Effect of Graphene on Differentiation and Mineralization of Dental Pulp

May 13, 2019 - ... X-ray (EDX) analyses indicated that the DPSCs differentiated along odontogenic lineages only on the nano- and microelectrospun scaf...
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Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2435−2443

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Effect of Graphene on Differentiation and Mineralization of Dental Pulp Stem Cells in Poly(4-vinylpyridine) Matrix in Vitro Linxi Zhang,†,‡,# Kuan-Che Feng,†,# Yingjie Yu,†,∥ Ya-Chen Chuang,†,‡ Chung-Chueh Chang,‡ Sahith Vadada,† Rushikesh Patel,† Vedant Singh,† Marcia Simon,§ and Miriam Rafailovich*,† †

Department of Materials Science and Chemical Engineering, Stony Brook University, Stony Brook, New York 11794, United States ThINC Facility, Advanced Energy Center, Stony Brook University, Stony Brook, New York 11794, United States § Department of Oral Biology and Pathology, University School of Dental Medicine, Stony Brook University, Stony Brook, New York 11794, United States ∥ Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States

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ABSTRACT: We have investigated the influence of graphene nanoplatelet scaffolds for dental pulp cells (DPSCs) made from poly(4-vinylpyridine) (P4VP) either via spin-casting flat films or electrospinning nano- and microscale fibers. We found that graphene predominated over other factors in promoting differentiation of DPSCs. In the absence of graphene, real-time-polymerase chain reaction (RT-PCR) and energy dispersive X-ray (EDX) analyses indicated that the DPSCs differentiated along odontogenic lineages only on the nano- and microelectrospun scaffolds. Closer scanning electron microscopy (SEM) imaging revealed formation of banded collagen structures, which nucleated on the electrospun fibers in the absence of graphene. Biomineral deposition was templated on these fibers, with mineral to protein ratios similar to dentin. In the microfibers, the graphene was completely encapsulated and appeared to hinder biomineralization. Previously minimal biomineralization and banded collagen were observed on flat spun cast substrates. Addition of graphene appeared to induce nucleation of banded collagen fibers and template biomineral deposition. Addition of graphene did not affect the outcome of the DPSCs cultured on the nanofibers, which biomineralized regardless of graphene inclusion. Based on these results, we hypothesize that direct contact with graphene is the primary factor determining differentiation of the DPSCs. On the flat surface and nanoscale electrospun fibers, the graphene protrudes from the sample enabling direct contact with the extracellular matrix (ECM) and cells, while on the microfibers, the graphene is fully encapsulated within the matrix. TUNA imaging with scanning force microscopy showed enhanced conductivity on fibers with encapsulated graphene, which we hypothesize may change the conformation of adsorbed ECM proteins, affecting DPSCs differentiation. KEYWORDS: P4VP, graphene, biomineralization, collagen, dentin

1. INTRODUCTION Graphene and graphene-based materials have been developed and widely used in tissue regeneration engineering, due to their excellent physical properties.1−4 Recent comprehensive reviews by Kenry et al. and Halim et al. have shown the exciting potential of graphene and graphene nanocomposites to influence multilineage differentiation of stem cells in vitro.5,6 Graphene and graphene-oxide are commonly used in most studies as substrates, which have direct contact with cells. Lee et al. have shown that the strong monovalent binding ability of graphene can assist the penetration of an osteogenic mediator, and hence mesenchymal stem cells’ (MSC) differentiation is accelerated on graphene sheets.7 Nayak et al. reported similar results, showing graphene can accelerate the differentiation of MSC into bone cells in vitro.8 Furthermore, graphene has been compounded with polymer to form composite materials for © 2019 American Chemical Society

tissue engineering studies. Many studies have shown the enhanced differentiation and proliferation of multiple cell types on graphene loaded polymer substrates.9−13 As we know, graphene is solely composed of carbon atoms and the unique structure of which allows graphene to exhibit unusual chemical, physical, and electrical properties. On the other hand, the surface morphology is highly affected by the aggregations of graphene. Since cells are capable of responding to various substrate conditions, including surface chemistry, surface mechanics, dielectrical conductivity, what remains unclear is the mechanism behind the cell−graphene interaction. Received: February 24, 2019 Accepted: May 13, 2019 Published: May 13, 2019 2435

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residual solvent, sterilize the substrates, and ensure attachment of the fibers to the films. 2.4. Characterization of P4VP Scaffolds. For determining the morphology, the electrospun fiber samples were sputter-coated with 4 nm gold/palladium (70/30) (EM ACE600, Leica) and imaged with scanning electron microscopy (Crossbeam340, Zeiss). Optical microscopy and transmission electron microscopy (Jeol 1400) were used to determine the distribution of graphene in the P4VP polymer fibers. P4VP/graphene thin films were scanned by atomic force microscopy (DimensionIcon, Bruker) to visualize the distribution of graphene. 2.5. In Vitro Cell Culture. A strain, AV3, of dental pulp stem cells (DPSCs) was isolated at the Stony Brook School of Dental Medicine, from discarded third molar teeth under IRB exemption for deidentified surgical waste (CORIHS Project ID 20076778, approval no. 00000125).23 The DPSCs were cultured in MEM Alpha (GIBCO, Invitrogen) which was supplemented with 10% fetal bovine serum (HyClone), 10 mM β-glycerophosphate, 200 μM L-ascorbic acid 2phosphate, and 100 units/mL penicillin, where the supplements were obtained from Sigma-Aldrich. The glass coverslips coated with the P4VP thin films or film/fiber scaffolds were placed in 24-well tissue culture plates, where they were plated with the DPSCs at a density of 5000 cells/cm2 and incubated for 28 days at 37 °C under 5% CO2 in a humidified environment. 2.6. Cell Staining and Confocal Laser Scanning Microscopy. The morphology and density of the DPSCs were imaged with confocal microscopy (Leica TCS SP2) after 1, 7, and 28 days in culture. Preparation for imaging was performed by washing the DPSCs twice with calcium and magnesium-free phosphate buffer saline (PBS) and then fixed in 3.7% (w/v) formaldehyde, permeabilized with 0.4% Triton X-100 solution in PBS, stained with Alexa Fluor 488 (Molecular Probes, Eugene, OR) to visualize the actin filaments and 4′,6-diamidino-2-phenylindole (DAPI, SigmaAldrich) to image the nuclei. Focal adhesion contacts were imaged via immunohistochemical staining. A primary monoclonal antibody (Sigma Chemical Co., St. Louis, MO) against human vinculin was first added, and then the samples were incubated with a green fluorescent conjugated goat antimouse secondary antibody (Sigma Chemical Co., St. Louis, MO) for 1 h at room temperature. 2.7. Scanning Electron-EDS and Raman Microscopy. The mineral disposition was imaged using scanning electron microscopy (backscattered mode) with energy dispersive X-ray (EDX) detection (Crossbeam340, Zeiss). DPSCs cultured for 28 days were gently washed with DI water at least twice to remove salt deposits and then air-dried for 24 h. Prior to SEM imaging, samples were inserted into a vacuum chamber (EM ACE600, Leica) where a 4 nm gold/palladium (70/30) layer was deposited via sputter coating. Raman spectra of the samples were collected using a 514 nm laser with an inVia, Renishaw spectrometer. 2.8. Real-Time-Polymerase Chain Reaction (qRT-PCR). The mRNA expressions of alkaline phosphatase (ALP) and osteocalcin (OCN) at 14 and 28 days were evaluated by qRT-PCR. Cells were rinsed twice with PBS after which RNA was extracted using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. To prepare cDNA, 2 μg of RNA from each sample was transcribed using Superscript II Reverse Transcriptase with random primers (Invitrogen). To quantify the amount of cDNA, qRT-PCR was then carried out using the QuantiTect SYBR Green PCR kit (QIAGEN) with primers against 18s, F: 5′-GTAACCCGTTGAACCCCATT-3′, R: 3′-CCATCCAATCGGTAGTAGCG-5′, ALP F: 5′-AATGCTGGAGCCACAAAC-3′, R: 3′-GCTTCCTTAGTCCCATTTC-5′ and OCN, F: 5′-ATGAGAGCCCTCACACTCCTCG-3′, R: 3′GTCAGCCAACTCGTCACAGTCC-5′ and analyzed using the MJ Research Opticon System (MJ Research, Waltham, MA) at the DNA sequencing facility at Stony Brook University. 2.9. Statistical Analysis. A minimum of three replicate tests were obtained for each data point in order to determine the mean and standard deviation values quoted. The p values were then calculated using the Prism 6 data analysis package. The p values were

Proteins derived from media and secreted by cells can absorb to surfaces influencing cell adhesion and function. Our previous study demonstrates a correlation between mineralization and the conformation of adsorbed extracellular matrix (ECM) proteins.14 Therefore, it is important to understand the influence of graphene on these proteins.5,15 Since these proteins are complex charged structures, and are diamagnetic, they can be impacted by both the morphologic as well as the electromagnetic properties of graphene. Poly(4-vinylpyridine) (P4VP) is a transparent polymer, which is glassy at ambient temperatures (glass transition, Tg = 137 °C), and which has been shown to be equivalent to tissue culture plastic for cell culture, supporting adhesion, and proliferation of multiple cells types. In addition, P4VP does not require surface treatments and has better uniformity than tissue culture plastic of surface properties.16 The results indicated that differentiation was determined by the ability of the collagen fibrils formed by the cells to cluster together and form the classical banded collagen fibers. Spuncast flat films and electrospun fiber scaffolds were compared, and banded collagen seemed to form only on the electrospun fibrous substrates. This system therefore provides an ideal platform for studying the additional effect of graphene. Graphene can be dispersed into P4VP matrixes either via spin-casting or electrospinning, and hence the identical substrates can be formed where the only additional variable would be the presence of graphene.17−20 We chose to use DPSCs, which have been shown to have the ability to differentiate into many cell types, including osteoblasts, odontoblasts, neural cells, etc.19−28 DPSCs were cultured on the graphene-loaded scaffolds and their response compared with that of cells on the identical neat polymer surfaces.

2. MATERIALS AND METHODS 2.1. Materials. The polymer, poly(4-vinylpyridine), P4VP, (average Mw ∼160 000) and the solvent, N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich, while ethyl alcohol (200 proof, A.C.S/USP) was purchased from Pharmco-Aaper, and the graphene nanoplatelets were purchased from XG Sciences (Lansing, MI). 2.2. Fabrication of P4VP and P4VP/Graphene Thin Films. Solutions for spin-casting P4VP thin films were prepared by dissolving 7 mg/mL of P4VP in DMF. Solutions for spin-casting P4VP/ graphene thin films were prepared by suspending 150 ng/mL graphene into DMF followed by stirring motion at constant speed overnight, and then P4VP is dissolved in graphene/DMF solution at 7 mg/mL concentration. Both types of thin film were spin coated with 2500 rpm for 30 s. The thickness is around 40 nm, which is measured by ellipsometry. The samples were annealed in a vacuum oven at 150 °C, 20 mTorr, for 24 h. 2.3. Electrospinning P4VP and P4VP/Graphene Fibers. Solutions for electrospinning P4VP fiber scaffolds were prepared as described previously.14 Solutions for electrospinning P4VP/graphene fibers were prepared by suspending graphene into DMF/ethanol (5:1, w/w), followed by stirring motion at constant speed overnight, and then P4VP is dissolved in graphene/DMF solution at the concentration of 17% and 25% (w/v). The graphene added to each solution is 3% P4VP polymer by weight. All the solutions were stirred at ambient conditions for 24 h before electrospinning. Solutions were pumped out from the syringe at a rate of 5 μL/min. P4VP thin films, spun cast on glass coverslips, were used as substrates for fiber deposition. Electrospinning was conducted using an applied voltage of 15 kV across a distance of 15 cm between the tip and the collector. The resulting P4VP fibers on the glass coated with the spun cast films were annealed at 150 °C for 24 h in a vacuum of 20 mTorr to remove 2436

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ACS Applied Bio Materials determined using an unpaired t test analysis, and the results were considered to be statistically significant if p < 0.05.

3. RESULTS 3.1. Scaffolds Characterization. Electrospun P4VP and P4VP/graphene fibers were imaged with SEM, as shown in Figure 1A. The mean diameters of the fibers were measured to

Figure 2. Graphene distribution in P4VP fibers. (A) TEM images show that the graphene is encapsulated in microsized fiber and exposed to the surface on nanosize fiber. (B) Laser microscopy confirms that graphene in microfiber is confined and the aggregations protrude out from the fiber. The scale bars are 10 μm.

3.2. Cell Morphology. Cell attachment and morphology on different scaffolds were evaluated days after 24 h of postplating. For this experiment, cells were stained with Alexa Fluor 488, DAPI, and green fluorescent conjugated goat antimouse secondary antibody for vinculin and imaged by confocal microscopy. We can see that in Figure 3A, cells adhere on the flat film of P4VP regardless of the presence of graphene and the actin filaments, emanating from the nucleus and terminating at focal adhesion points (Figure 4) on the edges of the cell membrane without preferred orientation. Since many researchers have shown that graphene is not toxic to cells when they are directly seeded on the pure graphene sheets, we believe that the aggregation of graphene increases the local roughness in the region, which is not preferable to the DPSCs.29−32 On the other hand, cells cultured on the P4VP fiber coated surface were seen to attach only to the fiber, with focal adhesion points following the contours of the fibers (Figure 4) and no cell attachment on the flat region observed. The actin filaments were elongated by the fiber structure and follow the orientation of the fibers. It is also seen that the graphene platelets encapsulated in the fibers do not affect the cell attachment to the fibers. In Figure 3B, we show the confocal images of the culture at day 7, where we can clearly see that this pattern is well established, with more of the micro- or nanofiber surfaces being covered by cells, regardless of the presence or absence of graphene. Similarly, no difference in cell morphology can be observed between days 1 and 7 on the flat surface regardless of the presence or absence of graphene. In Figure 5A, we show confocal images after 28 days in culture, where we find that confluent tissue has formed on all substrates and no difference in cell morphology is observed between the fibrous and flat surfaces or those with and without graphene. In Figure 4B we plot the doubling times of the cells cultured on the flat and microfiber surfaces, between days 1 and 7, where we can see that no difference in doubling time is observed between the microfibers with and those without graphene. A significant increase is observed between both microfiber surfaces and the flat surface without graphene as previously observed.14 It is

Figure 1. Morphology study of the scaffolds. (A) Nano- and microsized electrospun fiber mesh with and without additional graphene. The smoothness of fiber surface and fiber diameter is independent of graphene. The scale bars at low magnification are 10 μm. (B) Average diameter of the nano- and microfibers. (C) AFM image of spun cast thin film loaded with graphene. The histogram at the cross section is also shown. The aggregation of graphene is about nanometers. The scale bar is 2 μm.

be 361.9 ± 93.6 nm and 2271.4 ± 519.3 nm (Figure 1B), which correspond to curvatures of 181 ± 46.8 nm and 1135.7 ± 259.7 nm, for the nano- and microfibers, respectively. We can see that the values for both nano- and microfibers, as well as the surface smoothness of the fibers with and without the addition of graphene, did not reveal significant differences under SEM. In order to further understand the distribution of graphene in the fiber matrix, samples were imaged with optical microscopy and TEM. As shown in Figure 2, graphene was uniformly distributed in the microfibers and remains well encapsulated. In contrast, in nanofibers, aggregates of graphene protruded out of the fibers. These aggregates of graphene were about 500 nm in size. However, the aggregation of graphene did not change the overall surface morphology of the electrospun fibers. The graphene distribution in P4VP thin film was determined by AFM. The result is shown in Figure 1C, where we see a nonuniform distribution of graphene aggregates ranging from 10 nm to a few micrometers. 2437

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Figure 4. Location of focal adhesion points on the cell membrane imaged with florescent stain for vinculin (green), actin (Alexa Fluor, red), and nucleus (DAPI, blue). Note: Locus of focal adhesion are at the edge of the cell membrane on the flat surface and follow the electrospun fibers on the micro P4VP fibers. No change in the distribution is discerned upon introduction of graphene. Figure 3. Cell morphology study at (A) day 1 and (B) day 7 by confocal microscopy. Cells are elongated by the fiber structure, regardless of the addition of graphene. The scale bars are 50 μm.

interesting to note that an increase in doubling time is also observed for the flat surface with graphene, though the significance is relatively low (P < 0.1). 3.3. Biomineralization and ECM Deposition Study. Biomineralization was evaluated by SEM and Raman at 28 days postplating. The SEM images are shown in Figure 6 at low and high magnifications, respectively. On the flat film with no graphene, only a few isolated roundshaped mineral particles are observed to be embedded in the organic matrix. On the flat film with additional graphene, a large amount of mineral aggregates are observed. The minerals have a flakelike shape, which is different from the ones observed on the film containing no graphene. The mineral depositions are seen on both electrospun fibers with and without graphene. However, on the fiber containing graphene, we see a significant amount of deposition organized and templated exclusively on the P4VP fiber, while the depositions on the P4VP/graphene fiber are “ball-like” and more randomly located. In the higher magnifications in Figure 7, we see that on the P4VP fibers, minerals are templated within organized fibrillar bundles, which have the unique banded structure of collagen. This phenomenon is not observed on the graphene containing P4VP microfibers, where the deposition is tightly embedded in a layer of organic matrix. In order to identify the chemical composition of the mineral deposition, the EDS spectrum was used to image the microfiber sample. It is shown that the mineral deposition consists of phosphate and calcium,

Figure 5. (A) Confocal microscopy images of dental pulp stem cells after 28 days in culture on flat and microfiber scaffolds without and with graphene. (B) Doubling time measured for the cells incubated on microfiber and flat surfaces with and without graphene. P > 0.1 and hence no significant difference is observed with and without graphene on the fiber surfaces. A difference of P < 0.1 is observed only between the doubling time on the flat surface and the other surfaces.

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Figure 7. Collagen observation. The banded collagen structure is observed on the flat surface with graphene and microfiber without graphene after 28 days of incubation of the DPSCs. The scale bars are 500 nm.

Figure 8. Raman study of the biomineralization of DPSCs on the scaffolds after a 28-day incubation. (A) Raman spectra confirms the presence of hydroxyapatite with the characteristic peak of PO43− v1 mode at ∼960 cm−1. (B) Calculated mineral to matrix ratio determined by the ratio of PO43− v1 peak to amid I peak at ∼1660 cm−1. The full width at half-maximum (fwhm) of PO43− v1 peak at ∼960 cm−1 is also determined. The microfiber without the addition of graphene has a comparable value with natural dentin.

mod, are presented. It confirms that the structure of DPSCs is capable of producing hydroxyapatite on all scaffolds, regardless of the surface morphology and chemical composition. However, as we know, the organic matrix structure is critical for proper tissue formation. In addition to forming hydroxyapatite, the dentin or bone formation in vivo requires the presence and proper structure of relative protein groups, such as amid I and amid III.26,36 In Figure 8A, we can see that even though the peak representing amid I group at 1660 cm−1 is observed on all scaffolds, the amid III group in the region of 1200 cm−1 to 1450 cm−1 is not detectable on the P4VP/ graphene electrospun fibers. In order to further explore the structure of the mineral deposition and matrix deposition by DPSCs on different scaffolds, we also obtained the mineral to matrix ratio by calculating the peak intensity ratio of the PO43− v1 band at 960 cm−1 to the amid I band at 1660 cm−1. The result is shown in Figure 8B. The mineralized nodule on the electrospun fiber of P4VP has the most comparable ratio of mineral to matrix to that measured from natural dentin.26 The full width at half-maximum of the PO43− v1 peak on the P4VP fiber and the P4VP/graphene flat films are close to that of natural dentin. On the nanoscale fibers, the value seems not be affected by the presence of graphene. On the other hand, the value is high on P4VP/graphene fiber and P4VP flat film,

Figure 6. Biomineralization of DPSCs on the scaffolds after 28-day incubation. SEM images show that DPSCs form different structures of mineral deposition on the scaffolds. Minimal deposition is observed on the flat surface without graphene and electrospun microfiber with graphene. The scale bars are 10 μm for the left column and 200 nm for the right column, respectively.

which is consistent with the chemical composition of hydroxyapatite, the main component of human bone and tooth tissue. Raman is a powerful tool to determine the structure of biomineral deposition as well as the cell-sequenced organic matrix.33−35 We have obtained the Raman spectra from the mineralized deposition by DPSCs on the scaffolds and plotted it in Figure 8A. The characteristic peaks of hydroxyapatite at 960 cm−1 for PO43− v1 vibrational mode, 425 cm−1 for PO43− v2 vibrational mode, and 562 cm−1 for PO43− v4 vibrational 2439

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barrier required for the assembly of triple helical collagen fibrils into highly organized microbundles.44−46 Here we show that the addition of graphene completely reverses the outcome obtained with pure P4VP. In this case, extensive biomineralization was observed on the flat graphene filled surface. Raman spectra indicated that the deposits had mineral to protein ratios similar to dentin, and RT-PCR results showed upregulation of OCN, a marker associated with odontogenic differentiation. In contrast, the addition of graphene to the microfiber surfaces did not enhance the previously observed templated mineralization. Rather, mineralization was significantly decreased, and RT-PCR indicated suppression of OCN gene expression. Furthermore, in contrast to the previous results where the response of the cells was similar on the micro- and nanofibers, in this case there were significant differences. Robust biomineralization, upregulation of OCN, and formation of the banded collagen fibers were all observed for cells cultured on the nanofibers, regardless of the presence of graphene. For cells plated on the microfibers, very little biomineral deposition was observed. No banded collagen fibers were detected, and there was no upregulation of OCN expression. Previous groups had reported that graphene was able to concentrate osteogenic differentiation precursors via noncovalent π bonding, thereby encouraging osteogenic differentiation in mesenchymal stem cells. In our case, this mechanism may also be responsible for the enhanced biomineralization by DPSCs on the graphene/P4VP thin film scaffolds. As shown in the Figure 1C, the aggregation of graphene in the P4VP flat surface introduces the surface roughness and allows graphene to be exposed to the cells during the culture. Similarly, on the nanofibers, graphene aggregates protrude from the fibers, since the fiber diameters are much smaller than the aggregates. Direct contact between the graphene and the cell, in both flat and nanofiber scaffolds, is thus achieved. Lee et al.7 further suggest that the unique π bonding of graphene enables growth factors released by DPSCs to be better absorbed to the surface and thereby accelerate the biomineralization on the scaffold. On the other hand, as explained earlier, the curvature produced by the fiber decreases the activation energy for collagen folding. It promotes the construction of proper structure of the ECM proteins to induce the DPCSs to differentiate along an odontogenetic pathway and form a biomineralized structure similar to dentin. As graphene is introduced and well encapsulated in the electrospun P4VP microfibers, the cells have no direct contact with the graphene. Therefore, the π bonding is not exposed to cells or ECM, preventing graphene from enhancing the binding of odontogenic factors to the substrate. The results indicate that the differentiation and biomineralization of DPSCs are suppressed by the addition of graphene, compared with the microfibers of pure P4VP with no graphene. As the graphene does not change the diameter or surface smoothness of the P4VP microfibers, we hypothesize that the effect of curvature on collagen folding is overcome by a change of other internal properties of the microfibers due to the addition of graphene. As we know, graphene has excellent electrical and mechanical properties, so we tested the electrical conductivity of the P4VP and P4VP/graphene fibers with peak force tunneling AFM (TUNA). In Figure 10, we see that the conductivity is significantly improved by the addition of graphene. It was known that the cell-secreted peptide chains of collagen carry a certain amount of ionizable residues, which makes electrostatic interactions very important in stabilizing

indicating a wider band of the peak. The result could be caused by the higher degree of disorder of hydroxyapatite. 3.4. Expression of Related Genes. In general, biomineralization is correlated to the differentiation process of stem cells.37−39 In order to determine, in our case, the differentiation activity of DPSCs on different scaffolds, we performed RT-PCR to evaluate the regulation of mRNA expression of two gene markers, ALP and OCN, from day 14 to day 28. The expression of ALP was increased 5−35-fold compared to day 1. As we see in Figure 9, these increases were

Figure 9. RT-PCR study of DPSCs during 28-day incubation on all scaffolds.

transient in cells cultured on nanofibers without graphene, microfibers with graphene, and on a flat surface with or without graphene. The expression of OCN was increased 3− 10-fold compared to day 1 in cells cultured on nanofibers with and without graphene, microfiber without graphene, and flat surfaces with graphene. The pattern of OCN increases mimics the mineralization detected by SEM and Raman shown in Figures 6 and 8.

4. DISCUSSION It has been well established that stem cells can respond to multiple growth factors40−42 and to the structure and composition of ECM. Current studies have shown that graphene, as one of the most popular new materials developed in recent years, can have an impact on stem cell proliferation and differentiation.5,8,43 The mechanism whereby graphene affects cell processes though remains unclear. Therefore, we have chosen to conduct a study where we use the P4VP system, which has been shown to be indistinguishable from tissue culture plastic in its ability to support cell proliferation but can be used without additional surface treatment. In a previous study, we demonstrated that curvature had a profound effect on differentiation, where cells differentiated along an odontogenic lineage when the P4VP was fibrillar but not flat.14 This was explained in terms of the structure of the ECM secreted by the cells where banded collagen structures were nucleated only on the P4VP fibers and not on the flat substrates. This indicated that curvature reduced the activation 2440

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mineral deposits templated on banded collagen structures. Based on Raman data, mineral to protein ratios were similar to natural dentin. Addition of graphene into the scaffolds completely reversed these results and led to robust odontogenic differentiation on the flat films containing graphene. On these scaffolds, biomineral deposits were nucleated on banded collagen fibers. In contrast, minimal differentiation or biomineralization were observed on the graphene containing micrometer sized electrospun fibers; these microfibers contained fully encapsulated graphene platelets. In contrast, incorporation of graphene into the nanofiber did not impact mineralization or OCN expression. We can conclude that two factors independently contribute to banded collagen nucleation and stem cell differentiation, namely, surface curvature and/or direct cell/graphene contact. The former reduces the barrier to the formation of banded collagen fibers, while the latter is responsible for π−π bonding which promotes adsorption of various induction factors. When the graphene is completely encapsulated, as is the situation with the micrometer sized fibers, the π−π bonding does not contribute to adsorption of induction factors. The encapsulated graphene appears to prevent the formation of the banded collage structures and therefore suppress differentiation. TUNA imaging of the substrates indicates that an increase in sample conductivity may be a consequence of embedded graphene, which somehow interferes with absorbed protein conformation.

Figure 10. Electrical conductivity by peak force tunneling AFM (AFM TUNA): (A) microfiber and flat scaffolds with the addition of graphene, applied voltage = 0 V and (B) microfiber and flat scaffolds with the addition of graphene, applied voltage = 5 V. The scale bars are 1 μm.

the folding of chains.47 Therefore, it is possible that the change in the electrical conductivity of the fiber interferes with collagen folding and overcomes the effect of curvature. In this case biomineralization is not favored. In contrast, on the nanofibers, the graphene aggregates either protrude or are excreted due to fiber diameters and hence do not affect the electrical properties of the polymer. However, since the graphene is not encapsulated, it can make direct π bonding contact with the ECM or the cells and hence induce biomineralization. In this case, both curvature and π bonding contribute to the formation of the banded collagen structures which serves as a template for biomineralization. These results are summarized in Table 1.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kuan-Che Feng: 0000-0001-7999-7605 Author Contributions #

L.Z. and K.-c.F. contributed equally to this paper.

Notes

The authors declare no competing financial interest. In contrast to carbon nanotubes, which have known cell toxicity, graphene has been found to promote cell adhesion and growth. Less is known though on its influence on cell function. Here we explored the effect of graphene on dental pulp stem cell differentiation, and found that it has a very significant role, which dominates over other factors. Furthermore, its influence is not intuitive and direct contact with its surface vs noncontact induction of electric fields has opposite effects on odontogenic or osteogenic differentiation. Our data highlights that there are still many factors that are poorly understood regarding its interactions with cells and ECM, and hence no clear pronouncement regarding its nanotoxicity can yet be drawn.

Table 1. Summary of the Two Factors That Have Impact on Cell Biomineralization and Differentiation graphene direct contact with cells and ECM

substrate curvature

nanofiber

no

yes

yes

microfiber

no

yes

yes

flat film nanofiber/ graphene microfiber/ graphene flat film/ graphene

no yes

no yes

suppressed yes

no

yes

suppressed

yes

no

yes

P4VP scaffolds

biomineralization

OCN gene expression upregulation upregulation suppressed upregulation suppressed

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upregulation

ACKNOWLEDGMENTS We gratefully acknowledge the support of the National Science Foundation (Grant NSF-INSPIRE no. 1344267) and the Morin Foundation Trust.

5. CONCLUSION We have investigated the influence of graphene inclusion on dental pulp stem cells cultured on P4VP flat and electrospun scaffolds. We found that graphene had a profound effect on the differentiation of the stem cells. In the absence of graphene, the DPSCs differentiated along odontogenic lineage only on the nano- or microsized electrospun scaffolds, producing bio-

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REFERENCES

(1) Li, N.; Zhang, Q.; Gao, S.; Song, Q.; Huang, R.; Wang, L.; Liu, L.; Dai, J.; Tang, M.; Cheng, G. Three-Dimensional Graphene Foam as a Biocompatible and Conductive Scaffold for Neural Stem Cells. Sci. Rep. 2013, 3 (1), 1604.

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DOI: 10.1021/acsabm.9b00127 ACS Appl. Bio Mater. 2019, 2, 2435−2443