Different Sialoside Epitopes on Collagen Film Surfaces Direct

Dec 23, 2015 - Because no osteogenic or chondrogenic supplements in culture media were added, the inductive effect in terms of increased gene expressi...
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Different Sialoside Epitopes on Collagen Film Surfaces Direct Mesenchymal Stem Cell Fate Antonella Sgambato,† Laura Russo,† Monica Montesi,‡ Silvia Panseri,‡ Maurilio Marcacci,§ Elena Caravà,# Mario Raspanti,# and Laura Cipolla* †

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy Bioceramics and Bio-hybrid Composites Group, Institute of Science and Technology for Ceramics, National Research Council, Via Granarolo 64, 48018 Faenza, Ravenna, Italy § Laboratory of Biomechanics and Technology Innovation, Rizzoli Orthopaedic Institute, via di Barbiano, 1/10, 40136 Bologna, Italy # Department of Surgical and Morphological Sciences, Insubria University, Via Guicciardini 9, 21100 Varese, Italy ‡

S Supporting Information *

ABSTRACT: 3′-Sialyllactose and 6′-sialyllactose have been covalently linked to collagen films. Preliminary in vitro study on the behavior of mesenchymal stem cells (MSCs) in terms of cell viability, proliferation and induction of osteogenic and chondrogenic related genes has been performed. Results indicate that sialoside epitopes on collagen surface represent a suitable support for MSCs adhesion and cell proliferation, moreover, the neoglycosylation provide MSCs with different and specific stimuli, saccharide-type depending, in term of expression of osteogenic and chondrogenic related genes. In particular, 3′-sialyllactose significantly upregulate the expression of RUNX2 and ALP, well-known markers of osteogenesis, whereas 6′-sialyllactose up-regulate the expression of chondrocyte marker ACAN. Because no osteogenic or chondrogenic supplements in culture media were added, the inductive effect in terms of increased gene expression has to be ascribed uniquely to collagen surface functionalization. These results support the promising role of sialosides in the regulation of stem cells fate and open brilliant perspective for the future use of the presented approach toward osteochondral tissue engineering applications. KEYWORDS: collagen, sialic acid, carbohydrates, osteogenesis, chondrogenesis, mesenchymal stem cells

1. INTRODUCTION Innovative bioactive materials are designed to interact with the body, carry constructive signals and modulate biological properties in order to favor tissue regeneration. Recent improvements in biomaterial engineering and scaffold fabrication enable the development of ex vivo cell expansion systems as valuable tools for advanced cell-therapies, allowing differentiation of stem cells into specific lineages.1,2 It is wellrecognized that cells perceive and respond to their microenvironment;3,4 as a consequence, biomolecular and biophysical stimuli are an obvious need for the design of the new generation of biomaterials in order to mimic cell microenvironment and stem cell niche.,5,6 Several nanoscale fabrication technologies have been developed over the years to achieve spatial, topographical, biochemical, and biophysical control over cellular functions and differentiation, and for the delivery of soluble bioactive molecules7,8 at the site of interest and therefore create more functional tissue engineered constructs.9 In principle, the fine regulation of stem cell fate might allow the engineering of complex organs comprising multiple cell types, and in the future mesenchymal stem cells (MSCs) will probably find use in several autologous regenerative therapies. The cellular behavior and the molecular pathways involved in the interaction with the biomaterial must be deeply studied in order to understand the key issues needed to mimic the native © XXXX American Chemical Society

microenvironment and modulate cell fate. It is well-known that cell activity is strictly dependent upon material surface features, such as wettability, and chemistry (i.e., charge, functional groups). A possible approach to tune biomaterial surface characteristics in order to control cell behavior by providing adequate signaling is the surface immobilization of bioactive molecules in vivo, several bioactive molecules are embedded into the extra-cellular matrix (ECM),10 thus ECM proteins may be considered as biomaterials of choice for tissue regeneration.11,12 Among them, collagen is attracting great interest for tissue regeneration applications, since it is the main structural protein in the extracellular space in various connective tissues.13 In addition, several molecular signals may be used for biomaterial surface modification, in order to mimic the extracellular microenvironment: they comprise soluble macromolecules (i.e., growth factors, cytokines, or chemokines), proteins or glycans usually exposed on the surfaces of neighboring cells, or signaling fragments of insoluble macromolecules, such as laminin Special Issue: Current Trends in Functional Surfaces and Interfaces for Biomedical Applications Received: September 3, 2015 Accepted: December 9, 2015

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ACS Applied Materials & Interfaces or fibronectin,14 the most widely studied being the adhesive peptide arginine-glycine-aspartate, or “RGD”.15 It is well-established that glycans play an essential role in a plethora of biological events including cellular adhesion and migration, organism development,16 disease progression,17,18 and modulation of immunological responses.19 Their use as signaling molecules for material surface functionalization to control stem cell growth and differentiation is presently limited, due to the high complexity of their structure, synthesis and chemical manipulation. However, recent data highlight them as promising cues for tissue engineering and regenerative medicine applications.20,21 Given these premises, we propose herein a preliminary in vitro study on the behavior of MSCs grown on chemically modified surfaces of collagen film with relevant saccharidic structures (1 and 2, Figure 1). Cell viability, proliferation, and induction of osteogenic and chondrogenic related genes has been evaluated.

delay was 5 s. Water contact angle. Hydrophilic characteristics of the functionalized and pristine collagen films were evaluated by static contact angle (WCA) measurements, using a Data Physics OCA 20 instrument. 2.2. In Vitro Cell Culture. Mouse mesenchymal stem cells (C57BL/ 6 mMSCs, GIBCO) were cultured in DMEM Glutamax medium (Gibco) containing 10% Fetal Bovine Serum and 1% penicillinstreptomycin (100 U/mL to 100 μg/mL) and kept at 37 °C in an atmosphere of 5% CO2. Cells were detached from culture flasks by tripsinization, centrifuged and resuspended. Cell number and viability were assessed with the trypan-blue dye exclusion test. The films 3, 4, and pristine collagen (used as control group, CT) were previously washed in EtOH 70% for 10 min, followed by a wash in PBS 1× for 10 min, airdried and sterilized by UV irradiation for 15 min per side under laminar flow hood. In detail 10.0 mm × 10.0 mm films for cell morphology, cell viability and proliferation assay and 50.0 mm × 30.0 mm films for gene expression profiling were used. Samples were placed one per well in a 24multiwell plate or in 90.0 mm-Petri dish (depending on dimension) and presoaked in PBS 1× for 4 h. Then cells were plated at a density of 2.5 × 103 cells/cm2 on collagen films. In detail, a drop of 1 mL and 200 μL of culture medium, depending of the membrane dimension, containing cells were seeded on the upper collagen films allowing cell attachment for 1 h in the incubator before adding 9.0 or 1.0 mL of standard culture medium (αMEM Glutamax, 10% Fetal Bovine Serum and 1% penicillinstreptomycin 100 U/mL−100 μg/mL) without any osteogenic and chondrogenic supplements. All cell-handling procedures were performed in a sterile laminar flow hood. All cell culture incubation steps were performed at 37 °C with 5% CO2. The media were changed every 2 days. 2.3. Cell Morphology. Samples were washed with 1x PBS for 5 min, fixed with 4% (w/v) paraformaldehyde for 15 min and washed with 1x PBS for 5 min. Cellular permeabilization was performed with 1x PBS with 0.1% (v/v) Triton X-100 for 5 min. FITC-conjugated Phalloidin (Invitrogen) 38 nM in 1× PBS was added for 20 min at room temperature in the dark. Cells were washed with 1× PBS for 5 min and incubated with DAPI (Invitrogen) 300 nM in 1× PBS for 5 min. Images were acquired by inverted microscope (Ti-E fluorescence Nikon). One sample per group was analyzed at day 1. 2.4. Cell Viability and Proliferation Assay. The MTT reagent (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was prepared at 5 mg/mL in 1x PBS. Cells were incubated with the MTT reagent 1:10 for 2 h at 37 °C. Medium was collected and cells incubated with 1 mL of dimethyl sulfoxide for 15 min. In this assay, the metabolically active cells react with the tetrazolium salt in the MTT reagent to produce a formazan dye that can be observed at λmax of 570 nm, using a Multiskan FC Microplate Photometer (Thermo Scientific). Using a calibration curve, we represented the mean of the total cells detected on each film (N. Five films per group) at day 1, 2, 3, 7, and 14. 2.5. Quantitative Real-Time Polymerase Chain Reaction (qPCR). At day 7 and 14, cells grown on 3, 4 and pristine collagen films (CT), used as calibrator, were homogenized and total RNA extraction was performed by use of the Tri Reagent, followed by the Direct-zol RNA MiniPrep kit (Euroclone) kit according to manufacturer’s instructions. RNA integrity was analyzed by native agarose gel electrophoresis and quantification performed by the Qubit 2.0 Fluorometer together with the Qubit RNA BR assay kit, following manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed to cDNA using the high-capacity cDNA reverse transcription kit, according to manufacturer’s instructions. Quantification of gene expression, using Taqman assays (Applied Biosystems), for Runtrelated transcription factor 2 (RUNX2, Mm01340178), alkaline phosphatase (ALP, Mm00475834), transcription factor SOX-9 (SOX9, Mm00448840), Aggrecan (ACAN, Mm00545794) and glyceraldehyde 3-phosphate dehydrogenase, used as housekeeping gene, (GAPDH, Mm99999915) was performed by use of the StepOne Real-Time PCR System (Applied Biosystems). Experiment was done in triplicate, using three technical replicates for each experiment. Data were collected using the OneStep Software (v.2.2.2) and relative quantification was performed using the comparative threshold (Ct) method (ΔΔCt), where relative gene expression level equals 2−ΔΔC.23

Figure 1. Saccharidic structures used for collagen neoglycosylation: 6′sialyllactose (1, an α-2 → 6 sialoside) and 3′-sialyllactose (2, an α-2 → 3sialoside).

2. EXPERIMENTAL SECTION 2.1. Collagen Neoglycosylation, Functionalization, and Hydrophilicity Quantification. Insoluble Collagen Type I from bovine Achilles tendon was used for the preparation of films by solvent casting method as previously described.22 Briefly, the collagen was dissolved in acetic acid 0.5 M for 4 h at 40 °C. The suspension was homogenized with a mixer for 2 min at maximum speed. After removal of the aggregates, 40 mL of collagen solution was poured into a 8.5 × 12.5 cm2 culture multiwell lid and the solvent evaporated in the fume hood for 2 days. The collagen matrices were produced as thin transparent films (1 mg/cm2). Under the SEM, the film surface was rough and irregular, party covered by collagen fibril-like structures, randomly oriented. The cross-section, where visible, was consistent with the surface and contained other fibril-like fragments interspersed in an amorphous matrix. The film thickness is in the 6−7 μm range, as determined by SEM. A collagen patch (80 mg, 12 cm × 7 cm) was immersed in 20 mL of 0.006 M 6′-sialyllactose or 3′-sialyllactose solution followed by the sequential addition of 0.003 M NaBH3CN in citrate buffer (pH 6.00) and reacted overnight. After this time, the collagen film was thoroughly washed first with 20 mL of Milli-Q (mQ) H2O three times for 20 min, and finally with 20 mL of ethanol for 20 min. NMR quantif ication. Functionalization of the native and neoglycosylated collagen films with maleic anhydride was performed to label and quantify unreacted -NH2 groups of lysine residues. Collagen matrices (32 mg) were immersed in a THF solution (0.04 M) of maleic anhydride in the presence of NaHCO3.The reaction was carried out at room temperature overnight. After washing with mQ H2O (three times for 15 min) and with ethanol (one time for 10 min) collagen films were dried under vacuum and then dissolved in 0.6 mL of 2 N NaOD in D2O. 1 H NMR spectra were recorded using a Varian 400 MHz, at room temperature. The 90° pulse-width (pw90) was calibrated, the number of scans varied depending on the signal-to-noise ratio, and the recycling B

DOI: 10.1021/acsami.5b08270 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces 2.6. Atomic Force Microscopy. All specimens prepared as described required no further treatment. Specimens were simply immobilized onto standard steel stubs with conductive biadhesive tape and observed with a Digital Instruments Multi-Mode Nanoscope IIIa equipped with a Digital Instruments Phase Extender and fitted with Nanosensors Tesp-SS silicon probes (k ≈ 42 N m−1 and f ≈ 300 kHz). All images were obtained in tapping-mode atomic force microscopy in air, at a scan speed of 1.5−2 Hz and at a resolution of 512 by 512 pixel. 2.7. Scanning Electron Microscopy. The collagen films were inspected by scanning electron microscopy (SEM). Fragments of film were mounted on suitable stubs with conductive biadhesive tape, coated with 10 nm gold in an Emitech K550 sputter-coater (Quorum Technologies, Laughton, UK) and visualized with a FEI XL-30 FEG high-resolution scanning electron microscope (FEI, Eindhoven, The Netherlands) operated in secondary electrons imaging (SE) at an acceleration voltage of 7−10 kV. Images were obtained in digital form as 8bpp grayscale TIFF files. 2.8. Statistical Analysis. Results of MTT assay were expressed as mean ± sd plotted on graph and the analysis of the effect of the films on cell culture was made by two-way ANOVA, followed by Bonferroni’s post hoc. Gene expression analysis was made by Multiple t test and correct the multiple comparisons using the Holm-Sidak methods and the data were expressed as mean and upper and lower value. Statistical analysis was performed by the GraphPad Prism software (version 5.0), with statistical significance set at p ≤ 0.05.

3. RESULTS AND DISCUSSION The combination of material science and cell-based therapies, in the past decades, led to enormous progress in the optimization of strategies for tissue engineering of functional osteochondral tissue regeneration.24 However, there are still many issues to be addressed. In this context, we propose a preliminary study of saccharidic structures containing sialoside epitopes, such as 1 and 2 (Figure 1) providing to the MSCs, considered the most valid candidates for osteochondral tissue engineering, a comfortable niche which stimulates cells to proliferate and to differentiate toward to osteogenic or chondrogenic lineage.25 In detail, we investigated whether these saccharidic structures might influence MSCs behavior and affect the early stage of osteogenesis and chondrogenesis process, in the search for new strategies for functional osteochondral tissue regeneration. It is worth of note that the saccharidic structures differs by the linkage between the sialic acid and the galactose unit, being α-2 → 6 in structure 1 and α-2 → 3 in structure 2. Sialic acids26 are found in human as the outermost residues on glycoproteins of the cell surface and possess carboxylate groups able to coordinate cations. Many studies have been published on the function of α-2 → 6 and α-2 → 3 sialosides found on glycoproteins exposed on cell surfaces, as for examples in osteogenesis,27 in angiogenesis,28,29 or in the regulation of complement system response by cancer and pathogens.30 Thus, to gain insights in the possible role of sialic acids in osteochondral tissue regeneration, MSCs were cultured on collagen-based films 3 and 4 (Figure 2A), obtained by chemical functionalization of collagen with saccharides 1 and 2 (Figure 1). The functionalization was achieved treating the collagen films with sialosides 1 and 2, in the presence of the reducing agent NaCNBH3.20 The degree of surface functionalization was quantified by NMR, and resulted 6 nmol/cm2. Collagen functionalization with carbohydrates, due to their polyhydroxylated nature, and moreover to the presence of the carboxyl groups characteristic of sialic acid residues causes a relevant increase in the hydrophilicity of the surface, as determined by contact angle measurement (Figure 2B). In both cases, contact angle decreases from 102.0° ± 1.4° for the

Figure 2. (A) Schematic representation of sialoside epitopes on collagen films; (B) variation in surface hydrophilicity as determined by water contact angle.

unfunctionalized collagen to 59.7° ± 4.5° for 3 and 72.8°± 6.0° for 4. A morphological evaluation of the collagen surface after the functionalization with sialosides was carried out by Atomic Force Microscopy (AFM). With this technique the surface of both specimens 3 and 4 was mostly represented by numerous, randomly oriented tactoids, a few micrometers long and up to 500 nm wide, with the distinctive 67 nm cross-banding of collagen (Figure 3B, C). The space among the tactoids was occupied by a finely grainy, featureless surface. No significant differences were found between the 6′-sialyllactose (3) or the 3′sialyllactose collagen (4). At higher magnification, the course of the collagen molecules along the tactoids became visible as a fine longitudinal texture (Figure 3A−C, right image, and Figure S1); an even higher magnification revealed the disordered, wavy course of individual collagen molecules filling up the space among the tactoids (Figure S2). Biological evaluation of MSCs morphology, viability, proliferation and the expression of the osteogenic and condrogenic C

DOI: 10.1021/acsami.5b08270 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. (A) Analysis of cell morphology by immunofluorescence image. Cells were spread with good morphology and firmly attached to the surface (day 1, CT group). Phalloidin in green stains for actin filaments and DAPI in blue stains for cell nuclei. Scale bars 50 μm. (B) Analysis of cell proliferation by the MTT assay, after 1, 2, 3, 7, and 14 days of MSCs culture on collagen films. *p ≤ 0.05, *** p ≤ 0.001; n = 5.

respect to the other groups. However, these differences were not observed after 7 and 14 days of culture, when films 3 and 4 showed comparable cell number to CT group. Results indicate that the collagen films positively influence the MSCs behavior in term of viability and proliferation, and that, in the long term culture (d7 and d14), the surface functionalization does not have detrimental effect on cell proliferation. To gain insights into the role sialo-functionalized collagen films as osteogenic and chondrogenic stimuli, the principal markers of these cell differentiative pathways have been evaluated. The progression of osteogenic differentiation was assessed quantifying RUNX2 and ALP gene expression, which are considered the main markers of osteoblast commitment.31 During early osteogenic process, RUNX2 acts as a transcriptional downstream activator of bone morphogenetic protein signaling, essential for osteoblast differentiation.32,33 In that phase, cells initiate the synthesis of extracellular matrix (ECM), which consists primarily of collagen type I. Subsequently, cells produce ALP and a variety of noncollagenous proteins, followed by induction of ECM calcification.34 The quantification of mRNA showed that film 4 significantly up-regulate the expression of RUNX2 and ALP after 14 days of culture: ∼ 3.29 and ∼2.88 fold change relative to CT, respectively (p = 0.0042 and p = 0.024). Moreover, a significant increase of both the osteogenic genes was observed (Figure 5) compared to film 3 (RUNX2 p = 0.035 and ALP p = 0.023). The potential effect of sialylated collagen films in the chondrogenic inductions of MSCs was evaluated by the relative quantification of both early and late chondrocyte markers, SOX9 and ACAN. SOX9 is a transcription factor that plays a key role in chondrogenesis and skeletogenesis and it has been shown to directly regulate the expression of ACAN, that codify for the predominant proteoglycan of cartilage extracellular matrix.35,36 The results showed that, although no differences were found in the expression level of SOX9, film 3 significant up-regulated the

Figure 3. AFM micrograph of (A) unfunctionalized collagen as control; (B) functionalized collagen with 6′-sialyllactose (3); (C) functionalized collagen with 3′-sialyllactose (4). On film surfaces is appreciable the ordered longitudinal course of collagen molecules as well as the crossbanding. As usual for AFM images, for each sample, the left image contains the height channel and the right one the corresponding Amplitude channel. The field of view in each image is 3 × 3 μm (for sake of clarity, higher dimension figures can be found in the Figure S1A−C).

related genes was then performed. In particular, organization of the cytoskeletal structure of actin filaments is an essential element in maintaining and modulating cellular morphology and structural integrity. The morphological analysis showed MSCs well adhered to the collagen films after 1 day with their typical spindle/fibroblast-like morphology without any difference among the groups (Figure 4A). Moreover the number of metabolically active cells was evaluated by MTT assay. An overall increase in cell proliferation was observed from day 1 to day 14 for all groups, proving that cell viability was not negatively affected by neoglycosylation (Figure 4B). CT group (cells seeded on pristine collagen film) exhibited the higher cell number after the first 3 days (p ≤ 0.05 at day 2 and p ≤ 0.001 at day 3), in D

DOI: 10.1021/acsami.5b08270 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 6. Relative quantification (2−ΔΔCt) of chondrogenic related gene expression after 7 and 14 days of MSCs cultured on the films. Mean, upper and lower value of the technical triplicate of SOX9 and ACAN in respect to CT, were indicated. Statistical significant differences among the samples are indicated in the graph: * p = 0.0015 and ** p = 0.0003.

Figure 5. Relative quantification (2−ΔΔCt) of osteogenic related gene expression after 7 and 14 days of MSCs cultured in direct contact with all the films. Mean, upper, and lower value of the technical triplicate of RUNX2 and ALP respect to the expression of the CT, were indicated. Statistical significant differences among the samples are indicated in the graph. RUNX2: * p = 0.035, **p = 0.0042. ALP: *p = 0.025, **p = 0.023, ***p = 0.024.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08270. Enlarged AFM images and AFM images with higher magnification (PDF)

expression of ACAN (Figure 6), compared to CT, after 7 and 14 days of culture (p = 0.0015 and p = 0.0003, respectively). It is worth noting that cells were grown without any osteogenic or chondrogenic supplements in culture media, thus, the inductive effect in terms of increased gene expression are uniquely a consequence of the sialoside moieties exposed on collagen surface. Overall, these data clearly indicate that MSCs are able to perceive the two different surface functionalization of collagen films, despite they differ only by a glycosidic linkage. The two sialosides are thus able to convey different molecular signals. Proliferation data highlight that in general collagen-based films represent a suitable support for rapid cell adhesion and cell proliferation. In fact, it was observed a great increase of cell number since day 1 after seeding (Figure 4). Moreover, the chemical functionalization with sialoside epitopes seems to provide MSCs with different and specific stimuli, saccharidedependent, in terms of osteogenic and chondrogenic related gene expression.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +390264483460. Fax: +390264483565. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

The authors gratefully acknowledge Fondazione CARIPLO, grant n° 2010−0378, 2011−0270 and MIUR, under project PRIN 2010/L9SH3K. Notes

The authors declare no competing financial interest.

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5. CONCLUSIONS In this work different collagen films exposing on their surface two different sialosides have been evaluated in vitro with MSCs for their ability to influence gene expression toward osteogenesis or chondrogenesis. Preliminary results suggest that sialylated collagen films provide a “functional network” for suitable MSCs/material interactions and cell stimulations for osteochodral tissue engineering. Deeper biological studies are needed in order to clarify the critical role of different carbohydrates in the commitment process of precursors stem cells.

ACKNOWLEDGMENTS The authors gratefully acknowledge Fondazione CARIPLO, under grant 2010-0378, 2011-0270 and MIUR, under project PRIN 2010/L9SH3K and Dr. Stefano Zanini, Department of Physics “G. Occhialini”, University of Milano-Bicocca, for contact angle measurements.

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ABBREVIATIONS ACAN, aggrecan gene DOI: 10.1021/acsami.5b08270 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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(22) Taraballi, F.; Zanini, S.; Lupo, C.; Panseri, S.; Cunha, C.; Riccardi, C.; Marcacci, M.; Campione, M.; Cipolla, L. Amino and Carboxyl Plasma Functionalization of Collagen Films for Tissue Engineering Applications. J. Colloid Interface Sci. 2013, 394, 590−597. (23) Livak, K. J.; Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-DeltaDelta C(T)). Methods 2001, 25, 402−408. (24) Kock, L.; van Donkelaar, C. C.; Ito, K. Tissue Engineering of Functional Articular Cartilage: the Current Status. Cell Tissue Res. 2012, 347, 613−627. (25) Bianco, P.; Riminucci, M.; Gronthos, S.; Robey, P. G. Bone Marrow Stromal Stem Cells: Nature, Biology, and Potential Applications. Stem Cells 2001, 19, 180−192. (26) Chen, X.; Varki, A. Advances in the Biology and Chemistry of Sialic Acids. ACS Chem. Biol. 2010, 5, 163−176. (27) Xu, L.; Xu, W.; Xu, G.; Jiang, Z.; Zheng, L.; Zhou, Y.; Wie, W.; Wu, S. Effects of Cell Surface α2−3 Sialic Acid on Osteogenesis. Glycoconjugate J. 2013, 30, 677−685. (28) Kitazume, S.; Imamaki, R.; Ogawa, K.; Komi, Y.; Futakawa, S.; Kojima, S.; Hashimoto, Y.; Marth, J. D.; Paulson, J. C.; Taniguchi, N. α2,6-Sialic Acid on Platelet Endothelial Cell Adhesion Molecule (Pecam) Regulates its Homophilic Interactions and Downstream Antiapoptotic Signaling. J. Biol. Chem. 2010, 285, 6515−6521. (29) Kitazume, S.; Imamaki, R.; Ogawa, K.; Taniguchi, N. Sweet Role of Platelet Endothelial Cell Adhesion Molecule in Understanding Angiogenesis. Glycobiology 2014, 24, 1260−1264. (30) Langford-Smith, A.; Day, A. J.; Bishop, P. N.; Clark, S. J. Complementing the Sugar Code: Role of GAGs and Sialic Acid in Complement Regulation. Front. Immunol. 2015, 6, 25−32. (31) Orlando, B.; Giacomelli, L.; Ricci, M.; Barone, A.; Covani, U. Leader Genes in Osteogenesis: a Theoretical Study. Arch. Oral Biol. 2013, 58, 42−49. (32) Franceschi, R. T.; Ge, C.; Xiao, G.; Roca, H.; Jiang, D. Transcriptional Regulation of Osteoblasts. Ann. N. Y. Acad. Sci. 2007, 1116, 196−207. (33) Gersbach, C. A.; Byers, B. A.; Pavlath, G. K.; Garcia, A. J. Runx2/ Cbfa1 Stimulates Transdifferentiation of Primary Skeletal Myoblasts into a Mineralizing Osteoblastic Phenotype. Exp. Cell Res. 2004, 300, 406−417. (34) Malaval, L.; Liu, F.; Roche, P.; Aubin, J. E. Kinetics of Osteoprogenitor Proliferation and Osteoblast Differentiation in Vitro. J. Cell. Biochem. 1999, 74, 616−627. (35) Ito, A.; Nagai, M.; Tajino, J.; Yamaguchi, S.; Iijima, H.; Zhang, X.; Aoyama, T.; Kuroki, H. Culture Temperature Affects Human Chondrocyte Messenger RNA Expression in Monolayer and Pellet Culture Systems. PLoS One 2015, 10, e0128082. (36) Diekman, B. O.; Christoforou, N.; Willard, V. P.; Sun, H.; Sanchez-Adams, J.; Leong, K. W.; Guilak, F. Cartilage Tissue Engineering Using Differentiated and Purified Induced Pluripotent Stem Cells. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19172−19177.

AFM, atomic force microscopy ALP, alkaline phosphatase MSCs, mesenchymal stem cells RUNX2, Runt-related transcription factor 2 SOX9, chondrogenic transcription factor

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DOI: 10.1021/acsami.5b08270 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX