Impact of RGD Nanopatterns Grafted onto Titanium ... - ACS Publications

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Impact of RGD Nanopatterns Grafted onto Titanium on Osteoblastic Cell Adhesion Minh Ngoc Nguyen,† Thomas Lebarbe,† Omar F. Zouani,‡ Loïc Pichavant,†,‡ Marie-Christine Durrieu,‡ and Valérie Héroguez*,† †

CNRS UMR5629, Laboratoire de Chimie des Polymères Organiques, IPB-ENSCBP, Université de Bordeaux 1, 16 avenue Pey Berland F-33607 Pessac, France ‡ CNRS UMR 5248 CBMN, Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux 1, 2 rue Robert Escarpit F-33607 Pessac Cedex, France ABSTRACT: This work reports on the synthesis of titanium bone implants functionalized with nanoparticles (NPs) containing Arg-Gly-Asp-Cys peptide (RGDC) and shows the adhesion behavior of cells seeded on these materials. RGDC peptides were first conjugated to a norbornenyl-poly(ethylene oxide) macromonomer (Nb-PEO). Then, functional NPs with a size of ∼300 nm and constituted of polynorbornene core surrounded by poly(ethylene oxide) shell were prepared by ring-opening metathesis polymerization in dispersed medium. The grafting density of these NPs on the titanium surface is up to 2 NPs·μm−2 (80 pmol of RGDC per cm−2 of NP surface). Cell adhesion was evaluated using preosteoblast cells (MC3T3-E1). Results of cells cultured for 24 h showed that materials grafted with NPs functionalized with RGDC peptides enhance specific cell adhesion and can create filopodia-like structures among NP sites by stressing the cells.

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INTRODUCTION Titanium and its alloys, which present good mechanical strength, high corrosion resistance, and good biocompatibility,1 are widely used in bone implants. Nevertheless, the modification of titanium surface is, in most cases, indispensable to improve its surface properties to enhance biointegration between the implant and the bone tissue.2,3 Therefore, various bioactive substances that can induce adhesion, proliferation and differentiation of cells were studied. In particular, proteins and peptides such as collagen, transforming growth factors-β1, bone morphogenetic proteins (BMPs), and Arg-Gly-Asp peptide (RGD) were grafted or coated onto titanium,4−11 polymer,12,13 and hydrogel14−16 surface. Among these bioactive molecules, RGD peptide that was discovered by Pierschbacher in the 1980s17 is known for inducing cell adhesion and accelerating bone formation.11,18 Cell adhesion plays an integral role in cell communication and regulation and is of fundamental importance in the development and maintenance of tissues. Cell adhesion is involved in stimulating signals that regulate cell differentiation, the cell cycle, and cell survival. The adhesion of cells to each other or to the extracellular matrix (ECM) is responsible for a wide range of normal and aberrant cellular activities. Cell adhesion constitutes the first stage of cell−material interactions, and the quality of this stage will influence the capacity of cells to proliferate and differentiate in contact with materials. Up to now, the immobilization of RGD peptides onto materials is performed by physical adsorption (through van der © 2012 American Chemical Society

Waals, hydrophobic, or electrostatic forces) or by chemical and electrochemical methods.18 The physical adsorption may not provide long-term implantation because of several drawbacks including low stability and uncontrolled desorption of biomolecules under physiological environments.19 On the contrary, chemical and electrochemical routes lead to the creation of a stable link between substrate and RGD peptides. The most common approach for achieving this goal is to graft RGD peptides either directly onto the surface via small organic molecules such as aminosilane and phosphonic compounds8,9 or through polymer spacer.11,20,21 Obviously, the aim is not to alter peptide conformation and orientation and then to disturb its bioactive center and hence to reduce its bioactivity.18 Among the studied polymers, poly(ethylene oxide) (PEO) and its copolymers are often the most widely used because of their excellent biocompatibility and biodegradability. It has also been reported that inserting a PEO chain between substrate and RGD peptide can favor the recognition of RGD peptide by osteoblastic cells.11 So far, however, no research has been found that examined the role of RGD-functionalized nanoparticles (NPs) to study cell attachment on Ti surface (various RGD densities, distributions on a nanometer scale could be achieved). Received: December 20, 2011 Revised: January 26, 2012 Published: January 28, 2012 896

dx.doi.org/10.1021/bm201812u | Biomacromolecules 2012, 13, 896−904

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polymerization of EO was carried out under an anhydrous inert atmosphere. ROMPs were performed in an inert glovebox. Average molar masses (M̅ n and M̅ W) and molar mass distribution (MMD) were determined using a size-exclusion chromatography (SEC) equipment consisting of Tosohaas TSK columns, a Jasco HPLC pump type 880-PV, a Varian refractive index detector, and a Jasco 875 UV/vis absorption detector. THF was used as the mobile phase at 1 mL·min−1. Polystyrene samples were used as standards to calibration. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a spectrometer Brüker 400 MHz in CDCl3 or D2O at room temperature. Particle sizes were measured by dynamic light scattering (DLS) on a Malvern Zetasizer 3000 HSA equipped with He−Ne laser. Samples for size measurements were prepared by dilution (∼100 times) of the CH2Cl2/Et−OH (v/v 35/65) mixture to minimize multiple scatterings caused by high concentration. Scanning electron microscopy (SEM) observations were performed as previously described.31 In brief, cells were washed with PBS 1× and then fixed with paraformaldehyde in PBS (4%) for 20 min at 4 °C. Samples were dehydrated in increasing concentrations of ethanol (30, 70, 80, 90, 95, and 100%) and critical point dried. Replicas were goldcoated and observed with a scanning electron microscope (SEM Hitachi S2500) at 10 kV. Synthesis of Functionalized Macromonomers and Nanoparicles. Synthesis of α-Norbornenyl-ω-hydroxyl Poly(ethylene oxide) (Nb-PEO-OH). α-Norbornenyl-poly(ethylene oxide) (Nb-PEO-OH) was synthesized by anionic polymerization using Nb-CH2OH activated by DPMK as an initiator as reported in a previous article.29 In our case, the synthesis features were chosen so as to target a molar mass of 3000−4000 g·mol−1. The yield was up to 90%, as evaluated by comparing the weight of recovered Nb-PEOOH to the theoretical one. 1H NMR (CDCl3, 400 MHz): δ = 5.85−6.04 (m, −CHCH−cycle), 3.40−3.80 (m, −CH2− O−), 2.72−3.30 (m, −CH−cycle), 1.04−1.74 (m, −CH2−cycle). Synthesis of α-Norbornenyl-ω-carboxylic Acid Poly(ethylene oxide) (Nb-PEO-COOH). Nb-PEO-OH (5 mmol) was dissolved in anhydrous THF (50 mL); then, a succinic anhydride solution (25 mmol in 5 mL of anhydrous THF) was added. After the addition of a solution of DMAP (0.5 mmol in 5 mL of anhydrous THF) under an inert atmosphere, the reaction mixture was stirred for 15 h at 60 °C. The solvent was then evaporated under reduced pressure. The obtained product was dissolved in dichloromethane and finally purified by precipitation three times in diethyl ether. 1H NMR (CDCl3, 400 MHz): δ = 5.85−6.04 (m, −CH CH−cycle), 4.10 (m, −CH2−OCO−), 3.40−3.80 (m, −CH2−O−), 2.60 (m, −CH2−COO−), 2.72−3.30 (m, −CH−cycle), 1.04−1.74 (m, −CH2−cycle). Synthesis of α-Norbornenyl-ω-Succinimidyl Poly(ethylene oxide) (Nb-PEO-SC). In a typical experiment, Nb-PEO-OH (1.5 mmol of powder) was dissolved in 25 mL of dry dioxane and then DSC (9 mmol in 20 mL of dry acetone) was added. DMAP (9 mmol in 15 mL of dry acetone) was added slowly under magnetic stirring, and the reaction was carried out at room temperature for 6 h. Nb-PEO-SC was directly precipitated from the reaction mixture by diethyl ether; then, several cycles of redissolving of the product in acetone and precipitation in diethyl ether were carried out to remove excess DSC and DMAP. The activated product was stored dry at 4 °C in fridge. 1H NMR (CDCl3, 400 MHz): δ = 5.85−6.04 (m, −CHCH−cycle), 4.40 (m, −CH2−OCO−), 3.40−3.80 (m, −CH2−O−), 2.72−3.30 (m, −CH−cycle), 2.77 (m, −CH2−CON−), 1.04−1.74 (m, −CH2−cycle). Synthesis of α-Norbornenyl-ω-RGDC Poly(ethylene oxide) (NbPEO-RGDC). RGDC peptide (45 mg, 0.1 mmol) was dissolved (final aqueous concentration of 1 mg/mL) in 30 mL of sodium bicarbonate buffer (50 mM, pH 8.2). Nb-PEO-SC (0.2 mmol) was dissolved separately in 15 mL of sodium bicarbonate buffer (50 mM, pH 8.2). Then, the Nb-PEO-SC solution was added dropwise to the peptide solution and reacted at room temperature under stirring for 2 h. NbPEO-RGDC was separated from the reaction medium by dialysis (using a dialysis membrane with a molecular weight cutoff of 1000 Da) and finally lyophilized. 1H NMR (D2O, 400 MHz): δ = 5.85−6.04 (m, −CHCH−cycle), 4.30 (m, −CH2−OCO−), 3.40−3.80 (m, −CH2− O−), 2.72−3.30 (m, −CH−cycle), 1.04−1.74 (m, −CH2−cycle).

In this study, we proposed the development of bone bioimplants enhancing cell adhesion by using polymer NPs functionalized with Arg-Gly-Asp-Cys peptides (NPs-RGDC) and grafted onto Ti-6Al-4V surface. This innovative technique permits the synthesis of materials having (i) a tunable density of grafted active principles onto materials, (ii) a high density of ligands taking into account the size and the specific surface of the NPs. Moreover, an interesting prospect using these NPs could be a multifunctionalization of materials using various active principles able to control cell adhesion, migration, proliferation and differentiation. NPs are synthesized by ring-opening metathesis polymerization (ROMP) in dispersion and have an average size of ∼300 nm. Particles are of the core−shell type with a polynorbornene (PNb) core and a PEO shell. To obtain this structure, norbornene (Nb) is copolymerized with norbornenyl-poly(ethylene oxide) macromonomers (Nb-PEO) in the presence of Grubbs first catalyst. The PEO-based macromonomer permits us to stabilize particles during their formation and also permits us to functionalize NPs easily with bioactive ligands or chemical functions.22−24 Moreover, PEO chains give furtivity properties in vivo.25 Such NPs have already been reported in our group26 and give rise to promising applications in the field of health sciences. For example, they are currently developed as nanocarriers for cancer therapy25,27 or as new bone implants grafted with drugs to avoid inflammatory reactions.28 To graft RGDC peptides onto the NPs surface, we first synthesized a Nb-PEO macromonomer activated by N,N′disuccinimidyl carbonate, which presents a high reactivity toward the amine functions of the peptide. The synthesis of a macromonomer terminated by a carboxylic acid function was also necessary to graft the particles onto the titanium surface activated by aminosilane molecules. This Article has been divided into three parts. The first part deals with the synthesis of RGDC-ended PEO macromonomer and RGDC-functionalized NPs (NPs-RGDC). In the second part, grafting of NPs onto Ti-6Al-4V surface has been investigated. Finally, cell adhesion has been evaluated using preosteoblast cells (MC3T3).

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EXPERIMENTAL SECTION

Materials. Nb (Aldrich, 99%), 5-hydroxymethyl-5-norbornene (NbCH2OH, Aldrich, 98%), N,N′-dicyclohexylcarbodiimide (DCC, Aldrich, 99%), disuccinimidyl carbonate (DSC, Acros, 98%), 4-dimethylaminopyridine (DMAP, Acros, 99%), N-hydroxysuccinimide (NHS, Alfa Aesar, 98%), 3-aminopropyltriethoxysilane (APTES, Aldrich, 99%), and Nsuccinimidyl-3-maleimido propionate (SMP, Aldrich, 95%) were used as received without further purification. Ethylene oxide (EO, Aldrich, 99.5%) was dried over sodium at −30 °C for 2 h and subsequently cryodistilled. Arg-Gly-Asp-Cys (RGDC, Genecust Europe, 98%) was stored at −20 °C and used as received. Bis(tricyclohexylphosphine) benzylidine ruthenium(IV) chloride (GC1, first generation Grubbs catalyst, Aldrich) was stored in a glovebox filled with argon prior to use. Diphenylmethyl potassium (DPMK) solution in THF was prepared as previously described.29 Dioxane, acetone, dimethylformamide (DMF), ethanol, and dichloromethane (CH2Cl2) were dried and cryodistilled prior to use. Water was deionized on milli-Qplus 185 and subsequently degassed by nitrogen bubbling. Rhodamine-terminated norbornenyl-poly(ethylene oxide) macromonomer (Nb-PEO-rhodamine) was prepared according to one of our previous reports.25 Ti-6Al-4V disks (8 mm in diameter) were produced by Good Fellow (France) with an Ra of 5 to 6 μm. Immobilization of RGDC directly onto Ti-6Al-4V surface was realized as described in our previous work.8 Methods. Monomer conversion was measured using gas chromatography (GC) as previously described.30 Anionic ring-opening 897


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Principal characteristic proton peaks of RGDC moiety: 4.6 ppm (−NHCH(CO)CH2COOH of aspartic acid), 4.05 ppm (−NHCH2C(O)− of glycine), and 1.9 ppm (−CH2CH2CH2NHC(NH)NH2 of arginine). Synthesis of Nanoparticles. In a 100 mL Schlenk round-bottomed flask, 380 mg (4.04 mmol) of Nb, 380 mg of macromonomers (NbPEO-COOH and Nb-PEO-RGDC or Nb-PEO-OH), 10 mg of a fluorescent macromonomer (Nb-PEO grafted with rhodamine, NbPEO-rhodamine), and some drops of dodecane (internal standard) were dissolved in 9 mL of a degassed solvent mixture composed of dichloromethane and ethanol (v/v 35/65). In a second 100 mL Schlenk round-bottomed flask, 20 mg (0.024 mmol) of Grubbs catalyst was dissolved in 5 mL of degassed dichloromethane/ethanol (v/v 50/50) mixture, and the solution was added quickly to the mixture of monomers. Reaction was carried out overnight under vigorous stirring at ambient temperature. The reaction was stopped by adding 0.2 mL of ethyl-vinyl ether as quenching agent. Grafting of NPs onto Ti-6Al-4V Surface: Biomimetic Modification. Silanization. The pretreatment of Ti-6Al-4V disk surface was performed as reported in our previous article.8 Then, Ti-6Al-4V disks were outgassed at 150 °C under secondary vacuum (10−5 Torr) for 20 h. In brief, each titanium disk was then put into a vial containing 2 mL of a 10−2 M APTES solution in anhydrous hexane under an Ar atmosphere, and the vial was stirred for 2 h. The silanized titanium disks were washed by putting them successively into several anhydrous hexane baths and by sonication for 30 min. Finally, they were degassed at 100 °C under secondary vacuum (10−5 Torr) for 4 h. Grafting of NPs on Titanium Surface. Two mL of NP suspension in dichloromethane/ethanol (v/v:35/65) were introduced in a roundbottomed flask with 2 mL of DMF. Dichloromethane and ethanol were removed by evaporating under reduced pressure. Each silanized disk was then put into a vial containing 2 mL of NPs suspension in DMF under inert atmosphere. An appropriate quantity of NHS/DCC was also added, and the vial was stirred for 4 h. Disks were taken out, washed successively with DMF and ethanol (several times for each solvent), dried, and stored in glovebox. Cell Adhesion Evaluation. Cell Culture. MC3T3-E1 (preosteoblast like) cells are a nontransformed cell line established from newborn mouse calvaria and exhibit an osteoblastic phenotype. Cells were grown on materials in α-MEM medium supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin. Cells were subcultured once a week using trypsin/EDTA and maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were used for experiments at passages 4 or 5. Throughout this study, cells were seeded at a density of 40 000 cells/cm2. The cells were seeded on surfaces without FCS during 12 h, which allows the NPs-RGDC to act on integrins without hassle of serum proteins. Immunostaining. After 24 h of culture, the cells on the surfaces were fixed for 30 min in 4% paraformaldehyde/PBS at 4 °C. After fixation, the cells were permeabilized in PBS solution of 1% TritonX100 for 15 min. Cell cytoskeletal filamentous actin (F-actin) was visualized by treating the cells with 5 U/mL Alexa Fluor 488 phalloidin (Sigma, France) for 1 h at 37 °C. Vinculin was visualized by treating the cells with 1% (v/v) monoclonal antivinculin (clone hVIN1 antibody produced in mouse) for 1 h at 37 °C. Then, we coupled to Alexa fluor 568 (F(ab′)2 fragment of rabbit antimouse IgG(H + L)) for 30 min at room temperature. The nucleus was visualized with DAPI. Images for this experiment were taken on a Leica SP5 confocal microscope with Metamorph software. For quantification of focal adhesion (FA) (vinculin) positive contacts areas, we used freeware image analysis ImageJ (NIH, http://rsb.info.nih.gov/ij/) described in previous works.32

structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth, and cell−cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable. A cell-adhesive peptide, Arg-Gly-Asp (RGD), was immobilized onto various material surfaces in an attempt to mimic an ECM structure.8,31−34 The RGD sequence is by far the most effective and most often employed peptide sequence for stimulated cell adhesion on synthetic surfaces,18 but, from the early days of RGD mediated cell adhesion, there has been an ongoing discussion about how many RGD molecules are required to induce not only cell attachment but also cell spreading and focal contact formation. As previously shown, there is clearly an impact of the RGD peptides density grafted onto materials on cell adhesion.32 Cells are likely to be able to respond to nanostructures because in vivo they live inside ECM containing nanoscale collagen fibrils and because their own surface is structured on the nanoscale level (receptors and filopodia). A crucial point in this field is understanding cell−material interactions. In this way, we propose here a strategy of nanostructured surface synthesis using polymer NPs functionalized with RGD peptides. The strategy of developing bioactive titanium materials is presented in Scheme 1. In the first part, the different macromonomers such as Nb-PEO-OH, Nb-PEO-COOH, NbPEO-SC, and Nb-PEO-RGDC that are necessary for the synthesis of multifunctional NPs (presenting at the same time COOH functions and RGD peptides) have been synthesized. Conjugating RGDC to Nb-PEO-OH was performed in two steps. Nb-PEO-OH was first activated to introduce a succinimidyl function at the end chain. Then, this compound was coupled to RGDC peptides. To finish, ring-opening metathesis copolymerization of Nb with Nb-PEO-OH, NbPEO-COOH, and Nb-PEO-RGDC has been carried out in dispersion. In the second part, the grafting of NPs onto Ti-6Al4V surface has been investigated. To ensure the efficient grafting of the NPs onto the Ti surface, we have added a PEO macromonomer terminated with a carboxylic function, and Ti surface has first been activated with aminosilane molecules. To finish, cell adhesion has been evaluated using preosteoblast cells. Synthesis of Functionalized Macromonomers and Nanoparticles. Synthesis of Nb-PEO-OH. Nb-PEO terminated by an OH function was synthesized by anionic polymerization of EO using Nb-CH2O− as initiator following a procedure, which we have previously developed (Scheme 1).29 Molar mass of PEO chain about 4000 g·mol−1 was targeted to induce good stabilization behavior as well as small particle size. Good control of molar mass and narrow molecular weight distribution was obtained as shown by SEC and NMR analysis and a high degree of functionalization is observed (Table 1). Synthesis of Nb-PEO-RGDC. As RGDC is an adhesion peptide, it is important to link it to NPs on a permanent bond to warrant its cell adhesion activity. In other words, this bond must be stable under bone physiologic environment (acidic medium). That is why the carbamate bond between RGDC and macromonomer is chosen. Macromonomer grafted by RGDC peptide was synthesized by a two-step protocol (Scheme 1). First, an activated norbornenyl-poly(ethylene oxide) (Nb-PEOSC) was formed by reacting Nb-PEO-OH macromonomer with

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RESULTS AND DISCUSSION ECM is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides 898


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Scheme 1. Synthetic Strategy to Access to Bioactive Titanium Bone Implants

N,N′-disuccinimidyl carbonate in the presence of DMAP as catalyst and in anhydrous dioxane/acetone solvent mixture according to the literature.35 1H NMR characterization indicates that polymer end chains were successfully modified by a succinimidyl carbonate group with its characteristic proton

peak (δ 2.7, 4H). Indeed, integration of the later peak over the peak area of Nb double-bond protons (δ 5.8 to 6.1, 2H) leads to a functionalization yield of 0.95 (Figure 1, Table 1). In the second step, reaction of RGDC peptide with Nb-PEOSC was carried out in aqueous buffer medium (NaHCO3) at a 899


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Table 1. Characteristics of Functionalized Macromonomers products Nb-PEO-OH Nb-PEO-SC Nb-PEO-COOH Nb-PEO-RGDC

M̅ n,NMR M̅ n,GPC M̅ w/ M̅ n,th (g mol−1)a (g mol−1)b (g mol−1)c M̅ n 3200

3900 4150 4100 4200

3700 3670 4030

1.07 1.09 1.09

yield (%)d

fe

95 75 60 92

0.90 0.95 0.90 0.33

Theoretical molar mass, M̅ n,th = (nEO)/(nNb−OH) × conv. × MEO × MNb‑OH with MEO = 44 g·mol−1 and MNb‑OH = 124 g·mol−1. bObtained by comparing the integration of the 1H NMR peaks corresponding to PEO (δ 3.58, 4H, Ib,b′, CH2CH2O) and that corresponding to Nb double bond (δ 5.8 to 6.1, 2H, Ia,a′, CHCH). cMeasured by SEC (RI detector, PS standards) and corrected by multiplying with a correction factor of 1.12 (for PEO of 2000−5000 g·mol−1). dMass yield obtained after purification. ef = Degree of functionalization. f Nb‑PEO‑OH = (M̅ n,NMR)/(M̅ n,GPC); f Nb‑PEO‑Su = (Ie,e′/4)/(Ia,a′/2); and f Nb‑PEO‑COOH = (Id,d′/4)/(Ia,a′/2). Ia,a′, Id,d′, and Ie,e′ are the NMR integrals of the peaks corresponding to double bond of Nb, four protons of carboxylic moiety, and four protons of succinimidyl moiety, respectively (details of 1H NMR peaks in Figure 1). Degree of functionalization of Nb-PEO-RGDC was calculated by comparing the 1H NMR integral of double bond of Nb to that of the peak at 1.9 ppm (−CH2CH2CH2NHC(NH)NH2 of arginine). a

Figure 2. 1H NMR (in D2O) spectra of RGDC and Nb-PEO-RGDC.

spectroscopy as well as revelation by ninhydrine (appearance of orange coloration characteristic of secondary amine function). A functionalization degree of 0.33 was found by comparing the integrals of characteristic peaks of grafted-RGDC and of Nb moiety (Table 1). This value means that 66% of RGDC used for the reaction were grafted to polymer chains (initial molar ratio of macromonomer to peptide of two). No efforts were made to remove unreacted Nb-PEO in this stage because it does not disturb the subsequent synthesis of NPs. Synthesis of Nb-PEO-COOH. To graft NPs onto the titanium surface, we synthesized macromonomer with carboxylic acid function at chain end. Previously, our group reported the synthesis of Nb-PEO-COOH macromonomer in two steps.24 In this contribution, a new route is proposed, allowing quantitative functionalization in only one step. For this purpose, reaction between Nb-PEO-OH and succinic anhydride was carried out in anhydrous THF using DMAP as catalyst (Scheme 1). FT-IR characterization (not presented here) showed a characteristic peak of carbonyl functions at 1740 cm−1, indicating that succinic anhydride efficiently reacted with Nb-PEO-OH. This statement was confirmed by 1H NMR analysis with the presence of characteristic signal of protons in α position of the ester function (δ 2.5, 4H, Hd,d′ Figure 1). Integration values for Hd,d′ and protons of Nb double bond

pH of 8.2. At this pH, the amine function of RGDC is in its basic form and potentially reactive toward the activated macromonomer. However, a molar ratio of 2 between macromonomer and the peptide is necessary to limit the competitive reaction of hydrolysis of the activated macromonomer. Figure 2 displays the 1H NMR spectra of RGDC and Nb-PEO-RGDC. Spectra were recorded in D2O, solvent in which RGDC and RGDC-grafted polymer have good solubility. The 1H NMR characterization of purified product (after dialysis to eliminate unreacted RGDC) confirmed the incorporation of the peptide into the macromonomer with the presence of characteristic proton peaks of RGDC (δ 1.9, 2H, −CH2CH2CH2NHC(NH)NH2 of arginine; δ 4.05, 2H, −NHCH2C(O)− of glycine; and δ 4.6, 1H, −NHCH(C O)CH2COOH of aspartic acid). The efficiency of the reaction was also proved by thin layer chromatography, MALDI-TOF

Figure 1. 1H NMR (in CDCl3) spectra of Nb-PEO-OH, Nb-PEO-SC, and Nb-PEO-COOH. 900


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the solvents of ROMP polymerization, we tried to use these solvents directly as medium for the grafting of NPs on titanium surface. As shown Figure 3, few NPs were grafted on the

(Ha,a′) gave a ratio corresponding to a functionalizing efficiency of 90% (Table 1). Nanoparticles Synthesis. NPs with and without RGDC functions were synthesized by ROMP in dispersion in dichloromethane/ethanol medium using Grubb’s catalyst as initiator (Table 2). As described in Scheme 1, particles obtained Table 2. Synthesis of NPs Using ROMP in Dispersion in Dichloromethane/Ethanol (v/v 35/65) Mixture at Room Temperature for 8 ha [Nb]/[M1]/ [monomers]/ conv. M̅ n,GPC M̅ w/ size of NPs [M2]/[M3] [GC1] (%)b (g·mol−1)c M̅ n (nm)d 90/0/1/1 90/1/1/0

140/1 140/1

100 100

105 000 25 000

1.58 2.28

330 280

PdI 0.052 0.062

a

Nb-PEO-rhodamine (10 mg) was used to obtain fluorescent NPs. Nb = Norbornene; M1= Nb-PEO-OH; M2 = Nb-PEO-COOH; M3 = Nb-PEO-GDC. bMeasured by gas chromatography with dodecane as internal standard. cMeasured by SEC (RI detector, PS standards) without correction of molar mass. dMeasured by DLS.

are composed of graft copolymer with a PNb main chain on which PEO functionalized grafts are randomly distributed. In both cases, total monomer conversions were obtained, as shown by GC. SEC indicated that macromonomer conversions were superior to 98%. These particles are monodisperse, spherical, and stable for several months, with an average particle sizes of 330 nm, which means a density of 80 pmol of RGDC per cm2 of NP (ca. 1.6 × 105 RGDC molecules per NP). This value is comparable to the density of RGD (55 pmol·cm−2) grafted on the surface of PEGhydrogels reported in the literature.15 Grafting of NPs onto Ti-6Al-4V Surface: Biomimetic Modification. The immobilization of NPs on Ti surface involves the activation of the inert Ti surface before binding of NPs. Using the surface activation process based on silanization reported in our previous paper,8 a surface with a homogeneous and high density of APTES molecules was obtained. Then, NPs were immobilized on titanium surface by reaction between carboxylic function of NPs and amine group of silane molecules available onto the titanium surface in forming stable amide bond. Different conditions of solvent, time, and surface state were examined (Table 3). As ethanol and dichloromethane are

Figure 3. SEM images of titanium surface before and after the immobilization of NPs under different conditions.

surface. This is probably due to the reaction of alcohol function with NHS. Replacing these solvents by anhydrous DMF avoids this problem and consequently leads to obtain a good immobilization of NPs onto the Ti surface with a density up to 2 NPs/μm2 (∼55 pmol/cm2 of RGDC peptides: nanospheres of 330 nm in diameter homogeneously functionalized with 80 pmol/cm2 of RGDC peptides are obtained). The distance between two NPs can be approximated by 0.8 μm, using the Poisson’s model. Given a previously publication,36 the RGDC density grafted onto the surface of the NPs as well as the global RGDC density on the biomaterial surface are sufficient to initiate interactions with integrins: the obtained densities are comparable to those naturally occurring in vivo.37 The roughness of the Ti surface does not seem to affect the grafting because the density of NPs grafted onto polished titanium and titanium with a Ra of 5 to 6 μm is similar. However, reaction time strongly affects the grafting. No particle aggregates were observed on the surface when the NP grafting duration is 4 or 12 h, whereas NPs tend to form clusters if the reaction time is longer (72 h), as shown in Figure 3. This phenomenon could be due to the linkage between NPs due to secondary reactions between carboxylic function (in excess to amine functions) and alcohol function (at the end-chain of NbPEO) available on the surface of NPs. Cell Adhesion Evaluation. The integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties.38 Indeed, the integrins play a key role in such interactions, mediating the assembly of multimolecular complexes that bridge between the ECM and actin cytoskeleton. Several intracellular anchor proteins are recruited to the adhesion site, ultimately inducing the assembly of complexes and FA and the associated cytoskeleton that create a different global organization. Herein, the potential of NPs functionalized with RGDC to promote the cells adhesion at the surface of Ti-6Al-4V was investigated using preosteoblast cells. SEM images of preosteoblast cells cultured for 24 h on Ti-6Al-4V functionalized with RGDC or NPs-RGDC

Table 3. Experimental Conditions and Results of the Immobilization of NPs Containing RGDC on Titanium Surfacea density time solvent (h) surface state ethanol DMF

4 4

DMF

4

DMF

72

observation

unpolishedb very little or no NPs unpolishedb NPs in the form of beads NPs in the form of polishedc beads coalescence of NPs polishedc (form of heap/ cluster)

of NPs of RGDC (NPs/μm2 of (pmol/cm2 Ti surface) of NP surface) ∼2

80

∼2

80

a

Mass of Nb-PEO-COOH = 26%. [NHS]/[Nb-PEO-COOH] 62/1. Titanium pellet was just cut (high roughness). cTitanium pellet was cut and polished (surface is like mirror after polishing process). Density of NPs on Ti surface was estimated from SEM images. Density of RGDC on NP surface was calculated by considering that NP has a spherical shape.

b

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Figure 4. Preosteoblast behavior on bioactive bioimplant. (A) SEM micrographs of Ti-6Al-4V functionalized with RGDC (1) and with NPs-RGDC (1′). In 1″ left, magnification of NPs-RGDC functionalized Ti-6Al-4V, (scale bar, 300 nm). In 1″ right, the scheme of this system. (B) SEM micrographs of preosteoblast cells on Ti-6Al-4V functionalized with RGDC and with NPs-RGDC after 24 h of culture (scale bar, 50 μm.). In (1) and (1′), the magnifications of cell topography on NPs-RGDC and the cytoplasmic extensions toward NPs-RGDC, respectively. (C) Immunofluorescence images of cells in Ti-6Al-4V functionalized with RGDC and with NPs-RGDC stained for F-actin (green), vinculin (red far), and nucleus (blue). The NPs-RGDC were stained in red. (D) Relative area of contacts on Ti-6Al-4V surfaces functionalized with RGDC and with NPs-RGDC.

compared with Ti-6Al-4V functionalized with RGDC (homogeneously). Cells are flat, spread out, and round-shaped on Ti-6Al4V substrate grafted with RGD peptides, and they become elongated on Ti-6Al-4V functionalized with NPs-RGDC. We observed that the projected areas of individual osteoblastic cells were maximal in the case of Ti-6Al-4V homogeneously functionalized

were realized (Figure 4B). (Ti-6Al-4V surface has been homogeneously functionalized with RGDC peptides following a wellestablished protocol described in a previous paper.39 The grafting density of such material is ∼600 pmol/cm2.) Results obtained from these images reveal that the NPs-RGDC complexes grafted onto Ti-6Al-4V clearly lead to a different cell adhesion for 24 h 902


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Notes

with RGDC. Regarding Ti-6Al-4V functionalized with NPsRGDC, the shape of cells is different with different attachment points, which are NPs-RGDC (Figure 4B,C). The interest of NPs is to guide cell extensions and induce novel cell shape because of their nanodistribution. Importantly, we clearly observe the presence of filopodia extensions for cells cultured on Ti-6Al-4V functionalized with NPs-RGDC. These filopodia extensions were characterized with greater FAs in their end than on Ti-6Al-4V functionalized with RGDC. Indeed, we observe the presence of a higher relative area of focal contacts per cell (Figure 4 D) on Ti-6Al-4V functionalized with NPsRGDC compared with Ti-6Al-4V functionalized with RGDC. These results suggest that cell adhesion could be manipulated by the distribution of NPs functionalized with RGDC. The process of the formation of filopodia structures formation was observed during cell culture (on the first 24 h, Figure 4C, right). We observe that cytoplasmic extensions toward NPs-RGDC are formed utilizing actin filaments (stage 1). Then, the cytoplasmic extentions interact with NPs-RGDC utilizing FAs (stage 2). Finally, the filopodia structures were stabilized by the maturation of the FAs induced by the RGDC peptide immobilized on the NPs (stage 3). In the Ti-6Al-4V functionalized with RGD, these filopodia structures were not observed (Figure 4C). The advantage of NPs-RGDC is the ability to manipulate the cell shape and thus the expression of selected cytoskeletal machinery. These results are promising from the induction of filopodia structures and by the maturation of FAs on these structures. These cellular characteristics are considered as a sign of a differentiation induction of cells and especially for adult stem cells toward osteoblast cells.40−42 These results suggest that cells interpret changes in the biomolecules distributions grafted onto material as changes in adhesion-ligand presentation and that cells themselves can adapt by creating filopodia extensions and maybe manipulate their fate. The biomaterials tested in this study may help us have a better understanding of the cell differentiation using functionalized implants instead of native ones. Further experiments in cell biology, such as expression of proteins responsible for cytoskeletal tension (myosin II and RhoA) and osteoblast differentiation (Runx2 and osteopontin), are currently ongoing.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the “Région Aquitaine”, the GIS “Advanced Materials in Aquitaine” as well as the “Agence Nationale pour la Recherche” (ANR) for their financial support. We thank Jérôme Kalisky, engineer within INSERM U1026, for the polishment of titanium and Dr. Floraine Collette, in postdoctoral position within LCPOIPB-ENSCBP, for supplying Nb-PEO-Rhodamine.

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CONCLUSIONS In summary, this work reports the synthesis of novel multifunctional NPs containing RGDC adhesion peptide and the evaluation of their role in osteoblast adhesion on Ti surface. The NPs with a size of ∼300 nm were prepared from Nb and macromonomers such as Nb-PEO-OH, Nb-PEO-COOH, and Nb-PEO-RGDC by ROMP in dispersion. Covalent immobilization of these NPs on the Ti surface was performed by reaction between carboxylic acid groups of NPs and amine groups of silanized Ti surface using NHS/DCC as coupling reagents. Results obtained from cell adhesion study clearly showed an impact of NPs-RGDC grafted onto titanium on cell adhesion with the creation of filopodia-like structures among NP sites. This provides a tool to study adult stem cells osteogenic capacity that is currently under investigation. Therefore, the biomaterials tested in this study may be exploited for bone tissue engineering and repair.

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*E-mail: [email protected]. 903


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Impact of RGD Nanopatterns Grafted onto Titanium ... - ACS Publications

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