Journal of Biomaterials and Nanobiotechnology, 2011, 2, 244-249
doi:10.4236/jbnb.2011.23031 Published Online July 2011 (http://www.SciRP.org/journal/jbnb)
Unbinding Process of Amelogenin and Fibrinogen
Adsorbed on Different Solid Surfaces Using AFM
——Unbinding of Adsorbed Amelogenin and Fibrinogen Using AFM
Ludovic Richert1,2,3, Abdessamad Boukari2, Simon Berner4, Michel Dard4, Joseph Hemmerlé1*
1Inserm National de la Santé et de la Recherche Médicale, Strasbourg, France; 2Dental School, University of Strasbourg, 1 place de
l’hôpital, Strasbourg, France; 3Centre National de la Recherche Scientifique, UMR 7213, Institute of Pharmacy, University of Strasbourg,
Illkirch, France; 4Institut Straumann AG, Peter Merian-Weg 12, Basel, Switzerland.
Received March 4th, 2011; revised April 10th 2011; accepted May 4th, 2011.
ABSTRACT
The interaction of proteins with solid surfaces is a fundamental phenomenon in the biomaterials field. We investigated,
using atomic force microscopy (AFM), the interactions of a recombinant amelogenin with titanium, a biphasic calcium
phosphate (BCP) and mica. The unbinding processes were compared to those of an earlier studied protein, namely fi-
brinogen. Force spectroscopy (AFM) experiments were carried out at 0 ms, 102 ms, 103 ms and 104 ms of contact time.
In general, the rupture forces increased as a function of interaction time. The unbinding forces of amelogenin interact-
ing with the BCP surface were always stronger than those of the amelogenin-titanium system. The unbinding forces of
fibrinogen interacting with the BCP surface were always much stronger than those of the fibrinogen-titanium system.
For the most part, this study provides direct evidence that recombinant amelogenin binds more strongly than fibrinogen
on the studied substrates.
Keywords: Amelogenin, Titanium, Calcium Phosphate, Force-Mode AFM
1. Introduction
The interaction of proteins with solid surfaces is the first
step in the integration of an implanted device [1]. Initial
interactions between macromolecules and a given sub-
strate influence cell response at the cell-biomaterial in-
terface [2].
Amelogenins are the major extracellular matrix protein
in developing dental enamel. They constitute 90% of the
proteins present during enamel formation [3]. Amelogen-
ins are a family of hydrophobic proteins derivable from a
single gene by alternative splicing and controlled post se-
cretory processing. The C-terminal region of the protein
is composed of a sequence of hydrophilic and charged
amino acids [4]. Amelogenin protein interacts, at the ex-
tra-cellular level, with calcium and phosphate ions to con-
trol the nucleation, growth and organization of the apatite
crystals of tooth enamel [1].
Protein adsorption is highly dependent on the individ-
ual nature of the protein and the surface involved [1,5].
Titanium and its alloys are widely used in orthopedic and
oral implants. They have polycrystalline structures with
different crystallographic orientations. Physical proper-
ties of polycrystalline materials strongly depend on the
distribution of the crystallographic orientations of the
surface grains [2]. Hydroxyapatite is the predominant in-
organic component of human bones and teeth [6]. It has
received much attention in materials science and medical
fields because of its special surface interaction properties
and biocompatibility [7,8]. Currently it is widely used in
many medical practices such as bone implants [9]. It was
found that when hydroxyapatite is exposed in the organ-
ism matrix in vivo, its surface is rapidly covered by a
proteinous layer [10]. The atomic order of a mineral sub-
strate such as hydroxyapatite can serve as an ordered
template to interact with the functional groups of the
proteins [2]. For instance, it has been shown that osteo-
calcin can only interact with calcium ions in specific
crystallographic planes in the hydroxyapatite lattice
[11].
Techniques that operate at the molecular scale provide
investigators with unique approaches to characterize
biomolecular mechanisms. Recent advances in the field of
scanning probe techniques, particularly in atomic force

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM 245
microscopy, have yielded insights into the protein self-
assembly and the mechanisms of protein unfolding. Mo-
nitoring the tip-sample interactions with an atomic force
microscope is a powerful tool for in situ measurements
and real-time assessments of biomolecular phenomena
[12].
Despite extensive studies, basic questions on the be-
havior of a protein in its adsorbed state are still difficult
to answer [1]. We used atomic force spectroscopy (AFM)
to produce new data about the molecular mechanisms
governing the interactions between amelogenin and dif-
ferent substrates. The aim of this investigation was to
analyze the adsorption process of a pure recombinant
amelogenin and how the physico-chemical surface prop-
erties of common implant surfaces (titanium and biphasic
calcium phosphate) can affect this process. As fibrinogen
plays a crucial role in protein adsorption on artificial
surfaces [13] and its adhesion mechanisms are available
onto different substrates [14,15], we used fibrinogen for
comparison with amelogenin. Since freshly cleaved mica
offers a very flat and monocrystalline surface, we used it
as a comparative adsorbent substrate. The ultimate goals
of these assessments were to measure, understand and
predict interfacial aspects of the protein-surface pheno-
mena on the molecular level.
2. Materials and Methods
2.1. Titanium Substrate
Commercially pure titanium discs were provided by In-
stitut Straumann AG (Institut Straumann AG, Peter
Merian-Weg 12, Basel, Switzerland). The discs were
prepared from 1 mm thick sheets of titanium grade 2.
Metal disks were polished mechanically to a mirror fin-
ish.
2.2. Biphasic Calcium Phosphate (BCP)
Biphasic calcium phosphate (BCP) was prepared by
mixing calcium hydroxide (Ca(OH)2) and phosphoric
acid (H3PO4) to ensure homogenous phase distribution.
The HA/TCP ratio (60:40) was reached by optimizing
the concentrations of the reagents and the synthesis con-
ditions (pH and temperature). Crystallographic data of
the HA/TCP material were documented by X-ray diffrac-
tion analysis (Rigaku CN 2005, Rigaku Corporation,
Tokyo, Japan). Biphasic calcium phosphate substrates
were chemically characterized by energy dispersive
X-ray analysis and disclosed a Ca/P atomic ratio of 1.48
(data not shown).
2.3. Mica
High quality muscovite mica sheets were purchased (Eu-
romedex, Souffelweyersheim, France). Freshly cleaved
mica surfaces were used.
2.4. Surface Treatment
All substrates were cleaned by sonication, at a frequency
of 40 kHz for 10 min, in ethanol and were subsequently
treated by ultraviolet-ozone irradiation (BioForce UV.
TC. EU 003, Nanosciences, Inc. Iowa, USA) for 20 min.
2.5. Atomic Force Microscopy
The experiments were performed with a NanoScope IV
(Veeco Metrology group, Santa Barbara, CA, USA) in-
strument equipped with a PicoForce device. All meas-
urements were realized in liquid environment (0.15 M
NaCl, 10 mM tris, pH 7.4) at room temperature. A ramp
size of 500 nm was used for the sample movement. Ap-
proach and retraction rates were equal to 1 µm·s–1. Con-
tact times varied between 0 and 104 ms. About 50 ap-
proaching-retracting cycles were carried out per experi-
mental condition, at three different places with the same
protein (amelogenin or fibrinogen) coated cantilever. The
average values of all forces, recorded for each experi-
ment, were used for further interpretations. All results are
expressed as mean ± standard error.
2.6. Cantilevers
MSCT cantilevers (Veeco Metrology group, Santa Bar-
bara, CA, USA) were used either with tip C (10 pN·nm–1)
or with tip D (30 pN·nm–1). The spring constants were
confirmed by a thermal fluctuations method [16]. An
incubation time of 30 min at room temperature was ap-
plied for protein adsorption at the cantilever tips. Curves
acquired with protein coated tips were compared to
non-coated tips to make sure that the tips were properly
coated. Force curves did not show noticeable differences
after coated cantilevers where left up to 60 min in buffer
solution, thus indicating the stability of the adsorbed
proteins.
2.7. Proteins
Recombinant human amelogenin was prepared as fol-
lows:
A gene encoding the X-chromosomal human 175 ami-
no acid amelogenin (NCBI accession number AAA51717,
excluding the 16 amino acid N-terminal signal peptide)
was synthesized by PCR. Nine oligonucleotides were
used to build the gene, which was codon optimized for E.
coli. The assembled amelogenin gene was amplified, by
using flanking primers and the assembly mixture as tem-
plate, and subsequently cloned into a cloning vector. The
gene was sequenced and point mutations were corrected.
The gene was finally inserted in the expression vector
pET11a (EMD4Biosciences, Novagen, Darmstadt, Ger-
many). All cultivation was carried out in shake flasks

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM 246
using the E. coli expression strain BL21. The purification
was conducted by a multistep centrifugation process and
reverse phase high performance liquid chromatography
[17]. This recombinant human amelogenin is nonglyco-
sylated.
Human fibrinogen (Sigma # F-4883) was purchased
from Sigma. The purity of the sample was checked by
AgNO3-stained SDS/PAGE. It was used without further
purification.
2.8. Protein Preparation
The protein solutions were prepared by dissolving 10 mg
of recombinant human amelogenin or human fibrinogen
in 1·mL of MilliQ water (ρ = 18.2 MΩ·cm). Working
solutions corresponded to 1 mg·mL–1 dilutions (0.15 M
NaCl, 10 mM tris, pH 7.4).
2.9. Statistical Analysis
Data analysis was performed using the Kruskal-Wallis
one way analysis of variance on ranks (ANOVA on ranks)
by means of the computer software package SigmaStat
(SPSS Inc., Chicago, IL, USA). All pairwise multiple
comparisons have been performed with the Dunn’s
method. The existence of significant differences was
determined with a P-value < 0.05 (p or probability to
have really the same median).
3. Results
The surface topography of the titanium, biphasic calcium
phosphate and mica substrates were analyzed by atomic
force microscopy. Table 1 sums up the roughness meas-
urements for the three surfaces at given scanning sizes.
For instance, for 5 × 5 µm2 scanning dimensions, the
measured height (Z) values were 6 ± 1 nm, 76 ± 7 nm
and less than 0.2 nm for titanium, BCP and mica surfaces
respectively.
Reproducible force-distance curves were obtained
throughout the different experiments. Figure 1 shows ty-
pical force vs. distance curves obtained for amelogenin
protein interacting with each substrate (respectively tita-
nium, biphasic calcium phosphate and mica for a contact
time of 104 ms. The effects of interaction time were as-
sessed at 0 ms, 102 ms, 103 ms and 104 ms. Consecutive
measurements were performed at different locations of
the substrate to confirm that we actually were measuring
protein-surface interactions. As the different approach-
retraction sequences showed equivalent force curves, that
were different from others obtained with a bare tip, we
definitely could attribute the collected data to protein-
surface interactions. Force measurements were carried
out by loading at a rate of 1 μm·s–1 followed by unload-
ing at the same rate.
When amelogenin coated cantilevers were brought
Table 1. Roughness measurements (Root mean square or
RMS), determined by AFM, of respectively titanium, BCP
and mica surfaces for different scanning sizes.
Scan size (µm²)Titanium BCP Mica
50 × 50 14 ± 1.5 nm 424 ± 30 nm <0.5 nm
10 × 10 6 ± 1.5 nm 176 ± 38 nm <0.5 nm
5 × 5 6 ± 1 nm 76 ± 7 nm <0.2 nm
Figure 1. Typical force vs. distance curves obtained for
amelogenin interacting with titanium (black curve), biphasic
calcium phosphate (red curve) and mica (green curve) for a
contact time of 10000 ms.
into contact with the three different substrates, it ap-
peared that the dissociation force increased as a function
of contact time for the three investigated systems (Figure
2). As depicted on the bar chart of Figure 2, in general,
the rupture forces were the strongest for the amelogenin-
mica pair. On the other hand, one can note that the un-
binding forces of amelogenin interacting with the BCP
surface were always stronger than those of the ame-
logenin-titanium system (Figure 2). For example, at a
contact time of 103 ms the average force needed to break
off the contact between amelogenin and the BCP surface
was equal to 1.38 ± 0.04 nN, whereas an average rupture
force of 0.58 ± 0.03 nN was calculated for the ame-
logenin-titanium interaction.
When fibrinogen coated cantilevers were brought into
contact with the three different substrates, it appeared
that the dissociation force increased as a function of con-
tact time for the three investigated systems (Figure 3).
The results depicted on the bar chart of Figure 3 clearly
show that fibrinogen binds strongly to the BCP substrate;
even at short contact times (0.57 ± 0.04 and 0.53 ± 0.03
nN) for 0 and 100 ms, respectively. Moreover, one can
note that the unbinding forces of fibrinogen interacting

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM 247
Contact time (ms)
01001000 10000
Force (nN)
0.0
0.5
1.0
1.5
2.0
2.5
Figure 2. Mean adhesion forces after interactions of ame-
logenin with titanium (black bars), BCP (red bars) and
mica (green bars) surfaces for respectively 0, 100, 1000 and
10000 ms contact times. Error bars represent the standard
error on the mean.
Contact time (ms)
01001000 10000
Force (nN)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Figure 3. Mean adhesion forces after interactions of fi-
brinogen with titanium (black bars), BCP (red bars) and
mica (green bars) surfaces for respectively 0, 100, 1000 and
10000 ms contact times. Error bars represent the standard
error on the mean.
with the BCP surface were always much stronger than
those of the fibrinogen-titanium system (Figure 3). For
example, at a contact time of 103 ms the average force
needed to break off the contact between fibrinogen and
the BCP surface was equal to 0.63 ± 0.04 nN, whereas an
average rupture force of 0.16 ± 0.03 nN was calculated
for the fibrinogen-titanium interaction.
A summary of the adhesion force of all investigated
protein-substrate pairs is plotted in Figure 4. As follows
from Figure 4, the recombinant amelogenin protein
binds stronger, compared to fibrinogen protein, and this
independently of the surface (titanium, BCP or mica) and
Contact time (m s)
01001000 10000
Force (nN)
0.0
0.5
1.0
1.5
2.0
(a)
Contact time (ms)
01001000 10000
Force (nN)
0.0
0.5
1.0
1.5
2.0
(b)
Contact time (ms)
01001000 10000
Force (nN)
0.0
0.5
1.0
1.5
2.0
(c)
Figure 4. Effect of contact times (respectively 0 ms, 10 ms,
100 ms and 1000 ms) after interactions of amelogenin ()
and fibrinogen () with (a) titanium, (b) BCP and (c) mica
surfaces. Error bars represent the standard error on the
mean.

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM 248
the considered contact times (except on BCP at 0 ms).
4. Discussion
The purpose of the present assessments was to analyze
how the physico-chemical surface properties of titanium
and calcium phosphate can affect the adsorption process
of recombinant human amelogenin. Fibrinogen was used
as a reference protein for comparison with amelogenin.
Mica was used as a reference surface since it exposes
always the same crystallographic plane of the crystal la-
ttice to the proteins.
AFM can measure very small (about 10 pN) forces be-
tween the tip and a surface. Force spectroscopy has pro-
vided new insights into protein-surface interactions [14].
By fastening a ligand of interest to the AFM tip and
bringing the modified tip into contact with a surface, one
can, in principle, directly measure the attractive and re-
pulsive intermolecular forces as a function of the tip-
sample separation distance on the molecular level [12].
In a conventional force spectroscopy experiment, the
deflection of a force microscope cantilever with a known
spring constant is monitored while a surface of interest is
moved towards the AFM tip until contact is made and
then retracted. Once the cantilever’s restoring force ex-
ceeds the attractive force between the protein- coated tip
and the surface, a pull-off event does occur. The vertical
jump during pull-off can be used to calculate the interac-
tion force on the basis of Hooke’s law [12].
The advantage of applying AFM for adhesion force
measurements is the possibility of deriving quantitative
data directly for the interaction phenomena of submicro-
scopic contact areas in aqueous solutions. Thus contact
experiments can be described on an atomistic level [18].
However, forces measured by AFM cannot be trivially
related to binding affinities [12]. As shown earlier, the
unbinding force, measured when the molecule separates
from the substrate, depends to a large extend on the dis-
sociation rate at which the pulling force is applied
[19,20]. Present measurements were carried out by load-
ing at a rate of 1 μm·s–1 followed by unloading at the
same rate.
Amelogenin proteins are hydrophobic in nature, with
most of the charged amino acids located in the N- and
C-terminal segments [3]. The C-terminal amino acids of
amelogenin are hydrophilic [21]. The loss of the charged
COO– terminal of amelogenin results in a reduction of
the affinity to hydroxyapatite [22]. Amelogenin mole-
cules spontaneously self-assemble into spherical struc-
tures called nanospheres [23]. Available data suggest that
the cooperative self-assembly of amelogenins is pH-de-
pendent [24]. The pH during the secretory stage of
amelogenesis is tightly regulated between pH 7.2 and 7.4.
A major challenge in working with enamel matrix pro-
teins in vitro is that their solubility under the latter phy-
siological conditions is very low [25]. Nevertheless, pre-
sent experiments were performed in liquid environ- ment
at pH 7.4 to approach physiological conditions.
Our investigations give new insight into the underlying
behaviors of amelogenin and fibrinogen adsorbed onto
substrates of interest in the biomaterials field. The force
curves we obtained are characteristic for protein-surface
unbinding processes. In all cases studied here, it is evi-
dent that the unbinding forces of the adsorbed proteins
increase steadily as a function of contact time. That is
consistent with previously published results [14,15]. Pre-
sent analyses demonstrate that the unbinding forces of
amelogenin interacting with the BCP surface are always
stronger than those of the same protein interacting with
the titanium substrate. It appears also that the rupture
forces of fibrinogen interacting with the BCP surface are
always much stronger than those of the same protein in-
teracting with the titanium surface. Interestingly our re-
sults clearly indicate that the recombinant human
amelogenin always binds more strongly than fibrinogen
independently of the surface (titanium, BCP or mica) and
the contact time (0, 102, 103 and 104 ms). These findings
emphasize the implication of the physico-chemical prop-
erties of the surface in the unbinding process of adsorbed
proteins.
5. Acknowledgements
We acknowledge support from the Région Alsace for
financial contribution to the AFM equipment. L.R. is
indebted to the Faculté de Chirurgie Dentaire of Stras-
bourg for financial support. M.D. greatly appreciated the
contributions of J. Svensson and L. Bülow (University of
Lund, Sweden).
REFERENCES
[1] J. J. Gray, “The Interaction of Proteins with Solid Sur-
faces,” Current Opinion in Structural Biology, Vol. 14,
No. 1, 2004, pp. 110-115. doi:10.1016/j.sbi.2003.12.001
[2] S. Faghihi, F. Azari, H. Li, M. R. Bateni, J. A. Szpunar, H.
Vali and M. Tabrizian, “The Significance of Crystallo-
graphic Texture of Titanium Alloy Substrates on Pre- os-
teoblast Responses,” Biomaterials, Vol. 27, No. 19, 2006,
pp. 3532-3539.
[3] A. G. Fincham, J. Moradian-Oldack and J. P. Simmer,
“The Structural Biology of the Developing Dental
Enamel Matrix,” Journal of Structural Biology, Vol. 126,
No. 3, 1999, pp. 270-299. doi:10.1006/jsbi.1999.4130
[4] J. Moradian-Oldak, “Amelogenins: Assembly, Processing
and Control of Crystal Morphology,” Matrix Boilogy, Vol.
20, No. 5-6, 2001, pp. 293-305.
[5] L. Richert, F. Variola, F. Rosei, J. Wuest and A. Nanci,
“Adsorption of Proteins on Nanoporous Ti Surfaces,” Sur-

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM 249
face Science, Vol. 604, No. 17-18, 2010, pp. 1445-1451.
doi:10.1016/j.susc.2010.05.007
[6] H. Zhou, T. Wu, X. Dong, Q. Wang and J. Shen, “Ad-
sorption Mechanism of BMP-7 on Hydroxyapatite (001)
Surfaces,” Biochemical and Biophysical Research Com-
munications, Vol. 361, No. 1, 2007, pp. 91-96.
doi:10.1016/j.bbrc.2007.06.169
[7] R. Goobes, G. Goobes, C. T. Campbell and P. S. Stayton,
“Thermodynamics of Statherin Adsorption onto Hydro-
xyapatite,” Biochemistry, Vol. 45, No. 17, 2006, pp.
5576-5586. doi:10.1021/bi052321z
[8] J. Hemmerlé, F. J. G. Cuisinier, P. Schultz and J.-C. Voegel,
“HRTEM Study of Biological Crystal Growth Mecha-
nisms in the Vicinity of Implanted Synthetic Hydroxya-
patite Crystals,” Journal of Dental Research, Vol. 76, No.
2, 1997, pp. 682-687.
doi:10.1177/00220345970760020901
[9] J. Hemmerlé, A. Önçag and S. Ertürk, “Ultrastructural
Features of the Bone Response to a Plasma-Sprayed Hy-
droxyapatite Coating in Sheep,” Journal of Biomedical
Materials Research, Vol. 36, No. 3, 1997, pp. 418-425.
doi:10.1002/(SICI)1097-4636(19970905)36:3<418::AID-
JBM17>3.0.CO;2-9
[10] K. Kandori, N. Horigami, H. Kobayashi, A. Yasukawa and
T. Ishikawa, “Adsorption of Lysozyme onto Various Syn-
thetic Hydroxyapatites,” Journal of Colloid and Interface
Science, Vol. 191, No. 2, 1997, pp. 498-502.
doi:10.1006/jcis.1997.4943
[11] Q. Q. Hoang, F. Sicheri, A. J. Howard and D. S. Yang,
“Bone Recognition Mechanism of Porcine Osteocalcin
from Crystal Structure,” Nature, Vol. 425, 2003, pp. 977-
980. doi:10.1038/nature02079
[12] C. M. Yip, “Atomic Force Microscopy of Macromolecu-
lar Interactions,” Current Opinion in Structural Biology,
Vol. 11, No. 5, 2001, pp. 567-572.
doi:10.1016/S0959-440X(00)00247-5
[13] S. M. Slack and T. A. Horbett, “Changes in the Strength
of Fibrinogen Attachment to Solid Surfaces: An Ex-
planation of the Influence of Surface Chemistry on the
Vroman Effect,” Journal of Colloid and Interface Science,
Vol. 133, No. 1, 1989, pp. 148-165.
doi:10.1016/0021-9797(89)90288-9
[14] J. Hemmerlé, S. M. Altmann, M. Maaloum, J. K. H.
Hörber, L. Heinrich, J.-C. Voegel and P. Schaaf, “Direct
Observation of The Anchoring Process During the Ad-
sorption of Fibrinogen on a Solid Surface by Force-
Spectroscopy Mode Atomic Force Microscopy,” Pro-
ceedings of the National Academy of Sciences of the USA,
Vol. 96, No. 12, 1999, pp. 6705-6710.
doi:10.1073/pnas.96.12.6705
[15] A. Boukari, G. Francius and J. Hemmerlé, “AFM Force
Spectroscopy of the Fibrinogen Adsorption Process onto
Dental Implants,” Journal of Biomedical Materials Re-
search A, Vol. 78, No. 3, 2006, pp. 466-472.
doi:10.1002/jbm.a.30778
[16] T. Thundat, R. J. Warmack, G. Y. Chen and D. P. Allison,
“Thermal and Ambient-Induced Deflections of Scanning
Force Microscope Cantilevers,” Applied Physics Letters,
Vol. 64, No. 21, 1994, pp. 2894-2896.
doi:10.1063/1.111407
[17] J. Svensson. C. Andersson, J. E. Reseland, P. Lyng-
sta-daas and L. Bülow, “Histidine Tag Fusion Increases
Expression Levels of Active Recombinant Amelogenin in
Escherichia Coli,” Protein Expression and Purification,
Vol. 48, No. 1, 2006, pp. 134-141.
doi:10.1016/j.pep.2006.01.005
[18] G. Jänchen, M. Mertig and W. Pompe, “Adhesion Energy
of Thin Collagen Coatings and Titanium,” Surface and In-
terface Analysis, Vol. 27, No. 5-6, 1999, pp. 444-449.
doi:10.1002/(SICI)1096-9918(199905/06)27:5/6<444::AI
D-SIA531>3.0.CO;2-3
[19] C. Gergely, J.-C. Voegel, P. Schaaf, B. Senger, M. Maa-
loum, J. K. H. Hörber and J. Hemmerlé, “Unbinding Proc-
ess of Adsorbed Proteins Under External Stress Studied by
Atomic Force Microscopy Spectroscopy,” Proceedings of
the National Academy of Sciences of the USA, Vol. 97,
No. 20, 2000, pp. 10802-10807.
doi:10.1073/pnas.180293097
[20] J. Hemmerlé, C. Picart, C. Gergely, P. Schaaf, J.-F. Stoltz,
J.-C. Voegel and B. Senger, “Modeling of The Detach-
ment of a Molecule from a Surface: Illustration of the
“Bell- Evans Effect,” Biorheology, Vol. 40, No. 1-3, 2003,
pp. 149-160.
[21] A. G. Fincham, J. Moradian-Oldack, T. G. Diekwish, D.
M. Layaruu, J. T. Wright and P. Bringas, “Evidence for
Amelogenin “Nanospheres” as Functional Components of
Secretory-Stage Enamel Matrix,” Journal of Structural
Biology, Vol. 115, No. 1, 1995, pp. 50-59.
doi:10.1006/jsbi.1995.1029
[22] J. Moradian-Oldak, N. Bouropoulos, L. Wang and N.
Gharakhanian, “Analysis of Self-Assembly and Apatite
Binding Properties of Amelogenin Proteins Lacking the
Hydrophilic C-Terminal,” Matrix Biology, Vol. 21, No. 2,
2002, pp. 197-205. doi:10.1016/S0945-053X(01)00190-1
[23] C. Du, G. Falini, S. Fermani, C. Abbott and J. Moradian-
Oldak, “Supramolecular Assembly of Amelogenin Nano-
spheres into Birefringent Microribbons,” Science, Vol.
307, No. 5714, 2005, pp. 1450-1454.
doi:10.1126/science.1105675
[24] X. He, W. Li, S. Habelitz, “The Cooperative
Self-Assembly of 25 and 23 kDa Amelogenins,” Journal
of Structural Biology, Vol. 164, No. 3, 2008, pp. 314-321.
doi:10.1016/j.jsb.2008.09.005
[25] F. B. Wiedemann-Bidlack, E. Beniash, Y. Yamakoshi, J.
P. Simmer, H. C. Margolis, “pH Triggered Self-Assembly
of Native and Recombinant Amelogenins under physio-
logical pH and Temperature in Vitro,” Journal of Struc-
tural Biology, Vol. 160, No. 1, 2007, pp. 57-69.
doi:10.1016/j.jsb.2007.06.007