Table 3: Studies on CS-based scaffolds for nerve tissue regeneration.

AuthorsScaffold typeStudy outlineResultsConclusion

Simões et al. 2011 [72]High MW CS membranes crosslinked with GPTMS. (DA: NA; ratio: NA)In vitro: CC in neuroblastoma clone cell culture (N1E-115); fluorescence microscopy for intracellular Ca++ 
In vivo: HT analysis of the subcutaneous tissue in adult Wistar rats. No control group 
Sample (in vivo):
CS membranes promoted cell adhesion and differentiation in vitro  
In vivo: slight to intense chronic inflammation was observed in HT analysis  
Presence of fibrous capsule
Authors concluded that CS membranes demonstrated biocompatibility and potential for use in the regeneration of nerve tissue However, the presence of chronic inflammation and fibrous capsules contradict the conclusion ()

Wei et al. 2011 [73]CS (MW 1800 kDa; DA 6.5%) + SF films (ratios: 50 : 50 or 70 : 30) impregnated with SCIn vitro: CC in SC culture and Schwann cells  
In vivo: implantation of scaffolds in lesions of the sciatic nerve in adult Sprague-Dawley rats; functional and HT evaluation  
Control group: nongrafted rats  
Sample:
In vitro: greater adhesion and proliferation with CS and SF scaffolds when compared to pure CS  
In vivo: Greater functional recovery and tissue regeneration in the groups with CS+SF impregnated with SC
CS+SF impregnated with SC promoted better regeneration in sciatic nerve lesions and lower proliferation of fibrous scar tissue ()

Chen et al. 2011 [74]CS conduits (DA 7.7%; MW 22 kDa) whether or not impregnated with BMMSCIn vivo: implantation of conduits in spinal cord defects in adult Sprague-Dawley rats; functional evaluation and electromyography;  
HT and IHC analyses  
Control group: untreated defects  
Sample:
Control:
Better motor and electromyographic response with CS+BMSC; better macroscopic and HT regeneration of defects filled with scaffolds with SCCS +SC scaffolds were capable of promoting axonal regeneration, remyelination, and functional recovery after sectioning of spinal cord ()

Liao et al. 2012 [75]CS in the form of conduits (DA 15–25%; MW: NA), either with or without SC impregnationIn vivo: implantation of scaffolds in sciatic nerve defects in adult Sprague-Dawley rats; evaluation of repair through magnetic resonance, functional evaluation, and HT analyses. Control group not specified  
Sample:
Nerves implanted with scaffolds impregnated with MSC demonstrated better functional recovery and better magnetic resonance results than acellular scaffoldsCS impregnated with SC promoted regeneration of nerve tissue; magnetic resonance was effective for evaluating regeneration of the sciatic nerve ()

Xue et al. 2012 [76]CS+PLGA (ratio NA) in the form of tubes, whether or not impregnated with SC (DA: NA; MW: NA)In vivo: grafting of conduits on to sciatic nerve defects in adult Beagle dogs; functional and electroneuromyographic evaluations and neuron count; morphometric analysis and HT analysis of associated muscles  
Control groups: nongrafted and autogenous grafted defects 
Sample:
Better functional recovery in CS+PLGA+SC group; remyelination and recovery of nerve diameter; histologically, greater regeneration in the autogenic and CS+PLA+SC groupsCS+PLGA scaffold, either with or without stem cells, favored regeneration of extensive sciatic nerve lesion and showed viability of carrying out a clinical study with this material ()

Xiao et al. 2013 [77]CS+COL (ratio: 1 : 4) in the form of conduits, whether or not combined with RGD peptide 
(DA: NA; MW: NA)
In vivo: implantation of scaffolds in segmental defects of adult Sprague-Dawley rats sciatic nerves; functional evaluation via electroneuromyography, neuron markers, and histology  
Control groups: untreated defects and autogenous grafted defects 
Sample:
CS+COL scaffolds showed better functional recovery than negative control; CS+COL+RGD showed greater management of nerve stimuli than negative control, but lower than the autogenous control. Scaffolds demonstrated greater tissue regeneration than negative control but less than the positive controlCS+COL+RGD was capable of accelerating the regeneration of the sciatic nerve, obtaining satisfactory results in 2 months ()

Biazar and Keshel 2013 [78]CS (medium MW; DA 15–25%) in the form of conduits, whether or not crosslinked with PHBVIn vitro: CC in Schwann cell culture 
In vivo: implantation of scaffolds in sciatic nerve defects of 4–8-week-old Wistar rats; macroscopic and microscopic analyses via HT and IHC  
Control groups: untreated defects and autogenous grafted defects  
Sample:
In vitro: CS+PHBV was found to exhibit greater cell viability and proliferation  
In vivo: CS (crosslinked or not with PHBV), produced regeneration results far superior to the negative control, though inferior to the autogenous control
CS+PHBV demonstrated capacity to regenerate lesions of the sciatic nerve in rats (), having potential for application in tissue engineering and clinical studies

Gu et al. 2014 [30]CS+SF (ratio NA) in the form of conduits impregnated with EMC (DA: NA; MW: NA)In vitro: isolation of Schwann cell EMC derived from rats 
In vivo: implantation in sciatic nerve defects in adult Sprague-Dawley rats; HT and IHC analyses; electrophysiological tests  
Control group: acellular xenogeneic nerve graft  
Sample: not specified
In vivo: better nerve tissue regeneration and density in the CS+SF+EMC group after 12 weeks. The electrophysiological tests got a response in all groups, though to a lesser extent in the CS+SF groupThe CS+SF+EMC scaffold was effective in regenerating nerve tissue ()

Wang et al. 2016 [79]CS conduits (DD 92.3%; MW 250 kDa) or chitooligosaccharides (COS) in silicon conduitsIn vitro: CS biodegradation and CC in Schwann cell culture 
In vivo: implantation of scaffolds in lesions of the sciatic nerves of adult Sprague-Dawley rats; HT and IHC analyses  
Control: saline group 
Sample: not specified
In vitro: COS promoted greater cell proliferation and differentiation  
In vivo: greater expression of nerve cell markers in the chitooligosaccharide groups
Chitooligosaccharides promote nerve cell proliferation and differentiation, stimulating regeneration of nerve tissue (/)

BMMSCs: bone marrow mesenchymal stem cells; CC: cytocompatibility; COL: collagen; COS: chitooligosaccharides; CS: chitosan; DA: degree of acetylation; ECM: extracellular matrix; GPTMS: glycidoxypropyltrimethoxysilane; HT: histological; IHC: immunohistochemical; kDa: kilodaltons; MW: molecular weight; PLGA: polylactic-co-glycolic acid; RGD: cell-adhesive peptide; SC/BMSC: stem cells/bone marrow stem cells; SF: silk fibroin.