| Peripheral nerve regeneration and functional recovery are current clinical challenges. For short nerve gap lesions, direct end-to-end nerve sutures are proposed as treatments. The standard technique for long-lesion gaps is to transplant autologous nerve grafts (autografts) from uninjured sites to the injured site to form a bridge between the two nerve stumps. However, the use of autografts has a number of disadvantages and limitations, including limited graft availability, secondary deformities, and potential differences in the tissue structures and sizes, etc. Therefore, it is imperative to develop alternatives to conventional nerve autografts.Bridging long nerve gaps with tissue engineering grafts produced promising results and has gained extensive attention. Tissue engineering grafts are generally developed by combining biomaterial scaffolds, seed cells and biologically active molecules. The inner microstructural properties of the scaffold are the predominant factors that determine the efficacy of a tissue engineering graft in 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. Oriented micro-structured scaffolds have been more advantageous than scaffolds with a random inner structure for guiding the linear growth of axons across nerve gaps, which indicates that physical guiding cues are essential for guiding axon regeneration. To date, several scaffolds with oriented structures, such as fibers and grooves, have been successfully fabricated and have been shown to be capable of physical guiding the linear growth of regenerated axons to some extent. However, these oriented structures still differ substantially from the guiding basal lamina micro-channels in nerve autografts, which are considered the gold standard technique for treating peripheral nerve lesions.Since the basal lamina micro-channels in normal nerves are known to play a significant guiding role in the linear growth of regenerated axons, extracellular matrix (ECM) based scaffolds with dimensions resembling the basal lamina micro-channels are expected to provide a promising alternative to autografts for briding nerve gaps. To date, however, little information has been obtained about scaffolds with similar inner microstructures, and the efficacy of such scaffolds in bridging peripheral nerve gaps in vivo has never been examined.In the current study, a collagen-chitosan scaffold (CCH) with longitudinally oriented pore channels and an interconnected porous structure was successfully fabricated by axially freezing and subsequently freeze-drying collagen-chitosan suspensions. The optimum parameters 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-drying technology. The mechanical property and degradationtion of the CCH scaffolds were modified via post-fabrication cross-linking with genipin. The biocompatibility of the scaffold was also fully examined according to the national standard before its usage in vivo. Subsequently, we evaluated its efficacy in bridging a 15 mm long sciatic nerve defect in rats by using a combination of morphological and functional techniques.Part one: Fabrication of CCH scaffolds and determination of the optimum parameters for 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 scaffolds from collagen/chitosan. The inner structure was observed under the scanning electronic microscope. The pore sizes and interval porosity were examined to determine the optimum parameters for the fabrication process. [Results] The novel CCH nerve guidance scaffold with microstructure resembling the basal lamina micro-channels of normal nerves was developed in the present study. The scaffold posesses structural advantages including longitudinally orientated micro-channels (mean channel diameter: 37.41±11.0μm) and extensive interconnected pores between the parallel micro-channels (mean pore size: 20.68±4.61μm). In addition, there was a relatively hermetic outer wall (thickness: 2.45±1.43μm) around the CCH scaffold.The optimum parameters for CCH fabrication are listed as following: blend ratio: 4:1, concentration of acetic acid: 3mg/ml, velocity of freezing: 2×10-5 m/s. The interval porosity was above 90% and the mean pore size was nearly 30μm. Part two: Property modification and biocompatibility evaluation of the CCH scaffolds[Objectives] To modify the mechanical property and degradation of the CCH scaffold, and further evaluate the biocompatibility of the modified CCH scaffolds[Methods] The tensile mechanical properties and degradation of the CCH scaffolds were modified via post-fabrication cross-linking with genipin. The degradation kinetics of the cross-linked scaffolds was evaluated by incubating the scaffolds in PBS with or without lysozyme. The biocompatibility of the modified scaffolds was determined according to the national standard tests.[Results] After cross-linking, the CCH scaffolds withstood an average ultimate stress ranging from 0.098±0.049 MPa to 0.476±0.088 MPa in wet condition (stiffness: 3.938±1.326 MPa), which meets the requirements of surgical procedures. After 8 weeks, the cross-linked scaffolds showed a 17.9±4.2% (without lysozyme) or 20.1±4.6% (with lysozyme) reduction in dry weight relative to the original weights. These indicate that the cross-linked CCH scaffolds possessed the required mechanical stiffness and stability for successful surgical implantation and nerve regeneration.Both an MTT assay and implantation test showed that the fabricated scaffolds displayed almost no evidence of cytotoxicity or tissue toxicity, indicating their suitability for implantation in vivo.Part three: The efficacy of CCH scaffolds in bridging a 15 mm long sciatic nerve defect in rats[Objectives] To investigation the efficacy of CCH scaffolds in bridging a long nerve gap in rats. [Methods] The efficacy of the CCH scaffolds in bridging a 15 mm long sciatic nerve defect in rats was investigated by using a combination of morphological and functional techniques, including immunohistology, transmission electron microscope, electrophysiology, retrograde-labelling and behavioral tests.[Results] The CCH scaffold maintained its microstructure integrity four weeks after implantation. Importantly, penetration and remarkably linear growth of axons within the longitudinal micro-channels of the CCH scaffolds was also observed. Twelve weeks after implantation, the implanted CCH scaffolds were almost completely absorbed, and they were replaced by a large bundle of densely packed regenerated nerve fibers. The in vivo animal study demonstrated that the CCH scaffolds achieved regeneration and functional recovery equivalent to that of an autograft, without exogenous delivery of regenerative agents or cell transplantation. These findings demonstrate that CCH scaffolds may be used as alternatives to nerve autografts for peripheral nerve regeneration.
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