Enhanced Osteogenic Differentiation of Stem Cells on Phase

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Biological and Medical Applications of Materials and Interfaces

Enhanced Osteogenic Differentiation of Stem Cells on Phase-Engineered Graphene Oxide Jia-Wei Yang, Kuan Yu Hsieh, Priyank V. Kumar, ShengJen Cheng, You-Rong Lin, Yu-Chih Shen, and Guan-Yu Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02225 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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ACS Applied Materials & Interfaces

Enhanced Osteogenic Differentiation of Stem Cells on PhaseEngineered Graphene Oxide Jia-Wei Yang1, 2⊥, Kuan Yu Hsieh1, 2⊥, Priyank V. Kumar3, Sheng-Jen Cheng1, 2, You-Rong Lin1, 2, Yu-Chih Shen1, 2 and Guan-Yu Chen1, 4* 1

Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan 2 Department of Electrical and Computer Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan 3 Optical Materials Engineering Laboratory, ETH Zurich, 8092 Zurich, Switzerland 4 Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan

⊥

These two authors contributed equally to this work

* Corresponding author Phone: (886) 3-573-1920 FAX: (886) 3-573-1672 Email: [email protected]

Keywords: graphene oxide, phase transformation, human mesenchymal stem cells, osteogenic peptide, cell differentiation

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Abstract Graphene oxide (GO) has attracted significant interest as a template material for multiple applications due to its two-dimensional nature and established functionalization chemistries. However, for applications toward stem cell culture and differentiation, GO is often reduced to form reduced graphene oxide, resulting in a loss of oxygen content. Here, we induce a phase transformation in GO and demonstrate its benefits for enhanced stem cell culture and differentiation, while conserving the oxygen content. The transformation results in clustering of oxygen atoms on the GO surface, which greatly improves its ability toward substance adherence and results in enhanced differentiation of human mesenchymal stem cell (hMSCs) towards the osteogenic lineage. Moreover, the conjugating ability of modified GO strengthened, which was examined by auxiliary osteogenic growth peptide conjugation. Overall, our work demonstrates GO’s potential for stem cell applications, while maintaining its oxygen content, which could be enable further functionalization and fabrication of novel nano-bio interfaces.


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INTRODUCTION Graphene oxide (GO), a derivative of graphene, and its related nanomaterials are recently showing valuable applications in the biomedical field. Their stable physicochemical properties and large surface area affluent with evenly distributed oxygen functional groups have boosted their applicabilities.1-6 One such area where GO-related materials can be potentially useful is in cell culture and differentiation. However, when comparing the efficiency of osteogenesis differentiation on GO-based platforms, nanosheets with low oxygen contents, namely reduced graphene oxide (rGO), showed better function and outcome.7-9 Therefore, researchers resort to a number of chemical and thermal methods to produce rGO, which have low oxygen contents (typically < 10 at. %).10-11 Although rGO is suitable for cell differentiation, such as for hMSCs, a reduction in oxygen functional groups substantially weakens rGO surface functionality, thus potentially affecting its functionalization capability as well as its mechanical strength.1, 5, 11-12

This imperfection may be a bottleneck when it comes to future applications in

forming a nano-interface, where the properties of oxygen-rich GO are desirable. For instance, alongside accelerating hMSCs proliferation and differentiation, the abundance of oxygen functional groups on GO surface, if preserved, could act as conjugation sites for extra functional entities such as chemicals, biomaterials, proteins and growth factors.13-17 To this end, it would be interesting to have a modified structure of GO,18-20 which could accelerate cell differentiation, and at the same time, preserves a large fraction of oxygen for further functionalization capabilities. This would enable further



development of GO as a nano-bio interface, to facilitate hMSCs differentiations when integrating other sophisticated nanomaterials. In this work, we produce such a modified structure of GO and demonstrate its impact on stem cell differentiation. We follow a simple recipe recently proposed in the literature to produce modified-GO structures. By annealing GO structures at 80°C, we promote the aggregation of oxygen functional groups on its surface, which in turn leads to the clustering of sp2 carbon domains, thus offering a coexistence of abundant oxidized and graphitic domains. This modified-GO structure not only showed enhanced hMSCs differentiation compared to as-synthesized GO structures, but also retained a high oxygen content (~30 at.%), which could be useful for adding functionality to GO (Scheme 1).

RESULTS AND DISCUSSION Characterization of novel functional bio-interface with graphene oxide GO substrates were fabricated by coating uniformly distributed GO on the coverslip (Fig. S1), followed by annealing at mild temperatures to induce a phase transformation in GO. Specifically, GO substrates (GO d0, GO d5, GO d9 and GO d18) were annealed at 80°C for a period of 0, 5, 9 and 18 days, respectively. The chemical structure and other functional properties were then analyzed. Our modified GO substrates exhibited both oxidized and graphitic domains (Fig. 1a), as confirmed by Auger electron spectroscopy (AES) maps (Fig. 1b), while retaining high oxygen content (as will be shown below), due to the alteration in chemical structure during its phase transformation. The coating of GO could be confirmed by observing microscopic images of modified GO substrates under bright field. Coverslips without GO coating are


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transparent, however, they became darker as annealing progressed. SEM images (Fig. 1c) show the condition and size of GO sheets, before and after annealing. They clearly remained unchanged after 5, 9 and 18 days of modification, maintaining high quality throughout. From the results of XPS analysis, elements and chemical bonding on the surface of GO coverslip could be observed. Results of the survey spectrum (Fig. S3) indicated that each GO coverslip contained the same kind of elements (Table S1). Besides, the C1s and the O1s spectra illustrated the major chemical configurations, i.e. C=C (sp2), C-O (sp3) and C=O of GO (Fig. S4). The C1s spectra for different modified GO samples presented in Fig. 1d show a significant difference in the peak intensity between the C=C (sp2) and the C-O (sp3) carbon bonding configurations. The sp2 content in GO increased with longer annealing time, implying that more graphitic domains opened up in GO. Changes in the oxygen content and the C/O ratio were calculated (Fig. 1e), oxygen content in GO substrate decreased only slightly from 34.15 at.% to 30.09 at.% (attributed to the loss of water molecules), with C/O ratio increasing from 1.63 to 1.92. To summarize this section, we showed that our annealed GO structures not only exhibited graphitic domains unlike as-synthesized GO, but also retained a significant amount of covalently-bonded oxygen. These results are consistent with our previous findings18-19, and point to the fact that GO undergoes a phase transformation, where mixed sp3-sp2 domains of the as-synthesized sample phase separate to yield distinct graphitic and oxidized domains, as depicted schematically in Fig. 1a.

Cell viability and cytotoxicity



We explore the potential of our modified-GO substrates in biomedical applications. First, to verify the effects of modified-GO substrates on cell viability and cytotoxicity, cells cultured on GO samples were observed under bright field microscopy (Fig. S6). hMSCs were found to be growing identically on glass coverslips as well as on modifiedGO-coated coverslips after 12-hour adhesion and 72-hour proliferation. A precise cell counting was conducted by trypsinizing cells with EDTA (Fig. 2a), which established the harmlessness of modified-GO substrates toward hMSCs’ proliferation. In addition, viability/cytotoxicity kit assay were performed to analyze cell viability after the 72-hour incubation, whose results are shown in Fig. 2b. On modified-GO substrates, fluorescence microscopy images revealed no obvious red fluorescence signals of propidium iodide (PI). To further quantify and investigate hMSCs’ cellular viability, FITC Annexin V Apoptosis Detection Kit was used for measurements with a flow cytometer. Cellular apoptosis and necrosis were examined and these results are shown in Fig. 2c. The ratio of cellular apoptosis on GO d0, d5, d9, d18 were 0.96%, 2.18%, 0.98% and 0.78%, whereas that of necrosis were 0.66%, 2.04%, 1.10% and 0.74%, respectively. The above results collectively confirmed that phase transformation in GO did not enhance the cytotoxicity of the cells, confirming high biocompatibility.

Osteogenic differentiation To investigate the effects of modified-GO on osteogenic differentiation of hMSCs were cultured in basal medium (BM) and osteogenic medium (OM) (Fig. 3a). Cells were observed after culturing for 7 days and 14 days under optical microscope. From the bright field images obtained, osteogenic differentiated cells were distinguished by spotting


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cuboidal-shaped cells from spindle-shaped cells. Experimental outcomes indicated that cells were conserved as spindle-shape for 14 days when cultured in BM. On the other hand, hMSCs showed spindle-like shapes on modified-GO coverslips after a 7 day-OMculture. After a 14 day-cell culture, more cuboidal-shaped cells were found on all GO substrates, indicating that cell mineralization began after 7 days. After the cells were cultured for 14 days, Alizarin Red S staining was performed to quantify the condition of mineralization in order to determine the osteogenic differentiation. From the staining results (Fig. 3b and Fig. S8), in BM, no significant red stains were observed, while various intensities of stained areas were found in OM. The potency of mineralization was found to be in the order: GO d18 > GO d9 > GO d5 > GO d0 > control. This demonstrates that our modified-GO substrates accompanied by OM, enhances osteogenic differentiation. Additionally, from the fluorescence images (Fig. 3c) captured by confocal-microscope which targets the two major indicators of osteogenic differentiation, i.e. runt-related transcription factor 2 (RUNX-2) and osteocalcin (OCN), higher level of RUNX-2 and OCN protein expression were found on the GO d18 glass slide. Once again, our modified-GO coverslips were shown to increase osteogenic differentiation. All the above results indicated that our modified-GO substrates enhanced osteogenic differentiation. This interesting feature was possibly provoked by mild-annealing of GO which resulted in its phase transformation toward distinct oxidized and graphitic domains. In order to verify this, we present both dexamethasone and ascorbic acid adherence ability on annealed-GO substrates. The results showed that while the dexamethasone adherence was uniformly maintained, the ascorbic acid adsorption



amount in the case of GO d18 was 1.42 times greater than that in the case of GO d0, thus correlating well with enhanced differentiation observed. Previous studies13, 21 have shown that hydrogen bonding could enhance the ascorbic acid adhesion performance in the sp3 carbon domains.13, 21 Along similar lines, we hypothesize that oxygen clustering in the oxidized domains might play a role in enhancing this effect, leading to higher adsorption of ascorbic acid. This would however require further clarification. Nevertheless, our experiment illustrates the benefits of the mild thermal annealing procedure caused by domain manipulation, which can enable higher adsorption of ascorbic acid on the surface of GO.

Surface functionalization of chemical modification We have shown that our modified-GO structures can enhance osteogenic differentiation while continuing to preserve the oxygen framework. Another approach to use our modified-GO substrates for enhanced osteogenic differentiation would be to functionalize our modified-GO structures with suitable peptides that could then promote differentiation. We demonstrate this interesting functionalization capability here by thiolmaleimide coupling22-23 of osteogenic growth peptide (OGP)23-24 onto GO cover coverslips (Fig. 4a). In this work, OGP (KareBay Biochem) with FITC functionalization was used to examine the conjugation efficiency of our modified-GO substrates by detecting the fluorescence signals (Fig. 4b and Fig. S9). Fig. 4c indicated that modifiedGO showed significant conjugation capability as compared to the GO day0 sample. This phenomenon is consistent with that observed previously during the grafting process of antibodies.25 There, the greater degree of functionalization was attributed to an increase


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in the number of C=O groups upon oxygen clustering, which in turn improved the functionalization capacity (Fig. 4d).

CONCLUSIONS In summary, this work demonstrates that a simple phase transformation leads to a modified structure of GO, which led to improved hMSC adhesion, proliferation and differentiation, while sustaining a large fraction of oxygen functional groups. This approach has tremendous potential toward stem cells application, and differs considerably from graphene and other related derivatives, in that it overcomes the challenges caused by having solely graphitic or oxidized domains in the material. Our study showed that the improvement was correlated to an increase in ascorbic acid absorption due to the modified structure. Our study elucidates the benefits of implementing modified-GO as a biocompatible and transferable platform for stem cell culture.

MATERIALS AND METHODS Graphene oxide (GO) coated substrate GO solution (4 mg/mL, sigma-aldrich) was used as stock solution. Stock solution was diluted with deionized water to 1 mg/mL before the coatinul of GO. The procedures of GO coating were as described previously.26-27 In brief, firstly, cleaned glass coverslips (sonicated for 30 min in acetone , 95% ethanol and deionized water washing) were subjected to amine-functionalization with 3% 3-aminopropyltriethoxysilane (APTES,



sigma-aldrich) into toluene for 30 min, followed by washing with 95% alcohol and deionized water, then dried by nitrogen gas. Functionalized glass coverslips were immersed in GO solution for 1 hour and were placed on a rocker for gentle shaking to allow uniform GO immobilization on its surface. Lastly, the preparation of GO substrate ended up with washing with DI water and drying with nitrogen gas. They were then placed in an 80 °C oven and annealed for 0, 5, 9 and 18 days (GO d0, GO d5, GO d9 and GO d18).

GO substrate characterization The GO substrates' surface morphology was determined using the cold field emission scanning electron microscope (FT-SEM, Hitachi SU8010). The surface properties of GO substrates were investigated by high-resolution Raman (HOROBA, Lab RAM HR), X-ray photoelectron spectroscopy (ULVAC-PHI PHI 5000 Versaprobe II), and Auger electron nanoscope (ULVAC-PHI, PHI 700).

Cell Culture Human bone marrow-derived mesenchymal stem cells (hMSCs) were obtained from Lonza (Walkersville, MD) and cultured in T75 flasks with mesenchymal stem cell growth basal medium (BM, Lonza PT-3001). Cells were maintained at 37°C in a humidified incubator equilibrated with 5% CO2 and were replaced with fresh medium every two to three days until 80%-90% confluency. After 6-7 days, once they had reached 80% confluence, they were detached from the plate by adding trypsin-EDTA solution, and were then incubated in a 37°C incubator for 5 minutes. Then, centrifuged


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for 3-5 minutes to pellet the cells. Cell counter (Nexcelom Auto T4) was used to count the number of cells. They were plated into appropriate plates/ flasks at a density of 5000 cells/cm2, or could be frozen in growth media plus DMSO at a density of 45,000 cells/vial. Cells were passaged till 5th - 8th passage for all experiments.

Cell viability analysis To confirm the effect of GO substrates on hMSCs, viability/cytotoxicity kit (A017, GeneCopoeia) was used for rapid distinguishing live cells from dead cells by showing two-color fluorescent (green and red) under a fluorescence microscope. GO substrates were placed in 12-well plates where hMSCs were seeded together at 5,500 cells/cm2 for overnight in BM to allow complete attachment. After the cells had attached, the medium was replaced by fresh medium and cell were maintained incubation for three days before staining, FITC Annexin V Apoptosis Detection Kit I (556547, BD) was also utilized for further quantification of hMSCs' necrosis and apoptosis response s by flow cytometer.

Osteogenic differentiation GO substrates were placed in 12-well plates and hMSCs were seeded at a density of 3,100 cell/cm2 for overnight in BM. After an overnight incubation, cells were attached, then replaced the culture medium from BM to osteogenic medium (OM, Lonza PT-3002) and fresh OM was replaced frequently, i.e every three days for two weeks. On the 14th day, Alizarin Red S (ARS, sigma-aldrich) stained hMSCs would show maximum expression

as

calcium

had

reached

maximum

deposition.

Moreover,

an

immunofluorescent analysis of osteoblastic molecules was performed to evaluate the



expression of osteoblast specific marker, including the runt-related transcription factor 2 (RUNX2, Cell Signaling Technology), which are critical for osteoblast differentiation and osteocalcin (OCN, Abcam), which is one of osteoblast marker genes. Cells were fixed in 4% paraformaldehyde in PBS for 15 min and then blocked with 10% serum for one hour. After permeabilization with 0.3% Triton X-100 (Sigma), the cells were incubated with primary antibodies in 4°C overnight and Cy3-labeled secondary antibody (EMD Millipore) or Alexa488 labeled secondary antibody (Jackson ImmunoResearch) for 2 h at room temperature.

Adsorption capacity of GO surface Ascorbic acid and dexamethasone with concentrations of 200 µM and 1000 µM were prepared separately in deionized water. A standard curve of chemical solutions were constructed in serial dilutions of the original solutions within wide range of concentrations and measured absorption at the wavelength of 266 nm and 242 nm using a spectrophotometer(Implen NanoPhotometer NP80, Germany). The adsorption of ascorbic acid and dexamethasone were calculated by changing in absorbance strength upon the 24h after the addition of GO substrates.

Immobilized growth peptide Carboxyl groups on GO substrates were activated through the EDC/NHS coupling reaction followed by immersing in 0.5 mM Maleimide-PEG-NH2 (PG2-AMML-400, Nanocs) in PBS for 6 hours for carboxyl groups and primary amines bonding covalently. Subsequently, maleimide functional groups of GO substrates were cross-linked by


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incubating

the

osteogenic

growth

peptide

with

FITC-labeled

cysteine

CALKRQGRTLYGFGGK(FITC)) in a concentration of 20 µg/mL in PBS containing 5 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) for 2 hours.27-28

Statistical analysis The data were analyzed via one-way ANOVA using the GraphPad Prism software and represented as the mean ± standard deviation based on at least 3 independent experiments and 8 representative fluorescent images.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxxxxx. See the Supporting information for details on the methods and experiments.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel. +886-3-5731920 Author Contributions J.W.Y. and K.Y.H. contributed equally to this work.



All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS G.Y.C. would like to acknowledge financial support from National Chiao Tung University (106W970), Ministry of Science and Technology (MOST 105-2628-B-009001-MY3, MOST 106-EPA-F006-001) and National Health Research Institutes (NHRI-EX107-10714EC), Taiwan.

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Figure Legends Scheme 1. The annealed-GO substrates exhibited a higher amount of molecular adsorption and peptide grafted content, thus enabled the differentiation of hMSCs towards osteogenic lineage.

Figure 1. Characterization of annealed-GO substrates. (a) Schematic illustration of phase separation in GO-coated substrates. (b)Auger electron spectroscopy (AES) oxygen mapping of coated GO with or without phase transformation process. The arrows with white spots indicate oxygen-rich regions and the black spots indicate oxygen-poor or carbon-rich regions. Scale bar, 500 nm. (c) SEM images of annealed-GO substrates (GO d0, GO d5, GO d9 and GO d18). Bar, 3 µm. (d) Investigate the structural manipulation


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of sp2 and sp3 on annealed-GO substrates by C1s XPS spectra. (e) The quantitative calculation of oxygen content and C/O ratio on annealed-GO substrates by stoichiometry. These results revealed that oxygen cluster induced after the mild thermal annealing procedure while contributing to the enlargement of sp2 carbons domain.

Figure 2. The effects of annealed-GO substrates on attachment, proliferation and viability of hMSCs. (a) Quantification of cells cultured for 12 h and 72 h. The values data represent the mean ± S.D. of at least 3 independent culture experiments; n.s., not significant. (b) Live/dead viability stainging of hMSCs culuted for 72 h, followed by fluorescence microscopy. Scale bar, 200 µm. (c) Quantification of apoptosis and necrosis by flow cytometry. The cells were harvested 72 h after culture with or without annealedGO substrates and analyzed for the percentage of cells emitting fluorescence. Glass coverslip without GO coating served as a control.

Figure 3. Annealed-GO substrates promote osteogenic differentiation of hMSCs. ((a) Schematic illustration of the substrate preparation and the procedure to differentiate hMSCs into osteogenic lineages.18,

29

(b) The alizarin red S staining of hMSCs on

annealed-GO substrates after 14 days of basal medium (BM) and osteogenic medium (OM) incubation. Scale bar, 200 µm. (c) Confocal microscopic observation of hMSCs stained with osteogenic marker Runt-related transcription factor 2 (RUNX-2, red) , osteocalcin (OCN, green), and DAPI (blue). The above images revealed that hMSCs cultured on GO d18 substrate showed higher expression of osteogenic marker compared to GO d0 substrate. Scale bar, 20 µm. (d) Annealed-GO increased the adsorption capacity



of ascorbic acid and dexamethasone. The 200 µM ascorbic acid and 1000 µM dexamethasone were prepared in deionized water, followed by addition to the annealedGO substrates for 24 h at room temperature and measurement at wavelength of 266 nm and 242 nm absorbance using a spectrophotometer (Implen NanoPhotometer NP80, Germany). ** p < 0.01 , *** p < 0.005.

Figure 4. Annealed-GO substrates enhances the ability to immobilize peptides. (a) Schematic illustration for the covalent immobilization strategy of FITC-labeled osteogenic growth peptide (OGP-FITC) via thiol-maleimide reaction. (b) Fluorescence microscopy observation of OGP-FITC conjugated on the annealed-GO substrates. Scale bar, 100 µm. (c) Quantification of the relative fluorescence intensity by fluorescence microscopy images showed a 1.6-fold increase in the OGP-FITC density between GO dO and GO d18. (d) Cartoon illustration of the surface chemistry redistribution caused by the phase separation and oxygen cluster, offering assistance to enhance peptide grafting and linking effects on the annealed-GO substrates. Quantitative data represent the mean ± S.D. of at least 3 independent culture experiments. ** p < 0.01 , *** p < 0.005.


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Enhanced Osteogenic Differentiation of Stem Cells on PhaseEngineered Graphene Oxide Jia-Wei Yang1, 2⊥, Kuan Yu Hsieh1, 2⊥, Priyank V. Kumar3, Sheng-Jen Cheng1, 2, You-Rong Lin1, 2, Yu-Chih Shen1, 2 and Guan-Yu Chen1, 4*

For Table of Contents Only



Scheme 1. The annealed-GO substrates exhibited a higher amount of molecular adsorption and peptide grafted content, thus enabled the differentiation of hMSCs towards osteogenic lineage. 151x94mm (300 x 300 DPI)


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Figure 1. Characterization of annealed-GO substrates. (a) Schematic illustration of phase separation in GOcoated substrates. (b)Auger electron spectroscopy (AES) oxygen mapping of coated GO with or without phase transformation process. The arrows with white spots indicate oxygen-rich regions and the black spots indicate oxygen-poor or carbon-rich regions. Scale bar, 500 nm. (c) SEM images of annealed-GO substrates (GO d0, GO d5, GO d9 and GO d18). Bar, 3 µm. (d) Investigate the structural manipulation of sp2 and sp3 on annealed-GO substrates by C1s XPS spectra. (e) The quantitative calculation of oxygen content and C/O ratio on annealed-GO substrates by stoichiometry. These results revealed that oxygen cluster induced after the mild thermal annealing procedure while contributing to the enlargement of sp2 carbons domain. 303x170mm (300 x 300 DPI)



Figure 2. The effects of annealed-GO substrates on attachment, proliferation and viability of hMSCs. (a) Quantification of cells cultured for 12 h and 72 h. The values data represent the mean ± S.D. of at least 3 independent culture experiments; n.s., not significant. (b) Live/dead viability stainging of hMSCs culuted for 72 h, followed by fluorescence microscopy. Scale bar, 200 µm. (c) Quantification of apoptosis and necrosis by flow cytometry. The cells were harvested 72 h after culture with or without annealed-GO substrates and analyzed for the percentage of cells emitting fluorescence. Glass coverslip without GO coating served as a control. 286x181mm (300 x 300 DPI)


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Figure 3. Annealed-GO substrates promote osteogenic differentiation of hMSCs. ((a) Schematic illustration of the substrate preparation and the procedure to differentiate hMSCs into osteogenic lineages.18, 29 (b) The alizarin red S staining of hMSCs on annealed-GO substrates after 14 days of basal medium (BM) and osteogenic medium (OM) incubation. Scale bar, 200 µm. (c) Confocal microscopic observation of hMSCs stained with osteogenic marker Runt-related transcription factor 2 (RUNX-2, red) , osteocalcin (OCN, green), and DAPI (blue). The above images revealed that hMSCs cultured on GO d18 substrate showed higher expression of osteogenic marker compared to GO d0 substrate. Scale bar, 20 µm. (d) Annealed-GO increased the adsorption capacity of ascorbic acid and dexamethasone. The 200 µM ascorbic acid and 1000 µM dexamethasone were prepared in deionized water, followed by addition to the annealed-GO substrates for 24 h at room temperature and measurement at wavelength of 266 nm and 242 nm absorbance using a spectrophotometer (Implen NanoPhotometer NP80, Germany). ** p < 0.01 , *** p < 0.005. 254x270mm (300 x 300 DPI)



Figure 4. Annealed-GO substrates enhances the ability to immobilize peptides. (a) Schematic illustration for the covalent immobilization strategy of FITC-labeled osteogenic growth peptide (OGP-FITC) via thiolmaleimide reaction. (b) Fluorescence microscopy observation of OGP-FITC conjugated on the annealed-GO substrates. Scale bar, 100 µm. (c) Quantification of the relative fluorescence intensity by fluorescence microscopy images showed a 1.6-fold increase in the OGP-FITC density between GO dO and GO d18. (d) Cartoon illustration of the surface chemistry redistribution caused by the phase separation and oxygen cluster, offering assistance to enhance peptide grafting and linking effects on the annealed-GO substrates. Quantitative data represent the mean ± S.D. of at least 3 independent culture experiments. ** p < 0.01 , *** p < 0.005. 257x230mm (300 x 300 DPI)


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Enhanced Osteogenic Differentiation of Stem Cells on Phase

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