Single or Mixed Tethered Peptides To Promote hMSC Differentiation

Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Single or Mixed Tethered Peptides To Promote hMSC Differentiation toward Osteoblastic Lineage Laurence Padiolleau,†,‡,§,⊥,¶ Christel Chanseau,†,‡,§ Steṕ hanie Durrieu,#,∥ Pascale Chevallier,⊥,¶ Gaet́ an Laroche,*,⊥,¶,△ and Marie-Christine Durrieu*,†,‡,§,△ †

Chimie et Biologie des Membranes et Nano-Objets (UMR5248 CBMN), Université de Bordeaux, Pessac, France CNRS and §Bordeaux INP, CBMN UMR5248, 33600 Pessac, France ⊥ Laboratoire d’Ingénierie de Surface (LIS), Département de Génie des Mines, de la Métallurgie et des Matériaux, Centre de Recherche sur les Matériaux Avancés (CERMA), Université Laval, Québec G1V 0A6, Canada ¶ Hôpital St-François d’Assise, Centre de Recherche du Centre Hospitalier Universitaire de Québec (CRCHUQ), Québec G1L 3L5, Canada # ARNA Laboratory, Université de Bordeaux, 33076 Bordeaux, France ∥ ARNA Laboratory, INSERM, U1212 − CNRS UMR 5320, 33000 Bordeaux, France

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ABSTRACT: The commitment and differentiation of human mesenchymal stem cells (hMSCs) are guided by bioactive molecules within the extracellular matrix. Among the various approaches to design biomaterials, the functionalization of biomaterial surfaces with peptides from the sequence of proteins from the extracellular matrix is quite common. The purpose of this functionalization is to recruit hMSCs and promote their differentiation into the appropriate lineage. The aim of this work was to investigate the influence of RGD and FHRRIKA peptides and peptide sequences taken from bone morphogenic protein (BMP-2) and histone H4 (osteogenic growth peptide; OGP) either tethered alone or as a mixture on the surface of a model material and to also examine the level of hMSC osteogenic commitment without using a differentiation medium. Grafting of the different peptides was assessed by X-ray photoelectron spectroscopy (XPS), while their surface density was quantified by fluorescence microscopy, and their surface properties were assessed by atomic force microscopy (AFM) and contact angle (CA). The osteogenic commitment of hMSCs cultured on the different surfaces was characterized by immunohistochemistry using Runx-2 as an earlier osteogenic marker and OPN, a late osteogenic marker, and by RT-qPCR through the expression of ColI-a1, Runx-2, and ALP. Biological results show that the osteogenic commitment of the hMSCs was increased on surfaces tethered with a mixture of peptides. Results indicate that tethered peptides in the range of pmol mm−2 were indeed effective in inducing a cellular response after 2 weeks of cell culture without using an osteogenic media. These findings contribute to the research efforts to design biomimetic materials able to induce a response in human stem cells through tethered bioactive molecules for bone tissue engineering. KEYWORDS: stem cells, biomimetic materials, bone tissue engineering, mimetic peptides, surface modification

1. INTRODUCTION

however, a material suitable for all applications has not yet been identified. The ideal material for tissue engineering displays perfect biocompatibility for cell seeding, homing, proliferation, and differentiation. The concept of “ideal material” can mainly be achieved by producing a very attractive surface for the cells. To engineer bone tissues, human mesenchymal stem cells (hMSCs) appear to be attractive cell candidates, as hMSCs are pluripotent

Tissue engineering has emerged from the biomaterials field and consists in restoring functional tissues with a combination of cells, scaffolds, and biologically active molecules.1 The objective of tissue engineering is thus to assemble functional constructs that repair, sustain, heal, or improve damaged tissues or whole organs. One key strategy to achieve this goal is the development of scaffolds to provide not only the early support for cell adhesion, but also a framework to organize the cells into the desired functional tissue.2 Until now, many natural and synthetic materials have been developed and used in tissue engineering;3 © XXXX American Chemical Society

Received: June 15, 2018 Accepted: November 15, 2018

A

DOI: 10.1021/acsabm.8b00236 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials

Figure 1. Peptide grafting procedure on PET surfaces.

could also be observed with OGP as one of the conjugated peptides.20,21 The goal of this study was therefore to develop surfaces with different peptides tethered onto the surface of PET material with a well-controlled density. PET material was chosen for its easy surface modification and as a model material. To assess the peptide grafting and their surface density, peptide-conjugated surfaces were characterized by X-ray photoelectron spectroscopy, fluorescence microscopy, atomic force microscopy, contact angle, and the Toluidine Blue O test. The biological impact of the tethered surfaces on the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage was assessed using RT-qPCR and immunochemistry.

and are thus able of proliferating and differentiating to various lineages including bone tissue.4 To produce materials capable of mimicking the stem cell niche, our approach was to functionalize a model material surface, namely, polyethylene terephthalate (PET) films, with molecules that would enhance the adhesion or induce the differentiation of hMSCs. As a result, the cell response toward the material would be controlled by intracellular mechanisms5 due to the interaction between transmembrane proteins and the modified surface.6 We investigated the impact of the grafting of adhesion peptides and growth factor mimetic peptides for hMSC differentiation into osteoblasts. For this purpose, we selected two pro-adhesive peptides containing either RGD or FHRRIKA (Phe-His-Arg-Arg-Ile-Lys-Ala), a heparin-binding domain peptide;7 the latter peptide is a more specific peptide toward osteoblast-like cells. Two peptides displaying potential for differentiation were also selected. The first peptide was taken from a bone morphogenetic protein-2 (BMP-2) sequence.8,9 BMPs are cytokines of the transforming growth factor beta family (TGF-β). This protein family is involved in bone formation,10−12 development, and remodeling.13 BMPs interact with their receptors I and II (BMPRI and BMPRII) through noncovalent bonds.14 Briefly, BMPs bind to BMPRII, after which the BMPRI receptors are phosphorylated. Subsequently, the activated BMPRI phosphorylates a group of SMAD proteins (SMAD1/5/8), which interact with a nuclear SMAD called SMAD4.9,10 As shown by Zouani et al., the concomitant grafting of the RGD sequence and a mimetic peptide of a growth factor, such as BMP, can further guide cells to differentiate.8 The second osteogenic peptide selected for this study was osteogenic growth peptide (OGP), an endogenous peptide found in the human blood system at concentrations in the micromolar level.15 Discovered in the early 90s, the peptide sequence (Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-GlyPhe-Gly-Gly) is taken from the C-terminal region of histone H4. However, it has been shown that the active sequence of this peptide is located between the 10−14 amino acid (OGP10−14) sequence.16,17 The signaling pathway of OGP is, however, not known with certainty. Moore et al. used this peptide tethered on polymer surfaces to evidence its activity on cell membranes.18 It appears that the specific orientation of the peptide has an impact, as the study by Panseri et al. showed that the best response was obtained when OGP10−14 was tethered by the N-terminal to the material.19 Furthermore, the effect of OGP10−14 has always been assessed individually. Given the biological synergy already observed between coconjugated peptides on surfaces, in terms of differentiation, it is suggested that this synergistic behavior

2. MATERIALS AND METHODS 2.1. Materials. PET samples were taken from a commercial crystalline biaxially oriented film obtained from Goodfellow (Lille, France). The bioriented film had a thickness of 75 μm. Inorganic reagents (NaOH, KMnO4, H2SO4, HCl, glacial acetic acid), acetone, acet oni trile , d imeth ylaminopropyl-3-e thylcarbodi imid eethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-(N-morpholino)-ethanesulfonic acid (MES), and Toluidine Blue-O (TBO) were purchased from Sigma-Aldrich (Lyon, France). GRGDSPC (RGD), GFHRRIKA (FHRRIKA), GYGFGG (OGP), RKIPKASSVPTELSAISMLYL, which is a BMP-2 mimetic peptide previously identified by our group (BMP),8,22 GRGDSPCTAMRA, and RKIPKASSVPTELSAISMLYL-FITC fluorescent peptides were synthesized by GeneCust, (Ellange, Luxembourg). 2.2. Methods. 2.2.1. Surface Preparation of PET and Covalent Grafting of Different Peptides. PET surfaces were functionalized according to the protocol described by Chollet et al.23 with some adjustments. Briefly, the PET surfaces were hydrolyzed and oxidized to generate carboxylic acid groups on the surface (referred to as PETCOOH). Oxidized surfaces were used as a control for the biological experiments. The surfaces were then immersed in a solution of ethylcarbodiimide hydrochloride (EDC) (0.2 M) + N-hydroxysuccinimide (NHS) (0.1 M) + 4-(morpholino)ethanesulfonic acid (MES) (0.1 M) in Milli-Q water to convert the surface carboxylic acid groups into activated esters. Because of the presence of MES during the activation step, the previously generated COOH group on the surface of the PET were not dissociated, as the pH of the solution remained acidic. Peptide immobilization onto the activated PET surfaces was subsequently performed by immersing the previous surfaces into the peptide solution in PBS, at 10−5 M (of one peptide or as 1:1 molar ratio of two peptides), for 16 h at room temperature. Following covalent immobilization, the surfaces were sonicated with Milli-Q water for 15 min to remove the physically attached peptides. Figure 1 illustrates the different surface modification steps. Surfaces with a single peptide and double grafted through the N-terminal to the surface were prepared. 2.2.2. Surface Characterization. 2.2.2.1. Toluidine Blue O. Following the oxidation step described previously, the concentration of carboxyl groups onto the film was assessed by means of the toluidine blue-O (TBO) protocol, in which the dye stains the negatively charged B

DOI: 10.1021/acsabm.8b00236 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials

each fluorescent peptide grafted was measured using the calibration curve (see Figure S2). 2.2.3. Cell Culture. Human MSCs from bone marrow (one donor) purchased from PromoCell (Heidelberg, Germany) were grown in mesenchymal stem cell basal media (MSCBM2) (PromoCell) in a humidified atmosphere containing 5% (v/v) CO2 at 37 °C. For each experiment, hMSCs between passages 4 and 5 were seeded on PET materials at a density of 5000 cells/cm2 in serum-free α-MEM during the first 6 h. The medium was then changed to α-MEM supplemented with 10% (v/v) fetal bovine serum FBS (Gibco) with no additional growth factors and was changed every 72 h. hMSC differentiation on the different PET substrates was evaluated after 14 days of cell culture. Because of the large number of materials required to perform the biological analyses, the decision was made to perform cell cultures at one specific time point. To define the optimal time to assess gene expression, exploratory experiments at 1, 2, and 4 weeks, using fluorescent microscopy, were performed with a smaller amount of materials. Oxidized PET was used as the control surface in each biological experiment. 2.2.4. hMSC Differentiation along Osteoblastic Lineage. 2.2.4.1. RT Quantitative Real-Time PCR. hMSCs were lyzed in TRIZOL reagent (Invitrogen) to isolate the total RNA, and a TurboDNA free kit (Ambion, Illkirch-Graffenstaden, France) was used to remove contaminating DNA from the RNA preparations. Two micrograms of purified total RNA was used to synthesize cDNA using Thermo Scientific Maxima Reverse Transcriptase (Thermo Scientific, Illkirch-Graffenstaden, France) and random primers (Thermo Scientific). cDNA aliquots (4 ng) were then amplified in a 10-μL reaction volume containing 500 nM primers and the SsoAdvancedTM Universal SYBR Green Supermix (BioRad, Marnes-la-Coquette, France) in a CFX96TM Real-Time PCR Detection System (BioRad). The PCR cycling parameters were as follows: denaturation at 95 °C for 30 s, followed by 40 cycles of PCR reactions (95 °C for 5 s and 60 °C for 10 s). Cq values for the gene of interest were normalized against RPC53 and PPIA. Bestkeeper software was used to determine the normalization effectiveness of each reference gene among all of the samples under study. The relative expression levels were calculated using the comparative method (2−ΔΔCt), with the controls arbitrarily set at 1. Table 1 lists the primers used for amplification.

carboxylic acid groups through ionic interaction. Briefly, a solution of 5.10−4 M of TBO was prepared by dissolving TBO powder in a NaOH solution (pH 10). The functionalized PET surfaces were individually placed in 15 mL tubes with 10 mL of TBO solution on a shaking plate for 6 h. Following TBO binding, the supernatant was removed from the tubes and the stained PET surfaces were successively washed twice with a NaOH solution (pH 10) and Milli-Q water. A solution of 1:1 v/v H2O:CH3COOH (5 mL) was added on the PET surfaces and left to react during 10 min to detach the TBO dye from the carboxyl groups. Thereafter, 200 μL of the washing solution from each sample was placed into a 96-well plate and the absorbance at 633 nm was measured using a UV−vis spectrometer. Nonspecific adsorption was assessed after 1 day of contact between the surface and the 1:1 v/v H2O/ CH3COOH solution. A calibration curve obtained with different dilutions of TBO in 1:1 v/v H2O/CH3COOH was used to determine the concentration of the carboxyl groups (see Figure S1). Calculation of the carboxylic acid surface concentration was based on the assumption of a 1:1 stoichiometric reaction between TBO and the carboxylic acid groups.24 The TBO assay was performed on spared surfaces and not on those used for further modification and biological assessment. 2.2.2.2. X-ray Photoelectron Spectroscopy (XPS). Surface chemical compositions were determined after each step of the peptide grafting by XPS using a PHI 5600-ci spectrometer (Physical Electronics, Eden Prairie, MN, USA). Survey spectra were recorded using a standard aluminum X-ray source (1486.6 eV) with charge neutralization, while high-resolution C 1s XPS spectra were recorded using a standard magnesium X-ray source (1253.6 eV) without charge neutralization. The detection angle was set at 45° with respect to the sample surface. The analyzed area was 0.5 mm2. The curve fitting procedures for C 1s were performed by means of a least-squares Gaussian−Lorentzian peak fitting procedure following Shirley background subtraction. The C−C and C−H contributions to the C 1s high-resolution spectra were referenced at 285 eV. Three measurements per sample on three different samples were carried out to ascertain the reproducibility of the surface chemistry. 2.2.2.3. Atomic Force Microscopy (AFM). AFM was used to characterize surface roughness and morphology (Dimension 3100, Digital Instruments, Veeco, Woodbury, NY, USA). It was performed in tapping mode with an etched silicon tip (OTESPA, tip radius