Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis.

Biomaterials xxx (2015) 1e11

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Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis Jia'En Jasmine Li, Naoki Kawazoe, Guoping Chen* Tissue Regeneration Materials Unit, International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 February 2015 Accepted 2 March 2015 Available online xxx

Stem cells exist in an in vivo microenvironment that provides biological and physiochemical cues to direct cell fate decisions. How the stem cells sense and respond to these cues is still not clearly understood. Gold nanoparticles (AuNPs) have been widely used for manipulation of cell behavior due to their ease of synthesis and versatility in surface functionalization. In this study, AuNPs with amine (AuNP eNH2), carboxyl (AuNPeCOOH) and hydroxyl (AuNPeOH) functional groups possessing different surface charge were synthesized. Human bone marrow-derived mesenchymal stem cells (hMSCs) were treated with the surface functionalized AuNPs and assessed for cell viability and osteogenic differentiation ability. The surface functionalized AuNPs were well tolerated by hMSCs and showed no acute toxicity. Positively charged AuNPs showed higher cellular uptake. AuNPs did not inhibit osteogenesis but ALP activity and calcium deposition were markedly reduced in AuNPeCOOH treatment. Gene profiling revealed an upregulation of TGF-b and FGF-2 expression that promoted cell proliferation over osteogenic differentiation in hMSCs. These results provide some insight on the influence of surface functionalized AuNPs on hMSCs behavior and the use of these materials for strategic tissue engineering. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Gold Nanoparticle Surface modification Stem cell

1. Introduction Human mesenchymal stem cells (hMSCs) are an important cell type and used in many tissue engineering strategies due to their great potential to differentiate into many cell types. The differentiation and fate of hMSCs are known to be governed and regulated by a variety of biological and physiochemical cues in the cellular microenvironment surrounding stem cells in vivo [1]. Recent studies have shown that specific chemical moieties may contribute significantly towards direction of stem cell fate [2,3]. Functional groups like amines (eNH2), carboxyl (eCOOH) and hydroxyl (eOH) are widely found on biomolecules such as proteins, nucleic acids and polysaccharides and are important factors in influencing stem cell behavior and differentiation [4]. Nanoscale materials have attracted broad attention in many tissue engineering studies because of their similarity to the nanostructured nature of microenvironment. Not only do nanomaterials possess a large surface area for interaction with biological molecules and cells (due to their high surface area to volume ratio),

* Corresponding author. Tel.: þ81 29 860 4496; fax: þ81 29 860 4706. E-mail address: [email protected] (G. Chen).

they are also able to traverse biological barriers and even enter the cell nuclei [5]. These factors imply a greater biological response from nanoscale materials in contrast with the bulk materials. Presenting functional chemical moieties on the surface of nanomaterials is anticipated to more closely mimic the microenvironment and to be appropriate to investigate the influence of the physiochemical cues on stem cell functions. Gold nanoparticles (AuNPs) are ideal candidates of choice for biological studies due to their ease of synthesis, relative biocompatibility and versatility in surface modification [5]. Functionalized AuNPs are especially useful for therapeutic strategies in drug delivery [6], diagnostic imaging [7] and antibody labeling and targeting [8]. Surface charge and different surface chemical moieties can influence many cell behaviors particularly uptake of the nanoparticles [9] as well as cytoskeletal remodeling [10]. Gold nanoparticles (AuNPs) have been found to promote osteogenesis in hMSCs [11]. However it is still unknown how the AuNPs functionalized with different functional groups affect the behaviors of hMSCs. Therefore in this study, AuNPs were surface modified with three differently charged alkanethiols to produce AuNPs with amine (AuNPeNH2), carboxyl (AuNPeCOOH) and hydroxyl (AuNPeOH) functional groups and investigated for their

http://dx.doi.org/10.1016/j.biomaterials.2015.03.001 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Li JJ, et al., Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis, Biomaterials (2015), http://dx.doi.org/10.1016/j.biomaterials.2015.03.001

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J.J. Li et al. / Biomaterials xxx (2015) 1e11

interactions with hMSCs. We aimed to investigate if such functional groups on nanomaterials will have an effect on hMSCs behavior and osteogenesis. 2. Materials and methods 2.1. Gold nanoparticle synthesis and characterization Depending on the type of surface modification, the AuNPs were synthesized under different methods. AuNPeNH2 was synthesized using the 1-step method while the AuNPeCOOH and AuNPeOH synthesis was conducted by 2-steps.

with 1 PBS and harvested by trypsinization. All media, cell suspension and wash solution were collected and centrifuged. The cells were resuspended in a known volume of medium and mixed in 0.4% trypan blue solution (Wako Chemicals, Japan) before counting with a hemocytometer (SLGC, Japan). Triplicate samples for each treatment group were used (n ¼ 3). The cell number data were collected and analyzed by calculating the total viable cells in a volume of resuspension medium. Cell viability was calculated by dividing the total viable cell count of AuNP treated samples with total viable cell count of the control and the values represented in percentage. 2.5. Inductively coupled plasma optical emission spectroscopy (ICP-OES)

(i) One-step process: Aminethanthiol surface modified AuNPs were synthesized according to the method as described by Niidome et al. and Techane et al. [12,13]. In brief, 56 mg of gold salt HCl4Au$4H2O (Wako Chemicals, Japan) was dissolved in 100 mL of ultrapure water and 2.42 mg of aminethanethiol (Wako Chemicals, Japan) was added to the gold salt solution. Next, 250 ml of 10 mM NaBH4 was added dropwise and the solution was left to stir in the dark for at least 2 h after which the AuNP solution was stored at 4 C in the dark. (ii) Two-step process: AuNPs were synthesized by the citrate reduction method [14] from gold salt HCl4Au$4H2O (Wako Chemicals, Japan). The resultant citrate coated AuNPs were centrifuged and resuspended in a solution of equal parts of water and ethanol adjusted to pH11. Next, 2 mM of 3-mercaptoethanoic acid (Dojindo, Japan) for carboxyl group functionalization or 2-mercaptoethanol (Wako Chemicals, Japan) for hydroxyl group functionalization were added into the solution and the mixtures were stirred for at least 24 h to ensure optimal ligand exchange was carried out. Size characterization of the AuNPs was measured by dynamic light scattering (DLS) (JASCO, Japan) and transmission electron miscroscopy (TEM) (Jeol, Japan). From the TEM images, diameter size of at least 100 individual nanoparticles was measured per sample type and recorded with the Image-J software (developed by Wayne Rasband, National Institutes of Health, USA). The surface modified AuNPs were resuspended in ultra-pure water and measured by a zeta-potential analyzer (Otsuka Electronics, Japan) to verify the surface charge of the synthesized AuNPs. 2.2. Cell culture and treatment with surface modified AuNPs Human bone marrow-derived mesenchymal stem cells (hMSCs) were obtained from LONZA (Walkersville MD, USA). The cells were cultured in 75 cm2 tissue culture flasks (BD Falcon, USA) with normal cell culture medium at 37 C in humidified air containing 5% CO2. The cell culture growth medium was Dulbecco's modified Eagle's medium (DMEM, Sigma, USA) supplemented with 10% fetal bovine serum, 4500 mg/ L glucose, 4 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.1 mM nonessential amino acids, 0.4 mM proline, 1 mM sodium pyruvate and 50 mg/mL ascorbic acid. The hMSCs were seeded into 24-well plates at a density of 5000 cells/cm2 with growth medium. After 1 day, the media in the wells were changed to fresh growth media with 0.1, 0.5 and 1.0 nM of AuNPeNH2, AuNPeCOOH and AuNPeOH. Media was changed every 2e3 days until the experimental time point. Cells were also treated with AuNPecitrate as a control.

hMSCs were cultured with surface modified AuNPs as described in 2.3 for 21 days in osteogenic induction medium. The cells were washed thrice in 1 PBS and then trypsinized and centrifuged to remove the supernatant. Then 3 mL aqua regia was added to each sample for digestion of biological material and dissolution of the AuNPs. Next, the samples were diluted in MilliQ water before measurement with the SII Nanotechnology SPS3520UV-DD ICP-OES system (Hitachi, Japan). Data were calculated to represent the amount of Au per sample normalized against the total viable cell number measured as described in 2.4. The experiment was repeated in triplicate for each treatment group (n ¼ 3). 2.6. Histological staining for osteogenic differentiation markers After 21 days of culture with AuNPs as described in 2.3, histological staining for alkaline phosphatase (ALP) was carried out. The cells were washed with PBS twice and subsequently fixed with 4% paraformaldehyde for 10 min at room temperature. The fixed cells were washed thrice with PBS and incubated with 0.1% naphthol ASMX phosphate (Sigma, USA) and 0.1% fast blue RR salt (Sigma, USA) in 56 mM 2amino-2-methyl-1,3-propanediol (pH 9.9, Sigma, USA) working solution at room temperature for 10 min, washed with PBS twice and observed using an optical microscope (Olympus, Japan). For calcium phosphate Alizarin Red S (ARS) staining, the cells were washed twice with PBS, followed by fixation with 4% paraformaldehyde for 20 min at room temperature. After that, the cells were incubated with 0.1% Alizarin Red S (Sigma, USA) solution at room temperature for 30 min, washed twice with PBS and observed using an optical microscope. After which, the samples were air dried and the ARS staining was eluted with 5% perchloric acid. The solution from each well was then transferred to a 96-well plate and the absorbance was read with a spectrophotometer (Biorad, USA) at an absorbance wavelength of 405 nm. The experiment was repeated in triplicate for each treatment group (n ¼ 3). 2.7. Alkaline phosphatase (ALP) activity assay Alkaline phosphatase activity was assayed with the Sensolyte® pNPP Alkaline phosphatase assay kit (Anaspec, USA). Cells were seeded in 24-well plates and treated with AuNP as stated in 2.3. After 21 days, cells were harvested according to the manufacturer's protocol and the color change was measured with a spectrophotometer (Biorad, USA) at absorbance wavelength of 405 nm. Cells from each treatment group were also counted with a hemocytometer to determine total cell number per well. The amount of ALP was calculated per well and normalized to the total cell number per well. The experiment was repeated in triplicate for each treatment group (n ¼ 3). 2.8. RNA extraction and RT2 gene profiler PCR array

2.3. Osteogenic induction of hMSCs The hMSCs were seeded onto 24-well plates as stated in 2.2 and then treated with 0.5 nM surface modified AuNPs in osteogenic induction medium. The osteogenic induction medium consisted of DMEM supplemented with 1000 mg/L glucose, 584 mg/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.1 mM nonessential amino acids, 50 mg/L ascorbic acid, 10% FBS, 10 nM dexamethasone and 10 mM b-glycerophosphate. The media were changed every 2e3 days for 21 days. 2.4. Cell viability studies of AuNP treated hMSC (i) Live/dead fluorescence staining Cells were cultured on 48-well plates and treated with AuNPs as stated in 2.2 and 2.3. The experiment was performed using the Cellstain LiveeDead Double Staining kit (Dojindo, Japan) as per manufacturer's instructions. After 3 days of culture with AuNPs, the cells were washed with 1 PBS thrice and incubated in 2 mM calcein-AM and 4 mM propidium iodide (PI) in PBS for 15 min. The live/dead images were observed with an inverted fluorescence microscope (Olympus, Japan). (ii) Trypan blue exclusion assay Cells were cultured on 24-well plates and treated with AuNPs in osteogenic induction medium as stated in 2.3. After 21 days of culture, the cells were washed

hMSCs were cultured in 6-well plates and treated with AuNPs according to the same procedure in 2.3. After 21 days of culture with AuNPs, the cells were washed once in PBS and harvested for RNA extraction with the RNAeasy Minikit (Qiagen, Netherlands) according to the manufacturer's protocol. Total RNA was then converted to cDNA by the proprietary first strand cDNA synthesis kit included in the PCR array system (Qiagen, Netherlands). The osteogenic pathway PCR array system (Cat #: PAHS-026Z, Qiagen, Netherlands) was selected for this study. The cDNA and SYBR green Master Mix were added to each well of the array plate according to the manufacturer's instruction. Real time PCR was performed on the 7500 Real-Time PCR system (Applied Biosystems, USA). All target gene expression results were normalized to GAPDH. Statistical analysis and fold change calculations were performed with the provided software at the Qiagen PCR Array Data Analysis web portal (www.SABiosciences.com/pcrarraydataanalysis.php). Gene expression changes of target genes were compared with control group using the student t-test and values of P < 0.05 were considered to be statistically significant. The experiment was repeated in triplicate for each treatment group (n ¼ 3). 2.9. Statistical analysis Statistical analysis was performed with the GraphPad Prism software (GraphPad Software, USA). All data were shown as mean ± standard error of the mean (SEM) with experiments repeated in triplicate (n ¼ 3). A one-way analysis of variance (ANOVA) with Tukey's post hoc test for multiple comparisons was used for statistical analysis. A value of P < 0.05 was considered to be a statistically significant difference.



3. Results

Table 1 AuNPs size characterization and zeta-potential values.

3.1. Characterization of AuNPs The AuNPs were functionalized with amine (eNH2), carboxyl (eCOOH) and hydroxyl (eOH) terminated alkanethiols. The features of the resulting AuNPs after ligand exchange were observed under TEM (Fig. 1). The AuNPs were well dispersed, homogenous in size and spherical. AuNPeNH2 generally showed a larger particle size distribution compared to the other samples. The zetapotentials of the AuNPs corresponded to their respective surface charges (Table 1) reflecting the functionalization of the alkanethiol groups on the AuNP surface. The sizes measured in the TEM images showed AuNPs with diameter sizes below 26 nm. The size measurement by DLS showed a higher mean diameter size reading as this reflected the hydrodynamic size of the AuNPs in aqueous solution. Energy-dispersive X-ray spectroscopy analysis showed the strong presence of gold element from the synthesized AuNPs in all samples (Supplementary Fig. 1). UVevis measurements were also taken showing the absorbance peak of the AuNPs around 520 nm (Supplementary Fig. 2). The absorbance peaks for all samples were around 520 nm wavelength which indicated the strong presence of the AuNPs in solution.

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TEM (nm) AuNPeNH2 AuNPeCOOH AuNPeOH AuNPecitrate

21.76 17.08 12.42 18.15

± ± ± ±

3.93 2.28 1.33 2.01

DLS (nm) 77.12 37.71 38.47 26.10

± ± ± ±

33.96 11.84 10.94 8.20

Zeta-potential (mV) 36.74 20.03 7.92 41.55

± ± ± ±

2.35 1.50 7.19 0.92

untreated control (Fig. 2). After 3 days of culture in AuNPs containing medium, the AuNPs were seen as dense and dark blue clusters in the cells (Fig. 2). The amount of AuNP uptake was dependent on concentration, the more AuNPs were observed in the cells treated with higher concentration of AuNPs. The hMSCs appeared to uptake the positively charged AuNPeNH2 more than the negatively charged AuNPeCOOH. However, there appears to be fewer cells in wells treated with 1.0 nM AuNPs after 3 days of culture compared to the control. Hence, 0.5 nM AuNP concentration was used in subsequent treatments to avoid adverse toxicity reactions. After treatment with the different surface modified AuNPs, the cells showed no differences in morphology after 3 days and 7 days of culture (Supplementary Fig. 3). To quantify these observations, cell viability and proliferation were further investigated for culture at the high concentration (0.5 and 1 nM) of AuNPs.

3.2. Cell culture with AuNP treatment 3.3. Effect of AuNPs on cell viability and proliferation The effect of AuNP surface charge and concentration on hMSCs was investigated. The hMSCs were cultured in growth medium supplemented with AuNPeNH2 and AuNPeCOOH at different concentrations (0, 0.1, 0.5, 1 nM). The cells were observed for up to 3 days in culture. Cell morphology did not appear different from the

Live/dead staining was carried out on samples treated with 0.5 nM concentration for 3 days and it showed good cell viability with few dead cells (Fig. 3A). As observed, the cells treated with 1.0 nM concentration showed some reduction in cell number as

Fig. 1. Transmission electron microscopy (TEM) of synthesized AuNPs with different surface modification. (A) AuNPeNH2 (B) AuNPeCOOH (C) AuNPeOH (D) AuNPecitrate (negative charge; unmodified control).


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Fig. 2. Photomicrographs of hMSCs after 3 days of culture with AuNPeNH2 and AuNPeCOOH at different concentrations 0 (control), 0.1, 0.5, 1 nM. Black arrowheads point to AuNP clusters in the cell. Scale bar ¼ 200 mm.

Fig. 3. (A) Live/dead fluorescence cell staining after hMSCs were cultured for 3 days without AuNPs (control) or with 0.5 nM AuNP concentration in growth medium (GM) and osteogenic induction medium (OM). Green staining indicates live cells and red staining indicates dead cells. Cell numbers were counted with a hemocytometer after hMSCs were cultured with (B) GM and (C) OM in 0.5 nM AuNPs for 1, 3 and 7 days. Data represent mean ± SEM, n ¼ 3. *, significant difference compared to control, P < 0.05. Scale bar ¼ 1 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

compared with the untreated controls (Supplementary Fig. 4A). The viable cell number was counted by trypan blue staining. The results showed that the cells treated with 1.0 nM surface modified AuNPs had lower than 90% viable cells with AuNPeNH2 and AuNPeOH showing lower than 80% cell viability compared with the control

(Supplementary Fig. 4B). Therefore, the 0.5 nM concentration was used in subsequent experiments to avoid adverse toxicity issues. Next, in order to verify that cell culture at 0.5 nM concentration do not adversely affect the cells, hMSCs were cultured in growth and osteogenic induction medium supplemented with 0.5 nM AuNPs



for 1, 3 and 7 days and the cells were collected and counted (Fig. 3B, C). The cell number showed no significant difference at day 1 and day 3 among the 4 treatment groups and control in both growth and osteogenic induction medium. Day 7 results showed some significant decrease in cell numbers for AuNPeNH2, eOH and ecitrate treatments in growth medium (Fig. 3B, n ¼ 3) but the difference was not significant when the cells were cultured in osteogenic induction medium (Fig. 3C). In both media, the cells cultured in AuNPeCOOH showed higher cell number than the control (Fig. 3B and C) although the difference was not statistically significant.

3.4. hMSCs with surface modified AuNPs in osteogenic induction medium The cells were cultured with AuNPeNH2, AuNPeCOOH, AuNPeOH and AuNPecitrate at 0.5 nM concentration to investigate cell response at a long period of culture in osteogenic induction medium (Fig. 4A). Again, the cells appeared to tolerate well under AuNP treatment and there were no obvious changes in cell morphology. Dense clusters of AuNPs could be seen after 21 days of culture in osteogenic induction medium (Fig. 4A) and the uptake of AuNPeNH2 and AuNPecitrate was greater due to the appearance of larger amount of dark dense blue clusters AuNPs inside the cell and on the extracellular matrix (ECM). The change in cell morphologies from the AuNPs treated samples was comparable to the untreated control. Over 3 weeks in osteogenic induction medium, the cells lost their fibroblastic appearance and appeared round in shape (Fig. 4A). To quantify the cell viability and proliferation, the cells were counted with a hemocytometer after 0.5 nM AuNP treatment in osteogenic induction medium. Total viable cell number was counted by the trypan blue exclusion assay method (Fig. 4B, n ¼ 3).

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There was no significant difference when measured against the control across the different AuNPs with surface modifications but cells treated with AuNPeCOOH appeared to have slightly higher cell numbers compared with the other treatment groups. The results indicated that the cells were able to tolerate the treatment of AuNPs under both growth medium and osteogenic induction medium at 0.5 nM concentration. For the purpose of quantifying the amount of Au uptaken by hMSCs in long term culture, ICP-OES was performed for each treatment sample. hMSCs were cultured in osteogenic induction medium containing 0.5 nM AuNPs for 21 days and the uptaken AuNP amount was measured. The results showed a significantly higher amount of AuNPeNH2 followed by AuNPecitrate (Fig. 4C). AuNPeCOOH and AuNPeOH treatment showed the lowest AuNP uptake in hMSCs.

3.5. Effect of AuNP treatment on osteogenic markers hMSCs were treated with AuNPs in osteogenic induction medium for 21 days to investigate AuNP effect on hMSC osteogenic differentiation. ALP staining results showed slightly less intense staining in the AuNPeNH2 and AuNPeCOOH treated groups than that of AuNPeOH, AuNPecitrate and control (Fig. 5A). After 21 days of culture, the cells were harvested and processed for measurement with an ALP activity assay kit. Treatment with AuNPeNH2 and AuNPeCOOH showed that ALP amount per cell was significantly lower than the untreated control (Fig. 5B). On the other hand, treatment with AuNPeOH showed an elevated level of ALP as compared with the control (Fig. 5B). Treatment with AuNPecitrate showed the same level of ALP as the control. Calcium deposition was evaluated by Alizarin Red S (ARS) staining (Fig. 5A). Dark red staining indicated presence of calcium phosphate deposition in the cellular matrix and they were

Fig. 4. (A) Optical micrographs of the cells treated with 0.5 nM surface modified AuNPs after 21 days in osteogenic induction medium (OM). Black arrow heads indicate AuNPs clusters in the cells and ECM. (B) Total viable cell count of hMSCs after 7, 14 and 21 days of AuNP treatment in OM. Cells were treated with 0.5 nM surface modified AuNPs. (C) Amount of Au uptake by the hMSCs after 21 days in OM normalized against the viable cell number. Amount of Au was undetected in control and data was represented as 0.00. Data represent mean ± SEM, n ¼ 3. ***, significant difference, P < 0.001. Scale bar ¼ 100 mm.


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Fig. 5. Alkaline phosphatase (ALP) activity and matrix mineralization after treatment with AuNPs in osteogenic induction medium for 21 days. (A) ALP staining and Alizarin Red S staining. Purple staining seen as patches over the cell cytoplasm indicated ALP staining. Dark blue spots were accumulation of AuNPs in cells or extracellular matrices. Red web-like staining indicated calcium deposits on the ECM while the blue/purple spots indicated aggregations of AuNPs. (B) ALP activity assay. Data represent mean ± SEM, n ¼ 3. **, significant difference compared to control, P < 0.01; *, significant difference, P < 0.05. (C) Quantification of ARS staining matrix mineralization per well. Absorbance OD values were normalized per cell. Data represent mean ± SEM, n ¼ 3. **, significant difference compared to control, P < 0.01. Scale bar ¼ 500 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

observed in all treatment samples with the exception of AuNPeCOOH treated cells which showed a marked reduction in staining. The ARS staining was eluted and quantified. The AuNPeCOOH treated sample showed significantly lower staining compared with the control (Fig. 5C). The result indicated that AuNPeCOOH treatment had an inhibitive effect on matrix mineralization of hMSCs while AuNPeNH2, AuNPeOH and AuNPecitrate treatment showed slightly lower absorbance readings but the results were not significantly different from the control.

3.6. Gene expression profile of hMSCs after AuNP treatment In order to better understand and to elucidate the pathways involved upon AuNP exposure, gene expression profile of the hMSCs cultured with AuNPs was performed. In the RT2 PCR array, 84 genes related to the osteogenesis pathway were assayed. The hMSCs treated with different surface modified AuNPs produced different gene expression profiles after 21 days of culture in osteogenic induction medium (Fig. 6 and Table 2). AuNPeNH2 and AuNPecitrate treatment induced significant down-regulation of osteogenesis genes while the AuNPeCOOH and AuNPeOH initiated some differential gene expression change. Key osteogenesis genes like ALPL were down-regulated in AuNPeNH2 and AuNPeCOOH treatments, which corresponded well with our ALP activity results. However, RUNX2 showed no significant difference among all treatment groups. AuNPeNH2 and AuNPecitrate treated cells showed a general down-regulation of cell adhesion genes

(cadherins and integrins) which may suggest changes in the cell interaction with the ECM due to the presence of these functional groups. Surprisingly, AuNPeOH treatment induced a significant upregulation of bone morphogenic protein 1 (BMP1) while it is significantly down-regulated in both AuNPeNH2 and AuNPeCOOH treated hMSCs (Fig. 6C and Table 2). These results also suggest that the hydroxyl moiety could be more favorable towards matrix development and osteoblast differentiation. Analysis of statistically significant results showed that AuNPeCOOH indeed caused a significant downregulation of several extracellular matrix genes such as the collagen genes COL 10A1, 14A1, 15A1, 3A1 and 5A1, fibronectin (FN) and biglycan (BGN), which correlates with the reduction in matrix mineralization seen previously. Moreover, genes involved with collagen biosynthesis like serpin peptidase inhibitor clade H (SERPINH1), SPP1 and BMP1 were also down-regulated. In contrast, genes for signaling molecules like integrin-alpha 1/2/M (ITGA1/2/M), fibronectin receptor integrin-beta 1 (ITGB1), SMADs 1/2/3/5, transforming growth factor (TGF-b 2/3), tumor necrosis factor (TNF) and Sox9 were strongly up-regulated in AuNPeCOOH treated cells. Taken together, the results suggest that while the AuNPs did not hinder the expression of the signaling molecules towards osteogenic differentiation, exposure to AuNPeCOOH inhibited some downstream pathways involved with the expression of ECM proteins and matrix mineralization. Also, the up-regulation of growth factors could imply a shift from osteogenic differentiation towards cell proliferation in this treatment group.



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Fig. 6. Gene expression profile results of hMSCs treated with (A) AuNPeNH2, (B) AuNPeCOOH (C) AuNPeOH and (D) AuNPecitrate. The graphs show fold changes of target genes with significant P-values (P < 0.05; mean ± SEM, n ¼ 3). Fold regulation value reflected is the mean of triplicate samples.

4. Discussion 4.1. hMSC interaction with surface modified AuNPs The nature of the charged surface renders an affinity for cellular uptake. The positively charged surface is known to be preferential for increased cellular uptake [9,15] as this is attributed to the largely negatively charged surfaces of cells. An increase in contact with the nanoparticles could also increase the biological effects [16] and risk for toxicity [17]. Knowledge of the uptake property opens the opportunity for strategic use of surface functionalization to enable gene transfection [18,19] or manipulation of endocytosis [20]. The type of functional group did not seem to affect the toxicity of hMSCs as there were no adverse effects on cell viability and proliferation

after 21 days of culture. Generally, surface functionalization does not appear to cause adverse toxicity [21,22] however such toxicity is also known to occur and may be specific to cell type or environment [23]. 4.2. Surface modified AuNPs on osteogenic ECM mineralization One of the interesting observations was that AuNPeCOOH treatment appeared to affect the deposition of the ECM upon osteogenesis while AuNPeNH2, AuNPeOH and AuNPecitrate showed matrix deposition comparable to control. Other studies have also shown that substrates coated with carboxylic functional groups also show reduced matrix mineralization [2,24]. It appears that exposure to the carboxyl moiety is enough to elicit such


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Table 2 RT2 PCR array gene profiler results for AuNPeNH2, eCOOH, eOH and ecitrate. Fold change with respective P-values. The P-values are calculated based on a Student's t-test of the triplicate 2^(DCt) values for each gene in the control group and treatment groups. Gene symbol ACVR1 AHSG ALPL ANXA5 BGLAP BGN BMP1 BMP2 BMP3 BMP4 BMP5 BMP6 BMP7 BMPR1A BMPR1B BMPR2 CALCR CD36 CDH11 CHRD COL10A1 COL14A1 COL15A1 COL1A1 COL1A2 COL2A1 COL3A1 COL5A1 COMP CSF1 CSF2 CSF3 CTSK DLX5 EGF EGFR FGF1 FGF2 FGFR1 FGFR2 FLT1 FN1 GDF10 GLI1 ICAM1 IGF1 IGF1R IGF2 IHH ITGA1 ITGA2 ITGA3 ITGAM ITGB1 MMP10 MMP2 MMP8 MMP9 NFKB1 NOG PDGFA PHEX RUNX2 SERPINH1 SMAD1 SMAD2 SMAD3 SMAD4 SMAD5 SOX9 SP7 SPP1 TGFB1

AuNPeNH2

P-value

AuNPeCOOH

P-value

AuNPeOH

P-value

AuNPecitrate

P-value

2.080 1.341 1.901 2.537 1.853 1.401 1.897 2.621 1.341 5.870 1.341 2.990 1.341 3.498 4.801 3.972 2.633 3.018 2.184 2.149 1.566 1.105 4.377 1.655 1.468 2.395 1.401 1.725 2.169 1.310 1.392 2.194 1.026 2.056 2.389 3.038 2.651 6.681 2.357 4.691 10.079 1.651 1.385 1.870 3.741 2.179 2.585 2.412 1.341 3.175 4.132 1.945 1.102 2.969 1.357 1.184 2.688 2.468 2.676 4.959 3.639 1.115 2.790 2.149 2.962 2.895 1.973 3.038 3.547 1.636 1.749 1.112 1.468

0.138 0.285 0.043 0.077 0.187 0.015 0.000 0.378 0.285 0.034 0.285 0.249 0.285 0.055 0.017 0.033 0.093 0.404 0.000 0.027 0.244 0.737 0.003 0.234 0.273 0.312 0.263 0.081 0.125 0.268 0.509 0.083 0.884 0.144 0.176 0.282 0.011 0.148 0.155 0.078 0.248 0.054 0.509 0.191 0.262 0.181 0.243 0.070 0.285 0.003 0.236 0.069 0.734 0.000 0.443 0.558 0.221 0.347 0.188 0.000 0.004 0.608 0.050 0.051 0.214 0.145 0.209 0.214 0.183 0.428 0.007 0.502 0.212

1.721 1.033 1.870 1.963 3.018 1.717 1.498 10.605 1.424 3.828 1.326 1.663 1.033 1.198 2.935 1.862 1.260 5.063 1.072 1.005 10.903 1.625 6.619 9.602 6.423 1.643 9.895 4.913 4.768 2.229 1.457 1.640 2.229 3.227 1.674 1.815 4.469 7.328 2.292 1.478 2.462 2.014 1.914 1.099 10.581 1.408 3.418 1.606 1.033 1.434 4.122 1.313 10.654 1.919 1.217 1.176 2.308 2.645 2.603 1.363 1.254 2.313 1.431 2.340 4.019 2.585 3.212 2.627 3.182 7.621 1.195 2.412 1.427

0.119 0.825 0.043 0.039 0.006 0.001 0.001 0.055 0.454 0.001 0.487 0.694 0.825 0.679 0.001 0.033 0.712 0.066 0.271 0.914 0.047 0.016 0.002 0.097 0.060 0.618 0.022 0.013 0.052 0.007 0.200 0.115 0.010 0.004 0.111 0.723 0.005 0.002 0.041 0.387 0.648 0.027 0.380 0.758 0.001 0.522 0.037 0.140 0.825 0.051 0.038 0.265 0.009 0.000 0.971 0.936 0.184 0.330 0.051 0.023 0.128 0.125 0.217 0.045 0.012 0.032 0.023 0.100 0.032 0.030 0.081 0.006 0.196

6.021 6.021 1.408 1.495 1.602 1.130 1.130 2.119 6.021 1.338 6.021 1.870 6.021 2.250 3.117 2.324 2.417 2.955 1.424 1.526 2.389 1.519 1.064 1.651 1.092 3.371 1.200 1.143 1.444 1.057 2.139 1.173 1.317 1.245 1.028 1.733 1.040 2.720 1.382 2.362 1.941 1.292 3.846 1.203 2.271 1.941 1.533 1.189 6.021 1.757 2.682 1.200 5.490 1.870 10.853 1.251 2.915 1.901 1.694 1.187 1.388 1.278 1.421 1.125 2.340 1.910 2.561 2.664 2.229 4.801 6.853 2.308 1.043

0.146 0.146 0.120 0.281 0.298 0.296 0.020 0.394 0.146 0.416 0.146 0.347 0.146 0.124 0.049 0.089 0.262 0.428 0.035 0.093 0.141 0.037 0.728 0.855 0.856 0.286 0.494 0.648 0.287 0.919 0.267 0.926 0.310 0.490 0.835 0.388 0.825 0.242 0.376 0.183 0.410 0.200 0.214 0.637 0.347 0.294 0.394 0.639 0.146 0.141 0.290 0.538 0.151 0.071 0.117 0.483 0.261 0.617 0.322 0.285 0.122 0.765 0.296 0.503 0.283 0.280 0.151 0.249 0.259 0.319 0.085 0.058 0.734

1.457 16.261 1.313 1.519 1.254 1.408 1.162 1.220 16.261 1.125 16.261 2.997 16.261 2.351 2.790 3.387 6.528 7.982 2.061 1.283 3.379 1.248 2.271 1.292 1.263 9.105 1.382 1.278 1.344 1.050 5.618 9.492 1.398 1.133 2.179 2.942 1.313 2.962 1.678 2.908 1.392 1.505 10.387 2.855 1.217 1.558 1.963 3.706 16.261 3.204 2.451 1.167 14.825 3.212 1.765 1.617 7.872 5.134 2.250 1.379 1.883 2.209 2.099 1.028 1.625 2.520 2.066 3.110 2.555 3.613 3.356 2.757 1.332

0.306 0.179 0.299 0.252 0.597 0.022 0.118 0.525 0.179 0.793 0.179 0.256 0.179 0.125 0.034 0.062 0.208 0.231 0.004 0.929 0.097 0.424 0.195 0.750 0.423 0.209 0.318 0.488 0.357 0.709 0.213 0.092 0.293 0.717 0.322 0.296 0.759 0.230 0.303 0.252 0.957 0.099 0.196 0.280 0.527 0.462 0.305 0.048 0.179 0.015 0.306 0.634 0.181 0.013 0.328 0.299 0.206 0.263 0.243 0.030 0.070 0.365 0.084 0.744 0.387 0.181 0.196 0.215 0.230 0.351 0.222 0.010 0.394



9

Table 2 (continued ) Gene symbol TGFB2 TGFB3 TGFBR1 TGFBR2 TNF TNFSF11 TWIST1 VCAM1 VDR VEGFA VEGFB

AuNPeNH2

P-value

AuNPeCOOH

P-value

AuNPeOH

P-value

AuNPecitrate

P-value

3.379 2.085 4.902 2.313 1.491 2.695 1.954 1.694 2.609 1.995 1.366

0.141 0.072 0.067 0.030 0.376 0.325 0.281 0.064 0.229 0.278 0.253

2.835 2.061 1.468 1.382 5.736 1.067 2.149 5.005 1.674 1.840 1.836

0.029 0.030 0.376 0.163 0.027 0.717 0.169 0.008 0.515 0.352 0.039

1.995 1.765 1.945 1.130 4.122 1.697 1.404 1.697 1.509 1.647 1.005

0.269 0.123 0.228 0.602 0.190 0.712 0.427 0.104 0.400 0.355 0.855

2.214 1.363 2.949 1.366 11.132 9.339 1.853 2.308 1.471 1.733 1.069

0.248 0.780 0.121 0.281 0.191 0.288 0.305 0.031 0.456 0.339 0.733

changes in differentiating hMSCs. For bone mineralization, there are different schools of thoughts regarding the exact mechanism of calcium deposition in osteogenesis. Internalization of AuNPs may affect the process. Recent studies have suggested that regulation of calcium deposition may also be internally controlled within the cellular vesicles [25] which are also important cell organelles for the uptake of AuNPs. Hence the presence of AuNPs in these intracellular spaces may cause disruption in the internal processes for calcium phosphate deposition in the ECM. In addition, calcium ions are known to preferentially adhere to acidic surfaces [26], hence the presence of many such carboxyl surfaces within the cell could cause dysregulation of the laying of calcium in the mineral matrix. Unlike AuNPeCOOH, the negatively charged AuNPecitrate did not hinder calcium deposition, so the differences between the two treatments might point to a functional group effect. Moreover, the citrate molecule was not conjugated to the AuNP surface like the carboxyl group, hence the surface groups might be affected by the presence of serum proteins in medium which could alter the surface charge and properties [27]. 4.3. Functional group effect on hMSC behavior and differentiation Surface modification and AuNP treatment did not seem to inhibit induction of osteogenic differentiation in hMSCs. Previous studies on unmodified AuNPs found that AuNP treatment promote osteogenesis in hMSCs [11,28,29] while no significant differences were observed in this study. It could be that these observations are influenced by AuNP concentrations and culture time. Most studies focus on the short term acute effects of AuNP exposure but long term culture with AuNPs could change gene expressions levels and cell behavioral responses [30,31]. Hence it is also important to investigate such exposure especially as AuNPs are being put to use for long term clinical applications [32,33]. 4.3.1. Carboxyl functionalization Treatment with AuNPeCOOH appeared to show the most interesting change in hMSCs. One study suggests that COOH functional groups on cell culture substrates may inhibit hMSC osteogenesis and matrix mineralization by changing the ECM structure leading to specific binding of integrin receptors on the cell surface [24] while others have found that COOH modified surfaces promoted chondrogenesis but not osteogenesis [3]. The regulation of osteogenesis differentiation is also a fine balance of osteogenic promotors and antagonists genes. Of particular note in this study are the FGF-2, TGF-b and TNF. FGF-2 and TGF-b are potent inducers of osteoblast proliferation but they could also inhibit alkaline phosphatase activity and matrix mineralization in osteoblasts [34e36]. The up-regulation of FGF-2 could imply a dedifferentiation or a reversal of emphasis on cell proliferation rather than bone mineralization in differentiating hMSCs [36]. Moreover, an increase in TGF-b expression, in association with SMAD 3, could

also hinder ALP activity and mineralization in osteoblasts [37] thereby promoting cell growth for a vast potential in many bone tissue regeneration strategies [38,39] (Fig. 7). TNF, while ordinarily considered to be a pro-inflammatory cytokine, may also have important roles in the wound healing process in bone by promoting vascularization and various supporting functions [40e42] and was also found to decrease collagen expression in hMSC cultures [43]. Such findings could warrant further investigation as such observation is dependent on cell type and environmental signals but it provides some insight into the cellular mechanisms on exposure to surface functionalized AuNPs. 4.3.2. Hydroxyl functionalizations Interestingly, our results showed that AuNPeOH increased ALP activity in differentiating hMSCs. Some studies have shown that hydroxyl functionalized surfaces could promote ECM growth and osteogenesis in hMSCs [44,45] more than amine or carboxyl surfaces. It has been reported that the hydroxyl functional group may help facilitate bone apatite formation and growth [46]. In addition, the up-regulation of BMP1 could support the cleaving of procollagen complexes for the development of the ECM. 4.4. Implications for tissue engineering Surface modified AuNPs showed differential responses to osteogenic differentiation in hMSCs. The promotion of cell growth by AuNPs could signify the usefulness of AuNPeCOOH on encouraging

Fig. 7. Schematic flowchart of the influence of AuNPeCOOH exposure on hMSCs. Long term exposure to carboxyl functionalized AuNPs in the cells could cause an upregulation of TGF-b (together with Smad3) and FGF-2 expression. This in turn impacts downstream pathways to shift the cells towards proliferation and inhibition of osteogenesis and matrix mineralization.


10


the repopulation of osteoblast cell density at sites of injury. Such ex vivo expansion of cells have also found to be able to retain their osteogenetic potential in the long term [36,47]. Hence subsequent matrix mineralization may also be possible by exposing a different surface moiety to encourage production of matrix proteins in these pre-osteoblasts. Therefore the manipulation of surface moieties must be managed to give further control over the osteogenesis of hMSCs. 5. Conclusion Mesenchymal stem cells cultured with surface modified AuNPs showed no adverse cell viability and were well tolerated for up to 21 days. The surface modified AuNPs were taken up by hMSCs but positive charged surfaces promoted higher uptake in hMSCs. Surface modification did not inhibit osteogenic differentiation of hMSCs however AuNPeCOOH treatment reduced ALP activity and matrix mineralization in hMSCs compared to the controls. Gene expression profiles of AuNPeCOOH treated hMSCs showed that an up-regulation of growth factors FGF-2 and TGF-b genes could promote the cells towards proliferation as well as inhibit ECM development. These results will contribute to our growing knowledge of nanoparticles in biomimetic cues on hMSC differentiation and shed light on the possible roadmap on the control of these cues for tissue regeneration therapies. Acknowledgment This work was supported by the World Premier International Research Center (WPI) Initiative on Materials Nanoarchitectonics from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.03.001. References [1] Jiang J, Papoutsakis ET. Stem-cell niche based comparative analysis of chemical and nano-mechanical material properties impacting ex vivo expansion and differentiation of hematopoietic and mesenchymal stem cells. Adv Healthc Mater 2013;2:25e42. [2] Benoit DS, Schwartz MP, Durney AR, Anseth KS. Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. Nat Mater 2008;7:816e23. [3] Curran JM, Chen R, Hunt JA. The guidance of human mesenchymal stem cell differentiation in vitro by controlled modifications to the cell substrate. Biomaterials 2006;27:4783e93. [4] Seong JM, Kim BC, Park JH, Kwon IK, Mantalaris A, Hwang YS. Stem cells in bone tissue engineering. Biomed Mater 2010;5:062001. [5] Alkilany AM, Lohse SE, Murphy CJ. The gold standard: gold nanoparticle libraries to understand the nano-bio interface. Acc Chem Res 2013;46:650e61. [6] Cheng Y, Meyers JD, Broome AM, Kenney ME, Basilion JP, Burda C. Deep penetration of a PDT drug into tumors by noncovalent drug-gold nanoparticle conjugates. J Am Chem Soc 2011;133:2583e91. [7] Hutter E, Maysinger D. Gold nanoparticles and quantum dots for bioimaging. Microsc Res Tech 2011;74:592e604. [8] Arvizo R, Bhattacharya R, Mukherjee P. Gold nanoparticles: opportunities and challenges in nanomedicine. Expert Opin Drug Deliv 2010;7:753e63. [9] Liu X, Huang N, Li H, Jin Q, Ji J. Surface and size effects on cell interaction of gold nanoparticles with both phagocytic and nonphagocytic cells. Langmuir 2013;29:9138e48. [10] Hoskins C, Cuschieri A, Wang L. The cytotoxicity of polycationic iron oxide nanoparticles: common endpoint assays and alternative approaches for improved understanding of cellular response mechanism. J Nanobiotechnol 2012;10:15. [11] Yi C, Liu D, Fong CC, Zhang J, Yang M. Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through p38 MAPK pathway. ACS Nano 2010;4:6439e48.

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