Natural Polyelectrolyte Self-Assembled Multilayers Based on Collagen

Jun 19, 2013 - Institute for Engineering and Medicine, Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/Biomac

Natural Polyelectrolyte Self-Assembled Multilayers Based on Collagen and Alginate: Stability and Cytocompatibility Wenxing Li,† Peng Zhao,‡,⊥ Chao Lin,‡ Xuejun Wen,‡,§ Eleni Katsanevakis,§ Decher Gero,∥ Olivier Félix,∥ and Yuehua Liu*,† †

Department of Orthodontics, School of Stomatology, Tongji University, 399 YanChangZhong Road, Shanghai 200072, China The Institute for Biomedical Engineering and Nanoscience, School of Medicine, Tongji University, 67 Chifeng Road, Shanghai 200092, China § Institute for Engineering and Medicine, Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia 23284-3028, United States ∥ Institut Charles Sadron, Université de Strasbourg, UPR CNRS 22, F-67034 Strasbourg, France ‡

ABSTRACT: Scientific interest in the self-assembly of collagen composite films has been increasing for their potential application in constructing bioactive materials. Here we report a highly stable and cytocompatible collagen/alginate (COL/ALG) ultrathin film, which was linearly fabricated via a layer-by-layer self-assembled technique. The variation in morphology and thickness of the films in air and in solutions with different pH and ion values were tested by atomic force microscopy. Results showed that the solutions with high pH values or solutions that contained electrolytes would disintegrate the film, while films with that were cross-linked for a long time prevented the dissolution and contributed to stability maintenance of the films. Interestingly, the COL/ALG coating not only improved the adhesion and proliferation of the human periodontal ligament cells, but also modified the morphology and migration of cells on the surface of glass and poly-L-lactic acid (PLA) electrospun scaffolds. In conclusion, the COL/ALG ultrathin films were highly stable and cytocompatible and could be easily fabricated by the cost-effective self-assembled technique presented. The findings of this study have the potential to play an important role in the surface modification of biomaterials.

■

INTRODUCTION As a key factor in regenerative medicine, scaffolds that encourage and sustain biological activities can be considered bioactive.1 Surface modifications of these scaffolds are becoming increasingly important in tissue engineering for its role in determining the outcome of biological-material interactions. Layer-by-layer (LbL)2,3 fabrication is an important technique for the surface modification of tissue engineering scaffolds. Traditionally, a liquid solution is applied by dipping, spraying, and brushing.4−6 This method is used to assemble various charged macromolecules onto the polymer surface in a designated way, without restricting the substrate shape, size, topography, or topology.7−11 Although there are several methods to create collagen-based biomaterials, the selfassembled process and the stability of the fabricated collagenbased biomaterials are still poorly understood.12 Type I collagen (COL) and alginate (ALG) have been used as a secondary material phase in order to encapsulate cells to increase cell seeding in polymeric scaffolds. This has been shown to improve seeding and cellular response in vitro when compared with statically seeded controls.13−19 Collagen has been shown to enhance cellular attachment, as well as perform © 2013 American Chemical Society

as a chemotactic agent to cells, owing to its more natural surfaces compared to synthetic polymers. However, the fast biodegradation rate and the low mechanical properties of the untreated collagen limit its further applications.20−23 Earlier methods for improving the biological activity of scaffolds using collagen coating mainly employed a general rapid-prototyping process or freeze-drying, critical-point-drying, and LbL techniques. COL-based films were produced with chondroitin sulfate,25 hyaluronic acid,24 polystyrene sulfonate,27 alginate,28 and so on. However, cells still remained on the top surface instead of migrating through the collagen foam in the previous research.19,29 Alginate, as a natural polysaccharide extracted from seaweed, has been demonstrated to be an ideal drug delivery vehicle, which has the ability to optimize the drug release in a controlled fashion.30−37 Although this material has also been extensively used as a scaffold for dental-derived cells, including periodontal ligament and gingival mesenchymal stem cells, the lack of cellular interactions limits its application.38,39 Received: April 11, 2013 Revised: June 6, 2013 Published: June 19, 2013 2647

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

to the COL/ALG film. The cross-linking agent solution of EDC (10.4 mM) and NHS (4.3 mM) was incubated with the multilayer films for 1, 2, and 25 h, respectively. Rinsing steps were then performed with Milli-Q water for 3 h. Ellipsometry Analysis and UV−Visible Spectroscopy. The film-thickness determination was carried out with a Plasmos SD 2000 ellipsometer at a wavelength of 632.8 nm. The UV−vis absorption spectra of the COL/ALG layers were recorded using a Varian Cary 500 scan spectrometer on a quartz slides. The quartz slides were first cleaned with a 2% Hellmanex solution and then thoroughly rinsed with Milli-Q water. Because of the peptide chain structure, UV absorption of collagen amino acids occurred near 195 nm, whereas no obvious UV absorption was shown in ALG. Quartz Crystal Microbalance (QCM-D). The (COL/ALG)n multilayer build-up on SiO2-coated crystal was monitored in situ by quartz crystal microbalance with dissipation monitoring. Measurements were performed with a Q-Sense D300 System (Gothenborg, Sweden) in static conditions at 25 °C The crystal was provided by QSense. It was cleaned for 15 min by UV/ozone treatment prior to use. Oscillations of the crystal at the resonant frequency (5 MHz) or at one of its overtones (15, 25, 35 MHz) were obtained when applying ac voltage. The variations of the resonance frequency (Δf) and of dissipation (ΔD) were monitored upon adsorption of the polyelectrolytes. Solutions were injected into the measurement cell using a peristaltic pump at a flow rate of 55 μL/min. Prior to the multilayer build-up, acetate buffer solution was injected to establish the baseline. The construction of COL/ALG multilayers was performed as follows: Step 1, collagen solution was brought into the measurement cell over 10 min; Step 2, rinsing was performed for 5 min using acetate buffer solution; Step 3, ALG and COL were then alternately injected according to the same procedure. When (1) ΔF < 2%F0, (2) the viscoelasticity of solvent is constant, and (3) the thickness of the film is basically well-distributed, the variance of the film mass ΔM can be obtained using Sauerbrey formula,49

The three-dimensional collagen-alginate scaffolds have been applied to overcome the shortcomings of collagen scaffolds, but the effect of utilizing a collagen-alginate surface modification for biomaterials has not yet been reported in the literature.40−42 Cell-scaffold interactions, which involves a series of proper biological responses of cells to the passive substratum, plays an important role in tissue engineering.43 In dentistry, the periodontal ligament is the soft connective tissue layer between the alveolar bone and the cementum.44 The fibroblasts within the periodontal ligament lie among the collagen fibers and are shaped like irregular flattened discs.45,46 An in situ study on the self-assembled and dynamic evolution of collagen gels indicated that collagen fibrils not only reinforce the mechanical properties of bone and tissues, but also influence cellular motility and morphology.47 It can be assumed that the application of collagen-based film in surface modification of biomaterials may provide a potential strategy for periodontal tissue engineering. From this background knowledge, we formulated a hypothesis that the combination of collagen and alginate into a COL/ALG coating might enhance cell-material interactions. To validate the hypothesis, a COL/ALG nano ultrathin film was fabricated linearly via an LbL assembled technique. The assembled process and stability of the film were investigated by various methods including ellipsometry analysis, ultraviolet (UV) absorption spectrum, quartz crystal microbalance (QCM), and atomic force microscope (AFM). This work provided a better understanding of the self-assembled process and stability of the COL/ALG ultrathin film. The COL/ALG film was then coated on both the silica glass (inorganic compound) and PLA electrospun scaffolds (biodegradable organic polymer),19 and cocultured with human periodontal ligament cells (hPDLC). The COL/ALG ultrathin film with high stability and cytocompatibility provides a new approach to construct new bioactive materials.

■

ΔM =

ΔFA − 2.6 × 105 × F02

(1)

in which F0 is the fundamental vibration frequency of quartz crystal with a value of 5 MHz, ΔF is the variance of the frequency, and A is active crystal area with a value of 1.54 cm2. AFM Imaging. AFM imaging was carried out in tapping mode either in Milli-Q water or in the air, using a commercial microsope (NanoScope 3, Digital Instruments, Santa Barbara, CA). In the liquid tapping mode, deflection images were acquired on the cross sections of the wet COL/ALG film using silicon nitride probes (DNP-S, Bruker Probes, Camerillo, CA) with a spring constant of 0.03 N/m, at a scan rate of 1 Hz for 5 min. In the air tapping mode, phase images were acquired on the air-dried COL/ALG film using silicon probes (NCHV, Bruker Probes, Camerillo, CA). Water Contact Angle Measurement. Static water contact angle measurements on the PLA scaffolds were performed using a contact angle analyzer (GBX-Digidrop, France). The water contact angles were recorded after the Milli-Q water droplets were balanced on the scaffold. Three tests on each film were analyzed and averaged. Scanning Electron Microscopy (SEM). Cell-seeded scaffolds were previously washed three times with phosphate buffered saline (PBS) and fixed in 3.7% methanal in PBS overnight. After dehydrating using graded alcohol solutions, samples were critical-point dried and sputter-coated with gold for 2 min at 20 mA. Surface morphologies of the cells and scaffold were characterized by a LEO 982 field emission SEM at 3.0 kV. Cell Culture. Following approved guidelines of the Ethics Committee of Tongji University, intact teeth were collected from a 20-years-old female who received orthodontic treatment with informed consent. Teeth were rinsed three times with PBS supplemented with Antibiotic-Antimycotic. Periodontal ligament tissue in the middle-third of the root was carefully removed with a surgical scalpel. Tissue was minced and enzymatically digested using

EXPERIMENTAL SECTION

Materials. COL (#07CBPE2) was purchased from Symatese, France; ALG (Pronova LVG-10) was bought from NovaMatrix, USA; polyethyleneimine (PEI, Mw = 70000), calcium nitrate (Ca(NO3)2·4H2O), trizma hydrochloride (T32253), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich (USA); sodium acetate aqueous solution (CH3 COONa), diammonium hydrogen phosphate ((NH4)2 HPO4), calcium chloride (CaCl2·2H2O), and acetic acid (CH3COOH) were obtained from Fluka. The salt solutions were prepared in Milli-Q water (CNRS, ICS). PEI solutions were prepared at a concentration of 0.56 mM in Milli-Q water. PLA was self-made. Acetate (acetic acid-sodium acetate) buffer (0.11 M, pH = 4) was prepared from acetic acid (89.2 mmol), sodium acetate (20.7 mmol), and NaCl (133.5 mmol). COL solutions were prepared at a concentration of 0.02% W/V in acetate buffer at pH 4 where it was positively charged. Tris-HCL (pH = 7.4) and Tris-NaOH (pH = 10) were prepared. A calcium solution of Ca(NO3)2·4H2O (0.32 M) and a phosphate solution of (NH4)2 HPO4 (0.19 M) were prepared in Tris buffer (pH = 10).48 Cyclic Spray Assembly and Cross-Linking. The spray deposition was carried out using air pump spray cans (“Air Boy”) purchased from Carl Roth (Karlsruhe, Germany). Each can was filled with the respective salt solution. The spray rate was about 0.6 mL/s with pump flasks. Spraying was carried out from a perpendicular angle to the receiving surface which was fixed in a vertical orientation. COL and ALG solutions were alternately sprayed for 3 s for the desired number of cycles. For each spraying, after a rest time of 15 s, a rinsing step with Milli-Q water or acetate buffer was applied to the COL/ALG film for 5 s, to rinse the polyelectrolyte, which was not closely attached 2648

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

Figure 1. The investigation of the thickness of COL/ALG self-assembled films as a function of pair layers: (a) in the acetic acid buffer and (b) in the Milli-Q water; (c) the investigation of UV absorption of COL/ALG self-assembled films with different pair layers; (d) UV absorption peak near 195 nm as a function of the number of the pair layers; (e) QCM-D data of COL/ALG self-assembled films; and (f) the quality of the films as a function of COL/ALG layers. type I collagenase (0.3% W/V, #17100−017, Gibco) and Dispase (Roche, Mannheim, Germany) for 1 h at 37.5 °C.50 A suspended cell solution was passed through a 70 mm-cell strainer to prepare single cell suspension. The cells were centrifuged and rinsed with PBS. The cell pellet was resuspended and was cultured in alpha-modified minimum essential medium (α-MEM; #32561037, Gibco) with 15% FBS (#10099−141, Gibco) at 37 °C in 5% CO2 in humidified air, until the cells proliferated and reached confluence. Cell Proliferation Assay and Seeding Efficacy. The COL/ALG film was coated on 70 mm × 70 mm × 1 mm silica glass slides. Pure glass slides (control group) and the coated slides (experimental group) were then placed in 96-well plates. The hPDLCs of the fourth passage were seeded at a density of 1000 cells per well. The MTT assay was performed at day 1, 3, 5, 7, 9, and 11 after induction. Briefly, 20 μL of MTT (12.08 mM, #57360-69-7, Sigma) was added to each well. After

incubation at 37 °C for 4 h, the supernatant was removed, and the formazan crystals were dissolved in 150 μL of dimethyl sulfoxide (DMSO). The optical density value (OD values) for each well were measured spectrophotometrically at 490 nm, and the assay was repeated three times. To assess seeding efficacy, a COL/ALG film was coating on 10 mm × 10 mm × 1 mm glass slides. The slides, with or without coating, were placed in multiwell plates, and a 0.5 mL cell suspension with a concentration of 105 per ml was added to the surface of the materials. Following a procedure by Kasten,51 the cells were allowed to attach for 8 h, after which the material was transferred to another plate. In this step, the cells that remained in the original well plate were considered the “lost cells”. After a further 24 h in culture, the new plate was placed on a shaker for 10 min, and the cells that were washed away from the surface of materials by culture fluid scouring were considered the 2649

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

Figure 2. The surface morphology of a (COL/ALG)n multilayer film on quartz substrates via AFM: (a−d) n = 1, 4, 6, and 25, respectively. The surface morphology of a (COL/ALG)n multilayer film on PEI-coated quartz substrates via AFM: (e−h) n = 1, 4, 6 and 25, respectively. “inadhesive cells”. After the cell/matrix constructs were harvested, the cells at the bottom of the dish were trypsinated and counted, to assess the seeding efficiency. The ratio between the lost/inadhesive cells and the total amount of seeded cells was calculated, respectively. Statistical Analysis. All quantitative results were obtained from triplicate samples. Data were expressed as the mean ± standard deviation (SD). Statistical analysis was assessed by the independent samples t test for the cell proliferation assay and seeding efficacy. A p value less than 0.05 was considered statistically significant.

■

spraying of the alginate electrolyte solution, the UV absorption intensity slightly reduced. As alginate had no UV absorption, it can be determined that the absorption was totally caused by the collagen. Thus, it was inferred that a small amount of collagen was eluted in alginate spraying process, due to the scouring action or surface tension. The COL/ALG multilayers growth was followed in situ, in the hydrated state, using QCM-D. The shifts of the resonant frequency (Δf) and of dissipation (ΔD) due to the adsorption of the collagen layer and then the alternated adsorption of alginate and collagen were recorded (Figure 1). For positive collagen (Figure 1e), Δf and ΔD were appreciably high compared to other common proteins, owing to the size, the extension in solution, and the rate of hydration of the collagen molecule. The high dissipation observed after adsorption of positive collagen indicates that the added collagen mass acts as a soft and viscoelastic layer.26 It is also shown that the adsorption of alginate induces a decrease of the dissipation, suggesting a contraction of the film. After the build-up of five bilayers, the response of the sensor could no longer be monitored for all harmonics, probably because the film became too thick. These observations demonstrate that collagen may be involved as a polycation for LbL assembly in the chosen conditions. After the COL/ALG multilayered film was constructed on the quartz substrate using the spraying technology, the film demonstrated well-proportioned mesh fiber structures (Figure 2) with an increase in the amount of structures with an increase in the number of layers. More fibers were observed on the substrate with a PEI coating (Figure 2e,f,g) than that without a PEI coating (Figure 2a,b,c). We believe coating of PEI on the substrate contributes to the initial (1−6 layers) adsorption of the film. As the amount of COL/ALG layers got progressively larger, the difference of the surface morphology between the blank substrate (Figure 2d) and the PEI-coated substrate (Figure 2h) gradually disappeared, and the diameter of the fibers increased from an average growth of (5.34 ± 2.49) nm to (67 ± 10.18) nm. This can be explained by the fact that the dendritic loose fiber bundles are built by sequential adsorptions

RESULTS AND DISCUSSION

Controllable Fabrication of (COL/ALG)n Multilayer Film. The LbL technique, which was based on the alternate adsorption of oppositely charged species, enabled the users to control the properties of the multilayer film, such as composition, thickness, and function.11 COL/ALG multilayer film was built on quartz substrates using the circulating spray coating method. The film was then rinsed using an acetate buffer (0.11 mol, pH = 4) and Milli-Q ultrapure water. As shown in Figure 1a (rinsed with buffer) and Figure 1b (rinsed with Milli-Q water), the film thickness curves rose linearly with an increase in layers. The thickness of the film increased faster when PEI was coated on the substrate as the first layer, because PEI has a structured form of branching, which favors the adsorption of polyelectrolytes. The multilayer film that used acetate buffer to rinse (Figure 1a) had worse uniformity compared with the films rinsed with Milli-Q water (Figure 1b). The higher ionic strength promotes the adsorption of polyelectrolyte, but gives rise to an easy formation of curled phase structures of the polyelectrolytes. The curled phase structures weaken the uniformity of the polyelectrolyte membrane. The UV absorption peak near 195 nm gradually increased with the layer growth of the COL/ALG multilayer film, as illustrated in Figure 1c. UV absorption of collagen amino acids occurred near 195 nm owing to the peptide chain structure, whereas no obvious UV absorption was shown in Alginate. UV absorption showed a linear growth with the number of layers, as seen in Figure 1d. These findings were consistent with the ellipsometry measurements in Figure 1a and 1b. After each 2650

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

Figure 3. The surface morphology and thickness of the (COL/ALG)n multilayer film in liquid after AFM scratching: (a−c) n = 5, 10, and 20, respectively.

Serious decays were found, and even the base was exposed, after the multilayer film was in contact with the HPO2− 4 ion (2 × 5 s), as shown in Figure 4b. Ca2+ and HPO2− 4 ions, which are commonly used in cell culture, were applied to assess the stability of the film. Figure 4c showed that the UV absorption of the multilayer film changed in the presence of Ca2+ and HPO2− 4 ions. The UV absorption of the multilayer film increased in the calcium ion solution, while it decreased in the hydrogen phosphate ions solution. Bicarbonate, a weak electrolyte, destroyed the balance of the original electropositive collagen solution by weakening or reversing the electric potential of the solution, and thus caused further dissolution of the multilayer film. Figure 4d shows that the pH value of the solution has an impact on the stability of the COL/ALG multilayer film. Once the film came into contact with high pH value solution, the multilayer film broke down regardless of the number of layers. As the multilayer film was formed by electrostatic attraction of polyelectrolytes with different electric potentials, an increase in pH value of solution weakened and even reversed the electric potential, and thus caused dissolution. The cross-linking process of the collagen is useful to slow down the biodegradation rate and to maintain the structural integrity of the material. 20 EDC and NHS are two biocompatible cross-linking agents, which reacted with the hydroxyl group and amino group in the molecular chain structure.53 A plastic compression technique combined with cross-linking results in the production of collagen films with

from the diminutive molecules to the larger ones. The quartz surface becomes rough due to the initial adsorption of small molecules and then adsorbs the larger ones. Figure 3 shows the surface morphology and thickness of the (COL/ALG)n multilayer film after the AFM scratching in the liquid phase condition. Before the AFM nanoscratching, the COL/ALG multilayer film thicknesses of a, b, and c equaled 8.8, 15.5, and 30.1 nm, respectively, in the dry state condition, which was measured by ellipsometry. After scratching, the thicknesses of a, b, and c turned into 14.5, 75.1, and 86.3 nm in the liquid condition, respectively. The thickness of the multilayer film in the liquid condition was much thicker than that in the dry condition, which can be attributed to swelling of the multilayer film in the liquid due to adsorption of water molecules and ions. It is believe that the swollen film will favor the adhesion and growth of cells. Stability of (COL/ALG)n Multilayer Film. As a major component of the extracellular matrix, collagen presents a unique hierarchical organization at multiple length scales ranging from nano to macroscale, and it has been broadly used as a substrate or scaffold for cell attachment, proliferation, and differentiation.19,52 Despite several methods we can use to create collagen-based biomaterials, the self-assembled process and stability of the collagen-based biomaterials are poorly understood.12 The (COL/ALG)n multilayer films fabricated in this study had good stability, and could be stored for long periods without decomposition in air, Milli-Q water, and a buffer solution with a pH value of 4, as shown in Figure 4a. 2651

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

Figure 4. (a) The thickness of (COL/ALG)n multilayer film in different conditions; (b) the surface morphology of the multilayer film in the 2− 2+ presence of HPO2− 4 ion(2 × 5 s); (c) UV absorption of the multilayer film in the presence of Ca (2 × 5 s) and HPO4 (2 × 5 s); and (d) the thickness of (COL/ALG)n multilayer film in the buffers with different pH values.

decreased, not because the film disintegrated, but because the film became dense. As shown in Figure 5b, many small fibrous structures were formed around the COL fibers after of 25 h cross-linking, which fixed part of the fibers on the quartz substrates.

high packing and mechanical integrity.17 Figure 5 shows the thickness change of the cross-linked (COL/ALG)5 film at different time points in the presence of HPO2− 4 (2 × 5 s) ion. The improvement of the stability was not obvious for 1 and 4 h cross-linking, while it became clear for 25 h of cross-linking time (Figure 5a). After 25 h cross-linking, the thickness 2652

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

Figure 5. (a) The thickness of (COL/ALG)5 in the presence of HPO2− 4 ion (2 × 5 s) as a function of the cross-linking time and (b) the surface morphology of the multilayer film in the presence of HPO2− 4 as a function of the cross-linking time.

Figure 6. Confocal laser scanning microscopy image of hPLCs: (a) the experimental group with (COL/ALG)20 film and (b) the control group with only glass surface; (c) the growth curves of hPLCs and (d) the percentage of the inadhesive cells and the lost cells in the experimental and control group. 2653

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

Figure 7. (a) Water contact angle measurements images: (a-1) PLA (COL/ALG)0, (a-2) PLA (COL/ALG)5, and (a-3) PLA (COL/ALG)10. (b) Pictures of hPDLCs adhering to the pure PLA scaffold and (c) pictures of hPDLCs adhering to (COL/ALG)20 coated PLA scaffold.

Cytocompatibility of (COL/ALG)n Multilayer Film. Fourth passage hPDLCs were observed on the fifth day of culture under a confocal laser scanning microscope. In the control group, the hPDLCs appeared polymorphic, but the predominant cells were fibroblastic in shape (Figure 6a) and were well distributed on the silica glass surface. In the (COL/ ALG)20 film group, after coculturing with the film for 5 days, most of the hPDLCs were cuboidal or polygonal (Figure 6b), and were arranged along the extension of the dendritic COL/ ALG fibers. The hPDLCs did not appear on the local surface without the (COL/ALG)20 film. It states that the COL/ALG film attracts the adhesion of hPDLCs. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was performed to investigate whether (COL/ALG)20 film affects cell proliferation in vitro. The OD value equals the mathematical quantity of light absorption of the cellular products at 490 nm. The OD value is positively correlated with the number of cells. As shown in Figure 6c, the cocultured hPDLCs exhibited significantly higher OD values

than the control cells after 11 days in culture. However, on days 1 to 9, there was no significant difference among the groups. These results suggest that the (COL/ALG)20 multilayer film has a stimulative effect on the proliferation of hPDLCs from the late stage of the logarithmic phase. As presented in Figure 6c, the inadhesive ratio of cells on the (COL/ALG)20 multilayer film (3.86%) was much lower than that of the control group (5.21%) (P < 0.05). Also, the amount of lost cells on the (COL/ALG)20 (4.91%) was significantly lower than that of the control group (9.23%) (P < 0.01)(Figure 6d). The ″lost ratio″ explains the attraction of materials to cells after cell seeding, and the ″inadhesive ratio″ explains the adhesiveness of materials to cells under fluid scouring. This further validates that the (COL/ALG)20 film coating favors the adhesion of hPDLCs. Tissue engineering will benefit from such a scaffold, as the predefined reproducible internal features running across the scaffold, such as these tiny channels formed by the dendritic fibers, can act as an artificial vascular system, potentially 2654

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

Notes

increasing the mass transport of oxygen and nutrients deep into scaffolds.20 A PLA coating was formed on the silica substrate by a spincoating method, and then the contact angle were measured. The angle of PLA(COL/ALG)0 (Figure 7a-1), PLA(COL/ ALG)5 (Figure 7a-2), and PLA(COL/ALG)10 (Figure 7a-3) equaled 75.43 ± 0.65°, 39.93 ± 2.60°, and 54.27 ± 2.83°, respectively. The decrease of the water contact angle demonstrates that the improvement of hydrophilicity as well as cell compatibility of the PLA surface is meliorated. The finding is in accordance with a previous report.43 We then assessed cell adhesion on the electrospun PLA fibers. Electrospinning is a fabrication process that polymer fibers are deposited onto a target substrate in an electric field.54 The SEM images showed the adherence of hPDLCs after 36 h coculture on the PLA electrospun film. In the PLA(COL/ ALG)0 group (Figure 7b), cells were not completely attached, and the contact zones between two cells were well-defined with a sleek borderline. While in the PLA(COL/ALG)20 group (Figure 7c), isolated flat cells attached to the sheet, demonstrating cellular contact zones that were interlaced with a great deal of pseudopods, which was a typical feature of active cells. In the above study, biodegradable polymers and the LbL technique were combined to construct a new kind of bioactive material. Moreover, many kinds of bioactive components can be deposited onto different matrix surfaces using the same process. The formation of nanofibers, such as the dendritic structure with tiny channels, allows for an incorporation of biologically active molecules which can be delivered deeper. Our findings suggest that COL/ALG is powerful for the surface modification of biomaterials.

■

CONCLUSIONS

■

AUTHOR INFORMATION

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study is supported by the following grants: the Ministry of Science and Technology of China (Grant No.2012CB966300), the National Natural Science Funds of China (Grant No.81271369), and Shanghai Municipal Natural Science Foundation (Grant No. 13ZR1443600). The authors would like to acknowledge their financial support.

■

REFERENCES

(1) Yunoki, S.; Matsuda, T. Biomacromolecules 2008, 9, 879−885. (2) Iler, R. J. Colloid Interface Sci. 1966, 21, 569−594. (3) Decher, G. Science 1997, 277, 1232−1237. (4) Verhoeven, M.; Driessen, A.; Paul, A.; Brown, A.; Canry, J.-C.; Hendriks, M. J. Controlled Release 2004, 96, 113−121. (5) Nakayama, Y.; Nishi, S.; Ishibashi-Ueda, H. Cardiovasc. Radiat. Med. 2003, 4, 77−82. (6) Aimé, C.; Mosser, G.; Pembouong, G.; Bouteiller, L.; Coradin, T. Nanoscale 2012, 4, 7127. (7) Luo, L. L.; Wang, G. X.; Li, Y. L.; Yin, T. Y.; Jiang, T.; Ruan, C. G. J. Biomed. Mater. Res. Part A 2011, 97A, 423−432. (8) Luo, C.; Zheng, Y.; Diao, Z.; Qiu, J.; Wang, G. J. Med. Biol. Eng. 2011, 31, 307−316. (9) Hammond, P. Adv. Mater. 2004, 16, 1271−1293. (10) Zhang, X.; Chen, H.; Zhang, H. Chem. Commun. 2007, 0, 1395− 1405. (11) Landoulsi, J.; Roy, C. J.; Dupont-Gillain, C.; DemoustierChampagne, S. Biomacromolecules 2009, 10, 1021−1024. (12) Köster, S.; Leach, J. B.; Struth, B.; Pfohl, T.; Wong, J. Y. Langmuir 2007, 23, 357−359. (13) Florczyk, S. J.; Leung, M.; Jana, S.; Li, Z.; Bhattarai, N.; Huang, J. I.; Hopper, R. A.; Zhang, M. J. Biomed. Mater. Res., Part A 2012, 100A, 3408−3415. (14) Shahin, K.; Doran, P. M. Biotechnol. Prog. 2011, 27, 191−200. (15) Malicev, E.; Radosavljevic, D.; Velikonja, N. K. Biotechnol. Bioeng. 2007, 96, 364−370. (16) Kleinman, H. K.; Klebe, R. J.; Martin, G. R. J. Cell Biol. 1981, 88, 473−485. (17) Li, Y.; Thula, T. T.; Jee, S.; Perkins, S. L.; Aparicio, C.; Douglas, E. P.; Gower, L. B. Biomacromolecules 2012, 13, 49−59. (18) Su, H.-W.; Ho, M.-S.; Cheng, C.-M. Appl. Phys. Lett. 2012, 100, 233703. (19) Lee, C. H.; Singla, A.; Lee, Y. Int. J. Pharm. 2001, 221, 1−22. (20) Sachlos, E.; Reis, N.; Ainsley, C.; Derby, B.; Czernuszka, J. Biomaterials 2003, 24, 1487−1497. (21) Smith, I. O.; Liu, X. H.; Smith, L. A.; Ma, P. X. Wiley Interdiscip. Rev.-Nanomed. Nanobiotechnol. 2009, 1, 226−236. (22) Ma, L.; Gao, C.; Mao, Z.; Zhou, J.; Shen, J. Biomaterials 2004, 25, 2997−3004. (23) Postlethwaite, A. E.; Seyer, J. M.; Kang, A. H. J. Cell Biol. 1978, 75, 871−875. (24) Zhang, J.; Senger, B.; Vautier, D.; Picart, C.; Schaaf, P.; Voegel, J.-C.; Lavalle, P. Biomaterials 2005, 26, 3353−3361. (25) Mhanna, R. F.; vörös, J.; Zenobi-Wong, M. Biomacromolecules 2011, 12, 609−616. (26) Fujie, T.; Furutate, S.; Niwa, D.; Takeoka, S. Soft Matter 2010, 6, 4672−4676. (27) Grant, G. G.; Koktysh, D. S.; Yun, B.; Matts, R. L.; Kotov, N. A. Biomed. Microdevices 2001, 3, 301−306. (28) Chaubaroux, C.; Vrana, E.; Debry, C.; Schaaf, P.; Senger, B.; Voegel, J.-C.; Haikel, Y.; Ringwald, C.; Hemmerle, J.; Lavalle, P. Biomacromolecules 2012, 13, 2128−2135. (29) Schoeters, G.; Leppens, H.; Gorp, U. V.; Heuvel, R. V. D. Cell Prolif. 1992, 25, 587−603. (30) Pawar, S. N.; Edgar, K. J. Biomaterials 2012, 33, 3279−3305.

This study investigated the stability and cytocompatibility of a COL/ALG nano ultrathin film constructed by a LbL assembled technique. The following conclusions can be drawn from the present study: (1) the multilayer film is stable in air and Milli-Q water; (2) cross-linking for an extended period of time prevents dissolution and contributes to the stability of the film in solutions with high pH value or electrolytes; (3) collagen fibers of the film form loose bundles, which improves the adhesion and proliferation of the hPDLCs and modifies the morphology and migration of cells; (4) the COL/ALG film is successfully applied as a surface modification to glass and PLA scaffold, and effectively enhances the cell-material interactions. These findings suggest that the COL/ALG self-assembed ultrathin film, which possessed high stability and cytocompatibility, can play an important role in the effective surface modification of biomaterials. It is foreseeable that the LbL assembly of other charged biomacromolecules, including cell growth factors, differentiation factors, and so on, can be applied and investigated for biocompatibility and biofunction of medical materials and tissue engineering applications.

Corresponding Author

*E-mail: [email protected]. Phone: +86-(0)2166313775. Fax: +86-(0)21-66524025. Author Contributions ⊥

These authors contributed equally to the article and should be co-first authors. 2655

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656

Biomacromolecules

Article

(31) Ramsey, D. M.; Wozniak, D. J. Mol. Microbiol. 2005, 56, 309− 322. (32) Lee, K. Y.; Mooney, D. J. Prog. Polym. Sci. 2012, 37, 106−126. (33) Elçin, Y. M.; Dixit, V.; Gitnick, G. Artif. Organs 2001, 25, 558− 565. (34) Kolambkar, Y. M.; Dupont, K. M.; Boerckel, J. D.; Huebsch, N.; Mooney, D. J.; Hutmacher, D. W.; Guldberg, R. E. Biomaterials 2011, 32, 65−74. (35) Tam, S. K.; Dusseault, J.; Bilodeau, S.; Langlois, G.; Jean-Pierre, H.; Yahia, L. J. Biomed. Mater. Res., Part A 2011, 98A, 40−52. (36) Boateng, J. S.; Matthews, K. H.; Stevens, H. N.; Eccleston, G. M. J. Pharm. Sci. 2008, 97, 2892−2923. (37) Man, Y.; Wang, P.; Guo, Y.; Xiang, L.; Yang, Y.; Qu, Y.; Gong, P.; Deng, L. Biomaterials 2012, 33, 8802−8811. (38) Moshaverinia, A.; Chen, C.; Akiyama, K.; Ansari, S.; Xu, X.; Chee, W. W.; Schricker, S. R.; Shi, S. J. Mater. Sci. 2012, 23, 3041− 3051. (39) Masuda, K. K.; Sah, R. L. R.; Hejna, M. J. M.; Thonar, E. J.-M. A. E. J. Orthop. Res. 2002, 21, 139−148. (40) Lee, B. R.; Hwang, J. W.; Choi, Y. Y.; Wong, S. F.; Hwang, Y. H.; Lee, D. Y.; Lee, S.-H. Biomaterials 2012, 33, 837−845. (41) Kim, G.; Ahn, S.; Kim, Y.; Cho, Y.; Chun, W. Chem. Mater. 2011, 21, 6165−6172. (42) Lee, H.-J.; Ahn, S.-H.; Kim, G. H. Chem. Mater. 2012, 24, 881− 891. (43) Zhang, Y. Z.; Venugopal, J.; Huang, Z. M.; Lim, C. T.; Ramakrishna, S. Biomacromolecules 2005, 6, 2583−2589. (44) Ho, S. P.; Kurylo, M. P.; Fong, T. K.; Lee, S. S. J.; Wagner, H. D.; Ryder, M. I.; Marshall, G. W. Biomaterials 2010, 31, 6635−6646. (45) Ahuja, T.; Dhakray, V.; Mittal, M.; Khanna, P.; Yadav, B.; Jain, M. Internet J. Microbiol. 2012, 10, DOI: 10.5580/2b4e. (46) Müller, H.-P. Periodontology; Thieme: New York, 2005. (47) Kö s ter, S.; Evans, H. M.; Wong, J. Y.; Pfohl, T. Biomacromolecules 2008, 9, 199−207. (48) Popa, G.; Boulmedais, F.; Zhao, P.; Hemmerlé, J.; Vidal, L.; Mathieu, E.; Félix, O.; Schaaf, P.; Decher, G.; Voegel, J.-C. ACS Nano 2010, 4, 4792−4798. (49) Srivastava, A. K.; Sakthivel, P. J. Vac. Sci. Technol. A 2001, 19, 97−100. (50) Seo, B.-M.; Miura, M.; Gronthos, S.; Mark Bartold, P.; Batouli, S.; Brahim, J.; Young, M.; Gehron Robey, P.; Wang, C. Y.; Shi, S. Lancet 2004, 364, 149−155. (51) Kasten, P. Biomaterials 2003, 24, 2593−2603. (52) Komaki, M.; Iwasaki, K.; Arzate, H.; Narayanan, A. S.; Izumi, Y.; Morita, I. J. Cell. Physiol. 2011, 227, 649−657. (53) Chang, J.-Y.; Lin, J.-H.; Yao, C.-H.; Chen, J.-H.; Lai, T.-Y.; Chen, Y.-S. Macromol. Biosci. 2007, 7, 500−507. (54) Matthews, J. A.; Wnek, G. E.; Simpson, D. G.; Bowlin, G. L. Biomacromolecules 2002, 3, 232−238.

2656

dx.doi.org/10.1021/bm4005063 | Biomacromolecules 2013, 14, 2647−2656