| Peripheral nerve defect can be directly lead to the loss of sensory or motor function, and increase the risk of secondary complications, and seriously affect the exercise of the body’s normal function, some even need a lifelong maintenance treatment, not only seriously affect the quality of patients life, and even cause social and economic effects of different level [1-4]. Repair of peripheral nerve defects with nerve injury is a difficult research.At present, autograft (autologous nerve transplantation, ANT) is still the "gold standard"for treatment of peripheral nerve defect [3], but its source is insufficient, and it can’t repair a wide range of neurological defects etc[5,6]. Acellular nerve scaffold prepared by the method and principle of tissue engineering, is applied to repair a wide range of peripheral nerve defects,which has become a hotspot in the field of research [7-10].This topic is based on the above ideas, to prepare a new type of artificial neural implants and to evaluate its biomechanical properties.Part I The preparation of the xenogeneic acellular nerve scaffold via MyroilysinObjective:To get a novel acellular nerve scaffold.Methods:After getting the bilateral sciatic nerve(15mm) from healthy Wistar rats, sciatic nerve was decellularized by using different concentration of Myroilysin enzyme, macroscopic observation and making tissue section,after HE staining and Masson staining, optical microscope was used to observe the acellular effect, retained nerve basement membrane structure,and the collagen structure of xenogeneic acellular nerve scaffold.Results:Macroscopic observation, the xenogeneic acellular nerve scaffold via high concentration of Myroilysin (500IU/mL,600IU/mL) had been almost completely dissolved, its length was significantly shortened than before experiment, the others had obviously volume expansion phenomenon, and colour and lustre was transparent.Histological observation, HE staining,under optical microscope the xenogeneic acellular nerve scaffold via low concentration of Myroilysin (200IU/mL, 300IU/mL) contained a large number of residual nuclei. Masson staining, under optical microscope the collagen fibers of xenogeneic acellular nerve scaffold via different concentration of Myroilysin arranged ruly,at the same time,we could see the gap between collagen fibers gradually increased. Through repeated experiments we finalized 400 IU/ML Myroilysin was the best.Conclusion:Myroilysin method was a new kind preparation of xenogeneic acellular nerve scaffold;Through Macroscopic observation and histological observation, Myroilysin 400IU/mL was the best.Methods:After getting the bilateral sciatic nerve(15mm) from healthy Wistar rats, sciatic nerve was decellularized by using different concentration of Myroilysin enzyme, macroscopic observation and making tissue section,after HE staining and Masson staining, optical microscope was used to observe the acellular effect, retained nerve basement membrane structure,and the collagen structure of xenogeneic acellular nerve scaffold.Results:Macroscopic observation, the xenogeneic acellular nerve scaffold via high concentration of Myroilysin (500IU/mL,600IU/mL) had been almost completely dissolved, its length was significantly shortened than before experiment, the others had obviously volume expansion phenomenon, and colour and lustre was transparent.Histological observation, HE staining,under optical microscope the xenogeneic acellular nerve scaffold via low concentration of Myroilysin (200IU/mL, 300IU/mL) contained a large number of residual nuclei. Masson staining, under optical microscope the collagen fibers of xenogeneic acellular nerve scaffold via different concentration of Myroilysin arranged ruly,at the same time,we could see the gap between collagen fibers gradually increased. Through repeated experiments we finalized 400 IU/ML Myroilysin was the best.Conclusion:Myroilysin method was a new kind preparation of xenogeneic acellular nerve scaffold;Through Macroscopic observation and histological observation, Myroilysin 400IU/mL was the best.Part Ⅱ The contrast of Myroilysin and Sondel acellular nerve scaffoldObjective:Compared the similarities and differences of Myroilysin acellular nerve scaffold and Sondell chemical acellular nerve scaffold.Methods:After getting the bilateral sciatic nerve(15mm) from healthy Wistar rats,respectively applied Myroilysin method (400 IU/ML) and classical Sondell chemical method for acellular process.Macroscopic observation and making tissue section, after HE staining and Masson staining, optical microscope was used to observe the acellular effect, retained nerve basement membrane structure,and the collagen structure of xenogeneic acellular nerve scaffold. And electron microscope samples were made to observe the surface physical properties of the nerve scaffold.Results:Compared with nerve scaffold gained by classical Sondell chemical method,nerve scaffold gained by Myroilysin method had more advantages,its diameter and volume was obviously larger.Compared with the natural nerve, theirs diameter and volume were larger than natural nerve. But Myroilysin acellular nerve scaffold was more obvious expansion than Sondell chemical acellular nerve scaffold, and colour and lustre was transparent. The diameter of Sondell chemical acellular nerve scaffold was only slightly larger than natural nerve. Histological observation, HE staining,under optical microscope both of the nerve scaffold didn’t contain cell nucleus, axon, myelin sheath; The neural structure of basement membrane was stained red,wavy,but compared with Sondell chemical acellular nerve scaffold, Myroilysin acellular nerve scaffold had order uniform and good continuity.Masson staining, collagen fiber arrangement of Sondell chemical acellular nerve scaffold was irregular, and its continuity was bad, fiber structure had been damaged,but Myroilysin nerve scaffold with relatively regular and continuous.Conclusion:Through the above comparison, Myroilysin method would become a new kind of preparation for nerve scaffold,and it had more advantages than others. collagen structure of xenogeneic acellular nerve scaffold. And electron microscope samples were made to observe the surface physical properties of the nerve scaffold.Results:Compared with nerve scaffold gained by classical Sondell chemical method,nerve scaffold gained by Myroilysin method had more advantages,its diameter and volume was obviously larger.Compared with the natural nerve, theirs diameter and volume were larger than natural nerve. But Myroilysin acellular nerve scaffold was more obvious expansion than Sondell chemical acellular nerve scaffold, and colour and lustre was transparent. The diameter of Sondell chemical acellular nerve scaffold was only slightly larger than natural nerve. Histological observation, HE staining,under optical microscope both of the nerve scaffold didn’t contain cell nucleus, axon, myelin sheath; The neural structure of basement membrane was stained red,wavy,but compared with Sondell chemical acellular nerve scaffold, Myroilysin acellular nerve scaffold had order uniform and good continuity.Masson staining, collagen fiber arrangement of Sondell chemical acellular nerve scaffold was irregular, and its continuity was bad, fiber structure had been damaged,but Myroilysin nerve scaffold with relatively regular and continuous.Conclusion:Through the above comparison, Myroilysin method would become a new kind of preparation for nerve scaffold,and it had more advantages than others.Part Ⅲ The biological properties of xenogeneic acellular nerve scaffold via MyroilysinObjective:To evaluate the biomechanical performance of xenogeneic acellular nerve scaffold via Myroilysin, to prepare for the later experiment in vivo.Methods:Used Endura TEC ELF 3200 mechanical test equipment, through testing normal nerve (group A) and Sondell chemical method (group B) and Myroilysin method (group C) received the biomechanical performance indexes,such as the limit load,elastic modulus, fracture toughness,limit stress,ultimate strain,fracture energy, and Compared the differences of biomechanical properties among the three groups,and judged the differences had or no statistical significance.Test results with mean+/-standard deviation, SPSS 16.0 was used for statistical analysis, comparison between groups using single factor analysis of variance and multiple comparisons between groups using LSD test. P<0.05 said the difference was statistically significant.Results:P values were greater than 0.05, that was, A, B, C three groups in the limit load, elastic modulus, toughness, limit stress, ultimate strain, fracture energy had no statistical significance.Conclusion:The biomechanical properties of Myroilysin xenogeneic acellular nerve scaffold was good, with no difference between the normal neural, accord with the requirement of the late in vivo. |
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