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。
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