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高仿真壳聚糖神经支架的制备及其桥接神经
缺损的实验研究
硕士研究生
:屈巍
     
罗卓荆教授
周围神经缺损是平时与战时最常见的创伤类型之一,常导致患者感觉和运动功能丧失,是目前尚未解决的临床难题之一。自体神经移植是治疗周围神经缺损的金标准,主要优势是其修复效果明确,但供体来源有限、供区功能损害以及供体和受体神经尺寸和类型不匹配等原因限制了自体神经移植的应用。组织工程学的发展为神经缺损的治疗带来了希望。目前已经有各种超微结构的神经支架被相继开发,并被尝试应用于神经缺损的治疗之中,但可惜的是其在体修复效果均不甚理想,主要的原因之一是神经支架的内部结构与正常神经的超微结构相差较大。目前国际公认的最理想的神经支架应该具有与正常神经基底膜高度仿真的轴向微管样结构。鉴于此,本研究以壳聚糖为主要原料,通过前期已获国家发明专利的梯度冷淋技术,成功构建了高仿真神经支架,并在大鼠坐骨神经缺损模型中,从形态学和功能学两方面将高仿真支架与自体神经移植进行对比,综合评估其修复效果。全文共分为三部分:
第一部分:高仿真壳聚糖支架材料的制备
目的:构建在超微结构上与正常神经基底膜结构高度仿真的组织工程神经支架。
方法:以壳聚糖为原料,应用改良的梯度冷淋技术制备高仿真神经支架,扫描电镜观察其内部结构。
结果:本实验制备的壳聚糖神经支架在纵截面上为相互平行的微管样结构,微管相对均一,管径为54.26 ± 12.1μm (范围:45 ~ 75 µm);神经支架的横截面类似于蜂窝样结构。
结论:利用前期开发的梯度冷淋技术制备的神经支架的超微结构高度仿真于正常神经的基底膜结构,有望成为神经缺损修复中自体神经的替代物。
第二部分:高仿真壳聚糖支架的基本性能测定
目的:对壳聚糖支架的性能进行优化和改进,并测定壳聚糖支架的相关特征和参数。
方法:测定壳聚糖支架的孔隙率、表观密度,在此基础上利用京尼平(genipin)对壳聚糖支架进行交联,确定壳聚糖材料的交联度与交联时间以及交联剂浓度的关系,同时评价交联后壳聚糖材料的细胞相容性,对比壳聚糖支架交联前后的降解性以及力学性能,以全面了解高仿真壳聚糖神经支架的基本性能。
结果:壳聚糖神经支架的孔隙率为85.13±2.89%表观密度为0.0623±0.0242mg/mm3;应用京尼平交联壳聚糖材料,材料的交联度随交联时间延长而增加,随交联剂浓度增大而增加。此外,壳聚糖支架经京尼平交联后,降解性以及力学强度有了明显改善,同时交联后的材料具有良好的细胞相容性,可以满足在体修复神经缺损的要求。
结论:壳聚糖支架经京尼平交联后,可以满足在体移植的要求。
第三部分:壳聚糖支架修复坐骨神经缺损的有效性评价
目的:评价高仿真壳聚糖支架在体桥接大鼠15mm坐骨神经缺损的修复效果。
方法:以前期制备的高仿真神经支架修复大鼠15mm坐骨神经缺损,应用形态计量学、逆行示踪、神经电生理以及行为学检测评价该高仿真支架的修复效果。
结果:本实验构建的壳聚糖支架具有轴向平行排列的微管样超微结构,该结构高度仿真于正常神经基底膜结构。在桥接大鼠15mm坐骨神经缺损的实验中,该仿真支架可取得与自体神经移植相似的修复效果。
结论:高仿真壳聚糖神经支架在体修复效果与自体神经移植相似,有望成为自体神经移植的替代产品。
 
关键词:仿真,壳聚糖,神经缺损,组织工程,神经再生
 
 
  
The fabrication of biomimicking chitosan scaffolds and its efficacy in bridging large nerve defect
 
Candidate for master: Qu Wei
Supervisor: Luo Zhuojing
Department of orthopaedics, Xijing hospital, Fourth Military Medical University,
Xi’an 710032, China
Peripheral nerve injury, one of the most common types of civilian and military trauma, always results in the loss of sensory and motor functions. Autograft transplantation is the gold standard treatment for large nerve gaps. Although the autograft transplantation achieved good nerve regeneration and functional recovery, the use of autograft was limited by secondary deformities, graft availability, and differences in structures and sizes, etc. Thus, it is necessary to develop an alternative to autografts. With the development of tissue engineering, numerous tissue-engineered nerve conduits with various micro-structures have been developed and tried in the treatment of nerve gap lesions. However, the nerve regeneration and functional recovery after their implantation has proved unsatisfactory. One of the main reasons is the difference between their inner microstructure and that in normal nerves, and the basal lamina micro-channels in normal nerves has been acknowledged as the ideal microstructure of nerve conduit. Therefore, in the present study, chitosan nerve conduits with longitudinal micro-channels were fabricated using a modified freeze-drying method. Its efficacy in guiding nerve regeneration was evaluated in the rat model of 15mm nerve gap. The whole experiment was divided into three parts:
Part one: fabrication of biomimicking chitosan scaffold
Aim: To fabricate a novel nerve conduit with longitudinal micro-channels.
Methods: Chitosan scaffolds were fabricated with a patented technique, which was developed from freeze-drying method. Scanning electron microscopy was adopted to examine the inner micro-structure of the chitosan scaffolds.
Results: The chitosan scaffold prepared in the present study showed a honey comb-like structure in the cross section, and longitudinal micro-channels in the longitudinal sections. Its mean diameter is 54.26 ± 12.1μm, ranging from 45µm to 75 µm. The inner microstructure of the chitosan scaffold resembles the basal lamina micro-channels in normal nerves.
Conclusion: A chitosan scaffold with the basal lamina micro-channels in its inner microstructure similar to those in normal nerves was successfully fabricated, which might be an ideal alternative to nerve autograft.
Part two: Property modification of the biomimicking chitosan scaffold
Aim: To modify the biodegradability and mechanical property of the chitosan scaffold, and further evaluate the basic characteristics of the modified chitosan scaffolds.
Methods: The porosity and apparent density of the chitosan scaffold were measured. Then the chitosan scaffold was cross-linked with genipin. The relationship between the cross-linking degree and cross-linking time (or the concentration of genipin) was determined in the present study. The cyto-compatability of the cross-linked scaffold was investigated and the comparisons between pre- and post cross-linking in the biodegradablity and mechanical properties were performed to comprehensively evaluate the basic characteristics of the chitosan scaffold.
Results: The porosity of the chitosan scaffold was 85.13±2.89%, and the apparent density was 0.0623±0.0242mg/mm3. The cross-linking degree of the chitosan scaffold increased when the cross-linking time was prolonged, or when the concentration of genipin was increased in the present study. The degradation rate was slowed down and the mechanical properties were strengthened by cross-linking with genipin. In addition, the genipin cross-linked chitosan scaffold showed good compatibility with Schwann cells.
Conclusion: The genipin cross-linked chitosan scaffold can meet the requirements of in vivo implantation.
Part three: The efficacy of biomimicking chitosan scaffold in guiding nerve regeneration
Aim: To investigate the efficacy of the biomimicking chitosan scaffolds in bridging 15 mm sciatic nerve gap in rats.
Methods: The chitosan scaffolds were used to bridge 15 mm nerve defect in rats, and their efficacy in bridging nerve gap was evaluated by morphometric analysis, retrograde labeling, electrophysiological studies and behavioral analysis.
Results: The chitosan scaffolds developed in the present study showed longitudinal oriented micro-channels, which resembled the dimensions of the basal lamina channels in normal nerves. Implantation of chitosan scaffold achieved similar axonal regeneration and functional recovery to autograft implantation in bridging 15 mm sciatic nerve gap in rats.
Conclusion: Implantation of chitosan scaffold achieved similar axonal regeneration and functional recovery to autograft implantation in vivo.The chitosan scaffold may be used as an alternative to autograft in bridging nerve gaps.
Key words:biomimicking; chitosan; nerve defect; tissue engineering; neural regeneration

 
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