Enhanced Osteogenic Commitment of Human Mesenchymal Stem

Article pubs.acs.org/journal/abseba

Enhanced Osteogenic Commitment of Human Mesenchymal Stem Cells on Polyethylene Glycol-Based Cryogel with Graphene Oxide Substrate Hwan D. Kim,† Jiyong Kim,† Rachel H. Koh,† Jimin Shim,† Jong-Chan Lee,† Tae-Il Kim,*,‡ and Nathaniel S. Hwang*,†,§ †

School of Chemical and Biological Engineering, N-Bio Institute, Institute of Chemical Process, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea ‡ Department of Periodontology, School of Dentistry, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul 03080, Republic of Korea § Interdisciplinary Program in Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea S Supporting Information *

ABSTRACT: Graphene oxide (GO) is considered a comparatively recent biomaterial with enormous potential because of its nontoxicity, high dispersity, and enhanced interaction with biomolecules. These characteristics of GO can promote the interactions between the substrates and cell surfaces. In this study, we incorporated GO in a cryogel-based scaffold system to observe their influence on the osteogenic responses of human tonsil-derived mesenchymal stem cells (hTMSCs). Compared to polyethylene glycol (PEG)-based cryogel scaffold, GO-embedded PEG-based (PEGDA-GO) cryogels not only showed improved cell attachment and focal adhesion kinase (FAK) signaling activation but also enhanced cell viability. Taken together, we demonstrated that PEGDA-GO cryogels can stimulate osteogenic differentiation under an osteoinductive condition and enhance osteogenic phenotypes compared to the control group. In summary, we demonstrate that GO embedded in cryogels system is an effective biofunctionalizing scaffold to control osteogenic commitment of stem cells. KEYWORDS: graphene oxide incorporation, polyethylene glycol based cryogel, mesenchymal stem cells, osteogenesis, bone tissue engineering

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INTRODUCTION

Recently, many studies have utilized graphene oxide (GO) for stem cell culture because of its superior biocompatibility.15,16 Human mesenchymal stem cells (hMSCs) showed increased cell growth and osteogenic differentiation on the GOfunctionalized surfaces17 or scaffolds.18,19 It has been demonstrated that GO has a potential to associate with a variety of surrounding biomolecules including serum proteins and soluble growth factors.20−22 Because ECM proteins such as fibronectin and integrin directly mediate cell adhesions and incorporation of GO can increase these protein adsorptions, it is hypothesized that incorporation of GO in scaffold design platform may facilitate cellular adhesion and potentiate integrin-related signaling cascades. Likewise, an increase in vinculin expression would promote osteogenic differentiation of stem cells via enhanced focal adhesion. Indeed, several studies have demonstrated that the upregulated vinculin expression

Stem cells constantly interact with their in vivo microenvironment through various physiochemical signals,1 where these signals directly regulate cellular fate determination.2,3 Therefore, it is important to incorporate these physiochemical signal components that provide factors for guided stem cell differentiation in scaffold design.4 In particular, hydrogels are considered to be adequate platforms for stem cell encapsulation.5 Polyethylene glycol (PEG)-based hydrogels have tunable mechanical properties and scaffold architecture,6−8 which makes it extremely attractive for tissue engineering applications. However, given its bioinert nature, PEGDA alone cannot provide adequate stimulus for stem cell differentiation. To endow PEGDA hydrogels with biofunctionality, several studies have incorporated extracellular matrix (ECM)-derived components9−11 to enhance cell−scaffold interactions and stimulate stem cell differentiation. Conjugating biofunctional materials with PEGDA backbone structure may be necessary to execute the proper application of PEGDA scaffolds to support stem cell behavior and promote stem cell differentiation.12−14 © XXXX American Chemical Society

Received: May 22, 2017 Accepted: August 25, 2017 Published: August 25, 2017 A

DOI: 10.1021/acsbiomaterials.7b00299 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

and filtration steps using 0.2 μm glass frit filter equipped with anodic aluminum oxide membrane (Whatman, Germany). After being vacuum-dried for 24 h (at room temperature), the preoxidized graphite flakes were vigorously stirred in a solution of NaNO3 (0.5 g) in H2SO4 (at 0 °C for 40 min). The flask was warmed up to 20 °C and 3.0 g of KMnO4 was gradually supplemented into the flask and stirred for an additional 45 min. After that, the temperature was elevated up to 35 °C while stirring the mixture for additional 2 h. Brown color solution appeared after the temperature was elevated up to 98 °C and 2.5 mL of H2O2 (30 wt %(aq)) was consequently supplied into the flask to complete the oxidation process, followed by several cleaning and filtration steps with HCl (250 mL, 10 wt %(aq)). For neutralization, the final product was comprehensively washed with distilled H2O until the pH value near 7.0. For long-term storage, GO was completely dehydrated under vacuum conditions at room temperature for 48 h. Swelling Ratio Measurement. For each cryogel, constructs were swollen with PBS for 24 h and their weight was measured in an equilibrium-swollen state. The cryogel constructs were subsequently lyophilized to eliminate the remaining H2O content to obtain the dry weight. Swelling ratio (Q) was calculated from the wet weight over the dry weight using the below equation

leads to enhanced osteogenic commitment of stem cells through increased focal adhesion-mediated cellular responses.18 As the several studies have reported the enhanced focal adhesion of stem cells interacted with GO,18,23,24 the GO incorporation to the scaffold is potentially an effective way to provide the osteoinductive platform for stem cells. In this study, we designed GO-incorporated PEG-based (PEGDA-GO) cryogel scaffolds. Human tonsil-derived stem cells (hTMSCs) were chosen as cell source because of their proliferative nature and multipotency.25,26 As a biofunctionalizing compound, the effect of PEGDA-GO cryogel scaffolds on cellular activities including cytotoxicity, adhesion, and osteogenic differentiation of hTMSCs was investigated and compared to that of control PEGDA cryogel. Here, we demonstrated the effect of PEGDA-GO cryogel on osteogenic responses of hTMSCs by investigating the interaction of stem cells with GO, osteogenic gene expression level, calcium deposition and bone regeneration profile on critical defect on mouse calvaria model.

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MATERIALS AND METHODS

swelling ratio, Q =

Fabrication of PEGDA Cryogels. PEGDA-based GO-incorporated (PEGDA-GO) cryogels were prepared by mixing 20% PEGDA (w/v) solution dissolved in phosphate buffered saline (PBS) and PBS with 10 and 20 μg/mL of grinded GO particle (1 μm, Figure S1) in a 1:1 volume ratio. Thus, the final concentration of PEGDA became 10% and 5 or 10 μg/mL of GO concentration. While storing this mixed solution at 4 °C, ammonium persulfate (10% (w/v), APS; Sigma-Aldrich, USA) and N,N,N′,N′- tetramethylethylenediamine (TEMED; Sigma-Aldrich, USA) were added to the final concentrations of 0.4% (w/v) and 0.25% (v/v) respectively.27 The polymer solution was then pipetted into the certain types of mold and placed in the −20 °C refrigerator for 20 h. After polymerization, macropores were created by thawing ice crystals in scaffold with freeze-drier. Prior to cell seeding, scaffolds were washed several times with sterile PBS to remove unreacted residues and then sterilized with UV for 1 h. Cells were seeded onto following types of scaffolds: 10% PEGDA (w/v), PEGDA + 5 μg/mL GO, and PEGDA + 10 μg/mL GO at a concentration of 1.5 × 104 cells/μL in a dropwise manner (Figure 1). Preparation of Graphene Oxide (GO) Particles. Graphene oxide (GO) was synthesized from graphite flakes using modified Hummers’ method.28 Concisely, gross graphite flakes (Sigma-Aldrich) were mixed in aqueous solution of both 0.5 g of K2S2O8 and 0.5 g of P2O5 in concentrated H2SO4 (at 80 °C for 6 h), followed by cleaning

weight of the equilibrated scaffold in PBS weight of the dried scaffold

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis. To identify the presence of different functional groups, we performed attenuated total reflectance Fourier transform infrared (ATR-FTIR, Bruker Tensor 27) analysis on phosphate buffer saline (PBS), graphene oxide (GO), poly ethylene glycol diacrylate (PEGDA), and PEGDA-GO cryogel at range between 4000 and 650 cm−1. Spectra of all of experimental groups including PBS, GO, PEGDA, and PEGDA-GO cryogel were recorded. Cell Culture method and Osteogenic Differentiation. Human-originated, tonsil-derived mesenchymal stem cells (hTMSCs, P6) were obtained from the Department of OtorhinolaryngologyHead and Neck Surgery, Ewha Woman’s University Medical Center (EWUMC, Seoul, Korea). hTMSCs were seeded at a density of 3 × 106 cells/cryogel scaffold and cultured with osteogenic medium (OM) for osteogenic differentiation. OM has a basis of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with glycerol-2phosphate (10 mM, Sigma-Aldrich, USA), L-ascorbic acid (50 mg/mL, Sigma-Aldrich, USA), dexamethasone (100 nM, Sigma-Aldrich, USA), Fetal Bovine Serum (FBS, 10%, Gibco, USA), Penicillin/streptomycin (Pen/Strep) (1%, Gibco, USA), L-glutamine (5%, Sigma-Aldrich, USA), and antibiotic-antimycotic (Anti-anti, 100 U/mL, ThermoFisher, USA) up to 2 weeks. Osteogenic differentiation medium was changed once a day. Cell Proliferation and Live/Dead Assay. Cellular proliferation was investigated by Alama Blue Assay Kit (Invitrogen, USA) following the manufacturer’s manual. Cells were seeded on the scaffold at 10 000 cells/μL density and incubated with 1:10 Alama blue solution diluted medium for 4 h. After incubation, Alama blue included medium was collected and its absorbance was measured with AT/Infinite M200 (TECAN, USA). Live/Dead assay was performed utilizing a Live/ Dead viability kit (Invitrogen, USA) after 3 days of cell seeding on PEGDA and PEGDA-GO scaffold. After 24 h, cells were incubated for 30 min with a Live/Dead solution kit containing calcein-AM and ethidium homodimer-1 (EthD-1). Then the images were obtained using LSM 720 confocal microscope (Zeiss LSM 720). Mechanical Properties. Prior to compression test, each group of cryogels was equilibrated in PBS for 24 h. Compression tests were demonstrated using Instron mechanical testing machine (Instron 5966, Instron Corporation) with a 10-kN-load cell. The load, stress and strain values were recorded and calculated into Young’s modulus. Scanning Electron Microscopy. After culturing cells with OM for 4 days, cells seeded on scaffolds were fixed with 4% paraformaldehyde (Polysciences., Korea) for 15−20 min, consecutively dehydrated in gradients of EtOH (70, 80, 90 and 100%, Daejung Chemical., Korea) each for 20 min, and switched to

Figure 1. Overall scheme of designing in vitro and in vivo study with hTMSCs and fabrication of PEGDA-GO cryogel scaffolds. B


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ACS Biomaterials Science & Engineering hexamethyldisilazane (HMDS; Daejung Chemical., Korea) for 5 min and dried. Scaffolds were projected with field-emission scanning electron microscopy (FE-SEM; JSM-6701F, JEOL) subsequently platinum sputtering at 20 mA for 100 μs. Cell Adhesion Measurement. To identify cell adhesion rate, DNA content of PEGDA, PEGDA + 5 μg/mL GO, and PEGDA + 10 μg/mL GO were quantified utilizing Quant-iT PicoGreen dsDNA assay kit (PicoG, Invitrogen, USA) according to the manufacturer’s protocol. We have then normalized the result with DNA content from the same number of cell seeded culture plate by standard curves. Cellular Distribution within Cryogel Scaffolds. In order to confirm the homogeneous cell distribution within cryogel scaffolds, GFP lentiviral transduced HeLa cells were seeded on PEGDA and PEGDA + GO cryogel scaffold groups with a density of 1 × 105 cells in a medium volume of 30 μL. After 24 h of seeding, the scaffold was briefly washed with PBS and slice vertically with the blade. The fluorescence image was then observed with a confocal microscopy (Zeiss LSM 720). Morphological Analysis. After culturing cells with OM for 4 days, cells were fixed with 10% (v/v) formalin and washed several times with PBS. After permeabilization with 0.1% of Triton X-100 for 30 min, each samples were stained with a mixed solution of 1:100 Phalloidin (Alexa Fluor 594, life technologies, USA) and 1:500 vinculin (Abcam, USA) for 1 h. Stained for another 1 h after secondary antibody treatment. Before 15 min to wash out secondary antibody solution, DAPI (6-diamidino-2-phenylindole, Sigma-Aldrich, USA) solution was added to a ratio of 1:200 and washed with PBS after 15 min. Then, fluorescence image was observed with a confocal microscopy (Zeiss LSM 720). Western Blot. After protein samples were collected by M-PER (Mammalian Protein Extraction Reagent, ThermoFisher), the protein level was analyzed using 10% (w/v) of SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. After that, proteins from SDS gel were transferred into an Immobilon-P membrane and blocked with 5% skim milk solution in PBS-T (1×, pH 7.5 within 0.1% Tween-20) then reacted with primary antibodies against vinculin (Abcam, USA) diluted in 1:1,000 and β-actin (#6276, Abcam, USA) diluted in 1:5,000 overnight in cold room under gentle agitation. The proteins were then reacted with the antirabbit IgG horseradish-peroxidase conjugated secondary antibody (#7074, Cell Signaling, USA) in a 1:2,000 dilution for 1 h at room temperature. After that, blot membranes were visualized by a chemiluminescence detection agents (Amersham Bioscience, USA). Real-Time PCR. RNA samples were harvested from the cell-laden PEGDA and PEGDA-GO scaffold (n = 3) with Trizol method (TRIzol, Life Technology, USA). A total RNA concentration was determined by a NanoDrop spectrometer (ND-2000, NanoDrop Technologies, USA). 1,000 ng of total RNA from each sample was reversely transcribed into cDNA using TOPscript Reverse Transcriptase Kit (Enzynomics, Korea) followed by manufacturer’s instruction. Real time PCR reactions were performed with SYBR Green PCR Master mix and ABI StepOnePlusTM real-time PCR system (Applied Biosystems, USA). cDNA samples were loaded and analyzed for four different gene (GAPDH, ALP, OCN, and RunX2) expressions to observe osteogenic gene expressions. Data were analyzed with −2ΔΔCt method as previously described.29 PCR primers sequences are listed as follow: GAPDH (forward: 5′-GTA TGA CTC CAC TCA CGG CAA A-3′, reverse: 3′-GAC GTG GTG GTT GAC GAA TC-5′) ALP (forward: 5′-ACG TGG CTA AGA ATG TCA TC3′, reverse: 3′- CTG GTA GGC GAT GTC CTT A −5′), OCN (forward: 5′-GCC TTT GTG TCC AAG C-3′, reverse: 3′-GGA CCC CAC ATC CAT AG-5′) RunX2 (forward: 5′-ACT GGG CCC TTT TTC AGA-3′, reverse: 3′-GCG GAA GCA TTC TGG AA-5′), Type I collagen (forward: 5′-GTC ACC CAC CGA CCA AGA AAC C-3′, reverse: 3′-AAG TCC AGG CTG TCC AGG GAT G-5′) Alizarin Red S Staining. For Alizarin Red S staining, 20 mg of Alizarin Red S powder (ARS, Sigma-Aldrich, USA) was mixed in distilled water (1 mL), and the pH was optimized around 4.1 to 4.2 with ammonium hydroxide. After OM treatment for 14 days, differentiated cells were fixed and stained with ARS solution for 20

min and washed several times with distilled water for 5 min. For the ARS quantification, 800 μL of 10% (v/v) acetic acid was added per well and incubated at room temperature for 30 min. To avoid isolated effect, we also have performed the ARS staining on empty GOincorporated cryogel (Figure SA2). Cells were collected using cell scraper and transferred to the 1.5 mL tube. Five hundred microliters of mineral oil was added to the 1.5 mL tube. We collected the cell with a cell scraper and moved it to the 1.5 mL Eppendorf tube and added 500 μL of mineral oil. The tube was heated at 85 °C for 10 min and cooled for 5 min with ice, and then centrifuged at 20 000g for 15 min. We then collected the 500 μL of supernatant and added 200 μL of 10% ammonium hydroxide. We measured the absorbance with AT/Infinite M200 (TECAN, USA). In Vivo Calvarial Defect Model. All animal protocols were approved by the Institute of Animal Care and Use Committee, Seoul National University. Female mice (6 weeks old, BALB/c-nude immunodeficient) were operated to make critical-size calvarial defects. Amounts of 30 mg/kg Zoletil and 10 mg/kg Rompun were used to anesthetize and the surgical incision was performed on the middle of the animals’ heads. After that, two of 4 mm diameter calvarial defects were created on both sides of mice skull using a hand drill (STRONG106, Saeshin Precision Co., LTD, Korea), and scaffolds with 4 mm diameter were inserted into the both critical defects. Before the implantation, hTMSCs were seeded onto each group (PEGDA, PEGDA + 5 μg/mL GO, PEGDA + 10 μg/mL GO) of scaffolds and cultured in OM for 2 weeks. Mouse skulls were collected after 8 weeks of implantation and analyzed for bone regeneration. Micro-CT Analysis. After fixation of mice skulls (4% Paraformaldehyde solution), collected samples were visualized with microcomputed tomography method (SkyScan1172, Bruker, USA). The operation voltage and the current of Micro-CT was around 59 kV, and 167 μA respectively. Every taken Micro-CT images were stacked and visualized by CTVol software (Bruker), and the area/volume of regeneration were evaluated by CTAn software (Bruker). Volume and surface area of bone regeneration was estimated as in the following equations:

regenerated volume(surface) BV(BS) = 100 TV(TS) total volume(surface) Histological Analysis. For decalcification, the collected skulls were immersed into 14% of ethylenediaminetetraacetic acid at pH 7.4 for 4 days, consecutively dehydrated with EtOH, and embedded in paraffin solution for 12 h. Paraffin-embedded tissue blocks were longitudinally microsectioned by 5-μm thickness (RM2145, Leica) and mounted onto prepared glass slides. 5-μm sectioning samples were deparaffinized with xylene solution and rehydrated with serial EtOH/ distilled H2O, and immersed in Bouin’s solution (Sigma-Aldrich, USA) at room temperature overnight. Sample slides were washed in running tap water, and the samples were stained with Masson’s trichrome solution (MTC) for 5 min. Stained samples were then placed in acetic acid (0.5%, 1 min). Images were taken by a bright-field microscope (Olympus, Japan) with a digital camera (ProgRes C14, Jenoptik, Germany). Immunofluorescence staining was accomplished following a manufacturer’s protocol. Sample slides were pretreated with TritonX100 (0.1%, Sigma-Aldrich, USA) for 15 min. For confirmation of protein expression, slides were primarily incubated with collagen type I antibody (1:200, rabbit polyclonal, Fitzgerald Industries International) for 2 h. After that, samples were reacted with secondary antibody against rabbit (1:500, Jackson Immuno Research) for additional 2 h. Staining samples were then visualized with confocal laser scanning microscopy (LSM700, Zeiss). Transmission Electron Microscopy Analysis. To analyze the actual phenotype of graphene oxide particle (or single flake), we separated and positioned a synthesized single graphene oxide particle on a TEM copper grid. Bright-field images of GO were visualized by HRTEM operating system (Tecnai F20, FEI). Statistical Analysis. The data were expressed as the mean ± standard deviation unless otherwise indicated. The statistical C


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Figure 2. (a) Gross images of dry and swelling states of PEGDA and 5−10 μg/mL of PEGDA-GO cryogels and (b) their quantified swelling ratio for each group. Swelling ratio of each group indicated similar porosity and structure of PEGDA/PEGDA-GO group.

1000 cm−1 and the intensity at 1500 cm−1 growths in the spectrum of PEGDA-GO by the increased carbonyl group content. According to these data, we confirmed GO incorporation into the PEGDA structure successfully. According to Wu et al., GO at a low concentration does not have any cytotoxic effects on the cells.31 To determine the optimal GO concentration for stem cell culture, we examined cell viability in scaffold using live/dead assay at day 3. Fluorescence images of the first three groups (PEGDA, PEGDA + 5 μg/mL GO, PEGDA + 10 μg/mL GO) indicated tolerable cell viability of over 95% (Figure 4). However, GO concentration over 0.5 mg/mL indicated high cytotoxicity and viability of less than 60%. Thus, in this study, GO concentration between 5 and 10 μg/mL was used for the PEGDA cryogel fabrication to prevent any potential cytotoxic effect caused by high GO concentrations. Furthermore, Young’s modulus measurement indicated no significant difference between mechanical properties of PEGDA and PEGDA-GO cryogels (Figure 5). On the stress vs strain graph, GO-incorporated cryogel, particularly 10 μg/mL GO, experienced greater stress at 60% strain. However, Young’s modulus at low strains are more relevant to cell growth; these three groups showed similar mechanical properties for this range. Conclusively, GO incorporation into the PEGDA cryogel did not present any noticeable differences in swelling and mechanical properties. Given these results, we concluded that the low density of GO concentrations could functionalize constructs without changing basic material properties of the PEGDA cryogel. Effect of GO Incorporation into PEGDA Cryogels on Structure, Cell Attachment, and Viability. SEM images showed highly porous surface and structure of each cryogel group (Figure 6A). The addition of GO into the PEG-based cryogel did not interfere with any other molecules and maintains in the overall inner structure. PEGDA with either 5 or 10 μg/mL GO maintained the similar shape as PEGDA cryogels. To confirm cell permeability of PEGDA and PEGDAGO cryogels, we seeded GFP-tagged HELA cells on each group of cryogels. After 24 h, we traced cells and imaged by confocal microscope. Diffuse GFP signal throughout each group of cryogels indicated that cells can easily penetrate and uniformly spread throughout the entire scaffolds (Figure 6B). However, when hTMSCs were seeded on the scaffold, cell adhesion densities were different. For PEGDA cryogels, cells were scattered throughout the entire scaffold. But cells in 5 or 10 μg/ mL GO-incorporated cryogels formed cell masses. These results point out that even low GO concentrations may substantially affect cell adhesion and even cell-to-cell interactions. GO enhanced initial cell attachment to cryogels. Compared to PEGDA cryogels (51.06 ± 2.28%), high cell attachment efficiency was measured at 5 μg/mL GO added

significances were analyzed by one-way analysis of variance (ANOVA, *p < 0.05, **p < 0.01).

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RESULTS Characterization of GO-Incorporated PEG-Based Cryogel Scaffolds. In this study, we utilized cryogel scaffolds made of 10% (w/v) PEGDA solution, 0.4% (w/v) APS solution, and 0.25% (w/v) of N,N,N′,N′-tetramethylethylenediamine (TEMED) containing GO for an intended concentration of 5 or 10 μg/mL. First, synthesized cryogels were characterized for porosity and swelling ratio. Because ice crystals form during polymerization, PEGDA-based cryogels contain highly porous structure. The high porosity of the scaffold allowed considerable water absorption. As expected, both PEGDA and PEGDA-GO cryogels had a similar swelling ratio of 9.8−10.8 (Figure 2). The structural features of Graphene Oxide (GO), PEGDA, and PEGDA-GO were examined by the FT-IR spectroscopy method in Figure 3. The FT-IR spectrum of GO presented a

Figure 3. Fourier Transform Infrared spectra analysis of PBS (orange), graphene oxide (GO, red), poly ethylene glycol diacrylate (PEGDA, black), and PEGDA-GO (blue) cryogel to confirm fabrication of GOincorporated cryogel.

different type of oxygen functionality by robust and broad absorption peak at 3392 cm−1 creating from Oxygen− Hydrogen stretching vibrations. Absorption band at around 1500 cm−1 is distinguished characteristic of the carbonyl group (CO) in the COOH unit located at the boundaries of the GO structure, whereas the bands of CC bond in graphitic sheet express around 1300 cm−1.30 After gelation, because of the low concentration of the GO particle, there were no dramatic changes in FTIR peaks. However, there might be some increase in intensity around ether groups (C−O−C) D


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Figure 4. Live/Dead assay of hTMSCs on fabricated PEGDA/PEGDA-GO cryogel (scale bar = 100 μm). (a) (i) PEGDA, (ii) PEGDA + 5 μg/mL GO, and (iii) PEGDA + 10 μg/mL GO group showed more than 95% cell viability. However, for the higher concentration like (iv) 0.5 mg/mL GO and (v) 1.0 mg/mL GO, (b) viability decreased to less than 60% and it showed toxicity for cell growth.

Figure 5. (a) Mechanical compression testing of GO-incorporated cryogel. (b) Compressive strain−stress curve for PEGDA/PEGDA-GO cryogels and Young’s modulus. Both of PEGDA/PEGDA-GO group showed similar strain−stress curve at below 40% stain. (c) Each PEGDA/PEGDA-GO cryogel group showed similar young’s modulus at approximately 70 kPa.

cryogels (57.78 ± 3.88%) and 10 μg/mL GO added cryogels (60.47 ± 4.37%) (Figure 7). Morphological Analysis and Focal Adhesion of hTMSCs on PEGDA-GO Cryogels. The morphological differences of hTMSCs at the cell-cryogel interface were assessed by F-actin staining analysis. Stem cells were uniformly seeded on each type of cryogels, and they displayed round morphology (Figure 8A). Because cells seeded on cryogels are usually located inside pores, we were not able to discover direct morphological responses on the cryogel surface. Instead, the expression level of vinculin protein, which is importantly related to focal adhesion of cell, was examined by Western blot analysis. The result indicated that vinculin expression level on GO-incorporated cryogels was higher than that of PEGDA cryogels (Figure 8B). From the results, we confirmed that GO incorporation into PEGDA cryogels can modulate cellular morphology and cell adhesion. Enhanced Osteogenic Gene Expressions and Osteogenic Responses of hTMSCs in vitro. Osteogenic gene

expression level was significantly increased in hTMSCs (Figure 9). Quantitative real-Time polymerase chain reaction (RTPCR) analysis showed that expression level of osteogenic genes (OCN, RunX2, Col I, and ALP) was increased in hTMSCs seeded on PEGDA + 5 μg/mL GO cryogels (n = 4, p < 0.05) and PEGDA + 10 μg/mL GO cryogels (n = 4, p < 0.01) compared with PEGDA cryogel group. Interestingly, at week 1, PEGDA + 5 μg/mL GO and PEGDA + 10 μg/mL GO showed a similar increase of gene expressions. However, data from week 2 showed many enhanced expressions of the osteogenic genes compared to PEGDA cryogel group. The expression level of osteogenic markers on PEGDA + 10 μg/mL GO group was much more upregulated compared to that of the other group at week 2 (n = 4, *p < 0.05, **p < 0.01). This suggests that GO incorporation increased osteogenic expression profile and induced osteogenic differentiation of hTMSCs. To determine whether hTMSCs differentiated into osteoblast-like cells, we measured the calcium content by Alizarin Red S staining (ARS, Figure 10). Red ARS staining was more E


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the effectiveness of GO-incorporated PEGDA cryogels scaffold in vivo, we applied our scaffold to the animal calvarial defect model. In advance of implantation, hTMSCs were seeded on 4 mm diameter PEGDA/PEGDA-GO scaffolds and cultured in osteogenic medium for a week. Scaffolds were then transplanted into the calvarial defect (4 mm diameter) of 6-week-old Balb/c-nude immunodeficient mouse. After 8 weeks of implantation, collected mice skulls were assessed by MicroCT and histochemical analysis (Figure 11A). Implantation of PEGDA-GO scaffold with hTMSCs increased bone regeneration area compared to PEGDA cryogel group; 5 μg/mL GOincorporated PEGDA cryogels showed a 2.3-fold times increased of bone regeneration volume compared with PEGDA cryogels and 10 μg/mL GO-incorporated PEGDA cryogel group displayed a 1.9-fold times increased regenerated bone volume compared to control groups (Figure 11b). Moreover, among the groups, 10 μg/mL GO-incorporated PEGDA cryogel had 4.8-fold times higher regenerated area than that in the controls by post mathematical pixel reconstruction (Figure 11c). However, there was no statistical difference of regenerated bone volume and surface between 5 and 10 μg/mL GO-incorporated PEGDA cryogel group. Furthermore, the group with either 5 or 10 μg/mL GO displayed a higher amount of collagen matrix as confirmed by Masson’s trichrome (MTC) staining (Figure 11d and Figure S3). Moreover, we have performed the immunostaining for the type 1 collagen (Figure 11e). The type 1 collagen was homogeneously distributed for 10 μg/mL GO-incorporated PEGDA cryogel throughout the entire regenerated area (white arrows), whereas in PEGDA cyrogel, with a minimum permineralization, the type I collagen immunostaining was mostly restricted to the scaffold surface. Besides, the visualized amount of collagen was insignificant compared to the 5 and 10 μg/mL GO-incorporated PEGDA cryogel. MTC and type I collagen immunostaining directly indicated an increase in bone matrix deposition over time for the different concentration of GO. As a result, GO-incorporated PEGDA cryogel scaffolds were able to regenerate bone tissue with high calcium and collagen content as indicated in MTC staining.

Figure 6. (a) Scanning electron microscope (SEM) images of PEGDA/PEGDA-GO cryogel at 150× without cells (scale bar = 100um) and 750× with cell (scale bar = 10 μm) to visualize the porous structure of the cryogels and cell adhesion morphology. (b) Confocal images of GFP-tagged HeLa cell seeded on the PEGDA/PEGDA-GO cryogels to demonstrate cell permeability throughout the scaffold.

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DISCUSSION Recently, cryogel-based scaffolds have shown enormous potential in tissue engineering, as they may provide a macroporous surface area for cellular growth.32−34 Furthermore, due to its tenability, biofunctionalized cryogel-based scaffolds have been utilized to investigate specific cell behaviors.34,35 Graphene oxide (GO) is a biomaterial with an enormous potential due to its high dispersity and increased hydrophilic interaction, which are characteristics that contribute to stimulating the interaction between cell and environment. Previous studies demonstrated that interaction with the environment is one of the most key factors in stem cell differentiation. For these reasons, effects of GO on stem cell differentiation have been actively studied. In this study, we modified PEG-based cryogels with GO particle to enhance biofunctionality. Our results demonstrated that GO incorporation did not affect either the shape or structure of cryogels. The unique morphological characteristic of cryogels with microporous and interconnected pores were also preserved. This suggests that GO incorporation does not change basic physical characteristics of PEGDA cryogels. Moreover, GO improved cell attachment, survival, and proliferation within cryogels, perhaps by providing cell attachment sites.

Figure 7. Cell adhesion rate on PEGDA/PEGDA-GO cryogel. Cell adhesion rates were analyzed by PicoG analysis. Increasing the GO concentration of PEGDA-GO cryogel group showed increased cell adhesion rate (%) compared to PEGDA group (*p < 0.05).

distinguished in 5 μg/mL GO-incorporated PEGDA cryogels and 10 μg/mL GO-incorporated PEGDA cryogel group compared to the control group. It demonstrated that incorporation of GO particle into PEGDA cryogels augmented calcium deposition of seeded stem cells. Furthermore, we facilitated ARS to quantify the calcium deposition level. Cells on the GO added cryogel scaffold had 2-fold, significantly higher ARS content compared to the control group. In agreement with osteogenic gene expression analysis by RTPCR, cells in GO-incorporated PEGDA cryogels deposited more calcium content than the control group. GO-Incorporated PEGDA Cryogels Promote Bone Regeneration of in Vivo Calvarial Defect. To evaluate F


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Figure 8. (a) Nucleus, vinculin, and F-actin staining for hTMSCs morphology on PEGDA/PEGDA-GO cryogel (scale bar = 10 μm). (b) Vinculin expression level of hTMSCs on PEGDA/PEGDA-GO cryogel analyzed by Western blot.

Figure 9. Osteogenic gene marker expression of hTMSCs seeded on PEGDA/PEGDA-GO cryogel. Real-time PCR was performed after 7 days and 14 days of osteogenic medium culture on each group. The expression level of osteogenic markers on PEGDA + 5 μg/mL GO and PEGDA + 10 μg/ mL GO was upregulated compared to PEG. (n = 4, *p < 0.05, **p < 0.01).

Figure 10. (a) Alizarin red staining of hTMSCs on PEGDA/PEGDA-GO cryogel. Alizarin red staining was performed after 14 days of osteogenic medium culture. (b) Calcium deposition level of PEGDA + 5 μg/mL GO and PEGDA + 10 μg/mL GO group was quantified by absorbance measurement (n = 3, p < 0.05).

Previous studies have demonstrated that GO encourages cellmatrix interactions by attracting ECM protein.23,24 Our results show that incorporating GO in PEGDA cryogels effectively improved cell adhesion, which may be attributed to the adsorbed ECM proteins.36 As presented in Figure 8B, comparably with the F-actin formation, the expression level of vinculin gene increased in hTMSCs seeded on the PEGDA-

GO cryogel. Considering that the enhancement in cell survival rate and osteogenic commitment of hTMSCs in PEGDA-GO cryogels coincided with the development of focal adhesion and interaction with the nearby environment, we assumed that the major working mechanism of GO arises from promoted cell adhesion via increased interaction between GO substrate and surrounding proteins. G


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Figure 11. (a) Micro-CT image for bone regeneration of mouse calvaria (defect diameter = 4 mm). (b) In the mouse calvarial defect model, percent bone volume of PEGDA + 5 μg/mL GO and PEGDA + 10 μg/mL GO group was higher than hTMSCs on PEGDA cryogel group (n = 3, p < 0.01) and (c) percent bone surface of PEGDA + 5 μg/mL GO and PEGDA + 10 μg/mL GO group was also higher than hTMSCs on PEGDA cryogel group. (d) Histological analysis of regenerated bone tissue with Masson’s trichrome staining, scale bar = 500 um. (e) Immunostaining of collagen type I in regenerated bone tissue for each groups, scale bar = 500 um (n = 3, *p < 0.05, **p < 0.01).

The GO incorporation into cryogel scaffolds promoted cell viability, proliferation, and differentiation. According to many literatures, enhancing cell adhesion plays an important role in communicating adhesion-dependent survival signaling pathways,37,38 and vinculin expression stimulates to focal adhesion kinase (FAK) signaling pathway as cell attaches to the proteins, both of which suppress the activity of apoptotic factors.38 Based on our experimental data, we confirmed that GO has a great potential for supporting cell survival and activating cellular interactions with its microenvironment. Our study, as well as numerous previous studies, has shown that GO induces osteogenesis of stem cells.17−19 Osteogenesisrelated genes (OCN, ALP, COL I, and RUN X2) were upregulated in GO-incorporating PEGDA cryogels on both weeks 1 and 2 (Figure 9). Characteristic osteoblast phenotypes including calcium deposition in ARS staining were increasingly detected in hTMSCs on GO-incorporating cryogels after 3 weeks of culture in osteogenic differentiation medium. Soluble and insoluble factors can accelerate GO-mediated osteogenic differentiation. Bone morphogenetic protein-2 (BMP-2) is a widely known potent osteoinductive growth factor that plays an important role in the development of bone. La et al. has demonstrated that GO attracts and localizes BMP-2 via hydrophobic π−π interactions, which enables sustained release of the growth factor for in vivo applications.22 Combined with substance P, which is known to recruit MSCs, sustained release of BMP-2 from GO-coated substrate can effectively promote in vivo bone formation by recruited MSCs. For in vivo analysis, PEGDA-GO and PEGDA cryogels were fabricated to fill the critical size defect in mouse calvarial defect model. After 8 weeks of implantation, new bone regeneration was observed at the defect sites in the all experimental groups. A degree of new bone regeneration was increased by incorporation of GO (Figure 11b, c). As for in vitro experiments, enhanced interaction with microenvironment by GO incorporation seemed to induce osteogenic differentiation of hTMSCs. These in vivo systems implanting GO-incorporated cryogels and hTMSCs were well-suited for tissue

regeneration with good biocompatibility. Moreover, the experimental groups displayed more collagen tissue formation compared to the control group in Masson’s trichrome staining and type I collagen immunostaining (Figure 11d, e). Especially, bone tissue with a high density of calcium and collagen was found in GO-incorporated PEGDA cryogel groups. Therefore, it was confirmed that GO incorporation into PEGDA cryogels has the ability to stimulate osteogenic differentiation of mesenchymal stem cells in a 3D environment and promote bone tissue regeneration.

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CONCLUSION In this sense, GO-functionalized PEGDA cryogels can be applied to stimulate hTMSCs to commit osteogenic lineage, and hTMSCs in combination with GO-incorporated PEGDA cryogels should be a prospective cell source in clinical use for bone tissue engineering and bone regenerative therapies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00299. TEM images of graphene oxide, alizarlin staining for acellular cryogel, and histological analysis of regenerated bone tissue with Masson’s trichrome staining (10×, scale bar = 500 um) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +82-2-2072-2642. Fax: +82-2744-1349. *E-mail: [email protected]. Tel.: +82-2-880-1635. Fax: +822-888-7295. ORCID

Nathaniel S. Hwang: 0000-0003-3735-7727 H


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H.D.K. and N.S.H. planned the work. H.D.K., J.K., R.H.K., and J.S. carried out the experiments. H.D.K. and J.K. analyzed the data, J.S. and J-C.L. provided advice and support. H.D.K. and J.K. prepared the draft and finalized manuscript for this submission, which was proofread and corrected by R.H.K., N.S.H., and T-I.K. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this work was provided from Seoul National University Dental Hospital Research Fund (01-20150006) and by the National Research Foundation of Korea (KNRF) grant funded by Ministry of Education, Science and Technology (NRF-2016R1E1A1A01943393). It is also supported by Global Ph.D. Fellowship Program from the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015H1A2A1029321).

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Enhanced Osteogenic Commitment of Human Mesenchymal Stem

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