Repair And Regeneration Of Damaged Sciatic Nerves By Tissue Engineering Scaffold Made By Nano-silver And Collagen Type-I

The regeneration of the defect or damaged peripheral nerves has been a big challenge of the neuroscience, and autotransplantation of the nerve is the major method in the clinical therapy. However, the source of the auto-nerves is limited, and allotransplantation could induce rejection. Recently, the use of biocompatible tissue engineering scaffolds for such purpose has become a hot research area. Bridging the nerve stumps through nerve conduits can induce the longitudinal outgrowth of the axons while avoiding the formation of neuroma. Since this approach provides a relatively isolated microenvironment for the nerve regeneration. The ideal engineered nerve tissues should have three main characters. First, the scaffolds are the temporary structure to support the adhesion and out-growth of the axons. Secondly, the seeding cells or proteins are the key components to govern the peripheral nerve functional regeneration. Thirdly, the construction technology is the key to combine the seeding cells or proteins with the scaffolds, to maximize the functionality of the final products and eventually replace the damaged tissues.We constructed axial arrayed micro-tubular structure scaffolds using collagen type I and gelatin as main materials in combination with nano-silver particles and the laminin protein by the cryochem. Microtubules with diameters of 20-80μm are highly biomimetic in both the composition and 3-D porous structure. Such structure is favorable to the induction of a directional growth of the axons. With the even distribution of positively charged nano-silver particles at the inter-surface of the microtubules, the nana-silver scaffold could improve the adherence of the nerve cells through the interaction between the positively charged nano-silver particles and the negatively charged nerve cells surface. Meanwhile, positively charged nano-silver particles could attract the extracellular matrix to distribute evenly at the internal surface of the scaffold. This kind of scaffold similar to the Schwann cells biomimeticly could improve the regeneration of the nerves.In the first part of the experiment, the collagen was mixed with the nano-silver solution and then collagen scaffolds containing nano-silver constructed by freeze-drying were in a cylinders shape. By using the scanning electron microscope to observe the inner structure of the scaffolds and testing the mechanical strength, the most suitable collagen scaffolds containing nano-silver were selected for nerve regeneration. The most suitable collagen scaffolds containing nano-silver were tested by degeneration and accumulation experiments in vivo, and bacteria resistant experiment in vitro. The results are as follows:1 Collagen scaffolds containing nano-silver constructed by freeze-drying were in a cylinders shape. The suitable concentrate of the nano-silver was 0-4mg/ml. Its cross section-showed microtubules of uniform diameter, and the longitudinal section displayed the parallel arrayed inter-structure of microtubules. Since the diameters of these microtubules were about 20-80 nm, similar to that of the sciatic nerve, they may provide the passage for the nerve signal transduction that is essential for the function of nerve fibers. When the concentrate of the nano-silver was above 0-4mg/ml, the inequality in the diameter of the microtubules occurred with a lot of wadding in them.2 Collagen scaffolds containing nano-silver were tested by the tensile stress. The tensile strength(MPa)= max breakage load(N)/ transverse section of the scaffold(mm2). The results showed that when the concentrate of nano-silver was 0-2mg/ml, there was no different in the tensile strength of the Collagen scaffolds which was 0.21-0.22MPa. When the concentrate of nano-silver was above 0-2mg/ml, the tensile strength of the Collagen scaffolds declined obviously.3 The 96 pores plate were paved by the Collagen membrane containing nano-silver (0-2mg/ml) . The Schwann cells suspension were added into the pores. MTT was used to detect the growth rate of the cells. The results showed that there was no different in growth rate of the Schwann cells between the different concentrate of the nano-silver.4 Staphylococcus, pseudomonas aeruginosa and bacterium coli were seeded on the agarose gel plate respectively. Then, the collagen scaffolds containing nano-silver were placed on the surface of the agarose gel for 24 hours in the 37℃incubator. The results showed that the collagen scaffolds containing 2mg/ml nano-silver had obvious bacteriostatic action, whereas, the collagen scaffolds containing 1mg/ml nano-silver and the collagen scaffolds without nano-silver had no bacteriostatic action. 5 The collagen scaffolds containing 2mg/ml nano-silver and the collagen scaffolds without nano-silver were implanted the space under the skin of the rats. One scaffold was taken out every month to observe the degradation. One month later, the tubal wall degraded partly and channel became wide. Two month later, the more tubal wall degraded and some channel collapsed. Four month later, only a little collagen remained. The degradation rate of the collagen scaffolds containing 2mg/ml nano-silver were 76.2%,51.9%,38.3% and 12.3% respectively in the past three months. The degradation rate of the collagen scaffolds without nano-silver were 79.9%,59.4%,33.7% and 10.1% respectively in the past four months. There was no different between the degradation rate of the collagen scaffolds without nano-silver and the degradation rate of the collagen scaffolds containing 2mg/ml nano-silver. The degradation rate of the collagen scaffolds was suitable for the regeneration of the nerve.6 The collagen scaffolds containing 2mg/ml nano-silver were used to connect the pre-broken ischiadic nerves of the rats. The brain, spinal marrow, liver and kidney were taken out every month to detect the silver. All the detected tissue was not found silver after three months, which proved that the implantation of the collagen scaffolds containing 2mg/ml nano-silver could not lead to accumulation of the silver.In the second part of the experiment, the collagen scaffolds containing nano-silver were attached by laminin and fibronectin which was extracellular matrix and could improve the growth of nerve through electrostatic attraction Then the collagen scaffolds were used to repair the 10cm defect of the ischiadic nerves of the rats and rabbits. The results are as follows:1) nano-silver were seen to distribute evenly on the inner surface of the nana-silver-collagen scaffold.2)Laminin proteins were seen to distribute unevenly on the inner surface of the collagen scaffold without nana-silver, form many aggregations of protein crystals and even obstruct the tubes, so that the nerve could not pass through. However, laminin proteins were seen to distribute evenly on the inner surface of the collagen scaffold with nana-silver.3) At 30 days after the collagen scaffolds containing nano-silver implantation into the damaged nerve tissues in the rabbit, the newly formed nerve fibers were detested by transmission electron microscope (TEM) and electrophysiological examination. The results showed that the ischiadic nerves were repaired partly by the the collagen scaffolds containing nano-silver. Nano-silver-collagen scaffold displayed superior functionality in comparison with the control collagen scaffold without nano-silver particles, and similar functionality in comparison with the self-transplantation of the nerve. Our results proved that the collagen scaffolds containing nano-silver could improve the regeneration of the nerve like the function of Schwann cells.Fibronectin as the main commponentod of the extracellular matrix play important role in the regeneration of the nerve. Laminin and fibronectin were attached laminatedly on the inner surface of the tubal wall. The collagen scaffolds containing nano-silver attached with laminin and fibronectin had the similar function of the Schwann cells and could partly repair the 10cm defect of the ischiadic nerves of the rats within 30 days.Our results showed that through electrostatic attraction nano-silver in collagen scaffolds could make the laminin distribute evenly on the inner surface of the tubes. This kind of inner surface of the tubes was similar to the microenvironment of the nerve regeneration and could improve the regeneration of the nerve. The results of the repair of the 10cm defect of the ischiadic nerves of the experiment animals by collagen scaffolds containing nano-silver was similar to the results of self-transplantation. Our experiment offered new evidence for the tissue engineering repair of the peripheral nerve.