| Spinal cord injury (SCI) often leads to permanent paralysis and loss of sensationbelow the site of injury because of the inability of damaged axons to regeneration in theadult central never system. Functional deficits following SCI result from damage to orseverance of axons, loss of neurons and glia, and demyelination. The axon growth is thekey to SCI repair. Olfactory ensheathing cells (OECs) in adults remains the ability toinduce axonal growth, making lifelong regeneration ability of olfactory nerve. Therefore,OECs have become a popular candidate for the transplant-mediated repair of the damagedSCI. However, the inhibited molecules formed after SCI will result in the formation ofchemical barrier which inhibited the axonal regeneration. Additionally, glial scar formedin the injured site due to the immigration of activating glial cell, and the nerve defectsbecome the physical barrier suppress the axonal regeneration. In this negative environment,the directional migration and orderly distribution of transplanted cells is impossible, andthe role of transplanted cells in supporting and guiding the axons regeneration is alsodifficult to play, these problems is the main reason that cell transplantation in SCItreatment is unsuccessful. Thus, the directional distribution of transplanted cells throughtissue engineering bionic scaffolds can overcome the adverse effects of the inhibitedmolecules in injury site, scar tissue, and nerve defects, providing support and guidance fornerve axon regeneration, which is not only the important direction for healing SCI, but alsoa hot topic in the research of SCI repair.The presupposition of tissue engineering approaches for management of SCI is to finda biological material with excellent performance and construct a bionic structure. Silkfibroin (SF), a kind of natural protein produced by silkworm, is well known for itsapplication in biomedical areas due to its excellent properties such good biocompatibility,outstanding mechanical properties, controllable degradation, biodegradability, and versatileprocessability. The key to construct biomimetic structure is the control on the nanostructures of scaffolds. Recently, the self-assembly mechanism and regulation onstructure of SF has been further clarified. Based on these progresses, our group controls SFto assemble into homogeneous nanofibrils which will significantly affect the diameter ofelectrospun SF nanofiber, making the fiber diameter control available. Therefore, SF is apromising candidate as a matrix material for tissue engineering scaffolds.To explore the feasibility of SF scaffold as a carrier to directional distributestransplanted OECs, the project plans to study the following aspects:（1）Establishing OECsculture system, inverted phase contrast microscope is employed to observe morphologicalchanges of cell within its cycle; On this basis, the cell station is used to monitor the OECs’migration dynamically, reveal the regularities of OECs’ behavior under the same cultureconditions.（2）Based on the above recognitions, further study is designed to clarify theeffect of culture conditions (such as with or without serum; different proliferation agents:Forskolin, bFGF) on the morphology and migration behavior of cell, and analyze theregulatory mechanisms on OECs migration.（3）Based on bombyx mori silk fibroin (BSF)and tussah silk fibroin (TSF), we prepare SF nanofiber by using electrospinning technique,and culture OECs on these nanofibrous materials. This experiment is designed to study theeffect of chemical composition of SF on the morphology, proliferation, survive, andphenotype of OECs, and clearly define the superior biological performance of TSF insupporting the growth of OECs.（4）Scanning electron microscope, optical microscope,immunostaining, cell station are employed to research the effect of silk fibroin types (TSFand BSF), fiber diameters (400nm and1200nm), and fiber orientations (parallel anddisordered arrangement) of nanofibrous scaffold on the ultrastructure, morphology anddistribution, cell phenotype and cytoskeleton, cell migration behavior (migratory path,migration speed) of OECs during culture in vitro, clearly define the role of topologicalstructure of silk fibroin scaffolds in regulating cell behavior and its regulation mechanism.（5）We employed RT-PCR to detect the gene expression, such as MBP, NGF, GDNF,BDNF, NCAM, and ELISA to detect related protein expression of OECs cultured on silkfibroin scaffolds. Through the investigation, further reveal the role of scaffold in regulatingcell behavior in a molecule and protein level.（6）To explore the effect of SF nanofibrousscaffold on the directional arrangement of OECs, clarify the biological role of SF scaffoldsin targeted distributing OECs, analyze the technique and its development direction inconstruct bionic scaffold, providing theoretical basis and experimental evidence for further scaffold design and its application. Through the above research the conclusions are asfollows:（1）This study employed cell station to continuous and dynamic observe themorphological changes of OECs during their migration, in this situation, the morphologicaldiversity is the result of OECs’ migration, which is attributed to the different functionalstates. The morphological diversity of OECs cultured in vitro is the result of cell migration,which shows the different function states of OECs. It is difficult to find a reasonableexplain for the morphology difference of OECs observed by light microscope and electronmicroscope, in which cell is observed in a static and fixed state, making it difficult todistinguish and correct understanding of cell morphology diversity. The clearly define therelationship between the morphology of OECs cultured in different conditions and cellmigration behavior is helpful to understanding the regulation mechanism of OECsmigration.（2）The strong deformability, active migration ability, and unique role in nerverepair of OECs are attributed to their morphological plasticity. The morphological changes,the formation of pseudopodia, and the migration ability of OECs are regulated by thesignaling molecules in the culture conditions, suggesting the response of OECs tomicroenvironment is sensitive. However, the microenvironment generated by cultureconditions has limited impact on cell migration rate, migration path, and migrationdirection, indicating that orderly distribution of signaling molecules or construction ofnanostructure mimicking ECM is the effective method to guide and regulate cell migrationunder designed direction.（3） SF (especially TSF) nanofiber scaffolds possess good biocompability.Electrospun BSF and TSF fibers are promising growth matrices for OECs due to highsurface to volume ratio and excellent hydrophilicity. The fiber diameter and orientationhave a significant effect on cell adhesion, proliferation, and arrangement. In addition, TSFfibers show more excellent biological properties might be perpetuated by the presence ofthe Arg-Gly-Asp (RGD) sequence and the hydrophilicity.（4）Regulation of the biological behavior of OECs through fiber diameter,orientation, and topological structure of SF nanofibers. The different cell responses tomicro and nano fibers of SF and TSF demonstrated the significant effect of fiber diameteron cell biological behavior. It is found that the fibrous scaffold with small fiber diameter (400nm) is favourable for cell adhesion, proliferation, growth, migration, and expressionof the gene protein. The aligned nanofiber scaffolds is more conducive to cell migration toform targeted arranged cell morphology. OECs on aligned SF nanofibers showed orderedand parallel arrangement, and particularly slender projections, and formed string-structureon a single nanofiber due to the ecphyma connection between cells. The process of findingmigration direction and way of cell ecphyma is disturbed uneasily by other fiber and cell,which keep the migration direction very well during the migration process, resulting in thetargeted and ordered distribution of OECs. At present, the research team has the ability tocontrol the diameter of SF nanofibers down to50nm, providing a platform for furtherinvestigate the effects of fiber with more smaller diameter on cell behavior, thuscontributing to the repair of damaged nerve functions.（5）The control of OECs’ migration and directional distribution by SF nanofiber isclose related to cell cytoskeleton. The biological responses of OECs to fiber diameter, fiberorientation, and3D topology of BSF and TSF nanofibrous scaffolds are different. OECsgrow very well (tubulin in the correct order) on SF nanofiber according to cellcytoskeletonand its arrangement, indicating the ability of cell to respond environment. Thefibrous scaffolds control cell morphology (such as the extension and retraction of theprotrusions, soma deformation), promote cell migration, induce the migration direction,and then control the arrangement by regulating the formation and structure of the cellcytoskeleton. TSF nanofibers exhibited more strong guidance role in regulating cellmigration behavior.（6）TSF nanofiber promote the expression of functional gene and protein of OECs.TSF nanofibers not only have no negative influence upon the expression and secretion ofthese factors of OECs, but promote some protein expression. The reason as follow: TSFnanofibers provide a environment mimicking ECM structure that is conducive to proteinsynthesis, secretion of extracellular matrix components, cell adhesion molecules andsoluble chemokines, making cell migration and aggregation on scaffolds, and formation ofmore communication like between cells, which is important for tissue regeneration andrepair. Meanwhile, the secretion of extracellular matrix by cells induced by TSF nanofiberscaffold would further promote cell growth and proliferation.（7）Directional distribution of OECs support the growth and development of neuron,guide axon extension of neuron. It is the key for OECs as substrates cell to support and guide the growth of neurons and their axons to promote the growth and development ofolfactory nerve and repair of damaged nerve in vivo. The directional arrangement of OECsby TSF nanofibrous scaffolds constructed in vitro is the core values of this research torepair injured neuron and its axons. The results showed that TSF nanofiber scaffold notonly support and promote OECs adhesion, growth, proliferation and gene expression andprotein secretion, but also guide cell orientation align the nanofibrous direction, theorientation distribution OECs support the growth of neurons, and promote the growth ofneurons and their axons.Natural extracellular matrix composed of collagen fiber with diameter size in therange of50-500nm, thus control the diameter and orientation of nanofibers precisely toconstruct scaffolds with maximum bionic ECM structure is an important part of futureresearch. In addition, based on the research breakthroughs of silk fibroin self-assemblymechanism, it is another important direction to form SF hydrogel with controllable gelbehavior and bionic structure. Silk fibroin nanofibers containing drug or related proteinfactor given the biological activity scaffold, in combination of OECs as transplanted cellwould be one of the feasible strategy to repair SCI, and achieve functional recovery. |
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