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Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study
Journal of Electron Spectroscopy and Related Phenomena 216 (2017) 33–38
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Journal of Electron Spectroscopy and Related Phenomena journal homepage: www.elsevier.com/locate/elspec
Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study Andrey Shchukarev a,∗ , Behnosh Öhrnell Malekzadeh b,c , Maria Ransjö b , Pentti Tengvall c , Anna Westerlund b a
Department of Chemistry, Umeå University, Umeå SE-90187, Sweden Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530, Sweden c Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, SE-40530, Sweden b
a r t i c l e
i n f o
Article history: Received 13 December 2016 Received in revised form 21 February 2017 Accepted 2 March 2017 Available online 6 March 2017 Keywords: XPS Titanium implant Insulin Cell culture medium Proteins at interface
a b s t r a c t Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in ␣-minimum essential medium (␣-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze the insulin behavior at the implant – ␣-MEM interface. In the absence of serum proteins in cell culture medium, the coated insulin layer remained intact, but experienced a time-dependent structural transformation exposing hydrophobic parts of the protein toward the solution. The presence of fetal bovine serum (FBS) in the medium resulted in partial substitution of insulin by serum proteins. In spite of some insulin release, the remaining coated layer demonstrated a direct surface effect by stabilizing the structure of protein competitors, and by supporting the accumulation of calcium and phosphate ions at the interface. A structurally stable protein layer with incorporated calcium and phosphate ions at the implant–tissue interface could be an important prerequisite for enhanced bone formation. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The understanding of interface phenomena and corresponding surface modifications is crucial for the improvement of the bone formation adjacent to the implant. The surface properties and interface processes are thus largely responsible for the bone response [1]. Insulin, a hormone known to regulate glucose metabolism, has additionally demonstrated anabolic effects in bone tissue [2–4]. In a recent study, locally delivered exogenous insulin enhanced bone formation in non-diabetic rats [5]. In order to provide a local delivery, insulin was immobilized to titanium surfaces. The mechanism, whereby an insulin coated implant surface exerts its effect remains unclear. Possible scenarios include a direct surface effect and/or an insulin release. The retained biological activity of insulin, which was released from titanium, has been confirmed in a separate study [6]. However, only one third of the immobilized insulin was released and the remaining two thirds remained bound to the titanium surface with an unknown composition. The aim of this
∗ Corresponding author. E-mail address: [email protected] (A. Shchukarev). http://dx.doi.org/10.1016/j.elspec.2017.03.001 0368-2048/© 2017 Elsevier B.V. All rights reserved.
study was therefore to characterize, by X-ray photoelectron spectroscopy (XPS), immobilized insulin behavior on titanium surfaces, after incubation in a biologically relevant cell culture medium, without and with the addition of serum proteins. 2. Experimental 2.1. Immobilization of insulin onto titanium discs Ti6Al4V discs (Ti – 89.6%, Al – 6.3%, V – 4.1%) with 8 mm in diameter and 1 mm thickness and with a turned surface were used (Part Precision AB, Mariestad, Sweden). The discs and null ellipsometry reference Silicon (Si) samples (1 cm × 1 cm) were cleaned during four minutes in an ultrasonic bath in 70% ethanol (Solveco, Rosersberg, Sweden), rinsed in distilled water, and dried by flowing nitrogen. The insulin immobilization protocol was similar to that in previous studies [5,6]: in brief, the discs were incubated for 30 min in 1% (3-Aminopropyl) triethoxysilane (APTES; Sigma–Aldrich, Steinheim, Germany), in xylene (VWR, Stockholm, Sweden), and rinsed in 99% xylene, 70% ethanol, and distilled water, respectively. The discs were subsequently incubated for 30 min in a 6% glutaraldehyde solution (GA; Sigma–Aldrich, Steinheim, Germany) in phosphate buffered saline (PBS; Sigma–Aldrich, Stein-
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heim, Germany), at pH 9 at room temperature, and rinsed in PBS (pH 9). Recombinant human insulin (10 mg/ml in 25 mM HEPES, pH 8.2, Sigma–Aldrich, Steinheim, Germany) was diluted to 1 mg/ml in PBS at pH 5.7. The aldehyde coated discs were incubated in this solution for 15 minutes at room temperature and rinsed with PBS (pH 5.7). To achieve a carboxyl group activation and surface cross-linking, the discs were incubated in a mixture of 0.2 M N-(3-dimethyl aminopropyl)-N -ethylcarbodiimide (EDC; Sigma–Aldrich, Steinheim, Germany), and 0.05 M N-hydroxysulfosuccinimide sodium salt (NHS; Sigma–Aldrich, Steinheim, Germany) in a PBS, pH 5.7 at room temperature for 15 min. The discs were subsequently incubated 10 times in insulin solution and EDC-NHS in between. Since the EDC solution is unstable, new solutions were made every 2½h. Finally, the discs were rinsed with PBS (pH 5.7), distilled water, and dried in flowing nitrogen. The null ellipsometry reference Silicon samples revealed an insulin thickness of approximately 3.5 nm. The assumed refractive index of insulin was nf = 1.465, as described elsewhere [5]. The Ti6Al4V alloy surface may not have a homogeneous distribution of insulin molecules although titanium is known to quickly adsorb proteins [7]. Therefore, the thickness at different surface locations is not expected to differ significantly. 2.2. XPS characterization of insulin-coated discs XPS was used to monitor changes in surface chemical composition when coated insulin layer interacts with cell culture medium. The XPS spectra were acquired with Kratos Axis Ultra DLD electron spectrometer using a monochromated Al K␣ source operated at 150 W, a hybrid lens system with a magnetic lens providing an analysis area of 0.3 mm × 0.7 mm, and a charge neutralization system. The binding energy (BE) scale was referenced to the C 1s line of aliphatic carbon, set at 285.0 eV. The processing of spectra was accomplished with Kratos software and CasaXPS package. The curve fitting of high-resolution C 1s and N 1s spectra was performed after Shirley background subtraction with a fixed number of spectral components corresponding to expected chemical states, two for N 1s and 3–4 for C 1s spectra, as described below. The BE position and FWHM of the components were not fixed, although the FWHM did not allow exceeding 1.6–1.7 eV. The accuracy in BE determination is 0.1 eV, and in atomic ratio – 8–10% rel. It is important to note that XPS sampling depth for organics, 7–9 nm [8], is larger than the thickness of deposited insulin, and therefore the entire protein layer was analyzed. In order to ascertain that the results were reproducible, all measurements were repeated at least in triplicate with different Ti discs. Reference XPS spectra of fast-frozen drops of ␣-MEM + 10% FBS were reported earlier [9,10] and for pure insulin powder (Insulin from Bovine Pancreas, I-6634, Sigma–Aldrich, Steinheim, Germany) especially acquired in the present study. 2.3. Sample preparation protocol The titanium (Ti) discs (non-coated and insulin-coated) were placed, one disk per well, in 24-well plates (NunclonTM Surface, Roskilde, Denmark). 1 ml/well of ␣-Minimum Essential Medium (␣-MEM; Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 1% benzyl-penicillin/streptomycin (ASTRA Zeneca AB, Sweden/Sigma–Aldrich, Steinheim, Germany), without or with 10% FBS (Life technology, Sweden) was added to the wells. The discs were incubated one or 10 days at 37 ◦ C, in a humid atmosphere containing 5% CO2 . In order to obtain reliable information about the surface composition that was formed in the cell culture medium, a sample preparation protocol was elaborated as follows. After the incubation, three different sample treatments for XPS analysis were tested: First, an excess of cell culture medium on the surface was
Fig. 1. Survey XPS spectra taken with three different treatments of insulin-coated Ti discs after incubation in cell culture medium. (a) After soaking up ␣-MEM cell culture medium (b) After rinsing in distilled water (c) After rinsing in distilled water and followed by ultrasonic treatment.
shaken off, and the rest (if present) gently soaked up by filter paper. This procedure resulted in a significant precipitation of NaCl from the medium and no Ti 2p signal could then be observed (Fig. 1a). Second, short washing (up to 30 s) in distilled water removed all precipitates and loosely bound organics, but did not alter the insulin binding (Fig. 1b). Third, further ultrasonic washing (1 min) in distilled water resulted in surface composition similar to that after a short washing (Fig. 1c), meaning that the proteins were tightly bound onto titanium. In the last two cases, XPS survey spectra are very similar to those obtained with insulin-coated Ti discs (Fig. 2). Therefore, these steps were applied to all samples treated in ␣MEM. 3. Results and discussion 3.1. Insulin deposition XPS spectra of non-coated and insulin-coated Ti discs are presented in Fig. 2. The Ti 2p spectra are very similar, consist of two Ti 2p doublets, and reveal that a continuous TiO2 layer formed in air (corresponding dominant Ti 2p3/2 component at 458.8 eV [11]) covers the surface. The insulin immobilization onto the surface was confirmed by the appearance of intensive nitrogen N 1s photoelectron line in the survey spectrum and by both high-resolution spectra of C 1s and N 1s (Fig. 2). The surface composition of non-coated discs differed remarkably in atomic compositions between the discs and was dominated by C (from 50 to 76 at.%), O (18–37 at%), and Ti (4–13 at.%). Only minor contributions of Al (1.5–2 at.%) and no V from alloy composition were observed (Table 1). Other surface contaminations including F, N, Bi, Ba, and Pb (total <0.5–2 at.%) showed no significant contributions and appeared possibly due to insufficient cleaning after the cutting processes. Observed high concentrations of adventitious carbon/oxygen likely correspond to organic contaminations within the thickness range of 2–4 nm. Irrespective of the contaminations, similar Ti implants enhanced (after insulin immobilization) bone formation in rat tibia [5]. Therefore, in order
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Fig. 2. XPS spectra of non-coated and insulin-coated titanium discs. Left: Survey spectra of non-coated and insulin-coated titanium. Upper right: Reference insulin powder C 1s and N 1s spectra. Lower right: C 1s and N 1s spectra of insulin-coated titanium.
Table 1 Atomic concentrations (at.%) of main elements at the surface of Ti6Al4V discs after different treatments. Samples
C
O
N
Ti
Non-coated discsa Reference insulin powderb
50–76 69.56
18–37 15.46
0–1.5 13.86
4–13 No
Insulin-coated discs Disk insulin-coated +␣-MEM without FBS, 1 dayc +␣-MEM without FBS, 10 daysc +␣-MEM with FBS, 1 dayc +␣-MEM with FBS, 10 daysc
55.9 ± 1.5 44.0 ± 2.5 37.7 ± 4.0 52.0 ± 2.1 58.0 ± 1.0
29.3 ± 0.7 38.2 ± 2.0 40.5 ± 3.5 30.1 ± 3.0 26.4 ± 1.0
9.1 ± 0.9 5.4 ± 0.1 5.4 ± 0.7 9.7 ± 0.3 10.3 ± 0.9
5.7 ± 0.3 9.4 ± 0.5 11.5 ± 1.5 6.5 ± 0.3 4.2 ± 0.4
Non-insulin coated discs ␣-MEM with FBS, 1 dayc ␣-MEM with FBS, 10 daysc
55.3 ± 1.4 62.4 ± 1.2
30.2 ± 1.1 25.3 ± 0.8
6.3 ± 0.3 5.7 ± 0.4
6.7 ± 0.1 5.3 ± 0.3
a Al atomic concentration – 1–2 at.%, no V was detected. Other surface contaminations – F, Bi, and Pb. b Minor elements: S – 1.13 at.%, Zn – 0.06 at.% (H is not detectable by XPS). c Total Ca and P atomic concentrations were within the 1–2 at.% range.
to follow similar interfacial processes, further surface cleaning of non-coated discs was not performed. However, after insulin-coating, the surface compositions became similar for all coated discs, with average concentrations of C (55.9 ± 1.5 at.%), O (29.3 ± 0.7 at.%), N (9.1 ± 0.9 at.%), and Ti (5.7 ± 0.3 at.%) (Table 1). Moreover, the intensity of the Ti 2p3/2 component at 454.1 eV, corresponding to Ti metal [11], correlated well to the estimated (≈ 3.5 nm) thickness of the deposited insulin layer. The active form of insulin is a monomer, although when stored in the body it is a hexamer. This insulin hexamer is torus shaped and 3.5 nm high and 5 nm in diameter [12]. When the protein is surface-bound, it may exist in different shapes and sizes. Insulin dominated the surface composition, although the metallic component of the Ti 2p spectrum was still detectable, suggesting that the deposited insulin may not form a homogeneous layer. These observations, together with the reproducible surface composition of the insulin-coated discs, indicate that protein immobilization removes most of the normally observed surface contaminations, organic as well as inorganic, although some adventitious carbon might still be observable.
3.2. Surface composition The average surface compositions of all discs, after incubation in the medium (␣-MEM without and with FBS), are given in Table 1. Minor elements such as Al, Ca, and P are not included because of low observed concentrations and their absence in the protein composition. Traces of sulfur (≈ 0.5 at.%) and no zink (below detection limit of 0.1 at.%) from adsorbed insulin molecules were found on insulin-coated discs. Insulin release from the surface after incubation in ␣-MEM without FBS [6] was observed as a decrease of the carbon and nitrogen concentrations, and followed by a corresponding Ti increase. This process continued for up to 10 days with the ␣-MEM treatment. The addition of FBS caused a remarkable rise in the surface nitrogen content, already after one day of incubation, in spite of a small decrease in carbon and an insignificant increase in titanium. This made it possible to assume that an additional adsorption of serum proteins from the medium occurred on top of the remaining coated insulin layer, thereby compensating the released insulin. Prolonged incubation in ␣-MEM with FBS resulted in noticeably higher carbon and nitrogen concentrations, and lower titanium, compared to insulin-coated discs, which were not incubated in the medium. Since the serum proteins were the only substantial sources of C and, especially, N in the medium, the increase in carbon and nitrogen signals indicated a complimentary serum adsorption onto the surfaces. 3.3. N 1s and C 1s spectra The N 1s spectra of insulin-coated Ti discs, incubated in the cell culture medium (Fig. 3), show predominantly two types of N bonds; the main line corresponds to nitrogen in amide and nonprotonated amine groups (400.2 eV), and the minor component is due to amine group protonation (401.8–402.0 eV) [13]. The spectra are typical for nitrogen in proteins, as seen when comparing them with corresponding insulin and ␣-MEM without and with FBS reference spectra (Fig. 3). Important to note is that the original ␣-MEM cell culture medium contains no proteins. Corresponding N 1s spectrum therefore differs significantly in intensity (low concentration of aminoacids in the media) and shape originating from both protonated (N 1s component at 402.0 eV) and non-protonated (N 1s component at 400.2 eV) amino-groups [9]. The N 1s line
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Fig. 3. Reference N 1s spectra (top) of insulin powder and fast-frozen drops of ␣-MEM without and with FBS. The N 1s spectra (bottom) of insulin-coated discs incubated for one day in ␣-MEM without FBS (a), 10 days in ␣-MEM without FBS (b), and with FBS (c) respectively. N 1s spectrum of insulin-coated disk incubated for one day in ␣-MEM with FBS is similar to others and not shown.
position and shape for insulin-coated Ti discs is independent on ␣-MEM composition and treatment time, although a weak variation in amine group protonation occurs in the medium without FBS. The similarity between N 1s spectra indicates that the surface protein layer remained intact upon all the treatments. Unfortunately, this similarity does not allow discrimination between the insulin and serum proteins in the layer after treatment in ␣-MEM with FBS. In contrast to N 1s, the C 1s spectra of insulin and ␣-MEM with FBS differ significantly (Fig. 4, top) in the intensities of spectral components related to hydrocarbon chains (285.0 eV), carbon singly bound to oxygen or nitrogen (≈ 286.4 eV), and the protein’s amide bonds (≈ 288.3 eV). Insulin-coated Ti discs incubated in both cell culture medium display C 1s spectra (Fig. 4, bottom) similar to that of the reference insulin powder and do not demonstrate any carbonate (≈ 290.3 eV) adsorbed from the medium. This confirms that the immobilized proteins remain intact at the surface, with insulin being the major component even after treatments in ␣-MEM containing FBS. However, C 1s spectra (Fig. 4, bottom) display noticeable changes in the relative intensity of spectral components, indicating structural re-arrangements or conformational alterations within the protein layer, all depending on medium composition and incubation time. These “unfolding” transformations also cause changes in atomic C/N and C (C, H)/Ctotal ratios, given in Table 2. The incubation of insulin-coated discs in ␣-MEM without FBS resulted in a remarkable increase in the surface carbon concentration, compared to nitrogen (C/N atomic ratio growth from 5 to 7–8). A simultaneous intensity raise in the hydrocarbon component of C 1s spectra (from ≈40 to ≈60%) implies that the top surface layer of insulin can experience a structural re-arrangement exposing hydrophobic parts of the molecules toward the aqueous medium. The changes in C 1s spectra, caused by incubation of insulin-coated discs in ␣MEM without FBS might also be attributed to removal of insulin from the surface and increased exposure of the Ti substrate, with the accompanying original organic contaminants which are domi-
Table 2 Atomic ratios at the surface of Ti6Al4V discs after different treatments. Samples
C/N
C (C, H)/Ctotal
Ca/P
Insulin powder reference
5:1
0.42:1
–
Insulin-coated discs Disk as coated +␣-MEM without FBS, 1 day +␣-MEM without FBS, 10 days +␣-MEM with FBS, 1 day +␣-MEM with FBS, 10 days
4.8 ± 0.1:1 8.0 ± 0.6:1 7.0 ± 0.3:1 5.6 ± 0.2:1 5.7 ± 0.5:1
0.5 ± 0.01:1 0.62 ± 0.01:1 0.57 ± 0.05:1 0.58 ± 0.01:1 0.51 ± 0 02:1
– 0.9 ± 0.02:1 1.4 ± 0.05:1 0.9 ± 0.02:1 1.1 ± 0.2:1
Non-coated discs +␣-MEM with FBS, 1 day +␣-MEM with FBS, 10 days
8.5 ± 0.2:1 11.1 ± 0.8:1
0.62 ± 0.01:1 0.68 ± 0.03:1
1.9 ± 0.3:1 1.4 ± 0.1:1
nated by C (C H) hydrocarbon bonds and do not contain nitrogen. If this is the case, the C/N and C (C, H)/Ctotal atomic ratios have to increase significantly, e.g., via a prolonged incubation time, due to more released insulin. In contrast, a decrease is observed (see Table 2); consequently, a reasonable rise in Ti atomic concentration (Table 1) supports the study’s assumption that the insulin immobilization procedure removes most of the surface organic contaminants, although the deposited protein layer does not seem homogeneous in thickness. After incubation of insulin-coated discs in ␣-MEM containing FBS, the additional adsorption of serum proteins (Table 1) resulted in C/N atomic ratio (5.7–5.8), close to the value of insulin powder (Table 2). The corresponding decrease in C (C, H)/Ctotal atomic ratio supports the hypothesis of a possible serum protein adsorption mechanism due to hydrophobic interactions between serum proteins and immobilized insulin. As a result, the protein layer seems to restore its “folding”, similar to one of the “as coated” disk (i.e., insulin-coated disk prior incubation). Unfortunately, XPS cannot be used to differentiate between individual proteins [14], even in a simple two-component mixture, and is not able to distinguish structural molecular mechanisms of the protein adsorption and re-
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Fig. 4. Reference C 1s spectra (top) of insulin powder and fast-frozen drops of ␣-MEM, and C 1s spectra (bottom) of insulin-coated discs incubated for one day in ␣-MEM without FBS (a), and 10 days without FBS (b) and with FBS (c), respectively. C 1s spectrum of insulin-coated disk incubated for one day in ␣-MEM with FBS is similar to spectrum (c) and not shown.
arrangement. Moreover, ultra-high vacuum conditions of the XPS experiment, which can easily change interfacial equilibrium, both compositional and structural, have to be taken into account. However, considering a similar chemical composition of the samples and the medium used, i.e., the sample treatment and the identical sample preparation protocol for the measurements, the extent of the surface-induced structural alterations is expected to be very close for all Ti discs studied. Therefore, noticeable consequences of interfacial processes should be evident at all interfaces. Nevertheless, to confirm described XPS findings, complimentary IR [15], circular dichroism spectropolarimetry [16], TOF-SIMS, and MALDITOF SIMS experiments are needed. To clarify the role of insulin at the titanium surface and the FBS behavior at the interface, additional experiments with noncoated Ti discs, incubated in ␣-MEM with FBS were performed. Without insulin coating, adsorbing serum proteins experienced similar structural changes by exposing their hydrophobic parts/hydrocarbon chains toward the medium. C 1s spectra, dominated by the C (C, H) component at 285.0 eV (Fig. 5a, b), and C/N and C (C, H)/Ctotal atomic ratios (Table 2) confirm this interfacial conformational change, which seems to be even more apparent than for insulin and, in contrast to insulin, became more pronounced after 10 days of incubation. It is important to note that corresponding N 1s spectra (Fig. 5c, d), similar in absolute intensity and shape to those acquired for insulin-coated discs incubated in ␣-MEM without FBS (Fig. 3a, b), suggest a close protein apposition toward the surface. Furthermore, the C/N and C (C, H)/C atomic ratios, measured after one day of incubation are practically the same as for insulin-coated discs after ␣-MEM without FBS incubations (Table 2). Similar alterations of the structure for individual proteins at the interface thereby implies a direct surface effect of the insulin-coated layer, largely preserving the structure of secondary adsorbed FBS. Finally, in spite of low concentrations of CaCl2 (1.8 mM) and NaH2 PO4 ·2H2 O (1.01 mM) in cell culture media, all insulin-coated
surfaces contained detectable amounts of calcium and phosphate ions (total atomic concentration of Ca and P – 1–2 atomic%, Table 1) which, in contrast to Na and Cl, were not removed, even after ultrasonic washings. The incorporation of structurally bound Ca2+ and PO4 3− ions into the protein layer may be an important prerequisite for hydroxyapatite nucleation, which is substantial in osseointegration. Literature shows contradictory opinions regarding interactions between adsorbed proteins on a biomaterial, and proteins in the surrounding media. A partial exchange process is suggested, but it is also reported that the artificial surface with an immobilized protein layer behaves similar to an inert non-adsorbing surface [17,18]. However, in a recent study, an increased insulin release was observed from insulin-coated discs upon incubation in ␣-MEM containing FBS, compared to ␣-MEM without FBS [6], indicating that serum adsorption via interfacial exchange process resulted in a partial release of insulin by serum proteins. Such an exchange agrees with the protein’s interfacial behavior, as discussed above, and may be, in addition to insulin’s biological effects, advantageous for osseointegration in vivo. 4. Conclusions The present study has increased the understanding of the immobilized insulin behavior after incubation in the cell culture medium. In absence of serum proteins, the main part of the immobilized insulin layer remained intact in buffer, but experienced timedependent structural changes and exposed hydrophobic domains outward from the Ti-surface. The observed increase in the carbon aliphatic component of C 1s spectra may be explained by the adsorption of adventitious carbon from the surrounding medium, i.e. ␣-MEM, or insulin removal from Ti discs exposing the adventitious carbon remaining at the surface. Both interface processes should cause a clear time-dependent increase in the C H/Ctotal and C/N atomic ratios, for all treatments performed. For insulin-
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Fig. 5. The C 1s spectra (top) and N 1s spectra (bottom) of non-coated Ti disk incubated in ␣-MEM containing FBS after one day (a, c) and 10 days (b, d).
coated discs, in contrast, a decrease in C/N atomic ratios in ␣-MEM without FBS and no changes in ␣-MEM with FBS were observed. Although these sources of aliphatic carbon cannot be excluded, their contribution to interface composition and long-term interfacial phenomena seems to be insignificant. FBS adsorption from the medium to non-coated Ti discs supported this study’s hypothesis on proteins’ structural rearrangements. In the presence of FBS and immobilized insulin layers, a partial replacement of insulin by serum proteins occurred, with insulin remaining as the major constituent at the surface. In spite of this substitution, chemically immobilized insulin demonstrated a direct surface effect by stabilizing the structure of all (insulin and adsorbed FBS) proteins, and by supporting the incorporation of calcium and phosphate ions into the layer. A structurally stable protein layer at the implant surface with incorporated noticeable amounts of Ca2+ and PO4 3− ions may be an important condition for the bone response at the implant–tissue interface. Acknowledgments The authors would like to acknowledge the Hjalmar Svensson Foundation, Swedish Dental Association, and Gothenburg’s Diabetes Society for their support by grants. Part of this work was presented at the 5th International Symposium of Surface and Interface of Biomaterials (ISSIB-V), 7–10 April 2015, Sydney, Australia. References [1] A. Göransson-Westerlund, in: M. Rizzo, G. Bruno (Eds.), Surface Coatings, vol. 1, Nova Science Publishers, Inc., 2009, pp. 1–49. [2] K. Fulzele, R.C. Riddle, D.J. DiGirolamo, X. Cao, C. Wan, D. Chen, M.C. Faugere, S. Aja, M.A. Hussain, J.C. Bruning, et al., Cell 142 (2) (2010) 309.
[3] J. Yang, X. Zhang, W. Wang, J. Liu, Cell Biochem. Funct. 28 (4) (2010) 334. [4] M. Ferron, J. Wei, T. Yoshizawa, A. Del Fattore, R.A. DePinho, A. Teti, P. Ducy, G. Karsenty, Cell 142 (2) (2010) 296. [5] B. Malekzadeh, P. Tengvall, L.-O. Öhrnell, A. Wennerberg, A. Westerlund, J. Biomed. Mater. Res. A 101 (1) (2013) 132. [6] B. Öhrnell Malekzadeh, M. Ransjö, P. Tengvall, Z. Mladenovic, A. Westerlund, J. Biomed. Mater. B (2016), http://dx.doi.org/10.1002/jbmb33717 (in press). [7] D.G. Castner, B.D. Ratner, Surf. Sci. 500 (2002) 28–60. [8] D. Briggs, Surface Analysis by Auger and X-ray Photoelectron Spectroscopy, in: D. Briggs, J.T. Grant (Eds.), IM Publications and Surface Science Spectra Ltd., 2003, p. 53. [9] Z. Mladenovic, A. Sahlin-Platt, A. Bengtsson, M. Ransjö, A. Shchukarev, Surf. Interface Anal. 42 (2010) 452. [10] A. Shchukarev, Z. Mladenovic, M. Ransjö, Surf. Interface Anal. 44 (2012) 919. [11] Handbook of X-ray Photoelectron Spectroscopy, in: J. Chastain, R.C. King Jr. (Eds.), Physical Electronics, Inc., USA, 1999, p. 261. [12] T.L. Blundell, J.F. Cutfield, E.J. Dodson, G.G. Dodson, D.C. Hodgkin, D.A. Mercola, Cold Spring Harb. Symp. Quant. Biol. 36 (1972) 214–233. [13] P.G. Rouxhet, M.J. Genet, Microbial Cell Surface Analysis and Physicochemical Methods, in: N. Mozes, P.S. Handley, H.J. Busscher, P.G. Rouxhet (Eds.), VCH Publishers, New York, 1991. [14] S.L. McArthur, G. Mishra, Ch.D. Easton, Surface Analysis and Techniques in Biology, in: V.S. Smentkowski (Ed.), Springer International Publishing AG, 2014, pp. 9–36. [15] A. Barth, Biochim. Biophys. Acta 1767 (2007) 1073. [16] A.A. Thyparambil, Y. Wei, R.A. Latour, Biointerphases 10 (1) (2015) 019002. [17] E.A. Vogler, Biomaterials 33 (5) (2012) 1201. [18] M. Lindblad, M. Lestelius, A. Johansson, P. Tengvall, P. Thomsen, Biomaterials 18 (15) (1997) 1059.
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Journal of Electron Spectroscopy and Related Phenomena journal homepage: www.elsevier.com/locate/elspec
Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study Andrey Shchukarev a,∗ , Behnosh Öhrnell Malekzadeh b,c , Maria Ransjö b , Pentti Tengvall c , Anna Westerlund b a
Department of Chemistry, Umeå University, Umeå SE-90187, Sweden Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530, Sweden c Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, SE-40530, Sweden b
a r t i c l e
i n f o
Article history: Received 13 December 2016 Received in revised form 21 February 2017 Accepted 2 March 2017 Available online 6 March 2017 Keywords: XPS Titanium implant Insulin Cell culture medium Proteins at interface
a b s t r a c t Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in ␣-minimum essential medium (␣-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze the insulin behavior at the implant – ␣-MEM interface. In the absence of serum proteins in cell culture medium, the coated insulin layer remained intact, but experienced a time-dependent structural transformation exposing hydrophobic parts of the protein toward the solution. The presence of fetal bovine serum (FBS) in the medium resulted in partial substitution of insulin by serum proteins. In spite of some insulin release, the remaining coated layer demonstrated a direct surface effect by stabilizing the structure of protein competitors, and by supporting the accumulation of calcium and phosphate ions at the interface. A structurally stable protein layer with incorporated calcium and phosphate ions at the implant–tissue interface could be an important prerequisite for enhanced bone formation. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The understanding of interface phenomena and corresponding surface modifications is crucial for the improvement of the bone formation adjacent to the implant. The surface properties and interface processes are thus largely responsible for the bone response [1]. Insulin, a hormone known to regulate glucose metabolism, has additionally demonstrated anabolic effects in bone tissue [2–4]. In a recent study, locally delivered exogenous insulin enhanced bone formation in non-diabetic rats [5]. In order to provide a local delivery, insulin was immobilized to titanium surfaces. The mechanism, whereby an insulin coated implant surface exerts its effect remains unclear. Possible scenarios include a direct surface effect and/or an insulin release. The retained biological activity of insulin, which was released from titanium, has been confirmed in a separate study [6]. However, only one third of the immobilized insulin was released and the remaining two thirds remained bound to the titanium surface with an unknown composition. The aim of this
∗ Corresponding author. E-mail address: [email protected] (A. Shchukarev). http://dx.doi.org/10.1016/j.elspec.2017.03.001 0368-2048/© 2017 Elsevier B.V. All rights reserved.
study was therefore to characterize, by X-ray photoelectron spectroscopy (XPS), immobilized insulin behavior on titanium surfaces, after incubation in a biologically relevant cell culture medium, without and with the addition of serum proteins. 2. Experimental 2.1. Immobilization of insulin onto titanium discs Ti6Al4V discs (Ti – 89.6%, Al – 6.3%, V – 4.1%) with 8 mm in diameter and 1 mm thickness and with a turned surface were used (Part Precision AB, Mariestad, Sweden). The discs and null ellipsometry reference Silicon (Si) samples (1 cm × 1 cm) were cleaned during four minutes in an ultrasonic bath in 70% ethanol (Solveco, Rosersberg, Sweden), rinsed in distilled water, and dried by flowing nitrogen. The insulin immobilization protocol was similar to that in previous studies [5,6]: in brief, the discs were incubated for 30 min in 1% (3-Aminopropyl) triethoxysilane (APTES; Sigma–Aldrich, Steinheim, Germany), in xylene (VWR, Stockholm, Sweden), and rinsed in 99% xylene, 70% ethanol, and distilled water, respectively. The discs were subsequently incubated for 30 min in a 6% glutaraldehyde solution (GA; Sigma–Aldrich, Steinheim, Germany) in phosphate buffered saline (PBS; Sigma–Aldrich, Stein-
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heim, Germany), at pH 9 at room temperature, and rinsed in PBS (pH 9). Recombinant human insulin (10 mg/ml in 25 mM HEPES, pH 8.2, Sigma–Aldrich, Steinheim, Germany) was diluted to 1 mg/ml in PBS at pH 5.7. The aldehyde coated discs were incubated in this solution for 15 minutes at room temperature and rinsed with PBS (pH 5.7). To achieve a carboxyl group activation and surface cross-linking, the discs were incubated in a mixture of 0.2 M N-(3-dimethyl aminopropyl)-N -ethylcarbodiimide (EDC; Sigma–Aldrich, Steinheim, Germany), and 0.05 M N-hydroxysulfosuccinimide sodium salt (NHS; Sigma–Aldrich, Steinheim, Germany) in a PBS, pH 5.7 at room temperature for 15 min. The discs were subsequently incubated 10 times in insulin solution and EDC-NHS in between. Since the EDC solution is unstable, new solutions were made every 2½h. Finally, the discs were rinsed with PBS (pH 5.7), distilled water, and dried in flowing nitrogen. The null ellipsometry reference Silicon samples revealed an insulin thickness of approximately 3.5 nm. The assumed refractive index of insulin was nf = 1.465, as described elsewhere [5]. The Ti6Al4V alloy surface may not have a homogeneous distribution of insulin molecules although titanium is known to quickly adsorb proteins [7]. Therefore, the thickness at different surface locations is not expected to differ significantly. 2.2. XPS characterization of insulin-coated discs XPS was used to monitor changes in surface chemical composition when coated insulin layer interacts with cell culture medium. The XPS spectra were acquired with Kratos Axis Ultra DLD electron spectrometer using a monochromated Al K␣ source operated at 150 W, a hybrid lens system with a magnetic lens providing an analysis area of 0.3 mm × 0.7 mm, and a charge neutralization system. The binding energy (BE) scale was referenced to the C 1s line of aliphatic carbon, set at 285.0 eV. The processing of spectra was accomplished with Kratos software and CasaXPS package. The curve fitting of high-resolution C 1s and N 1s spectra was performed after Shirley background subtraction with a fixed number of spectral components corresponding to expected chemical states, two for N 1s and 3–4 for C 1s spectra, as described below. The BE position and FWHM of the components were not fixed, although the FWHM did not allow exceeding 1.6–1.7 eV. The accuracy in BE determination is 0.1 eV, and in atomic ratio – 8–10% rel. It is important to note that XPS sampling depth for organics, 7–9 nm [8], is larger than the thickness of deposited insulin, and therefore the entire protein layer was analyzed. In order to ascertain that the results were reproducible, all measurements were repeated at least in triplicate with different Ti discs. Reference XPS spectra of fast-frozen drops of ␣-MEM + 10% FBS were reported earlier [9,10] and for pure insulin powder (Insulin from Bovine Pancreas, I-6634, Sigma–Aldrich, Steinheim, Germany) especially acquired in the present study. 2.3. Sample preparation protocol The titanium (Ti) discs (non-coated and insulin-coated) were placed, one disk per well, in 24-well plates (NunclonTM Surface, Roskilde, Denmark). 1 ml/well of ␣-Minimum Essential Medium (␣-MEM; Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 1% benzyl-penicillin/streptomycin (ASTRA Zeneca AB, Sweden/Sigma–Aldrich, Steinheim, Germany), without or with 10% FBS (Life technology, Sweden) was added to the wells. The discs were incubated one or 10 days at 37 ◦ C, in a humid atmosphere containing 5% CO2 . In order to obtain reliable information about the surface composition that was formed in the cell culture medium, a sample preparation protocol was elaborated as follows. After the incubation, three different sample treatments for XPS analysis were tested: First, an excess of cell culture medium on the surface was
Fig. 1. Survey XPS spectra taken with three different treatments of insulin-coated Ti discs after incubation in cell culture medium. (a) After soaking up ␣-MEM cell culture medium (b) After rinsing in distilled water (c) After rinsing in distilled water and followed by ultrasonic treatment.
shaken off, and the rest (if present) gently soaked up by filter paper. This procedure resulted in a significant precipitation of NaCl from the medium and no Ti 2p signal could then be observed (Fig. 1a). Second, short washing (up to 30 s) in distilled water removed all precipitates and loosely bound organics, but did not alter the insulin binding (Fig. 1b). Third, further ultrasonic washing (1 min) in distilled water resulted in surface composition similar to that after a short washing (Fig. 1c), meaning that the proteins were tightly bound onto titanium. In the last two cases, XPS survey spectra are very similar to those obtained with insulin-coated Ti discs (Fig. 2). Therefore, these steps were applied to all samples treated in ␣MEM. 3. Results and discussion 3.1. Insulin deposition XPS spectra of non-coated and insulin-coated Ti discs are presented in Fig. 2. The Ti 2p spectra are very similar, consist of two Ti 2p doublets, and reveal that a continuous TiO2 layer formed in air (corresponding dominant Ti 2p3/2 component at 458.8 eV [11]) covers the surface. The insulin immobilization onto the surface was confirmed by the appearance of intensive nitrogen N 1s photoelectron line in the survey spectrum and by both high-resolution spectra of C 1s and N 1s (Fig. 2). The surface composition of non-coated discs differed remarkably in atomic compositions between the discs and was dominated by C (from 50 to 76 at.%), O (18–37 at%), and Ti (4–13 at.%). Only minor contributions of Al (1.5–2 at.%) and no V from alloy composition were observed (Table 1). Other surface contaminations including F, N, Bi, Ba, and Pb (total <0.5–2 at.%) showed no significant contributions and appeared possibly due to insufficient cleaning after the cutting processes. Observed high concentrations of adventitious carbon/oxygen likely correspond to organic contaminations within the thickness range of 2–4 nm. Irrespective of the contaminations, similar Ti implants enhanced (after insulin immobilization) bone formation in rat tibia [5]. Therefore, in order
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Fig. 2. XPS spectra of non-coated and insulin-coated titanium discs. Left: Survey spectra of non-coated and insulin-coated titanium. Upper right: Reference insulin powder C 1s and N 1s spectra. Lower right: C 1s and N 1s spectra of insulin-coated titanium.
Table 1 Atomic concentrations (at.%) of main elements at the surface of Ti6Al4V discs after different treatments. Samples
C
O
N
Ti
Non-coated discsa Reference insulin powderb
50–76 69.56
18–37 15.46
0–1.5 13.86
4–13 No
Insulin-coated discs Disk insulin-coated +␣-MEM without FBS, 1 dayc +␣-MEM without FBS, 10 daysc +␣-MEM with FBS, 1 dayc +␣-MEM with FBS, 10 daysc
55.9 ± 1.5 44.0 ± 2.5 37.7 ± 4.0 52.0 ± 2.1 58.0 ± 1.0
29.3 ± 0.7 38.2 ± 2.0 40.5 ± 3.5 30.1 ± 3.0 26.4 ± 1.0
9.1 ± 0.9 5.4 ± 0.1 5.4 ± 0.7 9.7 ± 0.3 10.3 ± 0.9
5.7 ± 0.3 9.4 ± 0.5 11.5 ± 1.5 6.5 ± 0.3 4.2 ± 0.4
Non-insulin coated discs ␣-MEM with FBS, 1 dayc ␣-MEM with FBS, 10 daysc
55.3 ± 1.4 62.4 ± 1.2
30.2 ± 1.1 25.3 ± 0.8
6.3 ± 0.3 5.7 ± 0.4
6.7 ± 0.1 5.3 ± 0.3
a Al atomic concentration – 1–2 at.%, no V was detected. Other surface contaminations – F, Bi, and Pb. b Minor elements: S – 1.13 at.%, Zn – 0.06 at.% (H is not detectable by XPS). c Total Ca and P atomic concentrations were within the 1–2 at.% range.
to follow similar interfacial processes, further surface cleaning of non-coated discs was not performed. However, after insulin-coating, the surface compositions became similar for all coated discs, with average concentrations of C (55.9 ± 1.5 at.%), O (29.3 ± 0.7 at.%), N (9.1 ± 0.9 at.%), and Ti (5.7 ± 0.3 at.%) (Table 1). Moreover, the intensity of the Ti 2p3/2 component at 454.1 eV, corresponding to Ti metal [11], correlated well to the estimated (≈ 3.5 nm) thickness of the deposited insulin layer. The active form of insulin is a monomer, although when stored in the body it is a hexamer. This insulin hexamer is torus shaped and 3.5 nm high and 5 nm in diameter [12]. When the protein is surface-bound, it may exist in different shapes and sizes. Insulin dominated the surface composition, although the metallic component of the Ti 2p spectrum was still detectable, suggesting that the deposited insulin may not form a homogeneous layer. These observations, together with the reproducible surface composition of the insulin-coated discs, indicate that protein immobilization removes most of the normally observed surface contaminations, organic as well as inorganic, although some adventitious carbon might still be observable.
3.2. Surface composition The average surface compositions of all discs, after incubation in the medium (␣-MEM without and with FBS), are given in Table 1. Minor elements such as Al, Ca, and P are not included because of low observed concentrations and their absence in the protein composition. Traces of sulfur (≈ 0.5 at.%) and no zink (below detection limit of 0.1 at.%) from adsorbed insulin molecules were found on insulin-coated discs. Insulin release from the surface after incubation in ␣-MEM without FBS [6] was observed as a decrease of the carbon and nitrogen concentrations, and followed by a corresponding Ti increase. This process continued for up to 10 days with the ␣-MEM treatment. The addition of FBS caused a remarkable rise in the surface nitrogen content, already after one day of incubation, in spite of a small decrease in carbon and an insignificant increase in titanium. This made it possible to assume that an additional adsorption of serum proteins from the medium occurred on top of the remaining coated insulin layer, thereby compensating the released insulin. Prolonged incubation in ␣-MEM with FBS resulted in noticeably higher carbon and nitrogen concentrations, and lower titanium, compared to insulin-coated discs, which were not incubated in the medium. Since the serum proteins were the only substantial sources of C and, especially, N in the medium, the increase in carbon and nitrogen signals indicated a complimentary serum adsorption onto the surfaces. 3.3. N 1s and C 1s spectra The N 1s spectra of insulin-coated Ti discs, incubated in the cell culture medium (Fig. 3), show predominantly two types of N bonds; the main line corresponds to nitrogen in amide and nonprotonated amine groups (400.2 eV), and the minor component is due to amine group protonation (401.8–402.0 eV) [13]. The spectra are typical for nitrogen in proteins, as seen when comparing them with corresponding insulin and ␣-MEM without and with FBS reference spectra (Fig. 3). Important to note is that the original ␣-MEM cell culture medium contains no proteins. Corresponding N 1s spectrum therefore differs significantly in intensity (low concentration of aminoacids in the media) and shape originating from both protonated (N 1s component at 402.0 eV) and non-protonated (N 1s component at 400.2 eV) amino-groups [9]. The N 1s line
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Fig. 3. Reference N 1s spectra (top) of insulin powder and fast-frozen drops of ␣-MEM without and with FBS. The N 1s spectra (bottom) of insulin-coated discs incubated for one day in ␣-MEM without FBS (a), 10 days in ␣-MEM without FBS (b), and with FBS (c) respectively. N 1s spectrum of insulin-coated disk incubated for one day in ␣-MEM with FBS is similar to others and not shown.
position and shape for insulin-coated Ti discs is independent on ␣-MEM composition and treatment time, although a weak variation in amine group protonation occurs in the medium without FBS. The similarity between N 1s spectra indicates that the surface protein layer remained intact upon all the treatments. Unfortunately, this similarity does not allow discrimination between the insulin and serum proteins in the layer after treatment in ␣-MEM with FBS. In contrast to N 1s, the C 1s spectra of insulin and ␣-MEM with FBS differ significantly (Fig. 4, top) in the intensities of spectral components related to hydrocarbon chains (285.0 eV), carbon singly bound to oxygen or nitrogen (≈ 286.4 eV), and the protein’s amide bonds (≈ 288.3 eV). Insulin-coated Ti discs incubated in both cell culture medium display C 1s spectra (Fig. 4, bottom) similar to that of the reference insulin powder and do not demonstrate any carbonate (≈ 290.3 eV) adsorbed from the medium. This confirms that the immobilized proteins remain intact at the surface, with insulin being the major component even after treatments in ␣-MEM containing FBS. However, C 1s spectra (Fig. 4, bottom) display noticeable changes in the relative intensity of spectral components, indicating structural re-arrangements or conformational alterations within the protein layer, all depending on medium composition and incubation time. These “unfolding” transformations also cause changes in atomic C/N and C (C, H)/Ctotal ratios, given in Table 2. The incubation of insulin-coated discs in ␣-MEM without FBS resulted in a remarkable increase in the surface carbon concentration, compared to nitrogen (C/N atomic ratio growth from 5 to 7–8). A simultaneous intensity raise in the hydrocarbon component of C 1s spectra (from ≈40 to ≈60%) implies that the top surface layer of insulin can experience a structural re-arrangement exposing hydrophobic parts of the molecules toward the aqueous medium. The changes in C 1s spectra, caused by incubation of insulin-coated discs in ␣MEM without FBS might also be attributed to removal of insulin from the surface and increased exposure of the Ti substrate, with the accompanying original organic contaminants which are domi-
Table 2 Atomic ratios at the surface of Ti6Al4V discs after different treatments. Samples
C/N
C (C, H)/Ctotal
Ca/P
Insulin powder reference
5:1
0.42:1
–
Insulin-coated discs Disk as coated +␣-MEM without FBS, 1 day +␣-MEM without FBS, 10 days +␣-MEM with FBS, 1 day +␣-MEM with FBS, 10 days
4.8 ± 0.1:1 8.0 ± 0.6:1 7.0 ± 0.3:1 5.6 ± 0.2:1 5.7 ± 0.5:1
0.5 ± 0.01:1 0.62 ± 0.01:1 0.57 ± 0.05:1 0.58 ± 0.01:1 0.51 ± 0 02:1
– 0.9 ± 0.02:1 1.4 ± 0.05:1 0.9 ± 0.02:1 1.1 ± 0.2:1
Non-coated discs +␣-MEM with FBS, 1 day +␣-MEM with FBS, 10 days
8.5 ± 0.2:1 11.1 ± 0.8:1
0.62 ± 0.01:1 0.68 ± 0.03:1
1.9 ± 0.3:1 1.4 ± 0.1:1
nated by C (C H) hydrocarbon bonds and do not contain nitrogen. If this is the case, the C/N and C (C, H)/Ctotal atomic ratios have to increase significantly, e.g., via a prolonged incubation time, due to more released insulin. In contrast, a decrease is observed (see Table 2); consequently, a reasonable rise in Ti atomic concentration (Table 1) supports the study’s assumption that the insulin immobilization procedure removes most of the surface organic contaminants, although the deposited protein layer does not seem homogeneous in thickness. After incubation of insulin-coated discs in ␣-MEM containing FBS, the additional adsorption of serum proteins (Table 1) resulted in C/N atomic ratio (5.7–5.8), close to the value of insulin powder (Table 2). The corresponding decrease in C (C, H)/Ctotal atomic ratio supports the hypothesis of a possible serum protein adsorption mechanism due to hydrophobic interactions between serum proteins and immobilized insulin. As a result, the protein layer seems to restore its “folding”, similar to one of the “as coated” disk (i.e., insulin-coated disk prior incubation). Unfortunately, XPS cannot be used to differentiate between individual proteins [14], even in a simple two-component mixture, and is not able to distinguish structural molecular mechanisms of the protein adsorption and re-
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Fig. 4. Reference C 1s spectra (top) of insulin powder and fast-frozen drops of ␣-MEM, and C 1s spectra (bottom) of insulin-coated discs incubated for one day in ␣-MEM without FBS (a), and 10 days without FBS (b) and with FBS (c), respectively. C 1s spectrum of insulin-coated disk incubated for one day in ␣-MEM with FBS is similar to spectrum (c) and not shown.
arrangement. Moreover, ultra-high vacuum conditions of the XPS experiment, which can easily change interfacial equilibrium, both compositional and structural, have to be taken into account. However, considering a similar chemical composition of the samples and the medium used, i.e., the sample treatment and the identical sample preparation protocol for the measurements, the extent of the surface-induced structural alterations is expected to be very close for all Ti discs studied. Therefore, noticeable consequences of interfacial processes should be evident at all interfaces. Nevertheless, to confirm described XPS findings, complimentary IR [15], circular dichroism spectropolarimetry [16], TOF-SIMS, and MALDITOF SIMS experiments are needed. To clarify the role of insulin at the titanium surface and the FBS behavior at the interface, additional experiments with noncoated Ti discs, incubated in ␣-MEM with FBS were performed. Without insulin coating, adsorbing serum proteins experienced similar structural changes by exposing their hydrophobic parts/hydrocarbon chains toward the medium. C 1s spectra, dominated by the C (C, H) component at 285.0 eV (Fig. 5a, b), and C/N and C (C, H)/Ctotal atomic ratios (Table 2) confirm this interfacial conformational change, which seems to be even more apparent than for insulin and, in contrast to insulin, became more pronounced after 10 days of incubation. It is important to note that corresponding N 1s spectra (Fig. 5c, d), similar in absolute intensity and shape to those acquired for insulin-coated discs incubated in ␣-MEM without FBS (Fig. 3a, b), suggest a close protein apposition toward the surface. Furthermore, the C/N and C (C, H)/C atomic ratios, measured after one day of incubation are practically the same as for insulin-coated discs after ␣-MEM without FBS incubations (Table 2). Similar alterations of the structure for individual proteins at the interface thereby implies a direct surface effect of the insulin-coated layer, largely preserving the structure of secondary adsorbed FBS. Finally, in spite of low concentrations of CaCl2 (1.8 mM) and NaH2 PO4 ·2H2 O (1.01 mM) in cell culture media, all insulin-coated
surfaces contained detectable amounts of calcium and phosphate ions (total atomic concentration of Ca and P – 1–2 atomic%, Table 1) which, in contrast to Na and Cl, were not removed, even after ultrasonic washings. The incorporation of structurally bound Ca2+ and PO4 3− ions into the protein layer may be an important prerequisite for hydroxyapatite nucleation, which is substantial in osseointegration. Literature shows contradictory opinions regarding interactions between adsorbed proteins on a biomaterial, and proteins in the surrounding media. A partial exchange process is suggested, but it is also reported that the artificial surface with an immobilized protein layer behaves similar to an inert non-adsorbing surface [17,18]. However, in a recent study, an increased insulin release was observed from insulin-coated discs upon incubation in ␣-MEM containing FBS, compared to ␣-MEM without FBS [6], indicating that serum adsorption via interfacial exchange process resulted in a partial release of insulin by serum proteins. Such an exchange agrees with the protein’s interfacial behavior, as discussed above, and may be, in addition to insulin’s biological effects, advantageous for osseointegration in vivo. 4. Conclusions The present study has increased the understanding of the immobilized insulin behavior after incubation in the cell culture medium. In absence of serum proteins, the main part of the immobilized insulin layer remained intact in buffer, but experienced timedependent structural changes and exposed hydrophobic domains outward from the Ti-surface. The observed increase in the carbon aliphatic component of C 1s spectra may be explained by the adsorption of adventitious carbon from the surrounding medium, i.e. ␣-MEM, or insulin removal from Ti discs exposing the adventitious carbon remaining at the surface. Both interface processes should cause a clear time-dependent increase in the C H/Ctotal and C/N atomic ratios, for all treatments performed. For insulin-
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Fig. 5. The C 1s spectra (top) and N 1s spectra (bottom) of non-coated Ti disk incubated in ␣-MEM containing FBS after one day (a, c) and 10 days (b, d).
coated discs, in contrast, a decrease in C/N atomic ratios in ␣-MEM without FBS and no changes in ␣-MEM with FBS were observed. Although these sources of aliphatic carbon cannot be excluded, their contribution to interface composition and long-term interfacial phenomena seems to be insignificant. FBS adsorption from the medium to non-coated Ti discs supported this study’s hypothesis on proteins’ structural rearrangements. In the presence of FBS and immobilized insulin layers, a partial replacement of insulin by serum proteins occurred, with insulin remaining as the major constituent at the surface. In spite of this substitution, chemically immobilized insulin demonstrated a direct surface effect by stabilizing the structure of all (insulin and adsorbed FBS) proteins, and by supporting the incorporation of calcium and phosphate ions into the layer. A structurally stable protein layer at the implant surface with incorporated noticeable amounts of Ca2+ and PO4 3− ions may be an important condition for the bone response at the implant–tissue interface. Acknowledgments The authors would like to acknowledge the Hjalmar Svensson Foundation, Swedish Dental Association, and Gothenburg’s Diabetes Society for their support by grants. Part of this work was presented at the 5th International Symposium of Surface and Interface of Biomaterials (ISSIB-V), 7–10 April 2015, Sydney, Australia. References [1] A. Göransson-Westerlund, in: M. Rizzo, G. Bruno (Eds.), Surface Coatings, vol. 1, Nova Science Publishers, Inc., 2009, pp. 1–49. [2] K. Fulzele, R.C. Riddle, D.J. DiGirolamo, X. Cao, C. Wan, D. Chen, M.C. Faugere, S. Aja, M.A. Hussain, J.C. Bruning, et al., Cell 142 (2) (2010) 309.
[3] J. Yang, X. Zhang, W. Wang, J. Liu, Cell Biochem. Funct. 28 (4) (2010) 334. [4] M. Ferron, J. Wei, T. Yoshizawa, A. Del Fattore, R.A. DePinho, A. Teti, P. Ducy, G. Karsenty, Cell 142 (2) (2010) 296. [5] B. Malekzadeh, P. Tengvall, L.-O. Öhrnell, A. Wennerberg, A. Westerlund, J. Biomed. Mater. Res. A 101 (1) (2013) 132. [6] B. Öhrnell Malekzadeh, M. Ransjö, P. Tengvall, Z. Mladenovic, A. Westerlund, J. Biomed. Mater. B (2016), http://dx.doi.org/10.1002/jbmb33717 (in press). [7] D.G. Castner, B.D. Ratner, Surf. Sci. 500 (2002) 28–60. [8] D. Briggs, Surface Analysis by Auger and X-ray Photoelectron Spectroscopy, in: D. Briggs, J.T. Grant (Eds.), IM Publications and Surface Science Spectra Ltd., 2003, p. 53. [9] Z. Mladenovic, A. Sahlin-Platt, A. Bengtsson, M. Ransjö, A. Shchukarev, Surf. Interface Anal. 42 (2010) 452. [10] A. Shchukarev, Z. Mladenovic, M. Ransjö, Surf. Interface Anal. 44 (2012) 919. [11] Handbook of X-ray Photoelectron Spectroscopy, in: J. Chastain, R.C. King Jr. (Eds.), Physical Electronics, Inc., USA, 1999, p. 261. [12] T.L. Blundell, J.F. Cutfield, E.J. Dodson, G.G. Dodson, D.C. Hodgkin, D.A. Mercola, Cold Spring Harb. Symp. Quant. Biol. 36 (1972) 214–233. [13] P.G. Rouxhet, M.J. Genet, Microbial Cell Surface Analysis and Physicochemical Methods, in: N. Mozes, P.S. Handley, H.J. Busscher, P.G. Rouxhet (Eds.), VCH Publishers, New York, 1991. [14] S.L. McArthur, G. Mishra, Ch.D. Easton, Surface Analysis and Techniques in Biology, in: V.S. Smentkowski (Ed.), Springer International Publishing AG, 2014, pp. 9–36. [15] A. Barth, Biochim. Biophys. Acta 1767 (2007) 1073. [16] A.A. Thyparambil, Y. Wei, R.A. Latour, Biointerphases 10 (1) (2015) 019002. [17] E.A. Vogler, Biomaterials 33 (5) (2012) 1201. [18] M. Lindblad, M. Lestelius, A. Johansson, P. Tengvall, P. Thomsen, Biomaterials 18 (15) (1997) 1059.