The objective of this study was to investigate the effect of a laminin coating on calcium phosphate precipitation on three potentially bioactive titanium surfaces in simulated body fluid.
Blasted titanium discs were prepared by alkali and heat treatment (AH), anodic oxidation (AO) or hydroxyapatite coating (HA) and subsequently coated with laminin. A laminin coated blasted surface (B) served as a positive control while a blasted non coated (B-) served as a negative control. Surface morphology was examined by Scanning Electron Microscopy (SEM). The analysis of the precipitated calcium and phosphorous was performed by Energy Dispersive X-ray Spectroscopy (EDX).
The thickness of the laminin coating was estimated at 26 Å by ellipsometry. Interferometry revealed that the coating process did not affect any of the tested topographical parameters on µm level when comparing B to B-. After 2 weeks of incubation in SBF, the alkali-heat treated discs displayed the highest calcium phosphate deposition and the B group showed higher levels of calcium phosphate than the B- group.
Our results suggest that laminin may have the potential to be used as a
coating agent in order to enhance the osseoinductive performance of
biomaterial surfaces, with the protein molecules possibly functioning as
nucleation centres for apatite formation. Nevertheless,
Bone anchored titanium implants are widely used in the rehabilitation of edentulism.
In order to enhance bone growth around implants, various chemical modifications of
titanium surfaces have been proposed. Some techniques are alkali-heat treatment (AH)
[
The simulated body fluid (SBF) model has been extensively utilized for
Laminin is an organic biomolecule previously utilized with success for improving the
attachment of mesenchymal stem cells on TiO2 nanotubes [
The aim of the present study was to investigate the effects of laminin coating on titanium surfaces modified by three methods claimed to provide bioactivity in terms of Ca/P precipitation, surface morphology and surface chemistry in simulated body fluid.
Ninety discs (diameter = 8 mm, thickness = 1 mm) of titanium grade 4 were included in the study. The samples were blasted with Al2O3 powder with an average particle size of 120 µm with a force of 3.5 kg and from a distance of 15 mm and subsequently ultrasonically cleaned in diluted Extran MA01 and absolute ethanol and dried at 60 °C for 24 h. The specimens were then divided into five equally sized groups (n = 18). One group of blasted discs was coated with laminin and served as a positive control (B), while a non laminin-coated group of blasted specimens served as a negative control (B-). The other three groups were treated as follow and were ultimately coated with laminin.
Alkali and heat treatment was performed as described in the literature [
The samples were prepared in a mixed electrolyte containing calcium ions by the Micro
Arc Oxidation (MAO) method in a galvanostatic mode as described in the literature
[
A thin hydroxyapatite layer (< 50 nm) was obtained by dipping the titanium discs
(Ti-discs) into a solution containing surfactants, water, organic solvent and
crystalline hydroxyapatite particles with a Ca/P ratio of 1.67. The diameter of the
hydroxyapatite particles was approximately 10 nm. After the dipping procedure, the
discs were let to dry in open air for 30 min, allowing the organic solvent to
evaporate. To remove all dispersing agents, the discs were subjected to heat
treatment at 550 °C for 5 min [
Laminin (Sigma-Aldrich, L2020, Stockholm, Sweden) was diluted to a concentration of 100 µg/ml in PBS containing 0.15M NaCl, at pH 7.4 at room temperature. The titanium discs belonging to groups B, AH, AO and HA were subsequently incubated for 1 h at room temperature in 48 well plates (Nunclon Surface, Nunc, Roskilde, Denmark) containing 250 µl per well of the laminin solution. The discs were then rinsed with Milli-Q water and blown dry in order to avoid deposition of salts and remove non-adsorbed proteins.
For quantification of adsorbed laminin layer optically smooth titanium surfaces were
prepared as described by Linderbäck et al. [
The amount adsorbed laminin was calculated on the optically smooth titanium surfaces.
The surface treated discs were not possible to analyze, since these surfaces did not
reflect the laser beam in a measurable manner. Optically smooth titanium surfaces
were fixed in the ellipsometric quvette filled with PBS at room temperature. The
ellipsometry angles Δ0 and Ψ0 were measured with a Rudolph Research AutoEL
III ellipsometer operating in a wavelength of 632.8 nm at a 70° angle of incidence.
Thereafter, the quvette was emptied and filled with laminin solution and new angles
Δ and Ψ calculated. The protein layer thickness was iterated from the
ellipsometer angle changes under the assumption that the protein refractive index
was n = 1.465. The McCrackin algorithm was used for the calculations [
The revised SBF (r-SBF) used in this study was prepared according to the literature
[
The discs were immersed in 25 ml r-SBF in separate sealed polystyrene vials at 37 °C. After immersion for 1 h, 1 day, 3 days, 1 week and 2 weeks, the r-SBF immersion was interrupted and the specimens rinsed with distilled water in order to remove any loosely attached calcium phosphate. Thereafter, the specimens were left to dry at room temperature and ultimately sealed in dry vials. Three samples of each type of surface were not immersed in r-SBF (0 hours).
The specimens were topographically characterized after immersion in r-SBF with an interferometer MicroXam (Phase-Shift, Tucson, Arizona, USA) operating in a wave length of λ = 550 nm.
A Gaussian filter with size 50 × 50 µm2 was applied to separate roughness
from form and waviness. Thereafter, the surface roughness was calculated by using
the following topographical parameters defined as essential for describing the
topography of biomaterial surfaces [
Sa = Arithmetic mean height deviation from a mean plane (µm).
Sds = Density of summits, i.e. the number of summits of a unit sampling area (µm-2).
Sdr = Developed interfacial area ratio, i.e. the ratio of the increment of the interfacial area of a surface over the sampling area (%).
Calculations of group means and standard errors for each surface preparation and time point were performed.
For the SEM analysis, a LEO Ultra 55 FEG SEM equipped with an Oxford Inca EDX system, operating at 8 and 10 kV was used. The samples were examined without surface sputtering. Micrographs were recorded at different magnifications to investigate both the surface coverage and the morphology of the crystals. EDX analysis at a magnification of 150 times was performed to describe the atomic composition. Two samples from each surface composition. Three titanium discs for each preparation and incubation time were analyzed and a mean value calculated.
The normal distribution of the variables was confirmed by Kolmogorov - Smirnov test. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows, version 18 (SPSS®, Chicago, Illinois, USA) using one-way ANOVA (Analysis of Variance). The multiple paired comparisons were performed by Bonferroni Post-Hoc test. The statistical significance level was defined at 0.05.
As demonstrated by Table 1, the laminin coating elicited no difference on the examined topographic parameters Sa, Sds and Sdr when comparing group B and group B- (P > 0.05). Treatment of the titanium discs with alkali and heat resulted in the lowest Sa and the highest density of summits (P < 0.05) among the tested surface modifications. As a total, the AH – group possessed the highest developed interfacial area ratio resulting in larger total surface
Sa, Sds and Sdr for the five different surface groups. Mean values and standard errors are presented
|
|
|
|
|
1.36 ± 0.05 | 153873.2 ± 2585.8 | 60.99 ± 2.33 |
|
1.30 ± 0.07 | 157884.7 ± 6021.2 | 60.13 ± 5.85 |
|
1.24 ± 0.09a | 234634.8 ± 8454.7a | 84.61 ± 2.32a |
|
1.38 ± 0.05 | 171155.8 ± 5768.1 | 73.84 ± 3.14 |
|
1.33 ± 0.09 | 168952.5 ± 3574.2 | 66.00 ± 6.07 |
aSignificant at the level P < 0.05 (Bonferroni Post-Hoc test).
B- = uncoated blasted titanium; B = blasted and laminin coated;
AH = alkali heat treated and laminin coated; AO = anodic oxidized and laminin coated; HA = hydroxyapatite and laminin coated.
The thickness of the adsorbed protein was estimated to 26 Å, approximating 180
ng/cm2 [
SEM-images were acquired with a × 5000 magnification prior to and after 2 weeks of
incubation in r-SBF. The images prior to incubation demonstrated no differences in
surface morphology when comparing the uncoated (
SEM images of titanium disks prior to incubation in SBF (× 5000): (a) B- = uncoated blasted; (b) B = blasted and laminin coated; (c) AH = alkali heat treated and laminin coated; (d) AO = anodic oxidized and laminin coated; (e) HA = hydroxyapatite and laminin coated.
The bar presents 10µm.
After 2 weeks of incubation in SBF, the surfaces B, AH and AO were fully covered with
a homogenous calcium phosphate layer (
SEM image of a titanium disks after incubation in SBF for 2 weeks (× 5000): (a) B- = uncoated blasted; (b) B = blasted and laminin coated; (c) AH = alkali heat treated and laminin coated; (d) AO = anodic oxidized and laminin coated; (e) HA = hydroxyapatite and laminin coated. The bar presents 10 µm.
The total amount of calcium phosphate on surfaces of titanium discs was assessed
with EDX by measuring and adding the relative elemental amount of calcium (Ca) and
phosphorous (P) present on the surface. AH surfaces showed the highest Ca and P
content after 72 h, 1 week and 2 weeks. After 2 weeks no significant differences
were detected among the test surfaces AO, HA and the positive control B. At the same
time, the negative control B- demonstrated the lowest Ca and P precipitation (
Total amount of precipitated calcium and phosphate on titanium disks calculated by EDX: B- = uncoated blasted; B = blasted and laminin coated; AH = alkali heat treated and laminin coated; AO = anodic oxidized and laminin coated; HA = hydroxyapatite and laminin coated. Mean values and standard errors are presented.
The proposed bioactive surfaces, i.e. AH, AO and HA treated samples, demonstrated a
higher Ca/P ratio than both blasted control samples (B and B-) during the first 24
hours. Noteworthy, calcium and phosphate signals were detected at an earlier time
point on bioactive surfaces compared to blasted surfaces, depending on the bioactive
modification process. The high early Ca content on the AH surface contributed to a
high Ca/P ratio. After 2 weeks of SBF immersion, all the surface groups presented a
Ca/P ratio around 1.67, corresponding to hydroxyapatite crystalline formation (
Calcium/phosphorous ratio on titanium disks calculated by EDX: B- = uncoated blasted titanium; B = blasted and laminin coated; AH = alkali heat treated and laminin coated; AO = anodic oxidized and laminin coated; HA = hydroxyapatite and laminin coated. Mean values and standard errors are presented.
Protein coatings have previously been used as a method to stimulate bone formation
around implants in different experimental models with promising results [
The results demonstrate that all laminin coated surfaces induced a higher final CaP
deposition as compared to uncoated blasted titanium after 2 weeks. The fact that all
the "bioactively" modified surfaces, i.e. AH, AO, HA, demonstrate a higher
CaP formation is in agreement with the findings of Arvidsson et al. [
The SEM image demonstrating a laminin coated blasted disc after 2 weeks in SBF (
The results of a previous study from our group [
The results of the present study demonstrate that after 2 weeks of incubation in SBF
all the laminin coated titanium surfaces, including the laminin coated blasted
controls, induced a higher CaP deposition as compared to uncoated blasted titanium
discs. Among the tested surface modifications, alkali and heat treatment seemed to
induce a more rapid CaP precipitation. Our study demonstrates that laminin may have
the potential to be used as a coating agent in order to enhance the osseoinductive
performance of biomaterial surfaces with the protein molecules possibly functioning
as nucleation centres for apatite formation.
The authors thank Agneta Askendal from the department of Applied Physics in Linköping University, Sweden for her assistance on the protein coating process. This study was supported by the Swedish National Graduate School in Odontological Science. The authors also acknowledge the Swedish Research Council (K2009-52X-06533-27-3), Hjalmar Svenson Research Foundation, Sylvan Foundation, Wilhelm and Martina Lundgren Science Foundation, the Royal Society of Arts and Sciences in Göteborg and the Council for Research and Development in Södra Älvsborg, Sweden for funding the project.