Download ORS 2017 Annual Meeting Poster No.0397

Novel Bio-functional Materials for Orthopedic Implants: Surface Chemistry, Corrosion and Biocompatibility Aspects
Trino, L.D.1; Bronze-Uhle, E.S.1; Ramachandran A.2; Lisboa-Filho, P.N.1; Mathew, T. M.3, George, A.2
State University of São Paulo, Bauru, SP, Brazil 2University of Illinois at Chicago, Chicago, IL, USA 3Rush University Medical Center, Chicago, IL
Email: [email protected]
DISCLOSURES: Trino, L.D. (N), Bronze-Uhle, E.S. (N), Ramachandran A. (N), Lisboa-Filho, P.N. (N), Mathew, T. M. (N), George, A. (N).
INTRODUCTION: Bones are part of a special group of mineralized organic materials in the human body, known as hard tissue. These tissues are prone to
injury due to accident or illnesses. When the tissue cannot be recovered an internal, external fixation or a joint replacement is a part of their treatment.
Metallic biomaterials, as titanium, possess the combination of high strength, flexibility, ductility, corrosion resistance, and biocompatibility required for
load-bearing applications. However, despite the good bulk characteristics, there are still an undesirable number of implants failures and subsequent clinical
side effects on the patients. In order to overcome such concerns, improvements could be achieved by designing biomaterials where the bulk and the surface
are independently tailored with regenerative capabilities. The conjugation of organic molecules onto metal oxide surface can improve the biocompatibility
and bioactivity of the metallic implants and accelerate the osseointegration process [1]. These functionalized metal oxide surfaces have functional groups
that can interact with amino or carboxyl groups from peptides which may act as a translator between the surface properties of the material and the cell
receptors, highly mediating the behavior of the implanted device. Therefore, the goal of this study is to immobilize organic and biomolecules onto titanium
surface in order to evaluate their attachment by X-ray Photoelectron Spectroscopy (XPS) and analyze cell attachment, proliferation and differentiation upon
these functional surfaces. We hypothesize that the functionalization of TiO2 with organic molecules and DMP1 peptides will enhance cell adhesion,
mechanical properties and implant success.
METHODS: Titanium cp discs were polished until Ra≈150 nm then cleaned with deionized water. The samples were etched and hydroxylated in Piranha
solution. TiO2 deposition was performed by spin coating technique and then annealed at 830°C. The samples were divided in five groups. The control group
consists of Ti substrate coated with TiO2, and the other four TiO2 surface were functionalized with 3-mercaptopropionic acid (MPA), 3-4 aminophenyl
propionic acid (APPA), 3- aminopropyltrimetoxysilane (APTMS) and polyethylene glycol (PEG). Peptide pA (ESQES) and peptide pB (QESQSEQDS)
were diluted in the ratio 1:4, respectively, in order to have a concentration of 1mg/mL. White Light Interferometry (WLI) was used to determine average
surface roughness. To determine the chemical states of the elements XPS analysis was carried out. Human mesenchymal stem cells were cultured upon the
functional surfaces on which proliferation and differentiation assays were performed. Corrosion and tribocorrosion tests were developed in order to analyze
the mechanical properties.
RESULTS: The average surface roughness of the samples increased based on the substrate surface. The titanium surface modified only with the TiO 2
presented low roughness than the titanium functionalized with organic molecules and the peptides. The XPS analysis revealed that the organic molecules
were successfully attached to the surface (Figure 1). For both molecules with amino groups (APPA and APTMS) the N 1s peak appeared around 400 eV and
a peak in 103 eV for APTMS corresponding to Si 2p is shown. For MPA a peak referent to S 2p is shown in 165 eV. The increased intensity of C 1s along
with the decrease of Ti 2p peak evidences that PEG polymerized. Proliferation assay showed that the sample functionalized with MPA and the peptides
presented a higher cell viability. Quantitative analysis of the mRNA levels by the real-time RT-PCR method revealed that the marker genes for osteoblast
were upregulated in the presence of the peptides (Figure 2). Corrosion and tribocorrosion testes indicated that the surface functionalization enhances the
corrosion resistance (Figure 3).
DISCUSSION: Surface measurements using WLI demonstrated that the functionalization homogeneously recovered the titanium dioxide surface, increasing
the surface roughness from 300 nm with TiO2 to around 700 nm. The surface roughness plays an important role during cell attachment, proliferation and
differentiation. Surface roughness between 0.5 and 1.5 µm, as the presented ones, has shown improved cell adhesion [2]. XPS results showed an increase in
the intensity of C 1s for all samples when compared with the sample coated with TiO 2, indicating the success in the functionalization process. Different
contributions of oxygen can be observed in the high resolution spectra of O 1s from TiO 2 specimen, which can be divided into three peaks: the first two
located at lower energy are typical for the metal oxide bonds in TiO2, the other one located in 531.4 eV is assigned to the oxygen of Ti-OH bonds [3]. This
XPS results prove that the surface contains hydroxyl groups that act as anchoring points for the formation of densely packed monolayers. The results
confirmed that APTMS has a preferential attachment upon TiO2 surface trough silane groups due to the formation of covalent bond between silanes and the
underlying substrate that stabilizes the monolayer [4]. APPA grafting favored a covalent bond with TiO2 surface trough carboxylic group, presenting free
amine groups and the absence of N-Ti bond. The mercapto group from MPA binds covalently with titania showing SO4-2 species that coordinate bidentate
with Ti4+ sites. PEG polymerized due to the several hydroxyl groups available for derivatization that cross linked with each other. PCR results showed that
TiO2MPAP and TiO2P were the samples with enhanced gene expressions. It indicates that the surface functionalization had a positive influence in hMSC
osteogenic differentiation. The corrosion resistance was increased with the addition of TiO 2 layer. The functionalized samples presented a lower corrosion
resistance when compared with bare TiO2, however it is still higher when compared with titanium. The findings suggest that the biofunctionalization of the
Ti based implants with organic molecules and peptides can minimize the current clinical concerns in the orthopedic patients and possibly open door for the
application of regenerative medicine for the better future implants.
SIGNIFICANCE: The functionalization of orthopedic implants can improve the biocompatibility of the metal and prevent adverse tissue reactions such as
infection, inflammation, the foreign body response and other events. In this way is important to better understand the type of interaction of the anchoring
groups with the oxide layer in the metallic surface and the properties of this new functional materials in order to properly design functionalized oxide
coatings for a large variety of implant devices.
REFERENCES: [1] P. Silva-Bermudez et al. Surf. Coat. Technol. 233, 147–158 (2013). [2] K. Balani et al. Biosurface. John Wiley & Sons Inc.: New
Jersey, 2015. [3] J. F. Moulder et al. Perkin-Elmer Corporation: Minnesota, 1992. [4] S.P. Pujari, et al. Angew. Chem. Int. Ed. 53, 6322–6356 (2014).
1
0.20
70
Ti
TiP
TiO2
Col1a1
0.15
DMP1
60
OCN
25000
O 1s TiO2
Ti 2p
N 1s C 1s
40
20000
APPA
APTMS
TiO2 P
ALP
BSP
Intensity (Counts/s)
OPN
30
15000
20
TiO2 APPA P
0.05
TiO2 MPA P
0.00
-0.05
-0.10
10000
MPA
O-H
10
-0.15
0
-0.20
Ti-O
5000
528
526
Binding Energy (eV)
.
Figure 1: XPS survey spectra for the samples analyzed and high
resolution spectra for the element O 1s in TiO2.
e
+P
at
pl
PA
PA
+P
P
2
O
2+
O
TC
530
M
532
2+
534
O
0
AP
200
Ti
Binding Energy (eV)
400
2+
600
O
800
Ti
1000
Ti
TI
1200
Ti
+P
PEG
Ti
Intensity (a.u.)
0.10
Runx2
50
TiO2
Vf (V vs Ref)
O 1s
TiO2
Figure 2: Gene expression in
osteogenic
media
for
the
functionalized samples.
ORS 2017 Annual Meeting Poster No.0397
0
500
1000
1500
2000
2500
3000
3500
Time (s)
Figure 3: Open Circuit Potential (OCP)
results for the samples during corrosion
test.