Odontology DOI 10.1007/s10266-014-0162-5
ORIGINAL ARTICLE
Acidic pH resistance of grafted chitosan on dental implant Doris M. Campos • Be´renge`re Toury • Me´lanie D’Almeida • Ghania N. Attik • Alice Ferrand • Pauline Renoud • Brigitte Grosgogeat
Received: 15 January 2014 / Accepted: 10 March 2014 Ó The Society of The Nippon Dental University 2014
Abstract Over the last decade, access to dental care has increasingly become a service requested by the population, especially in the case of dental implants. However, the major cause of implant failure is an inflammatory disease: peri-implantitis. Currently, the adhesion strength of antibacterial coatings at implant surfaces remains a problem to solve. In order to propose a functionalized implant with a resistant antibacterial coating, a novel method of chitosan immobilization at implant surface has been investigated. Functionalization of the pre-active titanium (Ti) surface was performed using triethoxysilylpropyl succinic anhydride (TESPSA) as a coupling agent which forms a stable double peptide bond with chitosan. The chitosan presence and the chemical resistibility of the coating under acid pH solutions (pH 5 and pH 3) were confirmed by FTIR-ATR and XPS analyses. Furthermore, peel test results showed high adhesive resistance of the TESPSA/chitosan coating at the substrate. Cytocompatibility was evaluated by cell morphology with confocal imaging. Images showed healthy morphology of human gingival fibroblasts (HGF-1). Finally, the reported method for chitosan immobilization on Ti surface via peptide bindings allows for the D. M. Campos B. Grosgogeat UFR d’Odontologie, Universite´ Claude Bernard Lyon 1, 11 rue Guillaume Paradin, 69372 Lyon, France D. M. Campos (&) B. Toury M. D’Almeida G. N. Attik A. Ferrand P. Renoud B. Grosgogeat Laboratoire des Multimate´riaux et Interfaces (UMR CNRS 5615), Universite´ Lyon 1, Villeurbanne, France e-mail: [email protected] B. Grosgogeat Centre de Soins, d’Enseignement et de Recherche Dentaires (Public Health Department), Lyon, France
improvement of its adhesive capacities and resistibility while maintaining its cytocompatibility. Surface functionalization using the TESPSA/chitosan coupling method is noncytotoxic and stable even in drastic environments as found in oral cavity, thus making it a valuable candidate for clinical implantology applications. Keywords Dental implant Chitosan Cytocompatibility Peptide bond Chemical resistance
Introduction Titanium (Ti) implant-coated surfaces have been proposed over the last decades to improve biocompatibility, antimicrobial properties and greater tissue–implant integration in dentistry [1–3]. Despite the high success of metallic implants in replacement of tooth loss, peri-implantitis infection affects the soft and hard tissues surrounding an osseointegrated implant leading to implant failure [4–6]. Even with antiseptic and antibiotic treatments applied during and after surgery, peri-implantitis is the most common reason for secondary failures in risk groups such as diabetics or smokers [7, 8]. As a preventive measure for this important social–economic issue, surface functionalization by natural bioactive polymers has been suggested with great results in reduction of microbial accumulation and plaque formation using antibacterial coatings [9–11]. Chitosan is a low-cost polysaccharide derived from chitin which is largely found in nature in exoskeletons of insects and crustaceans [3, 9, 12]. It has suitable antiseptic and biological properties such as biodegradability, nontoxicity and cytocompatibility. Their antimicrobial properties have already been demonstrated by different groups
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in implantology [2, 13, 14]. However, the attachment, stability and bioactivity maintenance of chitosan coatings at implant surfaces under drastic environments such as the oral cavity have proven to be highly influenced by different factors such as pH value variations [15, 16]. To improve the strength of the covalent adhesion bindings and consequently the immobilization of chitosan molecules on the Ti surfaces, organic coupling agents such as glutaraldehyde [17, 18] or silanated intermediaries such as 3-aminopropyltriethoxysilane (APTES) [19, 20] or triethoxysylilbutyraldehyde (TESBA) molecules [21] have been employed. Currently, a novel chemical functionalization of Ti surfaces by peptide bonds has provided greater mechanical and immobilization properties of chitosan coatings [22]. Following a surface activation process by chemical oxidation, organosilane molecules such as triethoxysilylpropyl succinic anhydride (TESPSA) are able to react with chitosan amino groups by stabilizing peptide bonds. TESPSA/chitosan coating at Ti implants results in higher adhesivity and chemical resistibility making it ideal for oral implantology applications [23, 24]. In this work, attention was mostly devoted to elaborating and evaluating a novel bioactive coating process to maintain greater chitosan surface immobilization to withstand variations in the oral environment. This new method to functionalize titanium surface has two particular strengths: an uncomplicated chemistry (no different intermediaries are used) and the chitosan is coated thanks to a stable double peptide bond. This strong link between the coating and the Ti surface plays a key role in the success of these implants. Without this anchoring, the bioactivity of these implants could be lost. Using TESPSA/chitosancoated surfaces, chemical composition and resistibility
properties under acidic condition were observed by infrared and X-ray photoelectron spectroscopies; coating adhesive strength was observed by peel test. Human gingival fibroblast cells were cultured on TESPSA/chitosancoated surfaces and cytocompatibility was evaluated by confocal imaging. TESPSA/chitosan coating is a stable uncomplicated method to functionalize titanium surfaces by suitable strength of chitosan immobilization for implantology.
Materials and methods Functionalization of titanium surface Titanium (Ti) foils were supplied by Global D (France). Samples were cleaned in an ultrasonic bath with a solution mixture (ethanol/acetone, v/v, 50/50) for 20 min. The surface was oxidized in fresh piranha solution (sulfuric acid/ hydrogen peroxide, v/v, 70/30) during 10 min, rinsed in deionized water and dried at room temperature. The silanation step of the surface was achieved by immersion of the sample in a solution of triethoxysilylpropyl succinic acid anhydride (TESPSA) (Sigma–Aldrich, France) in extra-dry toluene (v/v, 10/90) for 24 h (Fig. 1, step 1). Then the sample was cleaned successively in extra-dry toluene, dimethylformamide and deionized water by ultrasound for 15 min each. A biopolymer solution containing 4 wt% chitosan (Sigma–Aldrich, France), 3 % (v/v) acetic acid, and 97 % (v/ v) deionized water was grafted on the surface by dip coating (v = 3 mm/s) (Fig. 1, step 2). The chitosan-coated samples were then dried at 80 °C for 24 h.
Fig. 1 Schematic image of covalent graft of chitosan on oxidized titanium surface by TESPSA coupling agent method
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Adhesive properties of coating The adhesion strength of the TESPSA/chitosan coating on the Ti surface was observed by peel test. Cross-cut marks were made directly on the coating surface using a scalpel. Standard adhesive tape (ISO 2409 Adhesive Tape, Elcometer, USA) was put on the pattern and pulled off at an angle of 180°. Results were compared with surfaces coated by simple dip deposition. Titanium-coated surface chemical properties To evidence the chitosan presence on the substrate, infra-red spectra were recorded in an attenuated total reflectance mode with a Fourier transform infra-red (ATR-FTIR) spectrometer FTIR 300E (Jasco, France) at wavenumbers ranging from 4000 to 600 cm-1 (64 scans). The chemical bonds within the sample surface were investigated by X-ray photoelectron spectroscopy (XPS, PHI Quantera SXM, ULVAC-PHI, Inc., Japan) using a NANOscan 100 (cameca-RIBer) apparatus with Al K_ X-ray line at 1486.6 eV. In order to evaluate the resistance of the chitosan coating on the Ti surface in physiological media, tests using artificial saliva (Fusayama Meyer solution) were performed at different pH levels (3 and 5). The composition of artificial saliva is similar to natural saliva [25]. In order to reduce the pH, drops of acetic acid solution were added. The samples were placed in the acid solution for 24 h at room temperature. The ATR-FTIR and XPS analyses were compared with the original coated surface.
Samples were washed three times with PBS (pH 7.4) and fixed for 1 h by incubating in 4 % formaldehyde in PBS, followed by further washing. HGF-1 cells were permeabilized with 1 % Triton X100 in PBS and then blocked with 1 % bovine serum albumin (BSA) in PBS. Actin microfilaments were stained by Alexa Fluor 488 phalloidin (green fluorescence) to visualize HGF-1 F-actin. Cell nuclei were identified by Propidium Iodide (red fluorescence) at room temperature. 1024 9 1024 pixel regular confocal images were obtained with a 609 lens and a 0.231 9 0.231 lm pixel size. Images were stored as 12 bits/pixel TIFF files and analyzed with FV10- ASW 3.1 Software (Olympus, France).
Results Adhesive properties of coating Peel test was used in order to estimate the adhesion between the chitosan coating and the Ti substrate. Chitosan (simple dip deposited) and TESPSA/chitosan-coated substrates were tested and the images recorded after the peel test are given in Fig. 2a, b, respectively. In Fig. 2a, it is possible to observe that a large part of the coating has been removed, thus revealing the naked Ti substrate. By contrast, the TESPSA/chitosan grafting is entirely stuck to the Ti surface (Fig. 2b). This observation clearly shows the effectiveness of the method via TESPSA grafting at implant surfaces. Chemical resistibility
In vitro analysis Cell culture Human gingival fibroblast-like (HGF-1) cells were purchased from American Type Culture Collection (ATCC, USA). HGF-1 cells were cultured in essential medium (Dulbecco’s Modified Eagle’s medium (DMEM), with stable L-glutamine supplemented with 10 % fetal bovine serum, 100 U/mL penicillin G ? 100 lg/mL streptomycin sulfate and 2 mL/L amphotericin B, PAA GE healthcare, Austria) at 37 °C in a humidified atmosphere of 5 % CO2 in air. Ti samples were sterilized by UV radiation (at 254 nm) placed 15 cm for 20 min. HGF-1 cells were seeded (1 9 104 cells/scaffold) by adding 500 lL of cell suspension media onto Ti surfaces (uncoated and TESPSA/ chitosan coated) and incubated for 24 and 72 h. Cell morphological analysis Image series were obtained with FV10i, a confocal laser scanning biological inverted microscope (Olympus, France).
The chemical composition of the surface at various steps of the functionalization was determined by XPS. The changes in the elemental composition at the surface are quantified in Table 1. The first step of the reaction was the TESPSA silanation of the titanium surface as described in Fig. 1. Successful grafting of TESPSA on the Ti surface was indicated by the decrease in the Ti components and the arrival of Si components (Table 1). The second step of the reaction was the coating of the chitosan (Fig. 1). By XPS analysis, the presence of chitosan is confirmed since the recorded spectrum (Fig. 3a) presents three signals centered at around 285.5, 399.3 and 532.5 eV which are characteristic of the C1s, N1s and O1s photoelectron peaks, respectively, expected for the chitosan [26]. The increase in the percentages of C and N components and the disappearance of Ti elements are summarized in Table 1. The chitosan coating was also highlighted by ATRFTIR. The ATR-FTIR spectrum proposed in Fig. 4a displays all the expected bands for chitosan [21] assuming that the Ti surface is well covered by this biopolymer. The overlapped peaks of N–H stretching and O–H stretching
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Fig. 2 Optical images of chitosan-coated Ti substrate by simple dip deposition (a) and TESPSA/chitosan-coated substrate (b) after peel test. Scale bar 200 lm
Table 1 Elemental composition (%) and N/Ti atomic ratio at the surface of uncoated Ti sample, Ti/TESPSA, TESPSA/chitosan-coated sample and TESPSA/chitosan-coated samples immersed in acidic solution at pH 5 and pH 3 Substrate
C1s (%)
N1s (%)
O1s (%)
Ti2p (%)
Si2p (%)
Others (%)
N/ Ti
Ti (uncoated sample)
36
0.5
45.5
11.8
–
6.2
0.04
Ti/TESPSA
53
–
34.5
2.5
6
4
–
TESPSA/ chitosancoated sample
66.8
5.7
27.3
–
–
–
–
TESPSA/ chitosancoated sample immersed in acidic solution at pH 5
61.6
4.9
30.6
–
0.3
2.6
–
TESPSA/ chitosancoated sample immersed in acidic solution at pH 3
40.7
1.5
42.7
8.7
0.4
6
0.17
modes that occur around 3251 cm-1 indicate the presence of hydroxyl groups. The medium intensity peak at 1414 cm-1 corresponding to O–H bending confirms the existence of this O–H functional group. The weak intensity band at 2879 cm-1 corresponds to C–H stretch [27]. Peaks at 1633, at 1537 and at 1383 cm-1 are assigned to –C=O
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amide, –NH2, and –NHCO amide, respectively [28, 29]. The peaks at 1070, at 1011 and at 887 cm-1 are attributed to the saccharide structure of chitosan [30]. About the acidic chemical resistibility test, two other spectra were recorded from the TESPSA/chitosan-coated sample (Figs. 3b, c, 4b, c). XPS spectra were recorded on both samples after 24 h of immersion in pH 5 and pH 3 solutions (Fig. 3b, c, respectively). In Fig. 3, the peak at 399.3 eV which was assigned to N1s was still present for both immersions. The N1s percentage found on the TESPSA/chitosan-coated surface before and after the pH 5 acidic test was very close: 5.7 and 4.9 %, respectively, confirming chitosan preservation (Table 1). Otherwise, the percentage of nitrogen on the sample submitted to the pH 3 acidic test was lower (1.5 %), confirming the lower amount of chitosan in this case. This result was emphasized by the detection of an additional peak at 579 eV, characteristic of the Ti2p photoelectron peak. On one hand, part of the chitosan coating was dissolved under the more severe acidic conditions. On the other hand, at pH 3, the presence of the chitosan coating was also confirmed by the atomic ratio N/Ti. The FTIR-ATR analyses also confirm these results. Both of the added FTIR-ATR spectra showed equivalent signatures, meaning that chitosan chains were still present on the sample surface even after acidic immersion (Fig. 4). However, comparing both latter spectra, we verified that the chitosan band intensity decreased significantly, from the sample immersed in pH 5 solution to the sample immersed in pH 3. Cell morphology Confocal imaging was used to assess the cell morphology on the TESPSA/chitosan-coated surface. The ability of the cells to spread on Ti substrates was shown by cytoskeletal
Odontology Fig. 3 XPS spectra of TESPSA/chitosan-coated samples a; spectra of TESPSA/ chitosan-coated samples immersed in acidic solution at pH 5 b and pH 3 c for 24 h
Fig. 4 FTIR-ATR spectra of TESPSA/chitosan-coated samples a; spectra of TESPSA/chitosan-coated samples immersed in acidic solution at pH 5 b and pH 3 c for 24 h
and nucleus staining (Fig. 5). HGF-1 cell growth on uncoated and coated Ti implant surfaces respected their normal morphology following the 3 days of incubation (Fig. 5b, d). The morphology of cells in contact with the TESPSA/chitosan-coated substrate (Fig. 5c, d) was similar to that in contact with the uncoated titanium (Fig. 5a, b). No morphological alterations were obtained through experimental periods on the TESPSA/chitosan-coated surface showing no toxicity of chitosan coating. The HGF-1 cells appeared and maintained their proliferative capacity to cover the implant surfaces.
Discussion Titanium (Ti) and titanium alloys are key dental implants because of their great biocompatibility, surrounded tissue integration and mechanical properties. Nonetheless, bacterial infection on and around implants still remains a health problem which medically compromise patients. In some cases of peri-implantitis inflammation, antibiotic therapy is not sufficient to control the disease, leading to eventual implant removal [4–6]. Investigations about implant surface improvements such as the immobilization of antibacterial
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Odontology Fig. 5 Confocal images of human gingival fibroblast (HGF-1) morphology. HGF-1 cells were cultured on uncoated Ti surface and TESPSA/ chitosan-coated surface samples for 24 (a, c) and 72 h (b, d), respectively. HGF-actin microfilaments in green and cell nuclei in red stain. Scale bar 50 lm
molecules or the introduction of trough coupling agents have been studied by several research groups [9, 21, 29]. The current challenge is to produce bioactive coatings which resist to drastic environments, such as those seen in the oral cavity, while maintaining antibacterial and high biological performance. Described methods could be complicated, using two or three intermediaries and resulting in undesirable properties for implant clinical applications. The quality of a bioactive coating depends in part on the strength of the biomolecule adhesion at implant surfaces. We performed a simple and stable chitosan layer immobilized via one-step sinalation reaction at Ti substrates using an intermediary organosilane molecule—triethoxysilylpropyl succinic anhydride (TESPSA) (Fig. 1). We demonstrated that the adhesion strength of the chitosan coating on the surfaces treated with a TESPSA molecule was stronger than on the surfaces coated by simple dip chitosan deposition by peel test (Fig. 2). To immobilize chitosan molecules on the Ti surfaces, the chemical oxidation reaction created hydroxide groups on the Ti surface that reacted with the ethoxy groups of the TESPSA, resulting in Ti–O–Si groups. Therefore, during this step, Si–O–Si bonds could be formed. In the
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second reaction, the functional groups of TESPSA (anhydride) reacted with the amino groups of chitosan and this biopolymer became linked covalently via stable peptide bindings on the Ti surface (Fig. 1). The stability of this sequence of reactions may guarantee the high performance of this type of coated implant after implantation. The performance of coatings in the oral cavity has been previously evaluated using similar conditions such as pH value variation tests [25]. The presence and preservation of chitosan after the immobilization process and submitted to acidic environment has been observed by semi-quantitative techniques. By the XPS measurements (Fig. 3) and the FTIR-ATR analysis (Fig. 4) measurements, the spectra of the TESPSA/chitosan coating contained several characteristic peaks of chitosan with the presence of N–H stretching and O–H stretching modes and amide groups [21, 30]. These results tend to prove that the proposed grafting was efficient in keeping chitosan at the Ti substrate. Similar analysis was compared to materials under acidic conditions. On the chitosan-coated surface by simple dip deposition, we verified that all polymers were removed from the sample in contrast to the TESPSA/chitosan-coated sample. Moreover,
Odontology
when submitting TESPSA/chitosan-coated substrates to more drastic acidic conditions (at pH 3), the residual chitosan amount was less than the highest pH value treatment. Moreover, it should be mentioned that a naked eye observation allows for observing the chitosan coating that is still visible on the substrate in both cases. Finally, since the thickness probes with the ATR crystal were around 3 lm, residual chitosan thickness can be estimated up to 3 lm for the sample obtained after pH 5 immersion and lower than this value for the other sample (pH 3 condition). As has been seen from different groups focused in implantology, the use of numerous steps and chemicals for the implant development may lead to certain toxicity. Implant’s integration depends on the ability of surrounding cells to adhere and create a new extracellular matrix [1, 3]. The fact of having a biocompatible polymer as chitosan and a tridimensional structure provided by the layer plays crucial roles in the healing process. Lastly, from confocal imaging, we demonstrated the cytocompatibility with spread gingival fibroblasts by cytoskeletal actin microfilaments immunostaining (Fig. 5). In the current work, we performed and evaluated the chitosan immobilization on pre-active titanium surfaces through double peptide bonds using nontoxicity, one step less and stable triethoxysilylpropyl succinic anhydride (TESPSA) molecules. The aim of this study was to highlight a novel method to graft chitosan chains on titanium implant surfaces to reinforce the biocompatibility and prolong the presence of chitosan layer. Recently, this method was published as part of a patent document (WO PCT/FR2013/ O50132) [31] opening new clinical perspectives. The suitable results obtained by graft adhesion and cytocompatibility tests showed that the TESPSA/chitosan-coated surface was mechanically stable while maintaining a good biological performance. The higher chemical resistibility due to stable peptide bindings shows that these proposed functionalized implants would be resistant in a hostile environment such as in the oral cavity. Its bioactivity and antibacterial properties remain to be validated in future. Acknowledgments The authors wish to acknowledge Global D for providing titanium samples and Science et surface for XPS analysis; the authors wish to express their appreciation to the Lyon Science Transfert Department (LST 784 BTO) of the University of Lyon and the Institut Franc¸ais de la Recherche (IFRO 2011) for their financial support. Conflict of interest of interest.
The authors declare that they have no conflict
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ORIGINAL ARTICLE
Acidic pH resistance of grafted chitosan on dental implant Doris M. Campos • Be´renge`re Toury • Me´lanie D’Almeida • Ghania N. Attik • Alice Ferrand • Pauline Renoud • Brigitte Grosgogeat
Received: 15 January 2014 / Accepted: 10 March 2014 Ó The Society of The Nippon Dental University 2014
Abstract Over the last decade, access to dental care has increasingly become a service requested by the population, especially in the case of dental implants. However, the major cause of implant failure is an inflammatory disease: peri-implantitis. Currently, the adhesion strength of antibacterial coatings at implant surfaces remains a problem to solve. In order to propose a functionalized implant with a resistant antibacterial coating, a novel method of chitosan immobilization at implant surface has been investigated. Functionalization of the pre-active titanium (Ti) surface was performed using triethoxysilylpropyl succinic anhydride (TESPSA) as a coupling agent which forms a stable double peptide bond with chitosan. The chitosan presence and the chemical resistibility of the coating under acid pH solutions (pH 5 and pH 3) were confirmed by FTIR-ATR and XPS analyses. Furthermore, peel test results showed high adhesive resistance of the TESPSA/chitosan coating at the substrate. Cytocompatibility was evaluated by cell morphology with confocal imaging. Images showed healthy morphology of human gingival fibroblasts (HGF-1). Finally, the reported method for chitosan immobilization on Ti surface via peptide bindings allows for the D. M. Campos B. Grosgogeat UFR d’Odontologie, Universite´ Claude Bernard Lyon 1, 11 rue Guillaume Paradin, 69372 Lyon, France D. M. Campos (&) B. Toury M. D’Almeida G. N. Attik A. Ferrand P. Renoud B. Grosgogeat Laboratoire des Multimate´riaux et Interfaces (UMR CNRS 5615), Universite´ Lyon 1, Villeurbanne, France e-mail: [email protected] B. Grosgogeat Centre de Soins, d’Enseignement et de Recherche Dentaires (Public Health Department), Lyon, France
improvement of its adhesive capacities and resistibility while maintaining its cytocompatibility. Surface functionalization using the TESPSA/chitosan coupling method is noncytotoxic and stable even in drastic environments as found in oral cavity, thus making it a valuable candidate for clinical implantology applications. Keywords Dental implant Chitosan Cytocompatibility Peptide bond Chemical resistance
Introduction Titanium (Ti) implant-coated surfaces have been proposed over the last decades to improve biocompatibility, antimicrobial properties and greater tissue–implant integration in dentistry [1–3]. Despite the high success of metallic implants in replacement of tooth loss, peri-implantitis infection affects the soft and hard tissues surrounding an osseointegrated implant leading to implant failure [4–6]. Even with antiseptic and antibiotic treatments applied during and after surgery, peri-implantitis is the most common reason for secondary failures in risk groups such as diabetics or smokers [7, 8]. As a preventive measure for this important social–economic issue, surface functionalization by natural bioactive polymers has been suggested with great results in reduction of microbial accumulation and plaque formation using antibacterial coatings [9–11]. Chitosan is a low-cost polysaccharide derived from chitin which is largely found in nature in exoskeletons of insects and crustaceans [3, 9, 12]. It has suitable antiseptic and biological properties such as biodegradability, nontoxicity and cytocompatibility. Their antimicrobial properties have already been demonstrated by different groups
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in implantology [2, 13, 14]. However, the attachment, stability and bioactivity maintenance of chitosan coatings at implant surfaces under drastic environments such as the oral cavity have proven to be highly influenced by different factors such as pH value variations [15, 16]. To improve the strength of the covalent adhesion bindings and consequently the immobilization of chitosan molecules on the Ti surfaces, organic coupling agents such as glutaraldehyde [17, 18] or silanated intermediaries such as 3-aminopropyltriethoxysilane (APTES) [19, 20] or triethoxysylilbutyraldehyde (TESBA) molecules [21] have been employed. Currently, a novel chemical functionalization of Ti surfaces by peptide bonds has provided greater mechanical and immobilization properties of chitosan coatings [22]. Following a surface activation process by chemical oxidation, organosilane molecules such as triethoxysilylpropyl succinic anhydride (TESPSA) are able to react with chitosan amino groups by stabilizing peptide bonds. TESPSA/chitosan coating at Ti implants results in higher adhesivity and chemical resistibility making it ideal for oral implantology applications [23, 24]. In this work, attention was mostly devoted to elaborating and evaluating a novel bioactive coating process to maintain greater chitosan surface immobilization to withstand variations in the oral environment. This new method to functionalize titanium surface has two particular strengths: an uncomplicated chemistry (no different intermediaries are used) and the chitosan is coated thanks to a stable double peptide bond. This strong link between the coating and the Ti surface plays a key role in the success of these implants. Without this anchoring, the bioactivity of these implants could be lost. Using TESPSA/chitosancoated surfaces, chemical composition and resistibility
properties under acidic condition were observed by infrared and X-ray photoelectron spectroscopies; coating adhesive strength was observed by peel test. Human gingival fibroblast cells were cultured on TESPSA/chitosancoated surfaces and cytocompatibility was evaluated by confocal imaging. TESPSA/chitosan coating is a stable uncomplicated method to functionalize titanium surfaces by suitable strength of chitosan immobilization for implantology.
Materials and methods Functionalization of titanium surface Titanium (Ti) foils were supplied by Global D (France). Samples were cleaned in an ultrasonic bath with a solution mixture (ethanol/acetone, v/v, 50/50) for 20 min. The surface was oxidized in fresh piranha solution (sulfuric acid/ hydrogen peroxide, v/v, 70/30) during 10 min, rinsed in deionized water and dried at room temperature. The silanation step of the surface was achieved by immersion of the sample in a solution of triethoxysilylpropyl succinic acid anhydride (TESPSA) (Sigma–Aldrich, France) in extra-dry toluene (v/v, 10/90) for 24 h (Fig. 1, step 1). Then the sample was cleaned successively in extra-dry toluene, dimethylformamide and deionized water by ultrasound for 15 min each. A biopolymer solution containing 4 wt% chitosan (Sigma–Aldrich, France), 3 % (v/v) acetic acid, and 97 % (v/ v) deionized water was grafted on the surface by dip coating (v = 3 mm/s) (Fig. 1, step 2). The chitosan-coated samples were then dried at 80 °C for 24 h.
Fig. 1 Schematic image of covalent graft of chitosan on oxidized titanium surface by TESPSA coupling agent method
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Adhesive properties of coating The adhesion strength of the TESPSA/chitosan coating on the Ti surface was observed by peel test. Cross-cut marks were made directly on the coating surface using a scalpel. Standard adhesive tape (ISO 2409 Adhesive Tape, Elcometer, USA) was put on the pattern and pulled off at an angle of 180°. Results were compared with surfaces coated by simple dip deposition. Titanium-coated surface chemical properties To evidence the chitosan presence on the substrate, infra-red spectra were recorded in an attenuated total reflectance mode with a Fourier transform infra-red (ATR-FTIR) spectrometer FTIR 300E (Jasco, France) at wavenumbers ranging from 4000 to 600 cm-1 (64 scans). The chemical bonds within the sample surface were investigated by X-ray photoelectron spectroscopy (XPS, PHI Quantera SXM, ULVAC-PHI, Inc., Japan) using a NANOscan 100 (cameca-RIBer) apparatus with Al K_ X-ray line at 1486.6 eV. In order to evaluate the resistance of the chitosan coating on the Ti surface in physiological media, tests using artificial saliva (Fusayama Meyer solution) were performed at different pH levels (3 and 5). The composition of artificial saliva is similar to natural saliva [25]. In order to reduce the pH, drops of acetic acid solution were added. The samples were placed in the acid solution for 24 h at room temperature. The ATR-FTIR and XPS analyses were compared with the original coated surface.
Samples were washed three times with PBS (pH 7.4) and fixed for 1 h by incubating in 4 % formaldehyde in PBS, followed by further washing. HGF-1 cells were permeabilized with 1 % Triton X100 in PBS and then blocked with 1 % bovine serum albumin (BSA) in PBS. Actin microfilaments were stained by Alexa Fluor 488 phalloidin (green fluorescence) to visualize HGF-1 F-actin. Cell nuclei were identified by Propidium Iodide (red fluorescence) at room temperature. 1024 9 1024 pixel regular confocal images were obtained with a 609 lens and a 0.231 9 0.231 lm pixel size. Images were stored as 12 bits/pixel TIFF files and analyzed with FV10- ASW 3.1 Software (Olympus, France).
Results Adhesive properties of coating Peel test was used in order to estimate the adhesion between the chitosan coating and the Ti substrate. Chitosan (simple dip deposited) and TESPSA/chitosan-coated substrates were tested and the images recorded after the peel test are given in Fig. 2a, b, respectively. In Fig. 2a, it is possible to observe that a large part of the coating has been removed, thus revealing the naked Ti substrate. By contrast, the TESPSA/chitosan grafting is entirely stuck to the Ti surface (Fig. 2b). This observation clearly shows the effectiveness of the method via TESPSA grafting at implant surfaces. Chemical resistibility
In vitro analysis Cell culture Human gingival fibroblast-like (HGF-1) cells were purchased from American Type Culture Collection (ATCC, USA). HGF-1 cells were cultured in essential medium (Dulbecco’s Modified Eagle’s medium (DMEM), with stable L-glutamine supplemented with 10 % fetal bovine serum, 100 U/mL penicillin G ? 100 lg/mL streptomycin sulfate and 2 mL/L amphotericin B, PAA GE healthcare, Austria) at 37 °C in a humidified atmosphere of 5 % CO2 in air. Ti samples were sterilized by UV radiation (at 254 nm) placed 15 cm for 20 min. HGF-1 cells were seeded (1 9 104 cells/scaffold) by adding 500 lL of cell suspension media onto Ti surfaces (uncoated and TESPSA/ chitosan coated) and incubated for 24 and 72 h. Cell morphological analysis Image series were obtained with FV10i, a confocal laser scanning biological inverted microscope (Olympus, France).
The chemical composition of the surface at various steps of the functionalization was determined by XPS. The changes in the elemental composition at the surface are quantified in Table 1. The first step of the reaction was the TESPSA silanation of the titanium surface as described in Fig. 1. Successful grafting of TESPSA on the Ti surface was indicated by the decrease in the Ti components and the arrival of Si components (Table 1). The second step of the reaction was the coating of the chitosan (Fig. 1). By XPS analysis, the presence of chitosan is confirmed since the recorded spectrum (Fig. 3a) presents three signals centered at around 285.5, 399.3 and 532.5 eV which are characteristic of the C1s, N1s and O1s photoelectron peaks, respectively, expected for the chitosan [26]. The increase in the percentages of C and N components and the disappearance of Ti elements are summarized in Table 1. The chitosan coating was also highlighted by ATRFTIR. The ATR-FTIR spectrum proposed in Fig. 4a displays all the expected bands for chitosan [21] assuming that the Ti surface is well covered by this biopolymer. The overlapped peaks of N–H stretching and O–H stretching
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Fig. 2 Optical images of chitosan-coated Ti substrate by simple dip deposition (a) and TESPSA/chitosan-coated substrate (b) after peel test. Scale bar 200 lm
Table 1 Elemental composition (%) and N/Ti atomic ratio at the surface of uncoated Ti sample, Ti/TESPSA, TESPSA/chitosan-coated sample and TESPSA/chitosan-coated samples immersed in acidic solution at pH 5 and pH 3 Substrate
C1s (%)
N1s (%)
O1s (%)
Ti2p (%)
Si2p (%)
Others (%)
N/ Ti
Ti (uncoated sample)
36
0.5
45.5
11.8
–
6.2
0.04
Ti/TESPSA
53
–
34.5
2.5
6
4
–
TESPSA/ chitosancoated sample
66.8
5.7
27.3
–
–
–
–
TESPSA/ chitosancoated sample immersed in acidic solution at pH 5
61.6
4.9
30.6
–
0.3
2.6
–
TESPSA/ chitosancoated sample immersed in acidic solution at pH 3
40.7
1.5
42.7
8.7
0.4
6
0.17
modes that occur around 3251 cm-1 indicate the presence of hydroxyl groups. The medium intensity peak at 1414 cm-1 corresponding to O–H bending confirms the existence of this O–H functional group. The weak intensity band at 2879 cm-1 corresponds to C–H stretch [27]. Peaks at 1633, at 1537 and at 1383 cm-1 are assigned to –C=O
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amide, –NH2, and –NHCO amide, respectively [28, 29]. The peaks at 1070, at 1011 and at 887 cm-1 are attributed to the saccharide structure of chitosan [30]. About the acidic chemical resistibility test, two other spectra were recorded from the TESPSA/chitosan-coated sample (Figs. 3b, c, 4b, c). XPS spectra were recorded on both samples after 24 h of immersion in pH 5 and pH 3 solutions (Fig. 3b, c, respectively). In Fig. 3, the peak at 399.3 eV which was assigned to N1s was still present for both immersions. The N1s percentage found on the TESPSA/chitosan-coated surface before and after the pH 5 acidic test was very close: 5.7 and 4.9 %, respectively, confirming chitosan preservation (Table 1). Otherwise, the percentage of nitrogen on the sample submitted to the pH 3 acidic test was lower (1.5 %), confirming the lower amount of chitosan in this case. This result was emphasized by the detection of an additional peak at 579 eV, characteristic of the Ti2p photoelectron peak. On one hand, part of the chitosan coating was dissolved under the more severe acidic conditions. On the other hand, at pH 3, the presence of the chitosan coating was also confirmed by the atomic ratio N/Ti. The FTIR-ATR analyses also confirm these results. Both of the added FTIR-ATR spectra showed equivalent signatures, meaning that chitosan chains were still present on the sample surface even after acidic immersion (Fig. 4). However, comparing both latter spectra, we verified that the chitosan band intensity decreased significantly, from the sample immersed in pH 5 solution to the sample immersed in pH 3. Cell morphology Confocal imaging was used to assess the cell morphology on the TESPSA/chitosan-coated surface. The ability of the cells to spread on Ti substrates was shown by cytoskeletal
Odontology Fig. 3 XPS spectra of TESPSA/chitosan-coated samples a; spectra of TESPSA/ chitosan-coated samples immersed in acidic solution at pH 5 b and pH 3 c for 24 h
Fig. 4 FTIR-ATR spectra of TESPSA/chitosan-coated samples a; spectra of TESPSA/chitosan-coated samples immersed in acidic solution at pH 5 b and pH 3 c for 24 h
and nucleus staining (Fig. 5). HGF-1 cell growth on uncoated and coated Ti implant surfaces respected their normal morphology following the 3 days of incubation (Fig. 5b, d). The morphology of cells in contact with the TESPSA/chitosan-coated substrate (Fig. 5c, d) was similar to that in contact with the uncoated titanium (Fig. 5a, b). No morphological alterations were obtained through experimental periods on the TESPSA/chitosan-coated surface showing no toxicity of chitosan coating. The HGF-1 cells appeared and maintained their proliferative capacity to cover the implant surfaces.
Discussion Titanium (Ti) and titanium alloys are key dental implants because of their great biocompatibility, surrounded tissue integration and mechanical properties. Nonetheless, bacterial infection on and around implants still remains a health problem which medically compromise patients. In some cases of peri-implantitis inflammation, antibiotic therapy is not sufficient to control the disease, leading to eventual implant removal [4–6]. Investigations about implant surface improvements such as the immobilization of antibacterial
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Odontology Fig. 5 Confocal images of human gingival fibroblast (HGF-1) morphology. HGF-1 cells were cultured on uncoated Ti surface and TESPSA/ chitosan-coated surface samples for 24 (a, c) and 72 h (b, d), respectively. HGF-actin microfilaments in green and cell nuclei in red stain. Scale bar 50 lm
molecules or the introduction of trough coupling agents have been studied by several research groups [9, 21, 29]. The current challenge is to produce bioactive coatings which resist to drastic environments, such as those seen in the oral cavity, while maintaining antibacterial and high biological performance. Described methods could be complicated, using two or three intermediaries and resulting in undesirable properties for implant clinical applications. The quality of a bioactive coating depends in part on the strength of the biomolecule adhesion at implant surfaces. We performed a simple and stable chitosan layer immobilized via one-step sinalation reaction at Ti substrates using an intermediary organosilane molecule—triethoxysilylpropyl succinic anhydride (TESPSA) (Fig. 1). We demonstrated that the adhesion strength of the chitosan coating on the surfaces treated with a TESPSA molecule was stronger than on the surfaces coated by simple dip chitosan deposition by peel test (Fig. 2). To immobilize chitosan molecules on the Ti surfaces, the chemical oxidation reaction created hydroxide groups on the Ti surface that reacted with the ethoxy groups of the TESPSA, resulting in Ti–O–Si groups. Therefore, during this step, Si–O–Si bonds could be formed. In the
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second reaction, the functional groups of TESPSA (anhydride) reacted with the amino groups of chitosan and this biopolymer became linked covalently via stable peptide bindings on the Ti surface (Fig. 1). The stability of this sequence of reactions may guarantee the high performance of this type of coated implant after implantation. The performance of coatings in the oral cavity has been previously evaluated using similar conditions such as pH value variation tests [25]. The presence and preservation of chitosan after the immobilization process and submitted to acidic environment has been observed by semi-quantitative techniques. By the XPS measurements (Fig. 3) and the FTIR-ATR analysis (Fig. 4) measurements, the spectra of the TESPSA/chitosan coating contained several characteristic peaks of chitosan with the presence of N–H stretching and O–H stretching modes and amide groups [21, 30]. These results tend to prove that the proposed grafting was efficient in keeping chitosan at the Ti substrate. Similar analysis was compared to materials under acidic conditions. On the chitosan-coated surface by simple dip deposition, we verified that all polymers were removed from the sample in contrast to the TESPSA/chitosan-coated sample. Moreover,
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when submitting TESPSA/chitosan-coated substrates to more drastic acidic conditions (at pH 3), the residual chitosan amount was less than the highest pH value treatment. Moreover, it should be mentioned that a naked eye observation allows for observing the chitosan coating that is still visible on the substrate in both cases. Finally, since the thickness probes with the ATR crystal were around 3 lm, residual chitosan thickness can be estimated up to 3 lm for the sample obtained after pH 5 immersion and lower than this value for the other sample (pH 3 condition). As has been seen from different groups focused in implantology, the use of numerous steps and chemicals for the implant development may lead to certain toxicity. Implant’s integration depends on the ability of surrounding cells to adhere and create a new extracellular matrix [1, 3]. The fact of having a biocompatible polymer as chitosan and a tridimensional structure provided by the layer plays crucial roles in the healing process. Lastly, from confocal imaging, we demonstrated the cytocompatibility with spread gingival fibroblasts by cytoskeletal actin microfilaments immunostaining (Fig. 5). In the current work, we performed and evaluated the chitosan immobilization on pre-active titanium surfaces through double peptide bonds using nontoxicity, one step less and stable triethoxysilylpropyl succinic anhydride (TESPSA) molecules. The aim of this study was to highlight a novel method to graft chitosan chains on titanium implant surfaces to reinforce the biocompatibility and prolong the presence of chitosan layer. Recently, this method was published as part of a patent document (WO PCT/FR2013/ O50132) [31] opening new clinical perspectives. The suitable results obtained by graft adhesion and cytocompatibility tests showed that the TESPSA/chitosan-coated surface was mechanically stable while maintaining a good biological performance. The higher chemical resistibility due to stable peptide bindings shows that these proposed functionalized implants would be resistant in a hostile environment such as in the oral cavity. Its bioactivity and antibacterial properties remain to be validated in future. Acknowledgments The authors wish to acknowledge Global D for providing titanium samples and Science et surface for XPS analysis; the authors wish to express their appreciation to the Lyon Science Transfert Department (LST 784 BTO) of the University of Lyon and the Institut Franc¸ais de la Recherche (IFRO 2011) for their financial support. Conflict of interest of interest.
The authors declare that they have no conflict
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