具有管中管结构复合神经导管的制备及材料性能研究
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摘要
随着神经损伤发生率的大幅度上升,神经移植已不能很好地满足需要,新方向的探索变得尤为重要,神经导管作为一种新的治疗方法应运而生。神经导管组织工程支架可以为神经的生长提供良好的微环境,理想的神经导管还能促进受损神经的愈合或为神经的生长提供导向作用。本文设计了一种管中管新构型的神经导管,详细讨论了各部分材料的基本性能,对管中管结构神经导管的可行性进行初步探索,期望复合结构的神经导管会对神经再生起到积极作用。
通过静电纺丝法制备得到的纳米纤维,由于具有较高的孔隙率、较大的比表面积及高表面能等优势,能够从纳米尺度来模仿天然细胞外基质,被广泛地应用于组织工程支架材料、伤口敷料及药物载体等方面。因此本文首先采用静电纺丝技术制备神经导管的外管支架,通过旋转接收装置、优化纺丝参数,制备得到仿细胞外基质的有序纳米纤维支架,作为细胞生长的支撑,促进细胞的迁移及增殖。另外,湿法中空纤维成型是一步法连续制备中空纤维导管的有效途径,在制备管内部纤维导管时采用湿法纺丝技术。
本文选用脂肪族聚碳酸丁二脂(PBC)、甲壳素纳米晶须(ChW)、高密度壳聚糖(HCS)及表面功能化的多壁碳纳米管(f-MWNTs)作为基本支架材料,通过静电纺丝接收装置的改进、材料组合、表面改性等,系统地研究评价了这类材料用作神经修复组织工程材料的力学性能、细胞相容性及降解性能等。主要研究工作如下：
1.通过静电纺丝法制备无规和有序PBC纳米纤维,研究了接收线速度对纳米纤维基本性能的影响。之后研究了低温等离子表面改性技术对纤维表面亲水性的影响,并通过等离子改性方法诱导纤维表面接枝明胶,以增强纤维表面的生物相容性。研究表明,PBC可以均匀地溶解于甲酸、DMF、六氟异丙醇及氯仿等有机溶剂中,但仅溶解于甲酸能够得到表面光滑、粗细均匀的纳米级纤维；通过转轴法可以成功制备得到PBC有序纳米纤维,随着旋转线速度的增大,纤维的排列有序度、晶区及分子链的取向度、结晶度以及力学性能都有所增大。
2.为了进一步提高外管有序纳米纤维的力学性能。首先通过酸解法制备得到纳米级甲壳素晶须,然后将其与PBC进行共混复合,通过静电纺丝法制备得到纳米级复合纤维,研究发现利用酸解法制得的甲壳素晶须的长度范围为180-680nm,直径分布范围为15-30nm,平均长径比为14.7,且将晶须分散到甲酸中24h后对表面形貌的观察发现,在短时间内甲酸并不会影响到晶须形貌,为下一步实验提供了依据；对将不同含量的晶须添到PBC中制得复合纳米纤维的研究表明,当晶须含量为5.0wt%时,制得的纳米纤维表面光滑、直径分布均匀且随着晶须的加入结晶度、热稳定性及力学性能得到显著提高。之后采用低温等离子技术对表面进行改性处理并用明胶接枝以赋予纤维表面新的生物相容性,使其亲水性得到了很大的提高,且更有利于神经细胞RSC96的黏附与增殖。
3.采用高密度壳聚糖(HCS)来制备导管内部的中空纤维。首先研究了HCS质量分数以及温度等对纺丝浆液稳定性的影响；而后进行湿法纺丝制备了中空纤维,并对HCS中空纤维的化学结构、晶体结构和热性能进行了研究。结果表明,HCS的固含量为5wt%时,纺丝过程顺利进行,挤出的浆液在凝固浴中形成的初生纤维结构均匀,不会出现断丝情况。纺丝温度应控制在20-30℃；凝固浴浓度为3wt%时所得的中空纤维热力学及结构性能较好。因此,在后续的实验研究中,将采用质量分数为5wt%HCS纺丝浆液,以质量分数为3wt%的NaOH-乙醇溶液作为凝固浴制备中空纤维。
4.采用碳纳米管来增强HCS中空纤维的力学和电学性能。为了提高碳管在HCS溶液中的分散性及与HCS基体之间的相容性,通过表面沉积交联法对碳管进行表面修饰得到f-MWNTs。然后与HCS进行混合制得复合中空纤维,并对其性能进行了研究分析。研究发现,经过表面修饰得到的f-MWNTs的管身变得平直,缠结状态也有所缓解,且在水中的分散性就明显优于MWNTs;对复合中空纤维的研究发现当f-MWNTs的含量达到0.5wt%时,f-MWNTs在HCS基体中的分布最为均匀,中空纤维的断面形貌也最为致密；且复合纤维的拉伸强度和弹性模量均达到最大值,分别为9.33MPa和2.34GPa；在含水率相同的条件下,随着碳管含量的增加复合纤维的电导率也增加。经过等离子预处理和明胶接枝改性后,复合膜在各个压力下的水通量都有了明显的提高,且更有利于细胞的黏附与增殖。
5.管中管结构的神经导管外层通过将静电纺有序纳米纤维沿一定直径的芯棒卷绕成管状结构,内部填充中空纤维,得到内部通道不同的导管。对导管的压缩性能的研究表明,5-通道神经导管在形变量为25%时,负荷力高达201cN,完全能够满足神经导管径向支撑力的要求；管中管结构的神经导管在受到外界压力时,形状发生变化后能够自然恢复,即形变后也能承担一定的支撑作用,符合神经导管力学性能的要求。
因此本文所制备的管中管复合型神经导管在亲水性、降解性及细胞相容性上都有所提高,同时具有良好的机械性能,有望成为新一代的神经修复组织工程支架。
In recent years, with the increasing in the incidence of nerve injury, nerve graft could not satisfied the nerve regeneration for the restrictions of distance and defect in donor's functions. So the restoration, regeneration and functional recovery in nerve injury have become a primary research direction in neuroscience and the nerve conduit is put forward. The nerve conduit is the connection of fracture nerves to provide suitable microenvironment for nerve growth and the idea conduit can also provide orientation for proliferation of nerve cells. In this paper, a novel nerve conduit with a tube in tube structure was designed and detailed experimental studies of fundamental properties were made. In addition, technical feasibility and reasonableness of the method were also discussed based on the results, and expected to bring the nerve conduit closer to being a realistic possibility.
Nanofibrous scaffolds, prepared by electrospinning with high porosity and large surface area, could imitate the natural extracellular matrix in the nano-scale, so it can be used as a porous scaffold to promote cell migration and proliferation in tissue engineering, wound dressing and pharmaceutical carriers. In this paper, the outward scaffolds were prepared by electrospinning and aligned nano-fibers that obtained by optimizing the spinning parameters and receiving apparatus could promote the cells'migration and proliferation. What's more, wet spinning was an effective method to prepare continuous hollow fibers, so inside hollow fibers were gained by this method.
Poly(butylene carbonate)(PBC), chitin nano-whiskers (ChW), high-density chitosan (HCS) and functionalized multi-walled carbon nanotubes (f-MWNTs) were chosen as the basic materials for scaffolds. Through the improvement of electrospinning technology, materials composition and surface modification, the mechanical properties, cell compatibility and degradation rate were also deeply studied. Main researches are listed as follows:
1. Random nano-fibers and aligned nano-fibers were prepared by electrospinning and the relationship of rotating speed of the receiving instrument and the basic performance of nano-fibers were discussed in detail. After plasma pretreatment and gelatin graft, hydrophilicity and biocompatibility of the aligned fiber mats could be improved significantly. Studies show that PBC could dissolve in some organic solvent such as formic acid (FA), dimethylformamide (DMF), hexafluoroisopropanol (HFIP) and chloroform, but the uniform and smooth fibers could only obtain from the FA solution; During the process of preparing aligned PBC nano-fibers, the order degree of fibers, the crystallinity and the orientation of crystalline region including mechanical strength are all increased correspondingly with the increment of rotating speed.
2. Chitin nano-whiskers made by acid decomposition method were added into PBC solutions to obtain compound nano-fibers in order to improve mechanical properties. The studies found that, the length and diameter of ChW were in the range of180-680nm and15-30nm respectively and the average length diameter ratio was14.7. In addition, the morphology of ChW in FA solvent was not changed and this has provided basis for the further experiment. The nano-fibers with uniform diameter distribution and smooth surface could be prepared with the addition of ChW up to5.0wt%; the crystallinity, thermal stability and mechanical properties are increased correspondingly with the increasing of ChW contents. After plasma pretreatment, hydrophilicity of the aligned fiber mats was improved significantly, and the fiber mats was then immersed into gelatin solution for grafting. The aligned nano-fibers after gelatin grafting could stimulate the adhesion and proliferation of RSC96cells.
3. In the paper, the spinning solutions were prepared by using acetic acid as solvent; the rheology of the solutions were studied to investigate the effect of the solid content and the spinning temperature on the stability of the spinning solutions and the spinning process. Then hollow fibers were obtained by the method of wet spinning and chemical structures, crystal structures, thermal properties including mechanical property were researched. The results showed that:we could obtain hollow fibers smoothly with the concentration of the spinning solution in5wt%at20-30℃the fibers had excellent properties that regenerated within the coagulation bath of3wt%sodium hydroxide-ethanol (mass ratio of1:1).
4. The dispersivity and compatibility with HCS of f-MWNTs that obtained via a controlled surface deposition and crosslinking process have been improved a lot; most of the tube body spread out that close to single dispersion, and the tangle also was eased after deposition and crosslinking process. The hollow fibers with the f-MWNTs content of0.5wt%exhibit the optimal tensile property with the maximum tensile strength and elasticity modulus of9.33MPa and2.34GPa, respectively. The fiber was then immersed into gelatin solution for grafting after pretreatment. Under the condition of same moisture content, the conductivity of composite hollow fibers was also improved with the increase of the content of f-MWNTs. XPS results also showed that some oxygen element was introduced to the surface of the pretreated samples for subsequently exposing to air (atomic fraction rising from29.07%to38.28%); in following grafting reaction, the active groups from gelatin introduce a large amount of nitrogen (from1.73%to7.54%) and the C-C bond broke down to form C=O and C=N bonds or other polar groups to realize the grafting reaction. Form biological characterization such as cell attachment, cell morphology and proliferation, these results demonstrate following two aspects. First, the addition of a small amount of f-MWNTs in fibers with the cell adhesion rate of77.44%which equal to HCS fibers (78.29%) after24h have no affect on cell behavior; second, cells seeded in fibers grafted with gelatin have higher adhesion rate and cells can proliferate faster in the same time which proved that the superior ability of fibers after surface modification with gelatin can well support Schwann cells (RSC96) growth and proliferation.
5. The aligned nano-fibers prepared from electrospinning was rolled on the core bar with right diameter to form the outer tube of nerve conduit, and then hollow fibers were filled into the tube to receive the nerve conduit with a tube in tube structure. The results of compression properties of nerve conduit indicated that the load force of5-channel conduit could reach up to210cN with the deformation of25%, so the conduit could meet the requirements of the supporting force of nerve regeneration.
通过静电纺丝法制备得到的纳米纤维,由于具有较高的孔隙率、较大的比表面积及高表面能等优势,能够从纳米尺度来模仿天然细胞外基质,被广泛地应用于组织工程支架材料、伤口敷料及药物载体等方面。因此本文首先采用静电纺丝技术制备神经导管的外管支架,通过旋转接收装置、优化纺丝参数,制备得到仿细胞外基质的有序纳米纤维支架,作为细胞生长的支撑,促进细胞的迁移及增殖。另外,湿法中空纤维成型是一步法连续制备中空纤维导管的有效途径,在制备管内部纤维导管时采用湿法纺丝技术。
本文选用脂肪族聚碳酸丁二脂(PBC)、甲壳素纳米晶须(ChW)、高密度壳聚糖(HCS)及表面功能化的多壁碳纳米管(f-MWNTs)作为基本支架材料,通过静电纺丝接收装置的改进、材料组合、表面改性等,系统地研究评价了这类材料用作神经修复组织工程材料的力学性能、细胞相容性及降解性能等。主要研究工作如下：
1.通过静电纺丝法制备无规和有序PBC纳米纤维,研究了接收线速度对纳米纤维基本性能的影响。之后研究了低温等离子表面改性技术对纤维表面亲水性的影响,并通过等离子改性方法诱导纤维表面接枝明胶,以增强纤维表面的生物相容性。研究表明,PBC可以均匀地溶解于甲酸、DMF、六氟异丙醇及氯仿等有机溶剂中,但仅溶解于甲酸能够得到表面光滑、粗细均匀的纳米级纤维；通过转轴法可以成功制备得到PBC有序纳米纤维,随着旋转线速度的增大,纤维的排列有序度、晶区及分子链的取向度、结晶度以及力学性能都有所增大。
2.为了进一步提高外管有序纳米纤维的力学性能。首先通过酸解法制备得到纳米级甲壳素晶须,然后将其与PBC进行共混复合,通过静电纺丝法制备得到纳米级复合纤维,研究发现利用酸解法制得的甲壳素晶须的长度范围为180-680nm,直径分布范围为15-30nm,平均长径比为14.7,且将晶须分散到甲酸中24h后对表面形貌的观察发现,在短时间内甲酸并不会影响到晶须形貌,为下一步实验提供了依据；对将不同含量的晶须添到PBC中制得复合纳米纤维的研究表明,当晶须含量为5.0wt%时,制得的纳米纤维表面光滑、直径分布均匀且随着晶须的加入结晶度、热稳定性及力学性能得到显著提高。之后采用低温等离子技术对表面进行改性处理并用明胶接枝以赋予纤维表面新的生物相容性,使其亲水性得到了很大的提高,且更有利于神经细胞RSC96的黏附与增殖。
3.采用高密度壳聚糖(HCS)来制备导管内部的中空纤维。首先研究了HCS质量分数以及温度等对纺丝浆液稳定性的影响；而后进行湿法纺丝制备了中空纤维,并对HCS中空纤维的化学结构、晶体结构和热性能进行了研究。结果表明,HCS的固含量为5wt%时,纺丝过程顺利进行,挤出的浆液在凝固浴中形成的初生纤维结构均匀,不会出现断丝情况。纺丝温度应控制在20-30℃；凝固浴浓度为3wt%时所得的中空纤维热力学及结构性能较好。因此,在后续的实验研究中,将采用质量分数为5wt%HCS纺丝浆液,以质量分数为3wt%的NaOH-乙醇溶液作为凝固浴制备中空纤维。
4.采用碳纳米管来增强HCS中空纤维的力学和电学性能。为了提高碳管在HCS溶液中的分散性及与HCS基体之间的相容性,通过表面沉积交联法对碳管进行表面修饰得到f-MWNTs。然后与HCS进行混合制得复合中空纤维,并对其性能进行了研究分析。研究发现,经过表面修饰得到的f-MWNTs的管身变得平直,缠结状态也有所缓解,且在水中的分散性就明显优于MWNTs;对复合中空纤维的研究发现当f-MWNTs的含量达到0.5wt%时,f-MWNTs在HCS基体中的分布最为均匀,中空纤维的断面形貌也最为致密；且复合纤维的拉伸强度和弹性模量均达到最大值,分别为9.33MPa和2.34GPa；在含水率相同的条件下,随着碳管含量的增加复合纤维的电导率也增加。经过等离子预处理和明胶接枝改性后,复合膜在各个压力下的水通量都有了明显的提高,且更有利于细胞的黏附与增殖。
5.管中管结构的神经导管外层通过将静电纺有序纳米纤维沿一定直径的芯棒卷绕成管状结构,内部填充中空纤维,得到内部通道不同的导管。对导管的压缩性能的研究表明,5-通道神经导管在形变量为25%时,负荷力高达201cN,完全能够满足神经导管径向支撑力的要求；管中管结构的神经导管在受到外界压力时,形状发生变化后能够自然恢复,即形变后也能承担一定的支撑作用,符合神经导管力学性能的要求。
因此本文所制备的管中管复合型神经导管在亲水性、降解性及细胞相容性上都有所提高,同时具有良好的机械性能,有望成为新一代的神经修复组织工程支架。
In recent years, with the increasing in the incidence of nerve injury, nerve graft could not satisfied the nerve regeneration for the restrictions of distance and defect in donor's functions. So the restoration, regeneration and functional recovery in nerve injury have become a primary research direction in neuroscience and the nerve conduit is put forward. The nerve conduit is the connection of fracture nerves to provide suitable microenvironment for nerve growth and the idea conduit can also provide orientation for proliferation of nerve cells. In this paper, a novel nerve conduit with a tube in tube structure was designed and detailed experimental studies of fundamental properties were made. In addition, technical feasibility and reasonableness of the method were also discussed based on the results, and expected to bring the nerve conduit closer to being a realistic possibility.
Nanofibrous scaffolds, prepared by electrospinning with high porosity and large surface area, could imitate the natural extracellular matrix in the nano-scale, so it can be used as a porous scaffold to promote cell migration and proliferation in tissue engineering, wound dressing and pharmaceutical carriers. In this paper, the outward scaffolds were prepared by electrospinning and aligned nano-fibers that obtained by optimizing the spinning parameters and receiving apparatus could promote the cells'migration and proliferation. What's more, wet spinning was an effective method to prepare continuous hollow fibers, so inside hollow fibers were gained by this method.
Poly(butylene carbonate)(PBC), chitin nano-whiskers (ChW), high-density chitosan (HCS) and functionalized multi-walled carbon nanotubes (f-MWNTs) were chosen as the basic materials for scaffolds. Through the improvement of electrospinning technology, materials composition and surface modification, the mechanical properties, cell compatibility and degradation rate were also deeply studied. Main researches are listed as follows:
1. Random nano-fibers and aligned nano-fibers were prepared by electrospinning and the relationship of rotating speed of the receiving instrument and the basic performance of nano-fibers were discussed in detail. After plasma pretreatment and gelatin graft, hydrophilicity and biocompatibility of the aligned fiber mats could be improved significantly. Studies show that PBC could dissolve in some organic solvent such as formic acid (FA), dimethylformamide (DMF), hexafluoroisopropanol (HFIP) and chloroform, but the uniform and smooth fibers could only obtain from the FA solution; During the process of preparing aligned PBC nano-fibers, the order degree of fibers, the crystallinity and the orientation of crystalline region including mechanical strength are all increased correspondingly with the increment of rotating speed.
2. Chitin nano-whiskers made by acid decomposition method were added into PBC solutions to obtain compound nano-fibers in order to improve mechanical properties. The studies found that, the length and diameter of ChW were in the range of180-680nm and15-30nm respectively and the average length diameter ratio was14.7. In addition, the morphology of ChW in FA solvent was not changed and this has provided basis for the further experiment. The nano-fibers with uniform diameter distribution and smooth surface could be prepared with the addition of ChW up to5.0wt%; the crystallinity, thermal stability and mechanical properties are increased correspondingly with the increasing of ChW contents. After plasma pretreatment, hydrophilicity of the aligned fiber mats was improved significantly, and the fiber mats was then immersed into gelatin solution for grafting. The aligned nano-fibers after gelatin grafting could stimulate the adhesion and proliferation of RSC96cells.
3. In the paper, the spinning solutions were prepared by using acetic acid as solvent; the rheology of the solutions were studied to investigate the effect of the solid content and the spinning temperature on the stability of the spinning solutions and the spinning process. Then hollow fibers were obtained by the method of wet spinning and chemical structures, crystal structures, thermal properties including mechanical property were researched. The results showed that:we could obtain hollow fibers smoothly with the concentration of the spinning solution in5wt%at20-30℃the fibers had excellent properties that regenerated within the coagulation bath of3wt%sodium hydroxide-ethanol (mass ratio of1:1).
4. The dispersivity and compatibility with HCS of f-MWNTs that obtained via a controlled surface deposition and crosslinking process have been improved a lot; most of the tube body spread out that close to single dispersion, and the tangle also was eased after deposition and crosslinking process. The hollow fibers with the f-MWNTs content of0.5wt%exhibit the optimal tensile property with the maximum tensile strength and elasticity modulus of9.33MPa and2.34GPa, respectively. The fiber was then immersed into gelatin solution for grafting after pretreatment. Under the condition of same moisture content, the conductivity of composite hollow fibers was also improved with the increase of the content of f-MWNTs. XPS results also showed that some oxygen element was introduced to the surface of the pretreated samples for subsequently exposing to air (atomic fraction rising from29.07%to38.28%); in following grafting reaction, the active groups from gelatin introduce a large amount of nitrogen (from1.73%to7.54%) and the C-C bond broke down to form C=O and C=N bonds or other polar groups to realize the grafting reaction. Form biological characterization such as cell attachment, cell morphology and proliferation, these results demonstrate following two aspects. First, the addition of a small amount of f-MWNTs in fibers with the cell adhesion rate of77.44%which equal to HCS fibers (78.29%) after24h have no affect on cell behavior; second, cells seeded in fibers grafted with gelatin have higher adhesion rate and cells can proliferate faster in the same time which proved that the superior ability of fibers after surface modification with gelatin can well support Schwann cells (RSC96) growth and proliferation.
5. The aligned nano-fibers prepared from electrospinning was rolled on the core bar with right diameter to form the outer tube of nerve conduit, and then hollow fibers were filled into the tube to receive the nerve conduit with a tube in tube structure. The results of compression properties of nerve conduit indicated that the load force of5-channel conduit could reach up to210cN with the deformation of25%, so the conduit could meet the requirements of the supporting force of nerve regeneration.
引文
[1]Lin M Y, Manzano G, Gupta R. Nerve Allografts and Conduits in Peripheral Nerve Repair[J]. Hand Clinics.2013,29(3):331-348.
[2]Rivlin M, Sheikh E, Isaac R, et al. The Role of Nerve Allografts and Conduits for Nerve Injuries[J]. Hand Clinics.2010,26(3):435-446.
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