WO2023082214A1 - 掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法 - Google Patents

掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法 Download PDF

Info

Publication number
WO2023082214A1
WO2023082214A1 PCT/CN2021/130487 CN2021130487W WO2023082214A1 WO 2023082214 A1 WO2023082214 A1 WO 2023082214A1 CN 2021130487 W CN2021130487 W CN 2021130487W WO 2023082214 A1 WO2023082214 A1 WO 2023082214A1
Authority
WO
WIPO (PCT)
Prior art keywords
collagen
hap
selenium
solution
gbr
Prior art date
Application number
PCT/CN2021/130487
Other languages
English (en)
French (fr)
Inventor
陈填烽
罗学仕
Original Assignee
暨南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 暨南大学 filed Critical 暨南大学
Priority to PCT/CN2021/130487 priority Critical patent/WO2023082214A1/zh
Publication of WO2023082214A1 publication Critical patent/WO2023082214A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/12—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/26—Phosphates
    • C01B25/32—Phosphates of magnesium, calcium, strontium, or barium

Definitions

  • the patent relates to the field of medical technology, in particular to a selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane and a preparation method thereof.
  • GBR Guided bone regeneration
  • the GBR membrane should have good biocompatibility and a double-layer structure.
  • An ideal GBR membrane needs to meet the following conditions: 1. Barrier membrane. This is the most important point, the GBR membrane should have sufficient strength, be able to guarantee the spatial environment for bone regeneration, and have good retention and stability. At the same time, it has a degradation rate that matches the bone regeneration cycle, ensuring enough time and good space to wait for the growth of bone regeneration tissue. 2. Good osteogenic microenvironment.
  • the bone defect area for bone regeneration must be healthy and free from potential sources of infection. During bone healing, infection caused by pathogenic bacteria colonizing the wound is considered to be one of the main reasons for the failure of guided bone regeneration.
  • bacteriostasis is also an essential property of GBR membranes.
  • immune responses and bacterial infections affect the success of bone regeneration.
  • Antibacterial GBR membranes are a promising way to guide bone regeneration.
  • Promote bone regeneration Before being replaced by new bone, it can provide a scaffold for new bone growth and help new bone grow. Therefore, it is still a difficult problem to realize all the desired functions of GBR at the same time, that is, how to realize the unification of structure and function, and it is yet to be developed a barrier membrane with bone-promoting ability, soft tissue compatibility and antibacterial properties.
  • Collagen Bio-Gide membrane (Geistlich Pharma, Wolhusen, Switzerland) is the most widely used biodegradable membrane with many advantages: good biocompatibility and degradability, ability to promote cell adhesion and proliferation, as GBR membrane It has achieved certain curative effect clinically.
  • Bio-Gide biofilms have shortcomings such as high degradation rate, poor mechanical properties, and poor antibacterial properties, which are not conducive to the long-term regeneration and repair of bone defects.
  • the primary purpose of the present invention is to provide a preparation method of a selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane.
  • Another object of the present invention is to disclose the selenium-doped hydroxyapatite nano-reinforced collagen GBR film prepared by the above method, which is used in guiding bone regeneration and rehabilitation, and has antibacterial properties and the ability to promote osteogenesis and regeneration.
  • Se-HAP SeO-doped hydroxyapatite
  • Se-HAP is due to the doping of selenium Therefore, it has anti-bone tumor and antibacterial effects, and low-doped Se-HAP can promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts.
  • selenium can significantly affect the immune system, enhance cellular immunity, humoral immunity and non-specific immune function.
  • Se-HAP has nanostructures such as nanoneedles, nanowires, and nanosheets
  • Se-HAP nanoneedles, nanowires, or nanosheets can well enhance the strength of collagen scaffolds and improve their mechanical properties. Therefore, the use of Se-HAP nanowires to enhance the collagen double-layer GBR can well enhance the strength of the collagen GBR membrane and improve its mechanical properties. At the same time, it is expected to endow the GBR membrane with good antibacterial properties and promote osteogenesis and regeneration.
  • the selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane has more excellent bone regeneration and repair performance, and shows good prospects in guiding bone regeneration and related diseases.
  • the invention provides a method for preparing a selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane, comprising the steps of:
  • step (1) Se/(Se+P) with a mole fraction of 0.05%-0.4% is synthesized by a solvothermal method to synthesize Se-HAP nanowires, or nanoneedles, or nanosheet structures.
  • the molar fractions of the Se/(Se+P) are respectively 0.05%, 0.1%, 0.2% and 0.3%, which are respectively named as 0.5Se-HAP, 1Se-HAP, 2Se-HAP and 3Se-HAP, by solvothermal Se-doped Se-HAP nanostructures were synthesized by method.
  • step (1) specifically includes, dissolving NaOH in deionized water, stirring mechanically in an ice-water bath and adding deionized water, methanol and oleic acid, adding CaCl 2 aqueous solution drop by drop and mixing to obtain a mixed solution, after stirring, and then NaH 2 PO 4 ⁇ 2H 2 O and Na 2 SeO 3 were added dropwise to the mixture, the resulting mixture was transferred to an autoclave, sealed and heated at 170°C-200°C, then the resulting slurry was added A large amount of absolute ethanol was stirred, and collected by filtration; then washed several times with ethanol and deionized water, and finally the Se-HAP nanostructure was obtained.
  • step (2) specifically on a directional freezing device, the collagen solution is cooled from room temperature to negative 50°C-negative 60°C until it is completely frozen, and then the frozen collagen solution is stored in a negative After further freezing for a period of time in a refrigerator at a temperature below 70° C., freeze-drying can obtain the collagen GBR membrane reinforced with selenium-doped hydroxyapatite nanometers.
  • step (2) add animal type I collagen to 0.5% (v/v) acetic acid solution and stir to prepare a 40 mg/mL collagen solution, mechanically stir in an ice-water bath until completely dissolved; use interval low-temperature ultrasonic method to make Collagen is uniformly dispersed; add Se-HAP nanowire powder into the aqueous solution, stir fully to form Se-HAP slurry, mix the collagen solution and the Se-HAP slurry in an ice-water bath with mechanical stirring, and obtain collagen
  • the concentrated collagen solution was poured into a cylindrical copper mold, air-dried and centrifuged, the copper mold was placed on a directional freezer, and the collagen solution was cooled from room temperature to negative 60° C. until it is completely frozen for a period of time, and freeze-dried to obtain the selenium-doped hydroxyapatite nano-reinforced collagen GBR film.
  • the present invention also provides a selenium-doped hydroxyapatite nano-reinforced collagen GBR film, which is obtained according to the above preparation method. It is a collagen/Se-HAP double-layer GBR membrane with a porous and orderly surface and a dense and smooth bottom surface.
  • the present invention also provides the application of selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane in bone regeneration and repair.
  • the selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane is obtained according to the above preparation method and is used in guiding bone regeneration and rehabilitation. , has antibacterial properties and promotes osteogenesis and regeneration.
  • the present invention has the following advantages and effects:
  • the present invention is based on the material transformation of chemical composition and structure, and the invented selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane has a double-layer structure, and one side is designed to be compact and smooth, preventing non-osteoblasts from interfering with bone defects The aggregation of osteoblast-related cells and bone repair, while the other side is porous and rough, which facilitates the adhesion and recruitment of osteoblasts near the bone defect. Greatly improve the mechanical properties, antibacterial properties, and osteogenic properties of the collagen double-layer GBR membrane.
  • the construction of multifunctional dense smooth/porous ordered bilayer structure collagen/Se-HAP GBR membrane provides a microenvironment conducive to osteogenesis for the study of GBR membrane-induced repair of bone defects.
  • the present invention explores the osteogenic differentiation performance of bone marrow mesenchymal stem cells based on chemical composition, different polymer stabilized selenium nanoparticles (Col I-SeNPs, CS-SeNPs, LET-SeNPs, PVP-SeNPs), ⁇ 10uM concentration Among the selenium nanoparticles, Col I-SeNPs of collagen and selenium have the best biocompatibility and osteogenic properties. Se-HAP can release calcium ions, phosphate and selenite ions slowly, and 3Se-HAP with a selenium concentration of ⁇ 10uM has good biocompatibility. In terms of promoting bone marrow mesenchymal osteogenic differentiation: 3SeHAP>Col I -SeNPs>LET-SeNPs>CS-SeNPs>PVP-SeNPs.
  • the present invention strengthens the collagen GBR membrane by selecting selenium-doped hydroxyapatite nanowires, and the collagen/Se-HAP GBR membrane with dense smooth/porous and ordered double-layer structure has multi-effect properties such as antibacterial and osteogenesis, revealing
  • the physiological process of GBR membranous bone repair has created a new application field of bone regeneration and repair, and provides new technical guidance for the synthesis of better bone repair materials.
  • the present invention provides a method for preparing a selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane.
  • the raw materials are cheap and easy to obtain, the synthesis and production steps are highly operable, the synthesis scale is easy to expand, and the commercialization and application of new biological materials are realized.
  • a unique preparation step such as the synthesis of Se-HAP nanostructures from Se/(Se+P) by solvothermal method, is to add NaH 2 PO 4 ⁇ 2H 2 O and Na 2 SeO 3 dropwise to the mixture, and the The obtained mixture is transferred to an autoclave, sealed and heated at 170°C-200°C, and then the obtained slurry is stirred with a large amount of absolute ethanol, so that nano-selenium can be better incorporated into the microstructure of the GBR membrane under high temperature and high pressure. In the pores, the antibacterial ability and the strength of the collagen scaffold are greatly enhanced, and its mechanical properties are improved.
  • step (2) the collagen solution and the Se-HAP slurry are mechanically stirred and mixed in an ice-water bath to obtain a collagen/Se-HAP mixed solution.
  • the concentrated collagen solution is poured into a cylinder
  • the copper mold is placed on a directional freezer, and the collagen solution is cooled from room temperature to minus 60°C until it is completely frozen and then placed in a minus 80°C refrigerator until it is further frozen for a period of time
  • a unique double-layer structure is formed, one layer is compact and smooth, which prevents non-osteoblasts from interfering with the aggregation of osteoblast-related cells and bone repair at the bone defect site, while the other side is porous and rough, which facilitates the formation of osteoblasts near the bone defect.
  • Adhesion and recruitment are the collagen/Se-HAP mixed solution.
  • Figure 1 Transmission electron microscope images and elemental analysis images of hydroxyapatite (HAP) and selenium-doped hydroxyapatite (3Se-HAP), where (A) photo corresponds to HAP, and (B) photo corresponds to 3Se-HAP.
  • HAP hydroxyapatite
  • 3Se-HAP selenium-doped hydroxyapatite
  • FIG. 2 Cytocompatibility of Se-HAP with different amounts of selenium (Se/(Se+P)) on bone marrow mesenchymal stem cells (mBMSCs) (A: MTT assay and B: live-and-dead fluorescent staining), and osteogenesis Effect of differentiation (14 days) test results.
  • mBMSCs bone marrow mesenchymal stem cells
  • Figure 3 Scanning electron micrograph (A), infrared spectrum (B) of Col@Se-HAP GBR membrane, the ability to promote osteogenic differentiation of mBMSCs cells (C: alkaline phosphatase staining) and antibacterial properties (D: grape aureus Bacterial zone of inhibition).
  • FIG. 4 The shielding effect of the Col@Se-HAP double-layer GBR membrane of the present invention (2weeks): laser confocal image after immunofluorescent staining of cells: Col I (type I collagen), nucleus (nucleus), F-actin (F actin), NIH3T3 (fibroblasts), mBMSCs (mouse bone marrow mesenchymal stem cells).
  • Figure 5 Micro-CT of the Col@Se-HAP double-layer GBR membrane of the present invention after 4 weeks of repairing a rat skull defect.
  • Control group Commercial Bio-gide collagen membrane.
  • the invention provides a selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane, which is applied to guide bone regeneration and rehabilitation, has antibacterial properties, and has the performance of promoting osteogenesis differentiation and regeneration.
  • Embodiment 1 provides a method for preparing a selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane, comprising steps:
  • Se-HAP selenium-doped hydroxyapatite
  • Se/(Se+P) is synthesized into a Se-HAP nanostructure through a solvothermal method
  • the Se/(Se/( Se+P) Se-doped Se-HAP nanowires, or nanoneedles, or nanosheet structures were synthesized by a solvothermal method.
  • step (1) specifically includes, dissolving 9-10g NaOH in 140-150mL deionized water, stirring mechanically in an ice-water bath and adding 120-140mL deionized water, 50-60mL methanol and 100-110mL oleic acid, and dissolving 2.5 -3.5g of CaCl 2 aqueous solution was added dropwise and mixed to obtain a mixed solution.
  • Embodiment 2 provides a method for preparing a selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane, including:
  • Step (1) preparing selenium-doped hydroxyapatite (Se-HAP), using Se/(Se+P) to synthesize Se-HAP nanostructures by solvothermal method.
  • the molar fractions of the Se/(Se+P) are respectively 0%, 0.05%, 0.1%, 0.2% and 0.3%, which are named HAP, 0.5Se-HAP, 1Se-HAP, 2Se-HAP and 3Se-HAP respectively.
  • HAP, HAP nanowires and Se-HAP nanostructures with different Se-doped amounts were synthesized by solvothermal method. In this way, comparative experiments can be done to screen out the Se-HAP nanowires with the best selenium content.
  • the synthesis process of Se-HAP nanowires with a mole fraction of 0.3% is as follows. Dissolve 10.05g NaOH in 150mL deionized water, mechanically stir in an ice-water bath, add 135mL deionized water, mix 60mL methanol and 105mL oleic acid, add 120mL CaCl 2 (3.33g) aqueous solution dropwise to the above solution, stir for 30min, take 180mL NaH 2 PO 4 2H 2 O (9.33 g) and Na 2 SeO 3 (0.031 g) were added dropwise to the mixture, and the resulting mixture was transferred to a Teflon-lined stainless steel autoclave, sealed and heated at 180 °C 24h.
  • HAP nanowires and Se-HAP nanowires with different selenium doping amounts by referring to the above steps, and prepare Se-HAP nanowires according to different Se/(Se+P) mole fractions to explore different selenium doping amounts , to observe the effect on the osteogenic performance of bone marrow mesenchymal stem cells.
  • Step (2) preparing a collagen/SeHAP double-layer GBR membrane with a porous and orderly surface and a dense and smooth bottom surface.
  • mBMSCs mae bone marrow mesenchymal stem cells
  • 3Se-HAP was screened out as the optimal Se-HAP nanowire-reinforced collagen scaffold as the composite scaffold.
  • animal type I collagen was added to 0.5% (v/v) acetic acid solution and stirred to prepare a 40 mg/mL collagen solution, mechanically stirred in an ice-water bath until completely dissolved.
  • Interval low-temperature ultrasonic method every ultrasonic 10min, stay 10min makes the collagen disperse evenly.
  • Se-HAP nanowire powder in the aqueous solution according to different concentrations, fully stir to form a slurry, mix the collagen solution with the Se-HAP slurry, so that the quality of HAP/SeHAP is 3%, mechanically stir in the ice-water bath to make the collagen and Se-HAP Se-HAP mixed well.
  • the high-concentration collagen/Se-HAP mixed solution was concentrated into a collagen solution (0.5mL, 40mg/mL), which was poured into a cylindrical copper mold, and 0.5mL of the mixed solution was added after air drying .
  • the copper mold was placed on a directional freezer, and the solution was cooled from 20°C to -60°C at a rate of -5°C/min. The final temperature was maintained at -60 °C until the samples were completely frozen. The samples were then stored in a -80°C refrigerator until further frozen for 12 hours, and then freeze-dried to obtain the selenium-doped hydroxyapatite nano-enhanced collagen GBR membrane (ie, Col@Se-HAP GBR membrane).
  • rat tail collagen type I (Col I)
  • CS chitosan
  • LET lentinan
  • PVP polyvinylpyrrolidone
  • Figure 2 is the cytocompatibility results of Se-HAP with different selenium doping amounts (Se/(Se+P)) on bone marrow mesenchymal stem cells (mBMSCs) (A: MTT experiment and B: dead-live fluorescent staining), and the results of Effect on bone differentiation (14 days):
  • the effect concentration of HAP/SeHAP was 50 ⁇ g/mL.
  • 50 ⁇ g/mL selenium-doped hydroxyapatite has no toxicity to mBMSCs cells, while promoting the osteogenic differentiation of mBMSCs cells.
  • Figure 3 is the scanning electron micrograph (A), infrared spectrum (B), the ability to promote the osteogenic differentiation of mBMSCs cells (C: alkaline phosphatase staining) and antibacterial properties of Col@Se-HAP GBR membrane (D: grape aureus Bacterial zone of inhibition).
  • A scanning electron micrograph
  • B infrared spectrum
  • C alkaline phosphatase staining
  • D grape aureus Bacterial zone of inhibition
  • Fig. 4 is the shielding effect of the Col@Se-HAP double-layer GBR membrane of the present invention (2weeks): laser confocal image after immunofluorescent staining of cells: Col I (type I collagen), nucleus (nucleus), F-actin (F actin), NIH3T3 (fibroblasts), mBMSCs (mouse bone marrow mesenchymal stem cells).
  • Col@Se-HAP double-layer GBR membrane of the present invention can well prevent the invasion of NIH3T3 cells.
  • the results showed that the dense and smooth surface on one side could prevent non-osteoblasts from interfering with the aggregation of osteoblast-related cells and bone repair at the bone defect site, while the other side was porous and rough, which facilitated the adhesion and recruitment of osteoblasts near the bone defect.
  • FIG. 5 Micro-CT of the Col@Se-HAP double-layer GBR membrane of the present invention after 4 weeks of repairing a rat skull defect.
  • Control group Commercial Bio-gide collagen membrane. It can be seen that the Col@Se-HAP double-layer guided bone regeneration GBR membrane, based on the barrier membrane properties covering the bone defect area, prevents the invasion of fibroblasts from the surrounding soft tissue, allowing the osteoblasts on the bone surface to have enough time to adhere, proliferate, Osteogenic differentiation, and ultimately achieve the purpose of bone tissue regeneration and repair. The performance of bone regeneration and repair is better than that of the existing commercial Bio-gide collagen membrane.
  • the synthesized selenium-doped hydroxyapatite nano-reinforced collagen GBR membrane has a better therapeutic effect on promoting osteogenesis and regeneration, providing a new strategy for clinical bone regeneration surgery and choose.
  • the inventors have shown through transmission electron microscopy and elemental analysis that we have successfully prepared selenium-doped hydroxyapatite nanowires.
  • the inventors verified the bone regeneration performance of Col@Se-HAP GBR membrane in the rat skull defect model, and found that Col@Se-HAP GBR membrane has excellent bone defect repair ability compared with commercial Bio-gide membrane, and can Rapidly promote bone regeneration and repair of skull defects.
  • the present invention not only demonstrates the preparation method of selenium-doped hydroxyapatite nanowires and Col@Se-HAP double-layer GBR membrane, but also provides a basis for the bone regeneration mechanism of GBR membrane and its clinical application in bone defect regeneration treatment in the future. Apps provide more evidence.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Surgery (AREA)
  • Vascular Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Inorganic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Composite Materials (AREA)
  • Materials Engineering (AREA)
  • Materials For Medical Uses (AREA)

Abstract

涉及一种掺硒羟基磷灰石纳米增强的胶原GBR膜及其制备方法，制备方法包括步骤：(1)制备掺硒羟基磷灰石(Se-HAP)，将Se/(Se+P)通过溶剂热法合成Se-HAP纳米结构；(2)将动物皮胶原加到醋酸溶液中搅拌配制胶原溶液，机械搅拌至完全溶解，利用间隔低温超声方式使得胶原分散均匀；加入Se-HAP纳米线粉末于水溶液中，充分搅拌制成Se-HAP浆体，将所述胶原溶液与所述Se-HAP浆体混合均匀得到胶原/Se-HAP混合溶液超声处理后，再将浓缩成胶原蛋白溶液离心处理后放在冷冻装置上冷却至0℃以下，冷冻干燥即可得到所述掺硒羟基磷灰石纳米线增强的胶原GBR膜，可应用在引导骨再生的医疗康复，具有抗菌性、促成骨分化再生优良性能。

Description

掺硒羟基磷灰石纳米增强的胶原GBR膜及其制备方法 技术领域
本专利涉及医药技术领域，特别涉及一种掺硒羟基磷灰石纳米增强的胶原GBR膜及其制备方法。
背景技术
颅、颌面骨缺损的修复在整形外科和口腔颌面外科的治疗中仍是挑战，通常需要使用骨粉和引导组织再生膜进行修复，但较大的缺损修复仍然是临床中的难点。引导骨再生(guided bone regeneration，GBR)技术：基于覆盖骨缺损区域的屏障膜特性，阻止来自周围软组织的成纤维细胞侵袭，让骨面处的成骨细胞有足够的时间黏附、增殖、成骨分化，最终实现骨组织再生和修复的目的。在复杂骨缺损区如牙槽骨萎缩区，抗菌性也是GBR膜必不可少的性能，生物材料植入骨缺损区域后，免疫反应和细菌感染影响骨再生的成功。
GBR技术若要完成骨缺损区域的修复，GBR膜除了应该具有良好的生物相容性和双层结构，理想的GBR膜需要具备以下条件：1、屏障膜。这是最重要的一点，GBR膜应具有足够的强度，能够保证骨再生的空间环境，并具有良好的固位和稳定。同时具有骨再生周期匹配的降解速率，保证有足够的时间和良好的空间等待骨再生组织成长。2、良好的成骨微环境环境。进行骨再生的骨缺损区域，必须是健康的，并保证没有潜在的感染源。在骨愈合过程中，病原菌在创面处定植引起的感染被认为是引导骨再生失败的主要原因之一。在复杂骨缺损区，抑菌性也是GBR膜必不可少的性能，生物材料植入骨缺损区域后，免疫反应和细菌感染影响骨再生的成功，抗菌GBR膜引导骨再生是一种很有前途的预防感染和引导骨再生的治疗方法。3、促骨再生。被新骨置换前，可以为新骨生长提供支架，帮助新骨生长。因此，GBR要同时实现所有的理想功能即如 何实现结构与功能的统一仍是一个难以解决的问题，有待研发具有促骨能力以及软组织相容性和抗菌性的屏障膜。
胶原Bio-Gide膜(Geistlich Pharma,Wolhusen，瑞士)是使用最广泛的生物可降解膜，具有许多优点：具有良好的生物相容性和降解性，能够促进细胞的粘附和增殖，作为GBR膜在临床上已取得一定的疗效。然而临床应用中，Bio-Gide生物膜存在降解率高、机械性能差、抗菌性能差等不足，不利于骨缺损的远期再生修复。
发明内容
针对现有技术的不足，本发明的首要目的是提供掺硒羟基磷灰石纳米增强的胶原GBR膜的制备方法。
本发明的另一目的在于公开了上述方法制备得到的掺硒羟基磷灰石纳米增强的胶原GBR膜，应用在引导骨再生康复，具有抗菌性、促成骨分化再生性能。
发明人在实验发现，可通过SeO 3 2-或者SeO 4 2-离子取代HAP中OH -或PO 4 3-可制备掺硒羟基磷灰石(Se-HAP)，Se-HAP由于硒元素的掺入而具有具有抗骨肿瘤和抗菌作用，同时低掺杂量的Se-HAP能够促进骨髓间充质干细胞向成骨细胞的分化。人们通过硒的生物学作用研究发现硒能显著地影响免疫系统，增强细胞免疫、体液免疫及非特异性免疫功能。研究表明，Se-HAP具有纳米针、纳米线、纳米片等纳米结构，Se-HAP纳米针、纳米线或纳米片能够很好地增强胶原支架的强度，改善其力学性能。故采用Se-HAP纳米线增强胶原双层GBR，能够很好地增强胶原GBR膜的强度，改善其力学性能，同时期待赋予GBR膜良好的抗菌性、促成骨分化再生性能。相对于临床应用的Bio-Gide生物膜，掺硒羟基磷灰石纳米增强的胶原GBR膜具有更加优异的骨再生修复性能，在引导骨再生及其相关疾病方面显示出良好的前景。
本发明提供掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，包括步骤：
(1)制备掺硒羟基磷灰石(Se-HAP)，将Se/(Se+P)通过溶剂热法合成 Se-HAP纳米结构体；
(2)将动物I型胶原加到醋酸溶液中搅拌配制胶原溶液，机械搅拌至完全溶解，利用间隔低温超声方式使得胶原分散均匀；加入Se-HAP纳米粉末于水溶液中，充分搅拌制成Se-HAP浆体，将所述胶原溶液与所述Se-HAP浆体混合均匀得到胶原/Se-HAP混合溶液超声处理后，再将浓缩成胶原蛋白溶液离心处理后放在冷冻装置上，所述胶原蛋白溶液从室温冷却至0℃以下，直到完全冻结一段时间，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜(即Col@Se-HAP GBR膜)。
进一步，步骤(1)中：是将摩尔分数为0.05％-0.4％的Se/(Se+P)通过溶剂热法合成掺硒的Se-HAP纳米线、或纳米针、或纳米片结构体。
所述Se/(Se+P)的摩尔分数分别取0.05％、0.1％、0.2％和0.3％，分别对应命名为0.5Se-HAP、1Se-HAP、2Se-HAP和3Se-HAP，通过溶剂热法合成掺硒的Se-HAP纳米结构体。
进一步，步骤(1)具体包括，将NaOH溶于去离子水中，冰水浴中机械搅拌并加入去离子水、甲醇和油酸,将CaCl 2水溶液逐滴一起加入混合得到混合液，搅拌后，再将NaH 2PO 4·2H 2O和Na 2SeO 3逐滴加入所述混合液中，将得到的混合物转移到高压釜中，密封并在170℃-200℃下加热，然后得到的浆体加入大量的无水乙醇搅拌，并过滤收集；再用乙醇和去离子水洗多次，最后得到Se-HAP纳米结构体。
进一步，步骤(2)中，具体是在定向冷冻装置上，所述胶原蛋白溶液从室温冷却到负50℃-负60℃冷却，直到完全冻结，然后将冻结的所述胶原蛋白溶液保存在负70℃以下温度的冰箱中直到进一步冷冻一段时间后，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜。
更进一步，步骤(2)中，是将动物I型胶原加到0.5％(v/v)醋酸溶液中搅拌配制40mg/mL的胶原溶液，冰水浴机械搅拌至完全溶解；利用间隔低温超声法使得胶原分散均匀；加入Se-HAP纳米线粉末于水溶液中，充分搅拌制成Se-HAP 浆体，将所述胶原溶液与所述Se-HAP浆体，在冰水浴中机械搅拌混合均匀，得到胶原/Se-HAP混合溶液超声处理后，将浓缩成胶原蛋白溶液倒入圆柱形的铜模具中，风干后离心后,将铜模具放在定向冷冻装置上，所述胶原蛋白溶液从室温冷却至负60℃，直到完全冻结一段时间，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜。
本发明还提供一种掺硒羟基磷灰石纳米增强的胶原GBR膜，是根据上述制备方法得到。是表面多孔有序、底面致密光滑的胶原/Se-HAP双层GBR膜。
本发明还提供掺硒羟基磷灰石纳米增强的胶原GBR膜在骨再生修复的应用，所述掺硒羟基磷灰石纳米增强的胶原GBR膜是根据上述制备方法得到，应用在引导骨再生康复，具有抗菌性、促成骨分化再生性能。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明基于化学组成和结构的材料改造，发明的掺硒羟基磷灰石纳米增强的胶原GBR膜，它具有双层结构，一侧设计紧凑光滑，防止非成骨细胞干扰骨缺损部位成骨相关细胞的聚集与骨修复，而另一侧则多孔且粗糙，便于骨缺损附近成骨细胞黏附与募集。大大提高胶原双层GBR膜的力学性能、抗菌性能、成骨性能。多功能的致密光滑/多孔有序双层结构的胶原/Se-HAP GBR膜的构建，为GBR膜诱导修复骨缺损研究提供了利于成骨的微环境。
(2)本发明基于化学组成对骨髓间充质干细胞的成骨分化性能探究，不同高分子稳定硒纳米粒子(Col I-SeNPs,CS-SeNPs，LET-SeNPs，PVP-SeNPs)，＜10uM浓度的硒纳米粒子中，胶原与硒的Col I-SeNPs具有最优的生物相容性和促成骨性能。而Se-HAP能缓释钙离子、磷酸根与亚硒酸根离子，＜10uM硒浓度的3Se-HAP具有良好的生物相容性,在促进骨髓间充质成骨分化性能方面：3SeHAP＞Col I-SeNPs＞LET-SeNPs＞CS-SeNPs＞PVP-SeNPs。
(3)本发明通过选取掺硒羟基磷灰石纳米线增强胶原GBR膜，致密光滑/多孔有序双层结构的胶原/Se-HAP的GBR膜具有抗菌和促进成骨等多效性能，揭示GBR膜骨修复的生理过程，开创了一个新的骨再生修复应用领域，为合成 更好的骨修复材料提供新的技术指导。
(4)本发明提供掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法原料廉价易得，合成和生产步骤可操作性强，容易扩大合成规模，实现新生物材料的商业化和应用。
独特的制备步骤，比如将Se/(Se+P)通过溶剂热法合成Se-HAP纳米结构，是将NaH 2PO 4·2H 2O和Na 2SeO 3逐滴加入所述混合液中，将得到的混合物转移到高压釜中，密封并在170℃-200℃下加热，然后得到的浆体加入大量的无水乙醇搅拌，这样高温密闭高压下可以更好的将纳米硒融入到GBR膜微孔中，大大增强抗菌能力和胶原支架的强度，改善其力学性能。
步骤(2)中，将所述胶原溶液与所述Se-HAP浆体，在冰水浴中机械搅拌混合均匀，得到胶原/Se-HAP混合溶液超声处理后，将浓缩成胶原蛋白溶液倒入圆柱形的铜模具中，风干后离心后,将铜模具放在定向冷冻装置上，所述胶原蛋白溶液从室温冷却至负60℃，直到完全冻结再在负80℃的冰箱中直到进一步冷冻一段时间，这样方式形成独特的双层结构，一层紧凑光滑，防止非成骨细胞干扰骨缺损部位成骨相关细胞的聚集与骨修复，而另一侧则多孔且粗糙，便于骨缺损附近成骨细胞黏附与募集。
(5)通过对比动物实验和商用的Bio-Gide生物膜进行骨再生修复证明，掺硒羟基磷灰石纳米线增强的胶原GBR膜具有更加优异的骨再生修复性能，在引导骨再生及其相关疾病方面显示出良好的前景。
(6)通过探究不同质量分数(wt％,10、20、30、40、50、60、70、80％Se-HAP)胶原/Se-HAP复合膜中，30-50％Se-HAP的复合膜的综合性能最优，具有良好的力学性能、抗菌性能和促成骨性能。
附图说明
图1：羟基磷灰石(HAP)和掺硒羟基磷灰石(3Se-HAP)透射电镜图与元素分析图，其中(A)照片对应HAP，(B)照片对应3Se-HAP。
图2：不同掺硒量(Se/(Se+P))的Se-HAP对骨髓间充质干细胞(mBMSCs)的细胞相容性(A：MTT实验和B：死活荧光染色)，以及促成骨分化的影响(14天)试验结果。
图3：Col@Se-HAP GBR膜的扫描电镜图(A)、红外图谱(B)、促mBMSCs细胞成骨分化性能(C:碱性磷酸酶染色)和抗菌性能研究(D：金黄色葡萄球菌抑菌圈)。
图4：本发明Col@Se-HAP双层GBR膜的屏蔽作用(2weeks)：细胞免疫荧光染色后的激光共聚焦图：Col I(I型胶原)、nucleus(细胞核)、F-actin(F肌动蛋白)、NIH3T3(成纤维细胞)、mBMSCs(小鼠骨髓间充质干细胞)。
图5：本发明Col@Se-HAP双层GBR膜在大鼠颅骨缺损修复4weeks后的Micro-CT。对照组：商用Bio-gide胶原膜。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
本发明提供掺硒羟基磷灰石纳米增强的胶原GBR膜应用在引导骨再生康复，具有抗菌性、促成骨分化再生性能。
实施例一，本发明提供掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，包括步骤：
(1)制备掺硒羟基磷灰石(Se-HAP)，将Se/(Se+P)通过溶剂热法合成Se-HAP纳米结构体；是将摩尔分数为0.05％-0.4％的Se/(Se+P)通过溶剂热法合成掺硒的Se-HAP纳米线、或纳米针、或纳米片结构体。
进一步，步骤(1)具体包括，将9-10g NaOH溶于140-150mL去离子水中，冰水浴中机械搅拌并加入去120-140mL离子水、50-60mL甲醇和100-110mL油酸,将2.5-3.5g的CaCl 2水溶液逐滴一起加入混合得到混合液，搅拌后，再将180-190mL的NaH 2PO 4·2H 2O(8-9.5g)和Na 2SeO 3(0.02-0.035g)一起逐滴 加入所述混合液中，将得到的混合物转移到高压釜中，密封并在170℃-200℃下加热，然后得到的浆体加入大量的无水乙醇搅拌，并过滤收集；再用乙醇和去离子水洗多次，最后得到Se-HAP纳米结构体。
(2)将动物I型胶原加到醋酸溶液中搅拌配制胶原溶液，机械搅拌至完全溶解，利用间隔低温超声方式使得胶原分散均匀；加入Se-HAP纳米线粉末于水溶液中，充分搅拌制成Se-HAP浆体，将所述胶原溶液与所述Se-HAP浆体混合均匀得到胶原/Se-HAP混合溶液超声处理后，再将浓缩成胶原蛋白溶液离心处理后放在冷冻装置上，所述胶原蛋白溶液从室温冷却至0℃以下，直到完全冻结一段时间，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜(即Col@Se-HAP GBR膜)。
实施例二，本发明提供掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，包括，
步骤(1)、制备掺硒羟基磷灰石(Se-HAP)，将Se/(Se+P)通过溶剂热法合成Se-HAP纳米结构体。
所述Se/(Se+P)的摩尔分数分别取0％、0.05％、0.1％、0.2％和0.3％，分别对应命名为HAP、0.5Se-HAP、1Se-HAP、2Se-HAP和3Se-HAP，分别通过溶剂热法合成HAP纳米线和不同掺硒量的Se-HAP纳米结构。这样可以做对比试验，筛选出最佳硒含量的Se-HAP纳米线。
比如摩尔分数0.3％的Se-HAP纳米线合成过程如下。将10.05g NaOH溶于150mL去离子水中，冰水浴中机械搅拌加入135mL去离子水，60mL甲醇和105mL油酸混合,120mL CaCl 2(3.33g)水溶液逐滴加入上述溶液，搅拌30min，取180mL NaH 2PO 4·2H 2O(9.33g)和Na 2SeO 3(0.031g)逐滴加入混合液中，将得到的混合物转移到特氟龙内衬不锈钢高压釜中，密封并在180℃下加热24h。然后得到的浆体加入大量的无水乙醇搅拌，并用200目筛过滤收集。再用乙醇和去离子水洗多次，再分散在去离子水(15mg/g)中，得到Se/(Se+P)摩尔分数0.3％的Se-HAP纳米线(即3Se-HAP)。
参见图1-2，可以参照上述步骤制备HAP纳米线和不同掺硒量的Se-HAP纳米线，并按照不同的Se/(Se+P)摩尔分数制备Se-HAP纳米线探究不同掺硒量，观察对骨髓间充质干细胞成骨性能的影响。
步骤(2)、制备表面多孔有序、底面致密光滑的胶原/SeHAP双层GBR膜。
通过HAP/Se-HAP对mBMSCs(小鼠骨髓间充质干细胞)成骨分化的影响(见图2)，筛选出3Se-HAP作为复合支架的最优Se-HAP纳米线增强胶原支架。首先，将动物I型胶原加到0.5％(v/v)醋酸溶液中搅拌配制40mg/mL的胶原溶液，冰水浴机械搅拌至完全溶解。利用间隔低温超声法(每超声10min,停留10min)使得胶原分散均匀。按照不同浓度加入Se-HAP纳米线粉末于水溶液中，充分搅拌制成浆体，将胶原溶液与Se-HAP浆体混合，使得HAP/SeHAP的质量为3％，冰水浴中机械搅拌使得胶原和Se-HAP混合均匀。将混合溶液超声处理后，将高浓度胶原/Se-HAP混合溶液浓缩成胶原蛋白溶液(0.5mL,40mg/mL)被倒入圆柱形的铜模具中，风干后再加入0.5mL所述混合溶液。离心后,将铜模具放在定向冷冻装置上，溶液以-5℃/min速率从20℃至-60℃冷却。最终温度保持在-60℃，直到样品完全冻结。然后将样品保存在-80℃的冰箱中直到进一步冷冻12h后，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜(即Col@Se-HAP GBR膜)。
作为实验对比，采用不同高分子：鼠尾I型胶原(Col I)、壳聚糖(CS)，香菇多糖(LET)、聚乙烯吡咯烷酮(PVP)制备得到的硒纳米粒子，实验结果发现通过采用鼠尾I型胶原(Col I)与硒结合制备的复合材料，其促干细胞成骨分化效果最优。
通过上述方法制备的掺硒羟基磷灰石纳米增强的胶原GBR膜试验结果如下。
图2是不同掺硒量(Se/(Se+P))的Se-HAP对骨髓间充质干细胞(mBMSCs)的细胞相容性结果(A：MTT实验和B：死活荧光染色)，以及促成骨分化的影响(14天)：HAP/SeHAP的作用浓度为50μg/mL。50μg/mL掺硒羟基磷灰石对mBMSCs细胞没有毒性，同时促进mBMSCs细胞的成骨分化。
图3是Col@Se-HAP GBR膜的扫描电镜图(A)、红外图谱(B)、促mBMSCs细胞成骨分化性能(C:碱性磷酸酶染色)和抗菌性能研究(D：金黄色葡萄球菌抑菌圈)。通过实验证明，与HAP纳米线对比，3Se-HAP具有更加优异的成骨性能和抗菌性能；同时与纯胶原支架相比，复合支架Col@Se_HAP具有更加优异的成骨性能和抗菌性能。
图4是本发明Col@Se-HAP双层GBR膜的屏蔽作用(2weeks)：细胞免疫荧光染色后的激光共聚焦图：Col I(I型胶原)、nucleus(细胞核)、F-actin(F肌动蛋白)、NIH3T3(成纤维细胞)、mBMSCs(小鼠骨髓间充质干细胞)。
可见，本发明Col@Se-HAP双层GBR膜能很好阻止NIH3T3细胞的侵袭。结果说明一侧致密光滑面可以防止非成骨细胞干扰骨缺损部位成骨相关细胞的聚集与骨修复，而另一侧多孔且粗糙，便于骨缺损附近成骨细胞黏附与募集。
图5：本发明Col@Se-HAP双层GBR膜在大鼠颅骨缺损修复4weeks后的Micro-CT。对照组：商用Bio-gide胶原膜。可见Col@Se-HAP双层引导骨再生GBR膜，基于覆盖骨缺损区域的屏障膜特性，阻止来自周围软组织的成纤维细胞侵袭，让骨面处的成骨细胞有足够的时间黏附、增殖、成骨分化，最终实现骨组织再生和修复的目的。骨再生修复性能优于现有产品商用Bio-gide胶原膜。
本发明通过合成的掺硒羟基磷灰石纳米增强的胶原GBR膜，经发现相对于商用的Bio-Gide胶原膜具有更好促进成骨再生的治疗效果，为临床骨再生手术提供新的策略及选择。
发明人经过透射电镜和元素分析显示我们成功制备了掺硒羟基磷灰石纳米线。同时我们使用小鼠骨髓间充质干细胞验证Se-HAP和Col@Se-HAP GBR膜促进成骨分化的能力和屏蔽成纤维细胞侵袭的能力，同时通过金黄色葡萄球菌的抑菌圈实验验证Se-HAP和Col@Se-HAP的良好的抗菌性能。最终，发明人在大鼠颅骨缺损模型中验证Col@Se-HAP GBR膜促骨再生的性能，并发现Col@Se-HAP GBR膜相对商用的Bio-gide膜具有优异的骨缺损修复能力，能够快速地促进颅骨缺损的骨再生修复。
综上所述，本发明不仅展示了掺硒羟基磷灰石纳米线和Col@Se-HAP双层GBR膜制备方法，而且为GBR膜的促骨再生机制及今后在骨缺损再生治疗中的临床应用提供了更多的证据。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，其特征在于，包括步骤：
    (1)制备掺硒羟基磷灰石(Se-HAP)，将Se/(Se+P)通过溶剂热法合成Se-HAP纳米结构体；
    (2)将动物I型胶原加到醋酸溶液中搅拌配制胶原溶液，机械搅拌至完全溶解，利用间隔低温超声方式使得胶原分散均匀；加入Se-HAP纳米粉末于水溶液中，充分搅拌制成Se-HAP浆体，将所述胶原溶液与所述Se-HAP浆体混合均匀得到胶原/Se-HAP混合溶液超声处理后，再将浓缩成胶原蛋白溶液离心处理后放在冷冻装置上，所述胶原蛋白溶液从室温冷却至0℃以下，直到完全冻结一段时间，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜。
  2. 根据权利要求1所述掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，其特征在于，步骤(1)中：是将摩尔分数为0.05％-0.4％的Se/(Se+P)通过溶剂热法合成掺硒的Se-HAP纳米线、或纳米针、或纳米片结构体。
  3. 根据权利要求2所述掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，其特征在于，步骤(1)中：所述Se/(Se+P)的摩尔分数分别取0.05％、0.1％、0.2％和0.3％，分别对应命名为0.5Se-HAP、1Se-HAP、2Se-HAP和3Se-HAP，通过溶剂热法合成掺硒的Se-HAP纳米结构体。
  4. 根据权利要求1所述掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，其特征在于，步骤(1)具体包括，
    将NaOH溶于去离子水中，冰水浴中机械搅拌并加入去离子水、甲醇和油酸,将CaCl 2水溶液逐滴一起加入混合得到混合液，搅拌后，再将NaH 2PO 4·2H 2O和Na 2SeO 3逐滴加入所述混合液中，将得到的混合物转移到高压 釜中，密封并在170℃-200℃下加热，然后得到的浆体加入大量的无水乙醇搅拌，并过滤收集；再用乙醇和去离子水洗多次，最后得到Se-HAP纳米结构体。
  5. 根据权利要求1所述掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，其特征在于，步骤(2)中，具体是在定向冷冻装置上，所述胶原蛋白溶液从室温冷却到负50℃-负60℃冷却，直到完全冻结，然后将冻结的所述胶原蛋白溶液保存在负70℃以下温度的冰箱中直到进一步冷冻一段时间后，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜。
  6. 根据权利要求1所述掺硒羟基磷灰石纳米增强的胶原GBR膜制备方法，其特征在于，步骤(2)中，
    是将动物I型胶原加到0.5％(v/v)醋酸溶液中搅拌配制40mg/mL的胶原溶液，冰水浴机械搅拌至完全溶解；利用间隔低温超声法使得胶原分散均匀；加入Se-HAP纳米线粉末于水溶液中，充分搅拌制成Se-HAP浆体，将所述胶原溶液与所述Se-HAP浆体，在冰水浴中机械搅拌混合均匀，得到胶原/Se-HAP混合溶液超声处理后，将浓缩成胶原蛋白溶液倒入圆柱形的铜模具中，风干后离心后,将铜模具放在定向冷冻装置上，所述胶原蛋白溶液从室温冷却至负60℃，直到完全冻结一段时间，冷冻干燥即可得到所述掺硒羟基磷灰石纳米增强的胶原GBR膜。
  7. 一种掺硒羟基磷灰石纳米增强的胶原GBR膜，其特征在于，是根据权利要求1-6任意一项的所述制备方法得到。
  8. 根据权利要求7所述一种掺硒羟基磷灰石纳米增强的胶原GBR膜，其特征在于，是表面多孔有序、底面致密光滑的胶原/Se-HAP双层GBR膜。
  9. 一种掺硒羟基磷灰石纳米增强的胶原GBR膜在骨再生修复的应用，其 特征在于，所述掺硒羟基磷灰石纳米增强的胶原GBR膜是根据权利要求1-6任意一项的所述制备方法得到。
  10. 根据权利要求9所述一种掺硒羟基磷灰石纳米增强的胶原GBR膜在骨再生修复的应用，其特征在于，具体是应用在引导骨再生康复，具有抗菌性、促成骨分化再生性能。
PCT/CN2021/130487 2021-11-13 2021-11-13 掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法 WO2023082214A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/130487 WO2023082214A1 (zh) 2021-11-13 2021-11-13 掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/130487 WO2023082214A1 (zh) 2021-11-13 2021-11-13 掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法

Publications (1)

Publication Number Publication Date
WO2023082214A1 true WO2023082214A1 (zh) 2023-05-19

Family

ID=86334878

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/130487 WO2023082214A1 (zh) 2021-11-13 2021-11-13 掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法

Country Status (1)

Country Link
WO (1) WO2023082214A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080220148A1 (en) * 2004-10-28 2008-09-11 Clarkson Brian H Methods for Production and Use of Synthetic Hydroxyapatite and Fluorapatite Nanorods, and Superstructures Assembled from the Same
CN103071190A (zh) * 2013-01-25 2013-05-01 广州华美康联生物科技有限公司 一种组织引导再生用胶原基复合生物膜的制备方法
CN106063947A (zh) * 2016-05-25 2016-11-02 西北大学 一种硒掺杂纳米羟基磷灰石的制备方法
CN110078979A (zh) * 2019-05-21 2019-08-02 中国科学技术大学 一种用于引导骨再生的双层纳米复合膜及其制备方法
CN113018519A (zh) * 2021-03-15 2021-06-25 武汉亚洲生物材料有限公司 矿化引导组织再生膜及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080220148A1 (en) * 2004-10-28 2008-09-11 Clarkson Brian H Methods for Production and Use of Synthetic Hydroxyapatite and Fluorapatite Nanorods, and Superstructures Assembled from the Same
CN103071190A (zh) * 2013-01-25 2013-05-01 广州华美康联生物科技有限公司 一种组织引导再生用胶原基复合生物膜的制备方法
CN106063947A (zh) * 2016-05-25 2016-11-02 西北大学 一种硒掺杂纳米羟基磷灰石的制备方法
CN110078979A (zh) * 2019-05-21 2019-08-02 中国科学技术大学 一种用于引导骨再生的双层纳米复合膜及其制备方法
CN113018519A (zh) * 2021-03-15 2021-06-25 武汉亚洲生物材料有限公司 矿化引导组织再生膜及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PAJOR KAMIL, PAJCHEL LUKASZ, KOLODZIEJSKA BARBARA, KOLMAS JOANNA: "Selenium-Doped Hydroxyapatite Nanocrystals–Synthesis, Physicochemical Properties and Biological Significance", CRYSTALS, vol. 8, no. 5, pages 188, XP093064986, DOI: 10.3390/cryst8050188 *

Similar Documents

Publication Publication Date Title
WO2018072679A1 (zh) 一种仿生生物矿化人工骨修复材料及其制备方法与应用
Peng et al. Graphene oxide as an interface phase between polyetheretherketone and hydroxyapatite for tissue engineering scaffolds
Torgbo et al. Bacterial cellulose-based scaffold materials for bone tissue engineering
Antebi et al. Biomimetic collagen–hydroxyapatite composite fabricated via a novel perfusion-flow mineralization technique
CN106729928B (zh) 一种聚乙烯醇/海藻酸钠/羟基磷灰石复合纤维膜及其制备方法、应用
Chen et al. Robust silk fibroin/bacterial cellulose nanoribbon composite scaffolds with radial lamellae and intercalation structure for bone regeneration
Zhong et al. Degradation pattern of porous CaCO3 and hydroxyapatite microspheres in vitro and in vivo for potential application in bone tissue engineering
Sheikh et al. Electrospun titanium dioxide nanofibers containing hydroxyapatite and silver nanoparticles as future implant materials
Yang et al. Bioinspired porous octacalcium phosphate/silk fibroin composite coating materials prepared by electrochemical deposition
CN105148324B (zh) 一种由矿化的纳米纤维素晶须构建的骨仿生材料及其制备方法
Zhang et al. Preparation of chitosan/hydroxyapatite guided membrane used for periodontal tissue regeneration
CN106860915B (zh) 一种透明质酸寡糖修饰的矿化胶原仿生骨修复材料及其制备方法
Mobika et al. Substantial effect of silk fibroin reinforcement on properties of hydroxyapatite/silk fibroin nanocomposite for bone tissue engineering application
Ghomi et al. A novel investigation on characterization of bioactive glass cement and chitosangelatin membrane for jawbone tissue engineering
Fan et al. A new composite scaffold of bioactive glass nanoparticles/graphene: Synchronous improvements of cytocompatibility and mechanical property
CN104001208B (zh) 一种生物可降解高分子/甲壳素纳米晶复合支架材料的制备方法
CN101264341A (zh) 三维多孔组织工程支架材料、其制备及应用
Van Ho et al. Novel TOCNF reinforced injectable alginate/β-tricalcium phosphate microspheres for bone regeneration
Mobika et al. Fabrication of bioactive hydroxyapatite/silk fibroin/gelatin cross-linked nanocomposite for biomedical application
CN110772663A (zh) 一种微纳米等级结构仿生支架及其制备方法
CN112206353B (zh) 一种甲壳素晶须液晶弹性体修饰的聚乳酸复合材料及其制备方法与应用
Wang et al. A scaffold with zinc-whitlockite nanoparticles accelerates bone reconstruction by promoting bone differentiation and angiogenesis
Rama et al. Influence of silk fibroin on the preparation of nanofibrous scaffolds for the effective use in osteoregenerative applications
WO2023082214A1 (zh) 掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法
CN114177368A (zh) 掺硒羟基磷灰石纳米增强的胶原gbr膜及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21963662

Country of ref document: EP

Kind code of ref document: A1