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Composite System of Graphene Oxide and Polypeptide Thermogel As an Injectable 3D Scaffold for Adipogenic Differentiation of TonsilDerived Mesenchymal Stem Cells Madhumita Patel, Hyo Jung Moon, Du Young Ko, and Byeongmoon Jeong* Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, Korea S Supporting Information *
ABSTRACT: As two-dimensional (2D) nanomaterials, graphene (G) and graphene oxide (GO) have evolved into new platforms for biomedical research as biosensors, imaging agents, and drug delivery carriers. In particular, the unique surface properties of GO can be an important tool in modulating cellular behavior and various biological sequences. Here, we report that a composite system of graphene oxide/ polypeptide thermogel (GO/P), prepared by temperature-sensitive solto-gel transition of a GO-suspended poly(ethylene glycol)-poly(Lalanine) (PEG-PA) aqueous solution significantly enhances the expression of adipogenic biomarkers, including PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, and HSL, compared to both a pure hydrogel system and a composite system of G/P, graphene-incorporated hydrogel. We prove that insulin, an adipogenic differentiation factor, preferentially adhered to GO, is supplied to the incorporated stem cells in a sustained manner over the three-dimensional (3D) cell culture period. On the other hand, insulin is partially denatured in the presence of G and interferes with the adipogenic differentiation of the stem cells. The study suggests that a 2D/3D composite system is a promising platform as a 3D cell culture matrix, where the surface properties of 2D materials in modulating the fates of the stem cells are effectively transcribed in a 3D culture system. KEYWORDS: graphene oxide, sol−gel transition, hybrid system, stem cell, 3D culture
1. INTRODUCTION Reconstructive surgery by using autologous fat tissues from other parts of the body needs careful maintenance of graft volume and cell viability. In addition, the limited proliferative capability of mature adipocytes, insufficient angiogenesis, and inflammation around the transplanted tissue lead to failure of the graft and malignancy of the grafted site.1−3 In this regard, adipose-tissue-derived mesenchymal stem cells (ASCs) have recently been suggested as an alternative to the transplantation of fat tissues. Despite some tissue damage during the procedure, adipose tissues have been considered to be a goldmine of stem cells because of the rather facile availability of the ASCs from liposuction.4 However, because the biofunctionalities of ASCs are significantly age-dependent, ASCs isolated from adults often have limited capacity for proliferation and differentiation.5 In addition, the differentiation capability of ASCs also strongly depends on the tissue location and patient gender.6 ASCs can be reprogrammed to promote the adipogenic differentiation through the forced expression of adipogenic genes or retinoic acid treatments.7 However, instead of adipogenic differentiation, ASCs can undergo osteogenic or chondrogenic differentiation, and thus cyst formation or tissue calcification has been reported in 5% of patients following the ASC injection.8 Therefore, finding a new stem cell resource for © XXXX American Chemical Society
adipogenic differentiation is an urgent issue in reconstructive research. Recently, tonsil-derived mesenchymal stem cells (TMSCs) have been reported as a new source of stem cells with the capability of multiple differentiation.9,10 TMSCs are isolated through tonsillectomy, a procedure performed about 40 000 annually in Korea.9 The tonsil tissues have been wasted, otherwise. Tonsil tissues contain MSCs at a population density of 10−100-fold more than bone marrow does, and the proliferation rate of TMSCs is 2−3 times faster than that of bone marrow-derived mesenchymal stem cells (BMSCs).11 TMSCs have been proven to exhibit multiple differentiation capabilities to chondrocytes, osteocytes, hepatocytes, and neuronal cells, similar to ASCs and BMSCs.10−13 In particular, as tonsillectomy patients are usually young, the age-related problems of stem cells are relatively minor for TMSCs. Traditionally, most stem cell differentiation studies have been carried out on 2D surfaces of polystyrene plate. However, the stem cells cultured in 3D culture systems tend to exhibit biomarker and phenotype expressions different from the stem cells cultured on 2D systems.14,15 The differences in mass Received: December 17, 2015 Accepted: February 4, 2016
A
DOI: 10.1021/acsami.5b12324 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
PEG-PA Synthesis. PEG-PA was synthesized by the ring opening polymerization of the N-carboxy anhydrides of L-alanine in the presence of α-amino-ω-methoxy PEG.11,35 Briefly, α-amino-ωmethoxy PEG (5.0 g, 5.0 mmol; MW 1000 Da) and N-carboxy anhydrides of L-alanine (10.2 g, 88.7 mmol) were used. The polymerization was carried out at 40 °C for 24 h under anhydrous nitrogen conditions. The polymer was purified by fractional precipitation by using chloroform (solvent) and diethyl ether (nonsolvent). The polymer was dialyzed in water using a membrane with a molecular weight cutoff of 1000 Da and freeze-dried. 1 H NMR Spectroscopy. 1H NMR spectra of the polymer in CF3COOD (500 MHz NMR spectrometer; Varian, USA) were used to determine the composition and average molecular weight (Mn) of the polymer. Gel Permeation Chromatography (GPC). The gel permeation chromatography system (Waters 515) with a refractive index detector (Waters 410) was used to obtain the molecular weights and molecular weight distribution of the polymer. N,N-Dimethylformamide was used as an eluting solvent. PEGs with a molecular weight range of 400− 20000 Da were used as the molecular weight standards. An OHPAK SB-803QH column (Shodex) was used. Dynamic Mechanical Analysis. The modulus of the polymer aqueous solution (6.0 wt %) was investigated by dynamic rheometry (Rheometer RS 1; Thermo Haake, Germany) as a function of temperature. The aqueous polymer solution was placed between circular, 25 mm diameter, parallel plates separated by a gap of 0.5 mm. During the dynamic mechanical analysis, the samples were placed inside a chamber with water-soaked cotton to minimize water evaporation. The data were collected under a controlled stress (4.0 dyn/cm2) and frequency of 1.0 rad/s. X-ray Photoelectron Spectroscopy (XPS). The XPS machine (Thermo U.K.) adopted a monochromatic source of Al K-alpha. Any possible shift of the XPS peak caused by the charging effect was calibrated by referencing it to the C 1s peak at 284.8 eV. Transmission Electron Microscopy (TEM). The TEM images of G and GO were obtained by using a JEM-2100F microscope (JEOL, Japan) with an accelerating voltage of 200 kV. 3D Cell Culture. Human TMSCs were received from Ewha Womans University Medical School. The cells were cultured on a polystyrene plate at 37 °C in high glucose Dulbecco’s modified eagle media (DMEM, Hyclone, USA) supplemented with 10.0% fetal bovine serum (FBS, Hyclone, USA), 1.0% penicillin/streptomycin (Hyclone, USA), and 1.0% antibiotic-antimitotic (Gibco, USA) in 5% CO2 atmosphere. The TMSCs proliferated as adherent cells with fibroblast-like morphology on a 2D culture of polystyrene plate and their surface markers exhibited negative for CD14, CD34, and CD45, but positive for CD73, CD90, and CD105.13 Therefore, they were confirmed to be true MSCs. Harvested TMSCs (passage 6, 4.0 × 105 cells) were mixed with GO (1.0 wt %)-suspended PEG-PA aqueous solution (6.0 wt %, 0.2 mL) and were incubated at 37 °C in 24 well to prepare a 3D cell culture composite system of GO/P. TMSCs were encapsulated in the GO/P system by sol-to-gel transition of the polymer aqueous solution. Cells were 3D cultured for 2 weeks in an adipogenic induction medium prepared by high glucose DMEM with 10% FBS, 10 μg/mL insulin (Wako, Japan), 1 μM dexamethasone (Sigma, USA), 0.5 mM isobutyl-methyl xanthine (Sigma, USA), and 100 μM indomethacin (Sigma, USA). A composite system of TMSCencapsulated G/P was prepared for comparison by using G instead of GO by the above GO/P system protocol. In addition, a 3D cell culture system of TMSC-encapsulated in pure hydrogel (P) was also prepared by the same protocol in the absence of G or GO as a control. Cell Proliferation. The cell viability and morphology of TMSCs were evaluated by the Live/Dead kit (Life Technologies, USA). Cells were incubated at room temperature for 30 min in the solution of 4 μM ethidium homodimer-1 (EthD-1) and 2 μM calcein acetoxymethyl ester (AM). Image of cells were captured under a Nikon Eclipse E600 microscope using Lucia software. Live cells were stained with calcein AM (green) and dead cells were stained with EthD-1 (red). Real Time RT-PCR. Total RNA was extracted from the TMSCs encapsulated in hydrogels using 1 mL TRIZOL, (Invitrogen, USA)
transport, cell adhesion, mechanotransduction, cell−cell contacts, and cell communications between 2D and 3D systems are responsible for such behavior.16−18 In addition, stem cell differentiation can be affected by various physicochemical factors such as stiffness, functional groups, shape, and size of patterned culture substrates.19−23 For example, G and GO exhibit different biofunctions because of their difference in chemical functional groups. G enhanced the osteogenic differentiation while GO enhanced the chondrogenic and adipogenic differentiation of MSCs, respectively.22,23 The differences in interactions between materials surfaces and proteins in the culture media were suggested to be a plausible reason for the different cellular behaviors. When ASCs were cultured as a pellets in the presence of GO, the chondrogenic biomarkers were enhanced.24 The enhancement of chondrogenic differentiation of ASCs was attributed to the proteins of TGF-β3 and fibronectin adsorbed on GO. Recently, composite systems consisting of hydrogel and 2D nanomaterials have emerged as a frontier biomaterial in tissue engineering because of their controllable mechanical, biological, and chemical properties.25,26 Hydrogels not only provide high water content and high permeability for nutrients and oxygen, but also lower the mechanical tissue irritation around the implanted site.27,28 The 2D nanomaterials can provide the biological properties to the hydrogel. Considering that the cells in living tissues are in 3D environments, the transcription of information on traditional 2D cell culture research into 3D systems is expected to be realized by 2D material-incorporated 3D composite systems. In this regard, here we prepared a composite system consisting of GO and poly(ethylene glycol)-poly(L-alanine) (PEG-PA) thermogel (GO/P) for adipogenic differentiation of TMSCs. Thermogel is a thermosensitive polymer, the aqueous solution of which turns into a semisolid hydrogel as the temperature increases.29−32 The PEG-PA is unique in that the polymer is degraded by peptidases, however it is stable in the absence of proteolytic enzymes, and the final degradation products are amino acids, and thus neutral pH is maintained during the degradation.28 TMSCs and GOs were suspended in the PEG-PA aqueous solution, and were then entrapped into a hydrogel by increasing the temperature of the solution to a cell culture temperature of 37 °C. The in situ formed composite system provides a 3D cell culture environment for TMSCs toward adipogenic differentiation. The adipogenic biomarkers were investigated at mRNA and protein levels. The biofunction of the differentiated adipocytes was also investigated for lipid globule formation by the oil-red O assay. In addition, the mechanism of enhanced/suppressed adipogenic differentiation, if any, was investigated by assuming that the adhesion of a differentiation factor, insulin, might be involved. The adipogenic differentiation of TMSCs in a pure thermogel (P) and in a composite system consisting of graphene and thermogel (G/P) was also compared.
2. EXPERIMENTAL SECTION Materials. α-amino-ω-methoxy poly(ethylene glycol) (PEG) (MW = 1000 Da, IDB Chem, Korea), N-carboxy anhydrides of L-alanine (KPX life, Korea), and anhydrous N,N-dimethylformamide (SigmaAldrich,USA) were used as received. Toluene (Daejung, Korea) was distilled over sodium before use. The aqueous colloidal suspension of exfoliated GO nanosheets was synthesized by the modified Hummers’ method.33 Another precursor, the colloidal suspension of exfoliated G nanosheets, was prepared by a chemical reduction of GO using hydrazine.34 B
DOI: 10.1021/acsami.5b12324 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces and 200 μL of chloroform after 14 days of culture. The samples were centrifuged at 12,000 rpm for 10 min. After being washed with 75% ethanol and air-dried RNA pellets were dissolved in RNase free water. The concentration of RNA was determined by Nano drop 2000 spectrophotometer (Thermoscientific, U.S.A) and cDNA was synthesized by the ReverTra Ace qPCR RT kit (Toyobo, Japan). Then real-time PCR was performed with a CFX 96 system using the SYBR green super mix. The relative expression level of the target genes was calculated as 2−ΔΔCt, where target gene expression was normalized as ΔΔCt = (Gene A−GAPDH)t − (Gene A−GAPDH)t0. t0 indicates the zeroth day. As typical adipogenic differentiation biomarkers, PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, HSL, and UCP1 were investigated after incubation for 0, 3, 7, and 14 days. Primer sequences of PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, HSL, UCP1, and glyceraldehyde-3phosphate-dehydrogenase (GAPDH) are shown in the Table 1.
Determination of Loading Capacities. In a homogeneous dispersion of G or GO (0.025 mg/mL) in 1 mL of PBS, dissolved insulin (0.057 mg/mL) was added. During different time intervals (0, 1, 3, 5, and 7 days) the unabsorbed protein present in PBS was determined using a UV−vis spectrophotometer (Nano drop 2000 spectrophotometer). Energy-Dispersive X-ray Spectroscopy (EDS) Analysis. Protein adsorption on G or GO was evaluated by EDS analysis, after incubation of the insulin solution with G and GO for 1 day. The samples were prepared by fixing with 2.5% glutaraldehyde for 30 min and washed with distilled water. The samples were observed under EDS (INCA energy, Oxford Instruments Analytical Ltd., Bucks, UK). Statistical Analysis. The data were expressed as the mean ± standard error of the mean (SEM). The differences in the mean values were evaluated using the one-way ANOVA with Tukey tests. Differences were considered significant when the p value was less than 0.05 (marked as asterisk).
Table 1. PCR Primers Used in This Study
3. RESULTS AND DISCUSSION Polymer Synthesis and Characterization. PEG-PA was prepared by ring-opening polymerization of N-carboxy anhydrides of L-alanine onto α-amino-ω-methoxy PEG.11,35 Briefly, the molecular weight (Mn) of the each block of PEG-PA calculated by 1H NMR spectral peaks of the methoxy end group (CH 3 O−) of PEG, the ethylene glycol group (−CH2CH2O−) of PEG, and the methyl group (CH3−) of PA at 3.60−3.65, 3.90−4.10, and 1.40−1.90 ppm, respectively was to be 1000−970 Da (Figure S1a). Gel permeation chromatography exhibited the unimodal distribution of the polymer molecular weights with an average molecular weight of 1100 Da and polydispersity index (Mw/Mn) of 1.1. The PEGPA aqueous solution undergoes sol-to-gel transition as the temperature increases. Typically, a polymer aqueous solution (6.0 wt %) is a low viscous solution with a modulus of 0.01 Pa at 4 °C, whereas it turns into a hydrogel with a modulus of 650 Pa at 37 °C (Figure 1a). The gel modulus at 37 °C slightly decreased from 650 to 610 Pa by changing the solvent from DMEM to H2O (Figure S2). Ideally, a thermogelling polymer aqueous solution and cells are to be mixed at room temperature, followed by formation of a gel at 37 °C. However, there should be a compromise between the gel modulus and transition temperature in practical application of the thermogel
gene
primer sequence
PPAR-γ
sense 5′-GAA GAC GGA GAC AGA CAT GAG-3′ antisense 5′-GCA ACT GGA AGA AGG GAA ATG-3′ sense 5′-GGC ACG AAT CAG ACT CAT CTA C-3′ antisense 5′-TCG TCC CTC TCC AAG TTA CA-3′ sense 5′-CAT CCT CTT TGC CCA CTT CT-3′ antisense 5′-CCT GGA TCA TTG TTC TCT-3′ sense 5′-GCT GGC TCC TGT TGA GTT ATA G-3′ antisense 5′-ACC ACA GCA ATC ACC TTA CAT-3′ sense 5′-CAT GAA CTT TGA GGT GCC ATT-3′ antisense 5′-GAC ATC ATG GTC GCT GTA GTT-3′ sense 5′-CCC GGC AAC TCT AGT ATT TAG G-3′ antisense 5′-AAT GAC AAG GCA CGA TTT GC-3′ Sense 5′-TCT ACG ACA CGG TCC AGG −3′ antisense 5′-GTC TGA CTT TCA CGA CCT CTG −3′ sense 5′-CTC CTC ACA GTT GCC ATG TA-3′ antisense 5′-GTT GAG CAC AGG GTA CTT TAT TG-3′
LPL ELVOL3 HSL AP2 CEBP-α UCP1 GAPDH
Immunocytochemistry and Histology. The immunocytochemistry and histology were studied after 2 weeks of induction. To determine the protein expression, we immunostained samples with adipogenic markers like PPAR-γ and AP2 (Abcam, UK). The samples were fixed in acetone, permeabilized by Triton (0.5%), incubated with primary antibodies for 1 h and blocked with bovine serum albumin (Sigma, USA). After washing, the antibodies were detected by staining with secondary antibody Dylight 488 (Abcam, UK) according to the manufacturer’s protocol. The nucleus and actin were visualized using 4′6′-diamidino-2-phenyle indole (DAPI) (Molecular probes, USA) and phalloidin (Abcam, UK), respectively. Fluorescence images were taken by an Olympus 1 × 71 fluorescence microscope using Olympus DP2-DSW software. To assess the adipogenic differentiation of TMSCs, we analyzed lipid globules by the Oil Red O staining method. Cells were fixed with 10% formaldehyde solution for 10 min, and costained with Oil Red O solution (Sigma, USA) (in 60% isopropyl alcohol) and DAPI for 20 min, after being rinsed with 1X phosphate buffered saline (PBS) for three times. The cells were washed with distilled water and the stained cells were captured using light microscopy (Olympus 1 × 71 fluorescence microscope using Olympus DP2-DSW software). The relative fluorescence intensity was calculated as per image. Circular Dichroism (CD) Spectroscopy. The structure of insulin adsorbed on GO and G was examined after incubation in PBS for 1 and 3 days using CD spectrum (J-810, JASCO, Japan) and compared with the structure of native insulin. Insulin (0.057 mg/mL) and G or GO (0.025 mg/mL) were mixed in 1 mL of PBS for this study. The CD spectrum and its corresponding high voltage tension voltage curve were recorded using a quartz cell with an optical path length of 1 cm.
Figure 1. (a) Storage modulus (G′) and loss modulus (G″) of polymer aqueous solution (6.0 wt % in DMEM) as a function of temperature. The gel modulus (G′) increases from less than 0.01 Pa at 4 °C to 650 Pa at 37 °C. (b) Photo images of sol (4 °C) and gel (37 °C) states of the polymer aqueous solution (6.0 wt %). C
DOI: 10.1021/acsami.5b12324 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces because stiffness of a cell culture matrix affects the differentiation of MSCs.36 Neurogenesis and adipogenesis are preferred in a soft hydrogel (10 kPa).36−38 Polyacrylamide (PAAm) hydrogels with a modulus of 600 Pa was reported for preferential adipogenic differentiation of MSCs.38 Therefore, we selected 6.0 wt % polymer aqueous solution in current study. The modulus of the in situ formed gel at 37 °C was 650 Pa and the gel maintained its physical integrity as a 3D matrix during the cell culture period. The photo images of the sol (4 °C) and gel (37 °C) states prepared from the polymer aqueous solution (6.0 wt %) are shown in Figure 1b. GO Characterization. X-ray photoelectron spectroscopy (XPS) spectra of GO exhibited carbon−oxygen bonds of C− OH, C−O−C, CO, and O−CO groups at 285−292 eV, which were significantly diminished for G. (Figure S3a).19,39 GO and G were several micrometers in size as shown in the TEM images (Figure S3b). Cell Proliferation. The GO/hydrogel (GO/P) composite system encapsulating TMSCs was prepared by increasing the temperature of a polymer aqueous solution (6.0 wt %), in which GO (1.0 wt %) and TMSCs (passage 6, 4.0 × 105 cells cells/well) were suspended, to the cell culture temperature of 37 °C. The sol-to-gel transition of the system provided for the facile entrapment of the GO and TMSCs. The G/P composite system was similarly prepared by using G instead of GO. The TMSC-incorporated G/P composite system and TMSCincorporated pure hydrogel system (P) were used as control 3D culture systems for comparison. TMSCs in the P, G/P, and GO/P systems were 3D cultured by using adipogenic induction media of high glucose DMEM enriched with insulin (10 μg/ mL), FBS (10%), dexamethasone (1 μM), isobutyl-methyl xanthine (0.5 mM), and indomethacin (100 μM). The TMSCs exhibited a spherical shape at day 0 (0d) in all of the 3D culture systems of P, G/P, and GO/P (Figure 2a). Excellent cell viability was confirmed by the Live and Dead assay during the cell culture period, where the live cells and dead cells appeared as green and red, respectively. At day 14 (14d) after the 3D culture started, cell aggregation and change in cell morphology were also observed. Cell spreading on soft substrates is less than on stiff substrate.37,40 Rounded cells with small spreading promotes signaling process associated with adipogenesis.41 Similar cell morphologies were observed in 3D culture systems of ASCs, BMSCs, and TMSCs by using polypeptide thermogels, indicating that the thermogels with a low modulus
