CN116036369A - 一种仿生纳米支架及其制备方法和应用 - Google Patents
一种仿生纳米支架及其制备方法和应用 Download PDFInfo
- Publication number
- CN116036369A CN116036369A CN202310279121.3A CN202310279121A CN116036369A CN 116036369 A CN116036369 A CN 116036369A CN 202310279121 A CN202310279121 A CN 202310279121A CN 116036369 A CN116036369 A CN 116036369A
- Authority
- CN
- China
- Prior art keywords
- growth factor
- biomimetic
- concentration
- insulin
- scaffold
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 230000003592 biomimetic effect Effects 0.000 title claims description 54
- 239000002062 molecular scaffold Substances 0.000 title claims description 19
- 239000002071 nanotube Substances 0.000 claims abstract description 40
- 210000000845 cartilage Anatomy 0.000 claims abstract description 23
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims abstract description 19
- 102000013275 Somatomedins Human genes 0.000 claims abstract description 19
- 102000009618 Transforming Growth Factors Human genes 0.000 claims abstract description 19
- 108010009583 Transforming Growth Factors Proteins 0.000 claims abstract description 19
- 102000004127 Cytokines Human genes 0.000 claims abstract description 17
- 108090000695 Cytokines Proteins 0.000 claims abstract description 17
- 239000011543 agarose gel Substances 0.000 claims abstract description 14
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 13
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 11
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 9
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 9
- 210000001185 bone marrow Anatomy 0.000 claims abstract description 9
- 230000008439 repair process Effects 0.000 claims abstract description 9
- 230000012010 growth Effects 0.000 claims abstract description 8
- 238000011161 development Methods 0.000 claims abstract description 7
- 230000002950 deficient Effects 0.000 claims abstract description 5
- 210000001612 chondrocyte Anatomy 0.000 claims abstract description 4
- 210000005065 subchondral bone plate Anatomy 0.000 claims abstract 2
- 102000046299 Transforming Growth Factor beta1 Human genes 0.000 claims description 16
- 101800002279 Transforming growth factor beta-1 Proteins 0.000 claims description 16
- 101710127951 Extracellular matrix protein 3 Proteins 0.000 claims description 14
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 claims description 13
- 102100037852 Insulin-like growth factor I Human genes 0.000 claims description 13
- 239000000017 hydrogel Substances 0.000 claims description 13
- 229940099456 transforming growth factor beta 1 Drugs 0.000 claims description 13
- 108060003393 Granulin Proteins 0.000 claims description 12
- 208000013201 Stress fracture Diseases 0.000 claims description 11
- 229920000936 Agarose Polymers 0.000 claims description 10
- 102000055008 Matrilin Proteins Human genes 0.000 claims description 7
- 108010072582 Matrilin Proteins Proteins 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 230000013632 homeostatic process Effects 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 238000001338 self-assembly Methods 0.000 claims description 2
- 230000002980 postoperative effect Effects 0.000 claims 1
- 150000003384 small molecules Chemical group 0.000 claims 1
- 239000008280 blood Substances 0.000 abstract description 7
- 210000004369 blood Anatomy 0.000 abstract description 7
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 210000000130 stem cell Anatomy 0.000 abstract description 3
- 239000012620 biological material Substances 0.000 abstract description 2
- 230000003902 lesion Effects 0.000 description 8
- 238000001356 surgical procedure Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 210000001188 articular cartilage Anatomy 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002385 Sodium hyaluronate Polymers 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000003035 hyaline cartilage Anatomy 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229940010747 sodium hyaluronate Drugs 0.000 description 2
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 2
- 210000001258 synovial membrane Anatomy 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 230000022159 cartilage development Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000011227 chondrocyte hypertrophy Effects 0.000 description 1
- 230000009816 chondrogenic differentiation Effects 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 210000000968 fibrocartilage Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002608 insulinlike Effects 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000000733 zeta-potential measurement Methods 0.000 description 1
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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Transplantation (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
- Prostheses (AREA)
Abstract
本发明属于生物材料及生物医学技术领域，具体涉及一种仿生纳米支架及其制备方法和应用，其中，所述仿生纳米支架，其包含琼脂糖凝胶，所述琼脂糖凝胶负载有可注射的类DNA仿生纳米管以及细胞因子；所述细胞因子包括软骨外基质蛋白、转化生长因子和胰岛素样生长因子。本发明设置的仿生支架与渗血混合，形成适合骨髓间充质干细胞生长发育的微环境，在微环境中，干细胞可以精准的被调控向软骨细胞分化并维持稳态，产生软骨基质，形成高质量的透明样新软骨和软骨下骨修复，重建缺损的软骨区域，具有良好的实际应用价值。
Description
技术领域
本发明属于生物材料及生物医学技术领域，具体涉及一种仿生纳米支架及其制备方法和应用。
背景技术
本部分的陈述仅仅是提供了与本发明相关的背景技术信息，不必然构成在先技术。
微骨折手术是常用于修复膝关节软骨病损的技术，已被广泛用于临床，其具体操作为“在关节镜直视下，将软骨病损部位移除，并在骨头上钻几个孔，使骨髓中的血液与间充质干细胞一并渗出并在原位凝结，血凝块有一定软骨生成能力，进而产生软骨修复的作用”，因其技术简单、手术安全和成本效益低从而被美国食品药品监督管理局（FDA）和临床医生视为关节软骨修复的黄金标准。
然而，长期研究表明，由于纤维软骨的形成，微骨折手术的治疗效果通常不令人满意，纤维软骨的机械性能不如正常的透明软骨。这种不良结果是由手术后血凝块的微环境引起的，由于相对较低的承载能力和高流动性，这种微环境不适合骨髓间充质干细胞发育，导致募集能力不足和软骨分化效率低下。
发明内容
针对现有技术存在的不足，本发明提供一种仿生纳米支架及其制备方法和应用，从而可以构建合适的微环境，诱导干细胞，产生正常的透明软骨，重建缺损的软骨区域。基于上述研究成果，从而完成本发明。
为了实现上述目的，本发明是通过如下的技术方案来实现：
本发明的第一个方面，提供一种仿生纳米支架，其包含琼脂糖凝胶，所述琼脂糖凝胶负载有可注射的柔性类DNA仿生纳米管以及细胞因子；
所述细胞因子包括软骨外基质蛋白、转化生长因子和胰岛素样生长因子；其中，所述软骨外基质蛋白、转化生长因子和胰岛素样生长因子在等浓度情况下其用量体积比为14-17∶1-5∶0.1-1.5。
进一步地，所述类DNA仿生纳米管、软骨外基质蛋白、转化生长因子和胰岛素样生长因子在等质量浓度情况下其用量体积比为2-4∶14-17∶1-5∶0.1-1.5。
进一步地，所述类DNA仿生纳米管、软骨外基质蛋白、转化生长因子和胰岛素样生长因子在等质量浓度情况下其用量体积比为3∶16∶4∶1。
进一步地，所述的类DNA仿生纳米管（DNA-inspired biomimetic nanotubes,DBNTs）为亲水性柔性纳米管，由人工合成的带有氨基酸侧链的类碱基小分子单元通过分子间自组装作用形成；
进一步地，所述转化生长因子具体为转化生长因子-β1，所述软骨外基质蛋白具体为胞外基质蛋白-3，所述胰岛素样生长因子具体为胰岛素样生长因子-1。
本发明的第二个方面，提供上述仿生纳米支架的制备方法，包括如下步骤：配置琼脂糖凝胶浓度为1%-3%、可注射的柔性类DNA仿生纳米管浓度为1-2 mg/ml、软骨外基质蛋白浓度为0.5-1.5 μg/ml、转化生长因子浓度为0.5-1.5 μg/ml、胰岛素样生长因子浓度为0.5-1.5 μg/ml；将可注射的类DNA仿生纳米管与转化生长因子、软骨外基质蛋白、胰岛素样生长因子结合后与等量琼脂糖凝胶于38-40 ℃环境下混合得到可注射液体状态仿生纳米支架。
进一步地，所述琼脂糖水凝胶质量浓度为2%；所述可注射的类DNA仿生纳米管浓度为1mg/ml；所述软骨外基质蛋白浓度为1 μg/ml；转化生长因子浓度为1 μg/ml；胰岛素样生长因子浓度为1 μg/ml。
本发明的第三个方面，提供上述仿生纳米支架在制备微骨折手术后修复材料中的应用。
具体的，所述微骨折手术后修复材料具有至少如下任意一种或多种功能：
构建适合骨髓间充质干细胞生长发育的微环境；
促进骨髓间充质干细胞生长发育向软骨细胞分化并维持稳态，产生软骨基质，形成高质量透明样新软骨，重建缺损的软骨区域。
具体的，在实际应用时，在患者麻醉情况下置入关节镜器械，在镜头下寻找软骨病损区域，使用刨刀将病损区域清除，并清除关节腔内增生的滑膜，使用微骨折钻头，在关节软骨病损区域钻孔，直至有血液渗出；将可注射的类DNA仿生纳米管与细胞因子转化生长因子-β1、胰岛素样生长因子-1、蛋白胞外基质蛋白-3及琼脂糖凝胶结合，关节镜可视下注射到微骨折钻头所致软骨缺损区域，使其与渗出血液混合，待凝固后大量生理盐水冲洗关节腔，注射玻璃酸钠两支，退出关节镜器械。
上述本发明的有益效果如下：
本发明仿生纳米支架中的转化生长因子-β1 具有调节细胞增殖、促进充质干细胞向软骨分化的功能；已有研究证明，胰岛素样生长因子-1 与 转化生长因子-β1 联合使用可增强关节透明软骨的生成；胞外基质蛋白-3 作为软骨基质分子，具有抑制软骨细胞肥大，并向骨细胞分化的功能；胞外基质蛋白-3和转化生长因子-β1结合后不仅可更好地诱导软骨组织形成，还能起到维持软骨稳态的作用。类DNA仿生纳米管与多种细胞因子形成的可注射仿生细胞外基质支架具有立体网格结构，可增加细胞锚定并促细胞增殖，可为间充质干细胞生长提供结构上的支持。从而使得在微骨折手术产生的缺损中，本发明设置的仿生支架与渗血混合，形成适合骨髓间充质干细胞生长发育的微环境，在微环境中，干细胞可以精准的被调控向软骨细胞分化并维持稳态，产生软骨基质，形成高质量的透明样新软骨，重建缺损的软骨区域。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1是本发明实施例1的琼脂糖（Agarose）水凝胶电镜。
图2是本发明实施例1的类DNA仿生纳米管结合细胞因子后与水凝胶结合后电镜下照片。
图3是本发明实施例1的类DNA仿生纳米管结合细胞因子后与大鼠骨髓间充质干细胞紧密结合的冷冻扫描电镜照片。
图4是本发明实施例1紫外分光温度计（Nanodrop）的吸光光度测定。
图5是本发明实施例1的电动电位测定。
图6是本发明实施例1的类DNA仿生纳米管的透射电子显微镜（Transmissionelectron microscopy, TEM）图像。
图7是本发明实施例1的可注射仿生纳米支架的透射电子显微镜图像。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非本发明另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
实施例1
一种仿生纳米支架，其包含琼脂糖凝胶，所述琼脂糖凝胶负载有可注射的类DNA仿生纳米管以及细胞因子；所述细胞因子至少包括软骨外基质蛋白、转化生长因子和胰岛素样生长因子；
具体的，所述可注射的类DNA仿生纳米管（DBNTs）为亲水性自主装柔性纳米管；
所述转化生长因子具体为转化生长因子-β1（TGF-β1），所述软骨外基质蛋白具体为胞外基质蛋白-3（matrilin-3），所述胰岛素样生长因子具体为胰岛素样生长因子-1（IGF-1）。
具体的，所述仿生纳米支架的制备方法，包括如下步骤：
配置琼脂糖凝胶（Agarose水凝胶）浓度为2%；DNA仿生纳米管浓度为1mg/ml；胞外基质蛋白-3浓度为1 μg/ml；转化生长因子-β1浓度为1 μg/ml；胰岛素样生长因子-1浓度为1 μg/ml；使得类DNA仿生纳米管结合细胞因子∶胞外基质蛋白-3∶转化生长因子-β1∶胰岛素样生长因子-1用量体积比例为3∶16∶4∶1；类DNA仿生纳米管与细胞因子有序组装形成类DNA仿生纳米管/胞外基质蛋白-3/转化生长因子-β1/胰岛素样生长因子-1仿生细胞外基质支架(D/M/T/I Nano-Matrix)后，与等量琼脂糖凝胶（Agarose水凝胶）混合，此时琼脂糖凝胶（Agarose水凝胶）浓度为1%；类DNA仿生纳米管终浓度为62.5 μg/ml；胞外基质蛋白-3终浓度为0.33 μg/ml；转化生长因子-β1终浓度为83 ng/ml；胰岛素样生长因子-1终浓度为20ng/ml。
在单独进行水凝胶拍摄时，凝胶与等量双蒸水（ddH2O）混合，所拍摄琼脂糖凝胶（Agarose水凝胶）浓度为1%，得到如图1所示的琼脂糖凝胶（Agarose水凝胶）冷冻扫描电镜下照片，为光滑的网格状结构。
在拍摄类DNA仿生纳米管结合细胞因子后与水凝胶结合时，将仿生细胞外基质支架（D/M/T/I Nano-Matrix）溶液与2%水凝胶等量混合，然后进行拍摄。
图2为类DNA仿生纳米管结合细胞因子后与水凝胶结合后冷冻扫描电镜下照片，可以看到仿生纳米管可以填充于凝胶网格中，并形成更紧密的3D立体网格结构；图3为类DNA仿生纳米管结合细胞因子后与大鼠骨髓间充质干细胞紧密结合的电镜照片，可以观察到类DNA仿生纳米管可以与细胞结合将细胞固定。
实验分为8组进行紫外分光温度计（Nanodrop）吸光光度测定，结果如图4所示，类DNA仿生纳米管在220和280 nm处有两个吸收峰，加入转化生长因子-β1、胰岛素样生长因子-1，胞外基质蛋白-3三种细胞因子后，类DNA仿生纳米管吸收峰值显著降低，类DNA仿生纳米管与几种蛋白之间通过静电吸引力与生物亲和力结合。
实验分为3组进行电动电位（Zeta电位）测定，结果如图5所示，在生理条件下，胞外基质蛋白-3蛋白带负电荷；当与转化生长因子-β1及胰岛素样生长因子-1混合后，溶液表现出的点位值趋向中性，表明三种蛋白可以通过电荷作用后更加接近中性值；类DNA仿生纳米管在生理环境中带正电，胞外基质蛋白-3与转化生长因子-β1、胰岛素样生长因子-1混合物进一步与类DNA仿生纳米管结合，导致混合液由原本负电荷变为正电荷，结果表明三种成分可以有序组装成仿生细胞外基质支架（D/M/T/I Nano-Matrix）结构。
当使用时，在患者麻醉情况下置入关节镜器械，在镜头下寻找软骨病损区域，使用刨刀将病损区域清除，并清除关节腔内增生的滑膜，使用微骨折钻头，在关节软骨病损区域钻孔，直至有血液渗出；将可注射的类DNA仿生纳米管与细胞因子转化生长因子-β1、胰岛素样生长因子-1、蛋白胞外基质蛋白-3及琼脂糖凝胶结合，关节镜可视下注射到微骨折钻头所致软骨缺损区域，使其与渗出血液混合，待凝固后大量生理盐水冲洗关节腔，注射玻璃酸钠两支，退出关节镜器械。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种仿生纳米支架，其特征在于，其包含琼脂糖凝胶，所述琼脂糖凝胶负载有可注射的类DNA仿生纳米管以及细胞因子；
所述细胞因子包括软骨外基质蛋白、转化生长因子和胰岛素样生长因子；其中，所述软骨外基质蛋白、转化生长因子和胰岛素样生长因子在等质量浓度情况下其用量体积比为14-17∶1-5∶0.1-1.5。
2.如权利要求1所述的仿生纳米支架，其特征在于，所述可注射的类DNA仿生纳米管、软骨外基质蛋白、转化生长因子和胰岛素样生长因子在等质量浓度情况下的用量体积比为2-4∶14-17∶1-5∶0.1-1.5。
3.如权利要求2所述的仿生纳米支架，其特征在于，所述可注射的类DNA仿生纳米管、软骨外基质蛋白、转化生长因子和胰岛素样生长因子在等质量浓度情况下的用量体积比为3∶16∶4∶1。
4.如权利要求1所述的仿生纳米支架，其特征在于，所述可注射的类DNA仿生纳米管为亲水性柔性纳米管，由人工合成的带有氨基酸侧链的类碱基小分子单元通过分子间自组装作用形成。
5.如权利要求1所述的仿生纳米支架，其特征在于，所述转化生长因子为转化生长因子-β1。
6.如权利要求1所述的仿生纳米支架，其特征在于，所述软骨外基质蛋白为胞外基质蛋白-3。
7.如权利要求1所述的仿生纳米支架，其特征在于，所述胰岛素样生长因子为胰岛素样生长因子-1。
8.一种如权利要求1-7任一项所述仿生纳米支架的制备方法，其特征在于，包括如下步骤：配置琼脂糖凝胶浓度为1%-3%、可注射的类DNA仿生纳米管浓度为1-2 mg/ml、软骨外基质蛋白浓度为0.5-1.5 μg/ml、转化生长因子浓度为0.5-1.5 μg/ml、胰岛素样生长因子浓度为0.5-1.5 μg/ml；将可注射的类DNA仿生纳米管与转化生长因子、软骨外基质蛋白、胰岛素样生长因子结合后与等量琼脂糖凝胶混合得到仿生纳米支架。
9.如权利要求8所述的制备方法，其特征在于，所述琼脂糖水凝胶浓度为2%；所述可注射的类DNA仿生纳米管浓度为1mg/ml；所述软骨外基质蛋白浓度为1 μg/ml；转化生长因子浓度为1 μg/ml；胰岛素样生长因子浓度为1 μg/ml。
10.一种如权利要求1-7任一项所述仿生纳米支架在制备微骨折手术后修复材料中的应用；
具体的，所述微骨折手术后修复材料具有至少如下任意一种或多种功能：
构建适合骨髓间充质干细胞生长发育的微环境；
促进骨髓间充质干细胞生长发育向软骨细胞分化并维持稳态，产生软骨基质，形成高质量透明样新软骨和软骨下骨修复，重建缺损的软骨区域。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310279121.3A CN116036369A (zh) | 2023-03-22 | 2023-03-22 | 一种仿生纳米支架及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310279121.3A CN116036369A (zh) | 2023-03-22 | 2023-03-22 | 一种仿生纳米支架及其制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116036369A true CN116036369A (zh) | 2023-05-02 |
Family
ID=86127556
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310279121.3A Pending CN116036369A (zh) | 2023-03-22 | 2023-03-22 | 一种仿生纳米支架及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116036369A (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020151556A1 (en) * | 2001-01-17 | 2002-10-17 | Hicham Fenniri | Method and associated compounds for forming nanotubes |
| CN104307046A (zh) * | 2014-10-27 | 2015-01-28 | 王黎明 | 一种可注射骨髓间充质干细胞外基质/琼脂糖复合水凝胶及其制备方法和应用 |
| CN105255814A (zh) * | 2015-10-27 | 2016-01-20 | 上海科医联创生物科技有限公司 | 一种用于微骨折手术的软骨诱导基质及制备方法 |
| US20160361464A1 (en) * | 2015-06-12 | 2016-12-15 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Biomimetic Hydrogel Scaffolds and Related Methods |
| US20170112961A1 (en) * | 2014-05-16 | 2017-04-27 | Stemmatters, Biotecnologia E Medicina Regenerativa Sa | Products for repairing cartilage lesions, method of preparation and uses thereof |
| WO2022081721A1 (en) * | 2020-10-13 | 2022-04-21 | University Of Connecticut | Nanomaterial and methods of use thereof |
-
2023
- 2023-03-22 CN CN202310279121.3A patent/CN116036369A/zh active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020151556A1 (en) * | 2001-01-17 | 2002-10-17 | Hicham Fenniri | Method and associated compounds for forming nanotubes |
| US20170112961A1 (en) * | 2014-05-16 | 2017-04-27 | Stemmatters, Biotecnologia E Medicina Regenerativa Sa | Products for repairing cartilage lesions, method of preparation and uses thereof |
| CN104307046A (zh) * | 2014-10-27 | 2015-01-28 | 王黎明 | 一种可注射骨髓间充质干细胞外基质/琼脂糖复合水凝胶及其制备方法和应用 |
| US20160361464A1 (en) * | 2015-06-12 | 2016-12-15 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Biomimetic Hydrogel Scaffolds and Related Methods |
| CN105255814A (zh) * | 2015-10-27 | 2016-01-20 | 上海科医联创生物科技有限公司 | 一种用于微骨折手术的软骨诱导基质及制备方法 |
| WO2022081721A1 (en) * | 2020-10-13 | 2022-04-21 | University Of Connecticut | Nanomaterial and methods of use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 鄂征等: "《医学组织工程技术与临床应用》", 北京出版社, pages: 369 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | Silk fibroin-based biomaterials for cartilage/osteochondral repair | |
| Xue et al. | Recent advances in design of functional biocompatible hydrogels for bone tissue engineering | |
| Maihemuti et al. | 3D-printed fish gelatin scaffolds for cartilage tissue engineering | |
| Moses et al. | Chondroprotective and osteogenic effects of silk-based bioinks in developing 3D bioprinted osteochondral interface | |
| Ruvinov et al. | Articular cartilage regeneration using acellular bioactive affinity-binding alginate hydrogel: A 6-month study in a mini-pig model of osteochondral defects | |
| Lan et al. | Physicochemical properties and biocompatibility of the bi-layer polyvinyl alcohol-based hydrogel for osteochondral tissue engineering | |
| Li et al. | Biomimetic methacrylated gelatin hydrogel loaded with bone marrow mesenchymal stem cells for bone tissue regeneration | |
| Chen et al. | A single integrated osteochondral in situ composite scaffold with a multi-layered functional structure | |
| Xue et al. | Synthesis of injectable shear‐thinning biomaterials of various compositions of gelatin and synthetic silicate nanoplatelet | |
| CN116285386B (zh) | 一种软骨组织修复水凝胶及其制备方法 | |
| Wu et al. | Enhanced osteochondral regeneration with a 3D-Printed biomimetic scaffold featuring a calcified interfacial layer | |
| US20170056560A1 (en) | Implant comprising fgf-18 | |
| ES2515118T3 (es) | Materiales de relleno para huecos óseos y procedimientos de fabricación de los mismos | |
| Li et al. | Regional specific tunable meniscus decellularized extracellular matrix (MdECM) reinforced bioink promotes anistropic meniscus regeneration | |
| CA3201896A1 (en) | Particulate materials for tissue mimics | |
| Yao et al. | Multi-crosslinked hydrogels composed of hyaluronic acid, silk fibroin and chitosan nanoparticles with capacity of bioactive molecule delivery and degradation tolerance for cartilage tissue engineering | |
| CN115894985B (zh) | 具有生物诱导活性的组织修复水凝胶及其制备方法与应用 | |
| CN116036369A (zh) | 一种仿生纳米支架及其制备方法和应用 | |
| CN101085357A (zh) | 一种用于关节腔内注射治疗软骨缺损的非病毒纳米核酸转运复合物及其制备方法 | |
| Sanyal et al. | Three-dimensional bioprinting of articular cartilage using silk fibroin–gelatin bioink | |
| ES2361420T3 (es) | Procedimiento sencillo para trasplantar condrocitos inyectables para trasplante de condrocitos autologos. | |
| WO2022178907A1 (zh) | 多肽在促进软骨再生或修复中的用途 | |
| CN115105464B (zh) | 一种纳米复合水凝胶及其制备方法和应用 | |
| Zhao et al. | Nanohydroxyapatite and liposomes-coated integral bilayer scaffold for osteochondral repair via mimicking the dual differentiation microenvironment of BMSCs | |
| Liu et al. | Magnesium oxide nanoparticles modulate phase separation to form trabecular-structured cryogels for bone defect repair |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |