The Application Of A Novel Bionic Collagen-Chitosan (CCH) Scaffold In Bridging The Sciatic Nerve Defect In Beagles

The regeneration and functional recovery of Peripheral nerve injury are worldwide clinicalchallenges currently. Clinically, direct end-to-end nerve sutures are proposed as treatmentsfor short nerve gap lesions（<30mm）. The standard technique for long-lesion gaps（>30mm）is to transplant autologous nerve grafts (autografts) from uninjured sites to the injured site toform a bridge between the two nerve stumps. Nevertheless, the use of autografts has anumber of disadvantages and limitations, including limited graft availability, secondarydeformities, and potential differences in the tissue structures and sizes, etc. Accordingly, it isimperative to develop alternatives to the conventional nerve autografts. Tissue engineeringtechnology has developed rapidly in recent years and there has been an increasing focus onthe tissue engineering grafts since they produced promising results in bridging long nervegaps. Tissue engineering grafts are generally developed by combining biomaterial scaffolds,seed cells and biologically active molecules. The inner microstructural properties of thescaffold are the predominant factors that determine the efficacy of a tissue engineering graftin bridging nerve gap lesions. In recent years, a variety of scaffolds with either oriented or random inner structures have been developed to bridge nerve gaps. Orientedmicro-structured scaffolds have been more advantageous than scaffolds with a random innerstructure for guiding the linear growth of axons across nerve gaps, which indicates thatphysical guiding cues are essential for guiding axon regeneration. To date, several scaffoldswith oriented structures, such as fibers and grooves, have been successfully fabricated andhave been shown to be capable of physical guiding the linear growth of regenerated axons tosome extent. However, these oriented structures still differ substantially from the guidingbasal lamina micro-channels in nerve autografts, which are considered the gold standardtechnique for treating peripheral nerve lesions. Since the basal lamina micro-channels innormal nerves are known to play a significant guiding role in the linear growth ofregenerated axons, extracellular matrix (ECM) based scaffolds with dimensions resemblingthe basal lamina micro-channels are expected to provide a promising alternative to autograftsfor briding nerve gaps. To date, however, little information has been obtained about scaffoldswith similar inner microstructures, and the efficacy of such scaffolds in bridging peripheralnerve gaps in vivo has never been examined.In the current study, a collagen-chitosan scaffold (CCH) with longitudinally oriented porechannels and an interconnected porous structure was successfully fabricated by axiallyfreezing and subsequently freeze-drying collagen-chitosan suspensions. The optimumparameters for the fabrication process was determined regarding the raw material blend ratio,concentration of acetic acid and the velocity of freezing in the improved freeze-dryingtechnology. The mechanical property and degradationtion of the CCH scaffolds weremodified via post-fabrication cross-linking with genipin. The biomechanics of the scaffoldwas also fully examined before its usage in vivo. Subsequently, we evaluated its efficacy inbridging a30mm long sciatic nerve defect in Beagles by using a combination ofmorphological and functional techniques.Part one: Fabrication of CCH scaffolds and determination of the optimum parametersfor the fabrication process[Objectives] To fabricate the ECM based bionic scaffold with dimensions resembling the basal lamina micro-channels of normal nerve.[Methods] The freeze-drying technique was improved to fabricate nerve guidance scaffoldsfrom collagen/chitosan. The inner structure was observed under the scanning electronicmicroscope. The pore sizes and interval porosity were examined to determine the optimumparameters for the fabrication process.[Results] The novel CCH nerve guidance scaffold with different pore size andmicrostructure was fabricated using different velocity of freezing. The optimum velocity offreezing was examined to be2×10-5m/s (mean pore size:37.34±13.24μm). Considering themicrostructure and pore size of the CCH scaffold, the optimum concentration of acetic acidfor CCH fabrication was evaluated to be3mg/ml and the blend ratio was3:1subsequently.The pore size was ranging from24μm～102μm (mean pore size:49.85±19.85μm) andthe interval porosity was above90%.Part two: Property modification and biomechanics evaluation of the CCH scaffolds[Objectives] To modify the mechanical property and degradation of the CCH scaffold, andfurther evaluate the biomechanics of the modified CCH scaffolds.[Methods] The tensile mechanical properties and degradation of the CCH scaffolds weremodified via post-fabrication cross-linking with genipin. The degradation kinetics of thecross-linked scaffolds was evaluated by incubating the scaffolds in PBS with or withoutlysozyme.[Results] After cross-linking with genipin for48h, the CCH scaffolds showed a17.9±4.2%(without lysozyme) or20.1±4.6%(with lysozyme) reduction in dry weight relative to theoriginal weights after8weeks while the noncross-linked scaffolds showed29.6±4.8%(without lysozyme) or36.3±5.2%(with lysozyme) reduction. The cross-linked CCHscaffolds could withstand a higher average ultimate stress than the noncross-linked scaffoldsin either dry or wet condition, which meets the requirements of surgical procedures. Theseindicate that the cross-linked CCH scaffolds possessed the required mechanical stiffness andstability for successful surgical implantation and nerve regeneration. Part three: The efficacy of CCH scaffolds in bridging a30mm long sciatic nerve defectin Beagles[Objectives] To investigation the efficacy of CCH scaffolds in bridging a long nerve gap inBeagles.[Methods] The efficacy of the CCH scaffolds in bridging a30mm long sciatic nerve defectin Beagles was investigated by using a combination of morphological and functionaltechniques, including immunohistology, transmission electron microscope, electrophysiology,retrograde-labelling and behavioral tests.[Results] Twelve weeks after implantation, the implanted CCH scaffolds were almostcompletely replaced by a large bundle of densely packed regenerated nerve fibers and thepenetration and remarkably linear growth of axons within the longitudinal micro-channels ofthe CCH scaffolds was also observed.24weeks after implantation, the functional tests alsoconfirmed that the CCH scaffolds achieved regeneration and functional recovery equivalentto that of an autograft, without exogenous delivery of regenerative agents or celltransplantation. These findings demonstrate that CCH scaffolds may be used as alternativesto nerve autografts for peripheral nerve regeneration.