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XPS Study on the Use of 3-Aminopropyltriethoxysilane to Bond Chitosan to a Titanium Surface Holly J. Martin,* Kirk H. Schulz, Joel D. Bumgardner,† and Keisha B. Walters DaVe C. Swalm School of Chemical Engineering, James Worth Bagley College of Engineering, Mississippi State UniVersity, Box 9595, Mississippi State, Mississippi 39762 ReceiVed NoVember 9, 2006. In Final Form: March 7, 2007 Chitosan, a biopolymer found in the exoskeletons of shellfish, has been shown to be antibacterial, biodegradable, osteoconductive, and has the ability to promote organized bone formation. These properties make chitosan an ideal material for use as a bioactive coating on medical implant materials. In this study, coatings made from 86.4% deacetylated chitosan were bound to implant-quality titanium. The chitosan films were bound through a three-step process that involved the deposition of 3-aminopropyltriethoxysilane (APTES) in toluene, followed by a reaction between the amine end of APTES with gluteraldehyde, and finally, a reaction between the aldehyde end of gluteraldehyde and chitosan. Two different metal treatments were examined to determine if major differences in the ability to bind chitosan could be seen. X-ray photoelectron spectroscopy (XPS) was used to examine the surface of the titanium metal and to study the individual reaction steps. The changes to the titanium surface were consistent with the anticipated reaction steps, with significant changes in the amounts of nitrogen, silicon, and titanium that were present. It was demonstrated that more APTES was bound to the piranha-treated titanium surface as compared to the passivated titanium surface, based on the amounts of titanium, carbon, nitrogen, and silicon that were present. The metal treatments did not affect the chemistry of the chitosan films. Using toluene to bond APTES on titanium surfaces, rather than aqueous solutions, prevented the formation of unwanted polysiloxanes and increased the amount of silane on the surface for forming bonds to the chitosan films. Qualitatively, the films were more strongly attached to the titanium surfaces after using toluene, which could withstand the ultrahigh vacuum environment of XPS, as compared to the aqueous solutions, which were removed from the titanium surface when exposed to the ultrahigh vacuum environment of XPS.
Introduction One of the major issues with orthopaedic and dental/craniofacial implants today is the lack of interaction between the tissue surrounding the implant and the implant itself. This lack of osseointegration, or the incorporation of the implant into the surrounding bone, is being addressed in the biomedical literature through the modification of the implant surface by bonding bioactive coatings. Bioactive coatings help develop strong tissue attachments1 and include hydroxyapatite,2-4 calcium phosphate,5-6 bioactive glass,7 and biologically functional molecules, such as enzymes and proteins.8-10 Hydroxyapatite, calcium phosphate, and bioactive glass are ceramics or glassceramics, and their hard and brittle nature can lead to flaking and cracking caused by scratches produced during implantation.1,3,11 The loss of the coating reduces osseointegration and * To whom correspondence should be addressed. E-mail: hjp2@ msstate.edu. † Department of Biomedical Engineering, Herff College of Engineering, University of Memphis, 330 Engineering Technology Building, Memphis, TN, 38152. (1) Ratner, B. D. In Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, 1996; Foreword. (2) Kummer, F. J.; Jaffe, W. L. J. Appl. Biomater. 1992, 3, 211. (3) Cook, S. D.; Thomas, K. A.; Kay, J. F. Clin. Ortho. Relat. Res. 1992, 265, 280. (4) Friedman, R. J.; Bauer, T. W.; Garg, K.; Jiang, M.; An, Y. H.; Draughn, R. A. J. Appl. Biomater. 1995, 6, 231. (5) Yang, Y.; Agrawal, C. M.; Kim, K. H.; Martin, H.; Schulz, K.; Bumgardner, J. D.; Ong, J. L. J. Oral Implantology 2003, 29, 270. (6) Maxian, S. H.; Zawadsky, J. P.; Dunn, M. G. J. Biomed. Mater. Res. 1994, 28, 1311. (7) Schrooten, J.; Helsen, J. A. Biomaterials 2000, 21, 1461. (8) Nanci, A.; Wuest, J. D.; Peru, L.; Brunet, P.; Sharma, V.; Zalzal, S.; McKee, M. D. J. Biomed. Mater. Res. 1998, 40, 324. (9) Puleo, D. A. J. Biomed. Mater. Res. 1997, 37, 222. (10) Puleo, D. A. J. Biomed. Mater. Res. 1995, 29, 951. (11) Chen, F.; Wang, Z. C.; Lin, C. J. Mater. Lett. 2002, 57, 848.
leads to the growth of fibroblasts, which prevents orderly bone tissue construction.12 Ceramics, glass-ceramics, and biologically functional molecules are not the only materials that are considered bioactive. Chitosan, the de-acetylated derivative of chitin, is a bioactive polymer.13 Chitin is found in the exoskeletons of shellfish, arthropods, and the cell walls of some fungi.13-14 Chitosan is being investigated as an implantable polymer for a variety of reasons. First, chitosan has more amine groups than chitin. These amine groups become protonated in solution, thereby attracting and promoting cell adhesion.12,15 Second, chitosan has also been shown to promote the growth and cell shape retention of osteoblasts and prevent the growth of fibroblasts.12,16 Third, chitosan is biodegradable, and the byproducts of chitosan degradation are considered a normal part of cellular metabolism.15,17 Fourth, chitosan is considered nontoxic, with an LD50 of greater than 16 g/kg.14 Last, chitosan is considered bacteriostatic and antibacterial. In amounts as low as 1%, chitosan prevented the growth of Staphylococcus epidermis, Staphylococcus aureus, and Pseudomonas aeruginosa13. In higher amounts, such as 8 mg of chitosan per mL of water, chitosan killed members of the yeast family, Candida, and the bacteria, S. aureus and S. epidermis.18 (12) Klokkevold, P.; Vandemark, L.; Kenney, E. B.; Bernard, G. W. J. Periodont. 1996, 67, 1170. (13) Li, Q.; Dunn, E. T.; Grandmaison, E. W.; Goosen, M. F. A. J. Bioact. Compat. Polym. 1992, 7, 370. (14) Singla, A. K.; Chawla, M. J. Pharm. Pharmacol. 2001, 53, 1047. (15) Bumgardner, J. D.; Wiser, R.; Gerard, P. D.; Bergin, P.; Chestnutt, B.; Marini, M.; Ramsey, V.; Elder, S. H.; Gilbert, J. A. J. Biomater. Sci. Polym. Ed. 2003, 14, 423. (16) Lahiji, A.; Sohrabi, A.; Hungerford, D. S.; Frondoza, C. G. J. Biomed. Mater. Res. 2000, 51, 586. (17) Prasitsilp, M.; Jenwithisuk, R.; Kongsuwan, K.; Damrongchai, N.; Watts, P. J. Mater. Sci. Mater. Med. 2000, 11, 773.
10.1021/la063284v CCC: $37.00 © 2007 American Chemical Society Published on Web 05/09/2007
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Originally, chitosan was used for wound dressing19 and bone filler, such as in the holes produced by wisdom teeth extraction.20-23 Despite the positive attributes of chitosan as a biomedical material, there have been relatively few studies reported in the literature which have focused on the surface chemistry between the chitosan film and a metal surface. In fact, most of the tests performed on chitosan have been on films formed in plastic or glass dishes, without consideration of surface bonding.24-26 Of those research efforts that involved coating a substrate with chitosan, both uncomplicated methods and more intricate reactions exist. The most straightforward method to deposit chitosan on a substrate is evaporation, where a chitosan solution is poured over the material and the solvent allowed to evaporate.27 A more time-consuming and complicated method involves self-assembled layers which are created by reacting polycations and polyanions.28-29 With this technique, the substrate is dipped into polyethyleneimine followed by sodium hyaluronate and chitosan, respectively,28 or the substrate is dipped into polyethyleneimine followed by dipping into gelatin and chitosan, respectively.29 This procedure was repeated until the desired film thickness was obtained.28-29 Another extensive reaction method involves reacting the substrate with a silane molecule, followed by a linker molecule, and finally, the chitosan solution.15,30 However, only one of the methods of bonding chitosan to a substrate has been tested for bond strength. By comparing chitosan deposited by evaporation with chitosan deposited with a silane reaction, a significant increase in bond strength was seen, from 0.5 to 1.6 MPa, respectively.15 The bond strength of the chitosan deposited on the silanized metal was still significantly less than hydroxyapatite deposited on metal.15 One silane molecule commonly used in the literature to attach a variety of materials, such as proteins, is 3-aminopropyltriethoxysilane (APTES), which ends in a primary amine group.8-10 This amine group can be then be modified through a chemical reaction with a linker molecule, such as gluteraldehyde.15,30 This modification produces a different reactive group that can bond to an assortment of materials, such as proteins or biopolymers.8-10,15 However, the chemistry involved in using APTES is not well understood in the biomedical literature. In fact, most silane reactions found in the biomedical literature involved the use of APTES in solution with water.8-10,15 Unfortunately, APTES reacts with water, causing the release of nitrogen oxides, which (18) Muzzarelli, R. A. A.; Tarsi, R.; Filippini, O.; Giovanetti, E.; Biagini, G.; Varaldo, P. E. Antimicrob. Agents Chemother. 1990, 34, 2019. (19) Khor, E.; Lim, L. Y. Biomaterials 2003, 24, 2339. (20) Muzzarelli, R. A. A.; Mattioli-Belmonte, M.; Pugnaloni, A.; Biagini, G. In Chitin and Chitinases; Jolles, P., Muzzarelli, R.A.A., Eds.; Birkhauser Verlag Basel: Switzerland, 1990. (21) Muzzarelli, R. A. A.; Mattioli-Belmonte, M.; Tietz, C.; Biagini, R.; Ferioli, G.; Brunelli, M. A.; Fini, M.; Giardino, R.; Ilari, P.; Biagini, G. Biomaterials 1994, 15, 1075. (22) Muzzarelli, R. A. A.; Biagini, G.; Bellardini, M.; Simonelli, L.; Castaldini, C.; Fratto, G. Biomaterials 1993, 14, 39. (23) Muzzarelli, R.; Baldassarre, V.; Conti, F.; Ferrara, P.; Biagini, G.; Gazzanelli, G.; Vasi, V. Biomaterials 1998, 9, 247. (24) Marreco, P. R.; Moreira, P. d. L.; Genari, S. C.; Moraes, A. M. J. Biomed. Mater. Res. App. Biomater. 2004, 71B, 268. (25) Hwang, K. T.; Kim, J. T.; Jung, S. T.; Cho, G. S.; Park, H. J. J. Appl. Polym. Sci. 2003, 89, 3476. (26) Cervera, M. F.; Heinamaki, J.; Krogars, K.; Jorgensen, A. C.; Karjalainen, M.; Colarte, A. I.; Yliruusi, J. AAPS PharmSciTech 2004, 5, Article 15. http:// www.aapspharmscitech.org. (27) Lopez-Lacomba, J. L.; Garcia-Cantalejo, J. M.; Sanz Casado, J. V.; Abarrategi, A.; Correas, Magana, V.; Ramos, V. Biomacromolecules 2006, 7, 792. (28) Thierry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4, 1564. (29) Cai, K.; Rechtenbach, A.; Hao, J.; Bossert, J.; Jandt, K. D. Biomaterials 2005, 26, 5960. (30) Bumgardner, J. D.; Wiser, R.; Elder, S. H.; Jouett, R.; Yang, Y.; Ong, J. L. J. Biomater. Sci. Polym. ed. 2003, 14, 1401.
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results in the degradation of the reactive terminal amine ends.31 Also, the ethoxy ends react with water, forming a polymer composed of polysiloxane, best seen as white specks, ∼1 mm in diameter, on the surface.32-34 The removal of the reactive terminal amine ends and the formation of the polysiloxane polymer both result in the inadequate bonding of the desired coating, which could not withstand the ultrahigh vacuum of X-ray photoelectron spectroscopy (XPS).35 Therefore, this research used toluene as the solvent to prevent the loss of the reactive terminal amine ends and the formation of the polysiloxane polymer.32-34 XPS was used to examine the titanium surface following each reaction step to determine if the changes to the surface were consistent with the anticipated reaction series. XPS was also used to determine if the use of toluene as the solvent helped prevent the loss of the reactive terminal amine groups and if the metal treatment affected the amount of APTES that was bound to the titanium surface, as both can contribute to the low bond strengths reported for chitosan coatings. Qualitatively, chitosan coatings bonded to metal surfaces via aqueous silane reactions do not even withstand the ultrahigh vacuum of XPS machines, while the coatings produced using toluene withstood the ultrahigh vacuum. Therefore, the aim of this study was to evaluate the surface chemistry involved in the growth of a chitosan film on implant quality titanium, using APTES followed by the linker molecule, gluteraldehyde. Experimental Section Reagents. APTES (98%), 99.7+% ACS grade glacial acetic acid, gluteraldehyde, 35% aqueous solution hydrogen peroxide, 95-98% ACS grade sulfuric acid, 99% min. semiconductor grade toluene, and HPLC grade ultrapure water were purchased from Alfa Aesar (Ward Hill, MA). ACS grade acetone (99.5%) and 200 proof ethanol were purchased from Sigma Aldrich (St. Louis, MO). ACS grade nitric acid and ACS grade isopropyl alcohol were purchased from Acros Chemical (Morris Plains, NJ). Chitosan with a degree of deacetylation (DDA) of 86.4% was obtained from Vanson (Redmond, WA). Deionized water was created using a NANOpure Diamond ultrapure water system (Barnstead, Boston, MA) with a D3750 hollow fiber filter with a maximum operating pressure of 50 psi and a 0.2 µm pore size rating. Materials. A commercially pure titanium bar, grade 4 (ASTM F67) with nominal dimensions of 3 in. × 5 in. × 0.25 in. was purchased from Titanium Industries (Jacksonville, FL) and cut into 1 in. × 1 in. × 0.25 in. coupons using a Makita Cut-Off saw with a carbide blade (La Mirada, CA). Metal Polishing. The steps involved in polishing the titanium metal coupons to a 1200 grit finish, were modified from a procedure previously used at Mississippi State University.36 An electric belt sander (BR300, Type 1, Black and Decker, Towson, MD) with a grit of 120, width of 3 in. × 18 in., and speed of 656 ft/min was used to smooth out the roughest areas of the metal coupons. Next, 320 grit sandpaper (Norton, Worchester, MA) was used on a compressed air, dual-action sander (Nikota, Whitter, CA) to remove the scratches made from the coarse grit and to continuing the smoothing process. The samples were then sanded by hand for the remainder of the polishing with 600, 800, and finally 1200 grit sandpaper. The coupons were sanded in one direction, rotated 90°, and again sanded in one direction. Sanding continued from coarser (31) Acros Organics; Material Safety Data Sheet: 3-Aminopropyltriethoxysilane, 99% 2004, https://fscimage.fishersci.com/msds/85861.htm. (32) Heiney, P. A.; Gruneberg, K.; Fang, J. Langmuir 2000, 16, 2651. (33) Vandenberg, E. T.; Bertilsson, L.; Liedberg, B.; Uvdal, J.; Erlandsson, R.; Elwing, H.; Lunstrom, I. J. Colloid Interface Sci. 1991, 147, 103. (34) Qian, W.; Xu, B.; Wu, L.; Wang, C.; Yao, D.; Yu, F.; Yuan, C.; Wei, Y. J. Colloid Interface Sci. 1999, 214, 16. (35) Martin, H. J. Ph.D. Dissertation, Mississippi State University; Mississippi State, MS, 2006. (36) Lin, H. Y. Ph.D. Dissertation, Mississippi State University; Mississippi State, MS, 2002.
Using APTES to Bond Chitosan to Titanium to finer grit until all residual scratches had been removed (determined by visual inspection). Metal Preparations. One of two methods of chemical cleaning, passivation or piranha, was performed on the polished metal coupons before a reaction series, but never both on the same sample. PassiVation Method. Passivation was performed following the ASTM F86 standard.37 The coupons were sonicated for 10 min in each of the following chemicals in succession: acetone (70% by volume), ethanol, and deionized water, respectively. Following sonication in deionized water, the coupons were placed in a 3:7 (v/v) nitric acid-deionized water solution for 30 min at room temperature. Following the nitric acid treatment, the samples were rinsed with deionized water and placed in a covered ultrapure water bath for 24 h. Piranha Treatment. The second chemical treatment method, piranha treatment, can be extremely dangerous. Care must be taken as this reaction is highly exothermic and reacts strongly with carboneous materials. It can burn the skin from both the heat produced and the reaction of the chemicals with the skin. The coupons were first sonicated for 30 min in 70% isopropyl alcohol. Following sonication, concentrated sulfuric acid was poured into a beaker and 35% hydrogen peroxide slowly added at a 7:3 (v/v) ratio of sulfuric acid to hydrogen peroxide. The resulting mixture was then swirled gently to mix before being poured over the metal coupons. The coupons were left for 10 min before being removed and placed in a second piranha mixture for 5 min. Care should be taken that only a few samples at a time are placed in the piranha solution, as a runaway reaction can occur if too many samples are added at once. Also, care should be taken to remove the samples after 10 and 5 min, respectively. Piranha does react with the titanium and will etch the surface if the samples are left in the piranha solution for extended periods.38 After the second piranha treatment, the metal coupons were rinsed twice in ultrapure water before being placed in an ultrapure water bath for 24 h. To prevent contamination, a container with a lid was used to hold the ultrapure water and the titanium samples. Deposition of Chitosan. The following procedure was adapted from the previous work of Bumgardner et al.15 The procedure is a three-step process developed so that chitosan could be effectively bound to titanium surfaces. Scheme 1 shows the anticipated reaction steps. Reaction step 1 is the deposition of APTES on the titanium surface, reaction step 2 is the reaction between APTES and gluteraldehyde, and reaction step 3 is the reaction between gluteraldehyde and chitosan. The first reaction step involved the silane reaction where the dried coupons (either passivated or piranha-treated) were submerged in a 2% (v/v) solution of APTES in toluene in sealed individual containers and allowed to react for 24 h. Following the 24 h reaction time, the metal coupons were placed in pure toluene and sonicated for 30 min. The procedure of using fresh toluene with 30 min of sonication was repeated twice more, for a total sonication time of 90 min. To remove any residual toluene, the metal coupons were rinsed with ethanol followed by deionized water and then dried. Following this rinsing and drying process, the coupons were stored in individual containers. The second step in the reaction series involved reaction of a linker molecule, gluteraldehyde. A 2% (v/v) solution of gluteraldehyde in deionized water was prepared and stirred for 1 h. The watergluteraldehyde solution was poured over the Ti-APTES samples ensuring complete coverage of the metal coupon. The containers were then sealed and left for 24 h. Following the 24 h, the samples were rinsed thoroughly with deionized water and placed in a petri dish. The third step in the reaction series involved the chitosan film deposition. A solution of 1 wt % chitosan, 2 wt % acetic acid, and 97 wt % deionized water was prepared. The solution was stirred for 1 h to ensure that the chitosan had dissolved and then filtered through (37) ASTM F86-01, In Annual Book of ASTM Standards; ASTM International: West Conshohocken, PA, 2002; Vol. 13.01, p 10. (38) Williams, K. R.; Muller, R. S. J. Microelectromech. Syst. 1996, 5, 256.
Langmuir, Vol. 23, No. 12, 2007 6647 Scheme 1. Reaction Steps Involved in the Binding of Chitosan to Titanium Substrates: (1) 3-Aminopropyltriethoxysilane (APTES) Deposition, (2) Reaction of APTES with Gluteraldehyde, and (3) Reaction of Gluteraldehyde with Chitosan
several layers of cheesecloth to remove any undissolved particulate. The filtered chitosan solution was poured over the metal coupons in the petri dishes. The solution was then allowed to evaporate for 7-10 days, after which time a clear film was seen on the surface of the metal coupons (as the reflection of light was different than on an untreated metal coupon). X-ray Photoelectron Spectroscopy. A PHI 1600 XPS Surface Analysis System (Physical Electronics, Eden Prairie, MN) was used to obtain XPS data. The instrument used a PHI 10-360 spherical capacitor energy analyzer and an Omni Focus II small-area lens to focus the incident source to an 800 µm diameter surface analysis area. XPS data were obtained using an achromatic Mg KR X-ray source operating at 300 W and 15 kV. Survey spectra were gathered using an average of 10 scans with a pass energy of 26.95 eV and running from 1100 to 0 eV. High-resolution spectra were gathered using an average of 15 scans with a pass energy of 23.5 eV and a step size of 0.1 eV. The incident sample angle was held constant at 45°. For statistical analysis, measurements were taken on three samples per treatment and three spots per sample. The XPS data was collected and averaged using PHI Surface Analysis Software, Version 3.0 (Physical Electronics, Eden Prairie, MN). The XPS data was then analyzed using the Spectral Data Processor (SDP), Version 4.0 (XPS International LLC, Mountain View, CA). Statistical analyses were performed using SAS, Version 9.1 (SAS Institute Inc., Cary, NC). Comparison of the individual reaction steps was performed using completely randomized design with subsampling.
Results and Discussion The samples were scanned using XPS after each reaction step. The passivated or piranha-treated titanium surface was first scanned using XPS. APTES was then deposited (Scheme 1, reaction step 1), and XPS was performed on the APTES treated surface. Gluteraldehyde was then reacted with the APTES-Ti surface (reaction step 2), and XPS was again run on the treated surface. The chitosan film was then deposited (reaction step 3), and XPS was run on the final film. XPS Analysis of APTES and Gluteraldehyde Deposition on Passivated Metal. Table 1 shows the differences in the normalized elemental peak areas (per unit area) following each reaction step on the treated metal surfaces. The chitosan film surface is not shown, as the film was too thick to investigate the
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Table 1. Elemental Peak Areas for the Individual Reaction Steps on Treated Titanium Surfaces from XPS Survey Scans (per Unit Area)a element
passivated
APTES
gluteraldehyde
piranha
APTES
gluteraldehyde
carbon oxygen nitrogen silicon titanium
1.00 ( 0.07 1.12 ( 0.08a 0.00 0.00 0.61 ( 0.24
0.89 ( 0.05 1.10 ( 0.12a,b 0.19 ( 0.03 0.20 ( 0.03 0.27 ( 0.11f
1.09 ( 0.04 0.97 ( 0.06c 0.13 ( 0.03 0.15 ( 0.02e 0.11 ( 0.05f, g
0.64 ( 0.02 1.76 ( 0.07 0.00 0.00 1.71 ( 0.29
1.05 ( 0.04 1.06 ( 0.04b,d 0.26 ( 0.03 0.25 ( 0.02 0.12 ( 0.06h
1.21 ( 0.05 1.06 ( 0.07c,d 0.19 ( 0.04 0.15 ( 0.02e 0.06 ( 0.09g,h
a Values with the same superscript are not statistically different at the 5% significance level. All values are normalized on the basis of the passivated carbon peak area.
Figure 1. XPS high-resolution nitrogen spectra; passivated titanium surface following (a) APTES deposition and (b) gluteraldehyde reaction. Peaks with the same superscript are not statistically different at the 5% significance level.
bonding between the gluteraldehyde and the chitosan molecules with XPS. There were significant decreases in the composition of carbon and titanium following the reaction between the passivated metal and APTES. Two elements, silicon and nitrogen, were not present on the surface of the passivated metal but were present following the deposition of APTES (reaction step 1). Following the reaction of APTES with gluteraldehyde (reaction step 2), there was an increase in the presence of carbon. Decreases were seen in the nitrogen and silicon elements. No significant changes were seen in the oxygen composition following APTES deposition or following the reaction with gluteraldehyde, while no significant changes were seen in the titanium composition following the reaction with gluteraldehyde. A large amount of carbon was present on the titanium surface following passivation. This amount of carbon was the result of the solvents used in the cleaning phase of passivation, which were not removed during the passivation stage. The physisorbed carbon was then removed following reaction step 1, APTES deposition. The increase in the amount of carbon following reaction step 2, the reaction with gluteraldehyde, was expected, due to the presence of the pentyl group in the bound gluteraldehyde. The increase in nitrogen and steady oxygen levels following APTES deposition was indicative of the removal of the ethyl group during the bonding between the titanium surface and silicon in the silane molecule. The terminal amine group (away from the titanium substrate) can then be utilized for reaction with gluteraldehyde. Figure 1 shows the XPS spectra taken from the
APTES-treated surface and the gluteraldehyde-reacted surface. Peak identifications for nitrogen were made using literature values.39-43 Decreases were observed in the amount of C-N-H and NH4+ following the gluteraldehyde reaction which was consistent with a reaction between the terminal amine group and one of the aldehyde groups found in gluteraldehyde. NO was likely present at the end of the APTES molecule because of a reaction between the terminal amine group and an oxygen that was released from the triethoxy portion of the silane molecule. NH4+ was likely present because of a reaction between the amine groups and either the toluene solvent or ethyl groups that allowed hydrogen to bond. These two peaks, NO and NH4+, were very small and are not a critical part of the reaction pathway. The presence of silicon, in three different forms, as shown in Figure 2, was consistent with a reaction between the titanium surface and APTES. Peak identifications for silicon were made using literature values.44-46 The presence of Si-O-Si and SiO3 (39) Beguin, F.; Rashkow, I.; Manolova, N.; Benoit, R.; Erre, R.; Delpeux, S. Eur. Polym. J. 1998, 34, 905. (40) Qingliang, L.; Huaming, Y.; Xiong, Z.; Zude, Z. J. Mol. Struct. 1999, 478, 23. (41) Vanini, A. S.; Audouard, P;, J.; Marcus, P. Corros. Sci. 1994, 36, 1825. (42) Pashutski, A.; Folman, M. Surf. Sci. 1989, 216, 395. (43) Moulder, J. F.; Stickle, W. F.; Sobel, P. E.; Bomben, K. D. Handbook of X-Ray Photoelectron Spectroscopy; Perkin-Elmer Corporation: Ramsey, MN, 1992. (44) Roy, M.; Nelson, J. K.; MacCrone, R. K.; Schadler, L. S. IEEE Trans. Dielectr. Electr. Insul. 2005, 12, 629. (45) Xu, D. Ph.D. Dissertation, Virginia Polytechnic Institute and State University, Blacksburg, VA, 2004. (46) Graham, M. J. Corros. Sci. 1995, 37, 1377.
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Figure 2. XPS high-resolution silicon spectra; passivated surface following (a) APTES deposition and gluteraldehyde reaction. Peaks with the same superscript are not statistically different at the 5% significance level. Table 2. Titanium Peak Areas for the Individual Reaction Steps on Passivated Titanium Surfaces from XPS High-resolution Scans (per Unit Area)a chemical species
location (eV)
passivated
APTES
gluteraldehyde
piranha
APTES
gluteraldehyde
TiO247 TiO48 TiC49
458.4 ( 0.2 459.3 ( 0.1 460.3 ( 0.2
1.00 ( 0.18a,b 2.36 ( 0.55 0.36b
0.73 ( 0.36b,c 0.64 ( 0.18d 0.00
0.18 ( 0.09c 0.45 ( 0.09d,e 0.18b,g
0.91 ( 0.36a 6.27 ( 0.45 0.64 ( 0.18
0.00 0.55 ( 0.18f 0.27b,h
0.00 0.27 ( 0.27e,f 0.18b,g,h
a Values with the same superscript are not statistically different at the 5% significance level. All values are normalized on the basis of the passivated TiO2 peak area. b Only one observation at the given binding energy.
are consistent with the development of interlinking silane molecules over the metal surface.32 This layer developed when two or more silanes react with one another via residual ethoxy groups. The amount of SiO3 on the surface did not decrease following the gluteraldehyde reaction, demonstrating that, once formed, the APTES silane layers were stable in the aqueous environment (during gluteraldehyde deposition). Peak identifications for titanium were made using literature values.47-49 A decrease was seen in the amount of TiO present following reaction step 1 (APTES deposition), as shown in Table 2. This decrease was consistent with the bonding between the APTES molecule and the TiO groups present on the passivated surface. No significant changes were seen in the amount of TiO present following the gluteraldehyde reaction with the APTEStreated surface. No change in TiO247 was seen between the passivated surface and the APTES treated surface. An unreactive TiO2 on noncoated biomedical implants is highly desirable, as it increases the corrosion resistance of the implant by reducing the interaction between the implant surface and the surrounding body fluids.50 However, TiO2 is not desirable when modifying the surface of the titanium implant. Since TiO2 is highly unreactive, binding a coating to the surface is difficult. Therefore, (47) Huravlev, J. F.; Kuznetsov, M. V.; Gubanov, V. A. J. Electron Spectrosc. Relat. Phenom. 1992, 38, 169. (48) Fahlman, A.; Nordling, C.; Johansson, G.; Hamrin, K. J. Phys. Chem. Solids 1969, 30, 1835. (49) Luches, A.; Perrone, A.; Dubreuil, B.; Rousseau, B.; Boulmer-Leborgne, C.; Blondiaux, G.; Estrade, H.; Hermann, J.; Debrun, J. L.; Degiorgi, M. L.; Martino, M.; Brault, P. Appl. Surf. Sci. 1992, 54, 349. (50) Brunski, J. B. In Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, 1996; Chapter 2.
the lack of change in TiO2 between the APTES and gluteraldehyde reaction steps was likely due to the unreactiveness of TiO2, which does not react with the ethoxy groups of APTES. XPS Analysis of APTES and Gluteraldehyde Deposition on Piranha-Treated Metal. Table 1 shows the differences in the normalized elemental peak areas following each reaction step on the treated metal surfaces. XPS results following deposition of the chitosan film are not shown, as the film was too thick to investigate the bonding between the gluteraldehyde and the chitosan molecules. An increase in the presence of carbon was seen following reaction step 1, the reaction between the piranha-treated metal and APTES, while significant decreases in the compositions of oxygen and titanium were seen. Two elements, silicon and nitrogen, were not present on the surface of the piranha-treated metal but were present following the deposition of APTES, a trend seen previously on the passivated surfaces. Following the reaction of APTES with gluteraldehyde (reaction step 2), there was an increase in the presence of carbon. Decreases were seen in nitrogen and silicon, while no significant changes were seen for the oxygen and titanium peaks. An increase in carbon following the deposition of APTES was expected due to the propyl group present in the APTES molecule. The increase in the amount of carbon following the reaction with gluteraldehyde was also expected due to the addition of a pentyl group from gluteraldehyde. The smaller amount of carbon present on the surface of the piranha-treated metal, as compared to the passivated metal, was likely due to the piranha treatment. Piranha was designed to react with and remove carboneous materials,38 while the passivation protocol was designed to create nonreactive TiO2 groups.
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Figure 3. XPS high-resolution nitrogen spectra; piranha-treated surface following (a) APTES deposition and (b) gluteraldehyde reaction.
Figure 4. XPS high-resolution nitrogen spectra following APTES deposition: (a) passivated metal and (b) piranha-treated metal. Peaks with the same superscript are not statistically different at the 5% significance level.
As with the passivated metal, the increase in nitrogen following the deposition of APTES was consistent with the bonding of the ethoxy group of the silane molecule to the titanium surface, which would position the reactive amine end away from the metal surface. Peak identifications for nitrogen were made using literature values.39-43 As shown in Figure 3, following the reaction with gluteraldehyde, decreases in the C-N-H and the NH4+ peaks were seen, which was consistent with a reaction occurring between the terminal amine group and an aldehyde group of gluteraldehyde. As with the passivated surface, NO and NH4+ were likely present because of small side reactions. Comparing the nitrogen peak areas after the APTES deposition for the passivated and piranha-treated samples, we can see that there is a higher concentration of nitrogen in the piranha-treated samples (Figure 4). This may indicate a more densely packed silane layer and subsequently more terminal amine groups present for binding the gluteraldehyde.
Silicon was not present on the piranha-treated surface but was present following APTES deposition (reaction step 1). The presence of silicon further demonstrated a reaction occurred between the piranha-treated titanium surface and APTES. Peak identifications for silicon were made using literature values.44-46 Three different peaks for silicon were present (Figure 5), as was expected for the triethoxy silane. As with the passivated metal, the formation of interlinking silane layer occurred when the remaining ethoxy groups of each silane reacted with nearby ethoxy groups. As shown in Figure 5, the SiO3 peak did decrease following the gluteraldehyde reaction (reaction step 2) for the piranha-treated samples. Following the reaction with gluteraldehyde, there was a decrease in the overall amount of silicon (Table 1). This was likely due to the more densely packed APTES molecules which provided more complete gluteraldehyde coverage which allowed fewer photoelectrons to escape and be detected resulting in the decreased silicon peak area.
Using APTES to Bond Chitosan to Titanium
Langmuir, Vol. 23, No. 12, 2007 6651
Figure 5. XPS high-resolution silicon spectra; piranha-treated surface following (a) APTES deposition and (b) gluteraldehyde reaction. Table 3. Elemental Peak Areas of the Chitosan Films from XPS Survey Scans (per Unit Area) element
passivated
piranha-treated
carbon oxygen nitrogen calcium phosphorus silicon
1.00 ( 1.00 ( 0.06b 0.13 ( 0.03c 0.06 ( 0.04d 0.04 ( 0.05 0.01 ( 0.01e
1.03 ( 0.04a 0.97 ( 0.11b 0.13 ( 0.02c 0.08 ( 0.03d 0.02 ( 0.02 0.01 ( 0.01e
0.05a
a Values with the same superscript are not statistically different at the 5% significance level. All values are normalized on the basis of the carbon peak area.
A significant decrease in the amount of titanium was seen between the piranha-treated surface and the APTES-treated surface (Table 2). Peak identifications for titanium were made using literature values.47-49 As Table 2 shows, there was a large amount of TiO on the piranha-treated surface. A decrease following the deposition of APTES was seen in TiO, to a value lower than the amount of TiO present on the passivated surface. The decrease in TiO was consistent with significantly more APTES bound to the piranha-treated surface than on the passivated surface. No significant change was seen between the APTEStreated surface and the gluteraldehyde-treated surface. The lack of change in the amount of TiO present was not an indication that the APTES molecules dissociated from the surface due to the aqueous gluteraldehyde solution but an indication of the amount of photoelectrons that could be detected. TiO2 was present only on the piranha-treated surface and was not present following deposition of APTES. The disappearance of TiO2 was not due to a reaction between APTES and TiO2, but instead, was consistent with a more thorough coverage of the titanium metal by the APTES, which covered the small amount of TiO2 that was present on the piranha-treated surface. TiC was also present on the piranha-treated surface and following both the APTES and gluteraldehyde deposition reactions. The presence of TiC was consistent with contamination, which was not removed during any of the reaction steps, but instead, was hidden by the closely packed APTES molecules. XPS Analysis of the Chitosan Films. Unlike previous research, the chitosan films remained attached to the titanium
after exposure to the ultrahigh vacuum required for XPS.35 Qualitatively, this demonstrated that the bond between the chitosan film and the metal surface was greatly improved over published values,15 which were unable to remain attached to the titanium surface in the ultrahigh vacuum environment. Table 3 shows the normalized elemental peak areas of the chitosan films attached to the treated metal surfaces. There were no significant differences between the two films based on the normalized compositions of carbon, oxygen, nitrogen, calcium, and silicon. However, phosphorus was statistically different between the two metal surfaces. This may be due to the small sampling size and/ or possible variations in phosphorus concentration for the chitosan aliquots pulled for preparation of each chitosan solution. The films are ∼100 µm thick; since XPS can penetrate at most 10 nm into the sample, no conclusions about the bonding of the chitosan film to gluteraldehyde can be made from the XPS data.43 However, the lack of differences between the piranha-treated surface and the passivated surface, with respect to everything but phosphorus, demonstrated that there were no changes in the chemical composition of the chitosan film based on the metal treatment used.
Conclusions Unlike previous research, the chitosan films remained attached when stressed in the ultrahigh vacuum required for XPS qualitatively, indicating that the chitosan films were more tightly bound to the titanium surface. The chemical changes to the titanium substrates, documented by XPS, were consistent with the anticipated reaction steps showing significant changes in the amounts of nitrogen, silicon, and titanium. More APTES was bound to the piranha-treated surface as compared to the passivated surface, based on the lower amount of titanium and the higher amounts of carbon, nitrogen, and silicon. The surface composition of the chitosan films for the two metal treatments were statistically similar, demonstrating that differences in the metal treatments did not affect the chemistry of the chitosan films. Acknowledgment. Financial support from the Bagley College of Engineering at Mississippi State University is gratefully acknowledged. LA063284V
