Experimental Study On Repairing Sciatic Nerve Defects With GDNF Gelatin Microspheres Combined With Double-layer Collagen Nerve Conduits

Part one:Preparation of GDNF-loaded gelatin microspheresObjective To study the preparation of Glial cell-line derived neurotrophic factor(GDNF)-loaded gelatin microspheres.Methods B-type Gelatin microspheres were synthesized by means of emulsion technique,and then cross-linked by EDC.GDNF-loaded microspheres were prepared by a method of complexation occurred between positively charged GDNF(pI of 9.23)and negatively charged Type B gelatin(pI of 4.7-5.2).The shape,degree of cross-linking,fold swelling ratios,diameter distribution and in vitro release of gelatin microspheres were observed.Results GDNF-loaded gelatin microspheres(GM)had smooth surface and good dispersion.They were round with an average diameter of about(3.91±1.87)?m.The cross-linking rate could be modified by different concentrations of EDC and cross-linking time.The fold swelling ratios of gelatin microspheres,which with(56.0±1.9)%cross-linking degree,is(3.50±0.27)%.In vitro release studies with GM exhibited minimal initial burst release during the first three day(17.2%).Then the release slowed down in a linear manner(average release 0.1%/day for 4-32days).Conclusion Type B gelatin microsphere cross-linked by EDC can load GDNF via ion complexation.GDNF-loaded gelatin microspheres can be used for locally controlled release of GDNF.Part Two:Experimental Study of Gdnf-loaded Gelatin Microsphere Combine with Bi-layer Collagen Conduit for Sciatic Nerve Regeneration in Rat Objective to Find A Convenient Way to Combine Gdnf-loaded Gelatin Microspheres and Bilayer Collagen Membrane via Gelma Hydrogel for Promoting Peripheral Nerve Regeneration.Methods We Fabricated Glial Cell-line Derived Neurotrophic Factor(Gdnf)Loaded Microsphere,Then the Microspheres Seeded in Gelatin-methacrylamide(Gelma)Hydrogel Which Was Finally Integrated with the Commercial Bilayer Collagen Membrane(Bio-gide(?)).the Formed Novel Composite of Nerve Conduit Was Employed to Bridge A 10mm Long Sciatic Nerve Defect in Rat.A Combination of Sem,Electrophysiological Assessment,Histology of Regenerated Nerves for He Staining,Loyez Staining and Transmission Electron Microscopy(Tem)Observations for All Regenerated Nerves Were Conducted to Study Axonal Regeneration and Functional Recovery.Results the Nerve Was Regenerated in Both Conduit and Autograft(Ag)Groups.A Combination of Electrophysiological Assessment and Histomorphometry Analysis of Regenerated Nerves Showed That Axonal Regeneration and Functional Recovery in Collagen Tube Filled with Gdnf-loaded Microsphere(Gm+ct)Group Were Similar to Ag Group(P>0.05).in Ag and Gm+ct Groups,Most Myelinated Nerves Were Matured and Densely Arranged with A Uniform Structure of Myelin in A Neat Pattern Along the Long Axis,However the Absence of Regenerated Nerve in Blank Group and the Nerve Fiber Appeared Disordered Arrangement in Collagen Tube(Ct)Group.Conclusion the Hybrid System of Bilayer Collagen Conduit and Gdnf-loaded Gelatin Microsphere Combined with Gelatin-methacrylamide Hydrogels Can Serve as A New Nerve Conduit for Nerve Tissue Engineering and Promote Peripheral Nerve Regeneration.