replacing the mineralization solution every 2 - 3 days, and continuously mineralizing at 25 - 37 °C for 5 - 10 days.

[0025] 3. Use of a guided bone regeneration membrane in the preparation of a medical facility for promoting in-situ bone regeneration in vivo.

[0026] Optionally, the promotion of in-situ bone regeneration in vivo is selected from at least one of promoting in-situ bone regeneration in vivo by promoting cell recruitment, promoting in-situ bone regeneration in vivo by promoting M2 macrophage polarization, promoting in-situ bone regeneration in vivo by enhancing vascularization, and promoting in-situ bone regeneration in vivo by activating the BMP2-related pathway.

[0027] Compared with the GBR membranes currently used clinically, the ss-HMC / Zn membrane of the present invention has the following advantages:

[0028] Compared with the existing GBR collagen membranes, it has high mechanical strength, strong support performance, and a controllable degradation rate; compared with the existing GBR artificial synthetic polymer membranes such as polytetrafluoroethylene and titanium metal membranes, it has strong biocompatibility, can be degraded in vivo, does not require secondary removal, and can guide and promote in-situ bone regeneration without adding exogenous growth factors and cells. Brief Description of the Drawings

[0029] Figure 1 Shows A: A process flow chart of a method for preparing a GBR membrane according to an embodiment of the present invention; B: A schematic diagram of the preparation process of a GBR membrane according to an embodiment of the present invention.

[0030] Figure 2Respectively shown are: A: Morphologies of the Zn surface (lower part) of the GBR membranes prepared in Example 1 (covalent surface modification) and Example 2 (surface micro-polishing) of the present invention (from left to right are a schematic diagram, an SEM photograph of the sample of Example 1, and an SEM photograph of the sample of Example 2); B: Morphologies of the material interfaces of the GBR membranes prepared in Example 1 (covalent surface modification) and Example 2 (surface micro-polishing) of the present invention (from left to right are a schematic diagram, an SEM photograph of the sample of Example 1, and an SEM photograph of the sample of Example 2); C: Morphologies of the collagen surfaces of the materials of the GBR membranes prepared in Example 1 (covalent surface modification) and Comparative Example 2 of the present invention (from left to right are a schematic diagram, an SEM photograph of the sample of Example 1, and an SEM photograph of the sample of Comparative Example 1); D: FTIR spectra of the upper surfaces of the GBR membranes prepared in Comparative Example 1 (upper curve) and Example 1 (lower curve); E: Yield strength diagrams of the samples of Comparative Example 1, Comparative Example 2, and Example 1 measured by AFM; F: Ultimate tensile strength values of the samples of Comparative Example 1, Comparative Example 2, and Example 1 measured by AFM; and G: Contact angles of the surfaces of the samples of Comparative Example 1, Comparative Example 2, and Example 1 measured using a contact angle measuring instrument.

[0031] Figure 3 Respectively shown are: A: Scanning electron microscope photograph and measured energy-dispersive spectrum (EDS) of the GBR membrane prepared in Example 1; B: Transmission electron microscope (TEM) photographs of the GBR membrane samples of Example 1 and Comparative Example 1.

[0032] Figure 4 Shows the release profiles of Zn 2+ ions over time in physiological saline for the samples of Comparative Example 1 (pure zinc membrane), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn).

[0033] Figure 5Respectively shown are: A: Immunofluorescence photos of live / dead detection of human bone marrow stem cells inoculated on the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 1 day; B: Immunofluorescence staining photos of human bone marrow stem cells inoculated on the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 1 day; C: SEM photos of human bone marrow stem cells inoculated on the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn); D: Cell viability of human bone marrow stem cells inoculated on the samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) within 5 days through CCK-8 experiment; E: Immunofluorescence staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 7 days; F: ALP staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 7 days; G: Expression of RUNX2 and ALP in the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 7 days; H: Immunofluorescence staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 14 days; I: ARS staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 14 days; J: Expression of BMP2 and OCN in the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 14 days; K: ALP values of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 7 days; and L: Optical density values (OD values) of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after osteogenic induction culture for 14 days.

[0034] Figure 6Respectively shown are: A. Samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) were respectively implanted into rat cranial bone defects. After 6 weeks of observation, the in vivo osteogenesis conditions observed using Micro-CT, H&E, and Masson staining; B. Samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) were respectively implanted into rat cranial bone defects. After 12 weeks of observation, the in vivo osteogenesis conditions observed using Micro-CT, H&E, and Masson staining; C. The bone mineral density values (BMD) and bone volume values (BV) detected by Micro-CT 6 weeks and 12 weeks after samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) were respectively implanted into rat cranial bone defects.

[0035] Figure 7 It is shown that the GBR membrane of the present invention activates multiple pathways related to osteogenesis, angiogenesis, and cell recruitment. Detailed implementation manners

[0036] The implementation manners of the present invention are described in detail below. The implementation manners described below are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention. For those without specific technical or conditions noted in the implementation manners, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0037] In this article, collagen self-assembly refers to the ordered arrangement of collagen molecules through non-covalent intermolecular forces under static equilibrium conditions to form a collagen fiber structure.

[0038] In this article, collagen fiber mineralization refers to the deposition of nano-hydroxyapatite crystals inside the collagen fibers and the ordered arrangement to form mineralized collagen fibers.

[0039] In this article, the silicate cement is purchased from Lehigh Cement Company. The official website is: https: / / www.lehighwhitecement.com / en / library / product-data-sheets.

[0040] Guided bone regeneration membrane (GBR) is a widely used clinical method and a key treatment in the regeneration of bone defects. However, the existing GBR barrier membrane biomaterials still cannot fully meet the basic material characteristics of the ideal barrier membrane. In the clinical application of non-absorbable membranes, secondary infections caused by membrane exposure are common, which affects bone integration. Acidic products released during the degradation of aliphatic polymers may cause strong inflammatory reactions, leading to the absorption of regenerated bone. Compared with synthetic polymers, collagen has better biocompatibility and biodegradability, but its degradation rate is not easy to control, its mechanical properties are poor, and its ability to maintain effective space force is insufficient, thus limiting the application of collagen barrier membranes.

[0041] The present invention provides a combination of a pure zinc membrane (zinc content 99.00 wt% or higher, preferably 99.99% or higher) and a functionally layered mineralized collagen membrane (HMC), thereby providing an improved GBR membrane that not only has excellent mechanical properties and / or degradation rate, but also can guide and promote in-situ bone regeneration without adding exogenous growth factors and cells.

[0042] HMC has the hierarchy and nanostructure of natural bone, provides a good microenvironment for cell recruitment and osteogenic differentiation, and its degradation rate is adapted to the rate of bone ingrowth. Good results have been achieved when it is used as a bone filling material, and its porous structure makes it possible to slowly release zinc.

[0043] Therefore, in one aspect, the present invention provides a guided bone regeneration membrane, comprising: a substrate layer, the substrate layer comprising at least 99 wt% zinc; and at least one self-assembled mineralized collagen layer. The self-assembled mineralized collagen layer can be disposed on the substrate layer and comprises collagen fibers and apatite crystals bonded to the collagen fibers. The apatite can include hydroxyapatite, chlorapatite, etc.

[0044] Preferably, the substrate layer can be a pure zinc membrane or zinc foil, such as a pure zinc membrane with a purity of 99.990 to 99.998% and a thickness of 20 - 30 μm (preferably 30 μm).

[0045] Preferably, the mineralized collagen layer is formed by first forming a porous collagen scaffold layer on the substrate layer, and then catalyzing the mineralization solution in the pores of the collagen scaffold layer with polyacrylic acid as a catalyst. The composition of the mineralization solution can be:

[0046]

[0047] As Figure 1 shown in A, the substrate layer can be a zinc membrane subjected to surface pretreatment. The surface pretreatment can be selected from any one or both of acid etching pretreatment (pretreatment method one) in a phosphoric acid-containing solution with a pH value of 2 - 3 and mechanical grinding pretreatment (pretreatment method two) with sandpapers of various different particle sizes.

[0048] Specifically, the first preprocessing method may include surface covalent modification. The zinc film is pretreated by acid etching with a mixed solution of Zn(NO3)2 (0.07M) and H3PO4 (0.15M) (pH adjusted to 2.5 - 3) for 60 s.

[0049] The second preprocessing method may include micro - grinding. For example, the zinc film is mechanically ground with silicon carbide sandpaper of 600, 800, 1200, 1500 and 2000 grits in sequence for 3 minutes.

[0050] As Figure 1 As shown in B, the guided bone regeneration membrane may include at least one self - assembled mineralized collagen layer, preferably including 3 - 10 self - assembled mineralized collagen layers from bottom to top. The mineralized collagen layer may contain collagen fibers, as well as Ca and P. The mineralized collagen layer may contain apatite. The collagen fibers are bonded together by apatite. Mineralization is carried out by catalyzing the mineralization liquid in the pores of the collagen scaffold layer with polyacrylic acid as a catalyst. Therefore, the mineralization liquid may contain NaCl, KCl, Na2HPO4, K2HPO4·3H2O, Na3N, Portland cement and polyacrylic acid.

[0051] Relative to the total weight of the guided bone regeneration membrane, the content of the collagen fibers is 80 wt% - 85 wt%, and the content of the apatite crystals is 15 wt% - 20 wt%. Relative to the total thickness of the guided bone regeneration membrane, the thickness of the mineralized collagen layer accounts for 74 - 80%.

[0052] Preferably, the guided bone regeneration membrane has a yield strength of 45 - 80 MPa, an ultimate tensile strength of 55 - 80 MPa, and a water contact angle of 30 - 53°.

[0053] In a second aspect, the present invention provides a method for preparing a guided bone regeneration membrane, comprising the following steps:

[0054] S1 - Provide a substrate layer, the substrate layer containing at least 99 wt% of zinc;

[0055] S2 - Apply a collagen solution on the substrate layer, so that the collagen self - assembles into a collagen fiber layer, and is dried and formed to form a porous scaffold layer; and

[0056] S3 - Add a mineralization liquid into the pores of the porous scaffold layer, carry out mineralization treatment, and catalyze the mineralization liquid in the pores of the collagen scaffold layer to form apatite crystals, thereby obtaining the guided bone regeneration membrane.

[0057] Preferably, before step S2, the substrate layer is surface - pretreated, and then the collagen solution is applied to the pretreated substrate layer, wherein the surface pretreatment includes at least one of the following:

[0058] A) Pretreat the substrate layer by acid etching in a mixed solution of zinc nitrate and phosphoric acid with a pH value of 2 - 3, or

[0059] B) Mechanically grind the substrate layer successively with silicon carbide sandpapers of 600, 800, 1200, 1500 grit and 2000 grit.

[0060] Preferably, step S2 includes dropping a type I procollagen solution with a concentration of 0.5 - 2.0 mg / ml onto the substrate layer at a rate of 0.5 - 2 ml / min, self - assembling for 24 - 48 hours, and then freeze - drying for 12 - 24 hours to form a dry shape.

[0061] Preferably, the method for preparing the guided bone regeneration membrane further includes ultrasonic cleaning the surface - treated substrate with distilled water, acetone and absolute ethanol for 1 - 2 minutes at a temperature of 40 - 60 °C after surface pretreatment.

[0062] Optionally, step S3 includes adding 1 - 3 ml of the mineralization solution into each of the pores, replacing the mineralization solution every 2 - 3 days, and continuously mineralizing at 25 - 37 °C for 5 - 10 days.

[0063] In a specific embodiment, as Figure 1 shown in A, the method for preparing the guided bone regeneration membrane includes the following steps:

[0064] - Surface pretreatment of the pure zinc membrane, which may include pretreatment method one or pretreatment method two. Pretreatment method one includes immersing the pure Zn membrane (purity 99.998%) in a mixed solution composed of zinc nitrate (Zn(NO3)2, 0.07 M) and phosphoric acid (H3PO4, 0.15 M)), adjusting the pH to 2.5, and soaking for 60 s; Pretreatment method two may include mechanically grinding the pure Zn membrane (purity 99.998%) with silicon carbide sandpapers of 600, 800, 1200, 1500 grit and 2000 grit for 3 minutes.

[0065] - Ultrasonically clean the surface - pretreated Zn membrane in the order of distilled water, acetone and absolute ethanol for 1 - 2 minutes at 55 °C and a frequency of 36 kHz.

[0066] - Vacuum - dry the treated sample.

[0067] - Drop a type I procollagen solution with a concentration of 1 mg / mL onto the surface of the Zn membrane at a rate of 1 mL / min, and self - assemble for 24 - 48 hours to form collagen fibrils (ss - HC / Zn).

[0068] - Place the assembled ss-HC / Zn into a 24-well plate, freeze it in a refrigerator at -20 °C for 24 hours to form, and then freeze-dry it for about 12 hours to obtain a sponge-like three-dimensional porous collagen scaffold material.

[0069] - Mineralize the three-dimensional porous collagen scaffold material in a 24-well plate for 7 days at a temperature maintained at 37 °C. Add 1 mL of mineralization solution (Table 1) to each well, and change the mineralization solution every 2 - 3 days.

[0070] - Mineralize continuously for 7 - 10 days to obtain a double-layer GBR membrane ss-HMC / Zn.

[0071] As Figure 1 shown in B, in another embodiment, the method for preparing a guided bone regeneration membrane includes: providing a Zn foil, subjecting the Zn foil to surface covalent modification or micro-polishing to obtain a pretreated Zn foil; dropping a tropocollagen solution onto the pretreated Zn foil, and gradually performing self-assembly from bottom to top to obtain an ordered array layer of collagen fibers, applying a mineralization solution containing P and Ca onto the ordered array layer of collagen fibers for mineralization, and mineralizing continuously for 7 days to obtain ss-HMC / Zn.

[0072] In a third aspect, the present invention provides the use of a guided bone regeneration membrane in the preparation of a medical device for promoting in vivo in-situ bone regeneration.

[0073] Preferably, promoting in vivo in-situ bone regeneration is selected from at least one of promoting in vivo in-situ bone regeneration by promoting cell recruitment, promoting in vivo in-situ bone regeneration by promoting M2 macrophage polarization, promoting in vivo in-situ bone regeneration by enhancing vascularization, and promoting in vivo in-situ bone regeneration by activating the BMP2-related pathway.

[0074] Examples

[0075] In combination with the following specific examples, the invention is further described. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0076] This example includes the following aspects:

[0077] 1) Preparation and characterization of a novel double-layer GBR membrane ss-HMC / Zn;

[0078] 2) In vitro and in vivo experiments to detect the sustained-release effect of ss-HMC / Zn on zinc ions;

[0079] 3) In vitro experiments to detect the biocompatibility of ss-HMC / Zn;

[0080] 4) In vitro and in vivo experiments to detect the regulatory effect of ss-HMC / Zn on the osteogenic differentiation of human bone marrow mesenchymal stem cells;

[0081] 5) In vivo experiments were conducted to detect the internal mechanism of ss-HMC / Zn in promoting rat skull defects.

[0082] Example 1:

[0083] Zinc film pretreatment

[0084] Samples of pure Zn films (purity 99.998%, thickness 30 μm) were immersed in a mixed solution composed of zinc nitrate (ZnNO3[aq], 0.07 M) and phosphoric acid (H3PO4[aq], 0.15 M), with the pH adjusted to 2.5, and the samples were soaked for 60 s. After acid etching treatment, the zinc films were ultrasonically cleaned in the order of distilled water, acetone, and absolute ethanol for 1 - 2 minutes at 55 °C and a frequency of 36 kHz. The treated samples were dried in vacuo.

[0085] Functional mineralized collagen (HMC) assembly: After zinc film pretreatment, functional mineralized collagen (HMC) was self-assembled on the surface of the zinc film by a "bottom-up" method. Specifically, a type I procollagen solution (Corning, NY, USA, 3500 Da) with a procollagen molecule concentration of 1 mg / mL was dropped onto the Zn surface at a rate of 1 mL / min and self-assembled for 24 hours to form collagen fibrils (ss-HC / Zn); then, the assembled ss-HC / Zn was placed in a 24-well plate, frozen in a refrigerator at -20 °C for 24 hours to form a shape, and freeze-dried for about 12 hours to obtain a spongy three-dimensional porous collagen scaffold material. The three-dimensional porous collagen scaffold material was mineralized in a 24-well plate for 7 days at a temperature of 37 °C. 1 mL of mineralization solution, which was added with 0.25 mM of polyacrylic acid (Table 1), was added to each well of the three-dimensional porous collagen scaffold material, and the mineralization solution was changed every 2 - 3 days. Thus, ss-HMC / Zn of Example 1 was obtained. The preparation schematic diagrams are as shown in Figure 1 A and B.

[0086] Table 1

[0087]

[0088]

[0089] Example 2

[0090] Zinc film pretreatment

[0091] Samples of pure Zn films (purity 99.998%, thickness 30 μm) were mechanically polished with silicon carbide sandpapers of 600, 800, 1200, 1500 grit sizes and 2000 grit size. Then, the materials were ultrasonically rinsed in distilled water, ethanol, and acetone in sequence for 1 - 2 minutes at 55 °C and 36 kHz. The cleaned samples were dried in vacuo.

[0092] Functional Mineralized Collagen (HMC) Assembly: After the zinc film pretreatment, the functional mineralized collagen (HMC) was self-assembled on the zinc film surface by a "bottom-up" method. First, a type I procollagen solution (Corning, NY, USA, 3500 Da) with a procollagen molecule concentration of 1 mg / mL was dropped onto the Zn surface at a rate of 1 mL / min and self-assembled for 24 hours to form collagen fibrils (ss-HC / Zn); then the assembled ss-HC / Zn was placed in a 24-well plate, frozen in a refrigerator at -20 °C for 24 hours to form a shape, and freeze-dried for about 12 hours to obtain a sponge-like three-dimensional porous collagen scaffold material. The material was mineralized in a 24-well plate for 7 days at a temperature maintained at 37 °C, 1 mL of mineralization solution (Table 1) was added to each well of the three-dimensional porous collagen scaffold material, and the mineralization solution was changed every 2 - 3 days. Thus, the ss-HMC / Zn of Example 2 was obtained.

[0093] Comparative Example 1

[0094] A commercially available pure Zn film (purity 99.998%, thickness 30 μm) was used as a guided bone regeneration membrane.

[0095] Comparative Example 2 (without mineralization treatment)

[0096] A pure Zn film (purity 99.998%, thickness 30 μm) sample was immersed in a mixed solution composed of zinc nitrate (ZnNO3[aq], 0.07 M) and phosphoric acid (H3PO4[aq], 0.15 M), the pH was adjusted to 2.5, and the sample was soaked for 60 s. After the acid etching treatment, the zinc film was ultrasonically cleaned for 1 - 2 minutes in the order of distilled water, acetone, and absolute ethanol at 55 °C and a frequency of 36 kHz. The treated sample was dried in vacuo.

[0097] A type I procollagen solution (Corning, NY, USA, 3500 Da) with a procollagen molecule concentration of 1 mg / mL was dropped onto the Zn surface at a rate of 1 mL / min and self-assembled for 24 hours to form a collagen fibril layer. Thus, the guided bone regeneration membrane (ss-HC / Zn) of Comparative Example 2 was obtained.

[0098] Blank Group: The blank group was the group of rat cranial bone defects without covering the membrane and without adding bone powder.

[0099] Test Example 1: Characterization and Detection

[0100] The morphologies of the zinc film surface, interface, and collagen surface of the ss-HC / Zn of Comparative Example 2, Example 1, and Example 2 were observed by SEM (Hitachi S-4800) respectively, and the results are as Figure 2As shown in Figures A, 2B, and 2C. The results show that the covalently surface-modified zinc surface of Example 1 presents a rough, quadrilateral conical structure with a high binding surface area. The micro-polished zinc surface of Example 2 shows irregular protrusions and cracks; in terms of the cross-section, a tight interlocking structure is formed in the cross-section of the covalent surface modification, and obvious cracks can be seen in the cross-section of the micro-polishing. Figure 2 Figure C shows the hierarchical filamentous substructure of the collagen fibril clusters and the periodic banded pattern of the mineralized fibril characteristics (proving that the collagen fibers are bonded together by calcium phosphate), similar to natural bone.

[0101] The surface main element distribution of ss-HMC / Zn of Example 1 was detected by an energy dispersive spectrometer (EDS, Hitachi S-4800), and the results are as Figure 3 shown in Figure A. Figure 3 The energy dispersive X-ray spectroscopy (EDS) shown in Figure A proves the presence of Zn, P, and Ca ( Figure 3 Figure A).

[0102] The characteristic functional group structures of ss-HMC / Zn of Example 1 and ss-HC / Zn of Comparative Example 1 and Comparative Example 2 were determined by a Fourier transform infrared spectrometer (FTIR, LUMOS II of Bruker Corporation, USA), and the results are as Figure 2 shown in Figure D. Fourier transform infrared spectroscopy (FTIR) characterized the typical collagen (1577 - 1727 cm -1 ) and phosphate (900 - 1200 cm -1 ) functional group structures in the samples of Example 1 and Comparative Example 1 ( Figure 2 Figure D).

[0103] The yield strength and maximum tensile modulus of the samples of Comparative Example 1, Comparative Example 2, and Example 1 were measured respectively by an electronic universal testing machine (Z0207; ZwickRoell GmbH, Germany). The results are as Figure 2 shown in Figures E and 2F. In terms of mechanical strength performance, the film yield tensile strengths of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss HC / Zn), and Example 1 (ss HMC / Zn) are 52.48 ± 5.00, 41.78 ± 2.78, and 47.45 ± 1.58 MPa respectively; the maximum tensile strengths are 66.48 ± 0.87, 45.63 ± 1.17, and 58.1 ± 4.47 MPa respectively, far higher than that of the Bio-Gide film (3.7 MPa), indicating that ss-HMC / Zn has the ability to resist soft tissue collapse.

[0104] The hydrophilicity of the surfaces of the samples of Comparative Example 1, Comparative Example 2, and Example 1 was measured respectively by a contact angle measuring instrument (OCA15EC and SCA20 software, DataPhysics, Filderstadt, Germany), asFigure 2 as shown in G.

[0105] The presence of ss-calcium phosphate in the EDS spectrum of ss-HMC / Zn and the observation by transmission electron microscope (TEM, JEOL JEM-1011) proved the presence of apatite crystals ( Figure 3 ). Both EDS and TEM confirmed the presence of apatite crystals and abundant calcium and phosphorus elements in HMC ( Figure 3 ).

[0106] In terms of hydrophilicity, the contact angles of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss HC / Zn), and Example 1 (ss HMC / Zn) were 99.35±0.78°, 73.65±1.67°, and 55.87±4.5°, respectively. After the co-assembly of HMC and Zn, the contact angle of the surface was slightly smaller, indicating an increase in hydrophilicity (p<0.05) ( Figure 2 G).

[0107] Test Example 2: In vitro experiment to examine the effect of functionalized mineralized collagen coating on the degradation of zinc film and the sustained release of zinc ions

[0108] According to the GB / T 16886 standard, the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss HC / Zn), and Example 1 (ss HMC / Zn) were immersed in physiological saline at a standard of 1.25 cm 2 / mL in vitro for 28 days to observe the effect of self-assembled functional mineralized collagen on the degradation of zinc film and the sustained release of zinc ions. The results of zinc ion concentration measurement showed that both HMC and HC effectively alleviated the burst release of zinc ions ( Figure 4 ).

[0109] Test Example 3: In vitro experiment to detect biocompatibility

[0110] The biocompatibility of the samples of Comparative Example 1, Comparative Example 2, and Example 1 was detected in vitro by live / dead staining, CCK-8 assay, immunofluorescence staining, and electron microscopy observation. The cells used for detection were human bone marrow stem cells (Sciencell, USA, catalog number: 7500, batches: 6899, 6881, 6890). The cytotoxicity of the materials to cells was detected by live / dead staining within 24 hours after inoculation, the adhesion performance and morphology of the materials to cells were detected by immunofluorescence and electron microscopy observation, and the stretching performance of cells on the materials at the microscopic level was detected. The cell viability within 5 days after inoculating cells onto the materials was detected by CCK-8 assay.

[0111] Figure 5Respectively shown are: A: Immunofluorescence photos of live / dead detection of human bone marrow stem cells inoculated on the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 1 day; B: Immunofluorescence staining photos of human bone marrow stem cells inoculated on the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 1 day; C: SEM photos of human bone marrow stem cells inoculated on the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn); D: Cell viability of human bone marrow stem cells inoculated on the samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) within 5 days through CCK-8 experiment.

[0112] The results show that ss-HMC / Zn of Example 1 can promote cell adhesion and proliferation compared with the pure zinc film of Comparative Example 1, has no obvious toxic effect on cells, and cells show better cell morphology on the surface coated with functional mineralized collagen (HMC) ( Figure 5 A-D).

[0113] Test Example 4: Detection of the regulatory effect on osteogenic differentiation of human bone marrow stem cells in vitro and in vivo

[0114] The in vitro osteogenic experiment was verified by qPCR, immunofluorescence, ALP staining, and ARS staining. Human bone marrow stem cells were respectively inoculated (1.6×10 4 cells / well) on the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss HC / Zn), and Example 1 (ssHMC / Zn), and relevant detections were carried out after osteogenic induction culture for 7 days and 14 days.

[0115] Figure 5It shows the immunofluorescence staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 7 days of osteogenic induction culture; F: ALP staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 7 days of osteogenic induction culture; G: Expression of RUNX2 and ALP in the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 7 days of osteogenic induction culture; H: Immunofluorescence staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 14 days of osteogenic induction culture; I: ARS staining photos of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 14 days of osteogenic induction culture; J: Expression of BMP2 and OCN in the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 14 days of osteogenic induction culture; K: ALP values of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 7 days of osteogenic induction culture; and L: Optical density values (OD values) of the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) after 14 days of osteogenic induction culture.

[0116] The qPCR results showed that the expressions of RUNX2, ALP, BMP2, and OCN in ss-HMC / Zn were higher than those in ss-HC / Zn and the pure zinc film; the immunofluorescence results showed that the expressions of RUNX2 and OCN in ss-HMC / Zn were also significantly increased; both the ALP staining and ARS staining results showed that ss-HMC / Zn had a promoting effect on the osteogenic differentiation of human bone marrow stem cells ( Figure 5 F, 5I). In vivo experiments, the samples of Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) were implanted into the rat skull defects for 6 weeks and 12 weeks respectively. Micro-CT and immunohistochemical results showed that the osteogenic effect of the ss-HMC / Zn group of Example 1 was the best. Without adding exogenous growth factors or stem cells, it could guide and promote in-situ bone regeneration ( Figure 5 A-C).

[0117] Test Example 5: Detection of the internal mechanism for promoting rat skull defects in vivo

[0118] The internal mechanism of ss-HMC / Zn promoting osteogenesis was detected by transcriptome analysis and immunofluorescence exploration. Figure 6A: Samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) were respectively implanted into the cranial bone defects of rats. After 6 weeks of observation, the in vivo osteogenesis was observed by Micro-CT, H&E, and Masson staining; B: Samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) were respectively implanted into the cranial bone defects of rats. After 12 weeks of observation, the in vivo osteogenesis was observed by Micro-CT, H&E, and Masson staining; C: The bone mineral density values (BMD) and bone volume values (BV) detected by Micro-CT after 6 weeks and 12 weeks when samples of blank control, Comparative Example 1 (pure zinc film), Comparative Example 2 (ss-HC / Zn), and Example 1 (ss-HMC / Zn) were respectively implanted into the cranial bone defects of rats.

[0119] The analysis results show that ss-HMC / Zn can activate multiple pathways related to osteogenesis, angiogenesis, and cell recruitment; the immunofluorescence results show that ss-HMC / Zn can promote the high expression of VEGF, BMP2, and CD206 in the in vivo defect area, which further indicates that ss-HMC / Zn can promote the expression of angiogenesis, osteogenesis, and M2 macrophage polarization ( Figure 7 ).

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the embodiments of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Any changes or replacements that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A guided bone regeneration membrane, characterized in that Comprising: A substrate layer, said substrate layer comprising at least 99% by weight of zinc; And At least one self-assembled mineralized collagen layer, said self-assembled mineralized collagen layer being disposed on said substrate layer and comprising collagen fibers and apatite crystals bonded to the collagen fibers, wherein said mineralized collagen layer is formed by first forming a porous collagen scaffold layer on the substrate layer and then catalyzing the formation of a mineralization solution within the pores of the collagen scaffold layer with polyacrylic acid, wherein the composition of said mineralization solution is: Wherein, relative to the total amount of said mineralization solution, the concentration of polyacrylic acid is 0.20 - 0.30 mM.

2. The guided bone regeneration membrane according to claim 1, wherein: Said substrate layer is a surface-pretreated zinc film, wherein said surface pretreatment is selected from any one or both of acid etching pretreatment in a phosphoric acid-containing solution with a pH value of 2 - 3 and mechanical grinding pretreatment with sandpapers of various different grit sizes.

3. The guided bone regeneration membrane according to any one of claims 1 to 2, characterized in that: Relative to the total thickness of said guided bone regeneration membrane, the thickness of said mineralized collagen layer accounts for 74 - 80%, wherein, in said mineralized collagen layer, the content of said collagen fibers is 80% - 85% by weight, and the content of said apatite crystals is 15% - 20% by weight, and Said guided bone regeneration membrane has a yield strength of 45 - 80 MPa, an ultimate tensile strength of 55 - 80 MPa, and a water contact angle of 30 - 53°.

4. The preparation method of the guided bone regeneration membrane according to claim 1, characterized in that Comprising the following steps: S1 - Provide a substrate layer, said substrate layer comprising at least 99% by weight of zinc; S2 - Apply a collagen solution onto the substrate layer, allowing the collagen to self-assemble into a collagen fiber layer and drying and shaping it to form a porous scaffold layer; And S3 - Add a mineralization solution into the pores of the porous scaffold layer, perform mineralization treatment, and catalyze the formation of apatite crystals from the mineralization solution within the pores of the collagen scaffold layer, thereby obtaining said guided bone regeneration membrane.

5. The preparation method according to claim 4, characterized in that Before step S2, the substrate layer is subjected to surface pretreatment, and then said collagen solution is applied onto the pretreated substrate layer, wherein said surface pretreatment includes at least one of the following: A) Acid etching pretreatment of the substrate layer in a mixed solution of zinc nitrate and phosphoric acid with a pH value of 2 - 3, or B) Sequentially mechanically grinding the substrate layer with silicon carbide sandpapers of 600, 800, 1200, 1500, and 2000 mesh.

6. The preparation method according to claim 4 or 5, characterized in that Step S2 includes dropping a type I procollagen solution with a concentration of 0.5 - 2.0 mg / ml onto said substrate layer at a rate of 0.5 to 2 ml / min, self-assembling for 24 - 48 hours, and then freeze-drying for 12 - 24 hours to dry and shape.

7. The preparation method according to claim 6, characterized in that Said method further includes: After surface pretreatment and before step S3, ultrasonically cleaning the surface-treated substrate with distilled water, acetone, and absolute ethanol at a temperature of 40 - 60°C for 1 - 2 minutes, and / or Step S3 includes adding 1 - 3 ml of said mineralization solution into each of said pores, replacing the mineralization solution every 2 - 3 days, and continuously mineralizing at 25 - 37°C for 5 - 10 days.

8. Use of the guided bone regeneration membrane according to any one of claims 1 to 3 in the preparation of a medical material for promoting in-situ bone regeneration in vivo.

9. The use according to claim 8, characterized in that The promotion of in-situ bone regeneration in the body is selected from at least one of promoting in-situ bone regeneration in the body by promoting cell recruitment, promoting in-situ bone regeneration in the body by promoting M2 macrophage polarization, promoting in-situ bone regeneration in the body by enhancing vascularization, and promoting in-situ bone regeneration in the body by activating the BMP2-related pathway.

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