| BackgroundPeripheral nerve injury is a sort of ubiquitous nerve damage in modern life. So far, the transplantion of autologous nerves has always been the "gold standards" for the defects in long neurologic segments. However, the poor provision of transplantable autologous nerve tissues can be quite limited and meantime the organic functions of provisional areas could be severely sabotaged as well, all of which lead to a relatively high rate of mutilation to the damage on the peripheral nerves. So as to solve this obvious issue, there have been a number of experimental researches conducted for years and many an achievement has been yielded. It’s always the universal dream to develop artificial nerves with highly bionic structures and well-bridging functions. Especially in the last30years of reaching deeper in the field of life sciences and the cognitive principles on the regeneration of mechanic tissues, a variety of tissue-engineering nerve scaffolds have become the focal points in researches. With the rejuvenation of tissue engineering, scientists conduct much more in-depth experiments on the construction of structural model of artificial nerves, materials, manufacturing, generic cells, growing factors, drugs of pro-growing for nerves and repair of the rehabilitated neurotic functions, etc. On the preliminary researches, we used chitosans and I-typed collagens to develop peripheral nerve scaffolds of tissue engineering possessing highly bionic structures by means of lyophilization. The newly-built scaffold is characterized by high porosity, well degradability and low antigenicity and it has achieved great regeneration effect during the experiment on repairing the sciatic nerves of Sprague-Dawley rats. At the same time, we also noticed that there existed different levels of pain in the operative limbs during the process of rehabilitating the impaired nerves, which are mainly marked by reduction in drinking water, weight alleviation, emotional provocation and self-devour on more than half of the operative limbs. Pathologic pain followed by the peripheral nerve injury and bridging is incessantly the pestering problem. This experiment is furthering the development of effects on the rehabilitation in the new tissue engineering of neurotic scaffold bridging and peripheral nerve injury and laying emphasis on the new strategy provided by the triggering mechanism of pathologic pain caused by nerve rehabilitation.ObjectiveThis research is aimed at providing new thoughts on establishing peripheral nerve scaffold in the novel tissue engineering and making comprehensive use of tissue engineering, neuroethology, morphology and molecular biology to offer new strategies in repairing the defects in long neurologic segments, thus providing the empirical rationales of pathologic pains followed by the process of rehabilitation.Methods(1) Aaccording to the principle of cold crystallization, with I-typed collagens and chitosans as its major ingredients and added different strength of FK506, an improved process of lyophilization was used to manufacture the peripheral nerve scaffold of newly formed tissue engineering. (2)The structure of the peripheral nerve scaffold of newly formed tissue engineering was observed by the scanning electron microscope (SEM).(3) Animal models are divided into4groups as followed:Group1:autologous nerve controls; Group2:compound FK506(5mg/ml) scaffold repair group; Group3:compound FK506(10mg/ml) scaffold repair group; Group4:blank scaffold repair group. The peripheral nerve scaffold of newly formed tissue engineering was used to bridge the10mm gap of sciatic nerves of Sprague-Dawley rats.(4) Sciatic functional index were measured in4weeks,8weeks,12weeks,16weeks after operations.(5) In16weeks after surgery, a bipolar electrode was used to measure the latency of onset of compound muscle action potential (CMAPs), nerve conduction velocity (NCV) and peak amplitude of sciatic nerve on SD rat.(6)In16weeks after surgery, the materials were drawn and stained with toluidine blue to get bridge regenerated nerve fibers by transmission electron.(7) Von-Frey system was used to detect the threshold variation of mechanically operated side paw of SD rats in before surgery, after surgery in4weeks,8weeks,12weeks and16weeks, respectively.(8) Hargreaves Heat Radiation System was chosen to test the incubation changing in the caloric limb withdrawal in the post-operated SD rats in before surgery, after surgery in4weeks,8weeks,12weeks and16weeks, respectively.(9) Open-field experiments were used to observe and analyze the movement of motor function and behavioral changes of SD rats in16weeks after surgery.(10) To adopt immunofluorescence staining to examine the expression change of protein from the electric tension of cytolemma of post-operated DRG neuron, namely the gated tunnel of sodion, Navl.7and Nav1.8in16weeks after operation. Results(1) The peripheral nerve scaffold of newly formed tissue engineering adopted by advanced process generally looks ultraviolet in color and cylindrical in shape. Its elastic modulus is3.842±1.326MPa in the state of moisture and the three-dimensional sustainability also functions well.(2) The indices of the sciatic nerves of post-operated SD rats in the4th month are shown as follows: Group1-79.83±3.43,Group2-81.14±3.27,Group3-83.21±3.62,Group4-86.28±2.64. The indices of the sciatic nerves of post-operated SD rats in the8th month: Group1-64.29±3.61,Group2-65.51±2.97,Group3-67.21±3.47,Group4-71.03±3.18. The indices of the sciatic nerves of post-operated SD rats in the12th month: Group1-50.29±4.12,Group2-51.03±3.29,Group3-56.21±3.58,Group4-62.03±4.13. The indices of the sciatic nerves of post-operated SD rats in the16th month: Group1-38.74±2.19,Group2-39.07±2.26,Group3-42.53±2.71,Group4-56.28±2.64.(3) The incubation, transmission velocity and wave amplitude of CMAPs in the sciatic nerves of post-operated SD rats in the16th month are shown as follows:Group1:incubation1.24±0.12, transmission velocity17.01±1.13, wave amplitude26.12±2.46.Group2:incubation1.26±0.13, transmission velocity16.97±1.17, wave amplitude26.04±2.49.Group3:incubation1.54±0.15, transmission velocity16.04±1.87, wave amplitude24.25±2.54.Group4:incubation2.08±0.24, transmission velocity14.06±1.94, wave amplitude22.91±2.84.(4) The scaffold tissues are seen to be degraded and well absorbed in the16th week after the operation. The results of using toluidine blue to stain and bridge the regenerated neurotic fiber and employing the scanning electron microscope (SEM) to observe the structure of the peripheral nerve scaffold of newly formed tissue engineering are shown below:The respective groups of bridging and repairing neurotic tissues have regenerated on different levels. The regenerated neural myelin sheaths have outnumbered and thickened in Group1and Group2. The regenerated neural myelin sheaths in Group3are outnumbered and less thick than those in Group1and Group2. The regenerated neural tissues are finest with the regenerated neural myelin sheaths poorest in number and shape.(5) The indices of employing the von-Frey System to detect the threshold changing in the mechanical limb withdrawal in the post-operated SD rats are shown as follows:Pre-operation:Group13.20±1.17, Group23.31±1.21, Group33.21±1.18, Group43.12±1.08.Post-operation in the4th month:Group118.31±3.97, Group219.51±4.06, Group320.16±4.81, Group423.4±4.58.Post-operation in the8th month:Group18.62±3.72,Group29.82±3.78,Group311.71±4.16, Group413.2±4.29.Post-operation in the12th month:Group14.71±2.94;Grou25.80±2.48,Group36.78±3.84, Group48.13±3.94.Post-operation in the16th month:Group14.71±2.71,Group24.91±2.53,Group36.12±3.07, Group47.26±3.46.The results of indices in Group2are similar with the ones in Group1after the operation in the12th and16th month respectively, which is more effective in rejuvenation than the case in Group3and Group4.(6) The results of applying the Hargreaves Heat Radiation System into testing the incubation changing of the caloric limb withdrawal in the post-operated SD rats show that indices in Group2are similar with the ones in Group1after the operation in the12th and16th month respectively, which is more effective in rejuvenation than the case in Group3and Group4.(7) Outcome of the open-field locomotion in the16th week after the operation is analyzed as the following ones:i. the overall movement distance of rats in the blank group is lower than that in the other three groups, the groups between exist discrepancies (P<0.05); ii. no significant differences as for the central movement distance between the low density groups and the autologous groups (P>0.05); iii. no significance differences as for the average velocity between the low density groups and the autologous groups (P>0.05).(8) The16th week after the operation, the double-labeling of immunofluorescence is marked according to the cutting out of dorsal root ganglia of the rehabilitated neurotic tissues and adoption of Western Blot in examining the expression change of protein from the electric tension of cytolemma of post-operated DRG neuron, namely the gated tunnel of sodion, Navl.7and Nav1.8.Conclusion(1) In ways of manufacturing the peripheral nerve scaffold of newly formed tissue engineering with I-typed collagens and chitosans as its major ingredients, different concentrations of FK506and an improved process of lyophilization according to the principle of cold crystallization, the microtubule structures of which are highly similar to the height of the basement membrane in normal nerves, meantime they are greatly flexible in the state of moisture with a good function of three-dimensional sustainability. The scaffold tissues are seen to be degraded and well absorbed in the16th week after the operation. The scaffold constructed in this experiment has a good recovery through bridging, which proves to be a quite ideal peripheral neurotic scaffold in tissue engineering.(2) Inhibition from partial allergy and functional improvement of regeneration by bridging neurotic fibers as well as avoidance of side effects on the pantosomatous dosage can be achieved during the procedure of constructing by means of adding a definite strength of FK506which serves as the analogous effects of alleviation on repairing neurotic tissues. For the exorbitant density employed in dosage might lead to the pantosomatous inhibition from immunization, infection and other untoward effects; better effective improvement in bridging the regeneration of neurotic fibers would also be a failure. (3)The new-typed neurotic scaffold of tissue engineering with a definite strength of FK506is capable of alleviating the pathologic pain aroused by the rehabilitation in regenerating neurotic fibers during the process of bridging the rehabilitated peripheral nerves. In addition, the expression change of protein from the electric tension of cytolemma of post-operated DRG neuron rehabilitating the peripheral nerves, namely the gated tunnel of sodion, Navl.7and Nav1.8is quite likely to be one of the mechanisms which recondition the nerve pathological pain. The conclusions mentioned above thus have provided empirical evidences for a further study in the improvement and construction of new-typed neurotic scaffolds of tissue engineering. |
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