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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

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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

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

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

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

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

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Acknowledgments………..…………...……...31 References……….………...3

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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,

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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 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& E P ic ros ir iu s R ed /A lc ian Bl u e H& E P ic ros ir iu s R ed /A lc ian Bl u e a b c d e f g 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).

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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_1 FTIR19_237_1 FTIR19_ 227_1 Name

Amostra 006 por Administrator data terça-feira, maio 14 2019 Amostra 007 por Administrator data terça-feira, maio 14 2019

Description 4000 3500 3000 2500 2000 1500 1000 500 400 cm-1 100 71 72 74 76 78 80 82 84 86 90 92 94 96 98 %T 100 82 84 86 88 90 92 94 96 98 %T 103 74 76 78 80 82 84 86 88 90 92 94 96 98 100 %T

Native Chorionic Membrane

Decellularized Chorionic Membrane

Decellularized and Liophylized Chorionic Membrane

1634,00cm-1 1538,84cm-1 1448,36cm-1 3288,21cm-1 1398,90cm-1 1236,15cm-1 2928,59cm-1 1079,43cm-1 559,27cm-1 1634,00cm-1 1538,74cm-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-1 1538,57cm-1 3280,53cm-1 1447,43cm-1 1236,36cm-1 1078,48cm-1 667,87cm-1 1030,60 1400,44 1030,60 1078,49 1033,26 549,00 1397,78 FTIR19_234_1 FTIR19_235_1 FTIR19_ 224_1 Name

Amostra 002 por Administrator data terça-feira, maio 14 2019 Amostra 003 por Administrator data terça-feira, maio 14 2019

Description 4000 3500 3000 2500 2000 1500 1000 500 400 cm-1 100 77 78 80 82 84 86 88 90 92 94 96 98 %T 100 67 70 72 74 76 80 82 84 86 90 92 94 96 %T 101 87 88 89 90 91 92 93 94 95 96 97 98 99 100 %T

Native Amniotic Membrane

Decellularized Amniotic Membrane

Decellularized and Liophylized Amniotic Membrane

1635,06cm-1 1538,62cm-1 1448,49cm-1 2922,42cm-1 3282,77cm-1 1235,15cm-1 1079,46cm-1 559,76cm-1 1628,35cm-1 1539,19cm-1 1448,18cm-1 3288,02cm-1 1399,28cm-1 1236,37cm-1 1336,78cm-1 2933,17cm-1 555,17cm-1 1079,55cm-1 1634,61cm-1 1548,82cm-1 3287,72cm-1 1452,00cm-1 1236,82cm-1 1058,28cm-1 543,60cm-1 1395,12 1030,60 668,74 857,65 1400,44 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 Sz 0 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 Delta 0 2000 2500 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 Ta n D elt a

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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 10 0 100000 200000 300000 f (Hz) Pa G' G'' 0 2 3 4 6 8 10 12 14 16 18 0 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 Bila yer Sr Bila yer Sr + FM FM 0 20 40 60 CD14+/HLA-DR+ M0 M1 M2 Bila yer Sr Bila yer Sr + FM FM 0 20 40 60 80 100 CD14+/CD163+ M1 M2 Bilaye r Sr Bila yer Sr + FM FM 0 50 100 150 200 600 800 1000 1200 1400 MFI HLA-DR M1 M2 Bila yer Sr Bila yer Sr + FM FM 0.0 0.2 0.4 0.6 0.8 1.0 6 8 10 12 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 MFI CD163 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.005 0.010 0.25 0.50 0.75 1.00 Fold Change 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 mTNF-acrophTNF-ages presented higher levels of IL-10 in the medium, which wTNF-as 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 60 100 150 200 250 300 450 600 750 900 pg/ml M0 M1 M2 Bilayer Sr Bilayer Sr + FM FM

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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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