WO2022052150A1 - 一种定向引导视神经轴突再生复合支架的制备方法及其应用 - Google Patents

一种定向引导视神经轴突再生复合支架的制备方法及其应用 Download PDF

Info

Publication number
WO2022052150A1
WO2022052150A1 PCT/CN2020/116163 CN2020116163W WO2022052150A1 WO 2022052150 A1 WO2022052150 A1 WO 2022052150A1 CN 2020116163 W CN2020116163 W CN 2020116163W WO 2022052150 A1 WO2022052150 A1 WO 2022052150A1
Authority
WO
WIPO (PCT)
Prior art keywords
optic nerve
composite scaffold
directional
scaffold
gelatin
Prior art date
Application number
PCT/CN2020/116163
Other languages
English (en)
French (fr)
Inventor
南开辉
潘统鹤
陈杨军
王静洁
林森
李玲琍
Original Assignee
温州医科大学
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 温州医科大学 filed Critical 温州医科大学
Priority to US17/413,492 priority Critical patent/US11331413B1/en
Publication of WO2022052150A1 publication Critical patent/WO2022052150A1/zh

Links

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
    • A61L27/26—Mixtures of macromolecular compounds
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

  • the invention relates to the technical field of optic nerve repair scaffolds, in particular to a preparation method and application of a composite scaffold for directional guidance of optic nerve axon regeneration.
  • the cell bodies of retinal ganglion cells are located inside the optic nerve, and their axons project to the back of the eyeball and converge to form the optic nerve.
  • the optic nerve in each eye consists of approximately 1.2 million RGC axons and is perfused by branches of the ophthalmic artery. Damage to the optic nerve is collectively referred to as optic neuropathy neuropath), which can be caused by glaucoma, traumatic optic neuropathy (TON) and various ischemic, hereditary and neurological diseases.
  • TON is one of the complications of craniocerebral injury. It refers to the injury of the face, craniocerebral, orbital or back of the eye due to various external reasons, and the optic nerve is damaged at the same time.
  • the treatment principle for TON is mainly reflected in the protection of injured optic nerve to avoid apoptosis and the promotion of optic nerve axon regeneration and functional reconstruction.
  • the limiting factors of optic nerve regeneration can be divided into intrinsic and extrinsic factors.
  • the intrinsic factors are mainly manifested in two points: 1.
  • the apoptosis of RGCs caused by optic nerve injury will up-regulate the downstream factors of apoptosis-related signaling pathways (P53, Bax), increase the level of oxidative stress in RGCs, and further promote the apoptosis of RGCs. 2. While RGCs differentiate and mature, their intracellular program changes to inhibit proliferation.
  • myelin-associated protein which is highly expressed on CNS oligodendrocytes, is a major inhibitor of CNS axon regeneration.
  • myelin-associated proteins such as semaphorin 4D (semaphorin 4D), myelin-associated glycoprotein (MAG), oligodendrocyte myelin-associated glycoprotein (OMgp), tyrosine protein kinase B3 (ephrin B3) etc. have been shown to be related to the inhibition of axonal growth.
  • semaphorin 4D semaphorin 4D
  • MAG myelin-associated glycoprotein
  • OMgp oligodendrocyte myelin-associated glycoprotein
  • ephrin B3 tyrosine protein kinase B3
  • Myelin products can not only limit axon regeneration, but also activate the apoptosis cascade and further promote neuronal apoptosis.
  • Retinal glial cells including oligodendrocytes, astrocytes and retinal microglia are activated after optic nerve injury, up-regulate a variety of axon growth inhibitory factors, and promote chondroitin sulfate, proteoglycan and reactive astrocytes
  • Glial cells form glial scars. Glial scar is an unsuitable environment for optic nerve axon regeneration, and it is also a mechanical barrier for its growth. Studies have shown that reducing glial scar can promote axon regeneration.
  • Transplanting peripheral nerves improves the external inhibitory environment and promotes the regeneration of optic nerve axons.
  • the safety of gene therapy such as AAV is yet to be certified, and at the same time, axonal reentry often occurs during the proliferation of regenerated axons due to the lack of directional guiding factors, which affects the regeneration effect.
  • the provision of exogenous trophic factors or anti-inflammatory and anti-apoptotic drugs does not perform well in promoting optic nerve axon regeneration due to drug metabolism problems. Inflammatory stimuli can promote axonal regeneration and induce endophthalmitis easily to promote the apoptosis of RGCs.
  • the current clinical treatment methods for TON mainly include surgery, hormones and combined therapy.
  • the main surgical treatment is optic canal decompression, that is, by removing the optic canal fracture fragments and releasing the compression of the damaged and edematous optic nerve by the canal wall, reducing further damage to the optic nerve compression caused by external and internal blood vessel bleeding, increasing the blood supply of the optic nerve and relieving the pain.
  • the swelling of the optic nerve and the relative narrowing of the optic canal prevent further deterioration of the optic function, so as to restore or partially restore the conduction function of the optic nerve axon.
  • the present invention is based on the research of promoting the regeneration and repair of the optic nerve by improving the microenvironment of optic nerve injury and constructing a tissue engineering scaffold that promotes the directional regeneration of optic nerve axons, and provides a composite scaffold for directional guidance of optic nerve axon regeneration.
  • a directional pipeline composite stent with good three-dimensional directional penetrating pipeline structure, good biocompatibility, excellent biodegradability and suitable biomechanical properties can be prepared, which can be transplanted into the optic nerve injury site and can While replacing the local environment after optic nerve injury, it supports the injured optic nerve to avoid degeneration and collapse, and the through-pipe is conducive to the circulation of nutrients and guides the growth of axons, and has the function of promoting the directional regeneration of the optic nerve.
  • the technical solution adopted in the present invention is: a preparation method of a composite scaffold for directional guidance of optic nerve axon regeneration, comprising the following steps:
  • the concentration of the gelatin A solution obtained in the step (1) is 25 mg/ml.
  • the concentration of the sodium alginate solution obtained in the step (1) is 10 mg/ml.
  • the genipin solution in the described step (5) is that the concentration is 1 mg/ml of genipin in ethanol and stored in a brown bottle.
  • the present invention provides a preparation method and application of a composite scaffold for directional guidance of optic nerve axon regeneration, wherein the main components of the composite scaffold are combinations of one or more degradable biomedical materials in different proportions, Such as gelatin, sodium alginate, silk fibroin, chitosan, etc., but not limited to the materials listed above, prepared by gradient freezing method. Nipei is obtained by cross-linking.
  • the composite scaffold made by adding gelatin A has better mechanical properties and better biocompatibility.
  • Sodium alginate can adjust the different solubility problems of gelatin A during the gradient freezing process, which promotes the appearance of regular directional pipelines in the scaffold.
  • the stability of the composite scaffold was significantly enhanced after being cross-linked by genipin, and its directional channel morphology could provide a climbing site for optic nerve axon regeneration and guide optic nerve axon directional regeneration.
  • the composite scaffold provided by the invention has the ability to guide the optic nerve axon directional regeneration, can be transplanted into the optic nerve injury site to promote the optic nerve axon directional regeneration, and can be used as a scaffold material for optic nerve injury repair.
  • FIG. 1 is a schematic diagram of a composite stent for a directional pipeline after synthesis according to the present invention.
  • FIG. 2 is a schematic diagram of the application of the present invention to the optic nerve injury.
  • Fig. 3 is a visual observation diagram of the composite scaffold before and after cross-linking of the present invention.
  • Fig. 4 is a scanning microscope view of the oriented pipeline composite stent of the present invention, wherein picture A is a cross section, and picture B is a longitudinal section.
  • FIG. 5 is a scanning microscope image of the scaffold cross-linked by genipin according to the present invention, wherein picture A is a cross section, and picture B is a longitudinal section.
  • Figure 6 is a graph showing the results of transplanting a 2 mm-length optic nerve scaffold into a rat optic nerve injury model, where the red arrow points to the location of the scaffold.
  • Fig. 7 is a schematic diagram showing the removal of the rat optic nerve 3 weeks after stent transplantation.
  • Fig. 8 is an immunofluorescence image after 3 weeks of stent transplantation.
  • Picture A is an immunofluorescence image of the stent of the present invention after 3 weeks of transplantation.
  • the red arrow points to the transplanted stent, and the white arrow points to the optic nerve that newly grows into the stent.
  • Picture B shows the optic nerve injury. Immunofluorescence images of the injured optic nerve removed after model injury.
  • Fig. 9 is a composite scaffold synthesized by the present invention in comparison with different raw material ratios.
  • Fig. 10 is a graph of the morphology, porosity and degradation curve of the cross-linked composite scaffold in contrast to different cross-linking methods of the present invention.
  • the present invention consists of A-type gelatin and sodium alginate, wherein the A-type gelatin and sodium alginate mass ratio is 5:2, the A-type gelatin particle model is V900863, and the brand is Sigma-Aldrich.
  • the sodium alginate powder model is S817374 and the brand is Macklin.
  • the composite scaffold that can guide the directional regeneration of the optic nerve is prepared by mixing gelatin A particles with sodium alginate powder through a gradient freezing method, and is obtained by cross-linking with genipin. Specific steps are as follows:
  • the mixed solution was injected into a Teflon tubular mold with a diameter of about 12 mm, and liquid nitrogen was injected into a special freezing device for gradient freezing.
  • Gradient freezing needs to stabilize the temperature of the upper layer of the Teflon mold at -80 °C, the temperature of the lower layer is -180 °C, and the temperature difference is 100 °C.
  • the composite stent obtained by this temperature difference has a good pipeline morphology.
  • FIG. 4 The scanning microscope of the directional pipeline composite stent is shown in Figure 4, picture A is a cross section, and picture B is a longitudinal section, it can be seen that it has a good directional pipeline structure.
  • the composite scaffold of the present invention is prepared by using gelatin A and sodium alginate, and is formed by a gradient freezing method.
  • Cell growth and attachment from Section 3 Composition and Structure).
  • Gelatin A is produced by partial hydrolysis of collagen and is a natural protein polymer material with good biocompatibility.
  • Sodium alginate is a natural polysaccharide extracted from algae, which is stable, safe and has good biocompatibility.
  • gradient freezing method oriented ice crystals can be produced in gelatin A and sodium alginate solution, and then the ice crystals can be sublimated by a freeze dryer to obtain a composite scaffold with oriented pipes.
  • the composite scaffold has a relatively uniform directional channel with a diameter of about 20 ⁇ m, which is suitable for optic nerve axon ingrowth and nutrient circulation.
  • the composite scaffold has the best channel morphology.
  • Genipin is an excellent natural cross-linking agent that is far less toxic than glutaraldehyde and other commonly used chemical cross-linking agents. The use of genipin cross-linking ensures the good stability and mechanical properties of the composite scaffold, and also has good biocompatibility.
  • the synthetic directional pipeline composite stent is transplanted into the optic nerve injury site to replace the inhibitory microenvironment at the optic nerve injury site, thereby promoting the directional regeneration of the optic nerve axon.
  • the results of the animal test are shown in Figure 6 below.
  • An optic nerve scaffold with a length of 2 mm was transplanted into a rat optic nerve injury model.
  • the red arrow points to the stent graft site.
  • the optic nerve of the rat was taken out 3 weeks after the scaffold was transplanted. The results are shown in Figure 7 below.
  • Picture A is the immunofluorescence image of the stent of the present invention three weeks after transplantation, the red arrow points to the transplanted stent, and the white arrow points to the optic nerve that newly grows into the stent. It can be seen that stent implantation is beneficial to the regeneration of optic nerve axons.
  • Picture B is the immunofluorescence image of the injured optic nerve removed after the injury of the optic nerve injury model. It can be seen that there is no optic nerve regeneration at the injury site.
  • the scale bar is 150 ⁇ m.
  • the present invention synthesizes a composite support by comparing different ratios of raw materials, and selects the group with the best directional pipe shape as the ratio of raw materials.
  • group A gelatin A 50 mg/ml, sodium alginate 10 mg/mlA
  • group B gelatin A 25 mg/ml, sodium alginate 1 mg/ml
  • group C gelatin A 50 mg/ml /ml, sodium alginate 1 mg/ml
  • Group D was selected as the raw material ratio of the directional pipeline composite support.
  • the morphology, porosity and degradation curve of the cross-linked composite scaffolds were compared with different cross-linking methods.
  • the results are shown in Figure 10 below.
  • the porosity was about 94.1%.
  • the genipin cross-linking maintained the best shape of the directional pipe, and the genipin cross-linking expansion rate was 31.3% ⁇ 10.6%, which avoided the large water absorption and swelling after the stent was transplanted into the optic nerve, which would cause the optic nerve to be squeezed.
  • both genipin and glutaraldehyde showed good stability, so genipin was finally selected as the cross-linking method of the scaffold.
  • the invention prepares a directional three-dimensional directional through-channel structure, good biocompatibility, and suitable biomechanical properties.
  • the pipeline composite stent can support the damaged optic nerve to avoid degeneration and collapse while replacing the inhibitory environment of the damaged optic nerve, and the penetrating pipeline is conducive to the circulation of nutrients and the growth of axons, and has the function of guiding the directional regeneration of the optic nerve.
  • gelatin A and sodium alginate composite scaffolds prepared in the examples have good biocompatibility, certain mechanical properties and in vivo stability, and can simultaneously provide a medium for optic nerve axon regeneration and attachment, and guide its directional growth, so it is considered that It is a very potential optic nerve repair scaffold material.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Materials For Medical Uses (AREA)

Abstract

一种定向引导视神经轴突再生复合支架的制备方法及应用，复合支架主要组分为一种或多种可降解生物医用材料按不同比例组合通过梯度冷冻法制备，为增加支架力学性能或延长体内降解时间，可经生物交联剂交联得到，添加明胶后制成的复合支架具有更好的力学性能，且具有较好的生物相容性，海藻酸钠能调节明胶在梯度冰冻的过程中产生的溶解度不同问题，促使支架出现规整的定向管道形貌。复合支架在经京尼平交联后明显增强稳定性，其定向管道形貌能为视神经轴突再生提供攀附位点，引导视神经轴突定向再生。

Description

一种定向引导视神经轴突再生复合支架的制备方法及其应用 技术领域
本发明涉及视神经修复支架技术领域，具体涉及一种定向引导视神经轴突再生复合支架的制备方法及其应用。
 背景技术
视网膜神经节细胞（retinal ganglion cells，RGCs）胞体位于视神经内部，其轴突投射至眼球后补汇聚成视神经。每只眼的视神经大约有120万RGC轴突组成，并有眼动脉分支灌注。视神经功能受损统称为视神经病变（optic neuropath），可由青光眼、外伤性视神经病变（traumaticopticneuropathy，TON）以及各种缺血性、遗传性、神经性疾病等造成。TON是颅脑损伤的并发症之一，是指由于各种外力原因，造成面部、颅脑、眼眶或眼后部损伤的同时损伤视神经，损伤后可以没有外部或最初眼底镜下眼球或视神经损伤表现，却有不同程度视力的减退或视野的缺损，甚至视力的完全丧失。针对TON的治疗原则主要体现在保护损伤视神经避免凋亡以及促进视神经轴突再生及功能重建。视神经再生的限制因素可分为内在因素和外在因素。内在因素主要表现为2点：1.视神经损伤引起的RGCs凋亡会上调凋亡相关信号通路（P53、Bax）下游因子，增加RGCs氧化应激水平，进一步促进RGCs凋亡。2.RGCs在分化成熟的同时，其细胞内程序向抑制增殖转变。有报道表明许多的细胞内信号通路如cAMP、mTOR/PTEN、KLF4等可诱发转录级联和表观遗传改变，这些改变与中枢神经系统成熟密切相关。因此，在视神经损伤后RGCs轴突难以再生可能与这些分子的调控有关。外在因素主要包括缺乏神经营养因子、损伤端神经胶质瘢痕以及髓磷脂等。髓磷脂是胶质细胞产生的一种脂肪蛋白，作用是绝缘并加速电传导，其存在也被认为是中枢神经系统较外周神经系统再生能力差的一个原因。例如，Vajda F等人报道在中枢神经系统少突胶质细胞上高度表达的髓鞘相关蛋白（Nogo）是中枢神经系统轴突再生的一个主要抑制剂。此外，其他髓鞘相关蛋白如脑信号蛋白4D（semaphorin 4D）、髓鞘相关糖蛋白（MAG）、少突胶质细胞髓鞘相关糖蛋白（OMgp）、酪氨酸蛋白激酶B3（ephrin B3）等都被证明与抑制轴突生长相关。同时中枢神经系统损伤后髓磷脂清除效率降低容易导致外环境髓磷脂堆积，髓磷脂产物不仅能够限制轴突再生，更能够激活凋亡级联，进一步促进神经元凋亡。视神经损伤后视网膜胶质细胞包括少突胶质细胞、星形胶质细胞及视网膜小胶质细胞被激活，上调多种轴突生长抑制因子，并促进硫酸软骨素、蛋白多糖及反应性星形胶质细胞形成胶质瘢痕。神经胶质瘢痕是视神经轴突再生的不适宜环境，也是其生长的机械屏障，研究认为减少胶质瘢痕可以促进神经轴突再生。
针对以上因素，国内外做了较多的研究工作，主要包括以下几个方面：1、通过腺相关病毒（AAV）等基因治疗方式从基因层面打开细胞内程序的增殖抑制，从而促进视神经轴突再生；2、通过提供外源性神经营养因子，改善视神经损伤后营养因子缺乏的困境，从而促进轴突再生；3、通过抗炎抗凋亡药物应用，减少损伤后RGCs由于炎症及氧化应激造成的凋亡；4、通过炎症刺激激活RGCs细胞内在程序，促进视神经轴突再生。5、移植外周神经改善外界抑制环境，促进视神经轴突再生。但目前由于AAV等基因治疗安全性有待认证，同时再生轴突在增殖过程由于缺少定向引导因素常出现轴突折返现象，影响再生效果。提供外源性营养因子或抗炎抗凋亡药物由于药物代谢问题，在促进视神经轴突再生方面表现不佳。炎症刺激在促进轴突再生的同时，引起眼内炎容易促进RGCs细胞凋亡。而外周神经移植材料来源有限，异体移植容易存在排斥反应，因此以上方式均未向临床转化。目前临床对于TON的治疗方式主要包括手术、激素及联合治疗方式。手术治疗主要是视神经管减压术即通过清除视神经管骨折碎片和解除管壁对损伤和水肿的视神经的压迫，减少视神经外部及内部血管出血对视神经压迫的进一步损伤，以增加视神经血液供应，缓解视神经的肿胀和视神经管的相对缩窄，防止视功能进一步恶化，以达到恢复或部分恢复视神经轴束传导功能。但视神经发生损伤后，会启动内在的凋亡程序，即使及时通过手术解除视神经损伤的诱因并联合激素治疗，也无法阻止RGCs的慢性凋亡。因此目前亟需开发针对TON的新型治疗方式。
 技术解决方案
为了解决现有技术的缺陷，本发明立足于通过改善视神经损伤微环境、构建促进视神经轴突定向再生组织工程支架，从而促进视神经再生修复的研究，提供了一种定向引导视神经轴突再生复合支架的制备方法及其应用，制备出具有良好的三维定向贯通管道结构、良好的生物相容性、优秀的生物降解能力、适宜的生物力学性能的定向管道复合支架，可移植入视神经损伤部位，能够在替换视神经损伤后局部环境的同时，支撑损伤视神经避免退化塌陷，贯通管道有利于营养物质流通及引导轴突生长，具有促进视神经定向再生功能。
本发明采用的技术解决方案是：一种定向引导视神经轴突再生复合支架的制备方法，包括以下步骤：
（1）将明胶A溶解在70℃的去离子水中，得到明胶A溶液；将海藻酸钠溶解在70℃的去离子水中，得到海藻酸钠溶液；将两种溶液按照1:1比例混合，并搅拌均匀，静置去除气泡；
（2）将混合溶液注入直径约为12 mm的特氟龙管状模具中，注入液氮进行梯度冷冻，需要稳定保持特氟龙模具上层温度为-80±20℃，下层温度为-180±20℃，温度差为100±20℃；
（3）将冷冻后的混合溶液放入-80±2℃条件下复冻24小时；
（4）将复冻的混合溶液放入冷冻干燥机，干燥48小时；
（5）在京尼平溶液中放入明胶A、海藻酸钠复合支架，并在转膜摇盘上摇24小时进行交联；
（6）24小时后关闭转膜摇盘，静置6天即可交联复合支架。
所述的步骤（1）得到的明胶A溶液的浓度为25 mg/ml。
所述的步骤（1）得到的海藻酸钠溶液的浓度为10 mg/ml。
所述的步骤（5）中的京尼平溶液为浓度为1 mg/ml的京尼平乙醇溶液，并置于棕色瓶中保存。
一种定向引导视神经轴突再生复合支架在作为视神经损伤修复材料上的应用。
 有益效果
本发明的有益效果是：本发明提供了一种定向引导视神经轴突再生复合支架的制备方法及其应用，复合支架主要组分为一种或多种可降解生物医用材料按不同比例的组合，如明胶、海藻酸钠、丝素蛋白、壳聚糖等，但不限于上述所列材料，通过梯度冷冻法制备，为增加支架力学性能或延长体内降解时间，可经生物交联剂，如京尼平等交联得到。添加明胶A后制成的复合支架具有更好的力学性能，且具有较好的生物相容性。海藻酸钠能调节明胶A在梯度冰冻的过程中产生的溶解度不同问题，促使支架出现规整的定向管道形貌。复合支架在经京尼平交联后明显增强稳定性，其定向管道形貌能为视神经轴突再生提供攀附位点，引导视神经轴突定向再生。本发明所提供的复合支架具有引导视神经轴突定向再生的能力，可移植入视神经损伤部位促进视神经轴突定向再生，作为视神经损伤修复支架材料。
 附图说明
图1为本发明合成后定向管道复合支架示意图。
图2为本发明在视神经损伤处应用原理图。
图3为本发明交联前后复合支架肉眼观察图。
图4为本发明定向管道复合支架的扫描显微镜图，其中A图为横截面，B图为纵截面。
图5为本发明通过京尼平交联后的支架的扫描显微镜图，其中A图为横截面，B图为纵截面。
图6为将长度为2 mm的视神经支架移植入大鼠视神经损伤模型结果图，其中红色箭头指向支架移植位置。
图7为支架移植3周后将大鼠视神经取出示意图。
图8为支架移植3周后做免疫荧光图，A图为本发明支架移植三周后的免疫荧光图，红色箭头指向移植的支架，白色箭头指向新生长入支架的视神经，B图为视神经损伤模型损伤后移出损伤视神经的免疫荧光图。
图9为本发明对照不同原料配比合成复合支架。
图10位本发明对照不同交联方式交联复合支架形貌、孔隙率及降解曲线图。
 本发明的实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
下面结合附图以及具体实施例，可以更好地说明本发明。
本发明由A型明胶与海藻酸钠组成，其中A型明胶、海藻酸钠质量比为5:2，A型明胶颗粒型号为V900863，品牌为Sigma-Aldrich。海藻酸钠粉末型号为S817374，品牌为Macklin。
制备方法：
可引导视神经定向再生的复合支架是通过明胶A颗粒混合海藻酸钠粉末经由梯度冷冻法制备，并通过京尼平交联得到。具体步骤如下：
（1）将明胶A溶解在70℃的去离子水中，浓度为50 mg/ml；
（2）将海藻酸钠溶解在70℃的去离子水中，浓度为20 mg/ml；
（3）将两种溶液按照1:1比例混合，并搅拌均匀，静置去除气泡；
（4）将混合溶液注入直径约为12 mm的特氟龙管状模具中，通过特制的冷冻装置注入液氮进行梯度冷冻。梯度冷冻需要稳定特氟龙模具上层温度为-80℃，下层温度为-180℃，温度差为100℃，此温度差得到的复合支架具有较好的管道形貌。
（5）将冷冻后的混合溶液放入-80℃冰箱复冻24小时。
（6）将复冻的混合溶液放入冷冻干燥机，干燥48小时。
（7）将得到的明胶A、海藻酸钠复合支架通过京尼平进行交联。
（8）将京尼平溶于90%的乙醇溶液中，浓度为1 mg/ml，并注入棕色瓶中；
（9）在棕色瓶中放入复合支架，并在转膜摇盘上摇24小时；
（10）24小时后关闭转膜摇盘，静置6天即可交联复合支架。
定向管道复合支架的扫描显微镜图4所示，A图为横截面，B图为纵截面，可以看出具有较好的定向管道结构。
本发明复合支架采用明胶A和海藻酸钠制备，通过梯度冷冻法复合形成的，具有较好的定向管道取向，以及具有较好的生物相容性、极好的亲水性以及蛋白构架，适合细胞生长攀附（来源于第3部分组成及结构）。明胶A是通过胶原蛋白部分水解产生，是天然的蛋白高分子材料，具有较好的生物相容性。海藻酸钠是一种从藻类中提取的天然多糖，性质稳定、安全，具有较好的生物相容性。通过梯度冷冻方式，能够在明胶A、海藻酸钠溶液中制造定向冰晶，之后通过冷冻干燥机升华冰晶，得到具有定向管道的复合支架。复合支架具有较为均匀的定向管道，直径约为20 μm，适合视神经轴突长入及营养物质流通,明胶A：海藻酸钠质量比为5:2时，合成支架具有最好管道形貌。京尼平是一种优良的天然交联剂，其毒性远低于戊二醛和其他的常用化学交联剂。使用京尼平交联，保证复合支架酷游良好的稳定性和力学性能的同时，也具有较好的生物相容性。
实施例 .
本发明是通过将合成的定向管道复合支架移植入视神经损伤位置，替换视神经损伤处的抑制微环境，从而促使视神经轴突定向再生。动物试验结果如下图6所示，将长度为2 mm的视神经支架移植入大鼠视神经损伤模型当中。红色箭头指向支架移植位置。
支架移植3周后将大鼠视神经取出，结果如下图7所示，视神经损伤处移植支架降解，新生轴突长入填充。
支架移植3周后做免疫荧光，结果如下图8所示。A图为本发明支架移植三周后的免疫荧光图，红色箭头指向移植的支架，白色箭头指向新生长入支架的视神经。可见支架植入有利于视神经轴突再生。B图为视神经损伤模型损伤后移出损伤视神经的免疫荧光图，可见损伤处无视神经再生。标尺为150 μm。
对比例 1
本发明对照不同原料配比合成复合支架，选取定向管道形貌最佳的组别为原料配比。结果如下图9，A组（明胶A 50 mg/ml、海藻酸酸钠10mg/mlA）、B组（明胶A25 mg/ml、海藻酸酸钠1 mg/ml）、C组（明胶A 50 mg/ml、海藻酸酸钠1 mg/ml）、D组（明胶25 mg/ml、海藻酸酸钠10mg/ml）。Bar=80 μm。选取D组作为定向管道复合支架的原料配比。
对比例 2
本发明对照不同交联方式交联复合支架形貌、孔隙率及降解曲线，结果如下图10所示，A、B、C分别为京尼平、EDC+NHs、戊二醛三种方式交联后的支架SEM，孔隙率约为94.1%。京尼平交联对定向管道形貌保持最好，同时京尼平交联膨胀率为31.3%±10.6%，避免了支架移植入视神经后发生较大的吸水膨胀，引起对视神经的挤压。在降解实验中，京尼平与戊二醛均表现出了较好的稳定性，因此最终选择京尼平作为支架的交联方式。
本发明基于改善视神经损伤微环境、调控轴突再生不利因素、构建可引导轴突定向生长通道之思路，制备出具有三维定向贯通管道结构、良好的生物相容性、适宜的生物力学性能的定向管道复合支架，能够在替换损伤视神经抑制环境的同时，支撑损伤视神经避免退化塌陷，贯通管道有利于营养物质流通及轴突生长，具有引导视神经定向再生功能。实施例中所制备的明胶A、海藻酸钠复合支架具有良好生物相容性，一定的力学性能以及体内稳定性，能同时提供视神经轴突再生攀附的媒介，并引导其定向生长，因此被认为是一种极具潜力的视神经修复支架材料。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (5)

  1. 一种定向引导视神经轴突再生复合支架的制备方法，其特征在于，包括以下步骤：
    （1）将明胶A溶解在去离子水中，得到明胶A溶液；将海藻酸钠溶解在去离子水中，得到海藻酸钠溶液；将两种溶液按照1:1比例混合，并搅拌均匀，静置去除气泡；
    （2）将混合溶液注入特氟龙管状模具中，注入液氮进行梯度冷冻，需要稳定保持特氟龙模具上层温度为-80±20℃，下层温度为-180±20℃，温度差为100±20℃；
    （3）将冷冻后的混合溶液放入-80±2℃条件下复冻；
    （4）将复冻的混合溶液放入冷冻干燥机，干燥；
    （5）在京尼平溶液中放入明胶A、海藻酸钠复合支架，并在转膜摇盘上进行交联；
    （6）完成后关闭转膜摇盘，静置即可交联复合支架。
  2. 根据权利要求1所述的一种定向引导视神经轴突再生复合支架的制备方法，其特征在于，所述的步骤（1）得到的明胶A溶液的浓度为25 mg/ml。
  3. 根据权利要求1所述的一种定向引导视神经轴突再生复合支架的制备方法，其特征在于，所述的步骤（1）得到的海藻酸钠溶液的浓度为10 mg/ml。
  4. 根据权利要求1所述的一种定向引导视神经轴突再生复合支架的制备方法，其特征在于，所述的步骤（5）中的京尼平溶液为浓度为1 mg/ml的京尼平乙醇溶液，并置于棕色瓶中保存。
  5. 一种权利要求1所述的制备方法制备的复合支架在作为视神经损伤修复材料上的应用。
PCT/CN2020/116163 2020-09-10 2020-09-18 一种定向引导视神经轴突再生复合支架的制备方法及其应用 WO2022052150A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/413,492 US11331413B1 (en) 2020-09-10 2020-09-18 Preparation method and application of composite scaffold for directionally guiding regeneration of optic nerve axons

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010944499.7A CN112245659B (zh) 2020-09-10 2020-09-10 一种定向引导视神经轴突再生复合支架的制备方法及其应用
CN202010944499.7 2020-09-10

Publications (1)

Publication Number Publication Date
WO2022052150A1 true WO2022052150A1 (zh) 2022-03-17

Family

ID=74231685

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/116163 WO2022052150A1 (zh) 2020-09-10 2020-09-18 一种定向引导视神经轴突再生复合支架的制备方法及其应用

Country Status (3)

Country Link
US (1) US11331413B1 (zh)
CN (1) CN112245659B (zh)
WO (1) WO2022052150A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101564552A (zh) * 2009-03-20 2009-10-28 华南理工大学 高强度胶原组织修复材料制品的制备方法
CN106693067A (zh) * 2016-07-15 2017-05-24 温州生物材料与工程研究所 一种自愈合无模板多孔支架的制备
CN106729983A (zh) * 2016-12-30 2017-05-31 四川大学 一种促神经修复复合导管及其制备方法
CN106806941A (zh) * 2017-02-22 2017-06-09 中国人民解放军第四军医大学 一种易缝合阵列微管神经修复支架制备方法及其应用
CN108187148A (zh) * 2018-02-13 2018-06-22 中山大学附属第医院 一种能提高轴突再生有序性的神经导管及其制备方法
US20180221415A1 (en) * 2012-02-23 2018-08-09 Technion Research & Development Foundation Ltd. Scaffold for growing neuronal cells and tissue

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100591764C (zh) * 2005-02-25 2010-02-24 中国科学院化学研究所 列管式取向排列结构细胞支架及其制备方法
CN102671237B (zh) * 2009-01-16 2014-04-16 中国人民解放军第四军医大学 一种高仿真组织工程神经修复材料及其制备方法
CN208860768U (zh) 2018-09-07 2019-05-14 东莞精准通检测认证股份有限公司 一种滚珠球面检测器
CN109999222B (zh) * 2019-04-15 2021-08-13 大连工业大学 基于聚羟基脂肪酸酯/海藻酸钠电纺纳米纤维的神经导管材料的制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101564552A (zh) * 2009-03-20 2009-10-28 华南理工大学 高强度胶原组织修复材料制品的制备方法
US20180221415A1 (en) * 2012-02-23 2018-08-09 Technion Research & Development Foundation Ltd. Scaffold for growing neuronal cells and tissue
CN106693067A (zh) * 2016-07-15 2017-05-24 温州生物材料与工程研究所 一种自愈合无模板多孔支架的制备
CN106729983A (zh) * 2016-12-30 2017-05-31 四川大学 一种促神经修复复合导管及其制备方法
CN106806941A (zh) * 2017-02-22 2017-06-09 中国人民解放军第四军医大学 一种易缝合阵列微管神经修复支架制备方法及其应用
CN108187148A (zh) * 2018-02-13 2018-06-22 中山大学附属第医院 一种能提高轴突再生有序性的神经导管及其制备方法

Also Published As

Publication number Publication date
US11331413B1 (en) 2022-05-17
US20220143266A1 (en) 2022-05-12
CN112245659B (zh) 2021-11-30
CN112245659A (zh) 2021-01-22

Similar Documents

Publication Publication Date Title
Yan et al. Facile preparation of bioactive silk fibroin/hyaluronic acid hydrogels
CN103877617B (zh) 可注射蚕丝素蛋白-海藻酸盐双交联水凝胶及其制备方法和使用方法
CA2263361C (fr) Implant injectable par voie sous-cutanee ou intradermique
DE69828050T2 (de) Ein Verfahren zur Herstellung eines Polyvinylalkohol-Kryogels
DE69731959T2 (de) Implantierbares acrylamidcopolymer-hydrogel für therapeutische anwendungen
US20110150846A1 (en) Compositions and methods for tissue filling and regeneration
Hu et al. Stability and biodegradation of silk fibroin/hyaluronic acid nerve conduits
WO2010081408A1 (zh) 一种生物活性组织再生膜及其制备方法
CA2256327A1 (en) Polysaccharide sponges for cell culture and transplantation
CN101474424A (zh) 高仿真组织工程神经修复材料ngcs及其制备方法
CN110464874B (zh) 一种具有神经组织修复活性的聚合物材料及其制备方法和应用
Rao et al. Biomaterial-based Schwann cell transplantation and Schwann cell-derived biomaterials for nerve regeneration
CN108670946A (zh) 治疗关节软骨损伤的细胞凝胶制剂及其用途和所用的保持冻存细胞活性的凝胶溶液
JP2002541888A (ja) 超臨界抗溶媒技術により得られるヒアルロン酸誘導体含有三次元構造体
JP3848164B2 (ja) 軟部組織修復用の前脂肪細胞からなるバイオマテリアル
CN112138216B (zh) 一种高度仿生骨基质的杂化交联颅骨修复多孔膜及其制备方法
WO2022052150A1 (zh) 一种定向引导视神经轴突再生复合支架的制备方法及其应用
CN113877001A (zh) 一种注射用丝素蛋白复合凝胶
WO2021000641A1 (zh) 一种利用拓扑结构调控免疫微环境并引导再生的仿生可降解人工神经导管及其制备方法
EP3305340B1 (en) Cell-growing scaffold having structure memory property
Pedrosa et al. Scaffolds for peripheral nerve regeneration, the importance of in vitro and in vivo studies for the development of cell-based therapies and biomaterials: state of the art
WO2023130973A1 (zh) 一种用于骨修复的药物组合物
Alvites et al. Olfactory mucosa mesenchymal stem cells and biomaterials: A new combination to regenerative therapies after peripheral nerve injury
Qiu et al. Electrospun compliant heparinized elastic vascular graft for improving the patency after implantation
CN115645625A (zh) 一种基于丝素蛋白/透明质酸的可注射水凝胶及其制备方法与应用

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: 20952937

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20952937

Country of ref document: EP

Kind code of ref document: A1