| Background:Peripheral nerve injury remains a high incidence of life-long disability in the world wide. Among various types of peripheral nerve injuries, transection injuries where the nerve trunk is completely interrupted, especially those resulting in large neural gaps, may have a devastating impact on patients’ quality of life, and in these cases reconstructive surgery is required as a therapeutic management to achieve nerve regeneration and function restoration. In consequence, peripheral nerve repair represents a unique challenge and opportunity to clinical and translational neurosciences. After peripheral nerves are transected, a series of molecular and cellular events, collectively called Wallerian degeneration, is triggered throughout the distal stump of transected nerves and within a small zone distal to the proximal stump, resulting in the disintegration of axoplasmic microtubules and neurofilaments Within24h most axons along the distal stump of transected nerves are reduced to granular and amorphous debris; by48h the myelin sheath has begun to be transformed toward the short segment. Then macrophages and monocytes migrate into the degenerating nerve stumps to remove myelin and axon debris, while Schwann cells proliferate to form longitudinal cell columns, known as Bands of Bungner. Under the influences of neurotrophic factors and extracellular matrix molecules produced by Schwann cells, the proximal portion of transected nerves sprouts new daughter axons to generate a "regenerating unit" that is surrounded by a common basal lamina. New axonal sprouts usually emanate from the nodes of Ranvier, and undergo remyelination by Schwann cells. Functional reinnervation requires that the regenerating axons elongate under the mediation of growth cones until they reach their synaptic target, and in humans, axon regeneration occurs at a rate of about2-5mm/day; thus significant injuries may take many months to heal. To aid the repair of peripheral nerve injuries, clinical intervention has been attempted for several hundred years since as early as the17th century, when Ferrara first reported a suture technique for repairing a severed nerve. After the significant progress made in the19th and20th centuries, a wide range of surgical techniques has been put into use for the management of peripheral nerve injuries. Although there have been great advancements in the surgical repair of peripheral nerve injuries, autologous nerve grafting remains the gold standard technique to which other treatments are compared. Unfortunately, autologous nerve grafting is limited by the inherent drawbacks, such as limited availability of donor nerves, the need for a second surgery to obtain the donor nerve, donor site morbidity and secondary deformities, as well as mismatch between the injured nerve and the donor nerve. In addition, clinically functional recovery rates typically approach only80%for nerve injuries treated by autologous nerve grafts. It goes without saying that seeking promising alternatives to supplement or even substitute autologous nerve grafts constitutes a major challenge to peripheral nerve repair. In the last few decades, different types of biological or artificial grafts have been developed and investigated as compared with autologous nerve grafts in terms of the outcomes of nerve regeneration and functional recovery. More importantly, tissue engineering, an emerging multidisciplinary field, has grown at a significant rate in recent years. This offers great opportunities to neuroscientists and surgeons who have been collaborating to develop tissue engineered nerve grafts. Just like most of tissue engineered products, tissue engineered nerve grafts are typically composed of a physical scaffold with the introduction of support cells and/or growth factors or other biomolecular components.Attention has recently focused on the biological behaviors of Schwann cells, the principal glia of the peripheral nervous system. During development, Schwann cells migrate ahead of and along axonal tracts, ensheath several axons and eventually segregate to wrap around a single axonal segment. In response to peripheral nervous system injuries, Schwann cells proliferate, produce growth factors, remove debris, and lay down longitudinal tracks that provide guidance and support for regrowing axons. Additionally, aligned live Schwann cells and isolated Schwann cells topography have the capacity to direct axon growth in vitro. Because of their instrumental role in aiding peripheral nervous system injury repair, Schwann cells have received attention as an important cell type with therapeutic applications. It has been recently suggested that therapies to promote nerve repair, notably nerve guidance channels, will require transplantation of supportive cells such as Schwann cells.Schwann cells in spinal nerves originate from the neural crest, although the origin of cells in spinal roots is more complex. The end point of Schwann cell development is the formation of myelinating and non-myelinating cells that ensheath large and small diameter axons, respectively, throughout the PNS. Schwann cell formation is preceded by the generation of two other cell types:SCPs, which are the glial cells of embryonic day (E)14-15rat nerves (mouse E12-13), and immature Schwann cells, which are generated from the SCPs from E15to El7(mouse E13-15). The latter are the glial cells found in rat nerves from E17-18to about the time of birth. The postnatal fate of immature Schwann cells is determined by which axons they randomly associate with, with myelination being selectively activated in those cells that happen to envelop single large diameter axons. These events can be viewed as three main transitions, that is, the transition from migrating neural crest stem cells to SCPs, from SCPs to immature Schwann cells and, lastly, the divergence of this population to form the two mature Schwann cell types. These events are strikingly dependent on survival factors, mitogens and differentiation signals from the axons with which SCPs and Schwann cells continuously associate.Accumulating evidence suggests that cells interact with their environment not only biochemically but also physically. Although the biochemical interactions have been widely investigated in different cells systems, our understanding of how physical interactions affect cell behaviors is still in its infancy. The first patent that described the operation of electrospinning appeared in1934, when Formalas disclosed and apparatus for producing polymer filaments by taking advantage of the electrostatic repulsions between surface charges. Up until1993, this technique had been known as electrostatic spinning, and there were only a few publications dealing with its use in the fabrication of thin fibers. In the early1990s, several research groups revived interest in this technique by demonstrating the fabrication of thin fibers from a broad range of organic polymers. At this time the term electrospinning was coined and is now widely used in the literature. These timely demonstrations triggered a lot of experimental and theoretical studies related to electrospinning. It is notable that the number of publications in this field has been increasing exponentially in the past few years, on account of the remarkable simplicity, versatility, and potential uses of this technique. With the possibility of generating fibers in the nanoscale and the resulting scaffolds with organized architecture, electrospun fibrous meshes may mimic the extracellular matrix. RSC96cell line is naturally transformed from primary cell culture of rat Schwann cells. It could be served as an ideal class of Schwann cell analogues. Our study is mainly composed of two components. One is to compare the biological behaviors of RSC96cells with the ones of primary schwann cells, the other is to study the influence of electrospun fibers on the biologcial behaviors of RSC96cells based on the previous results.Objective1. To compare the biological behaviors of RSC96cells with primary culture of schwann cells.2. To fabricate different topography of electrospun fibers and varying diameter of aligned elecntrospun fibers.3. To study the influence of different topography of electrospun fibers on the biologcial behaviors of RSC96cells.4. To explore the influence of varying diameter of aligned electrospun fibers on the biologcial behaviors of RSC96cells. Methods1. The bilateral ischial nerves and brachial plexus nerves were taken from SD rats(1to3days). These were washed by D-Hanks solution several times, stripped the epineurium carefully under a dissecting microscope, and washed three times by D-Hanks solution. We cut the nerve tissue into small blocks in size of1mm3and isolated the tissue blocks with the dual-enzyme digestion. Cytarabine was used to inhibit fibroblast growth. Addition of forsoklin and pituitary extract bovine was to optional stimulate the proliferation of schwann cells and the pass was to further purify Schwann cells. Morphology was observed under light microscopy. Culture medium containing10%FBS,100U/mL penicillin,100μg/mL streptomycin, DMEM/F12was used to culture the RSC96cells.Then the culture dishes were placed in the CO2incubator at37℃. The medium was changed every other day. After the cell fusion rate got80%, the cells were digested and passed by0.25%trypsin with0.02%EDTA. The morphology was also observed under light microscopy. The two types of cells were digested by Trypsin and collected, Procedures were performed as follows:1) total RNA were extracted by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with (3-actin house-keeping gene expression.2) Proteins of RSC96cells were extracted from two groups by following the protocols of Total Cellular Protein Isolation Kit. Bradford Protein Assay Kit was used to detect the protein level. Equal quantities of total proteins from samples were mixed with equal volume of2×SDS gel loading buffer. After boiling at100℃for10min, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocked by primary antibodies of anti-NGF, anti-Laminin and anti-β-actin for2hours, membranes were rinsed3times and incubated with horseradish peroxidase-conjugated secondary antibodies. Films were digitally imaged using Image J software. 2. Firstly, In order to obtain thinner aligned PCL fibers, a solution of10wt%of PCL dissolved by Hexafluoroisopropanol was dispensed at a flow rate of4ml/h and electrospun under a voltage of10kv. The PCL fibers were collected on a grounded target rotating at-2800rpm. For scaffolds comprising randomly oriented PCL fibers,10wt%of PCL dissolved by Hexafluoroisopropanol was dispensed at a flow rate of3ml/h under a voltage of10kv and collected on rotating a target(-200rpm). The polymer supply-to-target distance were set15cm and13cm respectively. Secondly, In order to obtain thicker aligned PCL fibers, a solution of15wt%of PCL dissolved by a mixture of dichloromethane and methanol was dispensed at a flow rate of3ml/h under a voltage of10kv. The polymer supply-to-target distance was set10cm.3. After the conventional recovery of RSC96cells, proliferation was done for2-3generations. Group A (experimental):RSC96cells were seeded on the parallel electrospinning fibers. Group B (experimental):RSC96cells were seeded on the randomly electrospinning fibers. Group C (control):RSC96cells were seeded on the polycaprolactone film. Each group containing the equal amount of cells was then placed in the incubator under37℃,5%CO2for3days (medium was changed every other day). Procedures were performed as follows:1) Proteins of RSC96cells were extracted from three groups by following the protocols of Total Cellular Protein Isolation Kit. Bradford Protein Assay Kit was used to detect the protein level. Equal quantities of total proteins from samples were mixed with equal volume of2×SDS gel loading buffer. After boiling at100℃for10min, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocked by primary antibodies of anti-NGF, anti-Laminin and anti-β-actin for2hours, membranes were rinsed3times and incubated with horseradish peroxidase-conjugated secondary antibodies. Films were digitally imaged using Image J software.2) RNA of RSC96cells were extracted from three groups by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with β-actin house-keeping gene expression.4. After the conventional recovery of RSC96cells, proliferation was done for2-3generations. Group A (control):RSC96were seeded on the plain cell culture plate directly. Group B (experimental):Cells were seeded on the thinner aligned electrospun fibers. Group C (experimental):cells were seeded on the thicker aligned electro spun fibers. Each group containing the equal amount of cells was then placed in the incubator under37℃,5%CCO2for3days (medium was changed every other day). The total RNA of cells were extracted by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with β-actin house-keeping gene expression.Results1. The shape of the Schwann cells were bipolar spindle with bright edge, oval or oblong nucleis, protuberances around cell biopolar with multi-length ranges, cells growed side by side and arrayed in radial or nests under the light microscope, while RSC96cells were typically bipolar spindle or polygon shape and mostly extended long processes, and the shape was much similar with primary culture of Schwann cells. The results of qPCR and western blot analysis showed that RSC96cells cultured in vitro can also express NGF and Laminin similar with primary culture Schwann cells.2. Different topography of electrospun fibers were fabricated by different types of collectors and varying diameter of aligned elecntrospun fibers were created by the different intrinsic properties of the solution and distance between spinneret and collector.3. To detect mRNA expression by qPCR and protein expression by western blot: According to different culture conditions, cultures of RSC96cells were divided into three groups, group A(aligned), group B(randomly) and group C(film), and all the three groups were cultured for three days. NGF mRNA expression of RSC96was lower in group A, compared to group B and C (P<0.05). There was statistical differences between group B and group C (P<0.05). The laminin mRNA was expressed in all the groups, an decrease from group A to group C (P<0.05). What’s more, Laminin and NGF protein expressed in all groups. NGF protein expression had statistical differences in all the groups(P<0.05). The laminin protein expression decreased from group A to group C (P<0.05).4. To detect gene expression by qPCR:According to different culture conditions, cultured RSC96were divided into three groups, group A(control), group B(thinner diameter of aligned electrospun fibers) and group C(thicker diameter of aligned electrospun fibers), and all three groups were cultured for three days. The NGF gene expression of RSC96cells was higher in group A, compared to group B and C (P<0.05). There was no statistical difference between group B and group C (P>0.05). The Laminin gene expression of RSC96cells was lower in A, compared to group B and C (P<0.05). There was some statistical difference between group B and group C.Conclusion1. The growth pattern of RSC96cells cultured in vitro was much similar with the primary culture of Schwann cells, the cells also expressed neurotrophic factors and laminin and were an ideal substitute for the primary culture of Schwann cells, which avoided the long cycle and complicated primary schwann cells cultivation and long-term poor schwann cell function, and so on.2. Different topography of electrospun fibers and varying diameter of aligned elecntrospun fibers were fabricated dependent on a number of processing parameters.3. The different biological behaviours of RSC96cells were induced by aligned electrospun fibers and RSC96cells may be directed towards the more maturation state by topographic cues from electrospun fibers.4. Biologcial behaviours of RSC96cells were evaluated on different diameter of aligned electrospun fibers. There is some statistical difference in the expression of Laminin cDNA between the two groups. |
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