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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

NEW MULTILAYER HYBRID MATERIAL FOR ORAL AND MAXILLOFACIAL RECONSTRUCTION - RODRIGO OSÓRIO DE VALDOLEIROS PEREIRA DA

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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

Rodrigo Osório de Valdoleiros�


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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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