Materials Science and Engineering C 39 (2014) 2934

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Production and characterization of natural rubberCa/P blends for

biomedical purposes
R.M. Nascimento a,b, F.L. Faita a, D.L.S. Agostini c, A.E. Job c, F.E.G. Guimares d, I.H. Bechtold a
a
Departamento de Fsica,Universidade Federal de Santa Catarina, 88040-900 Florianpolis, SC, Brazil
b
Departamento de Fsica, Universidade Estadual Paulista, 13506-900 Rio Claro, SP, Brazil
c
Faculdade de Cincias e Tecnologia, UNESP, Departamento de Fsica, Qumica e Biologia, CP 467, CEP 19060-080, Presidente Prudente, SP, Brazil
d
Departamento de Fsica e Cincia dos Materiais,Universidade de So Paulo, Instituto de Fsica de So Carlos, 1356-6590, So Carlos, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This study presents the development of natural rubberCa/P blends, as promising candidates for biomedical pur-
Received 17 October 2013 poses. The specic objective was the incorporation of Ca/P into a natural rubber polymeric matrix. Ca/P crystalline
Received in revised form 17 January 2014 phases were synthesized by the solgel method and the polymeric matrices were produced using natural rubber
Accepted 16 February 2014
extracted from latex of the Hevea brasiliensis. The shape and size of natural rubber particles present in the
Available online 21 February 2014
NR membrane, as well as, the way the Ca/P powder grains aggregate in the polymeric matrix were investigated,
Keywords:
giving information about the interactions between the Ca/P and the natural rubber particles. Confocal uores-
Natural rubber cence scanning microscopy measurements allowed us to propose a structure where the Ca/P grains are
Calcium phosphate surrounded by natural rubber particles. This structure may mediate Ca2+ release for tissue regeneration. The sys-
Biomaterial tem investigated may open new horizons for development of a bandage which provides the controlled-release of
Tissue regeneration biomaterials.
2014 Elsevier B.V. All rights reserved.

1. Introduction to crystalline phases such as hydroxyapatite, tricalcium phosphate,

and octacalcium phosphates [16].
The loss of body parts has preoccupied humanity since ancient Natural latex obtained from the Hevea brasiliensis tree is a colloidal
times, stimulating the search for materials with functional surfaces system containing rubber-particles of poly-1,4-cis-isoprene (around
that can optimize the process of tissue regeneration [1,2]. The treatment 40% of the total latex volume) suspended in an aqueous medium. Latex
of bone defects around implants is a specic application of biomaterials can be separated into three distinct phases by centrifugation: rubber
in regenerative therapies, being used to isolate the wound, to prevent cream, serum and bottom fraction. The rubber cream is mainly composed
the invasion of epithelium cells at the site of injury and to accelerate of poly-isoprene rubber-particles, with small portions of Hevein proteins
the healing [38]. Considering the steps of bone regeneration, osteogen- (Hev. b1 and Hev. b3, with low allergenicity) and phospholipids [17,18].
ic particles can be released in a controlled manner by delivery systems The serum phase contains different types of compounds, including carbo-
to improve tissue regeneration, and the success of this process depends hydrates, several proteins, amino acids and enzymes. Finally, the bottom
on the surface properties [9,10]. Such delivery systems must fulll two fraction contains a high portion of monomers and metal ions [19,20].
specic functions: i) act as a physical barrier with sufcient strength Natural rubber (NR), obtained from the rubber cream, is emerging as
to maintain the original functionality at the local of the lesion through- a renewable, low-cost and biocompatible polymer [21,22] due to its low
out the period in which the osteoblasts migrate into the defect; ii) in- allergenicity, being employed as transdermal drug delivery systems
duce tissue regeneration by releasing particles [11,12]. where the NR is the controlling layer membrane [23,24]. Latex mem-
Calcium phosphates (Ca/P) can provide good biocompatibility branes have been used to promote the activity of bone regeneration
depending on the calcium/phosphorus ratio [13,14]. The Ca/P system through the Guided Bone Regeneration (GBR) model system by incor-
may present several crystalline phases, where hydroxyapatite porating bone morphogenetic proteins on NR membranes acting as pro-
(Ca10 (PO4 ) 6(OH) 2 ) and the tricalcium phosphate (Ca 3 (PO 4) 2 ) are tein delivery system [2528]. In this context, it is possible to combine
the most commonly studied biomaterials and play an important the NR characteristics as a physical barrier (to the epithelial cells or
role in tissue regeneration, in addition, pentacalcium octaphosphate other components) with particles of specic functionalities, such as
(Ca5P8) has been applied as efcient reservoir of Ca and P ions in bi- Ca/P, to produce a functionalized bandage.
ological environments [15]. When Ca/P is precipitated in supersatu- In this work, we describe the synthesis and characterization of Ca/P
rated aqueous solutions, a metastable amorphous phase forms particles, as well as, the development of self-sustaining membranes
quickly in the early stages of the reaction, being further converted where these particles are physically incorporated into the NR matrix.

http://dx.doi.org/10.1016/j.msec.2014.02.019
0928-4931/ 2014 Elsevier B.V. All rights reserved.
30 R.M. Nascimento et al. / Materials Science and Engineering C 39 (2014) 2934

The results suggest that the Ca/P particles are surrounded by proteins/ 2.4. Production of NR:Ca/P blend
phospholipids particles in the polymeric matrix. In addition, the interac-
tions at the NRCa/P interface are discussed. The Ca/P powder was dispersed in chloroform, at a concentration of
0.1 mg/mL, and submitted to sonication during 30 min. After this proce-
dure, the Ca/P solution was mixed with the NR solution (described in
2. Experimental Section 2.3) in the ratio NR:Ca/P of 9:1 considering the weight of the
solids and then submitted to magnetic stirring during 20 min. Finally,
2.1. Materials the NRCa/P blend membrane was produced by casting the NRCa/P so-
lution in Petri dishes followed by thermal annealing during 6 h at 45 C
Calcium nitrate tetrahydrate Ca(NO3)24H2O (99% purity), ortho- in an oven. The nal thickness of NRCa/P blend membrane was also
phosphoric acid H3PO4 (85 wt.% in H2O), ethylene glycol (PA, 99.8% around 200 m, checked with a digital micrometer.
purity), methyl alcohol (methanol 99.5%, PA), ammonium hydroxide
(PA-ACS) and chloroform (PA) were purchased from Sigma-Aldrich.
All the chemical products were used as received. NR was obtained 2.5. Characterization of the membranes
from the natural latex extracted from different rubber trees (RRIM
600 clone) in Indiana City, So Paulo State, Brazil. The mechanical properties were investigated on both membranes by
dynamic mechanical analyses (DMA) using a TA Instruments Analyzer,
model DMA-Q800, in single cantilever mode. Stressstrain curves
2.2. Synthesis and physicochemical characterization of the Ca/P powder were obtained at a temperature of 30 C with a strain rate of
2 mmmin 1 using a xed frequency of 1 Hz. Five samples of each
The Ca/P was synthesized by the solgel method using as precursors membrane were submitted to DMA measurements and the mean elastic
the calcium nitrate tetrahydrate and orthophosphoric acid in the pro- modulus (E) or Young's modulus of each sample was obtained using the
portion of 80:20 wt.%. The precursors were dissolved in 15 mL of a equation:
methanol:ethylene glycol solution (2:1 ratio) and maintained under
magnetic stirring and ambient conditions during 1 h to produce the .
F
sol solution. The obtained product was submitted to aging, drying
A
and annealing in three steps: i) 24 h at 50 C under constant magnetic E L . 0 ; 1

stirring, this procedure resulted in an opaque and viscose gel; ii) the L0
gel was poured into ceramic dishes and dried during 1 h at 100 C,
then grounded to powder with a mortar and pestle; and iii) the resultant
powder was annealed during 1 h at 700 C and stored under ambient where F is the force applied to the sample, L is the length variation, A0
conditions for the future analyses. The structural, morphological and and L0 are the original cross-sectional area and length of the object, re-
chemical properties of the Ca/P powder were investigated using X-ray spectively. Therefore, is the tensile stress (F/A0) and corresponds to
diffraction (XRD) with Rietveld analysis, scanning electron microscopy extensional strain (L/L0).
(SEM) and energy-dispersive X-ray spectroscopy (EDS). The incorporation of the Ca/P powder into the NR membrane was
The XRD pattern was recorded on a PANalytical X'Pert PRO Multi- veried by EDS (chemical color mapping) measurements. The shape
Purpose Diffractometer, using Cu-K radiation ( = 0.15418 nm), op- and size of the NR-particles (composed of proteins/phospholipids)
erating at 45 kV and 40 mA and equipped with the X'Celerator detector. present in the pure NR membrane and the interactions of the Ca/P pow-
The measurement was carried out at 2 angles ranging from 10 to 110 der with the polymeric matrix and/or NR-particles of the NRCa/P blend
with a scan step size of 0.033 using the BraggBrentano (2) geome- membrane were investigated using a Carl Zeiss LSM 780 confocal laser
try. Rietveld analysis [29] was performed from the complete XRD pattern scanning microscope equipped with a multi-line, diode laser of
using the X'Pert HighScore Plus software (version 2.2b of PANalytical, 405 nm, 6 mW and 20 MHz. Moreover, a water-immersion objective
Netherlands) and the starting structural models were based on the infor- (63 magnication) with the software Zen 2010 Light Edition was
mation given in the Inorganic Crystal Structure Database (ICSD) [30]. The used for image acquisition.
structural renement by the Rietveld method allowed the determination
of the main phase fractions. SEM images and EDS (X-ray from K energy
line emission) spectra were performed with a JEOLJSM-6390LV SEM mi-
croscope (under 15 kV typical tension acceleration) equipped with a
NORAN X-ray Micro-analysis System Six. ImageJ free software was
used to determine the particle size distribution from the SEM images.

2.3. NR solution and membrane production

The collected latex from the rubber trees was stabilized in ammoni-
um hydroxide at 2% (volume) and submitted to centrifugation using a
Cientec CT 5000R centrifuge at 6000 rpm at a temperature of 4 C
for 90 min. This centrifugation process allowed the separation of the
rubber cream phase fraction as previously reported [31]. The rubber
cream was dried over night in an oven at 60 C and dissolved in chloro-
form at a concentration of 30 mg/mL. The pure NR membrane was pro-
duced by casting the NR solution in Petri dishes followed by thermal
annealing during 6 h at 45 C in an oven. The nal thickness of the mem-
brane was measured with a digital micrometer, to be around 200 m. A Fig. 1. XRD pattern obtained for the Ca/P powder with the identication of the main in-
complete study about the morphological properties of NR membranes tense peaks and the estimated phase fractions by Rietveld method using the HighScore
was recently reported [31]. Plus software.
 R.M. Nascimento et al. / Materials Science and Engineering C 39 (2014) 2934 31

3. Results and discussion and therefore the XRD is not able to measure the interfacial component.
In addition, the amorphous and interfacial component atoms may act as
Fig. 1 shows the XRD pattern obtained for the Ca/P powder as syn- a reservoir of Ca2+ ions.
thesized, where crystalline phases were identied and attributed to In the biological environment, some of these phases, such as
monoclinic Ca5P8 (ICSD card #74854), trigonal CaCO3 (ICSD card Ca(PO4)3OH, can promote the optimization of tissue regeneration by
#28827) and two hexagonal Ca(PO4)3OH (ICSD card #81442) and Ca creating a gradient of cell proliferation. The Ca/P can also present different
(ICSD card #52747) phases. The Rietveld method was applied to estimate densities and porosities due to each specic polymorph phase and/or
the crystalline contribution of each main phases and the majority phase the mixing of different Ca/P phases. Moreover, mixing distinct Ca/P poly-
fractions were: Ca5P8 (47.8 wt.%), CaCO3 (36.3 wt.%), Ca(PO4)3OH morph phases allows different levels of degradation, caused by dissolu-
(9.5 wt.%) and Ca (6.3 wt.%). The nal adjusted lattice parameters tion, which is dependent on the solubility of the material, pH and
remained very close to those reported on the ICSD cards, indicating that temperature. The physical disintegration into smaller particles can also
the synthesis and thermal annealing processes provided a compound occur due to biological interactions. In this process, Ca2+ ions may be re-
with reduced strainstress lattice defects. Amorphous and crystalline leased into the environment and the proportion of calcium and phospho-
samples with structural defects are structurally and chemically metasta- rous altered [11,32].
ble. The properties of metastable samples can be altered by small rates Fig. 2 shows the SEM micrographs, the graphs resulting from the EDS
of energy and/or due to aging, thus samples with reduced structural de- measurement and the particles size distribution analyses. Fig. 2(a)
fects are desired to obtain crystalline samples stable for longer periods. shows the SEM image of the Ca/P powder as synthesized, where the
Atoms located at the boundaries of the interstices and surfaces of the graphs (I) and (II) show the histogram of the particles size distribution
grains do not contribute coherently in order to satisfy the Bragg's law (centered at 13 m) and the EDS spectrum of this image, respectively.

Fig. 2. SEM micrographs of the Ca/P powder as synthesized (a) and casted after dispersion and sonication in chloroform (b). Graphs I and II show the particle size histogram and EDS spec-
trum for the SEM image (a), respectively. Graph III shows the particle size histogram obtained for the SEM image (b). (c) represents the SEM image of the NRCa/P blend membrane and its
EDS chemical color map of Ca and P in (d) and (e), respectively.
32 R.M. Nascimento et al. / Materials Science and Engineering C 39 (2014) 2934

The EDS analysis showed a Ca/P ratio of 1.67. This value coincides with other factors, such as the adsorption of ions at the surface of the rubber
the range of Ca/P ratios typical of biological processes (0.5 to 2.0) [33]. particles [34].
Fig. 2(b) shows the Ca/P powder after dispersion in chloroform, sonica- From the DMA measurements we obtained elastic modules of 3.8
tion and casting on an NR membrane as substrate, while the new size 0.1 MPa and 3.9 0.1 MPa for the pure NR and NRCa/P blend mem-
distribution is shown in the graph (III). It can be observed that the dis- branes, respectively. Thus, the mechanical properties of the membranes
persion of Ca/P particle sizes is considerably reduced, ranging from 2 are not affected by the Ca/P doping, which is desired for biomedical ap-
to 16 m, being predominantly under 5 m. Fig. 2(c) shows the SEM plications. The low elastic modulus is because the membranes are not
image of the Ca/P particles incorporated into the latex membrane, and vulcanized.
Fig. 2(d) and (e) shows its EDS chemical color map for Ca and P, respec- The produced NR membranes were investigated by confocal uores-
tively. These results conrm the presence of Ca and P in the observed cent scanning microscopy and well-dened spherical rubber particles
particles and the presence of these particles homogeneously distributed with diameters ranging between 1 and 5 m were observed. Fig. 3(a)
in the membrane. shows the luminescence micrograph of a particle with a diameter of
In order to design controlled-release systems using bioactive parti- around 4 m. It is important to emphasize that this image was obtained
cles like Ca/P, a detailed understanding of the carrier properties is re- without additional dye doping for contrast. In Fig. 3(a), blue and green
quired. The latex membranes demonstrated good exibility and regions of emission can be seen throughout the particle surface,
mechanical resistance, which is an important characteristic for surgical resulting in the broadband spectrum presented in Fig. 3(d). Gaussian
manipulation. However, in this study, the priority was to investigate deconvolutions were applied to the emission spectra and two contribu-
the constituents of the Ca/PNR membranes. Concerning the NR obtain- tions were estimated at maximum wavelengths of 492 nm and 559 nm
ed from the rubber cream phase of the latex, models in the literature de- (dotted line) for a single NR particle (P1) and 484 nm and 536 nm
scribe the presence of rubber particles formed by a coreshell structure (dashed line) for NRCa/P (P2). These two specic emission bands can
comprised of a hydrophobic core (isoprene molecules) surrounded by a be attributed to the emission associated with two distinct structures
mixed layer of proteins (84%) and phospholipids (16%) with around 20 of the tryptophan present in the proteins of the NR particles. The struc-
nm thick [34,35]. The maximum diameter reported for these sphere- tural conformation of proteins has been studied [36], but in this case,
like structures is around 5 m. In addition, these negatively-charged our hypothesis is that solvent can change some aromatic ring chains
proteins control the colloidal stability of the latex, favoring several during the process of dispersion. On the other hand, the presence of

Fig. 3. Confocal luminescence micrographs of a single rubber particle captured from the unblended NR membrane (a); and the agglomerates in the NRCa/P blend samples (b) and (c).
(d) Luminescence spectra of the P1 and P2 regions. The dashed and dotted lines are the Gaussian deconvolutions of the emission spectra. (For interpretation of the references to color in
this gure legend, the reader is referred to the web version of this article.)
 R.M. Nascimento et al. / Materials Science and Engineering C 39 (2014) 2934 33

Ca/P particles could weakly interact with natural rubber compounds Calcium is a fusogenic agent and has a profound effect on the adsorption
mainly modifying only the structure of the proteins which is supposed of vesicles [41]. Calcium ions do not only participate in the screening of
to shift the emission bands evaluated from the NRCa/P composites. the charges, thereby modifying the electrostatic interactions, but they
Fig. 3(b) shows the confocal uorescent micrograph for the NRCa/P also directly interact with surfaces and lipids present in the NR [42].
membrane, where large agglomerates with sizes of up to 30 m can be These considerations may explain the increasing size of agglomerates ob-
clearly seen. In addition, the agglomerates with a focal plane at the center served in the NRCa/P membranes (see Fig. 3(b) and (c)). Moreover, sur-
of the particles display an emissive border with a non-emissive core. face interactions between the NR particles and the Ca/P grains may also
Fig. 3(c) shows two agglomerates in detail, one with a non-luminescent occur and contribute to the formation of NRCa/P agglomerates. A sche-
asymmetric center. The emission spectrum of the P2 region is very similar matic representation showing the possible conguration of the NRCa/P
to that obtained for the single rubber particle in Fig. 3(a), region P1. More- agglomerates is proposed in Fig. 4(a) and a schematic representation of
over, we tested by same confocal microscopy measurements the Ca/P the NR particles adapted from Ref. [34] is presented in Fig. 4(b). It is im-
particles dispersed in a poly(methyl methacrylate) (PMMA) polymeric portant to note that the non-spherical shape of the agglomerates, togeth-
matrix and any emission was observed under the same experimental er with the dimensions of the non-emitting center and the luminescent
conditions. shell of Fig. 3(b) and (c) is consistent with the proposed representation
By relating the two emission bands of the tryptophans present in the in Fig. 4(a).
proteins, the analysis of Fig. 3(a) suggests a random and homogeneous
distribution of these components over the NR particle embedded in the 4. Conclusions
bulk of the polymeric matrix. In contradiction with previous studies re-
ported in the literature, where models for the NR particles have been In summary, the solgel process was used to produce Ca/P micro-
proposed, considering the proteins and phospholipids having a hetero- grains with multi-crystalline phases, including the monoclinic Ca5P8 and
geneous distribution, with protein/phospholipid agglomerates on the the hexagonal Ca(PO4)3OH. The percentage contribution of each phase
NR particle surface [34,37]. was estimated by applying the Rietveld method to XRD measurements,
The NR particles show a resultant negative charge at around 56.7 where the major phase fractions obtained were: Ca5P8 (47.8 wt.%),
0.7 mV, as veried by zeta potential analysis, this result is in close agree- CaCO3 (36.3 wt.%), Ca(PO4)3OH (9.5 wt.%) and Ca (6.3 wt.%). The chemi-
ment with values previously reported for a latex solution at a pH of ~10 cal ratio between Ca and P was 1.67, as veried by EDS analysis, this value
[38]. Ca/P grains formed by regions with crystalline phases and boundary being within the bioactivity range. The spherical shape (with ~4 m of di-
atoms can present localized positive charges due to the presence of Ca2+ ameter) and homogeneous distribution of the proteins and phospholipids
on the surface of the grains. Thus, when in solution, these boundary on the shell of the NR particles were observed by luminescent confocal
atoms can promote electrostatic attraction due to the opposite charges microscopy. The interaction of the NR particles with the Ca/P grains was
of the NR particles and the Ca/P grains on the surface. In addition, during conrmed via confocal micrographs, supporting the proposed structure,
their diffusion to the polymeric matrix the Ca2+ ions can migrate around i.e., that the Ca/P grains are surrounded by NR particles, as shown in
the NR particles and promote a small degree of calcication. This calci- Fig. 4(a). This aspect represents the novelty of this study. This system
cation around NR particles has been previously observed and described may open new horizons in relation to its use as a biomaterial for applica-
in the literature [39]. Multivalent cations and/or anions (e.g., Ca2+ and tion in tissue regeneration. The results presented herein are the rst step
PO4) and NR latex anionic proteins could regulate their stability with toward obtaining a bandage which promotes the controlled-release of
this calcication [40]. Ca2+ ions have the ability to bridge negatively biomaterials. The degradation of natural rubber, the release of Ca2+ ions
charged entities, such as carboxylate groups and phosphate groups in in a simulated body uid and the biocompatibility of the material with tis-
charged and zwitterionic lipid species, or negative charges in the NR. sue regeneration are currently under investigation.

Fig. 4. (a) Suggested schematic representation of an NRCa/P agglomerate. (b) Detailed description of the proposed structural organization of the NR particles adapted from Ref. [34],
where the proteins and -terminals, as well as the phospholipids and -terminals, with their connections, are represented.
34 R.M. Nascimento et al. / Materials Science and Engineering C 39 (2014) 2934

Acknowledgments [19] M.M. Rippel, C. Leite, L.-T. Lee, F. Galembeck, Colloid Polym. Sci. 283 (2004)
570574.
[20] D.L.S. Agostini, C.J.L. Constantino, A.E. Job, J. Therm. Anal. Calorim. 91 (2008)
The authors are grateful to the Brazilian governmental agencies 703707.
CNPq (project #555100/2010-3), CAPES (project PNPD/2583/2011), [21] F. Mru, J.C. Netto, R. Ceneviva, J.J. Lachat, J.A. Thomazini, H. Tambelini, Mater. Res. 7
(2004) 277283.
INCT/INEO and NanoBiomed for nancial support and the Central Labo- [22] C.A. Balabanian, J. Coutinho-Netto, T.L. Lamano-Carvalho, S.A. Lacerda, L.G.
ratory of Electron Microscopy (LCMEUFSC) by SEM and EDS facilities. Brentegani, J. Oral Sci. 48 (2006) 201205.
The XRD experiment was carried out in the Laboratrio de Difrao de [23] E. Abraham, P.A. Elbi, B. Deepa, P. Jyotishkumar, L.A. Pothen, S.S. Narine, S. Thomas,
Polym. Degrad. Stab. 97 (2012) 23782387.
Raios-X (LDRX-CFM/UFSC). [24] W. Pichayakorn, J. Suksaeree, P. Boonme, W. Taweepreda, T. Amnuaikit, G.C.
Ritthidej, Chem. Eng. Res. Des. 91 (2013) 520529.
[25] R.D. Herculano, C.P. Silva, C. Ereno, S.A.C. Guimaraes, A. Kinoshita, C.F.D. Graeff,
References Mater Res. Ibero-Am. J. 12 (2009) 253256.
[26] C. Ereno, S.A.C. Guimaraes, S. Pasetto, R.D. Herculano, C.P. Silva, C.F.O. Graeff, O.
[1] M.M. Stevens, Mater. Today 11 (2008) 1825. Tavano, O. Baffa, A. Kinoshita, J. Biomed. Mater. Res. A 95A (2010) 932939.
[2] P. Broz, Knovel (Firm), RSC Nanoscience and Nanotechnology, 9, Royal Society of [27] M.A.C. Frade, I.B. Cursi, F.F. Andrade, J. Coutinho-Netto, F.M. Barbetta, N.T. Foss, Med.
Chemistry, Cambridge, UK, 2010, p. 1, (online resource, xv, 372 pp.). Cutan. Iber. Lat. Am. 32 (2004) 157162.
[3] S.Y. Shin, H.N. Park, K.H. Kim, M.H. Lee, Y.S. Choi, Y.J. Park, Y.M. Lee, Y. Ku, I.C. Rhyu, [28] M. Ferreira, R.J. Mendonca, J. Coutinho-Netto, M. Mulato, Braz. J. Phys. 39 (2009)
S.B. Han, S.J. Lee, C.P. Chung, J. Periodontol. 76 (2005) 17781784. 564569.
[4] N.S. Satarkar, J.Z. Hilt, Acta Biomater. 4 (2008) 1116. [29] H.M. Rietveld, J. Appl. Crystallogr. 2 (1969) 6571.
[5] N.S. Satarkar, D. Biswal, J.Z. Hilt, Soft Matter 6 (2010) 23642371. [30] Inorganic Crystal Structure Database (ICSD), Gmelin-Institut fr Anorganische
[6] W.F.P. Neves-Junior, M. Ferreira, M.C.O. Alves, C.F.O. Graeff, M. Mulato, J. Chemie and Fachinformationszentrum, FIZ, Karlsruhe, 2007.
Coutinho-Netto, M.S. Bernardes, Braz. J. Phys. 36 (2006) 586591. [31] F.L. Faita, M.E.R. Dotto, L.G. Frana, F.C. Cabrera, A.E. Job, I.H. Bechtold, Eur. Polym. J.
[7] A. Sculean, G.C. Chiantella, P. Windisch, N.B. Arweiler, M. Brecx, I. Gera, J. Clin. 50 (2014) 249254.
Periodontol. 32 (2005) 720724. [32] W.I. Abdel-Fattah, F.M. Reicha, T.A. Elkhooly, Biomed. Mater. 3 (2008) 034121.
[8] M. Kikuchi, Y. Koyama, T. Yamada, Y. Imamura, T. Okada, N. Shirahama, K. Akita, K. [33] R.Z. LeGeros, Monogr. Oral Sci. 15 (1991) 1201.
Takakuda, J. Tanaka, Biomaterials 25 (2004) 59795986. [34] K. Nawamawat, J.T. Sakdapipanich, C.C. Ho, Y. Ma, J. Song, J.G. Vancso, Colloids Surf.
[9] G.R. Owen, J. Jackson, B. Chehroudi, H. Burt, D.M. Brunette, Biomaterials 26 (2005) A Physicochem. Eng. Asp. 390 (2011) 157166.
74477456. [35] R. Wititsuwannakul, K. Rukseree, K. Kanokwiroon, D. Wititsuwannakul, Phytochem-
[10] S. Liao, W. Wang, M. Uo, S. Ohkawa, T. Akasaka, K. Tamura, F.Z. Cui, F. Watari, Bioma- istry 69 (2008) 11111118.
terials 26 (2005) 75647571. [36] L. Bateman, Natural Rubber Producers' Research Association, The Chemistry and
[11] H.H.K. Xu, M.D. Weir, L.M. Sun, Dent. Mater. 25 (2009) 535542. Physics of Rubber-like Substances, , Maclaren, London, 1963.
[12] R.D. Herculano, A.A.A. de Queiroz, A. Kinoshita, O.N. Oliveira, C.F.O. Graeff, Mater. Sci. [37] K. Berthelot, S. Lecomte, Y. Estevez, V. Zhendre, S. Henry, J. Thevenot, E.J. Dufourc,
Eng. C Mater. 31 (2011) 272275. I.D. Alves, F. Peruch, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/
[13] H.H. Xu, J.L. Moreau, L. Sun, L.C. Chow, Dent. Mater. 27 (2011) 762769. j.bbamem.2013.1008.1025.
[14] W. Thein-Han, J. Liu, H.H. Xu, Dent. Mater. 28 (2012) 10591070. [38] J. Sansatsadeekul, J. Sakdapipanich, P. Rojruthai, J. Biosci. Bioeng. 111 (2011) 628634.
[15] C. Hadenfeldl, F. Barlels, Z. Anorg. Allg. Chem. 620 (1994) 12471252. [39] M.M. Rippel, C.A. Leite, L.T. Lee, F. Galembeck, J. Colloid Interface Sci. 288 (2005)
[16] A.H. Aparecida, M.V.L. Fook, M.L.d. Santos, A.C. Guastaldi, Quim. Nova 30 (2007) 892896. 449456.
[17] J.M. Rolland, R.E. O'Hehir, Clin. Exp. Allergy 38 (2008) 898912. [40] B.Y. Ha, Phys. Rev. E. 64 (2001).
[18] A.M. Smith, H.S. Amin, R.E. Biagini, R.G. Hamilton, S.A. Arif, H.Y. Yeang, D.I. Bernstein, [41] R.P. Richter, R. Berat, A.R. Brisson, Langmuir 22 (2006) 34973505.
Clin. Exp. Allergy 37 (2007) 13491356. [42] I. Reviakine, A. Simon, A. Brisson, Langmuir 16 (2000) 14731477.