| Objective:(1)To study the optimal experimental conditions for the three-dimensional anatomy of the sciatic nerve of New Zealand white rabbits using high-resolution Micro-CT scan.(2)To develop a custom-designed digital model of multi-channel nerve conduit that guides nerve fascicles of nerve tissue to accurately dictate targeted distal nerve fascicles.The sciatic nerve of rabbits was taken as the "prototype" to fabricate bionic digital multichannel nerve conduits to evaluate the effect of customized multi-channel structure on the physical and biological properties of the conduit.(3)To explore the mechanism of the bionic muti-channel nerve conduit on repair of he New Zealand white rabbit sciatic nerve defect by implantation of bionic multi-channel nerve conduit into the New Zealand white rabbit sciatic nerve defect model.Method:(1)The specimens of the sciatic nerve tissue of the middle segment 10 mm in size of the adult New Zealand white rabbit were collected,the nerve samples of groups A and B were stained with 20% and 40% Lugol's fluid respectively.In the process of 1 to 3 days,the imaging changes of specimens from two groups were collected in every single day under light microscope and Micro-CT,and the imaging sequence of the micro-CT images was import into the Mimics software,and the threedimensional structure of rabbit sciatic nerve was visualized by 3D reconstruction software.(2)The middle segment 25 mm in size of sciatic nerve were taken from six adult New Zealand white rabbits,HE anatomical data of each inner nerve fascicles of nerves were obtained by Micro-CT scan and HE slice staining,the number and area of nerve fascicles,and the presence or absence of nerves integration or separation between bundles were obtained from the above two images for compare and observations.Based on the HE staining and micro-CT images of nerve tissue,four groups of neural conduit digital models with multi-channel structure were designed using Adobe Photoshop software.Four types of PLGA/gelatin microporous nerve conduits were constructed using techniques such as electrospinning,freeze-drying,and 3D printing technology.The physical properties of the nerve conduit were evaluated by physical features,experiments such as water absorption,thermal shrinkage and mechanical properties.The biological properties of the nerve conduit were evaluated by cell proliferation assays and enzyme degradation assays.(3)Multichannels nerve conduits were implanted into the 10-mm defect model of the sciatic nerve.Autologous nerve graft and 1-CANC groups were taken as the control groups,to study the effects of a customized bionic CANC on the axon regeneration of peripheral nerve by histology observation,electrophysiological test,simultaneous retrograde tracing,etc.Results:(1)The microscopic three-dimensional structure images of the group A and group B specimen were obtained with a Micro-CT scanning at 3 day and 2 day after Lugol's staining respectively.Micro-CT scans showed that the sciatic nerves of New Zealand rabbits had relatively fixed three-dimensional nerve structure and the obtained digital threedimensional model can visualize the internal microstructure of the sciatic nerve in any cross section.(2)The middle segment of the sciatic nerve of rabbit had 11±1 irregularly arranged nerve fascicles.Micro-CT scan showed no obvious integration or separation between nerve fascicles in the middle 25 mm segment of the sciatic nerve.The MI of 4 types of CANC measured by the Adobe Photoshop software showed the highest in 3-CANC group(77.1%),and the lowest MI(51.4%)in the 4-CANC group,The results show that the constructed all CANC have the same characteristics of appearance,porosity and heat shrinkage.In terms of water swelling,the 1-CANC is more prone to water absorption than other CANC group(p<0.05);the 1-CANC has the highest compressive strength in the lateral compression test,the 4-CANC group can withstand higher lateral compressive force than the 2-CANC group(p<0.05);The bending bending strength of 4-CANC is higher than 2-and 3-CANC group(p<0.05);Compared with PLGA biofilms,CANC have a higher degradation rate;The cell proliferation experiments suggest that the number of Schwann cells attached to the CANC group is excessive than in the PLGA biofilm group(p<0.05),there was no difference in the number of Schwann cells among CANC groups(p>0.05).(3)At the 12 th,and 24 th weeks after the implantation,the autologous group was superior to all nerve conduit implantation group in terms of histological observation,electrophysiological study,and functional recovery of the sensory and motor functions(p<0.01).The number of myelinated nerve fibers showed no difference among CANC group.However,a bigger and thicker myelin sheaths of axons were observed in the 2-and 3-CANC group compared to 1-and 4-CANC group.Similar experimental results were seen in electrophysiological study,autotomy behavior,and motor function assessment.The results of simultaneous retrograde tracing showed that the total number of labeled neurons among CANC group were not different(p>0.05),and the highest persentage of FB-NY labeled neurons was in the 1-CANC group(7.1% ± 2.4%),the lowest in the 4-CANC group(2.2% ± 1.2%,p<0.05).Conclusion:(1)The Micro-CT scan of the neural specimens stained with 20% Lugol's solution in 3 day or 40% Lugol's solution in 2 day can obtain a clear image of microscopic three-dimensional structure.The generated three-dimensional reconstruction model can by an important reference for designing three-dimensional bionic artificial nerve conduits.(2)Micro-CT scanning can accurately and clearly observe the three-dimensional structure of the middle segment of New Zealand white rabbits' sciatic nerve,combined with 3D printing model,freeze-drying and electrospinning techniques,simple bionics multi-channel nerve conduit can be made using anatomical structure of the rabbit sciatic nerve as a prototype.The CANC constructed by the above method has better physical and mechanical properties,which can meet the requirement of artificial nerve conduit implantation in animal experimental research.(3)The structure parameters of CANC have significant effects on the regeneration process of nerve tissue.The CANC have effect on reducing the mismatch of axons regeneration.The more similar of lumen diameter and lumen distribution of the CANC to the corresponding anatomical structure of repaired nerve fascicles of the targeted nerve lead to the better repair effect.The match index may can used for an evaluation indexes for the degree of bionics ratio of custom nerve conduit in furture. |
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