Improved Stability and Cell Response by Intrinsic Cross-Linking of

Article pubs.acs.org/Biomac

Improved Stability and Cell Response by Intrinsic Cross-Linking of Multilayers from Collagen I and Oxidized Glycosaminoglycans Mingyan Zhao,†,§ Lihua Li,† Changren Zhou,† Frank Heyroth,‡ Bodo Fuhrmann,‡ Karsten Maeder,§ and Thomas Groth*,§ †

Department of Materials Science and Engineering, Jinan University, Guangzhou 510630, China Interdisciplinary Center of Materials Science (IZM), and §Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale) D-06099, Germany

‡

S Supporting Information *

ABSTRACT: Stability of surface coatings against environmental stress, such as pH, high ionic strength, mechanical forces, and so forth, is crucial for biomedical application of implants. Here, a novel extracellular-matrix-like polyelectrolyte multilayer (PEM) system composed of collagen I (Col I) and oxidized glycosaminoglycans (oGAGs) was stabilized by intrinsic cross-linking due to formation of imine bonds between aldehydes of oxidized chondroitin sulfate (oCS) or hyaluronan (oHA) and amino groups of Col I. It was also found that Col I contributed significantly more to overall mass in CS-Col I than in HA-Col I multilayer systems and fibrillized particularly in the presence of native and oxidized CS. Adhesion and proliferation studies with murine C3H10T1/2 embryonic fibroblasts demonstrated that covalent cross-linking of oGAG with Col I had no adverse effects on cell behavior. By contrast, it was found that cell size and polarization was more pronounced on oGAG-based multilayer systems, which corresponded also to the higher stiffness of crosslinked multilayers as observed by studies with quartz crystal microbalance (QCM). Overall, PEMs prepared from oGAG and Col I give rise to stable PEM constructs due to intrinsic cross-linking that may be useful for making bioactive coatings of implants and tissue engineering scaffolds.

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surfaces like collagen IV and fibronectin has been found to enhance signal transduction into cells and promote cell spreading.8,9 Col I has been frequently used for making implant coatings, three-dimensional scaffolds, and hydrogels because of its general bioactivity toward different types of cells, biodegradability, and weak antigenicity.4,10,11 GAGs as further components of ECM and cell surface cooperate with numerous adhesive proteins (e.g., fibronectin) and cytokines (growth factors) that also impinge on essential biological processes such as cell growth and differentiation.12,13 In addition GAGs like chondroitin sulfate (CS) can promote the organization of soluble Col I precursors into fibrillar form, which is essential for ECM structure and function.14 While Col I represents a polycation below pH 4, GAGs like CS and hyaluronic acid (HA) are polyanions at physiological conditions having pKa values of 2−2.5 for CS15 and pKa of 2.9 for HA.16 Hence, Col I and either CS or HA are suitable PEL for making multilayers at a pH value of 4 that have an ECM-like composition characteristic for bone or fibrocartilage.4,17 Since the presence of Col fibrils affects cell response, important questions of this study were how the type of GAG affects fibrillization of Col I during multilayer formation and

INTRODUCTION Surface modification with biomolecules like oligopeptides or glycans that represent components of the extracelluar matrix (ECM) and address specific cell receptors has been applied frequently to achieve an improved healing response of medical implants.1 Among the variety of techniques used to modify biomaterial surfaces to improve their bioactivity, the so-called layer-by-layer (LbL) technique, a physical surface modification method, has emerged as a simple and versatile tool during the recent years.2 Based on the alternate adsorption of natural or synthetic polyelectrolytes (PEL) of opposite net charge, the LbL approach allows the built-up of polyelectrolyte multilayers (PEM) with tunable properties.3 Since matrix components like proteins and also glycosaminoglycans (GAGs) represent PEL, their application for formation of bioactive multilayer surface coatings by LbL technique has gained an increasing interest.4 The ECM is a complex support of cells composed of structural proteins such as collagens (Col) as well as a wide variety of proteoglycans and is crucial for the organization of tissues and organs.5 Fibril-forming collagen I (Col I) is one of most abundant matrix proteins and affects growth and differentiation of a variety of cells.6 Interaction of cells with ECM proteins like Col is translated by ligation of integrin cell adhesion receptors to the proteins followed by signal transduction, which regulates cell spreading, growth, and differentiation.7 Fibrillization of ECM proteins on biomaterial © 2014 American Chemical Society

Received: August 29, 2014 Revised: September 20, 2014 Published: September 23, 2014 4272

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dissolved in 0.15 M sodium chloride solution at a concentration of 0.5 mg mL−1. Col I from porcine skin (polycation, Mw ∼ 100 kDa, Sichuan Mingrang Bio-Tech, Sichuan, China) was dissolved in 0.2 M acetic acid (Roth) at a concentration of 2 mg mL−1 at 4 °C for overnight. The Col I solution was centrifuged at 9000g for 10 min before use to remove insoluble precipitates. A final concentration of 0.5 mg mL−1 was obtained by diluting the stock solution in 0.2 M acetic acid supplied with 0.15 M sodium chloride. Before use, the pH value of all PEL solutions was adjusted to pH 4.0 except for PEI. Preparation of Polyelectrolyte Multilayers (PEM). PEM were fabricated on cleaned glass coverslips and silicon substrates, respectively. PEI was used at pH 7.4 as anchoring base layer to obtain a positive net charge of the respective substrates. Multilayers were formed on top of the PEI layer using nGAGs (nCS, nHA) or oGAGs (oCS, oHA) as polyanions and Col I as polycation. The polyanions were adsorbed for 15 min at pH 4.0, while Col I was adsorbed for 20 min at the same pH value. Each adsorption step was followed by rinsing with 0.15 M sodium chloride solution (pH 4.0) for 3 × 5 min. As a result, PEM systems with eight layers on top of the PEI layer were obtained. The Col I terminated PEM systems (4 bilayers of Col I and nGAG or oGAG) were abbreviated as (nCS-Col I)4, (nHA-Col I)4, (oCS-Col I)4, and (oHA-Col I)4 Characterization of Multilayer Formation and Surface Properties. Ellipsometry. The thickness of PEM was measured with an M-2000 V scanning ellipsometer (J.A. Woollam Co. Inc., Lincoln, NE), which was equipped with a liquid cell (J.A. Woollam Co. Inc.) that can be used for solution injection with an angle of incidence of 70°. After four bilayers of PEM were obtained, measurement of multilayer thickness was performed with sodium chloride buffer. Thereafter, multilayers were challenged with pH 7.4 by rinsing with PBS (2.7 mM KCl, 137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, pH 7.4) to test stability of multilayers under physiological conditions. The thickness of PEM was obtained by fitting the experimental data to an additional Cauchy layer. A refractive index of 1.36 was used for thickness calculation of the native polymers, which corresponds to native PEM matrix values.17 The experimental data were analyzed using the software of the device (WVase32). Water Contact Angle (WCA) Measurements. Static WCA measurements were used to determine the wettability of multilayer surfaces using an OCA15+ device from Dataphysics (Filderstadt, Germany). The sessile drop method was applied using 1 μL of ultrapure water as test liquid with the Ellipse-fitting method. The experiments were run in triplicate with five droplets per sample. Means as well as standard deviations were calculated. Zeta Potential Measurements. Streaming potential measurements were carried out with a SurPASS device (Anton Paar, Graz, Austria) and special manufactured glass coverslips to obtain the zeta potential of PEM surfaces. Two identical PEM-modified coverslips were fixed on stamps and placed oppositely into the SurPASS flow cell. The gap of the flow cell was adjusted to a distance where a flow rate between 100 and 150 mL min−1 was reached at a maximum pressure of 300 mbar. A flow check was performed to achieve a constant flow in both directions. One mM potassium chloride (Roth) was taken as model electrolyte while hydrochloric acid (0.1 M) (Roth) was utilized for pH titration. Sodium hydroxide (1 M) (Roth) was used for adjusting the pH value of the model electrolyte to 10.5 before starting a measurement. Finally, the measurements were performed with an automated titration program from pH 10.5 to 2.25, and by using titration steps of 0.03 μL from pH 10.5 to 5.0 while 0.25 μL from pH 5.0 to 2.25. The buffer solution was purged with nitrogen throughout the whole measurement. Atomic Force Microscopy (AFM). The surface morphology of the PEM was investigated by AFM (Nano-R, Pacific Nanotechnology, Santa Clara, CA). Cleaned and PEM coated silicon wafers were probed in close-contact mode under ambient (air) laboratory conditions, and scans of (3 × 3) μm2 were recorded. The fibril diameter was measured by using the topography section analysis software “Gwyddion 2.30”. Determination of Collagen I (Col I) Content. The quantity of Col I in the four PEM systems was determined by a protein quantification assay. Briefly, after placing PEM coated glass coverslipes in 24-well

how cells respond to that. A further issue of this work was focused on improving the stability of multilayers made of ECM components. Multilayer films made of polysaccharides and proteins are sometimes unstable under physiological conditions.4,18 Since multilayer assembly is based primarily on ion pairing of PEL with different electric charges, a shift of pH value after implantation changes ionization state of weak PEL like proteins, which may result in dissolution of PEM. Therefore, a further question of this work was whether an intrinsic crosslinking of multilayers achieved by prior oxidation of GAGs to generate aldehyde groups to allow imine bond formation to Col I would contribute to improved stability of multilayers without loss of bioactivity. Results are reported herein.

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MATERIALS AND METHODS

Synthesis of Oxidized Glycosaminoglycans (GAGs). The vicinal hydroxyls of glucuronic acid rings of native GAGs (nGAGs) were oxidized using a slightly modified method reported previously.19,20 In brief, 0.5 g of native CS (nCS) (Mw ∼ 25 kDa, Sigma, Steinheim, Germany) and native HA (nHA) (Mw ∼ 1.3 MDa, Innovent, Jena, Germany) were dissolved in 100 mL of ultrapure water and allowed to react with sodium periodate at a molar ratio of 4:1 and 1:1, respectively (Sigma, Steinheim, Germany), at room temperature in the dark with constant stirring for 3 and 6 h, respectively. The oxidized GAGs (oGAGs) were purified by dialysis (Spectra/Por membrane, Mw cutoff = 3.5 kDa, Roth, Karlsruhe, Germany) against distilled water for 3 days. The final product was obtained by freezedrying (ALPHA 1-2 LD plus, Christ, Osterode am Harz, Germany). In preliminary experiments, it was found that the high molecular weight of nHA leads to a highly viscous solution, which partially inhibited the oxidation. This resulted in lower oxidation degrees as compared to nCS. Therefore, a higher molar ratio of sodium periodate was used here for nHA to compensate at least partly for that. Characterization of Oxidized Glycosaminoglycans (oGAG). The chemical structures of native and oxidized GAGs were analyzed by Fourier transform infrared (FTIR) spectroscopy (IFS 28, Bruker, Bremen, Germany). The quantity of aldehyde groups was determined using Schiff’s test.21 A volume of 2.5 mL of Schiff’s reagent was mixed with 0.5 mL of sample solution (4 mg mL−1). The absorbance of the colored complex was measured at 550 nm within 40 min using ultraviolet−visible (UV−vis) spectroscopy (Specord200, Analytik Jena, Jena, Germany). The concentration of aldehyde groups was calculated from the calibration curve plotted from a series of glutardialdehyde solution with known concentrations. The molecular weight and polydispersity of oGAGs were measured by asymmetrical flow field−flow fractionation (A4F) equipped with a Dawn EOS detector (Wyatt Technology Corporation, Goleta, CA) and a refractive index (RI)-detector (Shodex RI-101, Showa Denko Europe GmbH, Munich, Germany). All samples were measured in 50 mM sodium chloride (Sigma, Steinheim, Germany) containing 0.02% sodium azide (sigma) (w/v) to inhibit bacterial growth. Molecular weights were calculated using Astra software (Wyatt Technology Corporation). Substrate Preparation. Glass coverslips (⌀12 mm, Menzel, Braunschweig, Germany) were cleaned with 0.5 M sodium hydroxide (Roth, Karlsruhe, Germany) dissolved in 96% ethanol (Roth) at room temperature for 2 h followed by thorough rinsing with ultrapure water (10 × 5 min) and drying under nitrogen flow. Silicon wafers (Silicon materials, Kaufering, Germany) with a size of (10 × 10) mm2 and (37 × 17) mm2 were cleaned with a solution of ammonium hydroxide (25%, Roth), hydrogen peroxide (35%, Roth), and ultrapure water (1:1:5, v/v/v) at 75 °C for 15 min followed by extensive washing with ultrapure water.22 Glycosaminoglycans (GAGs) and Collagen I (Col I) Solution Preparation. PEL solutions were prepared as follows: poly(ethylene imine) (PEI, P, Mw ∼ 750 kDa, Sigma) was dissolved in 0.15 M sodium chloride (Roth) solution at a concentration of 5 mg mL−1. Native GAGs (nCS, nHA) and oxidized GAGs (oCS, oHA) were 4273

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incubated at 37 °C for another 2 h. Thereafter, 100 μL of supernatant of each well was transferred to a black 96-well plate (Greiner), and fluorescence intensities were measured with an excitation wavelength of 544 nm and at an emission wavelength of 590 nm using a plate reader (FLUOstar). Statistics. All data are represented as mean values ± standard deviations (SD). Statistical analysis was performed using Origin software with ANOVA test (one-way) followed by posthoc Tukey testing. The number of samples has been indicated in the respective figure and table captions. Statistical significance was considered for p < 0.05 and is indicated by asterisks in the figures.

plates (Greiner, Frickenhausen, Germany) (plain glass coverslips were used as the blank control), 400 μL of working reagent of the BCA Protein Assay Kit (Pierce, Rockford, IL) was added to each well and allowed to react at 37 °C for 5 h. Thereafter, 225 μL of supernatant of each well was transferred into a new 96-well plate (Greiner), followed by measuring the absorbance at 562 nm with a plate reader (FLUOstar, BMG LabTech, Ortenberg, Germany). The amount of Col I was calculated from the calibration curve plotted from a series of Col I solutions with known concentrations. Ultraviolet−Visible (UV−Vis) Spectroscopy. The UV−vis adsorption spectra of multilayers formed in a quartz cuvette were recorded using a UV−vis spectrophotometer (Specord200, Analytik Jena AG, Jena, Germany). Cell Culture. Cryopreserved C3H10T1/2 embryonic fibroblasts (ATCC, CCL-226, LGC Standards GmbH Wesel, Germany) were thawed and grown in Dulbecco’s modified Eagle’s medium (DMEM, Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS, Biochrom AG) and 1% antibiotic-antimycotic solution (AAS, Promocell, Heidelberg, Germany) at 37 °C in a humidified 5% CO2/95% air atmosphere. Cells of almost confluent cultures were washed once with sterile PBS followed by treatment with 0.25% trypsin/0.02% EDTA (Biochrom AG) at 37 °C for 5 min. Trypsin was neutralized with DMEM containing 10% FBS, and the cells were resuspended in DMEM after centrifugation at 250g for 5 min. Finally, the cells were seeded on plain and PEM coated glass coverslips at a concentration of 1 × 104 cells mL−1. Cell Adhesion and Spreading. The serum-free C3H10T1/2 embryonic fibroblast suspensions were prepared as described above. Cells were seeded on samples and incubated for 4 h. After incubation, the culture medium was removed, followed by rinsing with sterile PBS once. Thereafter, samples were stained with crystal violet (Roth) (0.5% (w/v) in methanol (Roth) at room temperature for 30 min. Finally, samples were carefully washed with water three times and dried in air. Images were taken in transmission mode with an Axiovert 100 (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) equipped with a CCD camera (Sony, MC-3254, AVT-Horn, Aalen, Germany). Cell count and morphology, such as cell area and aspect ratio, were evaluated from five images per sample using image processing software “ImageJ, NIH”. The experiments were run in triplicate. Focal Adhesion (FA) Complex Formation and Actin Organization. After incubation at 37 °C for 4 h, cells attached to the PEM were fixed with 4% paraformaldehyde solution (RotiHistofix, Roth) for 10 min. After rinsing with PBS twice, the cells were permeabilized with 0.1% Triton X-100 in PBS (v/v) (Sigma) for another 10 min. After rinsing twice again with PBS, nonspecific binding sites were blocked by incubation with 1% (w/v) bovine serum albumin (BSA, Merck, Darmstadt, Germany) at room temperature for 1 h. Antibodies were diluted in 1% (w/v) BSA in PBS. FA complexes were stained by incubation with a mouse monoclonal antibody raised against vinculin (1:100, Sigma) at room temperature for 30 min. Subsequently, after washing with PBS twice, samples were treated with a goat Cy2conjugated anti-mouse secondary antibody (1:100, Dianova, Hamburg, Germany) for another 30 min. In addition, actin cytoskeleton was visualized by incubating the samples with BODIPY-phalloidin (1:50, Invitrogen, Darmstadt, Germany) at room temperature for 30 min. The samples were then again washed with PBS twice, mounted with Mowiol (Calbiochem, Merck, Darmstadt, Germany), and examined with confocal laser scanning microscopy (CLSM 710, Carl Zeiss Micro-Imaging GmbH, Jena, Germany) using a 63× oil immersion objective. Images were processed with the ZEN2011 software (Carl Zeiss). Growth of C3H10T1/2 Embryonic Fibroblasts. A volume of 1 mL of serum-containing C3H10T1/2 embryonic fibroblast suspension (1 × 104 cells mL−1) was seeded on each sample. The quantity of cells was determined after 4, 24, and 48 h with a QBlue cell viability assay kit (BioChain, Newark, CA), which quantifies the amount of metabolic active cells. Before measuring, the old medium was carefully aspirated and the cells were washed with sterile PBS once. Then, 400 μL of prewarmed colorless DMEM supplemented with QBlue assay reagent at a ratio of 10:1 was added to each well, and the samples were again

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RESULTS AND DISCUSSION Synthesis and Characterization of Oxidized Glycosaminoglycans (oGAGs). Native HA and CS possess vicinal hydroxyl groups which can be oxidized to form aldehyde groups in the presence of sodium periodate. The degree of oxidation can be simply controlled by the relative amount of periodate added.19 Native GAGs and their derivatives were studied by FTIR (Figure 1). Native HA was distinguished by saccharide alkyls at

Figure 1. FTIR spectra of oHA (a), nHA (b), oCS (c), and nCS (d).

2920 and 1415 cm−1, and alkoxyls at 1030 cm−1. In contrast, the bands for nCS at 1377 and 1416 cm−1 represented CO and C−O stretching vibrations of ionized carboxyl groups, respectively, and the bands at 1066 and 1127 cm−1 represented the C−O stretching vibration of hydroxyl groups. The characteristic absorption peaks of nCS were located at about 1239 cm−1, which not only included the CO and O−H stretching vibration, but also the SO stretching vibration.23 After oxidation, the FTIR spectra of oHA and oCS presented new weak adsorption peaks/shoulders at around 1733 cm−1, which corresponded to the stretching vibration of the newly created aldehyde’s carbonyl group.20 The results of degree of oxidation and molecular weight of nGAGs and their derivatives are shown in Table 1. After oxidation, the molecular weight of nHA drastically decreased from 1.3 MDa to 55 kDa with an oxidation degree of 38.8%. The molecular weight of nCS moderately decreased from 25 to 13 kDa with a 13.9% degree of oxidation. Periodate oxidation not only breaks the vicinal glycols in polysaccharides to form their oxidized derivatives, but also hydrolyzes the glycosidic bond, which causes a decrease of molecular weight of GAG.19 Determination of Imine Bond Formation. The crosslinking between the aldehyde groups of oGAG and the amino groups of Col I is expected to produce −CN− bonds (Schiff 4274

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Table 1. Degree of Oxidation and Molecular Weight of Native and Oxidized Hyaluronic Acid (nHA and oHA) and Chondroitin Sulfate (nCS and oCS)a degree of oxidation D (%) sample nHA nCS oHA oCS

Dth

100 25

molecular weight (Da)

Dexp

Mw

Mn

PDI

38.8 13.9

1.3 × 106b 25 000 55 000 13 000

17 000 17 000 9900

1.5 3.2 1.3

a

Dth, theoretical degree of oxidation; Dexp, experimental degree of oxidation (the degree of oxidation was in relation to repeating disaccharide units); Mw, weight-average molecular weight; Mn, number-average molar mass; PDI, polydispersity index. (PDI is defined as the ratio of Mw Mn−1. If it approaches 1, the solution is monodisperse.) bValue provided by manufacturer of HA.

Figure 3. In situ thickness measurements of the four different PEM systems by ellipsometry. Results are means ± SD of two independent experiments. [Under pH = 4 condition: thickness of the PEM was measured in 0.15 M sodium chloride (pH = 4); after PBS washing: PBS (pH = 7.4) was injected into the cell, and measurement was done thereafter. Four bilayers of nHA, oHA, nCS, and oCS were prepared with collagen I (Col I) as polycation.]

19

base or imine bonds) in the multilayer systems, which should result in an absorbance at the UV region (see Figure 2).

7.4) done to simulate physiological conditions caused a noticeable decrease in thickness of native nGAG-Col I multilayer systems, which might correspond to a loss of material. The multilayer assembly was carried out at pH 4. Since the isoelectric point (pI) of Col I is around 5.5,27 it will acquire a net positive charge at this pH value. By contrast, both CS and HA represent polyanions with pKa values of about 2− 2.5 for CS15 and 2.916 for HA. Hence, it can be assumed that the formation of nGAG and Col I multilayers at pH 4 was predominantly based on ion pairing.28 After exposure to PBS and the raise of pH, Col I shall become negatively charged. One can assume that ion pairing will abolish under this conditions, which reduces the bonding of multilayer components and hence can be followed by a loss of material. This assumption is supported by findings of others that PEM assembled from polysaccharides and proteins have limited stability under physiological conditions.4,18 In contrast, the decrease in layer thickness was lower after exposure to PBS pH 7.4 for oGAGCol I systems, which indicates that intrinsic cross-linking provides higher stability of multilayers. To study also long-term stability of such multilayer systems, PEM were exposed to cell culture medium for 14 days. Here again, a significant decrease of layer thickness was found in multilayer systems made of native HA or CS while only a small insignificant change in thickness was observed for oGAG systems (see Figure S1 in the Supporting Information), which provides further evidence that intrinsic cross-linking of multilayers improves their stability. WCA measurements were conducted to monitor the surface wetting properties after adsorption of each single layer (Figure 4). Cleaned glass coverslips showed a WCA of about 22°. The adsorption of PEI led to a decrease in surface wettability, showing WCA of ∼48°, which was in accordance with previous results for amino-terminated surfaces.29 The adsorption of native and oxidized polyanions and Col I led to an oscillation of WCA, indicating a change of the terminating molecule after each adsorbed layer. Native HA and CS are known to be highly hydrophilic polysaccharides, while Col I is known to be more hydrophobic as pure film (WCA ∼ 110°).30 Hence, the lower wettability of Col I in comparison to both polyanions was also seen here. It is interesting to note that the WCA trend of the

Figure 2. (A) Schematic illustration of the proposed Schiff base formation and the participating groups on each macromolecule [GAG, glycosaminoglycan; Col I, collagen I]. (B) UV−vis spectra of nCS- and oCS-based multilayer systems. [Four bilayers of either nCS or oCS were prepared with Col I as polycation.]

Therefore, the expected covalent binding in oGAG-based systems was studied by UV−vis spectroscopy. In Figure 2B, UV−vis spectra of the CS-based systems are shown only because Schiff base formation was not detectable in the oHA system probably due to the lower quantity of Col I (see below). Obviously, no absorbance was found in the nCS-based system in the respective UV region. By contrast, the characteristic absorbance at 265 nm, which is attributed to the formation of imine bonds,24,25 was found in oCS-based system. Thus, covalent bond formation in addition to ion pairing was found in oGAG based systems, which could contribute to an improved stability and different mechanical properties of such multilayer systems. Characterization of Multilayer Formation and Surface Properties. Ellipsometry was used here to determine the thickness of PEM in situ in a hydrated state. Figure 3 shows the calculated layer thickness of PEM before and after exposure to PBS (pH 7.4). The overall averaged thickness before PBS washing was between 3 and 6 nm per bilayer, which corresponds well to the thickness of similar PEL multilayer films.26 Interestingly, the challenge of multilayers with PBS (pH 4275

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PEM systems at acidic pH values, while negative charges of polyanions are presented at basic pH values. The zeta potentials between HA- and CS-based PEM were comparable and did not differ significantly when the pH value was lower than pH 5. Additionally, after a steady increase of pH, HA-based PEM showed lower potentials in comparison to CS-based PEM, particularly in the alkaline regions. Theoretically, owing to the presence of sulfate groups, the plain CS film should be more negatively charged within the whole pH range if compared to HA.28 However, the lower values for the HA system indicate probably the dominant role of the polyanion in this multilayer system, which was also found by WCA measurements. Further, higher wettability was demonstrated on the oGAG-based PEM. In contrast, a lower zeta potential was found on nGAGcontaining PEM at pH values >6.5, which could indicate a loss of Col I during zeta potential measurements after exposure to electrolyte solutions of higher pH values. As mentioned above, the increase of pH value might lead to charge reversal of Col I, which could result in desorption of Col I from PEM. The BCA assay was applied here to quantify the Col I content in either the HA- or CS-based PEM systems (Figure 6). It was found that there was no significant difference in the

Figure 4. Static WCA measurement during multilayer formation up to eight layers. Results are means ± SD of three independent experiments. The initial layer is PEI, while odd numbers refer to native (nGAGs) or oxidized glycosaminoglycans (oGAGs) and even numbers refer to Col I. [nHA, oHA, nCS, and oCS each paired with Col I, adsorbed up to 4 bilayers.]

CS-based systems differed strongly from that of HA-containing systems from the fifth layer onward. There, the WCA increased with increasing layer number, meaning that the WCA of the following nCS or oCS layer was higher than the previous nCS or oCS layer, which held also for the Col I layers. This is contradictory to the HA-based systems, where the WCA of the PEM increased to a lesser extent after Col I adsorption, but decreased to a higher degree after subsequent HA polyanion deposition. There, the WCA of the terminal nHA or oHA layer was similar to that of the previous nHA or oHA layer. These findings indicate that GAGs are dominating in HA-based systems, while Col I contributed to much higher mass in CScontaining systems, which is also well in line with the conclusions drawn from BCA assay (see below). Figure 5 shows the zeta potential of the terminal Col I layers as a function of pH value. Previous studies have demonstrated that zeta potentials of PEM represent not only the charge distribution of outermost PEL layers, but also of preceding ones.28 Accordingly, polycations dominate the potential in

Figure 6. Amount of Col I within the four different PEM systems determined by BCA assay (n = 4, *p < 0.05). [Four bilayers of nHA, oHA, nCS, and oCS were prepared with Col I as polycation.]

amount of Col I within the same GAG systems. However, the amount of Col I in the CS-based systems (∼20.7 μg cm−2 for (nCS) and ∼20.5 μg cm−2 for (oCS), respectively) was almost twice as high as in the HA-based systems (∼10.2 μg cm−2 for (nHA) and ∼9.9 μg cm−2 for (oHA), respectively). Thus, these findings supported the idea that Col I adsorbed to a higher extent in CS-based systems, which is in accordance with the results found in WCA and zeta potential measurements. Tan et al.31 recently observed, when comparing to plain poly(ethylene terephthalate) (PET) surfaces, that the adsorption of Col I increased from 1.46 ± 0.13 to 4.77 ± 0.5 μg cm−2 after grafting with 12 nmol cm−2 carboxyl groups. There, the adsorption of Col I was likely driven by electrostatic attraction between negatively charged carboxylates in PET and the positively charged Col I, which is also in line with our findings. Col I fibrillogenesis is a sophisticated process, which is highly influenced by the microenvironment such as pH, electrolyte, polysaccharide, and so forth. The self-assembly of collagen fibrils, that is, in vitro reconstitution or fibrillogenesis, has received wide interest. The presence of GAG also plays an

Figure 5. Zeta potential measurements of the four different PEM systems with outermost Col I layer. Results are means ± SD of two independent experiments. [Four bilayers of nHA, oHA, nCS, and oCS were prepared with Col I as polycation.] 4276

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Figure 7. AFM observations of the outermost Col I layer of the four different PEM systems. (a) nHA and Col I based PEM; (b) oHA and Col I based PEM; (c) nCS and Col I based PEM; (d) oCS and Col I based PEM. Scan size of all images is (3 × 3) μm2.

Table 2. Averaged Diameter (mean ± SD) of Col I Microfibrils on Different PEM Surfaces [n ≥ 16, *p < 0.05]a

important role in the organization of soluble Col I precursors into a fibrillar form. Tsai and co-workers found that the type of polysaccharides affected the in vitro reconstitution rate, size, and morphology of Col I fibrils tremendously. 32 The topography of PEM surfaces with Col I as terminating layer was observed by AFM in a dry state. Figure 7 shows that the morphology of PEM was highly dependent on the type of GAG as well as the mechanism (covalent binding or ion pairing) of the multilayer formation. The fibrillar structure of the oGAGbased Col I (Figure 7b and d) was preserved and very similar to the structure of noncovalently bound nGAG-based PEM (Figure 7a and c) with Col fibrils randomly organized. These results indicated no impairment of Col I fibrillogenesis in the case of cross-linking between Col I and oGAG. It is also interesting to note that well-distinguished interconnected Col I fibrils were found in both nCS (Figure 7c) and oCS-based (Figure 7d) PEM. However, there was only a small quantity of short fibrils in both nHA (Figure 7a) and oHA-containing (Figure 7b) PEM. In HA-based PEM, Col I molecules seemed to assemble into elongated globules, which is similar to the results found by Jiang et al. (pH 4.5).33 Jiang et al. found that a pH value 5.5 led to pronounced fibrillar structures.33 In our work, the morphology and amount of the fibrils varied among the two different types of GAG-based PEM, which further demonstrated that the presence of GAG affects the assembly and structure of Col I fibrils. At room temperature, CS accelerated fibril formation while HA had no effect on it.14 Furthermore, the concentration of Col I also has an influence on fibril formation, where increased concentrations of Col I from 6 to 24 μg mL−1 drastically accelerate the fibrillogenesis.34 On the other hand, regardless of a high (40 to 300 mg mL−1) or low (0.1 to 1 mg mL−1) initial Col I concentration range, lower concentrations corresponded to fewer nucleation sites and, thus, resulted in fewer, but thicker fibrils by consecutive aggregation of the remaining collagen molecules.35,36 We could show with WCA and BCA investigations that larger amounts of Col I were present in PEM with CS as polyanion, while apparently less Col I was deposited in HA-based PEM. This is related to the observation that rather elongated globules than pronounced fibril structures exist in HA-based PEM while a distinguished fibril structure was detected on CS-based PEM. Another interesting phenomenon was the effect of polyanions used here on the diameter of the Col I fibrils. Cross-sectional analysis revealed that the averaged fibril diameter varied among the four different PEM systems (see Table 2). Fibril diameters of ∼84.61 and ∼81.37 nm were found in nHA and nCS-based PEM, which were larger than those in the oxidized PEM systems (74.5 nm (oHA) and 66.41 nm (oCS), respectively). Moreover, the fibrils were always smaller in CS-based

a

Four bilayers of nHA, oHA, nCS, and oCS were prepared with Col I as polycation.

multilayers in comparison to HA-based systems. It was found in a previous work that the number of fibrils formed determined also the fibril diameter; that is, the larger the amount of fibrils formed from a given concentration of soluble Col I, the smaller each fibril would be. Polyanions, which accelerated precipitation of Col I, led to a reduced diameter of fibrils.14 The smaller diameter of fibrils in CS-containing PEM was in accordance with these findings. From our point of view, the enhanced interaction between Col I and oGAGs by intrinsic cross-linking might inhibit the lateral aggregation of Col I fibril formation, which resulted in smaller fibril diameter. Overall, these findings indicate that by selection of GAG as polyanion one can obviously control quantity and fibrillization of Col I polycation, which may affect cellular responses. Biological Investigations. To investigate if the covalent binding and the nature of the terminal layers of PEM have an effect on cell adhesion and subsequent events, murine C3H10T1/2 embryonic fibroblasts were seeded in the absence of serum to study direct cell-surface interaction without additional protein adsorption. Quantitative results of cell count and morphology, such as cell area and aspect ratio visualized by crystal violet staining and image analysis, are shown in Figure 8. First, it was found that there was no significant difference of cell count in PEM systems with the same GAG either native or oxidized. By contrast, significantly more cells adhered to the terminal layers of CS-based PEM (Figure 8A), where Col I was dominating, compared to HAbased PEM. The determination of cell size (Figure 8B) and aspect ratio (Figure 8C) revealed that, unlike the round morphology of cells on plain glass coverslips, cells attached to PEM showed a superior spreading after 4 h incubation. Furthermore, cells were larger and more elongated on CSbased PEM when compared to HA-containing PEM, no matter if native or oxidized GAGs were used. Altogether, the results of adhesion experiment revealed that the use of oGAG with 4277

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Figure 8. Cell count (A), area (B), and aspect ratio (C) of C3H10T1/2 embryonic fibroblasts on different modified surfaces. Box−whisker diagrams are shown in panels (B) and (C), where the box indicates the 25th and 75th percentile and mean (black square) values, respectively. Results are means ± SD of three independent experiments. [Four bilayers of nHA, oHA, nCS, and oCS were prepared with Col I as polycation.]

Figure 9. Confocal laser scanning micrographs of C3H10T1/2 embryonic fibroblasts after 4 h incubation on different surfaces: (a) plain glass coverslips; (b) nHA and Col I based PEM; (c) oHA and Col I based PEM; (d) nCS and Col I based PEM; (e) oCS and Col I based PEM. The cells were stained for filamentous actin (red, upper row) and Vinculin (green, upper and lower row) present in focal adhesions. [Scale bar: 20 μm.]

adherent to PEM consisting of nCS and Col I (Figure 9d) showed a pronounced polymerization of actin, which was organized in bundles circumferentially. Further, FA plaques were distributed all over the ventral cell side. Interestingly, the number of these plaques was increased and much more pronounced on cells seeded on PEM with oCS (Figure 9e). Overall, adhesion, spreading and FA formation were increased in CS-containing systems in comparison to HAbased systems, which can be explained with the composition of the layers. First, more Col I was deposited in CS-based systems, as proven by WCA, zeta potential measurements and BCA assay. This was accompanied by a well-interconnected fibrillar structure of Col on the outermost layer, which also led to a moderately wettable surface. Col fibrils are known to enhance the adhesion of a variety of cells via an integrin-mediated interaction.9 In contrast, the GAG was obviously dominant in layer formation of HA-PEM leading to lower quantity of Col I and its fibrils in the terminal layer and higher hydrophilicity. Compared to highly hydrophilic or hydrophobic surfaces, cells

intrinsic cross-linking of Col I did not cause any loss of bioactivity of the terminal collagen layer. Additionally, C3H10T1/2 embryonic fibroblast adhesion was further studied by visualizing vinculin (green) present in FA complexes as well as the actin cytoskeleton (red) using CLSM after 4 h incubation (Figure 9). Vinculin is a structural protein present both in the FA plaque and in intercellular adherens junctions. Further, it plays an essential role in actin stress fiber organization as well as in cell morphology changes.37 As shown in Figure 9a, cells cultured on plain glass coverslips were round due to the lack of adsorbed adhesion-promoting proteins. Actin was condensed with low signs of fibrillar organization and organized predominantly circumferentially. Further, FA formation was weak in comparison to the other surfaces indicated by the short length of vinculin-positive streaks. Cells seeded on the HA-based PEM (Figure 9b, c) showed an increased spreading with F-actin fibers organized in bundles. In addition, FA complexes positive for vinculin were found mainly at the cell periphery, but also in central regions. However, cells 4278

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prefer to adhere on moderately wettable surfaces.38 Most probably, the increased hydrophilicity of HA-based PEM (WCA ∼ 28°−33°) reduced the adhesion and spreading of cells due to the increased water content of the layers because HA to binds large amounts of water.39 Similar findings on the effect of hydrophilicity of multilayers from polysaccharides on suppression of adhesion of C2C12 myoblast cells were made in one of our previous studies.28 On the other hand, Denuziere and the co-workers 40 found that the attachment of chondrocytes was higher on films complexed from chitosan and HA compared to those made of chitosan and CS. This shows that effect of multilayer compositions and physical properties is also depending on the type of cells. Except the composition and wettability of surface coatings, substrate stiffness is also an important feature on tailoring cell adhesion and spreading. Richert et al. found that chondrosarcoma cells could only adhere to and spread on cross-linked poly(L-lysine)/hyaluronan (PLL/HA) films, but not on native films of the same composition.41 Similarly, FA and actin organization of C2C12 cells were demonstrated to be affected by film stiffness as well. Numerous large, elongated FA plaques and well-organized F-actin stress fibers were observed when 50 and 100 mg mL−1 EDC were used for cross-linking PLL/HA multilayer films.42 We assumed here that the additional covalent cross-linking in oGAG multilayer systems should increase PEM stiffness as well as stability. In fact, additional studies with quartz crystal microbalance (QCM) shown in Supporting Information (see Figure S2) demonstrated a decrease of dampening shifts for cross-linked PEM made of oGAG and Col I compared to PEM with native GAGs. This indicates that multilayers with intrinsic cross-linking are stiffer, which corresponds also to finding of others.43 Thus, the increased cell size, elongation of FA plaques on oCS-based PEM can be related to different mechanical properties compared nCS-based PEM. The lack of such pronounced difference on HA-based PEM may be due to the higher hydrophilicity of the HA-based multilayers that reduces spreading, thus overriding the effect of different mechanical properties of native and oxidized HA multilayer systems. C3H10T1/2 proliferation over a period of 4, 24, and 48 h on the different PEM was monitored with QBlue assay, which determines the metabolic activity of cells used here to monitor cell growth (Figure 10). In correspondence to the cell adhesion results, the quantity of metabolic active cells on plain glass coverslips was always lower than that on the PEM, independent of the incubation time. Further, more metabolic active cells were determined on PEM containing CS in comparison to HAbased PEM, during the first 48 h of incubation. This indicates that cells attach and grow initially more on surfaces with higher amounts of Col I fibrils mimicking ECM structures. However, no significant differences in proliferation were found between both types of GAG based PEM after 48 h incubation. Presumably, the ability of cells to secrete matrix proteins like fibronectin may contribute to the lower differences. Altankov and Groth44 also found that fibroblasts were able to deposit significant amounts of fibronectin on relatively hydrophilic surfaces after 72 h incubation. It is well documented that fibronectin plays a critical role on increasing cell attachment and inducing subsequent cell growth.45 On the other hand, the low difference in cell growth between native and oxidized GAG-based systems demonstrated that cross-linking during the multilayer formation with oGAGs had no negative effect on the cell proliferation.

Figure 10. Proliferation of C3H10T1/2 embryonic fibroblasts plated on different surfaces and assessed by the QBlue assay for indication of metabolic active cells after 4, 24, and 48 h. Results are means ± SD of three independent experiments. [Four bilayers of nHA, oHA, nCS, and oCS were prepared with Col I as polycation.]

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CONCLUSIONS In the present work, we studied the effect of GAG on fibrillization of Col I during multilayer formation and of intrinsic cross-linking on multilayer stability. In addition, we were interested to learn how this affects behavior of cells. Previous methods for improving the stability of PEM, such as chemical cross-linking with small bifunctional cross-linkers like glutardialdehyde, caused significant changes in the physicochemical properties of films 46 and resulted partly in cytotoxicity.47 By contrast, the intrinsic cross-linking by reaction of oxidized GAGs with Col I had no major effect on Col I fibrillogenesis, led to long-term stabilization of PEM under physiological conditions in vitro, and did not negatively affect bioactivity toward cells. It was also found that PEM made of oGAG and Col I possessed a higher rigidity resulting from intrinsic cross-linking, which affected spreading of cells, which may pave the way for affecting subsequent cellular responses like differentiation of stem and other cells.42 A further important observation of this study was that the type of GAG played an important role in tuning multilayer composition and surface properties (topography, wettability, etc.) as well as Col I fibrillogenesis, which affected cellular behavior, too. In the presence of well-interconnected Col I fibrils in CS-based systems, C3H10T1/2 embryonic fibroblasts exhibited superior spread morphology with higher metabolic activity. The application of intrinsic cross-linking of multilayers based on oxidized GAGs and proteins like Col may be useful in the future to tailor mechanical properties of multilayers by change of oxidation degree or quantity of oxidized GAG. Furthermore, such multilayer systems can be used for covalent binding of growth factors, which is applicable in the area of tissue engineering and regenerative medicine.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information about the long-term stability and mechanical property of the four different multilayer systems were visualized by ellipsometry and QCM measurements. This material is available free of charge via the Internet at http:// pubs.acs.org. 4279

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49-(0)345-552 8460. Fax: +49-(0)345-552 7379. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was funded partly by the European Commission, the 7th Framework Program for Research (FP7-PEOPLE-2012IAPP) under grant agreement no. 324386 and the Chinese Scholarship Council program funded by Chinese government. The kind support by Dr. Marcus Niepel in manuscript proofreading and helpful discussions is greatly appreciated. We are thankful to Mrs. Marlis Porobin for doing zeta potential measurements.

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