CN104800885B - 一种具有趋化功能的生物活性支架的制备和应用 - Google Patents
一种具有趋化功能的生物活性支架的制备和应用 Download PDFInfo
- Publication number
- CN104800885B CN104800885B CN201510252317.9A CN201510252317A CN104800885B CN 104800885 B CN104800885 B CN 104800885B CN 201510252317 A CN201510252317 A CN 201510252317A CN 104800885 B CN104800885 B CN 104800885B
- Authority
- CN
- China
- Prior art keywords
- spinal cord
- scaffold
- cord injury
- endogenous
- cells
- 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.)
- Expired - Fee Related
Links
- 230000000975 bioactive effect Effects 0.000 title claims abstract description 23
- 230000035605 chemotaxis Effects 0.000 title claims abstract description 8
- 238000002360 preparation method Methods 0.000 title description 5
- 208000020431 spinal cord injury Diseases 0.000 claims abstract description 22
- 230000004069 differentiation Effects 0.000 claims abstract description 6
- 230000004083 survival effect Effects 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 30
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 claims description 10
- 241001465754 Metazoa Species 0.000 claims description 5
- 108010022355 Fibroins Proteins 0.000 claims description 4
- 230000008439 repair process Effects 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims description 2
- 239000005482 chemotactic factor Substances 0.000 claims 4
- 241000206672 Gelidium Species 0.000 claims 1
- 108010059642 isinglass Proteins 0.000 claims 1
- 229910052627 muscovite Inorganic materials 0.000 claims 1
- 238000002054 transplantation Methods 0.000 abstract description 21
- 210000000278 spinal cord Anatomy 0.000 abstract description 19
- 230000003399 chemotactic effect Effects 0.000 abstract description 18
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 abstract description 16
- 229930002330 retinoic acid Natural products 0.000 abstract description 16
- 229960001727 tretinoin Drugs 0.000 abstract description 16
- 108010010803 Gelatin Proteins 0.000 abstract description 10
- 229920000159 gelatin Polymers 0.000 abstract description 10
- 239000008273 gelatin Substances 0.000 abstract description 10
- 235000019322 gelatine Nutrition 0.000 abstract description 10
- 235000011852 gelatine desserts Nutrition 0.000 abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 9
- 208000027418 Wounds and injury Diseases 0.000 abstract description 9
- 230000006378 damage Effects 0.000 abstract description 9
- 208000014674 injury Diseases 0.000 abstract description 9
- 102000019034 Chemokines Human genes 0.000 abstract description 8
- 108010012236 Chemokines Proteins 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 241000255789 Bombyx mori Species 0.000 abstract description 5
- 230000012292 cell migration Effects 0.000 abstract description 5
- 230000000324 neuroprotective effect Effects 0.000 abstract description 4
- 238000004108 freeze drying Methods 0.000 abstract description 3
- 238000004132 cross linking Methods 0.000 abstract description 2
- 239000013078 crystal Substances 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 102100029268 Neurotrophin-3 Human genes 0.000 abstract 1
- 239000003431 cross linking reagent Substances 0.000 abstract 1
- 230000000508 neurotrophic effect Effects 0.000 abstract 1
- 235000003170 nutritional factors Nutrition 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 15
- 108090000742 Neurotrophin 3 Proteins 0.000 description 11
- 241000700159 Rattus Species 0.000 description 11
- 102000004230 Neurotrophin 3 Human genes 0.000 description 10
- 229940032018 neurotrophin 3 Drugs 0.000 description 10
- 239000000463 material Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 241000283707 Capra Species 0.000 description 8
- 238000011160 research Methods 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000012460 protein solution Substances 0.000 description 5
- 241000282472 Canis lupus familiaris Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 230000005012 migration Effects 0.000 description 4
- 238000013508 migration Methods 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- 210000004126 nerve fiber Anatomy 0.000 description 4
- 230000008961 swelling Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241000282465 Canis Species 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 101150117329 NTRK3 gene Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 2
- 102000000018 Chemokine CCL2 Human genes 0.000 description 2
- 102000006573 Chemokine CXCL12 Human genes 0.000 description 2
- 108010008951 Chemokine CXCL12 Proteins 0.000 description 2
- 241000729176 Fagopyrum dibotrys Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 2
- -1 Sangon) Chemical compound 0.000 description 2
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 238000002637 fluid replacement therapy Methods 0.000 description 2
- 230000023597 hemostasis Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 229960002275 pentobarbital sodium Drugs 0.000 description 2
- 238000009366 sericulture Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000019155 vitamin A Nutrition 0.000 description 2
- 239000011719 vitamin A Substances 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- 101000634196 Homo sapiens Neurotrophin-3 Proteins 0.000 description 1
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- GYDJEQRTZSCIOI-UHFFFAOYSA-N Tranexamic acid Chemical compound NCC1CCC(C(O)=O)CC1 GYDJEQRTZSCIOI-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001467 acupuncture Methods 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 1
- 150000001746 carotenes Chemical class 0.000 description 1
- 235000005473 carotenes Nutrition 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229920006237 degradable polymer Polymers 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000007646 directional migration Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 102000057714 human NTF3 Human genes 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 210000003007 myelin sheath Anatomy 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 230000007472 neurodevelopment Effects 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000010943 off-gassing Methods 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229940117828 polylactic acid-polyglycolic acid copolymer Drugs 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 description 1
- 150000002266 vitamin A derivatives Chemical class 0.000 description 1
Landscapes
- Materials For Medical Uses (AREA)
Abstract
一种用于修复脊髓损伤的具有趋化内源性细胞迁移、促进内源性细胞存活和分化，以及具后神经保护作用的新型生物活性支架。利用蚕茧中提取的天然丝蛋白作为媒介，利用冷冻干燥技术和乙醇交联剂使神经营养因子‑3(NT‑3)、维甲酸(RA)和丝蛋白晶形转变，交联固定在三维明胶海绵支架中。本发明提供的具有趋化功能的生物活性支架，可以在移植到脊髓后较长时间释放趋化因子吸引内源性细胞迁移到损伤/移植区，释放具有神经保护作用的营养因子，改善微环境，达到吸引内源性细胞施实自我修复脊髓损伤的目的。
Description
所属技术领域
本发明专利涉及一种用于修复脊髓损伤的支架材料，尤其是能够吸引内源性细胞迁移、促进内源性细胞存活和分化的生物活性支架。
背景技术
目前，公知的用于修复脊髓损伤的支架是由支架和外源性细胞组成的。主要是通过外源性细胞移植到受损伤处来达到替换丢失神经元和治疗脊髓损伤的效果，而支架仅仅是作为一种支撑细胞附着的载体。但是，临床应用过程中，外源性细胞的移植面临着来源、伦理和免疫排斥等重重困难，这使得脊髓损伤的外源性细胞移植治疗受到了极大的限制。因此，找到一种有效的方式即可规避细胞移植的细胞来源性问题，又能满足脊髓损伤/移植区对内源性细胞迁移、存活、分化和提供营养活性因子的需求是关键。
细胞的靶向迁移及其机制，一直是科学家们关注的焦点。目前的研究主要集中于外源性细胞体内移植之后的定向迁移。将能够刺激细胞迁移的趋化因子注射到病变部位，能够将移植的外源性细胞特异性吸引迁移过来，并能呈现治疗和替换的效果。常用的趋化因子包括单核细胞趋化蛋白-1(MCP-1)和基质细胞源性因子-1(SDF-1)等[Takeuchi H，etal.Intravenously transplanted human neural stem cells migrate to the injuredspinal cord in adult mice in an SDF-1-and HGF-dependent manner.Neurosci Lett，2007，426(2)：69-74]。近年来，利用具有神经保护作用的营养因子作为趋化因子，也展现出很好的趋化效果，同时这些因子还具有营养细胞和改善脊髓损伤微环境的作用，如神经营养素-3(NT-3)等[Chen YF，et al.Neurotrophin-3 stimulates migration ofmesenchymal stem cells overexpressing TrkC.Curr Med Chem，2013，20(24)：3022-3033]。
维甲酸(RA)是维生素A(Vit.A)的一种代谢产物。动物自身不能合成Vit.A，必须从植物中以胡萝卜素的形式和在动物中以视黄酸等形式摄取。RA在神经发育中扮演着主要角色。研究表明，RA能够诱导不同种类的特殊细胞用于移植治疗。本课题组前期研究发现，在体外，利用RA诱导骨髓间充质干细胞(MSCs)向神经组织细胞方向分化，提高相关受体mRNA表达水平[Zhang W，et al.Combination of adenoviral vector-mediatedneurotrophin-3 gene transfer and retinoic acid promotes adult bone marrowcells to differentiate into neuronal phenotypes.Neurosci Lett，2006，408(2)：98]。在体内，经过RA诱导的MSCs在NT-3和督脉电针的协同作用下，具有向神经元方向分化的能力[Zhang K，et al.Electro-acupuncture promotes the survival anddifferentiation of transplanted bone marrow mesenchymal stem cells pre-induced with neurotrophin-3and retinoic acid in gelatin sponge scaffold afterrat spinal cord transection.Stem Cell RevRep，2014，10(4)：612]。因此，RA在神经组织细胞的诱导和分化中有着关键性的作用。
目前在国内、外，一种自身具有生物活性并能够趋化内源性细胞迁移到脊髓损伤/移植处的支架材料仍未见报道。为此，我们设想构建一种具有趋化内源性细胞迁移功能的生物活性支架。拟将这种具有趋化活性支架移植入脊髓损伤处，调动内源性细胞迁移到损伤/移植处实施自我修复脊髓损伤。本发明的目的是想克服现有临床上治疗脊髓损伤的技术和方法上的不足，应用我们构建的具有趋化性能的生物活性支架为内源性细胞修复脊髓损伤提供崭新的思路和方法。
发明内容
为了克服现有的生物活性支架移植治疗脊髓损伤的方案不足，本发明专利提供一种具有趋化活性支架，该支架不仅能够吸引内源性相关细胞迁移到支架内部存活和分化，而且能够通过缓释神经营养因子和诱导因子来改善脊髓损伤/移植区的微环境。
本发明专利解决其技术问题所采用的技术方案是：
利用蚕茧中提取的天然丝蛋白作为媒介，利用冷冻干燥技术使神经营养素-3(NT-3)、维甲酸(RA)和丝蛋白混合液去水和除菌，乙醇促进趋化活性因子混合液的晶形转变，将趋化因子和诱导因子交联固定在三维支架中。移植后，支架上的趋化活性因子会缓慢释放到脊髓损伤区的微环境中，以支架移植部位为中心形成一种趋化因子富集区，促进相关的内源性细胞向该区域迁移，达到吸引内源性细胞迁移到支架内部存活和分化修复脊髓损伤的目的。
本发明专利的有益效果是：
这种具有功能性生物活性支架材料移植到脊髓损伤处后，可以较长时间释放趋化因子(NT-3)，持续吸引内源性相关细胞迁移到支架内，在诱导因子(RA)的协同作用下这些内源性相关细胞能够更好地存活，并且向神经组织细胞方向分化；同时释放的NT-3具有神经保护作用，持续改善脊髓损伤/移植区的微环境。
附图说明
下面结合附图和实施例对本发明专利进一步说明。
图1是具有趋化功能的生物活性支架的原理图(1示：趋化因子、诱导因子和丝蛋白复合物；2示：三维明胶海绵；3示：支架内部的不规则多孔隙结构；4示：PLGA外壳)。
图2是具有趋化功能的生物活性支架的溶胀率。
图3是具有趋化功能的生物活性支架的吸水率。
图4是具有趋化功能的生物活性支架移植到大鼠脊髓全横断处后，可观察到神经纤维生长和星形胶质细胞迁移进入损伤/移植区。
图5是具有趋化功能的生物活性支架移植到大鼠脊髓全横断处后，可观察到包绕着髓鞘结构的神经纤维生长进入损伤/移植区。
图6是具有趋化功能的生物活性支架移植到大鼠脊髓全横断处后，可观察到损伤/移植区有表达TrkC的细胞迁移进入。
图7是具有趋化功能的生物活性支架移植到犬脊髓半横断处后，可观察到血管样的结构延伸进入损伤/移植区。
图8是具有趋化功能的生物活性支架移植到犬脊髓半横断处后，可观察到大量细胞迁移进入损伤/移植区。
具体实施方式
下面通过具体实施例对本发明所用的主要仪器、趋化活性支架和试剂作详尽的描述：
1.主要仪器
超净工作台(苏州净化电子设备厂)；普通离心机(久保田日本)；恒温水浴箱(北京医疗设备厂)；5％CO2培养箱(Queue美国)；倒置相差显微镜(Olympus日本)；荧光显微镜(Leica德国)；扫描电镜(Philips荷兰)；透射电镜(Philips荷兰)；激光共聚焦成像系统(Carl Zeiss德国)；低温烤箱(上海跃进医疗器械厂)；高温烤箱(上海跃进医疗器械厂)；高压消毒锅(江阴滨江医疗设备厂)；恒冷箱切片机(Shandon英国)；超纯水仪(Molsheim法国)；酶联免疫检测仪(Bio-Rad美国)；电泳仪电源(Bio-Rad美国)；垂直板电泳槽(Bio-Rad美国)；电转仪(Bio-Rad美国)；超高速低温离心机(Beckman美国)；-80℃超低温冰箱(RevcoTech美国)；JY92-2D超声波细胞粉碎机(宁波新芝生物科技股份有限公司)。
2.趋化活性支架
制备支架的多孔隙明胶圆柱体支架材料是购自于南京金陵药业股份有限公司的产品---医用明胶海绵。包绕在多孔隙明胶圆柱体支架周围形成外壳薄壁的PLGA(50∶50)薄膜是购自于济南岱罡生物科技公司的产品。蚕茧是由浙江省农业科学院蚕桑研究所赠送。NT-3和RA均购自Sigma公司。
3.主要试剂
DMEM-LG(Gibico)，优级胎牛血清(TBD)，多聚赖氨酸(Sigma)，D-Hank’s平衡液(自配)，胰蛋白酶(Sigma)，EDTA(Sangon)，0.01mol/L PBS(中杉金桥)，MTT(Ameresco公司)，二甲基亚砜(DMSO，Sangon)，Hoechst33342(Sigma)，DAPI(Sigma)，山羊血清(中杉金桥)，小鼠抗BrdU单克隆抗体(Sigma)，Cy3标记羊抗小鼠IgG(Jackson ImmunoResearch)，calcein-AM/EthD-III Live/Dead kit(Biotium)，兔抗NT-3多克隆抗体(Santa Cruz)，鼠抗人TrkC单克隆抗体(RD)，兔抗大鼠NF单克隆抗体(Sigma)，小鼠抗大鼠NF单克隆抗体(Sigma)，兔抗大鼠GFAP多克隆抗体(Sigma)，小鼠抗大鼠MBP单克隆抗体(Millipore)，山羊抗小鼠FITC(Jackson Immunological Research)，Cy3标记的羊抗小鼠IgG(JacksonImmunoResearch)，Cy3标记羊抗兔IgG(Jackson ImmunoResearch)，Dylight 405标记的羊抗小鼠IgG(Jackson ImmunoResearch)，AMCA标记羊抗兔IgG(Jackson ImmunoResearch)，山羊抗兔HRP(Jackson ImmunoResearch)，蛋白定量检测试剂盒(鼎国)，细胞裂解液(Boster)，蛋白酶抑制剂cocktail(Sigma)，ECL发光底物检测试剂盒(康为世纪)，Epon-812(Ted Pella)，考马斯亮蓝(Bio-rad)，30％聚丙烯酰胺溶液(康为世纪)，X感光胶片(Kodak)。
本发明详细的具体操作技术说明如下：
1.趋化活性支架的构建
1.1支架外壳的制备
外壳薄壁是环绕圆柱体的聚乳酸-聚羟基乙酸共聚物(poly D，L-lactic-co-glycolic acid，PLGA)，其由可降解的高分子合成材料PLGA(聚乳酸与聚羟基乙酸比值为50∶50，分子量为100 000)构成。取一定量的PLGA溶解于二氯甲烷中，配成5％溶液，待PLGA完全溶解后，浇注与已调水平的聚四氟模具中，室温(控制温度在20℃)，挥发24小时，第2天小心揭下薄膜，反置于模具中24小时，剪裁到适宜大小后干燥保存。将薄膜包绕在直径为3mm的不锈钢圆柱磨具上一圈，边缘用丙酮粘贴，形成直径为3mm的圆筒状PLGA外壳。使用时将PLGA外壳剪成2mm长度，酒精浸泡15分钟，随后用无菌D-Hank’s平衡液浸洗3次，每次10分钟。
1.2三维材料的构建
无菌明胶海绵购自金陵药业公司，在超净工作台中将其剪成直径3mm，厚度2mm大小(约1.0mg)，其形貌呈圆柱体，其中的孔隙直径约200～600μm。无菌干燥保存待用。
1.3趋化活性微分子球的制备
将20g Na2CO3溶于4升水中，加热至100℃，放入75g家蚕蚕茧(由浙江省农业科学院蚕桑研究所赠送)，保持溶液微沸，并不断搅拌。1小时后，倒去溶液。再重复上述过程1次。将煮沸好的蚕茧自然冷却，用去离子水冲洗干净，放在烘箱内24小时，50℃烘干后备用。取CaCl244.40g、乙醇46.00ml，去离子水57.60ml制成混合溶液，在此溶液中放入烘干的蚕茧15.00g。使溶液充分浸没蚕茧后，80℃水浴加热，搅拌溶解。1小时后，蚕茧全部溶解为丝蛋白溶液。停止加热和搅拌，自然冷却丝蛋白溶液至室温。用透析袋(购自于广州市齐云生物技术有限公司)透析去除丝蛋白溶液中的盐离子。前2天用自来水浸泡含有丝蛋白溶液的透析袋，后1天改用去离子水浸泡，共透析3天。在透析期间，每隔3小时换1次自来水或去离子水。将透析后的丝蛋白溶液倒入量筒中，静置4小时，除去溶液中的固体杂质。用锥形瓶收集静置后的丝蛋白溶液。将0.5μg重组人NT-3和10-6mmol/L RA与3％丝蛋白混合，制备混合液。
1.4支架的组装
裁减好的天然明胶海绵吸附趋化活性微分子球至饱和，放入冻干瓶中，-80℃稳定晶形。在冷冻干燥机中，冷冻抽真空12小时，以除去水分和材料中的气体。平衡后，70％乙醇交联材料，D-Hanks洗3遍，并将材料放入PLGA套管中，制备成2mm的生物活性支架。
2.体外检测支架的性能
2.1溶胀率
一定时间内，支架在PBS溶液中的体积变化表示为其溶胀率，稳定的生物材料其溶胀率变化幅度较小。检测方法如下：将支架平行样各5个，分别测其直径R0和长度h0(精确到0.01mm)；将材料渗泡在PBS溶液中，测试样品在1h、2h、6h、12h和24h的尺寸变化Rt及ht。
2.2吸水率
支架放在PBS溶液中质量的变化，W0表示空白质量，Wt表示渗泡PBS溶液t时间后的质量。
3.体内检测支架的效能
3.1移植到大鼠体内的效能
大鼠脊髓全横断模型制备：选用成年雌性大鼠，体重约220g，每组大鼠各3-5只，在3组大鼠腹腔内注射戊巴比妥钠(30mg/kg)进行麻醉，在消毒条件下切开皮肤，分离肌肉，切除T9和T10脊柱椎板，并在T9椎板中位全横断脊髓(此处位于T9和T10脊髓节段分界处)，并切除其后2mm脊髓组织块，清除损伤腔内残留的神经纤维。在脊髓横断处分别移植入NT-3/丝素蛋白明胶海绵支架和丝素蛋白支架，明胶海绵大小为2×2×2mm3。充分止血后，逐层缝合肌肉和皮肤。术后每只动物腹腔内注射青霉素5万单位/d，连续注射3d，必要时给与补液。每天人工排尿，按常规饲养大鼠。
移植具有趋化功能的生物活性支架的大鼠饲养30d后，使用4％多聚甲醛固定。每只大鼠各取损伤/移植区前后共1cm长的脊髓进行纵切片，切片隔5取1。检测移植的生物活性支架内部迁移细胞的情况。
3.2移植到犬体内的效能
犬脊髓半横断模型制备：选用幼年比格犬，每组各5只，腹腔内注射戊巴比妥钠(30mg/kg)进行麻醉，在消毒条件下切开皮肤，分离肌肉，切除T9和T10脊柱椎板，并在T9椎板中位半横断脊髓(此处位于T9和T10脊髓节段分界处)，并切除右半侧2mm脊髓组织块，清除损伤腔内残留的神经纤维。在脊髓横断处移植入具有趋化功能的生物活性支架。充分止血后，逐层缝合肌肉和皮肤。术后每只动物腹腔内注射青霉素5万单位/d，连续注射3d，必要时给与补液。每天人工排尿。
移植具有趋化功能的生物活性支架的犬饲养30d后，使用4％多聚甲醛固定。每只犬各取损伤/移植区前后共1cm长的脊髓进行纵切片，切片隔5取1。检测移植的生物活性支架内部迁移细胞的情况。
Claims (1)
1.一种用于修复脊髓损伤的具有趋化功能的生物活性支架，其特征是PLGA导管中填充着吸附所述趋化因子NT-3、诱导因子RA和丝蛋白的三维明胶海绵，移植到脊髓损伤动物体内，能够捕获机体内干细胞，吸引那些表达与趋化因子NT-3相关受体的内源性细胞迁移到支架内存活和分化，修复脊髓损伤。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510252317.9A CN104800885B (zh) | 2015-05-13 | 2015-05-13 | 一种具有趋化功能的生物活性支架的制备和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510252317.9A CN104800885B (zh) | 2015-05-13 | 2015-05-13 | 一种具有趋化功能的生物活性支架的制备和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104800885A CN104800885A (zh) | 2015-07-29 |
| CN104800885B true CN104800885B (zh) | 2018-05-04 |
Family
ID=53686359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510252317.9A Expired - Fee Related CN104800885B (zh) | 2015-05-13 | 2015-05-13 | 一种具有趋化功能的生物活性支架的制备和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104800885B (zh) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109350767A (zh) * | 2018-09-17 | 2019-02-19 | 陈元峰 | 一种趋化内源性细胞及诱导成软骨分化的生物活性支架及其用途 |
| CN109432509A (zh) * | 2018-09-17 | 2019-03-08 | 陈元峰 | 一种抗炎及促成软骨分化的生物活性支架及其制备方法和用途 |
| CN112957316B (zh) * | 2021-03-29 | 2023-04-11 | 中山大学 | 一种引导t细胞趋化的水凝胶马达及其应用 |
| CN114246984B (zh) * | 2021-11-19 | 2022-10-14 | 浙江瑞谷生物科技有限公司 | 一种用明胶海绵载rhBMP-2的骨修复材料及其制备方法 |
| CN115671028B (zh) * | 2022-11-11 | 2023-07-14 | 西北工业大学 | 一种基于plga的局部眼用缓释复合制剂及其制备方法和应用 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008360B (zh) * | 2010-10-13 | 2015-12-16 | 中山大学 | 一种用于修复脊髓损伤的人工神经网络样导管的构建 |
| CN102727936A (zh) * | 2012-06-20 | 2012-10-17 | 中山大学 | 一种用于修复脊髓损伤的缓释nt-3明胶海绵圆柱体支架的构建 |
| CN104353113A (zh) * | 2014-08-29 | 2015-02-18 | 中山大学 | 一种用于修复脊髓损伤的类脊髓样组织的构建 |
| CN104258460A (zh) * | 2014-10-14 | 2015-01-07 | 中山大学 | 一种用于修复脊髓损伤的缓释TrkC受体配体明胶海绵圆柱体支架的构建 |
| CN204840394U (zh) * | 2015-05-13 | 2015-12-09 | 中山大学 | 一种具有趋化功能的生物活性支架的制备和应用 |
-
2015
- 2015-05-13 CN CN201510252317.9A patent/CN104800885B/zh not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104800885A (zh) | 2015-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pati et al. | Biomimetic 3D tissue printing for soft tissue regeneration | |
| CN104800885B (zh) | 一种具有趋化功能的生物活性支架的制备和应用 | |
| Lu et al. | Collagen scaffolds populated with human marrow stromal cells reduce lesion volume and improve functional outcome after traumatic brain injury | |
| Liu et al. | Local administration of stromal cell-derived factor-1 promotes stem cell recruitment and bone regeneration in a rat periodontal bone defect model | |
| Nie et al. | Targeted delivery of adipose‐derived stem cells via acellular dermal matrix enhances wound repair in diabetic rats | |
| Rioja et al. | Endothelial sprouting and network formation in collagen-and fibrin-based modular microbeads | |
| CN107281550A (zh) | 一种促进软骨损伤修复的共交联双网络水凝胶支架的制备方法 | |
| CN102600506B (zh) | Ngf壳聚糖微球-高仿生支架缓释系统及其制备方法 | |
| Wang et al. | Biomimetic collagen biomaterial induces in situ lung regeneration by forming functional alveolar | |
| CN104225667B (zh) | 一种促血管生成的温敏性水凝胶粉及用其制备的温敏性水凝胶 | |
| Li et al. | Subcutaneously engineered autologous extracellular matrix scaffolds with aligned microchannels for enhanced tendon regeneration: aligned microchannel scaffolds for tendon repair | |
| CN102143755A (zh) | 用于软组织工程化的方法和工具 | |
| Hao et al. | Biofabrication of cell-free dual drug-releasing biomimetic scaffolds for meniscal regeneration | |
| Xie et al. | Book‐shaped decellularized tendon matrix scaffold combined with bone marrow mesenchymal stem cells‐sheets for repair of achilles tendon defect in rabbit | |
| Liang et al. | Micronized acellular dermal matrix as an efficient expansion substrate and delivery vehicle of adipose‐derived stem cells for vocal fold regeneration | |
| Xu et al. | Decellularised nucleus pulposus as a potential biologic scaffold for disc tissue engineering | |
| Kambe et al. | Vascular induction and cell infiltration into peptide-modified bioactive silk fibroin hydrogels | |
| CN107007883A (zh) | 一种软骨修复支架及其制备方法 | |
| ES2989554T3 (es) | Sustituto de piel artificial biodegradable y biocompatible listo para uso y un método de preparación del mismo | |
| CN102416201A (zh) | 一种用于体内软骨修复的转化生长因子复合支架的制备方法及其应用 | |
| Li et al. | Human endometrium‐derived adventitial cell spheroid‐loaded antimicrobial microneedles for uterine regeneration | |
| Huang et al. | Collagen‐glycosaminoglycan matrix implantation promotes angiogenesis following surgical brain trauma | |
| Gindraux et al. | Human amniotic membrane: clinical uses, patents and marketed products | |
| Liu et al. | XT-type DNA hydrogels loaded with VEGF and NGF promote peripheral nerve regeneration via a biphasic release profile | |
| Chu et al. | Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180504 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |