MESTRADO INTEGRADO EM MEDICINA DENTÁRIA - ARTIGO DE INVESTIGAÇÃO CIENTÍFICA
New Multilayer Hybrid Material for Oral and Maxillofacial Reconstruction
Rodrigo Osório de Valdoleiros Pereira da Silva
FACULDADE DE MEDICINA DENTÁRIA DA UNIVERSIDADE DO PORTO
ÁREA CIENTÍFICA: ENGENHARIA DE TECIDOS, MEDICINA REGENERATIVA E CÉLULAS
ESTAMINAIS
PORTO 2019
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Faculdade de Medicina Dentária da Universidade do Porto
Mestrado Integrado em Medicina Dentária
New Multilayer Hybrid Material for Oral and
Maxillofacial Reconstruction
Monografia de Artigo de Investigação Científica
Área Científica: Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais
Autor: Rodrigo Osório de Valdoleiros Pereira da Silva 1, 2, 3
Orientadora: Professora Doutora Maria Cristina de Castro Ribeiro 2, 3, 4
Co-orientador: Dr. Germano Neves Pinto da Rocha 1
1 FMDUP - Faculdade de Medicina Dentária da Universidade do Porto, Porto, Portugal
2 INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal
3 i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
4ISEP - Instituto Superior de Engenharia do Porto, Porto, Portugal
Porto, 2019
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ÍNDICE Abstract…………………………………………………………………………………….……..I
1. Introduction…………………………………………………………………………................II
2. Materials and Methods………………………………………………………………………..III
2.1. Microspheres specifications and characterization……………………………….…………..4
2.1.1. Microspheres specifications………………………………………………………4
2.1.2 Microspheres characterization …………………………………………………….4
2.1.2.1 Environmental Scanning Electron Microscope (SEM/EDS) ...…………4
2.1.2.2. Zeta Potential Electro Kinetic Analyser (EKA) Electro Kinetic
Analyzer…………………………………………………………………………..……………...5
2.2. Alginate vehicle preparation and formation…………………….…………..………………..5
2.2.1. Alginate preparation……………………………………………….………………5
2.3. Fetal membranes collection, preparation and characterization …………..………………….5
2.3.1. Collection and storage …………………………………………..…………...……5
2.3.2. Preparation and decellularization of FM………………………..……………...….6
2.3.3. Tissue Histology analyze……………………………………..……………...……6
2.3.4. Transmission electron microscopy (TEM) …………………………..………...….6
2.3.5. Environmental Scanning Electron Microscope (SEM/EDS) ……….………….….7
2.3.6. Fourier Transform Infrared Spectroscopy (FT-IR) ………………………………..7
2.3.7. Atomic Force Microscopy (AFM) ………………………………………………..7
2.4. Bilayer hybrid system preparation …………………….……………………..………………7
2.5. Bilayer hybrid system characterization …………………………………….….……............8
2.5.1. Micro CT characterization ……..……………………………….…………………8
2.5.2. Tissue Histology analyzes…………………………………………………………8
2.5.3 Dynamical mechanical analysis - DMA tests…………………..……..…………....8
2.5.4. Rheometer analysis………………………………………………………………..9
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2.6. In vitro biological response………………………………………..…………………..……9
2.6.1. Cell culture ………………………………………………………………………9
2.6.2. Macrophages Polarization into pro-inflammatory - M1 or anti-inflammatory - M2.
…………………………………………………………..…………………..……………………9
2.6.3. Flow cytometry analysis……………………………………………….…………10
2.6.4. ELISA assays ……………………………………………………………………10
2.7. Statistical analysis……………………………………………………………..……………10
3. Results………………………………………………………………………………………..11
3.1. Microspheres characterization……………………………………………………………...11
3.1.1. Environmental Scanning Electron Microscope (SEM/EDS)…………………….11
3.1.2. Zeta Potential Electro Kinetic Analyser (EKA) Electro Kinetic
Analyzer……………………………………………………………………………….………..12
3.2. Fetal membranes characterization …………………………………………………………12
3.2.1. Tissue Histology analyze……………………………………………………...…12
3.2.2. Transmission Electronic Microscope (TEM)…………………………………….13
3.2.3. Fourier Transform Infrared Spectroscopy (FT-IR)………………………………14
3.2.4. Atomic Force Microscopy (AFM)………………………………………………..15
3.3 Multilayer Hybrid Biomaterial Characterization…………………………………..………..17
3.3.1. Micro-CT (μCT) reconstructed image of the morphometric 3D evaluation
scaffold……………………………………………………………………………….…………17
3.3.2. Dynamical mechanical analysis (DMA) tests ……………………………………18
3.3.3. Rheometer analyses………………………………………………………………19
3.3.5. Flow cytometry analysis………………………………………………………….21
3.3.6. ELISA assays…………………………………………………………………….23
4. Discussion…………………………………………………………………………………….24
5. Conclusion………………………………………………………………………..……...…...30
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Acknowledgments…………………………………………………………..…………...……...31
References…………………………………………………………………………….………...3
Abstract A big challenge in the development of scaffolds for oral and maxillofacial surgery
includes the engineering of materials that simultaneously promote hard and soft tissue
regeneration. The objective of the present work was to develop and characterize a bilayer hybrid
polymer-ceramic injectable system comprising a layer that will promote the formation of new
bone (layer 1) and another one that will induce the formation of mucosa (layer 2). Both layers
include as a vehicle a 3.5% (w/v) ultrapure sodium alginate solution being its in situ gelation
promoted by the addition of Sr carbonate and Glucone-δ-lactone, and also decellularized human
amniotic and chorionic membrane particles (1:1 ratio) in a concentration of 20 mg/mL. Layer 1
was mechanically reinforced with hydroxyapatite Sr-rich microspheres (35% w) with a diameter
between 500-560 μm. Sr incorporation in the system relies on the evidence that this metallic
element has beneficial effects in bone remodeling besides possessing antimicrobial properties.
Fetal membranes were used due to their potentially interesting properties for regenerative
medicine applications. The bilayer system and its components were physico-chemically and
morphologically characterized using different experimental techniques namely, Fourier transform
infrared spectroscopy, atomic force microscopy, transmission electron microscopy, micro
computing tomography, histology techniques, zeta potential evaluation, rheometry and dynamic
mechanical analysis. The capacity of the bilayer material to modulate the host immune response
was also investigated. Results showed that the decellularized protocol used was efficient since
no nuclei were identified in the membranes and the collagen matrix was preserved. The interface
between hybrid layers was very stable and mechanical tests showed that elastic behavior of the
hybrid is dominant over the viscous one being its storage modulus. Preliminary results show that
the bilayer material provides low macrophage activation. Further tests should be performed in
order to explore the immunomodulation capacity of the bilayer material.
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Key words: Bilayer scaffold, Fetal membranes, Strontium-Alginate, Nano-Hydroxyapatite,
Immune response modulation.
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1. Introduction
In the last decade there has been a substantial amount of innovation and research into
tissue engineering and regenerative approaches for the oral and maxillofacial region. A big
clinical challenge in this complex area is to develop implantable biodegradable scaffolds that
may simultaneously increase the novo bone formation and stimulate the formation of mucosa. Recently, our team has proposed an injectable system for bone regeneration consisting of
an alginate matrix crosslinked in situ in the presence of strontium (Sr), incorporating a ceramic
reinforcement in the form of Sr-rich microspheres (1-3) .Results showed that the developed Sr-
hybrid system
stands as an excellent biomaterial for bone regeneration. Based on that, in the present study we
decided to develop an injectable bilayer biomaterial composed of alginate as the vehicle, Sr-rich
ceramic microspheres and decellularized fetal membrane particles. The difference between the
two layers is the fact that the one in contact with bone includes the ceramic particles that will
favor the formation of new bone and will act as a mechanical reinforcement. The combination of
hydrogels and ceramics for bone regeneration strategies is a very interesting biomimetic approach
since bone is a composite material. Furthermore, the use of an in situ crosslinking hydrogel such
as alginate allows for the injectability in the defect of the composite material.
Alginate is a natural polymer extracted from brown algae that is biodegradable and
biocompatible and extensively used in biomedical applications. It is a linear co-polymer
composed of (1-4)-linked β-D-mannuronic acid (M units) and a-L-guluronic acid (G units)
monomers, arranged into M-blocks, G-bloks and/or MG-blocks, and is able to form hydrogels
under mild conditions in the presence of divalent cations such as calcium or strontium (4). The
grafting of cell-instructive moieties such as arginine-glycine-aspartic acid (RGD) peptides to the
polymer backbone by aqueous carbodiimide chemistry is a strategy widely used to provide
appropriate guidance signals to promote cell adhesion and cell matrix (3, 5, 6).
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Several studies show that strontium has the potential to increase the proliferation and
differentiation of osteoblasts and inhibit the formation, maturation and resorptive behavior of
osteoclasts. Besides that, it has antimicrobial properties (unpublished of our group) and
contributes to the modulation of the inflammatory response (ref nosso artigo).
Fetal membranes have been investigated for tissue engineering and regenerative medicine
applications due to their minimum inflammatory responses and scar formation (7, 8), biostability,
vasoactivity, thromboresistance (9), antibacterial and antiviral (10) properties, and the content of
biological species (growth factors, interleukins and tissue inhibitors of metalloproteases) (11).
In order to be used as a biomaterial, fetal membranes should be submitted to a
decellularization process to remove cell associated antigens while preserving the ultrastructure
and composition of the ECM. Decellularized fetal membranes can then act as a cell-guiding
template that contains the necessary cues and adequate three-dimensional set to facilitate cell
adhesion, modulate the immune host response, and promote tissue regeneration upon
biomaterial implantation and subsequent biodegradation (12). All decellularization methods
seem to inevitably alter the composition and ultrastructure of the ECM and remove some of
these desirable components. Therefore, the balance between decellularization processes (e.g.
chemical, enzymatic, and physical) and conservation of biologically active molecules that
favorably modulate the host response should be evaluated using the native ECM as a control.
When a biomaterial is inserted into the body, an inevitable inflammation process occurs
representing a first-line protective response of the host and it dictates the final outcome and
integration of an implant. The imune response to a biomaterial implant begins with the innate
imune system, which includes neutrophils and macrophages and the complex network of
cytokines they release that will trigger a cascade of several imune responders. Macrophages
exhibit a spectrum of transient polarization states related to their functional diversity, namely
the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. The design of
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immunomodulatory materials able to regulate the host inflammatory response is a recent and
exciting área of research in the biomaterials field.
The main goal of the present study was to develop and characterize a self-regenerative
inducer bilayer material for oral and maxillofacial applications that simultaneously promote hard
and soft tissue formation and modulate the immune response.
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2. Materials and Methods
Preparation of the injectable bilayer hybrid material formulations and procedures were
adapted and optimized from earlier studies of our group (2, 13-16).
2.1. Microspheres specifications and characterization
2.1.1. Microspheres specifications
Strontium hydroxyapatite microspheres (Sr-HAp microspheres) and Calcium
hydroxyapatite microspheres (Ca-HAp microspheres), with a diameter comprised between 540-
560 µm, were kindly donated by Fluidinova S.A. (Maia, Portugal) and prepared according to a
procedure described elsewhere (1-3). For the preparation of the microspheres a commercial
nanostructured synthetic hydroxyapatite powder was used (nanoXIM 202, Fluidinova S.A.) with
a particle size (d50) of 5,0+-1,0 µm, and a bulk density of 0,50 +- 0,10 g/cm3.
2.1.2 Microspheres characterization
Physical-chemical characterization of the Sr-HAp microspheres was measured using
different analytical techniques, following described.
2.1.2.1. Environmental Scanning Electron Microscope (SEM/EDS)
The exam was performed using a High resolution (Schottky) Environmental
Scanning Electron Microscope with X-Ray Microanalysis and Electron Backscattered Diffraction
analysis: Quanta 400 FEG ESEM / EDAX Genesis X4M.
Microspheres were coated with carbon, ink thin film by sputtering, using the SPI Module
Sputter Coater equipment.
2.1.2.2. Zeta Potential Electro Kinetic Analyser (EKA) Electro Kinetic
Analyser
Zeta potential of Sr-Hap and Ca-HAp hydroxyapatite microspheres was
determined from streaming potential measurements with a commercial electrokinetic analyzer
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(EKA, Anton Par GmbH), using a special cylindrical cell with an insert for granular samples. The
electrolyte used was 1 mM KCl and the pH was adjusted to normal and inflammatory
physiological values (7,4 and 5,4 respectively).
2.2. Alginate sterilization
Ultra-pure sodium alginate (NovaMatrix, FMC Biopolymers) with more than 60%
content of guluronic vs. mannuronic acid units was used in the study. Alginate was dissolved in
sterile distilled water at 0.5%, filtered in 0,22 µm Steriflip units (Milipore) followed by a
lyophilization process and storage at -20 °C.
2.3. Fetal membranes collection, preparation and characterization
2.3.1. Collection and storage
With proper informed consent, in accordance with the tenets of the Declaration
of Helsinki for research involving human subjects, placentas (n=3) were obtained at the time of
delivery. To ensure the highest quality, samples were collected after caesarean section under
sterile conditions through approved protocols by Ethic Committee of the Aveiro Hospital.
Samples were kept at 4 °C, in normal 0.9%, saline solution and processed in 24 h.
2.3.2. Preparation and decellularization of FM
Placentas were processed under sterile conditions. AM and CM were stripped from each
placenta and washed with sterile solutions supplemented with antibiotics and fungicide. In order
to obtain AM and CM as separated membranes, AM was peeled off from the CM. Cleaned
membranes were trimmed and placed individually in sterile DMEM:Glycerol (ratio 1:1) and
freeze-preserved at -80 °C for further analysis.
For the amniotic and chorionic membranes a decellularization protocol previously optimized in
our group was used (17).
Briefly, fetal membranes were incubated with hypotonic buffer A (10mM Tris, 0,1%
EDTA, pH 7.8) for 18 h. Tissue pieces were washed with PBS and decellularized for 24h with
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1% Triton X-100 + 1M DMSO. Following three washes with hypotonic buffer B (10 mM Tris,
pH 7.8) a 3 h digestion at 37 °C was performed using 100 U/ml DNase (Applichem) prepared in
20 mM Tris and 2 mM MgCl2, pH 7.8. Native FM were used as control.
After decellularization membranes freezed at -80 °C were lyophilized to be used in the
preparation of the bilayer biomaterial.
2.3.4. Tissue Histology analysis
Samples were processed and embedded in paraffin. 3 µm sections were stained
with Hematoxylin and Eosin (HE), slides were visualized under a light microscope and imaged.
2.3.5. Transmission electron microscopy (TEM)
In brief, samples were fixed overnight with 2.5% glutaraldehyde/ 2% paraformaldehyde
in cacodylate buffer 0.1 M (pH 7.4). Samples were washed in 0.1 M sodium cacodylate buffer
and fixed in 2% osmium tetroxide in the 0.1 M sodium cacodylate buffer overnight, followed by
new fixation in 1% uranyl acetate overnight.
Dehydration was performed in gradient series of ethanol solutions and propylene oxide
and included in EPON resin in a silicon mold, by immersion of samples in increasing series of
propylene oxide to EPON (till 0:1 ratio) for 60 min each. Sections with 60 nm thickness were
prepared on a RMC Ultramicrotome (PowerTome, USA) using a diamond knife and recovered to
200 mesh Formvar Ni-grids, followed by 2% uranyl acetate and saturated lead citrate solution.
Visualization was performed at 80 kV in a (JEOL JEM 1400 microscope) and digital images were
acquired using a CCD digital camera Orious 1100 W.
2.3.6. Environmental Scanning Electron Microscope (SEM/EDS)
FM were coated with gold ink thin film by sputtering, using the SPI Module
Sputter Coater equipment.
2.3.7. Fourier Transform Infrared Spectroscopy (FT-IR)
Native, decellularized and decellularized and lyophilized AM and CM
membranes from three donors were analyzed using a Perkin Elmer Frontier system in Attenuated
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Total Reflectance (ATR) mode. A resolution of 4 cm-1 was used and 200 scans were acquired in
each analysis.
2.3.8. Atomic Force Microscopy (AFM)
The native and decellularized AM and CM membranes were fixed using a double-face
tap. AFM images of membranes were obtained with a PicoPlus scanning probe microscope
interfaced with a Picoscan 2500 controller (Keysight Technologies, USA) using the PicoView
1.20 software (Keysight Technologies, USA) Each sample was imaged, in air, with a 50×50 µm2
piezoelectric scanner. All measurements were performed in Contact mode at RT, using triangular-
shaped cantilever silicon tips (AppNano, USA) with a spring constant of 0.389 N/m. The scan
speed was set at 1.5 l/s. The roughness measurements of Ra, Rq and Rmax.values was obtained
using the WSxM software (18).
2.4. Bilayer hybrid system preparation
For the hybrid system preparation, sterile alginate was dissolved in 0,9% (w/v) NaCl
solution under sterile conditions to yield a 4 % (w/v) solution and mixed with an aqueous
suspension of SrCO3 (Sigma) as a source of Sr ions at SrCO3/COOH molar ratio 1.6. A solution
of glucone delta-lactone (GDL/ Sigma) was added to trigger gel formation by a release of Sr2+
caused by an acidic pH. The final crosslinked gel had a concentration of 3,5% (w/v) and a
carbonate/GDL molar ratio of 0,125.
For the preparation of the layer with ceramic microspheres, Sr-HAp microspheres were added to
the alginate solution in a concentration of 35% /w/v). Lyophilized AM and CM membrane
particles in a 1:1 ratio and a concentration of 20 mg/ml were use in the preparation of the bilayer
hybrid material.
In resume, cylinders of 4 mm in diameter and 5 mm in height of the bilayer material used
in the different tests performed had the following composition:
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- Layer 1: alginate (3.5% (w/v) + Sr-HAp microspheres (35% /w/v) + Fetal membranes
(20 mg/ml)
- Layer 2: alginate (3.5% (w/v) + Fetal membranes (20 mg/ml)
2.5. Bilayer hybrid system characterization
2.5.1. Micro CT characterization
The sample was scanned in high-resolution µCT Skyscan 1276 scanner (Skyscan, Kntich,
Belgium) mode, using a pixel size of 3.99 and an integration time of 2,15 s. The X-ray source
was set at 70 kV of energy and 57 µA of current. A 0,5 mm-thick aluminium filter and a beam
hardening correction algorithm were employed to minimize beam-hardening artifacts (Skyscan
hardware/software).
Additionally, 3D virtual models were created, and visualized using image processing
software (NRecon 1.7.4.2 /Skyscan1276), with a total of 1922 slices. Fully automated computer
algorithms were applied for segmentation and analysis, using ITK and VTK toolkits.
2.5.2. Tissue Histology analyze
Samples were processed and embedded in paraffin. 3 µm sections were stained with
Hematoxylin and Eosin (HE), Slides were visualized under a light microscope and imaged.
2.5.3 Dynamical mechanical analysis (DMA)
DMA compressed mode (DMA 8000, Perkin Elmer) was used to study the viscoelastic
properties of the bilayer material. Cylinders of 4 mm in diameter and 5 mm in height of the bilayer
material were tested. The samples were subjected to compression cycles of 1 Hz frequency using
a displacement amplitude of 0,054 mm and a compression ratio of 1,5 (drive control mode), at
room temperature of 20 °C to prevent dehydration of the scaffolds. Samples were tested for 15
minutes.
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2.5.4. Rheometer analysis
Cylinders of the bilayer material were tested using a Kinexus Pro – Malvern rheometer.
Tests were performed at room temperature (25°C). A 0,5 Hz frequency sweep tests were applied,
by 15 min.
2.6. In vitro biological response
2.6.1. Cell culture
Human primary monocytes from four healthy blood donors were isolated from buffy
coats (BC), kindly donated by Instituto Português do Sangue from Hospital São João, Porto,
Portugal.
Isolation was performed following previous group developed protocol (19) by negative
selection, using a RosetteSep isolation kit (Stem Cell Technologies SARL). The mixture was then
diluted at a 1:1 ratio with PBS: 2% FBS, layered over Histopaque (Sigma Aldrich, System
Histopaque 1077) and centrifuged. The enriched monocyte portion was carefully collected and
washed with PBS and centrifuged. The pellet was resuspended in culture medium (CM-RPMI
1640 + Glutamax (invitogen, Paisly, UK)), supplemented with 10% FBS and 1% Pen/Strep
(Invitrogen).
2.6.2. Macrophages Polarisation into pro-inflammatory - M1 or anti-inflammatory
- M2.
Cell culture was stimulated with 10 ng/ml of lipopolysaccharide (LPS), and M2 was
stimulated with 10 ng/ml of IL-10.
To analyze the influence of the biomaterial in the macrophages polarization, a Transwell
Assay (Corning Tranwellâ Insert) system (3.0µm pore size) was used on top of 5 x 105
macrophages seeded onto 2D surface of cell culture dishes, of a 6-well compartment plate
maintained at 37 °C for 72 hours, in RPMI supplemented with 10 %FBS (Biowest), and 1% P/S
(Invitrogen).
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2.6.3. Flow cytometry analysis
Macrophages polarization was routinely checked by flow cytometry, as describes
previously (19). Fluorescence was measured using a FACS Canto II and 10,000 events were
collected per sample. Analysis were performed using FlowJo Software.
5.6.4. ELISA assays
For cytokine evaluation, different cytokines were quantified in cell culture supernatants
using commercially available ELISA MAXä (BioLegendâ, Dan Diego, CA, USA) kit. Human
IL-10, IL-4, IL-6 and TNF-a, were performed according to the manufacturer’s protocol. Samples
concentration (pg/ml) were determined from the mean absorbance values for each set of samples,
compared to a standard calibration curve.
2.7. Statistical analysis
Statistical analysis was performed using GraphPad Prism Program. To compare data from
two different groups Wilcoxon tests and Mann-Whitney tests (or its parametric counterpart
Student’s t-test) were used. When comparing three different groups, non-parametric Kruskal-
wallis followed by Dunn’s Multiple Comparison test (or their parametric counterpart ANOVA)
were used, Wilcoxon tests and Mann-Whitney tests, were used. A value of p < 0.05 was
considered statistically significant.
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3. Results
3.1. Microspheres characterization
3.1.1. Environmental Scanning Electron Microscope (SEM/EDS)
3.1.2. Zeta Potential analysis
Zeta Potential of a surface influences its interaction with proteins and cells.(39). At pH ~
7,4 and 5,4 the microspheres were negatively charged. ZP average values obtained were: -11,61
mV (Sr-HAp) and -12,08 (Ca-HAp) mV, -14,45 mV (Sr-Hap) and -12,55 (Ca-Hap) mV,
respectively.
Figure 1: SEM images of 500-560 um Nano Sr-HAp porous microspheres, with increasing magnification,
revealing the microstructure of the particles. General view of the microspheres showing their porous
interconnected structure. X-ray diffraction pattern of the porous Sr-hydroxyapatite microspheres, showing
components present in microspheres.
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3.2. Fetal membranes characterization
3.2.1. Tissue Histology Analyze
Histological analysis showed that the protocol used to decellularize the membranes was
efficient since no cells were observed after decellularization and the integrity of the membranes
was preserved (Fig.2).
Figure 2: Characterization of amniotic native membranes (AM) - (a; c) and chorionic native membranes (CM) - ((e;
g), and characterization of the resulted decellularized AM (b; d) and the decellularized CM (f; h). H&E stainings -
DNA quantification (blue) elastic fibers (red) -. Picrosirius Red/Alcian Blue staining – Collagen (red) and
sGAGs(blue). Overall, decellularization protocol preserved the ECM components of the membranes.
H&
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3.2.2. Transmission Electron Microscopy (TEM)
TEM results confirmed the efficacy of the decellularization method used showing empty nucleous
and the morphology of the membranes preserved (Fig. 3).
Figure 3: TEM analyze of the AM - native (a, b, c); AM - decellularized (d, e, f); CM - native (g, h, i); CM - decellularized (j, k, l)
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3.2.3. Fourier Transform Infrared Spectroscopy (FT-IR)
FTIR analysis of the membranes showed that no significant changes were observed in the
composition of the membranes after decellularization and liophylization (Fig. 4). Bands at
approximately 2960 cm-1 that are assigned to an asymmetric stretching mode of CH3 group
decreases after decellularization possible due to cells loss in the membrane matrices.
FTIR_236_1FTIR19_237_1FTIR19_ 227_1
NameAmostra 006 por Administrator data terça-feira, maio 14 2019Amostra 007 por Administrator data terça-feira, maio 14 2019
Description
4000 4003500 3000 2500 2000 1500 1000 500cm-1
100
7172747678808284869092949698
%T
100
8284
86
88
90
92
94
96
98
%T
103
74767880828486889092949698100
%T
Native Chorionic Membrane
Decellularized Chorionic Membrane
Decellularized and Liophylized Chorionic Membrane
1634,00cm-11538,84cm-1
1448,36cm-1
3288,21cm-1
1398,90cm-1
1236,15cm-1
2928,59cm-11079,43cm-1 559,27cm-1
1634,00cm-11538,74cm-1
3281,18cm-1
1447,97cm-1
1 2 3 7 , 2 4 c m - 1
5 5 5 , 4 3 c m - 1
1633,84cm-11538,57cm-1
3280,53cm-1
1447,43cm-1
1236,36cm-1
1078,48cm-1667,87cm-1
1030,60
1400,441030,60
1078,49
1033,26549,00
1397,78
FTIR19_234_1FTIR19_235_1FTIR19_ 224_1
NameAmostra 002 por Administrator data terça-feira, maio 14 2019Amostra 003 por Administrator data terça-feira, maio 14 2019
Description
4000 4003500 3000 2500 2000 1500 1000 500cm-1
100
777880828486889092949698
%T
100
67707274768082848690929496
%T
101
87888990919293949596979899100
%T
Native Amniotic Membrane
Decellularized Amniotic Membrane
Decellularized and Liophylized Amniotic Membrane
1635,06cm-11538,62cm-1
1448,49cm-1
2922,42cm-13282,77cm-11235,15cm-1
1079,46cm-1 559,76cm-1
1628,35cm-11539,19cm-1
1448,18cm-13288,02cm-1
1399,28cm-1
1236,37cm-11336,78cm-1
2933,17cm-1555,17cm-11079,55cm-1
1634,61cm-1
1548,82cm-1
3287,72cm-11452,00cm-1
1236,82cm-11058,28cm-1
543,60cm-1
1395,12
1030,60
668,74
857,651400,44
1339,25
1160,98
1206,21
937,47
2933,04
Figure 4: FTIR analysis of the membranes showed that no significant changes were observed in the
composition of the membranes after decellularization and liophylization
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3.2.4. Atomic Force Microscopy (AFM)
Evaluation of AFM showed that in the 2-D images of height data, bright area represents
a high peak or projection whereas dark area represents surface depressions. The 3-D image
confirmed the depression occurred by ridges visualization on the membrane. Membrane surface
was typically heterogenous in all samples, although with the decellularization process the surface
become more homogeneous. Significant differences in roughness (p < 0,05) weren’t seen between
AM and CM native and decellularized samples (Sa, Sq, Sz), however between AM & CM native
and decellularized samples (Sa, Sq, Sz) differences were observed, with more pronunciation at
Sz scale (Fig.5). The morphology of CM and AM decellularized samples was similar and the
irregularity surface were yet obvious (Fig.5). In the CM native membranes, the structural matrix
tended to have more irregularities and depression marks on the surface, compared to AM native
(Fig.5). 2-D and 3-D topography images confirmed differences noticed (Fig-5). The peaks were
prominent in the 2-D in Peak Force Error channel image, for both native membranes, which were
confirmed by more rough peaks in the 3-D image. On the other hand, with the decellularization
process we observed a clearance of the depressions.
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Figure 5: Surface analysis of decellularized AM and CM evaluated by AFM. Sets of high resolution topography ((image
scale: 50x50 μm) are depicted in (C) three-dimensional presentation, (A) height where dark areas represented
depressions and brighter areas were protrusions on the surface, and (B) amplitude showing the deflection generated by
cantilever tip when it encountered the sample topography.
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3.3. Multilayer Hybrid Biomaterial Characterization
Micro-CT images show that microspheres are well distributed inside the hydrogel, with
preserved size and shape. Although crosslinked alginate hydrogel is transparent, the presence of
denser particles distributed in the alginate and between the microspheres was observed, possibly
corresponding to non-dissolved Sr-carbonate. In Fig. 7 H&E staining’s of a cut of the bilayer
system is also represented for comparison. It is observed that Sr-HAp microspheres are embedded
within the crosslinked alginate transparent hydrogel and surrounded by fetal membrane particles.
Sa Sq Sz0
2
4
6
8
10
AM Native AM Decellularized CM Native CM Decellularized
µm
Figure 6: Mean and standard deviations of the surface analysis of decellularized
AM and CM evaluated by AFM.
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3.3.3. DMA tests
DMA tests were performed in order to evaluate the mechanical behavior of the bilayer
material in compression dynamic conditions. The Figure 8 illustrate the viscoelastic behavior of
the bilayer Sr-HAp hybrid system scaffold. The storage modulus (E’) - Pa represents the elastic
Figure 7: Micro-CT analysis of the scaffold. (1, 2) - 3D reconstructed image and respective orthogonal slices of micro-CT
acquisition. (1b; 2) - Sr-HAp microspheres are represented in white & yellow. (1, 1a, 1b, 2) -Denser particles are represented in
red, possibly corresponding to non-dissolved Sr-carbonate. (2a-d) - H&E stainings: Decellularized chorionic (CM) and amniotic
membranes (AM), these images show the histological structure analysis of the scaffold.
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component of the scaffold and reveals the capability of a material to accumulate energy during
deformation. The Loss factor (E’’) gives information about the viscoelastic properties of the
material and tan delta represents the ratio between the amount of energy dissipated by viscous
mechanisms and the energy stored in the elastic component. It is observed that the elastic behavior
of the hybrid is dominant over the viscous one.
Figure 8: DMA tests of bilayer Sr-hybrid system. In the scaffold preparations microspheres were sintered at 1200ºC.
Mean values of Storage (E’) Pa Modulus, loss modulus (E’’) Pa Loss Modulus, both left Y axis, and tan delta (right
axis) for bilayer Sr-hybrid.
3.3.4. Rheometric analyses
Figure 9 represent of display how shear storage moduli (G’) and shear loss moduli (G’’)
variated with frequency. For the analyses the values of G’ and G’’ are nearly independent, which
confirm solid-like mechanical behavior. The G’ values are superior than G’’ for all models,
indorsing that the elastic properties are dominant.
Pa Modulus Pa Loss Modulus Tan Delta0
20002500
3000
3500
4000
18000
20000
22000
24000
0.14
0.15
0.16
0.17
0.18
Time
Pa
Pa Modulus
Pa Loss Modulus
Tan Delta
Tan Delta
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Figure 9: Rheological tests of bilayer Sr-hybrid system. In the scaffold preparations microspheres were sintered at
1200ºC. Mean values of Storage (G’), loss modulus (G’’) for bilayer Sr-hybrid.
0 5 100
100000
200000
300000
f (Hz)
Pa
G'G''
0 2 4 6 8 10 12 14 16 1830
50000
100000
150000
200000
250000
Time (min)
Pa
G''G'
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3.3.5. Flow cytometry analysis
In order to understand the mechanisms involved in differentiation and activation of
monocyte-derived cells in tissue regeneration by the scaffold, flow cytometry analysis was
performed to evaluate the expression of macrophage surface markers for M1 (CD14+HLA-DR+)
and M2 (CD14+CD163+) phenotype (Figure 9). The results obtained indicate that M1 and M2
markers were decreased in all three conditions, in relation to the control groups. Control groups
include naive (M0), M1 and M2 macrophages, stimulated with LPS and IL-10, respectively. M1
control group has increased surface expression and MFI of HLA-DR, while M2 has increased
CD163 surface expression and MFI, in comparison with M0. Surprisingly, CD163 levels were
high in all the control macrophages of this blood donor. However, the low levels of HLA-DR
observed in the 3 conditions tested revealed that none of the conditions tested, the bilayer Sr alone,
the bilayer Sr with FM and the FM, promote significant macrophage activation.
Figure 10: Expression of cell surface markers of macrophages differentiation on Scaffolds (Bilayer Sr, Bilayer Sr + FM, FM) and the control groups
(M0, M1, M2). (A) Human primary monocyte derived macrophages, differentiated for 10 d, were harvested and cells surface stained with the indicated
antibodies before FACS analysis. Stimulation with LPS and IL-10 in control groups M1 and M2, respectively, were performed.
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M0 M1 M2
Bilaye
r Sr
Bilaye
r Sr +
FM FM0
20
40
60
CD14+/HLA-DR+
M0 M1 M2
Bilaye
r Sr
Bilaye
r Sr +
FM FM0
20
40
60
80
100
CD14+/CD163+
M1 M2
Bilaye
r Sr
Bilaye
r Sr +
FM FM
0
50
100
150
200
600
800
1000
1200
1400
MFI
HLA
-DR
M1 M2
Bilaye
r Sr
Bilaye
r Sr +
FM FM
0.00.20.40.60.81.0
68
1012
100020003000400050006000700080009000
10000
MFI
CD1
63
0.00000.00050.00100.00150.00200.00250.0030
0.005
0.0100.250.500.751.00
Fold
Cha
nge
Bilayer Sr + FM
FM
Bilayer Sr
HLA-DR CD163
Figure 11: Scatter charts represent the mean fluorescence intensity (MFI) for each cell surface markers from one donor and the fold
change for each cell surface markers from one donor.
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3.3.6. ELISA assays
In order to complement the analysis of cell surface expression by flow cytometry,
the production of pro and anti-inflammatory cytokines was analyzed by ELISA (Figure 11).
Results from figure 11, show that M1 macrophages produced increased levels of IL-6 and TNF-
a while M2 macrophages presented higher levels of IL-10 in the medium, which was expected
since these cultures were IL-10 supplemented. Nevertheless, IL-4 production was not detected in
M2 macrophages, which was surprisingly. Still, bilayer Sr biomaterials seems to promote IL-6
and TNF-a production, compared with naïve macrophages, although in lower levels than M1
macrophages. The presence of FM in the bilayer Sr hybrid did not affect IL-6 secretion but
decreased TNF-a production by macrophages. FM by itself seems to promote IL-6 and IL-4
production
Figure 12: Cytokine production by Scaffolds-differentiated macrophages and controls. Cytokine release
was determined by ELISA, using cell culture supernatants from differentiated human primary monocyte-
derived macrophages.
0
10
20
30
40
50
60
100150200250300450600750900
pg/ml
M0
M1
M2
Bilayer Sr
Bilayer Sr + FM
FM
IL-6 IL-10 IL-4 TNF-alpha
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4. Discussion
Hydrogel revealed a promising approach in regenerative responses acting on cell
behavior during the tissue reconstruction (3-5, 11, 12, 40).
Therefore alginate hydrogels are very useful in cell therapy approaches to achieve a
controlled release of cells in space and time, biocompatibility, non-toxicity, mild gelation and
retention of physical structure with similarity to ECM of the nature tissues, maintain a
physiologically moist environment, absorbing excess wound fluid and minimize bacterial
infections (10, 13, 41-43). By action of divalent cations, alginate can be prepared through various
cross-linking hydrogels methods, supplemented with the ability of delivering bioactive agents for
tissue regeneration. (44-46). The resulted hydrogels properties depend in type of crosslink
configurations, such elasticity, stability and stiffness (1, 10, 44). Studies (8, 45) reveal that a high
content of G provides stiff, stable and a high porosity gels, contrarily to low-G or high-M
proportion alginates, leading to a more elastic and less stable Ca gels (1, 44). In the present work
the guluronic vs mannuronic acid alginate units used had more than 60% of G content. However,
in this study we used as divalent ion Sr2+ cation with a high affinity toward alginate and a
beneficial bind to G and M blocks, suitable for the alginate immobilization of living cells in long-
term perfusion studies in relation to Ca2+. (47, 48). The integration of Sr in microspheres and in
the alginate as a hybrid system vehicle, allows the early stage release of Sr2+, at implantation
time, due to the different degradation kinetics of the polymer channels, in relation to ceramic
microspheres (15). Strontium will be released locally enhancing bone formation, and it wouldn’t
be expected relevant systemic effects (3, 15). The main disadvantage concerning load-bearing
applications is alginate low mechanical properties, which can be overwhelmed through the
reinforcement with ceramic constituents (49). Using the same average diameter of 540-560 µm,
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of micro HAp-microspheres, in the hybrid system, used in previous group studies (1, 3), allowing
an adequate mechanical resistance and space between particles to facilitate in situ cell
colonization and invasion by blood vessels, we expect, by using a nano-Hap synthesis, with
different shaping, porosity and rearrangement structure, for bone regeneration, that the biological
activity will be improved (50-54). A study of Hulbert and Young (55) showed that pores greater
than 100 µm promote bone growth through the material, allowing the formation of new bone and
developing a capillary system. Zeta Potencial (ZP) (39) interactions molecules and porous
materials may take place on an inorganic surface, such Van der Waals bonds, hydrogen,
hydrophobic and hydrophilic. At pH ~ 7,4 and 5,4 the microspheres ZP medium average value
obtained were: -11,61 mV (Sr-Hap) and -12,08 (Ca-Hap) mV, -14,45 mV (Sr-Hap) and -12,55
(Ca-Hap) mV, respectively. Although, we don’t have a statistically significance between the two
values, both of them represent a negative ZP, proving their favor to osseointegration, apatite
nucleation and bone formation facilitating bone cell activity, therefore a potential implant material
(56). A study of Cooper and Hunt (57) analyzed the selected expression of osteogenic markers
such: alkaline phosphatase, osteocalcin, osteopontin, collagen type I, and core binding factor
alpha-1 (CBFA1), by RT-PCR, with positive and negative ZP values. The ability of create a
microenvironment capable of absorb specific ECM proteins onto his surface providing an integrin
mediator for osteoblast attachment, on an in vivo studies (58, 59) a negative surface charge
biomaterial placed into healthy bleeding bone, provides an acceleration of the dynamic bone
growth cascade (58-61). In fact, a study (61), compared the negative and a positive surface
materials, and concluded, that negative surfaces materials have superior osteoconductive
properties, and grater osteogenic markers were expressed, showing that bone, on pH (6-8) has a
electronegativity in order of -80 mV and a osteoblast had a -29,4 to -52,4 mV on a range of 7,3
to 7,5 pH values, respectively, giving a significant bone electronegativity differences of 68,38
mV and a 67,92 mV at 7,4 pH, and 65,55 mV and 67,45 mV at 5,4 pH for Sr-Hap and Ca-Hap
microspheres, respectively. The hydroxyapatite powders used to produce the macroporus
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microspheres possess a round shape and organized themselves as aggregates. (Fig. x). The
particles present an average diameter of (…)
The micro and macroporosity in addition to interconnectivity of the Sr-Hap porous
microspheres were analyzed and the microporous scaffolds posses an oval shape and organized
themselves as aggregates (Fig.x). Average particles diameter of 500-560 µm (figx), with a pore
diameter ranging from x were observed.
In this new design we used amniotic and chorionic particles membrane are combined to
form a graft, which result in a more effective regeneration by delivering both growth factors (29),
by using an effective decellularization protocol of both membranes clarify a new view of tissue
engineering.
AFM seemed to offer an ultrastructural images of surface morphology information,
complementary the images obtained by SEM. The measurement of surface roughness can be used
as a guide for, not only for cell shipping agent, cellular response to apoptosis and chemical stress,
but also to an indicator in selecting and planning a scaffold design for tissue engineering.
Bone have a viscoelastic behavior and the dissipation of energy by bone is influenced by the
viscoelastic properties of the material in a bone filing defect and influenced by the response of
surrounding healthy and damaged tissue (1), exhibiting both creep and stress relaxation (62). This
viscoelastic property determines the capacity of dissipation of energy and may differ between
macroscopic and microstructural levels (1, 62). sDMA tests revel that E’ is higher than E’,
representing that the elastic behavior of the bilayer hybrid is ascending over the viscous one, plus
any of the layers disattached from the other, maintaining the interface between them intact. On a
group study (1) before, concluded that the storage modulus was proportional to the Hap
microspheres content, acting as reinforcement filler, on a sustained load from the hydrogel to the
microspheres. Although mechanical properties of hydrogels are lower, below cancellous bone
(20-5000 MPa), they can be used on non-load bearing sites, or used a supplementary internal
fixation (1, 63, 64). However, this material will provide an acceptable mechanical streght after
surgery with a gradual replacement by healthy, strong bone, maintaining of part, the
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microspheres. This is the reason we have a timepoint delivery Sr2+ flow greater in the hydrogel
matrix, than the microspheres, proved on a recent study group (3, 15, 63).
It is also important, the understanding of the biomaterials and biologics in craniofacial
reconstruction (65-67) and biomechanics (63), the necessity of have in consideration, not only,
the bone structure by an anisotropic level (compression, tension, shear), but also, the role of
muscle plays on bone strength, mechanical protection, preserving and repairing bone tissue, some
myokines secreted by muscle as growth factor which stimulate the bone formation (IGF-1, FGF-
2) or suppress their healing or formation (GDF-8, TGF-b1), matrix molecules promoting
mineralization, healing and remodeling (SPARC, MMP-2, BMP-1), and inflammatory factors,
causing bone resorption (IL-6, IL-7, IL-15) (68).
Biomaterials implantations causes injury that will lead to an acute inflammatory response,
the magnitude and duration of the inflammation process are controlled by active resolution
mediators, enhancing clearance of inflammation within the lesion and thus promoting tissue
regeneration (69). Macrophages are the predominant mediators throughout the immune response
to biomaterials. Studies shows that early macrophages phenotype determines the end stage
outcome in several biological matrices, particularity, an increased ratio of M2/M1 macrophages
correlates to enhances remodeling (70).
Chemistry surface/topographic features of biomaterials influence the function of different
cells, including macrophages and their interaction with the host and cell response by tailoring
chemical, temporal and mechanical characteristics (52, 54, 69, 71-76). Through biomimetic forms
and an immune-informed design, we can positively interact with the macrophages, by his pivotal
role, improving positively the response after biomaterial implantation, mediating tissue
remodeling (69, 77). The complexity of macrophages understanding lies on interaction between
the biomaterial and the host microenvironment, local and systemically. In reality, macrophages
adjust their polarization states in response to their microenvironment (69), studies suggest that
the presence of different states of polarization in the same microenvironment, induce a helpful
remodeling mechanisms (78, 79). The ideal high M2:M1 ratio is a key of the perfect design to
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enhance positive tissue integration, remodeling and regeneration, which integrin-ligand
interactions network of the material affect cell shape, function and mobility (80). Through RGD
hydrogels Blankney et al. (72) concluded that hydrogels are capable of a reduce inflammatory
response. Previous studies group (15), show that the using of a Sr-hybrid system (Hap
microspheres doped with Sr alginate vehicle crosslinked in situ), increase F4/80+/CD206+ cells
(M2- phenotype macrophages), in inflammatory exudates, with a decrease of inflammatory
cytokines (TNF-RI, MIP-1gamma and RANTES, at day 3, and concluded that Sr- releasing hybrid
system promoted an M2 regenerative response towards a reparative phenotype.
The Nano topography of the Sr-Hap microspheres module cell response, controlling cell
shape and elasticity (72, 81), pore size of 34 µm showed reduce fibrous encapsulation (82).
McWhroter et al. (74) identified that M1 cells assume a rounded shape and M2 cells adopt an
elongated shape using models of 50 and 20 µm, respectively. Adding that the elongation of M2,
synergized the cytokines induced (IL-4, IL-3) by these cells which by biophysical signals
presented in the biomaterials can have compliment effects on native environment. M1 initiate
angiogenesis by secreting VEGF, and M2 promote vessel maturation, by PDGF-BB and MMP9
in the later stages, as insulin-like growth factor (73, 83).
In this study, the data obtained indicates a low macrophage activation compared to M1
control. This conclusion was based essentially on the combined analysis of cell surface markers
and cytokine secretion profile. Regarding cytokines associated to M2 phenotype, we cannot rely
on IL-10 quantification, because it was exogenous added, and surprisingly IL-4 was not detected
in M2 macrophages. Nevertheless, the FM by itself seems to promote IL-6 and IL-4 production.
This effect was not observed when the FM was incorporated in the Sr hybrid system, probably
because it has not been released in significant amounts in such a short period of time (72h).
Interestingly, IL-6 is a pleiotropic cytokine with an important mediator of cellular
communication, orchestrating both pro- and anti-inflammatory response, by increasing and
reducing the inflammation process (84). Although several studies show us that M2/repair does
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not require T cell cytokines, depending the type of activity in resident macrophages, and IL-4
stimulated macrophages very closely resemble to resident macrophages (85, 86). So, the ratio of
M1/M2 balance needs to be carefully analyzed, which macrophages function needs to be
addressed to modulate inflammation (87, 88). Remember that inflammation markers can be
uncooperative because whenever macrophages are present there is inflammation, which by we
cannot measured the inflammation, only by inflammatory markers (87, 89)
Therefore, in the future, different studies involving the interaction between macrophages
and biomaterials need to be further explored.
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5. Conclusion
We have developed a new bilayer strontium rich viscoelastic hybrid system herein
described, biologically enriched with amniotic and chorionic membrane particles, that can be
manually injected and sets in situ at body temperature, providing a scaffold for cell migration and
tissue ingrowth. When implanted, the biomaterial layer in contact with bone will offer structural
support while providing a temporary scaffold onto which new bone can grow. The incorporation
of two Sr release kinetics (from the alginate and from the microspheres), may further improve
effective bone regeneration. In opposition, the fetal membrane enriched alginate layer will
promote soft tissue formation. Preliminary results concerning immunomodulatory properties of
the developed system indicate that the bilayer biomaterial provides low macrophage activation.
Additional tests should be performed (on going work) in order to further explore the
immunomodulation capacity of the bilayer material.
The results obtained till now indicate that the new developed biomaterial might provide
a promising multifunctional approach for oral and maxillofacial reconstruction.
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Acknowledgments
This work was financed by FEDER funds, through the Programa Operacional Factores
de Competitividade — COMPETE, and by Portuguese funds through FCT — Fundação para a
Ciência e a Tecnologia in the frame- work of the project PTDC/CTM/103181/2008 and of project
Pest-C/SAU/ LA0002/2011.
FLUIDINOVA, S.A. Tecmaia - Rua Eng. Frederico Ulrich, 2650, 4470-605 Moreira da
Maia - Portugal, is also acknowledge for kindly offering the Sr-Ha microspheres.
The Dynamic Mechanical Analysis (DMA) - Tritec DMA 2000 (Triton Technologies /
Perkin Elmer, USA), Zeta Potential Electro Kinetic Analyser (EKA), Rheometry analyses
(Kinexus Pro - Malvern, UK) was performed at the Biointerfaces and Nanotechnology (BN) core
facility with the assistance of Ricardo Vidal Silva.
Atomic Force Microscopy (AFM) was performed at the Biointerfaces and
Nanotechnology (BN) core facility with the assistance of Manuela Brás.
The Micro CT Skyscan 1074 scanner (Skyscan, Kontich, Belgium) - was performed at
the Bioimaging core facility with the assistance of María Gómez Lázaro.
The Histology preparations was performed at the Bioimaging core facility with the
assistance of Cláudia Machado.
The authors thankfully acknowledge the use of of microscopy services at Centro de
Materiais da Universidade do Porto (CEMUP) and to Daniela Silva, responsible for the
experimental techniques mentioned.
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