Neurogenic Differentiation of Human Dental Pulp Stem Cells on Graphene-Polycaprolactone Hybrid Nanofibers

Stem cells derived from dental tissues—dental stem cells—are favored due to their easy acquisition. Among them, dental pulp stem cells (DPSCs) extracted from the dental pulp have many advantages, such as high proliferation and a highly purified population. Although their ability for neurogenic differentiation has been highlighted and neurogenic differentiation using electrospun nanofibers (NFs) has been performed, graphene-incorporated NFs have never been applied for DPSC neurogenic differentiation. Here, reduced graphene oxide (RGO)-polycaprolactone (PCL) hybrid electrospun NFs were developed and applied for enhanced neurogenesis of DPSCs. First, RGO-PCL NFs were fabricated by electrospinning with incorporation of RGO and alignments, and their chemical and morphological characteristics were evaluated. Furthermore, in vitro NF properties, such as influence on the cellular alignments and cell viability of DPSCs, were also analyzed. The influences of NFs on DPSCs neurogenesis were also analyzed. The results confirmed that an appropriate concentration of RGO promoted better DPSC neurogenesis. Furthermore, the use of random NFs facilitated contiguous junctions of differentiated cells, whereas the use of aligned NFs facilitated an aligned junction of differentiated cells along the direction of NF alignments. Our findings showed that RGO-PCL NFs can be a useful tool for DPSC neurogenesis, which will help regeneration in neurodegenerative and neurodefective diseases.


Introduction
Many efforts have been made to secure stem cell sources [1]. Depending on the cell sources, the stem cells are classified as embryonic stem cells (ESCs), bone-marrow-derived stem cells (BMSCs), and adipose-derived stem cells (ADSCs). However, one critical limitation for acquiring these stem cells is the long and intense nature of the acquisition processes; this necessitates a second surgery coherency than the RFs ( Figure 1D). Furthermore, incorporation of RGO influenced the alignments of the NFs. One percent incorporation of RGO caused a significant decrease in the coherency of RF-1% compared with that of RF-0% and RF-0.1%. In Raman spectroscopy, the NFs with 0.1% and 1% RGO exhibited D (~1450 cm −1 ) and G peaks (~1600 cm −1 ), the representative peaks of RGO ( Figure 1E). The coherencies of AFs were significantly higher than those of RFs. Each of 25 images was used for the analysis. Error bars represent the standard deviation. Same alphabets represent non-significance (p < 0.05). (E) Raman spectroscopy results. The 0.1% and 1% RGO-incorporated NFs exhibited D (~1450 cm −1 ) and G peaks (~1600 cm −1 ), the characteristic peaks of RGO. (C) XRD results. The 0.1% and 1%incorporated NFs exhibited the characteristic peaks of RGO at approximately 23.5°.

Influence of the RGO-PCL NFs on DPSC Behavior
After characterization of the NFs, their influence on DPSCs was assessed. Two days after seeding the DPSCs, the cellular morphologies were observed by ICC (Figure 2A). The DPSCs on the RFs were aligned randomly, whereas those on the AFs were well-aligned following the orientation of the fibers. The alignments of the DPSCs according to the NFs were analyzed using image analysis [25]. Corresponding to the alignments of NFs, the cellular alignments were randomly distributed on the RFs, whereas those on the AFs were narrow ( Figure 2B). The AFs exhibited significantly higher coherency than the RFs, except for AF-1% ( Figure 2C). Based on the results, the cellular alignments were confirmed to be influenced highly by NF alignments. Furthermore, high incorporation of RGO (1%) decreased cellular alignments significantly. In the characterization study, high incorporation of RGO (1%) decreased the alignments of the NFs. Therefore, it was anticipated that high incorporation The coherencies of AFs were significantly higher than those of RFs. Each of 25 images was used for the analysis. Error bars represent the standard deviation. Same alphabets represent non-significance (p < 0.05). (E) Raman spectroscopy results. The 0.1% and 1% RGO-incorporated NFs exhibited D (~1450 cm −1 ) and G peaks (~1600 cm −1 ), the characteristic peaks of RGO. (C) XRD results. The 0.1% and 1%-incorporated NFs exhibited the characteristic peaks of RGO at approximately 23.5 • .

Neurogenic Differentiation
Dental pulp stem cells were placed at a density of 1 × 10 4 cells/cm 2

Characterization of the RGO-PCL NFs
First, the RGO-PCL NFs were characterized. The morphology of RGO-PCL NFs was assessed by SEM ( Figure 1B). The RFs were disordered without any alignment, whereas the AFs were aligned along with the revolution direction of the rotating collector. The alignments of the NFs were quantitatively analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). The box plot revealed that the orientations of the RFs were distributed broadly, whereas those of the AFs were concentrated in a narrow region ( Figure 1C). The AFs showed significantly higher coherency than the RFs ( Figure 1D). Furthermore, incorporation of RGO influenced the alignments of the NFs. One percent incorporation of RGO caused a significant decrease in the coherency of RF-1% compared with that of RF-0% and RF-0.1%. In Raman spectroscopy, the NFs with 0.1% and 1% RGO exhibited D (~1450 cm −1 ) and G peaks (~1600 cm −1 ), the representative peaks of RGO ( Figure 1E).

Influence of the RGO-PCL NFs on DPSC Behavior
After characterization of the NFs, their influence on DPSCs was assessed. Two days after seeding the DPSCs, the cellular morphologies were observed by ICC ( Figure 2A). The DPSCs on the RFs were aligned randomly, whereas those on the AFs were well-aligned following the orientation of the fibers. The alignments of the DPSCs according to the NFs were analyzed using image analysis [25]. Corresponding to the alignments of NFs, the cellular alignments were randomly distributed on the RFs, whereas those on the AFs were narrow ( Figure 2B). The AFs exhibited significantly higher coherency than the RFs, except for AF-1% ( Figure 2C). Based on the results, the cellular alignments were confirmed to be influenced highly by NF alignments. Furthermore, high incorporation of RGO (1%) decreased cellular alignments significantly. In the characterization study, high incorporation of RGO (1%) decreased the alignments of the NFs. Therefore, it was anticipated that high incorporation of RGO (1%) decreased the alignments of NFs, which further resulted in a corresponding decrease in cellular alignments. The ratio of the numbers of alive cells was also assessed ( Figure 2D), and it was found that the NF alignments and RGO concentration did not significantly affect the numbers of alive cells on day 3, except for AF-1%; the ratio of the numbers of cells with AF-1% was significantly decreased. Based on the results, we believe that the NF alignments and high concentration of RGO (1%) negatively affect initial cell proliferation. On the contrary, the NF alignments and RGO concentration appeared to affect the ratio of the numbers of cells on day 7. Cell numbers with AF-0.1% were significantly increased, whereas those with RF-0.1% and RF-1% were significantly decreased. It is well-known that the incorporation of nanomaterials usually has negative effects on cell viability. Concurrently, on day 7, we found that cell viability was decreased on RF-0.1% and RF-1%. On the contrary, the cell viability on AF-1% was similar to that on RF-0%. The cell viability on AF-0.1% was further significantly higher than that on RF-0.1%. Thus, we believe that incorporation of an appropriate percentage of RGO and AFs synergistically increases the cell numbers of DPSCs. of RGO (1%) decreased the alignments of NFs, which further resulted in a corresponding decrease in cellular alignments. The ratio of the numbers of alive cells was also assessed ( Figure 2D), and it was found that the NF alignments and RGO concentration did not significantly affect the numbers of alive cells on day 3, except for AF-1%; the ratio of the numbers of cells with AF-1% was significantly decreased. Based on the results, we believe that the NF alignments and high concentration of RGO (1%) negatively affect initial cell proliferation. On the contrary, the NF alignments and RGO concentration appeared to affect the ratio of the numbers of cells on day 7. Cell numbers with AF-0.1% were significantly increased, whereas those with RF-0.1% and RF-1% were significantly decreased. It is well-known that the incorporation of nanomaterials usually has negative effects on cell viability. Concurrently, on day 7, we found that cell viability was decreased on RF-0.1% and RF-1%. On the contrary, the cell viability on AF-1% was similar to that on RF-0%. The cell viability on AF-0.1% was further significantly higher than that on RF-0.1%. Thus, we believe that incorporation of an appropriate percentage of RGO and AFs synergistically increases the cell numbers of DPSCs.

Neurogenic Differentiation
The effects of RGO and NFs on the neurogenic differentiation were also observed. The DPSCs seeded on the RGO-PCL NFs were subjected to neurogenic differentiation. On the 3rd and 7th day, the DPSCs seeded on NFs with 0.1% and 1% RGO showed apparent changes in their morphologies ( Figure 3A). In contrast to the cells on tissue culture polystyrene (TCPS) (Figure S1), those grown on RGO-PCL NFs severely changed their morphology, representing that the RGO-PCL NFs promote neurogenic differentiation of DPSCs compared to the TCPS. In particular, NFs with 0.1% and 1% RGO resulted in high expression of Tuj-1, the early marker of neurogenesis, and NeuN, the late marker of neurogenesis. In particular, the expression of NeuN was visually compared in Figure S2. As a result, the intensity of NeuN expression seemed to be increased on higher RGO concentration groups or AF groups. Therefore, the neurogenic differentiation of the DPSCs can be influenced by the concentration of nanomaterials and alignment of adjacent ECM. In addition, the cells on the 1% group showed shorter axon-like legs. These results indicated that excessive incorporation of RGO (1%) might result in neurodegeneration of DPSCs. Therefore, incorporation of the appropriate concentration of RGO (0.1%) might promote the neurogenic differentiation of DPSCs. The alignments of the NFs also affected the orientation of the differentiated cells. The alignments of cells on the RFs were randomly distributed, whereas those on the AFs were highly organized. Based on the ICC results, we conducted image analyses to confirm the cellular alignments ( Figure 3B,C). The differentiated cells on the RFs exhibited randomly oriented alignments, whereas those on the AFs exhibited well-oriented alignments ( Figure 3B). The coherency also proved the result: those on the AFs showed significantly better results than those on the RFs ( Figure 3C). The neurite length of each group was assessed ( Figure 3D). The AF-0.1% group on day 7 showed significantly increased neurite length compared with other groups, except for RF-1% on day 7. Consequently, appropriate RGO incorporation and aligned ECM may increase neurite extension. The effects of RGO and NFs on the neurogenic differentiation were also observed. The DPSCs seeded on the RGO-PCL NFs were subjected to neurogenic differentiation. On the 3rd and 7th day, the DPSCs seeded on NFs with 0.1% and 1% RGO showed apparent changes in their morphologies ( Figure 3A). In contrast to the cells on tissue culture polystyrene (TCPS) (Figure S1), those grown on RGO-PCL NFs severely changed their morphology, representing that the RGO-PCL NFs promote neurogenic differentiation of DPSCs compared to the TCPS. In particular, NFs with 0.1% and 1% RGO resulted in high expression of Tuj-1, the early marker of neurogenesis, and NeuN, the late marker of neurogenesis. In particular, the expression of NeuN was visually compared in Figure S2. As a result, the intensity of NeuN expression seemed to be increased on higher RGO concentration groups or AF groups. Therefore, the neurogenic differentiation of the DPSCs can be influenced by the concentration of nanomaterials and alignment of adjacent ECM. In addition, the cells on the 1% group showed shorter axon-like legs. These results indicated that excessive incorporation of RGO (1%) might result in neurodegeneration of DPSCs. Therefore, incorporation of the appropriate concentration of RGO (0.1%) might promote the neurogenic differentiation of DPSCs. The alignments of the NFs also affected the orientation of the differentiated cells. The alignments of cells on the RFs were randomly distributed, whereas those on the AFs were highly organized. Based on the ICC results, we conducted image analyses to confirm the cellular alignments ( Figure 3B,C). The differentiated cells on the RFs exhibited randomly oriented alignments, whereas those on the AFs exhibited well-oriented alignments ( Figure 3B). The coherency also proved the result: those on the AFs showed significantly better results than those on the RFs ( Figure 3C). The neurite length of each group was assessed ( Figure 3D). The AF-0.1% group on day 7 showed significantly increased neurite length compared with other groups, except for RF-1% on day 7. Consequently, appropriate RGO incorporation and aligned ECM may increase neurite extension.  NeuN. The DPSCs on the RFs seemed randomly aligned, whereas those on the AFs seemed well-aligned. (B) Orientation of the alignments of the differentiated cells. Corresponding to the alignments of the RGO-PCL NFs, the cells on the RFs showed broad orientation, whereas those on the AFs showed narrow orientation. (C) Coherency of the alignments of the differentiated cells. The alignments of DPSCs on the AFs were significantly higher than those of the DPSCs on the RFs, except for AF-1%. Furthermore, the cells with high RGO incorporation (1%) showed significantly decreased coherency. Error bars indicate the standard deviation. Same alphabets mean a non-significant difference between samples (p < 0.05). (D) Neurite length of the differentiated cells. The AF-0.1% group showed significantly increased neurite length compared with other groups. Error bars indicate the standard deviation. Same alphabets mean a non-significant difference between samples (p < 0.05). (E) Distribution of neurite numbers on each group. RF groups and AF groups showed apparent changes in distribution of neurite numbers.
The alignments of the NFs seemed to influence not only the cellular alignments, but also cellular morphologies. The cells on RF-0.1% had multipolar structures, whereas those on AF-0.1% had bipolar structures ( Figure 4A). Therefore, it was proposed that the cells on RF-0.1% were connected with the neighbor cells, whereas those on AF-0.1% seemed to be connected along the direction of the fiber alignments. When we compare the ratio of neurite numbers, cells grown on RFs showed apparently higher neurite numbers compared to those grown on AFs ( Figure 3E). It is well-known that nanostructures affect cellular adhesion and alignments [26]. In particular, anisotropic nanopatterns give rise to well-ordered alignments with adjacent cells [27,28]. Cellular alignments influence not only cellular function, but also stem cell differentiation. Therefore, anisotropic nanopatterns have been used frequently in stem cell engineering, especially in neurogenic differentiation. To date, many types of techniques, such as self-assembly [29], lithography [30], and electrospun NFs [31], have been used in neurogenesis. Among them, electrospun NFs have been used widely due to their good biocompatibility and easy fabrication. Furthermore, specific cytokines or nanomaterials can be easily incorporated into the NFs, which results in enhanced functionality of the NFs. To date, many nanomaterials have been reported to affect cellular behaviors [32][33][34]. Among them, GO-and RGO-incorporated NFs have been reported to enhance the viability of neuronal cells and mesenchymal stem cells, respectively. However, the influence of RGO-incorporated NFs on stem cell differentiation, especially in neurogenic differentiation, has not been studied so far. The results of our study showed that the incorporation of an appropriate concentration of RGO (0.1%) increases cell viability and neurogenic differentiation. Furthermore, the alignments of the NFs influence the alignments of the DPSCs as well as the linkage of differentiated neurites. Based on these results, we suggest that the use of RF-0.1% is suitable for the generation of multidirectional neural networks, whereas the use of AF-0.1% is suitable for the generation of unidirectional neural networks ( Figure 4B).

Conclusions
In this study, RGO-PCL NFs were fabricated with different alignments and RGO concentrations and applied to the neurogenesis of DPSCs. The presence of RGO was confirmed by Raman spectroscopy and XRD. The alignments of the RGO-PCL NFs directly affected the alignments of the DPSCs: the DPSCs followed the alignments of the RGO-PCL NFs. Furthermore, the combination of the alignments and RGO increased the cell viability. In the neurogenic differentiation study, incorporation of an appropriate concentration of RGO (0.1%) enhanced the neurogenesis of the DPSCs. Furthermore, the alignments of NFs seemed to correlatively affect the tissue morphologies. In conclusion, our findings indicated that the application of RGO-PCL NFs with an appropriate concentration of RGO would open the gates for the use of DPSCs in neurological therapy and neurogenesis.
Supplementary Materials: The following are available online at: XXX(please add). Figure S1. Neurogenic differentiation of DPSCs on TCPS. Compared to the NF groups, the TCPS groups showed less differentiated cell shapes and the expression of neurogenic markers. Figure S2. NeuN expression of each condition. The more the RGO concentration, the higher the NeuN expression. Furthermore, the expression of NeuN was higher on the AF groups compared to the RF groups.

Conclusions
In this study, RGO-PCL NFs were fabricated with different alignments and RGO concentrations and applied to the neurogenesis of DPSCs. The presence of RGO was confirmed by Raman spectroscopy and XRD. The alignments of the RGO-PCL NFs directly affected the alignments of the DPSCs: the DPSCs followed the alignments of the RGO-PCL NFs. Furthermore, the combination of the alignments and RGO increased the cell viability. In the neurogenic differentiation study, incorporation of an appropriate concentration of RGO (0.1%) enhanced the neurogenesis of the DPSCs. Furthermore, the alignments of NFs seemed to correlatively affect the tissue morphologies. In conclusion, our findings indicated that the application of RGO-PCL NFs with an appropriate concentration of RGO would open the gates for the use of DPSCs in neurological therapy and neurogenesis.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/8/7/554/s1. Figure S1. Neurogenic differentiation of DPSCs on TCPS. Compared to the NF groups, the TCPS groups showed less differentiated cell shapes and the expression of neurogenic markers, Figure S2. NeuN expression of each condition. The more the RGO concentration, the higher the NeuN expression. Furthermore, the expression of NeuN was higher on the AF groups compared to the RF groups.

Conflicts of Interest:
The authors declare no conflict of interest.