Amniotic epithelial stem cell biocompatibility for

Materials Science and Engineering C 69 (2016) 321–329

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Amniotic epithelial stem cell biocompatibility for electrospun poly(lactide-co-glycolide), poly(ε-caprolactone), poly(lactic acid) scaffolds Valentina Russo a,b,1, Loredana Tammaro c,1,2, Lisa Di Marcantonio a,⁎, Andrea Sorrentino d, Massimo Ancora e, Luca Valbonetti a,b, Maura Turriani a, Alessandra Martelli a, Cesare Cammà e, Barbara Barboni a,b a

Faculty of Veterinary Medicine, University of Teramo, Campus Universitario Coste S. Agostino Via R. Balzarini 1, 64100 Teramo, Italy StemTeCh Group, Italy c Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, SA, Italy d Institute for Polymers, Composite and Biomaterials (IPCB), CNR, P.le Enrico Fermi 1, I-80055 Portici, Napoli, Italy e Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy b

a r t i c l e

i n f o

Article history: Received 22 December 2015 Received in revised form 28 May 2016 Accepted 29 June 2016 Available online 01 July 2016 Keywords: Electrospinning Biodegradable polymers Contact angle SEM Amniotic epithelial stem cells Biocompatibility

a b s t r a c t Three biodegradable thermoplastic polymers, poly(ε-caprolactone) (PCL), poly(L-lactide-co-D,L-lactide) (PLA) and poly(L-lactide-co-glycolide) (PLGA), have been used to produce nonwovens scaffolds with uniform micrometer fibres. Scaffolds' physical and morphological characterization was performed by X-ray diffraction, Scanning Electron Microscopy and Contact-Angle test. Morphological investigations revealed that all produced fibres were randomly orientated with interconnected pores ranging between 5 and 12 μm in diameter. An average fibre diameter of 1.5, 0.75 and 1.2 μm was found for PCL, PLA and PLGA, respectively. Moreover, experiments were designed to verify whether the fabricated electrospun substrates were biocompatible for ovine amniotic epithelial stem cells (oAECs) under in vitro conditions. Cell adhesion, survival, spatial organization on fibres, proliferation index, and DNA quantification after 48 h culture, showed an enhanced adhesion and proliferation, especially for PLGA scaffolds. The favourable interaction between oAECs and the fibrous scaffolds was attributed to the greatly improved porosity and pore size distribution of the electrospun scaffolds. In addition, AECs can be considered ideal for tissue engineering especially when using biocompatible and opportunely produced scaffolds. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Amniotic epithelial stem cells (AECs) are a promising alternative of stem cells to be used in tissue engineering, since they conjugate a remarkable plasticity with safety properties. Clear experimental

Abbreviations: PCL, poly(ε-caprolactone); PLA, poly(L-lactide-co-D,L-lactide); PLGA, poly(L-lactide-co-glycolide); AECs, amniotic epithelial stem cells; OCT-4, octamerbinding protein 4; SOX2, SRY-related HMG-box gene 2; NANOG, homeobox protein NANOG; TERT, telomerase reverse transcriptase; ECM, extracellular matrix; R.H., relative humidity; XRD, X-ray diffraction; SEM, scanning electron microscopy; EtOH, ethanol; dH2O, distilled water; PBS, phosphate buffered saline; FCS, fetal calf serum; EGF, epidermal growth factor; BSA, bovine serum albumin; PI, proliferation index; MHC, major histocompatibility complex. ⁎ Corresponding author. E-mail address: [email protected] (L. Di Marcantonio). 1 Equally contributed authors. 2 Present address: ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, SSPT-PROMAS-MATAS, S.S. 7 Appia, km 706-72100 Brindisi, Italy.

http://dx.doi.org/10.1016/j.msec.2016.06.092 0928-4931/© 2016 Elsevier B.V. All rights reserved.

evidences indicate that these kind of cells are a source of pluripotent stem cells [1–3]. These stem cells are obtained from the placenta, which is particularly interesting for two reasons: firstly, its immunological characteristics are fundamental for the maintenance of feto-maternal tolerance during pregnancy; secondly, placental tissues originate during the first stages of embryological development supporting the possibility that these tissues may still contain cells with an immature phenotype. These two features make placenta cells good candidates for a possible use in cell therapy approaches, with the possibility to provide immature cells with low immunogenicity properties, and thus being well tolerated after allo- and xenotransplantation [4–9]. In addition, in long-term transplantation experiments, amniotic tissues and cells did not cause any evidence of tumorigenesis, demonstrating the safety of this stem cell source [10–13]. Furthermore, given that the placenta is generally discarded after birth, the recovery of cells from this tissue does not involve any invasive procedure for the donor, and their use does not pose any ethical concerns.

322

V. Russo et al. / Materials Science and Engineering C 69 (2016) 321–329

Recent studies aimed at defining AECs characteristics, show that they express some of the surface markers associated with embryonic stem cells. These include OCT-4, SOX-2, TERT and NANOG pluripotency markers [1,14–16]. In sheep, these pluripotency associated transcription factors have been confirmed to be expressed [2,3,17], thus showing a conserved behaviour of this class of amniotic-derived cells. This allowed the use of ovine AECs (oAECs) in experimentally induced defects to evaluate their regenerative properties and safety on the ovine animal model of high translational value, in particular, for muscle skeleton diseases [6, 17–19] and in clinical studies on horses affected by spontaneous tendinopathies [7,9]. Thus, AECs retain great potential in cell-based therapies, although their performance in regenerative medicine would be increased by creating AECs engineered scaffolds allowing the regeneration of a tissue, and possibly develop future medicine therapies. Tissue engineering and regenerative medicine are commonly defined as being an interdisciplinary field that aims at the development of biological substitutes that restore, maintain or improve tissue function or a whole organ. Efforts have been directed to produce biocompatible scaffolds that physically support cells and provide conditions for cell adhesion and growth, mimicking the native extracellular matrix (ECM) of tissues. Those scaffolds can be obtained from different materials, including biodegradable polymers, which better support and promote cell viability and proliferation. In general, was found that both the high surface area and the interconnected porosity of the nonwoven structures allow a high percentage of cellular attachment. Additionally, was also found that fibres in the submicron range more closely resemble the size scale of extracellular components [20]. Electrospinning is a well-known method for production of micro and nanofibres through an electrically charged jet of polymer solution or melt. It can be modified in order to yield electrospun fibres with the desired morphology and properties. Furthermore, the large number of available polymeric materials potentially gives all possible morphologies, which in turn are able to mimic every type of natural extracellular matrix [21–27]. Poly(α-hydroxy esters), including poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and their copolymer poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone) (PCL) are the most commonly used synthetic polymers in tissue engineering, because of their well characterized biodegradable property and they are also FDA approved for clinical use. They have been the most extensively studied for obtaining nonwoven scaffolds in tissues regeneration. However, despite the huge literature present, only little attention was devoted to the correlation between fibre morphologies, surface properties and oAECs attachment, spatial organization and proliferation. In this work, three different microfibrous systems, based on PCL, PLA and PLGA, were fabricated by electrospinning technique and compared in terms of physical and morphological properties. Experiments were designed to verify whether the fabricated electrospun substrates were biocompatible for oAECs under in vitro conditions.

2. Materials and methods 2.1. Materials Poly(ε-caprolactone) (PCL, Mn = 80,000 Da, Capa™ 6800) was purchased from Perstorp (Sweden), poly(L-lactide-co-D,L-lactide) (PLA, 67:33 to 73:27 M ratio L-lactide: D,L-lactide, RESOMER 708) from Evonik Industries AG (Germany) and poly(L-lactide-co-glycolide) (PLGA, 85:15 L-lactide:glycolide, PURASORB PLG 8523) from Corbion Purac Biomaterials (The Netherlands). The solvents acetone and chloroform were supplied by Sigma-Aldrich (Italy) and used as received without further purification. 2.1.1. Scaffold preparation Polymeric solutions were prepared by dissolving the required amount of the polymer in hot solvent and stirring for about 2 h. Resulting solutions were then processed using a climate-controlled electrospinning equipment (EC-CLI by IME Technologies, The Netherlands). Several experiments were performed in order to optimize the electrospinning parameters for the three biodegradable thermoplastic polymers. Trials have been made by changing in sequence the applied high voltage, the needle-collector distance, the feeding rate, the temperature and the relative humidity (R.H.). In Table 1, the values, which guarantied materials to be processed into scaffolds showing a beadless fibrous morphology, are reported. The collected fibres were firstly placed in a vacuum oven overnight to fully eliminate any solvent residuals and moisture, and then stored over silica gel in a dryer. Control substrates in contact-angle tests were prepared by solvent casting. 10 ml polymeric solutions, previously soaked with the corresponding solvent, were poured into 80 mm diameter glass Petri dish and air dried overnight. After being peeled off, films were stored over silica gel in a dryer. 2.1.2. Methods 2.1.2.1. X-ray diffraction (XRD). XRD patterns data were collected in reflection geometry using an automatic Bruker D8 Advance diffractometer equipped with a continuous scan attachment and a proportional counter, with the nickel filtered Cu Kα radiation (λ = 1.54050 Å) and operating at 40 kV and 40 mA in the 2θ range of 5–40°, step size 0.05°, time per step 3 s. 2.1.2.2. Scanning electron microscopy (SEM). All substrates without cells were observed by scanning electron microscopy (FEI Quanta 200 FEG, Eindhoven, The Netherlands). Before the observation, the samples were directly coated with gold using the sputter coater (Agar Automatic Sputter Coater Mod. B7341, Stansted, UK) at 30 mA for 60 s. The diameter of the fibres was measured from the SEM micrographs. The average and standard deviation were reported. 2.1.2.3. Contact-angle test. To evaluate the wettability (or hydrophilicity) of the electrospun nonwoven materials, the water contact angle was

Table 1 Electrospinning parameters for PCL, PLA and PLGA nonwoven scaffolds. Material

Concentration [wt%]

Solvent

Flow rate [ml/h]

Voltage [kV]

Distance [mm]

Needle diameter [mm]

T [°C]

R.H. [%]

Fibre diameter [μm]

PCL PLA PLGA

14.0 2.5 8.0

Acetone Acetone Acetone/CHCl3 (3:2)

4.0 2.0 1.0

30 20 15

200 200 150

0.80 0.84 0.84

24 24 24

35 35 35

1.0–2.0 0.5–1.0 0.8–1.6


323

The cells were isolated from the amniotic epithelial layer after enzymatic digestion (Trypsin-EDTA, Sigma Chemical Co., St. Louis, MO, USA) as previously described by Barboni et al. [2,3]. Briefly, cell suspensions were seeded in flasks in minimum essential medium Eagle-α modification (α-MEM) supplemented with 20% fetal calf serum (FCS), 1% ultraglutamine, 1% penicillin/streptomycin plus 10 ng/ml epidermal growth factor (EGF) at a concentration of 3 × 103 cells/cm2. At 70% of confluence, the cells were dissociated by 0.05% trypsin-EDTA and plated again at 3 × 103/cm2 for three consecutive passages. Ovine AECs were characterized, confirming negativity for haemopoietic markers (CD14, CD58, CD31 and CD45), positivity for surface adhesion molecules (CD29, CD49f and CD166), and stemness markers TERT, SOX2, OCT4 and NANOG, a low expression for MHC class I molecule, and the absence of MHC class II (HLA-DR) antigens, as previously described in our reports [2,3,28,29]. Then, 2.5 × 106 oAECs were stored in liquid nitrogen in vials. Fig. 1. XRD patterns of PCL, PLA and PLGA electrospun scaffolds.

measured by a OCA 15pro contact angle meter from DataPhysics Instruments GmbH with SCA20 software (version 3.4.6 build 79). For comparison, casted films of the same polymer materials were characterized in the same conditions. Before test, samples were cut into a rectangular shape (10 × 10 mm2) and conditioned in a vacuum oven at 30 °C overnight. The contact angle was measured by the sessile drop method at room temperature. The water droplet size was set at 0.5 μl. Ten samples were used for each test. Due to the inherent porous architecture of samples, spontaneous liquid penetration might occur. The rate of penetration might vary largely depending on the wettability and the porous structure. However, for all analysed samples, after stabilization it occurred slowly enough that reproducible results (±0.3°) were obtained at least for 5 min. The average value was reported with standard deviation (±SD). 2.1.3. Scaffolds sterilization procedure PLGA, PCL and PLA electrospun scaffolds were sterilized with 70% ethanol (EtOH) in 0.9% NaCl/distilled water (dH2O) for few seconds and washed in sterile phosphate buffered saline (PBS), then rehydrated in culture medium containing minimum essential medium Eagle-α modification (α-MEM) supplemented with 10% fetal calf serum (FCS), 1% ultraglutamine, 1% penicillin/streptomycin for 24 h in incubator at 38 °C with 5% CO2. 2.2. Ovine amniotic epithelial stem cells (oAECs) ethics statement All cells and tissues were collected from slaughtered animals, and this does not require an ethic statement. 2.2.1. oAECs isolation and characterization Ovine AECs (oAECs) were collected from slaughtered sheep of Appenninica breed at 3 months of pregnancy (25–30 cm of length).

2.2.2. Scaffolds cells culture and seeding Before seeding, thawed oAECs were stained with the red fluorescent cell linker dye PKH26, according to manufacturer's instructions (Sigma Chemical Co., St. Louis, MO, USA) as previously described [3,17]. The PKH26 dye stably incorporates into lipid regions of the cell membrane. Due to this extremely stable fluorescence, PKH26 is the linker dye of choice for in vivo cell surviving and cell organization studies (http://www.sigmaaldrich.com/catalog/product/sigma/pkh26gl). All scaffolds were placed in Petri dishes, culture media was added containing Minimum Essential Medium (α-MEM) supplemented with 10% fetal calf serum (FCS), 1% ultraglutamine, 1% penicillin/streptomycin. Successively 0.05 × 106 oAECs were seeded on scaffolds previously sterilized as described, and cultured for 48 h in incubator at 38 °C with 5% CO2. 2.2.3. oAECs SEM engraftment analysis SEM was used to analyse oAECs engraftment within the analysed scaffolds. Scaffolds were fixed in 1% paraformaldehyde/0.5% glutaraldehyde/0.1 M cacodylate buffer pH 7.4 at 4 °C for about 1 h. Subsequently, after washing in 0.15 M cacodylate pH 7.4 at 4 °C, they were air dried overnight. After sputtering with gold palladium, SEM analysis was performed by using the ZEISS EVO scanning electron microscope (Carl Zeiss, Jena, Germany). 2.2.4. Cell survival and spatial distribution analysis After 48 h incubation of oAECs on scaffolds, these were fixed for 10 min in 4% paraformaldehyde. Ovine AECs biocompatibility for electrospun PCL, PLA and PLGA scaffolds was assed analysing oAECs survival and spatial distribution. The cells were stained with two vital dyes, the red fluorescent cell membranes marker PKH26 before cell seeding, as described above, and the cell nuclei vital blue fluorescent marker Hoechst 33342. This marker is a cell-permeable DNA stain that binds preferentially to adenine-thymine (A-T) regions of DNA into the minor groove of DNA. Ovine AECs within the scaffolds were allowed

Fig. 2. SEM micrographs of electrospun PLA, PCL and PLGA fibres at 500× magnification; scale bar: 10 μm.

324


Fig. 3. (A) Pore size distribution based on SEM image analysis in nonwoven scaffolds. (B) Statistical analysis of pore size frequency on analysed samples. PLA R2 = 0.953; PCL R2 = 0.7694; PLGA R2 = 0.9327.

to incorporate 0.5 μl of Hoechst33342 for 15 min according to manufacturer's instructions (https://tools.lifetechnologies.com/ content/sfs/manuals/MAN0011717_Hoechst_33342_UG.pdf). 2.2.5. oAECs proliferation index To quantify cell proliferation, firstly it was carried out an immunohistochemical analysis to reveal proliferating cells with the Ki-67 marker, a nuclear and nucleolar protein strictly associated with cell proliferation [30,31], and then Ki-67 positive cells cultured on scaffolds or on Petri dishes were quantified at 48 h, 96 h and 168 h. In detail, immunohistochemistry was performed with the anti-Ki-67 primary antibody (Ki-67; 1:50/PBS/BSA1%; Dako Cytomation, Denmark). Then, the primary antibody was revealed with an anti-mouse Alexa Fluor 488 secondary antibody (diluted 1:100 PBS/1%BSA; Molecular Probes), whereas cell nuclei were identified with DAPI (Sigma Chemical Co., St. Louis, MO, USA). As negative controls, the primary antibody was replaced with non-immune sera. To quantify the proliferation index (PI), counting of Ki-67 positive cells/100 cells counterstained with DAPI was performed. To this aim, for each experimental group three replicates were considered. Two trained observers blinded to the experiment counted at least 100 cells for each replicate. All samples were analysed using an Axioskop 2 Plus incident-light fluorescence microscope (Carl Zeiss) equipped with a CCD camera (Axiovision Cam; Carl Zeiss) with a resolution of 1300 × 1030 pixels, configured for fluorescence microscopy, and interfaced to a computer workstation provided with an interactive and automatic image analyser (Axiovision; Carl Zeiss). Digital images were acquired at × 100 and × 200 magnification using standard filter set up optimized for FITC, CY3 or DAPI. For quantitative purposes, digital images were consecutively captured immediately after immunoreactions. At the beginning of an imaging section, optimum exposure times were determined. Results were recorded for statistical evaluation.

D'Agostino and Pearson. The sets of data were compared using ANOVA test and thereafter, if necessary, by post-hoc Tukey test (GraphPad Prism 5, GraphPad Software, USA). Values were considered statistically significant for p b 0.05. 3. Results 3.1. Structure and morphology In Fig. 1 the XRD patterns of the three electrospun nonwoven scaffolds are shown. PCL fibres show very sharp diffraction peaks centred at about 2θ = 21.4° and 23.8° indicating a reduced amorphous fraction. On the contrary, the broad pattern of the nonwoven PLA can be attributed to the reflection of amorphous phase centred at about 2θ = 23°. In addition, the pattern of electrospun PLGA shows only the broad peak characteristic of the amorphous phase centred at about 2θ = 15.4°. In both cases, the process of evaporation of the solvent during electrospinning is so quickly not allowing for polymer crystallization. Fibre dimensions and pore analysis were measured quantitatively from the SEM images (Fig. 2) by using image analysis software. A substantial effort was made to characterize the nonwoven structure adequately. This involved measuring several images taken in different locations in each sample. Under the condition reported in Table 1, electrospinning led to PCL, PLA and PLGA defect-free cylindrical fibres with diameter ranging from 1.0 to 2.0 μm for PCL, 0.5 to 1.0 μm for PLA and 0.8 to 1.6 μm for PLGA. Fig. 3 shows the pore size distribution

2.2.6. DNA extraction and quantification Total genomic DNA was extracted from the seeded scaffolds at 4 h and 48 h by Maxwell 16 cell DNA purification kit, according to the manufacturer's instructions (Promega Corporation, Madison, Wisconsin). All samples were analysed using a fluorescence-based DNA quantification approach that utilizes the fluorescent property of nucleic acid binding dyes. The Qubit Quantitation Platform calculates concentration based on the fluorescence of the Qubit® dsDNA HS Assay (Life Technologies™), which binds to double-stranded DNA. 2.2.7. Statistical analysis All data were obtained in triplicate and expressed as mean ± standard error (± SE) and evaluated first by testing normal distribution

Fig. 4. Contact angles of nonwoven scaffolds (PCL_ES, PLA_ES and PLGA_ES) and relative casted film (PCL Cast, PLA Cast, PLGA Cast).


325

Fig. 5. oAECs engraftment on electrospun scaffolds: Scanning Electron Microscope ultrastructural analysis. Scale bars represent 10 μm and 20 μm.

and frequency of the three samples. The highest frequency of mean pore size is about 6 μm for PLA, 8 μm for PCL and 14 μm for PLGA, respectively (PLA R2 = 0.953; PCL R2 = 0.7694; PLGA R2 = 0.9327).

into the pores and around the microfibres of the scaffolds, especially in PLGA samples (Fig. 5). 3.4. Cell survival and spatial distribution on scaffolds

3.2. Contact angle It is well known that cells adhere and spread more effectively on surfaces with suitable hydrophilicity than on hydrophobic surfaces [32]. In our case, the contact angles of the electrospun nonwoven materials resulted higher than 120° for all the materials investigated (see Fig. 4). This very high contact angle indicates that the water droplet does not spread on the substrate due to the intrinsic hydrophobicity of the micro/nano nonwoven structure of the samples. In fact, compared with the contact angles measured with the respective casted films, these values are extremely higher and comparable, regardless the type of polymer analysed. This fact indicates that the micro/nano fibre morphology is the main responsible for the strong reduction of the wettability of these materials.

To analyse if oAECs survived within scaffolds, two supravital dyes, PKH26, a cell membrane linker molecule (red fluorescence), and Hoechst 33342 (blue fluorescence) that specifically links to DNA, were analysed. PKH26/Hoechst 33342-labelled oAECs were always retrieved within the scaffolds after 48 h of culture (Fig. 6). The fluorescent signals confirmed a good cell integration and survival. In detail, oAECs seeded on PLA, PCL and PLGA spread within the whole surface and especially around the microfibres. Although, the type of biomaterial influenced oAECs spatial distribution efficiency and PLGA showed the best result, as after 48 h cells coated almost all microfibres (Fig. 6). After 48 h culture, no evidence of nuclear pyknosis was observed in any analysed samples. 3.5. Ovine AECs proliferation within the engrafted scaffolds

3.3. Cell engraftment inside scaffolds SEM analysis demonstrated oAECs engraftment in all scaffolds. In particular, after 48 h incubation the ultrastructural examination showed that the seeded cells adhered to scaffolds indicating that PLA, PCL and PLGA scaffolds possessed biocompatibility for oAECs attachment. SEM investigation also revealed that oAECs extended their cellular processes

To evaluate oAECs PI within PLGA, PLA, and PCL scaffolds the quantification of a cell proliferation marker, Ki-67 (green fluorescence), and the DNA quantification were assessed. In detail, the immunohistochemical analysis on oAECs engineered scaffolds and oAECs cultured in Petri dishes, showed a specific positivity for Ki-67 (green fluorescence) in some cell nuclei (Fig. 7A, B) confirming their mitotic activity.

Fig. 6. Cell survival and spatial distribution on scaffolds: Merged images showing the PKH26-labelled (red fluorescence) and Hoechst 33342-labelled (blue fluorescence) oAECs within scaffolds. Scale bars represent 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

326


Fig. 7. oAECs proliferation index within the analysed scaffolds: (A) Merged images of oAECs within the analysed scaffolds and Petri dishes showing Ki-67 positivity (green fluorescence) and cell nuclei counterstained in blue (Hoechst 33342); Scale bars represent 20 μm. (B) oAECs proliferation index after engraftment within PLGA, PCL, and PLA scaffolds and oAECs Petri dishes. (C) Quantitative analysis of oAECs engineered scaffolds and oAECs cultured in Petri dishes after 48 h, 96 h, and 168 h culture. a statistically significant values among PLGA, PLA, and PCL (p b 0.05). *statistically significant values of PLGA, PLA, PCL compared to Petri dishes (p b 0.05). (D) Statistical analysis correlation between the mean pore size with the highest frequency within each analysed scaffold and the related % of oAECs proliferation index. R2 = 0.967. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Ovine AEC proliferation index after 48 h, 96 h, and 168 h culture were significantly higher in PLGA compared to PCL and PLA (p b 0.05 at alltime points; Fig. 7C). In order to verify the correlation between pore size and proliferation index, we statistically correlated the mean pore size with the highest frequency of the PLGA, PCL, and PLA scaffolds and oAECs proliferation index after 48 h culture on these biomaterials. This analysis demonstrated that there is a high correlation between the mean pore size and oAECs proliferation index (Fig. 7D; R2 = 0.967). Indeed, a high oAECs proliferation index was evident especially in PLGA having larger pore size than PCL and PLA. Instead, proliferation index in oAECs cultured in Petri dishes was higher respect to cells seeded within scaffolds at 48 h (p b 0.05), then at the end of culture period it significantly dropped respect to oAECs seeded in scaffolds (p b 0.05; Fig. 7C), as cells reached confluence. DNA quantification, after 4 h culture, demonstrated that the same amount of cells adhered on scaffolds. Instead, after 48 h, it was evident an increased DNA quantity respect to 4 h culture (p b 0.05; Table 2) and a different growth rate on cell population within the three seeded scaffolds, confirming the PI results. Indeed, data indicated in Table 2, confirm the influence of the different scaffold chemical composition on DNA quantity. In particular, after 48 h culture oAECs DNA quantity was significantly higher in PLGA (350.66 ± 1.17 pg/μl) compared to PCL and PLA (248 ± 1.44 pg/μl and 198.66 ± 1.31 pg/μl, respectively: p b 0.05; Table 2).

4. Discussion Stem cell scaffold engineering for a functional tissue substitute outcome depends on the right combination of scaffolds biocompatibility and stem cells ability to colonize and proliferate on it. Ovine AECs were chosen to test PCL, PLA and PLGA nonwoven electrospun scaffolds because of their multilineage differentiation potential [16], immunomodulatory properties in vitro and in vivo [2,5,6, 33,34], and low teratoma risk [12,14,35,36]. These cells with such features have already found application in scaffold engineering [19] and in allo- and xeno-transplantation preclinical and clinical settings [6, 17–19]. However, the biocompatibility grade and biologic activities of the seeded oAECs was influenced both by the scaffold's chemical and

Table 2 DNA quantification by Qubit® dsDNA HS Assay.

4h 48 h

PLA + oAECs

PCL + oAECs

PLGA + oAECs

89.66 ± 1.75 ⁎pg/μl

95,45 ± 1.29 ⁎pg/μl

198.66 ± 1.31 apg/μl

248 ± 1.44 apg/μl

82.66 ± 1.26 ⁎pg/μl 350.66 ± 1.17 apg/μl

⁎ Statistically significant values compared to the same type of scaffolds after 4 h and 48 h of cell culture (p b 0.05). a Statistically significant values compared to different scaffolds after 48 h of cell culture (p b 0.05).


physical structure. Indeed, all the obtained electrospun biopolymers showed similar morphologies characterized by random oriented and beadless fibres with micrometer scale diameters. Structural analysis showed that electrospun PLA and PLGA scaffolds were prevalently amorphous, whereas PCL scaffold showed a discrete level of crystallinity. The analyses of the contact angle confirmed that all samples presented similar wettability. In fact, regardless the type of polymer, contact angles ranged between 125 and 136°. It would be expected that the hydrophilic character is the key parameter for the cell adhesion and viability. Instead, the pore size distribution seemed to have a more important effect on the enhancement of the cell activity. By comparing oAECs adhesion ability and the pore size distribution results, the samples having higher percentage (electrospun PLGA) exhibited the highest cell viability in terms of cell migration and proliferation. Such interesting result can be explained by the fact that hydrophilic nature intervenes in the first contact with cells, whereas pores are basically responsible for the diffusion and the propagation through the webs [37,38]. Certainly, the hydrophilic nature of these scaffolds is essential for cell adhesion, which is one of the most important aspects of cell interaction with a biomaterial. It is the first cellular event to take place after cells are seeded onto a biomaterial surface; cell migration, proliferation, and/or differentiation take place only after cells are securely adhered [39]. Pore size is another essential aspect of scaffolds for tissue engineering. If pores are too small cell migration, diffusion of nutrients, and removal of waste products is limited. Conversely, if pores are too large there is a decrease in specific surface area available limiting cell attachment. Therefore maintaining a balance between the optimal pore size for cell migration is essential [40] improving cell survival within the scaffold [41]. In this study, ultrastructural analysis showed a strict interaction between oAECs and the microfibres. The cells appeared to be completely integrated into the structure of the scaffolds with oAECs cellular processes around microfibres of the scaffolds, especially in PLGA. Furthermore, the immunofluorescence analyses with the supravital dyes show that these stem cells are able to survive within the scaffolds and colonize them. Although, this analyses have also evidenced a pronounced difference in oAECs distribution and spatial organization within the three different analysed scaffolds. In particular, oAECs were randomly located on PLA and PCL, whereas they were more homogeneously aligned on PLGA scaffold microfibres showing higher spatial distribution efficiency compared to PLA and PCL. This occurrence could be attributable to the higher percentage of higher pore size of the PLGA scaffolds. As a result, oAECs are able to attach at multiple focal points on the surface of PLGA scaffolds. Ovine AECs integration and colonization within scaffolds was also confirmed by assessing their mitotic activity. Indeed, these cells at alltime points were proliferating. Instead, the PI of cells cultured in Petri dishes significantly dropped once they reached the confluence. In particular, the seeded cells revealed a significant increase in cell proliferation activity especially in PLGA scaffolds compared to PLA and PCL at all-time points. Statistical quantification demonstrated that pore size influenced cell proliferation. Indeed, a direct correlation between the mean pore size with the highest frequency and proliferation index was confirmed. Definitely, PLGA showed the best combination between the mean pore size frequency and oAECs proliferation index when compared to PCL and PLA. Consistent with oAECs PI, is the DNA quantification analysis of the seeded scaffolds. In detail, after 48 h culture, the amount of DNA resulted significantly higher in PLGA scaffolds compared to PCL and PLA. The obtained data show that the geometrical features, including pore size, and the chemistry of the scaffold can influence oAECs spatial infiltration and organization within the scaffold, supporting and promoting in a relatively efficient manner cell adhesion, survival and proliferation. Indeed, all these characteristics provide a bioactive environment, leading a cell proliferation and bio-synthesis improvement [42–45].

327

Li et al. [46] puts forward the view that a scaffold material must be carefully selected and the scaffold architecture should be properly designed to ensure biocompatibility with the seeded cells. He also states that ideal characteristics for a scaffold include (a) biocompatibility; (b) promotion of cellular activities such as cell adhesion; (c) biodegradability with a controlled rate of degradation that corresponds with tissue growth within the scaffold; (d) a 3D highly porous structure with an interconnected network of microscopic spaces to allow tissue growth and permeation of nutrient medium; (e) favourable mechanical properties; and (f) a highly reproducible and adaptable fabrication process for different shapes or sizes. These characteristics are determined by the material selection and the method of scaffold fabrication. The fabricated scaffolds used in our investigation seem to fulfil all these features. In fact, they result biocompatible for oAECs, indeed cells were vital and no signs of pyknosis were observed. Promoted cellular activities allowing a high degree of cell colonization and proliferation in only 48 h, probably due to their pore size distribution (especially for PLGA). AECs can be considered ideal for tissue engineering especially when using biocompatible and opportunely produced scaffolds, as in this study. Even though, PLGA gave the best results in terms of scaffold oAECs colonization, distribution and proliferation. 5. Conclusions In this work, PCL, PLA and PLGA nonwovens scaffolds were produced by electrospinning technique and successfully characterized through structural and morphological analyses. Experimental results demonstrated that oAECs were biocompatible for these scaffolds, validating the experimental parameters used to produce them. Indeed, oAECs were able to colonize and proliferate inside the analysed electrospun biopolymers. In conclusion, the investigated biocompatible electrospun matrices represent a promising approach for the fabrication of bioscaffolds in AECs-based regenerative tissue, which could be used in allo- and xeno-transplantation settings. Although, it is evident that the design of the ideal scaffold should take in consideration several factors, such as, chemical characteristics, microfibres spatial organization, and pore size distribution ideal for the stem cells source used, besides the biodegradation rate and the mechanical properties, before assessing their potential preclinical/clinical application. These last points will be examined in a forthcoming paper. Acknowledgments We thank Dr. Annunziata Mauro for her assistance in revising this manuscript, and Prof. Bernabò for his statistical analysis contribution. References [1] T. Miki, S.C. Strom, Amnion-derived pluripotent/multipotent stem cells, Stem Cell Rev. 2 (2) (2006) 133–142, http://dx.doi.org/10.1007/s12015-006-0020-0. [2] B. Barboni, V. Russo, V. Curini, A. Martelli, P. Berardinelli, A. Mauro, M. Mattioli, M. Marchisio, P. Bonassi Signoroni, O. Parolini, A. Colosimo, Gestational stage affects amniotic epithelial cells phenotype, methylation status, immunomodulatory and stemness properties, Stem Cell Rev. 10 (5) (2014) 725–741, http://dx.doi.org/10. 1007/s12015-014-9519-y. [3] B. Barboni, V. Curini, V. Russo, A. Mauro, O. Di Giacinto, M. Marchisio, M. Alfonsi, M. Mattioli, Indirect co-culture with tendons or tenocytes can program amniotic epithelial cells towards stepwise tenogenic differentiation, PLoS One 7 (2) (2012), e30974, http://dx.doi.org/10.1371/journal.pone.0030974. [4] A. Cargnoni, L. Gibelli, A. Tosini, P.B. Signoroni, C. Nassuato, D. Arienti, G. Lombardi, A. Albertini, G.S. Wengler, O. Parolini, Transplantation of allogeneic and xenogeneic placenta-derived cells reduces bleomycin-induced lung fibrosis, Cell Transplant. 18 (4) (2009) 405–422, http://dx.doi.org/10.3727/096368909788809857. [5] M. Magatti, S. De Munari, E. Vertua, C. Nassauto, A. Albertini, G.S. Wengler, O. Parolini, Amniotic mesenchymal tissue cells inhibit dendritic cell differentiation of peripheral blood and amnion resident monocytes, Cell Transplant. 18 (8) (2009) 899–914, http://dx.doi.org/10.3727/096368909x471314. [6] B. Barboni, V. Russo, V. Curini, A. Mauro, A. Martelli, A. Muttini, N. Bernabo, L. Valbonetti, M. Marchisio, O. Di Giacinto, P. Berardinelli, M. Mattioli, Achilles tendon regeneration can be improved by amniotic epithelial cell allotransplantation, Cell

328

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

V. Russo et al. / Materials Science and Engineering C 69 (2016) 321–329 Transplant. 21 (11) (2012) 2377–2395, http://dx.doi.org/10.3727/ 096368912x638892. A. Muttini, L. Valbonetti, M. Abate, A. Colosimo, V. Curini, A. Mauro, P. Berardinelli, V. Russo, D. Cocciolone, M. Marchisio, M. Mattioli, U. Tosi, M. Podaliri Vulpiani, B. Barboni, Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects, Res. Vet. Sci. 94 (1) (2013) 158–169, http://dx.doi.org/10.1016/j.rvsc.2012.07.028. A. Muttini, V. Salini, L. Valbonetti, M. Abate, Stem cell therapy of tendinopathies: suggestions from veterinary medicine, Muscles Ligaments Tendons J. 2 (3) (2012) 187–192. A. Muttini, V. Russo, E. Rossi, M. Mattioli, B. Barboni, U. Tosi, N. Maffulli, L. Valbonetti, M. Abate, Pilot experimental study on amniotic epithelial mesenchymal cell transplantation in natural occurring tendinopathy in horses. Ultrasonographic and histological comparison, Muscles Ligaments Tendons J. 5 (1) (2015) 5–11. B. Scaggiante, A. Pineschi, M. Sustersich, M. Andolina, E. Agosti, D. Romeo, Successful therapy of Niemann-pick disease by implantation of human amniotic membrane, Transplantation 44 (1) (1987) 59–61, http://dx.doi.org/10.1097/00007890198707000-00014. N. Sakuragawa, H. Yoshikawa, M. Sasaki, Amniotic tissue transplantation: clinical and biochemical evaluations for some lysosomal storage diseases, Brain Dev. 14 (1) (1992) 7–11, http://dx.doi.org/10.1016/S0387-7604(12)80272-5. M. Bailo, M. Soncini, E. Vertua, P.B. Signoroni, S. Sanzone, G. Lombardi, D. Arienti, F. Calamani, D. Zatti, P. Paul, A. Albertini, F. Zorzi, A. Cavagnini, F. Candotti, G.S. Wengler, O. Parolini, Engraftment potential of human amnion and chorion cells derived from term placenta, Transplantation 78 (10) (2004) 1439–1448, http://dx.doi. org/10.1097/01.TP.0000144606.84234.49. A.J. Marcus, T.M. Coyne, J. Rauch, D. Woodbury, I.B. Black, Isolation, characterization, and differentiation of stem cells derived from the rat amniotic membrane, Differentiation 76 (2) (2008) 130–144, http://dx.doi.org/10.1111/j.1432-0436.2007.00194.x. T. Miki, T. Lehmann, H. Cai, D.B. Stolz, S.C. Strom, Stem cell characteristics of amniotic epithelial cells, Stem Cells 23 (10) (2005) 1549–1559, http://dx.doi.org/10. 1634/stemcells.2004-0357. M.P. Dobreva, P.N. Pereira, J. Deprest, A. Zwijsen, On the origin of amniotic stem cells: of mice and men, Int. J. Dev. Biol. 54 (5) (2010) 761–777, http://dx.doi.org/ 10.1387/ijdb.092935md. S. Ilancheran, A. Michalska, G. Peh, E.M. Wallace, M. Pera, U. Manuelpillai, Stem cells derived from human fetal membranes display multilineage differentiation potential, Biol. Reprod. 77 (3) (2007) 577–588, http://dx.doi.org/10.1095/biolreprod.106.055244. M. Mattioli, A. Gloria, M. Turriani, A. Mauro, V. Curini, V. Russo, S. Tete, M. Marchisio, L. Pierdomenico, P. Berardinelli, A. Colosimo, A. Muttini, L. Valbonetti, B. Barboni, Stemness characteristics and osteogenic potential of sheep amniotic epithelial cells, Cell Biol. Int. 36 (1) (2012) 7–19, http://dx.doi.org/10.1042/cbi20100720. P. Niemeyer, T.S. Schonberger, J. Hahn, P. Kasten, J. Fellenberg, N. Suedkamp, A.T. Mehlhorn, S. Milz, S. Pearce, Xenogenic transplantation of human mesenchymal stem cells in a critical size defect of the sheep tibia for bone regeneration, Tissue Eng. Part A 16 (1) (2010) 33–43, http://dx.doi.org/10.1089/ten.TEA.2009.0190. B. Barboni, C. Mangano, L. Valbonetti, G. Marruchella, P. Berardinelli, A. Martelli, A. Muttini, A. Mauro, R. Bedini, M. Turriani, R. Pecci, D. Nardinocchi, V.L. Zizzari, S. Tete, A. Piattelli, M. Mattioli, Synthetic bone substitute engineered with amniotic epithelial cells enhances bone regeneration after maxillary sinus augmentation, PLoS One 8 (5) (2013), e63256, http://dx.doi.org/10.1371/journal.pone.0063256. A. Martins, J.V. Araujo, R.L. Reis, N.M. Neves, Electrospun nanostructured scaffolds for tissue engineering applications, Nanomedicine (London) 2 (6) (2007) 929–942, http://dx.doi.org/10.2217/17435889.2.6.929. S. Agarwal, J.H. Wendorff, A. Greiner, Use of electrospinning technique for biomedical applications, Polymer 49 (26) (2008) 5603–5621, http://dx.doi.org/10.1016/j. polymer.2008.09.014. H.R. Darrell, C. Iksoo, Nanometre diameter fibres of polymer, produced by electrospinning, Nanotechnology 7 (3) (1996) 216, http://dx.doi.org/10.1088/ 0957-4484/7/3/009. S.G. Kumbar, R. James, S.P. Nukavarapu, C.T. Laurencin, Electrospun nanofiber scaffolds: engineering soft tissues, Biomed. Mater. 3 (3) (2008) 034002, http://dx.doi. org/10.1088/1748-6041/3/3/034002. T.C. Lim, M. Kotaki, T.K.J. Yong, F. Yang, K. Fujihara, S. Ramakrishna, Recent advances in tissue engineering applications of electrospun nanofibers, Mater. Technol. 19 (1) (2004) 20–27, http://dx.doi.org/10.1039/C4RA05218H. Z. Ma, M. Kotaki, R. Inai, S. Ramakrishna, Potential of nanofiber matrix as tissue-engineering scaffolds, Tissue Eng. 11 (1–2) (2005) 101–109, http://dx.doi.org/10. 1089/ten.2005.11.101. D.H. Reneker, A.L. Yarin, H. Fong, S. Koombhongse, Bending instability of electrically charged liquid jets of polymer solutions in electrospinning, J. Appl. Phys. 87 (9) (2000) 4531–4547, http://dx.doi.org/10.1063/1.373532. T.J. Sill, H.A. von Recum, Electrospinning: applications in drug delivery and tissue engineering, Biomaterials 29 (13) (2008) 1989–2006, http://dx.doi.org/10.1016/j. biomaterials.2008.01.011. M. Mattioli, A. Gloria, M. Turriani, P. Berardinelli, V. Russo, D. Nardinocchi, V. Curini, M. Baratta, E. Martignani, B. Barboni, Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study, Theriogenology 77 (7) (2012) 1425–1437, http://dx.doi.org/10.1016/j.theriogenology.2011.11.008. A. Mauro, M. Turriani, A. Ioannoni, V. Russo, A. Martelli, O. Di Giacinto, D. Nardinocchi, P. Berardinelli, Isolation, characterization, and in vitro differentiation of ovine amniotic stem cells, Vet. Res. Commun. 34 (Suppl. 1) (2010) S25–S28, http://dx.doi.org/10.1007/s11259-010-9393-2. X. Lu, D. Chen, Z. Liu, C. Li, Y. Liu, J. Zhou, P. Wan, Y.G. Mou, Z. Wang, Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium, Mol. Vis. 16 (2010) 611–622.

[31] J.H. Yang, M.Y. Wu, C.D. Chen, M.J. Chen, Y.S. Yang, H.N. Ho, Altered apoptosis and proliferation in endometrial stromal cells of women with adenomyosis, Hum. Reprod. 22 (4) (2007) 945–952, http://dx.doi.org/10.1093/humrep/del493. [32] S.J. Cho, S.M. Jung, M. Kang, H.S. Shin, J.H. Youk, Preparation of hydrophilic PCL nanofiber scaffolds via electrospinning of PCL/PVP-b-PCL block copolymers for enhanced cell biocompatibility, Polymer 69 (2015) 95–102, http://dx.doi.org/10.1016/j. polymer.2015.05.037. [33] O. Parolini, M. Caruso, Review: preclinical studies on placenta-derived cells and amniotic membrane: an update, Placenta 32 (Suppl. 2) (2011) S186–S195, http://dx. doi.org/10.1016/j.placenta.2010.12.016. [34] M. Caruso, M. Evangelista, O. Parolini, Human term placental cells: phenotype, properties and new avenues in regenerative medicine, Int. J. Mol. Cell Med. 1 (2) (2012) 64–74. [35] A.J. Marcus, T.M. Coyne, I.B. Black, D. Woodbury, Fate of amnion-derived stem cells transplanted to the fetal rat brain: migration, survival and differentiation, J. Cell. Mol. Med. 12 (4) (2008) 1256–1264, http://dx.doi.org/10.1111/j.1582-4934.2008.00180.x. [36] T. Tamagawa, I. Ishiwata, S. Saito, Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro, Hum. Cell 17 (3) (2004) 125–130, http://dx.doi.org/10.1111/ j.1749-0774.2004.tb00028.x. [37] T. Bhardwaj, R.M. Pilliar, M.D. Grynpas, R.A. Kandel, Effect of material geometry on cartilagenous tissue formation in vitro, J. Biomed. Mater. Res. 57 (2) (2001) 190–199, http://dx.doi.org/10.1002/1097-4636(200111)57:23.3.CO;2-A. [38] J.H. Lee, H.W. Jung, I.K. Kang, H.B. Lee, Cell behaviour on polymer surfaces with different functional groups, Biomaterials 15 (9) (1994) 705–711, http://dx.doi.org/10. 1016/0142-9612(94)90169-4. [39] J.E. Sanders, B.S. Nicholson, S.B. Mitchell, R.E. Ledger, Polymer microfiber mechanical properties: a system for assessment and investigation of the link with fibrous capsule formation, J. Biomed. Mater. Res. A 67 (4) (2003) 1412–1416, http://dx.doi. org/10.1002/jbm.a.20049. [40] C.M. Murphy, F.J. O'Brien, Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds, Cell Adhes. Migr. 4 (3) (2010) 377–381, http://dx.doi.org/10.4161/cam.4.3.11747. [41] G.F. Muschler, C. Nakamoto, L.G. Griffith, Engineering principles of clinical cell-based tissue engineering, J. Bone Joint Surg. Am. 86-a (7) (2004) 1541–1558. [42] S.R. Chastain, A.K. Kundu, S. Dhar, J.W. Calvert, A.J. Putnam, Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM ligands and controls their osteogenic differentiation, J. Biomed. Mater. Res. A 78 (1) (2006) 73–85, http://dx.doi.org/10.1002/jbm.a.30686. [43] R. Lange, F. Luthen, U. Beck, J. Rychly, A. Baumann, B. Nebe, Cell-extracellular matrix interaction and physico-chemical characteristics of titanium surfaces depend on the roughness of the material, Biomol. Eng. 19 (2–6) (2002) 255–261, http://dx.doi.org/ 10.1016/S1389-0344(02)00047-3. [44] G. Marletta, G. Ciapetti, C. Satriano, F. Perut, M. Salerno, N. Baldini, Improved osteogenic differentiation of human marrow stromal cells cultured on ion-induced chemically structured poly-epsilon-caprolactone, Biomaterials 28 (6) (2007) 1132–1140, http://dx.doi.org/10.1016/j.biomaterials.2006.10.027. [45] S. Neuss, C. Apel, P. Buttler, B. Denecke, A. Dhanasingh, X. Ding, D. Grafahrend, A. Groger, K. Hemmrich, A. Herr, W. Jahnen-Dechent, S. Mastitskaya, A. Perez-Bouza, S. Rosewick, J. Salber, M. Woltje, M. Zenke, Assessment of stem cell/biomaterial combinations for stem cell-based tissue engineering, Biomaterials 29 (3) (2008) 302–313, http://dx.doi.org/10.1016/j.biomaterials.2007.09.022. [46] W.-J. Li, R.M. Shanti, R.S. Tuan, Electrospinning Technology for Nanofibrous Scaffolds in Tissue Engineering, Nanotechnologies for the Life Sciences, Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Valentina Russo has a Master Degree in Veterinary Medicine (cum laude; 1996), is an Associate Professor of Anatomy at the School of Veterinary Medicine of the University of Teramo. She firstly focused her research activity in Reproductive Biotechnologies. In the last decade her research interests include amniotic derived stem cell biology and their application in regenerative medicine studying their biological properties in vitro and investigating the mechanisms of action of amniotic cell-based therapies applied to musculoskeletal experimental and spontaneous diseases of medium (sheep) and large (horse) sized animal models.

Loredana Tammaro graduated in Chemistry (2002) and gained the PhD degree in Chemical Engineering (2006) from University of Salerno (Italy). Since December 2014 she is permanent researcher at Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA). Her experience concerns polymeric and composite materials. Her research interests include synthesis and characterization of inorganic-organic hybrid materials tailored for several applications, from food packaging to biomedical devices. In the last years her attention has been focused on the preparation of high surface-to-volume ratio micro- and nanoscale fibres using electrospinning and suitable as scaffolds for tissue engineering and drug delivery.


329

Lisa Di Marcantonio is a PhD Student in Cellular and Molecular Biotechnology at the University of Teramo, Italy. She achieved a Bachelor's Degree in Biotechnology (2011) and a Master Degree in Biotechnology of Reproduction (2013) at the University of Teramo. The main research activities are in the field of regenerative medicine studying the regenerative and immunomodulatory role of amniotic derived stem cells in preclinical experimental tendons defects, cell adhesion, survival and differentiation on electrospun bioscaffolds for tendon regeneration.

Maura Turriani is a technician at the University of Teramo, Italy. She achieved a Bachelor's Degree in Biology (1990) at the University of Pisa and she received her PhD in “Biochemistry” (1994) at the University of Florence. She focused her research on the biology of reproduction in mammals studying female gamete biology. The main current research focuses on amniotic derived stem cells, in particular on the following aspects: isolation, amplification and typing of stem cells of epithelial origin, evaluation of their differentiation capabilities in vitro and regenerative capabilities on preclinical models of tendon defects and bone lesions.

Andrea Sorrentino graduated in Chemical Engineering (2000) and received his PhD (2004) from the University of Salerno (Italy). Since November 2011 he is Researcher at the Institute for Polymers, Composites and Biomaterials (IPCB) of Italian National Research Council (CNR). He has extensive experience in various polymer processing including product design and process development. His main research activities include the effect of thermo-mechanical history on final morphology and properties of polymeric materials. Some of his areas of expertise include the development of nano-functional materials for barrier and electrical applications. Furthermore, he has experience in Polymer (bio/photo)-degradation and bio-nanocomposites material.

Alessandra Martelli is an Academic researcher in the SSD V30 A (Anatomy of the Domestic Animals), University of Teramo, Italy. She graduated in Veterinary Medicine (1994, University of Bologna, Italy), in Psychological Sciences Applied (2014, University of L'Aquila, Italy), and she's Counsellor (2015). The main lines of research are: angiogenesis mechanisms involved in ovarian folliculogenesis; biotechnology applied to animal reproduction; mechanisms underlying the interaction between stem cells and biomaterials; regenerative properties of amniotic stem cells tested on experimental animal models.

Massimo Ancora is a molecular biologist at the Istituto Zooprofilattico Sperimentale “G. Caporale” of Teramo, Italy, where he is involved in research and development in the field of animal health and food safety biotechnology. He is an expert for the National Reference Center and World Organization for Animal Health (OIE) Laboratory for Brucellosis and, as part of the PhD in Cellular and Molecular Biotechnology at the University of Teramo, Italy, his research activities are also in the field of biotechnology of reproduction especially in the identification of possible predictive molecular biomarkers of human oocyte quality by Next Generation Sequencing analysis.

Luca Valbonetti is Assistant Professor of Veterinary Surgery, at the University of Teramo, Italy. He received his degree in Veterinary Medicine (1998) and the Specialization in “Medicine and Surgery of the Horse” (2003) at the University of Teramo. His research activity is mainly focused on: Medical Imaging (micro-CT, SEM, radiology, confocal microscopy, software for image analysis); Regenerative medicine, use of stem cells for bone and tendon repair in translational medicine and experimental surgery.

Dr. Cesare Cammà is the head of the Research and Development Biotechnology Unit at the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (IZSAM). He is a Member of International Network “Global Microbial Identifier” and a Member of the OIE Group on Non Tsetse Transmitted Animal Trypanosomoses (NTTAT). The main activities of the unit are the development of molecular biology methods for diagnosis of infectious animal diseases and zoonoses, and the molecular characterization of viral and bacterial strains by using different DNA sequence-based subtyping techniques including whole genome sequencing through Next Generation Sequencing (NGS) technologies.

Barbara Barboni, PhD in Neuroendocrinology, is a full Professor and Chair of Physiology at the School of Veterinary Medicine of the University of Teramo. Engaged for more than two decades with research areas related to reproductive biotechnologies, she has recently moved the cell biology background towards the field of regenerative medicine studying the biological properties of amniotic derived cells in vitro and investigating the mechanism of action of amniotic cellbased therapies applied to musculoskeletal experimental and spontaneous diseases of medium (sheep) and large (horse) sized animal models.