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RESEARCH REPORT |
1 Institut National de la Santé et de la Recherche Médicale, Unité 595, 11, rue Humann, 67085 Strasbourg Cedex, France;
2 Faculté de Chirurgie Dentaire, Université Louis Pasteur, 1, Place de l’hôpital, 67000 Strasbourg, France; and
3 Institut Charles Sadron, UPR 22 CNRS, 6 rue Boussingault, 67083 Strasbourg Cedex, France
* corresponding author, new address, Division of Cancer Biology and Tissue Engineering, Tufts University, School of Dental Medicine, 55 Kneeland Street, Boston, MA 02111, USA; christophe.egles{at}tufts.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: surface treatment • polyelectrolyte multilayer film • denture base polymer • coating
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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A broad range of applications for these films has been considered, extending from drug delivery to specific bio-applications based on surface modifications. For example, the multilayer film technique has been used to create microcapsules (Wang et al., 1997), defined as micro- and nanocontainers for storage, transport, and release of active macromolecules (Ibarz et al., 2001). Coating of polystyrene microspheres was developed for application as biosensors in immunoassays (Yang et al., 2001). Other applications have been developed for the use of multilayer films in post-surgical regeneration (Elbert et al., 1999) as anti-coagulant coatings (Serizawa et al., 2002), and for protection against microbial biofilm formation (Boulmedais et al., 2004; Etienne et al., 2004).
Use of these types of multilayered film coatings may be of interest in the dental prosthetic field; however, for these coatings to be used for oral applications, specific issues must first be addressed. The first issue is the nature of the prosthetic polymer surface, whose roughness, low charge, hydrophobicity, and multi-component character could lead to an incomplete coating in comparison with that on glass or industrial metal surfaces. The second issue is related to the specific environment of the oral cavity, since such materials would be in constant contact with saliva, specific enzymes, and a low and changing pH, which could alter the structural integrity of multilayer films.
The purpose of this work was to characterize the thickness and wettability of polyelectrolyte multilayer film coating onto different oral prostheses polymers, and to study its behavior in natural saliva, in vitro and in vivo. The research hypothesis was that these films could be applied to denture base polymers, with clinical benefits in terms of wettability and antimicrobial bioactivity.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Polyelectrolyte Multilayer Film-coated Discs
We prepared solutions of poly(ethylene-imine) (PEI), poly(allylamine hydrochloride) (PAH), and Poly(L-lysine) (PLL) (as polycations), and poly(sodium 4-styrene sulfonate) (PSS) and poly(L-glutamic acid) (PGA) (as polyanions), at 1 mg/mL in 0.15 M NaCl. The multilayer film was constructed by successive dipping of specimen discs in alternating solutions of polycations and polyanions (Fig. 1A
). Each adsorption step was followed by specimen rinsing in 0.15 M NaCl. A precursor film of PEI-(PSS-PAH)2 (Pre) was followed with n (PGA-PLL) layer pairs (Pre-(PGA-PLL)n), where n varied from 6 to 20 adsorption steps, depending on the experimental method used. Dipping was achieved with a robotic device (DR3, Kirstein GmbH, Berlin, Germany). After preparation, the film-coated discs were stored in 0.15 M NaCl until used.
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f), with a v excitation frequency of the crystal on which macromolecule layers were deposited. A decrease in f/v is usually associated with an increase of the mass coupled to the quartz, whereas a degradation (or mass decrease) leads to an increase in f/v.
Film Thickness
PLL labeled with fluorescein isothiocyanate (PLLFITC, Sigma-Aldrich, St. Louis, MO, USA) was used to image the dye-labeled film in the green channel of a confocal laser scanning microscope (CLSM) (LSM510, Carl Zeiss AG, Oberkochen, Germany). PLLFITC was deposited as the last layer, since this compound is able to diffuse throughout the film (Picart et al., 2002). This application allowed us to measure the mean thickness (± standard deviation) of the film by acquiring 80 z-axis sections.
Surface Wettability Changes
The static contact angle measurement was performed with the use of a contact angle meter face instrument (CA-S-150, Kyowa, Tokyo, Japan) at room temperature in ambient conditions. Pre-(PGA-PLL)20 coated samples and bare samples were dried under nitrogen flow before being brought into contact with a 10-µL ultrapure water drop (Milli Q-plus system, Millipore Corporation, Billerica, MA, USA). The angle was determined by the relation
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with h corresponding to the height and r to the radius of the contact area of the drop on the sample (Berg, 1993). For each sample, the mean value of 3 measurements was taken.
Specimen Surface Topography
Topography differences between and among specimen discs were observed by scanning electron microscopy in the environmental mode (ESEM) (XL30, Philips electron optics, Eindhoven, The Netherlands). This mode enabled us to make direct visual observations of the roughness and homogeneity of the composition at the surface, without any further surface treatment.
In vitro Film Stability
After institutional approval for human saliva use and the informed consent of the individuals, specimens were immersed in fresh human saliva obtained by pooling from three individuals after stimulation by paraffin chewing. Surface-deposited film mass was measured over 1000 min by QCM. Using the same film construction as described for thickness measurement, we evaluated the integrity and thickness variations of the film after 48 hrs, using fluorescence microscopy (Eclipse TE2000, Nikon, 10X objective), and after 7 days by CLSM.
In vivo Film Stability
Biocompatibility of the coating was first tested with a fibroblast culture at the surface of a Pre-(PGA-PLL)20 film (see APPENDIX). In vivo studies were conducted on 6 three-month-old male Wistar rats, after institutional approval for animal use. Unpolished specimen discs (1 x 3 mm) were used and coated with the use of the dipping robot (Fig. 4A). The discs were placed at the center of the rat’s cheek to avoid tooth contact (Fig. 4B). After 4 days, 2 discs were lost and 4 were retrieved and observed with CLSM by three different operators to determine film thickness and integrity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). This growth profile has been explained by the PLL diffusion in and out of the film during the build-up steps (Picart et al., 2002). Film thickness, as measured by optical waveguide lightmode spectroscopy, is also related to the number of layers by an exponential law (data not shown). For a Pre-(PGA-PLL)20-PGA-PLLFITC film built on a glass surface, the film thickness approximates 1 µm, as observed by confocal microscopy (Fig. 1C).
Specimen Surface Topography and Surface Wettability Changes
ESEM analysis of PMMA- (Fig. 2A) and PDM-treated disc surfaces (Fig. 2C) indicated a relatively smooth surface when compared with VPS surfaces (Fig. 2E). For all specimens, the effect of polishing was noticeable. PMMA specimens presented a very homogeneous surface (Fig. 2B), whereas PDM surfaces were still heterogeneous, showing glass particles and polymer beads (Fig. 2D). Roughness topography of VPS surfaces, although decreased after polishing, was still quite prominent (Fig. 2F). Analysis of the Z-sections showed total coating of each surface, with great variation in thickness, especially for a non-polished specimen and particularly for VPS. Polished surfaces showed a homogeneous film deposition (Figs. 2B', 2D', 2F'), with mean thicknesses of 0.8 ± 0.1 µm, 1.5 ± 0.2 µm, and 2.1 ± 0.5 µm, respectively, for PMMA, PDM, and VPS. Non-polished specimens showed a coating with aggregates (Figs. 2A', 2C', 2E') and a mean thickness of 4.0 ± 0.3 µm, 4.1 ± 0.7 µm, and 4. 7 ± 1.7 µm, respectively, for PMMA, PDM, and VPS. Surface wettability of the film-coated polymers was drastically increased compared with that of uncoated surfaces. All samples showed at least a ten-fold increase in wettability, except for the polished VPS samples, for which an increase approximated only five-fold (Fig. 2G).
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). Thickness stability of Pre-(PGA-PLL)20-PGA-PLLFITC film was also assessed by confocal microscopy, and no changes (t test, p = 0.61, alpha = 0.05) between the control film in 0.15 M NaCl (1.2 ± 0.4 µm) and the film in human saliva (1.3 ± 0.5 µm) were observed after 48 hrs (Figs. 3B, 3C). Cross-scratch of the film made after the build-up was still visible by fluorescence microscopy after 7 days in saliva (Figs. 3D, 3E).
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). In contrast, tongue-side surfaces showed large spaces devoid of applied film, but were not completely degraded over the four-day period (Fig. 4D). Therefore, it seems that shear forces created by tongue friction were sufficient to alter the film coating, whereas saliva and friction between the mucosal surface and the disc were not. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Taken together, our results support the hypothesis that these polyelectrolyte multilayer films could be useful for coating denture base polymers. Importantly, this advance will provide clinical benefits in terms of increasing the wettability and the antimicrobial protection of oral prosthetic devices.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received May 19, 2004; Last revision August 17, 2005; Accepted September 8, 2005
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REFERENCES |
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Boulmedais F, Frisch B, Etienne O, Lavalle P, Picart C, Ogier J, et al. (2004). Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials. Biomaterials 25:2003–2011.[Medline]
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Etienne O, Picart C, Taddei C, Haikel Y, Dimarcq JL, Schaaf P, et al. (2004). Multilayer polyelectrolyte films functionalized by insertion of defensin: a new approach to protection of implants from bacterial colonization. Antimicrob Agents Chemother 48:3662–3669.
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Picart C, Mutterer J, Richert L, Luo Y, Prestwich GD, Schaaf P, et al. (2002). Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers. Proc Natl Acad Sci USA 99:12531–12535.
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| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
= 3). The exponential regression follows the function: y = y0 + Aexp(Bx) (with y0 = –398.7, A = 431.8, B = 0.1, R = 0.999). (C) Vertical section obtained by confocal laser scanning microscopy (CLSM) observation through a Pre-(PGA-PLL)20-PGA-PLLFITC film on a glass substrate. The film thickness is about 1 µm.
