Ryo Jimbo
Stefan St
übinger
Marcel Obrecht
Michel Dard
Simon Berner
Nanostructures and hydrophilicity
influence osseointegration: a
biomechanical study in the rabbit tibia
Authors’ affiliations:
Ann Wennerberg, Ryo Jimbo,Department of Prosthodontics, Faculty of Odontology, Malm€o University, Malm€o, Sweden
Stefan Stübinger, Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland
Marcel Obrecht, Michel Dard, Simon Berner, Research Department, Institut Straumann AG, Basel, Switzerland
Michel Dard,Department of Periodontology and Implant Dentistry, New York University, New York, NY, USA
Corresponding author: Ann Wennerberg
Department of Prosthodontics, Faculty of Odontology
Malm€o University Malm€o, Sweden Tel.: + 46 40 6658499 Fax: + 46 40 6658503
e-mail: ann.wennerberg@mah.se
Key words: bone healing, hydrophilicity, hydrophobicity, implants, nanostructures Abstract
Objective: Implant surface properties have long been identified as an important factor to promote osseointegration. The importance of nanostructures and hydrophilicity has recently been discussed. The aim of this study was to investigate how nanostructures and wettability influence
osseointegration and to identify whether the wettability, the nanostructure or both in combination play the key role in improved osseointegration.
Materials and Methods: Twenty-six adult rabbits each received two Ti grade 4 discs in each tibia. Four different types of surface modifications with different wettability and nanostructures were prepared: hydrophobic without nanostructures (SLA), with nanostructures (SLAnano); hydrophilic with two different nanostructure densities (low density: pmodSLA, high density: SLActive). All four groups were intended to have similar chemistry and microroughness. The surfaces were evaluated with contact angle measurements, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy and interferometry. After 4 and 8 weeks healing time, pull-out tests were performed.
Results: SLA and SLAnano were hydrophobic, whereas SLActive and pmodSLA were super-hydrophilic. No nanostructures were present on the SLA surface, but the three other surface modifications clearly showed the presence of nanostructures, although more sparsely distributed on pmodSLA. The hydrophobic samples showed higher carbon contamination levels compared with the hydrophilic samples. After 4 weeks healing time, SLActive implants showed the highest pull-out values, with significantly higher pull-out force than SLA and SLAnano. After 8 weeks, the SLActive implants had the highest pull-out force, significantly higher than SLAnano and SLA.
Conclusions: The strongest bone response was achieved with a combination of wettability and the presence of nanostructures (SLActive).
The hydrophilicity of an implant has been identified as an important factor that may influence the early bone response (Buser et al. 2004; Sawase et al. 2008), that is, with a high degree of hydrophilicity, more rapid healing occurs and consequently a better stability and possibility for early loading with good clinical predictability. Several in vitro investigations have demonstrated effects on cells in terms of attachment, proliferation, differentiation and gene expression in favour of super-hydrophilic surfaces compared with other hydrophilic and hydrophobic implant surfaces (Jimbo et al. 2008; Shibata et al. 2010; Li et al. 2012). From in vivoinvestigations, results have been pub-lished suggesting an enhanced bone healing for super-hydrophilic implants (Jimbo et al. 2011; Hirakawa et al. 2012).
Commercially available implants with a super-hydrophilic surface exist; the most well documented is the SLActive implant surface (Institut Straumann AG, Basel, Switzerland). SLActive is a surface achieved by blasting the implant surface with large corundum par-ticles and etching in a mixture of HCl and H2SO4. Thereafter, the implants are rinsed in
water under nitrogen protection and stored in NaCl aqueous solution. This surface has been investigated in vitro; several studies have demonstrated a strong increase in alka-line phosphatase, osteoprotegerin and osteo-calcin production when compared with SLA surface (Institut Straumann AG, Basel, Swit-zerland) (blasted and acid-etched, but packed dry under ambient conditions) (Brunner & Langer 1999; Masaki et al. 2005; Brunner Date:
Accepted 16 May 2013
To cite this article:
Wennerberg A, Jimbo R, St€ubinger S, Obrecht M, Dard M, Berner S. Nanostructures and hydrophilicity influence osseointegration– A biomechanical study in the rabbit tibia. Clin. Oral Impl. Res.25, 2014, 1041–1050
2010; Klein et al. 2010; Mamalis & Silvestros 2011; Mamalis et al. 2011). In vivo in differ-ent animal models, including pigs, dogs and sheep, SLActive has demonstrated faster bone formation than SLA. In particular, the early healing phase seems to be enhanced, while several publications have observed that after 4 weeks or longer, the difference between SLActive and SLA levels out (Buser et al. 2004; Bornstein et al. 2008; Abdel-Haq et al. 2011). Similar observations have been made in experimental studies performed in human bone. In the human mandibular retromolar region, SLActive exhibited significantly more bone in contact with the implant surface after 28 days of healing when compared with SLA. However, there was no difference after 7, 14 or 42 days between the two surfaces (Lang et al. 2011). Clinical studies investigat-ing SLA vs. SLActive are rather few; how-ever, both surfaces demonstrated very good clinical results with survival rates evolving between 96% and 100% after a follow-up time of about 1 year (Heberer et al. 2011; Ka-rabuda et al. 2011) without any significant differences in clinical outcome.
Although hydrophilicity was claimed to be the reason for improved cell and tissue response in experimental studies, the
mecha-nisms behind these findings are still
unknown. In a recent publication, the occur-rence of nanostructures on SLActive was reported (Wennerberg et al. 2013). The influ-ence of nanostructures on osseointegration has gained much interest in the last few years, and several studies showed that impor-tant steps in the osseointegration process are modulated by the presence of nanostructures (Mendonca et al. 2009). Furthermore, chemi-cal alterations, such as incorporation of mag-nesium, fluoride and calcium ions, may be
another cause for increased biological
response (Ellingsen et al. 2004; Sul et al. 2004, 2006; Monjo et al. 2008).
The purpose of this study was to separately assess the influence of nanostructures and wettability on osseointegration, while keep-ing the surface chemistry similar, and to identify whether the wettability, the nano-structure or both in combination play the key role for better osseointegration.
Material and methods
Material
In total, 140 discs with a diameter of 6.2 mm and a thickness of 2 mm were prepared for the in vivo experiments from commercially pure titanium grade 4. 104 discs were
inoper-ated in the rabbit bone; 36 discs were used for surface characterization. The discs had similar surface chemistry and microrough-ness and were divided in 4 groups.
Group A: SLA
Sandblasted with corundum (particle size 250–500 lm), acid-etched in a mixture of HCl and H2SO4, followed by cleaning in
nitric acid and rinsing in deionized water. Finally, the discs were air-dried and packed in aluminium foil. This group was used as a control.
Group B: SLActive
Same sandblasting and acid-etching process as for SLA, however, further treatment took place under nitrogen cover gas to prevent exposure to air. The discs were rinsed in 0.9% NaCl solution and finally stored in 0.9% NaCl solution at pH 4 to 6.
Group C: SLAnano
SLActive discs were stored for 3 months in NaCl solution and then thoroughly rinsed and ultrasonicated in ultrapure water to remove all traces of NaCl. Then, the discs were dried in a stream of N2, packed in
alu-minium foil like the SLA discs and stored for another 4 months prior to implantation. Group D: pmodSLA
SLA discs were subjected to oxygen plasma cleaning and subsequently stored in 0.9% NaCl solution for about 1 month prior to implantation. The storage of the discs in NaCl solution was identical to the storage of the SLActive discs.
The discs of all groups were c-sterilized (25–42 kGy) prior to all following evalua-tions.
All samples and surface modifications were prepared by Straumann (Institut Straumann AG). The surfaces of Ti SLA and Ti SLActive correspond to the commercially available SLAâand SLActiveâimplant surfaces. Surface characterization
Contact angle
The contact angle measurements were per-formed using a sessile drop test with
ultra-pure water (EasyDrop DSA20E, Kr€uss
GmbH). Three samples were measured for each surface modification. The dry samples
(SLA and SLAnano) were evaluated as
received, whereas the samples stored in liquid (SLActive and pmodSLA) were blown dry in a stream of argon prior to the measure-ment. The droplet size for the contact angle
measurements was 0.3ll for the samples
stored dry and 0.1ll for the samples stored in liquid. The contact angles were deter-mined by fitting a general conic section equa-tion to the contour of the droplet placed on the surface (tangent method).
Scanning electron microscopy (SEM)
High-resolution SEM images were performed with a Hitachi S-4800 (Hitachi High-Tech-nologies Corporation, Tokyo, Japan) equipped with a cold field emission electron source and an in-lens secondary electron detector. Three samples were analysed for each type of surface modification (SEM and XPS analyses). The measurements were performed at the Microscopy Center of the University of Basel, Basel, Switzerland. Additionally, the explant-ed (pullexplant-ed out) discs of two selectexplant-ed rabbits per healing time point were investigated by SEM/EDX. These discs were investigated at Straumann with a Zeiss Supra 55 SEM (Carl
Zeiss AG, Oberkochen, Germany). The
implant discs were coated by a thin Pt/Pd layer with a thickness of about 2 nm. X-ray photoelectron spectroscopy (XPS)
Chemical composition of the surface (outer-most 5–10 nm), chemical state analysis and oxide layer thickness were investigated with XPS. The measurements were performed by SuSoS AG, D€ubendorf, Switzerland. Spectra were acquired on a PHI5000 VersaProbe
spec-trometer (ULVAC-PHI, INC., Chigasaka,
Japan) equipped with a focused scanning monochromatic Al-Ka source (1486.6 eV). The photoelectrons were detected at an angle of 45° to the surface normal by means of a hemispherical analyser with a multichannel detection system with 16 channels. Three samples were analysed for each surface modi-fication. Each sample was analysed on one spot with an area of 1.49 0.5 mm2
. A survey scan and detailed spectra of the elements observed in the survey were acquired. The samples stored in saline solution were rinsed with ultrapure water and dried in a stream of nitrogen prior to the XPS measurements. The
samples stored dry were measured as
received.
The oxide layer thickness was calculated using the detailed analysis of the oxidation state of the Ti2p spectrum (Sittig et al. 1999). The calculation models a homogenous (in terms of thickness and composition) TiO2
layer on top of metallic titanium. The lim-ited probing depth of XPS of about 10 nm in the case of TiO2 might lead to systematic
layer, so the calculated values should there-fore be interpreted carefully. Nevertheless, the applied method typically allows a reasonable, relative comparison between different types of samples, that is, an order of the oxide layer thickness of the different surface modifica-tions can be established.
Interferometer
The surface roughness at the lm scale was measured with an interferometer (MicroXam, Phase-shift, Phoenix, AZ, USA). Three sam-ples from each surface modification were selected at random, and each was assessed at three positions with a measuring area of 2509 200 lm2. A Gaussian filter with a size
of 509 50 lm2was applied before parameter
calculation. A selection of four different parameters was made to characterize the surface topography in height and spatial direction:
Sa= average height deviation from the
mean plane, measured in lm and
repre-sents a pure height descriptive parameter. Ssk= skewness of the height distribution,
a parameter used to distinguish whether the height deviation is mainly due to dominating valleys or peaks. A positive value indicates distinct peaks, a negative distinct valleys.
Sds= density of summits, measured in
number of peaks per square mm and rep-resents a pure spatial parameter.
Sdr= developed surface area, measured in
% enlargement compared with a totally smooth reference area (equal to the mea-sured area).
Atomic force microscopy (AFM)
Surface topography on the nanometre level was characterized by AFM. From each sur-face, the three randomly selected discs were used after measurements with interferometry, and three randomly selected regions on each disc were measured (XE-100, Park Systems, Suwon, Korea) using a noncontact mode set-up in air and at room temperature (scan size 19 1 lm2). The parametric calculation was
performed after the form removal and
wavi-ness by the use of a Gaussian filter
(0.259 0.25 lm2). The evaluation parameters
were the same as for the topographical evalu-ation at thelm scale.
Animals
Twenty-six adult Swedish looped ear rabbits of both sexes with a weight of 3.3–4.3 kg were used. Thirteen rabbits were aimed for 4 weeks of healing and 13 for 8 weeks.
Ani-mals were housed in standard cages and fed with laboratory animal diet pellets. The animals had free access to tap water. The study was performed with permission of the Malm€o-Lund University ethical commit-tee (N° M 205-11.3).
Anaesthetics and analgesia
During surgery, general anaesthesia was
induced via intravenous injections of keta-mine (Ketalar Vet, Pfizer AB, Sollentuna, Sweden, 50 mg/ml, 0.35 ml/kgbw) and
mede-tomidine (Dormitor Vet, Orion Pharma,
Espoo, Finland, 1 mg/ml, 0.15 ml/kgbw).
Local anaesthesia was induced with 0.9 ml of lidocaine/epinephrine solution per site
(Xylocain Dental adrenalin 20 mg/ml+
12.5 mg/ml, Astra AB, S€odert€alje, Sweden). Postoperatively, the animals were given anal-gesics (buprenorfin, Temgesic, Schering-Plough AB, Stockholm, Sweden, 0.3 mg/ml, 0.3 ml/animal) for 3 days.
Surgical procedure
The operation was performed under aseptic conditions. The tibia was exposed through a skin incision and gently accessed through the aponeurosis tissue. Four guide holes were made with a 1.0-mm-diameter twist drill (Medartis, Basel, Switzerland) using a drill guide to ensure standardized and correct
posi-tioning. Using a custom-made 7.05-mm
diameter-bur mounted in a slow–speed dental
implant drill with copious physiological sal-ine solution irrigation, a platform was made for the disc implants (Fig. 1a–b). Each rabbit received 1 disc of each surface modification. Polytetrafluoroethylene (PTFE) caps were placed on the discs to prevent on- and over growth of bone. The implants were stabilized with a preshaped 0.25 mm titanium band, retained in the cortical bone with two 1.29 3 mm titanium screws (Medartis, Swit-zerland). The soft tissues and the flap were repositioned and sutured with resorbable sutures (Vicryl 4-0, FS2, Ethicon, Somervill, NJ, USA).
Biomechanical pull-out measurements
Twelve and thirteen animals were euthanized by an overdose of pentobarbital (Pentobarbital natrium, Apoteket AB; Stockholm, Sweden, 60 mg/ml) after 4 and 8 weeks, respectively. The soft tissues were completely removed from the tibia samples to expose the implan-tation site. The titanium band covering the implants was exposed and carefully removed. To avoid any disturbance of the interface between sample and bone during removal of the PTFE cap, a hole was made in the centre of the cap with a hollow needle and pressur-ized air was applied to remove the caps and expose the reverse of the implant. The tibia was fixated in a specially designed device that effectively stabilized it during the tensile test procedure (Fig. 2a–b).
(a) (b)
Fig. 1. (a) The prepared implant site in the rabbit tibia. (b) The discs are covered with polytetrafluoroethylene caps and secured by a titanium band.
(a) (b)
The set-up was adjusted in line and perpen-dicular with the load cell using a level tube. The tensile test was performed with an In-stron 8511 testing machine (InIn-stron, High Wycombe, UK) fitted with a calibrated load cell of 250 N. Cross-head speed range was set to 1.0 mm/min. Detailed information con-cerning the surgical procedure as well as the pull-out test has already been published else-where (Ronold et al. 2003).
Statistics
The measurements for the surface character-ization were summarized as means and stan-dard deviations, and the pull-out force as means, standard deviations, medians and quartile values. The surface roughness values were compared by one-way ANOVA followed by a post hoc Turkey–Kramer test with the value of statistical significance set at the 0.05 level. The pull-out force differences among the implants were first examined mixed-model regressions adjusted for animal, position of the implants in the tibiae and tibiae side to determine the respective com-parisons of implant types stratified by end-point. The factors implant, position and side were included in the model as fixed effects and the factor animal as a random effect. The P-values for all comparisons were calculated
using the nonparametric Brunner–Langer
method (Brunner & Langer 1999). A global
comparison (Brunner–Langer, F1_LD_F1
model) was first examined; then, the calcu-lated effects resulting from the above regres-sions were adjusted for multiple comparisons using Dunnett–Hsu’s correction (Brunner 2010). The level of significance was set at P< 0.05. SAS release 9.3 (SAS Institute, Cary, NC, USA) was used to perform the statistical analysis.
Results
Surface analyses Contact angle
The samples stored dry (SLA and SLAnano) were hydrophobic with comparable contact angles of about 120°–130°, whereas the sam-ples stored in liquid (SLActive and pmodSLA) were super-hydrophilic and showed complete wetting (contact angle 0°) (Table 1).
Scanning electron microscopy
No nanostructures were present on the SLA surface, whereas the three other surface mod-ifications clearly showed the presence of nanostructures. SLActive and SLAnano had
comparable nanostructures; however, lower nanoparticle density was observed on pmod-SLA. (Fig. 3a–d). Furthermore, small pits in the micrometre level were observed on all surface modifications (Fig. 4). Table 2 sum-marizes the results of the contact angle and SEM measurements.
X-ray photoelectron spectroscopy
The chemical composition of the different surfaces as determined by XPS is shown in Table 3. The XPS spectra correspond to those commonly obtained for titanium surfaces and show signals of Ti, O and C as well as low amounts of N, Al and F. The hydropho-bic samples SLA and SLAnano showed higher carbon contamination levels (around 35% C)
compared with the hydrophilic samples
(below 15% C). The production process and storage conditions protect the pmodSLA and
SLActive samples (nitrogen cover gas, storage in saline solution) from carbon-containing species and only low amounts adsorb on the Table 1. Contact angle measurements of the
four different surface modifications
Disc 1 Disc 2 Disc 3 Mean SD CA [0] CA [0] CA [0] CA [0] CA [0] SLA 127.2 124.0 126.5 125.9 1.7 SLAnano 132.1 129.3 125.8 129.1 3.2 pmodSLA 0 0 0 0 SLActive 0 0 0 0 SD, standard deviation (a) (b) (c) (d)
Fig. 3. Scanning electron microscopy (SEM) images of (a) SLA (b) SLAnano (c) SLActive and (d) pmod SLA. No nanostructures were present only on the SLA surface, whereas all other surface modifications clearly showed the presence of characteristic nanostructures.
Table 2. Summary of the contact angle and SEM results Hydrophobic Hydrophilic No nanostructure SLA Nanostructure, low density pmod SLA Nanostructure, high density SLAnano SLActive
Fig. 4. A representative SEM image of small pits that were observed on all types of surface modifications. These pits are occasionally observed on the disc mate-rial after acid-etching and are probably related to impu-rities located on grain boundaries. The three arrows are demonstrating three pits.
surface. On the contrary, the samples stored dry (SLA and SLAnano) are exposed to atmo-spheric conditions during the production
pro-cess and during storage, and thus,
hydrocarbons as well as other carbon-contain-ing species will adsorb on the surface leadcarbon-contain-ing to an increased amount of C in the XPS spec-trum. The low amounts of Al stem from Al2O3 residuals due to the sandblasting
pro-cess. The traces of F are most probably due to existing Teflon parts used in the reactor/ containers for sample preparation (acid-etching, rinsing) or due to the sample handling prior to the XPS analysis.
A detailed analysis of the Ti2p core level revealed that different oxidation states of the titanium were present (metallic [0], TiO [2+], Ti2O3 [3+], TiO2 [4+]). Table 4 presents the
average values of the relative amounts of these different Ti species as well as the TiO2
layer thickness. The thickness of the TiO2
layer showed the following trend:
SLActive> SLAnano > pmod SLA >> SLA. Interferometer
All surfaces were moderately rough accord-ing to the definition suggested by Wenner-berg & Albrektsson (2009) (WennerWenner-berg & Albrektsson 2009). The surface topography at the lm level demonstrated similar surface roughness for all four groups. However, the SLActive showed a statistically significant larger surface development, which may be explained by the density of nanoparticles. Although the lateral resolution of the instru-ment does not allow to resolve individual nanoparticles, still the vertical resolution is good enough to implement the height value in this parameter, thus the Sdr value may be increased by nano features. All surfaces were negatively skewed, that is, they all had more valleys than summits (Table 5). A descriptive 3D reconstructed image is presented on Fig. 5.
Atomic force microscopy
The surface topography at the nanometre level showed no statistically significant dif-ferences. However, a tendency was observed
for the SLActive surface towards larger height
deviation (Sa) while the SLAnano was
smoothest in this respect. The standard devi-ation was very large for all parameters. Simi-lar to the lm level resolution, all four surfaces included more pits than bumps as shown by the skewness parameter, although this finding was less pronounced at the nm level resolution (Table 6). A descriptive 3D reconstructed image is presented on Fig. 6. Biomechanical pull-out measurements
One rabbit, aimed for 4 weeks healing time, did not recover from anaesthesia, all other rabbits were healthy, and the healing of the implant sites was clinically uneventful. One disc implant was placed upside down in the 8 weeks healing time group; hence, in total, the maximum pull-out force value of 99 disc implants was measured. Table 7 shows the descriptive statistics for the pull-out mea-surements for the different implant types at the two different endpoints.
The pull-out force increased significantly for all four surface modifications from week 4 to week 8.
Fig. 7 shows the adjusted means of the pull-out measurements and the respective 95% confidence intervals. After 4 weeks, SLActive implants showed the highest values (85.3 [N], 95% CI: 73.3–97.3) with signifi-cantly higher pull-out force than SLA (40.4 [N], 95% CI 28.5–52.4; P = <0.0001) and
SLAnano (61.2 [N], 95% CI: 49.3–73.2;
P= 0.0100). However, the difference to
pmodSLA was not statistically significant (67.8, 95% CI: 55.9–79.8; P = 0.0976). In
addi-tion, pmodSLA (P= 0.0034) and SLAnano
(P= 0.0395) demonstrated significantly
higher pull-out values than the SLA group.
Similarly, after 8 weeks, the SLActive sam-ples (131.4 [N], 95% CI: 114.7–125.3) demon-strated the highest pull-out force and a statistically significant difference was observed compared with SLAnano (96.4 [N], 95% CI: 80.4–112.3; P = 0.0036) and SLA (93.7 [N], 95% CI: 67.8–99.6; P < 0.0001). Again SLActive vs. pmodSLA (121.4 [N], 95% CI: 105.6–137.4; P = 0.7479) demonstrated no significant difference, whereas pmodSLA pre-sented significantly higher pull-out forces than SLA (P< 0.0001).
The P -values for the global comparisons for the factors implant type, time and inter-action implant type by time were, respec-tively,<0.0001, <0.0001 and 0.7032.
After the pull-out experiment, SEM/EDX on the explanted discs showed the presence of calcium- and phosphate-containing parti-cles/structures on the discs. The SEM images demonstrated mineralized collagen fibres resembling those of mature bone (Fig. 8).
Discussion
In the present study, we investigated the bone response to two hydrophilic (SLActive and pmodSLA) and two hydrophobic (SLA and SLAnano) surfaces. The chemical composi-tion of the surface seems to be crucial for the degree of wettability. Both hydrophilic sur-faces had low concentrations of carbon, while the two hydrophobic surfaces displayed con-siderably higher amounts. Clean TiO2
sur-faces have high surface energies and are thus intrinsically hydrophilic. Adsorption of hydro-carbons lowers the surface energy and leads to an increase in the water contact angle. Thus, an increase in C-contamination on the surface Table 3. Apparent normalized (sum equals
100%) atomic concentration [%] of the
elements detected by XPS. The average values of three samples per group are presented. One sample of the group SLAnano contained about 1% of S (not shown here)
O% Ti% N% C% Al% F%
SLA 45.8 16.8 1.2 35.3 0.9 0.0
SLAnano 44.2 15.2 1.3 38.3 0.9 0.1 pmodSLA 59.5 24.4 1.0 13.5 1.3 0.4 SLActive 60.7 24.4 1.6 11.3 1.3 0.7
Table 4. Detailed analysis of the Ti2p3/2 core level. The different oxidation states are quantified as well as the TiO2layer thickness (d). The average values of three samples per group are
pre-sented. The standard deviation (SD) presented for d does not include the systematic error of the measurement, but represents a purely statistical error
Ti Met TiO Ti2O3 TiO2 D SD d
% % % % nm nm
SLA 6.5 3.3 6.5 83.6 5.7 0.3
SLAnano 2.7 2.3 4.1 90.9 7.7 0.0
pmodSLA 3.6 2.4 3.4 90.7 7.2 0.2
SLActive 2.1 2.0 3.2 92.7 8.3 0.3
Table 5. Topographical evaluation performed with an Interferometer, standard deviation in parentheses Sa lm Ssk Sds 103/mm2 Sdr % SLA 1.5 (0.1) 0.8 (0.2) 177 (7) 72 (5) SLAnano 1.5 (0.2) 0.9 (0.4) 183 (4) 74 (8) pmodSLA 1.4 (0.1) 0.8 (0.4) 201 (5) 77 (6) SLActive 1.6 (0.2) 1.0 (0.2) 203 (7) 93 (9)
leads to a reduction in the surface energy and finally to hydrophobic surfaces.
Among these four surface modifications, three had nanoparticles formed on the outer-most surface layer, while one (SLA) did not.
In a recent publication, it was demon-strated that nanostructures will develop on titanium surfaces if they are etched and stored in aqueous solution. Furthermore, it takes about 14 days for this process to be completed (Wennerberg et al. 2013). The degree of nanostructure formation was unexpected in case of pmodSLA. In the previous investiga-tion, pmodSLA only displayed rare
occur-rence of nanoparticles, while in the present evaluation, nanostructures were present at a higher density. Most probably, the increased formation of the nanostructure is related to the purity of the titanium: whereas the current discs were made of grade 4 material, the discs used in the previous publication were made from the purer grade 2 material. This indicates a high sensitivity of the nano-structure formation on, for example, the level of impurities. The slightly higher level of impurities in Ti grade 4 is apparently just enough to facilitate the diffusion processes forming the nanostructures and allow their
formation on the pmodSLA samples already within the first two months of storage in saline solution (Wennerberg et al. 2013). However, whereas SLActive and SLAnano displayed comparable densities of nanoparti-cles, as expected, pmodSLA displayed a clearly lower nanoparticle density, as can be observed in Fig. 3a–d.
The small pits observed on all types of sur-face modifications in this study are occasion-ally observed on the disc material after acid-etching and are probably related to impurities located on grain boundaries of the Ti base material. The presence of these pits is likely the main reason for all surfaces being nega-tively skewed.
The samples with nanostructures (SLAnano, SLActive, pmodSLA) had a thicker TiO2layer
compared with SLA, due to the nanostruc-tures that consist of TiO2. However, there
are small differences in the calculated oxide layer thickness of the samples having nano-structures: the pmodSLA samples had the
(a) (b)
(c) (d)
Fig. 5. Descriptive 3D images (a) SLA (b) SLActive (c) SLAnano and (d) pmodSLA from the interferometer measurements (scan area: 2609 200 lm).
Table 6. Topographical evaluation performed with Atomic force microscopy, standard deviation in parentheses Sanm Ssk Sds103/lm2 Sdr% SLA 21.7 (12.7) 0.5 (0.4) 4.1 (1.2) 370 (501) SLAnano 20.8 (5.1) 0.3 (0.3) 3.4 (2.1) 273 (217) pmodSLA 22.3 (3.9) 0.6 (0.4) 3.9 (0.9) 389 (290) SLActive 28.1 (7.5) 0.1 (0.3) 3.6 (0.9) 382 (234)
thinnest oxide layer, as expected, due to the lower density of the nanoparticles. There was also a small difference between SLAnano and
SLActive – SLAnano had a slightly lower
thickness compared with SLActive. Although not obvious from the SEM images, this may imply that SLAnano has a lower density of nanoparticles or smaller nanoparticles com-pared with SLActive. On the other hand, the measured thickness of the oxide layer corre-lates with the time of the samples in saline solution. For all sample modifications, sand-blasting and acid-etching were performed at the same time to minimize variations in the microstructure between the different groups. Thus, although all groups had the same total storage time of about 7 months, the time samples spent in the NaCl solution was different for SLActive (7 months), SLAnano (4 months) and pmodSLA (1 month). As a consequence, the differences in oxide layer thickness of the nanostructured samples may indicate some additional growth of the oxide layer while the samples were immersed in
(a) (b)
(c) (d)
Fig. 6. Descriptive 3D images (a) SLA (b) SLActive (c) SLAnano and (d) pmodSLA from the atomic force microscopy measurements (scan area: 19 1 lm).
Table 7. Descriptive statistics for the pull-out force measurements
Parameter Pull-out force [N] 4 weeks Pull-out force [N] 8 weeks
SLA N 12 13 Mean SD 40.4 11.5 82.4 39.8 Median (Range) 38.5 (25.0–58.8) 77.2 (21.2–194.0) SLAnano N 12 13 Mean SD 61.2 18.7 97.6 27.4 Median (Range) 62.7 (31.8–95.5) 95.0 (57.8–148.7) pmodSLA N 12 13 Mean SD 67.8 22.6 122.0 25.5 Median (Range) 67.5 (32.3–94.6) 120.9 (80.1–168.8) SLActive N 12 12 Mean SD 85.3 24.9 130.1 26.6 Median (Range) 83.9 (51.0–144.0) 136.3 (87.0–166.0) 4 Weeks 8 weeks PpmodSLAvs.SLActive 0.0976 0.7479 PSLAnanovs.SLActive 0.0100 0.0036 PSLAvs.SLActive <0.0001 <0.0001 PpmodSLAvs.SLA 0.0034 <0.0001 PSLAnanovs.SLA 0.0395 0.0964 PpmodSLAvs.SLAnano 0.6385 0.0244
The givenP-values for the different comparisons between implant types were calculated using the Brunner–Langer model (F1_LD_F1) (Brunner & Langer 1999).
the saline solution. However, although the limited accuracy of the method applied for the calculation of the oxide layer thickness allows for comparison between the groups, it should be borne in mind that the results indi-cate a trend rather than precise values.
The surface topography in the micrometre range showed much higher values than at the nanometre level. This is related to the larger measurement area and especially to the larger cut-off wavelength of the Gaussian filter. The cut-off wavelength determines the wave-lengths of the features that are included in the calculation of the roughness parameters. A larger cut-off wavelength includes larger features and therefore in general leads to increased roughness values.
At the nanometre level, no significant dif-ferences between the various surface modifi-cations were observed. The AFM method used to image the nanometre-sized features allows only the evaluation of relatively small areas, which resulted in large deviations between individual measurements.
The different surfaces showed clearly
different biological responses. All surfaces demonstrated a strong (significant) increase in pull-out forces from week 4 to week 8, with SLActive exhibiting the highest pull-out forces and thus the strongest bone ingrowth at both healing times.
The surface roughness, presence of nano-structures and the surface chemistry all influence the pull-out force. The surface chemistry, characterized by wettability and carbon content, had a pronounced influence on the bone healing. The two
super-hydro-philic surfaces, SLActive and pmodSLA,
showed low carbon content and higher pull-out values at 4 and 8 weeks compared with the two hydrophobic surfaces SLAnano and SLA. This difference was significant for SLActive vs. both SLAnano and SLA at 4 and 8 weeks as well as for pmodSLA vs. SLA at 4 and 8 weeks. Only in the case of pmod-SLA vs. pmod-SLAnano was the difference not significant.
However, the nanostructures also had a beneficial influence on the osseointegration. Of the hydrophobic surfaces with (SLAnano) and without nanostructures (SLA), the for-mer demonstrated higher pull-out values at both healing times, although only the differ-ence at 4 weeks was statistically significant. In addition, a trend to higher pull-out forces at both 4 and 8 weeks was observed for the surfaces with denser (SLActive) compared to that with a less dense nanostructure (pmod-SLA). This result suggests that the appearance (density, morphology) of the nanostructure is Fig. 7. Pull-out force mean values for the different implants by endpoint. Means were adjusted by position side and
animal effects. Whiskers represent the 95% confidence intervals.
Fig. 8. Discs explanted after 4 weeks in rabbit bone, SLA on the left side and SLActive on the right. Optical micro-graphs and SEM images of the same discs at two different magnifications are presented. The pull-out force of the SLA and SLActive discs were 27.5 N and 95.9 N, respectively. More bone was attached on the SLActive surface.
of importance for the bone response. However, to date, the optimal density and morphology of nanostructures are still not known.
In general, higher amounts of residual tis-sue were observed on the surfaces showing higher pull-out values, indicating that the fracture during pull-out test partly occurred within the bone and not always at the inter-face implant/bone tissue.
In summary, the results of the present study revealed that both hydrophilicity and the presence of nanostructures contribute to a favourable biological response. Therefore, the superior osseointegration of the SLActive surface is most probably due to its hydrophi-licity in combination with the presence of nanostructures.
Conclusion
•
All four surfaces had similar microtopog-raphy.•
Osseointegration was influenced by wet-tability, and super-hydrophilic surfacesdemonstrated stronger bone response
compared with hydrophobic surfaces.
•
Osseointegration was positively influ-enced by the presence of nanostructures.•
The strongest bone response was achieved with a combination of wettability and the presence of nanostructures.•
From the present study, it was not possi-ble to define the optimal density of nano-structures.•
Pull-out test caused fracture partly within the bone for all surfaces.Acknowledgements:
This study was partially funded both by Institut Straumann AG and the Swedish knowledge foundation. We would like to thank Jian-Sheng Wang (Lund University, Lund, Sweden) for all the help related to the pull-out tests and Leticia Grize (Swiss Tropical and Public Health Institute, Basel, Switzerland) for the help with the statistical analysis of the pull-out data.References
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