Materials Science and Engineering C 34 (2014) 130–139

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Design of 3D scaffolds for tissue engineering testing a tough polylactide-based graft copolymer R. Dorati a,c,⁎, C. Colonna a,c, C. Tomasi b, I. Genta a,c, G. Bruni b, B. Conti a,c a b c

Department of Drug Sciences, University of Pavia, V.le Taramelli 12, 27100 Pavia, Italy C.S.G.I., Department of Chemistry, Division of Physical Chemistry, University of Pavia, V.le Taramelli 16 I, 27100 Pavia, Italy Center for Tissue Engineering (CIT), University of Pavia, Via Ferrata 1, 27100 Pavia, Italy

a r t i c l e

i n f o

Article history: Received 10 January 2013 Received in revised form 29 July 2013 Accepted 29 August 2013 Available online 6 September 2013 Keywords: Scaffold Tissue regeneration Cell adhesion Biodegradation Tensile properties

a b s t r a c t The aim of this research was to investigate a tough polymer to develop 3D scaffolds and 2D films for tissue engineering applications, in particular to repair urethral strictures or defects. The polymer tested was a graft copolymer of polylactic acid (PLA) synthesized with the rationale to improve the toughness of the related PLA homopolymer. The LMP-3055 graft copolymer (in bulk) demonstrated to have negligible cytotoxicity (bioavailability N85%, MTT test). Moreover, the LMP-3055 sterilized through gamma rays resulted to be cytocompatible and non-toxic, and it has a positive effect on cell biofunctionality, promoting the cell growth. 3D scaffolds and 2D film were prepared using different LMP-3055 polymer concentrations (7.5, 10, 12.5 and 15%, w/v), and the effect of polymer concentration on pore size, porosity and interconnectivity of the 3D scaffolds and 2D film was investigated. 3D scaffolds got better results for fulfilling structural and biofunctional requirements: porosity, pore size and interconnectivity, cell attachment and proliferation. 3D scaffolds obtained with 10 and 12.5% polymer solutions (3D-2 and 3D-3, respectively) were identified as the most suitable construct for the cell attachment and proliferation presenting pore size ranged between 100 and 400 μm, high porosity (77–78%) and well interconnected pores. In vitro cell studies demonstrated that all the selected scaffolds were able to support the cell proliferation, the cell attachment and growth resulting to their dependency on the polymer concentration and structural features. The degradation test revealed that the degradation of polymer matrix (ΔMw) and water uptake of 3D scaffolds exceed those of 2D film and raw polymer (used as control reference), while the mass loss of samples (3D scaffold and 2D film) resulted to be controlled, they showed good stability and capacity to maintain the physical integrity during the incubation time. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Extracellular matrix (ECM) represents a support for cells providing a natural environment for the proliferation, the differentiation of cells and for the morphogenesis contributing to the organogenesis and cell-based tissue regeneration. In case of large tissue defects, it is unlikely that the damaged tissue initiates the regeneration process exclusively supplying cells to the diseased area because both cells and ECM as well as the surrounding environment are lost [1–3]. For this reason, the design and the development of local cell environment, namely artificial scaffolds, gain importance because they can initially assist the cell attachment and subsequently promote proliferation and differentiation, inducing cell-based tissue regeneration [1]. Several biomaterials have been studied and used to develop 3D scaffold and 2D film [4–6]. It is generally established that a biomaterial to be used

⁎ Corresponding author at: Department of Drug Sciences, University of Pavia, V.le Taramelli 12, 27100 Pavia, Italy. Tel.: +39 0382 987786; fax: +39 0382 422975. E-mail address: [email protected] (R. Dorati). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.08.037

in TE needs to have specific features such as biocompatibility, suitable surface chemistry, defined mechanical properties and biodegradability. The choice of biomaterials for TE applications is dictated by the final application and consequently requires severe considerations regarding the physical, chemical and mechanical properties of selected material [7–9]. The prolonged permanence of the polymer in contact with biological fluids and the response of the immediate surroundings (tissues) determine the choice between biostable (non-biodegradable) and biodegradable polymers. Moreover, the biological interactions seem to influence the selection of naturally derived polymers vs synthetic ones, encouraging the combination of several materials and eventually their functionalization with specific bio-molecules to promote cell adhesion and proliferation [10]. A massive number of different natural materials have been studied and proposed for the development of 3D scaffold for TE, they present good biodegradability, low toxicity, low manufacture and disposal cost, and renewability. Moreover, these natural materials offer several advantages in TE area such as biological signaling, cell adhesion, and cell response degradation and re-modeling. However, their use as a unique scaffold constituent is generally minimized by i) the high solubility of

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these materials and their ability to dissolve into the physiological fluids, ii) the rapid degradation and iii) the potential loss of intrinsic biological features during the formulation process. Furthermore, there are several concerns about the immune-rejection and disease transmission when natural materials are used [11]. Potential risks such as toxicity, immunogenicity and infections are reduced for pure synthetic materials which consist into well-known simple structures [12]. Synthetic materials exhibit predictable and reproducible physical, chemical and degradation properties that can be modified and tuned to satisfy the structural and functional requirements for the intended application. Moreover, they are easily processable into desired and specific shapes and sizes as well. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester, and it has extensively been used as a biomaterial for use in the human body because of its adsorbability and non-toxicity after degradation [13,14]. PLA basically exhibits brittleness and its fracture behavior strongly depends on the crystal structure. Therefore, improving the toughness of PLA appears to be an important topic in the field of biomaterial and in particular for TE application [15,16]. The aim of this research was to explore the feasibility of using the LMP-3055 in the biological environment and to investigate the capability of using LMP-3055 as substrates for TE. In particular, the interest was focused on the design and development of 3D scaffolds and 2D film for tissue engineering. The polymer studied and tested was a graft copolymer of polylactic acid (PLA) supplied by the Marc Hillmyer research group (University of Minnesota). The copolymer was synthesized with the rationale to improve the toughness and tensile properties of the related PLA homopolymer; indeed, the TE applications of the PLA homopolymer are limited by its brittle nature [15]. The approach used to improve the toughness tensile properties of PLA homopolymer consists in the addition of rubbery domains into the backbone of PLA homopolymer. The rubbery phase makes available supplementary dissipation energy during the deformation process, resulting into an increase of block copolymer toughness [13]. The Mark Hillmyer group synthesized a graft copolymer (LMP3055) containing 5% weight of the rubbery phase and 95% of PLA homopolymer with excellent toughness tensile properties. LMP-3055 is the poly(cyclooctadiene-co-norbornenemethanol-graft-lactide) with C200N3L95 composition, where the numbers respectively define the degree of polymerization of the cyclooctadiene (COD), the norborneneMeOH (N) copolymerized with COD and the D,L-lactide in the synthesized graft copolymer. The mechanical behavior of LMP-3055 graft copolymer was assessed by tensile testing of compression-molded bars using PLA samples as control. The PLA sample failed after a restricted deformation (13 ± 4%), with no evidence of neck formation and stress whitening: this behavior is intrinsic of a brittle material. On the opposite, LMP-3055 graft copolymer exhibited stress whitening, neck formation, and cold drawing with high elongation percentage (238 ± 43%) [15]. The toughness and tensile properties exhibited by LMP-3055 addressed the present study aimed to investigate on the use of LMP3055 as substrate for tissue engineering application, as urethral reconstruction. LMP-3055 polymers could be useful as raw material to formulate biodegradable scaffolds for urethral reconstruction. Even though several advancements have been made in the surgical techniques for the urethral reconstruction, the nature of material used as substitute substrate in the repair of urethral strictures or defects remains the most challenging problem in this field. Various autologous grafts or flaps from skin or mucosa have been proposed for urethra structure repair, and today the buccal mucosa (BM) is considered as the best tissue for urethral substitution [17]. It is defined as the gold-standard for its good tensile properties, its stability to urine and excellent resistance to infections. Limits related to the use of autologous tissue are donorsite morbidity and time-consuming harvesting. To mimic the complex structure of urethral tissue the substrate will be prepared combining LMP-3055 with other biocompatible and biodegradable polymers.

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In the first part of this work, attention was focused on the evaluation of the cytotoxicity of the LMP-3055 graft copolymer (in bulk) on adult fibroblasts as a healthy cell model by MTT assay. Moreover, the cytotoxicity of the graft copolymer was assessed after sterilization by gamma rays at 25 kGy dose, to verify the effects of the sterilization technique on biological performances, such as cell adhesion and growth. Gamma irradiation was selected because it is defined as the most appropriate technique for the sterilization of polymeric device, micro and nanoparticulate systems, implant and scaffolds [18]. The second part of the work was aimed to design and prepare 3D scaffold and 2D film. Scaffolds were produced using specific preparation procedures which were set up and optimized to attend the compulsory requirements of systems intended for the TE [12]. Scaffolds were prepared using different LMP-3055 concentrations and characterized in terms of shape, size, apparent density, pore size and porosity. The in vitro degradation properties of 3D scaffolds and 2D film were assessed through gel permeation chromatography (GPC) and monitoring the mass loss, water uptake and buffer pH shifts. The mechanical properties of LMP-3055 3D scaffolds and 2D film were measured by an electromagnetic testing machine. Biological tests were performed on 3D scaffolds and 2D film using human fibroblasts and evaluating both cell adhesion and growth. 2. Experimental section 2.1. Materials The polymer LMP 3055 (95% mol of D,L lactide and, 5% mol of poly(1,5 cyclooctadiene-co-5-norbornene-2-methanol)), Mw 500 kDa and polydispersity 1.5, inherent viscosity 0.7 dl/g (~0.5% w/v in CHCl3 at 30 °C) was synthesized by the Marc Hillmyer research group (University of Minnesota). Tetrahydrofuran (THF) and 1,4 dioxane, hexane, cyclohexane and ethanol were purchased from Sigma-Aldrich Corporation (Milan, Italy). Krebs–Ringers Hepes (KRS): sodium chloride (NaCl, 0.1 M), potassium chloride (KCl, 3.6 mM), sodium bicarbonate (NaHCO3, 5 mM), monosodium phosphate (NaH2PO4, 0.49 mM), magnesium chloride anhydrous (MgCl2, 0.01 M), glucose (C6H12O6, 2.5 mM), bovine serum albumin (BSA, 0.0007 mM), N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES, 9.98 mM), sodium azide (NaN3, 0.15 M) and monosodium phosphate (NaH2PO4, Mw 119.98 g/ mol, 0.49 mM) were obtained from Carlo Erba, Milan (Italy). Dulbecco modified Eagle's medium (DMEM) with 4.5 g/l glucose and glutamine was purchased from Lonza, Milan (Italy). Fetal bovine serum (FBS, Eu approved) was purchased from EuroClone, Milan (Italy). Human adult dermal fibroblasts as primary cells were purchased from International PBI, Milan (Italy). All the reagents were of analytical grade. 2.2. Methods 2.2.1. Cytotoxicity studies The cytotoxicity of the LMP-3055 polymer was evaluated on two sets of samples: i) polymer suspension in DMEM at different concentrations (ranging from 0.01875 to 0.6 mg/100 μl of the working cell medium) and ii) LMP-3055 films (corresponding to LMP-3055 polymer amounts from 0.84 to 3.18 mg/100 μl of the working cell medium and prepared as reported below). Two sets of samples (polymer suspension and films) were considered because LMP-3055 polymer (as raw material) was like heterogeneous agglomerates, thus making difficult sample preparation. The effects of LMP-3055 polymer on cell viability were assessed with the 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium (MTT) assay, using 96 Well Cell Culture Cluster with 10,000 fibroblasts plated in contact to 100 μl of polymer suspensions and polymer film samples [19]. Briefly, fibroblasts were cultured in wells in DMEM supplemented with FBS for 24 h at 37 °C, then media were removed and fresh

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DMEM (without serum) with the samples was added. After 48 h, 25 μl of MTT solution (5 mg/ml in DMEM) was added into wells. Cells were incubated for 2 h at 37 °C to allow MTT reduction by mitochondrial dehydrogenase in viable cells. After 2 h, a suitable detergent was added to dissolve the resulting blue formazane crystals. Results were revealed by a multiwell scanning spectrophotometer (Microplate Reader Model 680, Bio-Rad Laboratories, USA). The optical density was measured at 595 nm with 655 nm as reference wavelength. Cell viability was calculated as the percentage of untreated cells (control). Considering the intended use of LMP-3055 graft copolymer to produce sterile products useful for TE field, this preliminary cytotoxicity study was completed evaluating the cytotoxicity of LMP-3055 graft copolymer films previously sterilized by gamma rays (25 kGy). 60Co was used as irradiation source (Applied Nuclear Energy Laboratory (L.E.N.A.), University of Pavia). It was checked by thermometric control that sample temperature during the irradiation did not appreciably increase above room temperature. 25 kGy was selected as the minimum absorbed dose considered adequate for the purpose of sterilizing medical and pharmaceutical products without providing any biological validation (European Guidelines 3AQ4a).

into the pores, ii) it was a LMP-3055 non-solvent and iii) it did not induce polymer shrinkage or swelling. Briefly, a weighted LMP-3055 3D scaffold (W) was immersed in a graduated cylinder containing a known volume (V1) of ethanol. The sample was kept in the non-solvent for 10 min, and then a set of evacuation–repressurization cycles were conducted to force the ethanol into the pore structure. Cycling was continued until no air bubbles were observed from the surface sample. The total volume of the ethanol and ethanol-soaked sample was recorded as V2. The volume difference (V2 − V1) is the volume of the LMP-3055 3D scaffold skeleton. The ethanol-soaked sample was then removed from the cylinder and the residual ethanol volume was recorded as V3. The volume (V1 − V3) represents the ethanol volume retained in the porous 3D scaffold and is defined as the pore volume of the sample. The total volume of the sample was calculated as follows: V ¼ ðV2 −V1 Þ þ ðV1 −V3 Þ ¼ V2 −V3 : The density of the scaffold (d) was expressed as: d ¼ W=ðV2 −V3 Þ:

2.2.2. Preparation of 3D scaffold and 2D film 3D scaffolds were prepared by a modified solvent/casting particulate leaching method [20] using 1,4-dioxan as solvent. Porogen particles (paraffin spheres) were sieved onto 1000 and 600 mm sieves and 200 mg of sieved porogen spheres were inserted in a cylindrical Teflon mold with a diameter of 10 mm. 500 μl of LMP-3055 solution (7.5, 10, 12.5 and 15% w/v in 1,4-dioxan) was cast drop by drop into the Teflon mold filled with the porogen. The mold containing the porogen and the polymer solution was first maintained at room temperature (RT, 25 °C), overnight to permit the diffusion of the polymer solution through the porogen particles and then it was placed at −25 °C for 24 h. The frozen porogen/polymer mixture was freeze-dried at − 50 °C for 12 h to completely remove the solvent. Scaffolds were dialyzed in hexane and cyclohexane (200 ml) at RT for 4 days. The solvent was changed three times a day, and after dialysis the scaffolds were freeze-dried at −50 °C overnight. The prepared scaffolds (3D-1, 3D-2, 3D-3 and 3D-4, Table 1) were stored in desiccators at −25 °C until characterization. 2D films were prepared by a previously optimized casting method [21]. Briefly, the LMP-3055 polymer was solubilized in methylene chloride (CH2Cl2) in order to obtain a homogeneous solution (7.5, 10, 12 and 15% w/v in CH2Cl2). The solutions were cast into Teflon molds of 44 mm diameter. Homogeneous films were obtained after solvent evaporation at 25 °C (RT). The 2D films (2F-1, 2F-2, 2F-3 and 2F-4 prepared respectively from 7.5, 10, 12 and 15% w/v polymer solution) were removed from the Teflon molds, cut into 5× 5 mm test sections, and stored for 48 h at −25 °C prior to further experimentation.

And the porosity of the 3D scaffold (ε), expressed as percentage (%), was calculated by: eð% Þ ¼ ðV1 −V3 Þ=ðV2 −V3 Þ  100: The density and porosity were determined in triplicate (n = 3) and expressed as mean ± standard deviation. 2.2.4. Scanning electron microscopy (SEM) A Zeiss EVO MA10 (Carl Zeiss, Oberkochen, Germany) scanning electron microscope was used to perform SEM analyses on LMP-3055 scaffolds. In order to evaluate the internal morphology, scaffolds were included into an agarose matrix (3% w/v) and then fractured using a cryotome (Cryostat, Leica CM1850) to obtain slides of the scaffold internal matrix. The samples were then sputtered with gold for the observation. 2.2.5. Gel permeation chromatography Gel Permeation Chromatography (GPC) was used to perform the physico-chemical characterization, in terms of weight-average molecular weight (Mw), weight-average molecular number (Mn) and polydispersity index (PI) polymer chains constituting 3D scaffold and 2D film. The GPC system consisted of three Ultrastyragel columns connected in series (7.7 × 250 mm each, with different pore diameters: 104 Å, 103 Å and 500 Å), a pump (Varian 9010, Milan, Italy), a Prostar 355 RI detector (Varian Milan, Italy), and software for computing Mw, Mn distribution and PI (Galaxie Ws, ver. 1.8 Single-Instrument, Varian Milan, Italy). 3D scaffolds and 2D film were dissolved in tetrahydrofuran (THF) under stirring and the system was maintained into ice bath to prevent THF evaporation. Polymer solutions were filtered through a 0.45 μm filter (Millipore, Massachusetts, USA) before injection into the GPC system (50 μl), and eluted with THF at 1 ml/min flow rate. Mw, Mn and PI of

2.2.3. Porosity and apparent density measurements The density and porosity values of LMP-3055 3D scaffolds were measured by a modified liquid displacement method [20]. Ethanol was chosen as the displacement liquid because i) it penetrated easily

Table 1 Characterization of 3D scaffolds composed of LMP-3055. Scaffold #

3D-1 3D-2 3D-3 3D-4

LMP-3055 concentration (%, w/v)

Porosity (%)

Pore size (μm)

Apparent density (g/l)

GPC analyses

7.5 10 12.5 15

76.6 78.0 77.5 82.2

200–900 100–400 100–200 b100

0.08 0.12 0.83 1.19

450 490 680 650

± ± ± ±

2.9 5.2 3.5 2.9

± ± ± ±

0.01 0.02 0.01 0.03

Mw (kDa) ± ± ± ±

MDSC analyses Mn (kDa)

2.6 1.3 0.53 0.8

180 260 470 430

± ± ± ±

PI (kDa) 3.6 5.9 0.14 0.12

2.2 2.11 1.8 1.5

± ± ± ±

Tg (°C) 0.5 0.8 0.6 0.9

– 45.9 ± 0.5 45.1 ± 0.5 49.5 ± 0.5

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each sample was calculated using monodisperse polystyrene standards (Mw 1–950 kDa). The data were processed as weight-average molecular weight (Mw), average molecular number (Mn) and polydispersity index (PI). The results were expressed as average molecular weight and molecular number percentage variations (ΔMw, ΔMn%), and as polydispersity index (PI). 2.2.6. Differential scanning calorimetry (DSC) Thermal behavior of LMP-3055 scaffolds was determined by a differential scanning calorimeter, DSC 2910 (TA Instruments, USA), fitted with a standard DSC cell and equipped with a liquid nitrogen cooling accessory (LNCA). Samples of about 10 mg were hermetically sealed in aluminum pans and subjected to two cooling and heating cycles from −80 °C to 200 °C at 5 °C/min. The DSC cell was purged with dry nitrogen at 40 ml/min. The baseline correction was performed by recording a run with empty pans. The system was calibrated both in temperature and enthalpy with indium as standard material. The data were treated with Thermal Solutions software (TA Instruments, USA) and the results expressed as the mean of three determinations. 2.2.7. Cell culture and 3D scaffold seeding Adult dermal fibroblasts as primary cells were purchased from International PBI (Milan, Italy). The cells were cultured in DMEM containing 10% fetal bovine serum (FBS, Eu approved) and 1% antibiotic solution (100 U/ml penicillin, 100 ug/ml streptomycin, Sigma-Aldrich Corporation). After expansion, the cells were detached for the cell seeding experiments. To define the most appropriate cell-seeding procedure, four seeding methods were investigated: i) static surface seeding, ii) cell suspension injection, iii) cell seeding with a lab-scale 2D rocker and iv) cell seeding using a centrifuge [22]. A cell seeding of 5 × 105 cells/scaffold was used for all 3D scaffolds, all samples were sanitized by washing with ethanol (70% v/v) for three times followed by sterile physiological solution (0.9% w/v) prior to cell culture. For the static surface seeding a concentrated cell suspension (50 μl) containing complete DMEM dropped on the top of the scaffold. The scaffold was incubated for 3 h to allow the attachment of the cells. At the end of the incubation, the scaffolds were submersed into complete DMEM. Cell suspension injection was performed by injecting a concentrate suspension (5 × 105 cells/20 μl) into the scaffold matrix using a sterile 25-gauge needle. Dynamic cell seeding with the use of a lab-scale 2D rocker was assessed as follow: scaffolds were incubated with the cell suspension (in complete DMEM containing 5 × 105 cells/3 ml per scaffold) in a sterile 50 ml falcon that was placed on the 2D rocker for 2 h at room temperature. After incubation, the scaffold was returned into a 6-well plate and submersed in complete medium. For the cell seeding with centrifuge the scaffold was incubated with the cell suspension (in complete DMEM containing 5 × 105 cells/3 ml per scaffold) in a sterile 50 ml tube and centrifuged at 2000 rpm at room temperature for 2 min. The centrifuge cycle was repeated for three times resuspending cell after each cycle, the scaffold was then returned to a 6-well plate with complete medium. To determine the long-term effects of cellseeding techniques, the experiments were carried out for 7 days: at day 3 the 3D scaffolds were moved into a new 6-well plate to avoid cell migration from bottom culture plate to scaffold matrix. The medium was completely refreshed every two days until the 7-day time point was reached. At day 7, the MTT assay was assessed to evaluate the number of cells onto/into 3D scaffold following the protocol described above. 2.3. MTT assay To determine cell-seeding efficacy, cell numbers on seeded scaffold were determined after 3 h of incubation using MTT (3-(4,5-dimethyl2-thiazolyl)-2,5 diphenyl-2H-tetrazolium) assay [19]. Cells in 2D culture (bottom of culture plate) seeded with equivalent numbers were used as total cell number controls. After 6 h, the cell-seeded scaffolds

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and controls (cells seeded on culture plate) were thoroughly rinsed in PBS and then 300 μl of MTT solution (5 mg/ml in DMEM without serum) was added into wells. The scaffolds were submersed in fresh DMEM without serum. Cells were incubated for 2.5 h at 37 °C to allow MTT reduction by mitochondrial dehydrogenase in viable cells. Thereafter, a suitable detergent was added to dissolve the resulting blue formazane crystals. The results were revealed by a multiwell scanning spectrophotometer (Microplate Reader Model 680, Bio-Rad Laboratories, USA). The optical density (OD) was measured at 595 nm with 655 nm as reference wavelength. The formazan product OD produced by cells attached on the scaffold matrix was compared with that of control cultures seeded with cell numbers equivalent to the scaffold seeding density to determine the total number of cells. The percentage of cell-seeding efficiency (Eq. (1)): cell seeding efficiency ¼

OD of cell seeded scaffold  100: OD of total cells for cell seeding

ð1Þ

2.4. Cell proliferation study Cell proliferation studies were assessed on 3D scaffolds and 2D film. All samples were sanitized by washing with ethanol (70% v/v) for three times followed by a sterile physiological solution (0.9% w/v) prior to cell culture. Fibroblast cells were seeded on the sanitized samples at fixed cell concentration (5 × 105 cells/3D scaffold and 2 × 104 cells/2D film) following the cell-seeding technique. The samples were kept at 37 °C in an atmosphere of 5% CO2 for incubation periods up to 28 days. At scheduled times (7, 10, 14, 21 and 28 days) scaffolds were removed from their respective wells and placed in new wells, after each time point, in order to ensure that only cells attached to the test samples were considered for analysis. Control cultures were grown on the bottom of wells. The data are expressed as total number of cells/scaffold. 2.5. In vitro degradation study 3D scaffolds and 2D film were incubated at 37 °C in 10 ml of Krebs– Ringer Hepes buffer (KRH), pH 7.4. The samples were fixed to the bottom of the 6-multiwell by agarose solution (3% w/v) to ensure that the whole sample was immersed into KRH buffer (9 ml), and then incubated at 37 °C in static conditions. The incubation buffer from each test tube was withdrawn, collected and replaced with fresh KRH buffer at regular intervals (twice a week). At scheduled times (3, 7, 10, 14, 21, 28 and 35 days) scaffolds were recovered, washed with distilled water, weighted and then lyophilized (Lio 5P, Cinquepascal s.r.l., Milan, Italy) at −50 °C, for 24 h. Polymer degradation properties were evaluated by GPC and by monitoring the water uptake into the scaffold matrix, the weight loss of the scaffold and the shift of KRH pH during all the incubation times. To reduce experimental errors, all of the data presented in the figures of this paper are the average data from 3 parallel samples and expressed as mean ± standard deviation (SD). 2.5.1. Water uptake determination The amount of buffer diffused and interpenetrated (WU) into the 3D scaffold and 2D film incubated in KRH at 37 °C was determined gravimetrically (Mettler Toledo AG 245, Milan, Italy). Briefly, scaffolds submitted to the in vitro degradation test were weighed immediately after recovering and subsequently rinsed with distilled water, freeze-dried and weighed. Water content was calculated as follows (Eq. (3)): Water content E ð%Þ ¼

Wt Wo  100 Wt

ð3Þ

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where Wt is the weight of wet sample at time t, and W0 is the initial weight of the dry sample. Water uptake was determined in triplicate (n = 3). 2.5.2. Weight loss determination The weight loss (WL) of 3D scaffolds and 2D film was determined gravimetrically (Mettler Toledo AG 245, Milan, Italy) at scheduled times (3, 7, 10, 14, 21, 28 and 35 days). The samples incubated in KRH were washed to remove buffer residuals and freeze-dried before gravimetric analysis. Weight loss was determined in triplicate, and computed as follows (Eq. (4)): Weight loss ð% Þ ¼

W′ 0 −W″ t 100 W′ o

ð4Þ

where W′0 is the weight of samples before incubation, and W′t is the weight of samples after incubation and freeze drying (n = 3). 2.5.3. pH determination pH shifts of KRH containing the samples were measured with a pH meter (pH meter 827 pH Lab; Metrohm, Switzerland) twice a week during the in vitro degradation tests. 2.6. Mechanical testing Mechanical testing of 3D scaffolds and 2D film was performed on an electromagnetic testing machine (Enduratec Elf 3200, Bose Corporation, Eden Prairie, MN, USA) with a load cell of 220 N. Different grips were used with the machine depending on the test configuration. Compression measurements were performed under displacement control, at a velocity of 0.1 mm/s until the sample was 40% of initial height. The machine actuator displacement was converted in strain defined as (l − l0) / l0, where l is the displacement and l0 is the initial height. The measured force was converted in stress dividing the force by the area of the sample section normal to the direction of the load. The elastic moduli were extracted from the linear regions of the stress–strain curves. Average and standard deviations were calculated for scaffolds of the same geometry and material. 2.7. Statistical analysis Data were expressed as mean ± standard deviation (S.D.). A comparison of mean values was performed using one-way analysis of variance (ANOVA). A statistically significant difference was considered for P b 0.01. 3. Results and discussion 3.1. Cytotoxicity studies The most important requirement for a biodegradable polymer to be used in medical application as TE is the physical and chemical compatibility and the behavior when in contact to the body. Cytotoxicity mainly deals with the substances that leach out the biomaterials, such as residual monomers, stabilizers, many other additives and degradation products and subsequent metabolites [23]. Collected data showed the absence of cytotoxicity of the LMP-3055 polymer in short time testing both for suspensions and for films. A quite wide range of polymer concentrations was considered in these two sets of experiments: 0.0175– 0.6 mg/100 μl of medium for suspensions and 0.84–3.18 mg/μl for film samples (Fig. 1). For all samples, namely LMP-3055 in bulk suspended in DMEM without serum and graft copolymer formulated as films, the fibroblast viability was good ranging from 85 to 100%. No evidence of interaction with cellular metabolism was highlighted. In the case of polymeric films incubated with fibroblasts, films were recovered after treatment in order to get information about the possible

Fig. 1. Cytotoxicity studies performed on LMP-3055 as a) polymer suspension and b) polymeric film at different concentrations/well (0.84–3.18 mg/100 μl). Cell viability results were determined by MTT assay after 48 h of incubation in complete DMEM at 37 °C and 5% CO2. Assay of equivalent cell number (1 × 104/well) on well plates were used as control values for total available cells.

chance of cell growth onto film surfaces. This chance was confirmed both by microscopy investigation and by the MTT assay carried out directly on the films: the determined viability percentage ranged between about 24 and 36%. These encouraging preliminary findings suggested that LMP-3055 polymer not only was not interfering with cells seeded onto the well bottom, but also that it could be suitably considered as a surface for the direct cell growth. Considering the intended application of polymer to produce biocompatible, biodegradable and sterile substrates for the regenerative medicine in the tissue engineering field, the cytotoxicity of LMP-3055 graft copolymer was also assessed on LMP-3055 film sterilized through gamma irradiation. The samples were sterilized in glass vials, at room temperature and at 25 kGy dose and subsequently they were incubated onto fibroblasts cultured in wells in DMEM without FBS for 48 h. Surprisingly, the results showed that the irradiation treatment led to a statistical significant increment of the cell viability percentage. For the considered concentrations (from 0.98 to 2.94 mg/100 μl), cell viability was much higher for gamma-irradiated samples with respect to non-treated films (Fig. 2) resulting in Abs values of more than 0.5 (P b 0.01). These preliminary data could be attributed to the effect of gamma irradiation on polymer composition and the type of leachable substances induced by gamma rays. The LMP-3055 treated through gamma rays can be considered cytocompatible and non-toxic because it has a positive effect in the sense of biofunctionality promoting the cell growth. To confirm these preliminary results further experiments are in progress that are aimed to identify and study the gamma-irradiation induced effects both on the LMP-3055 film surface, structural properties and on polymer physico-chemical properties.

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Looking into the pore size (Fig. 3a and d), the results suggested that the 3D scaffolds, obtained using 10 and 12.5% w/v polymer solutions, were the most successful formulation showing relatively uniform macro- and micro-porous architecture. 3.4. Differential scanning calorimetry

Fig. 2. Cytotoxicity studies performed on non-irradiated and irradiated LMP-3055 films, irradiation dose 25 kGy. Cell viability results were determined by MTT assay after 48 h of incubation in complete DMEM at 37 °C and 5% CO2. Assay of equivalent cell number (1 × 104 cells) on well plates were used as control values for total available cells.

3.2. Preparation of 3D scaffold 3D scaffolds were prepared using a previously set-up protocol [20]. Scaffolds were prepared using specific LMP-3055 concentrations (7.5, 10, 12.5 and 15% w/v in 1,4-dioxane) in order to assess the effect of the polymer concentrations both on the structural features of scaffold (such as porosity, pore size and apparent density) and on biological properties (such as cell seeding ability and cell attachment and proliferation). Scaffolds were characterized in terms of porosity, pore size and apparent density; moreover, the glass transition temperature (Tg) and Mw, Mn and PI of LMP-3055 3D scaffolds were evaluated by DSC and GPC, respectively (Table 1). All 3D scaffolds displayed a high porous structure with a porosity percentage ranging from 76.6 to 82.25%. The pore size appeared to be influenced by the graft copolymer concentration of the initial polymer solution: indeed, 3D-4 scaffolds with nominal pore size lower than 100 μm were achieved using the most concentrate polymer solution (15% w/v). On the opposite, the largest pore size (200–900 μm) was observed for 3D-1 scaffold prepared with a 7.5% w/v starting polymer solution. Apparent density values seem to be congruent with the pore size results: indeed, a more compact and dense matrix, with a density of 1.19 g/cm3, was detected for 3D scaffold prepared with the most concentrated solution (15% w/v). 3D-1, -2 and -3 scaffolds with an apparent density ranging from 0.08 to 0.83 g/cm3 were developed using polymer solution concentrations of 7.5, 10 and 12.5% w/v. No proof of structural failure was observed during the dialysis and freeze-drying phases. Moreover, freeze-dried scaffolds presented suitable physical properties and good handling characteristics. Prototypes presented rough surface, with a dense and compact structure, solid in consistency. 2D film demonstrated good handling resistance and robustness, useful to be exploited during the transplantation. No evidence of rupture was highlighted in the working conditions even after rehydration in simulated physiological buffer (unreported data). 3.3. Scanning electron microscopy SEM microphotographs of 3D scaffold internal matrix are shown in Fig. 3. The images are referred to scaffolds 3D-2 and 3D-3, while the images of 3D-1 and 3D-4 are not reported because the density and pore size values did not satisfy the requirement needed for their use as support for tissue regeneration (Table 1) [24]. The structure clearly changed with the polymer concentration (Fig. 3a–c and d–f): 3D scaffolds obtained with 10% w/v solution (3D-2) possessed a fibrous network with well interconnected and widespread porosity (Fig. 3a–c) while scaffolds 3D-3 had a more dense and compact structure (Fig. 3d–f).

The DSC traces recorded on the 3D scaffolds, polymer as raw material and paraffin porogen are reported in Fig. 4. Data were collected up to 200 °C since above this temperature incipient decomposition of polymer takes place. The baseline deflection corresponding to the glass transition and occurring at 49.3 °C for LMP-3055 raw polymer is also evident in the 3D-2, 3D-3 and 3D-4 traces. Glass transition temperatures change according to the analyzed scaffold and are reported in Table 1. It is possible to note that Tg values recorded on 3D-2 and 3D-3 scaffolds are 3 ÷ 4 degrees lower than that of LMP-3055 raw polymer. Such evidence highlights how the high porosity could affect the polymer thermal properties leading to lower glass transition temperatures [25]. Conversely, more compact samples with little amount of pores, as for scaffold 3D4 produce no significant Tg changes with respect to the raw polymer. About scaffold 3D-1, the situation seemed different: the appreciable amount of paraffin still included in the sample can easily be detected by comparing 3D-1 DSC trace with trace recorded for porogen particles. The double melting peak of paraffin falls in the temperature range of 20 °C ÷ 65 °C and completely masks the glass transition pertinent to 3D-1 scaffold. 3.5. Cells studies The rationale of cell seeding studies was to evaluate the effect of polymer concentration on the cell seeding capacity. The results of the cell seeding optimization were plotted in Fig. 5. Scaffolds were seeded using four different methods with the same seeding density (5 × 105 cells/scaffolds), the cell seeding efficiency was determined using MTT assay 6 h after seeding. Surface seeding and injection seeding had efficiencies ranging between 16.41 and 82.04%, respectively. The cell seeding efficiency values determined with the dynamic techniques (cell-seeding with a lab-scale 2D rocker and with a centrifuge) range from 24.32 and 55.17%. In comparing all the seeding methods the best results were observed with the static techniques and in particular the cell-seeding surface. The differences between the scaffolds (3D-1, -2, -3 and -4) could be correlated to the pore size and porosity characterizing the 3D scaffolds: the large pores (200–900 μm) detected for 3D-1 scaffolds and the small pores (b100 μm) of the 3D-4 scaffolds negatively affected the cell seeding efficiency when surface and injection techniques were performed. The cell numbers on the scaffolds were determined after 7 days of incubation, to highlight the influence of cell-seeding technique on the cell growth in the scaffolds. A cell reduction was highlighted at day 7 for 3D-1 and 3D-4 demonstrating that the scaffold microenvironment was not suitable for cell growth. Cell seeding data resulted to be congruent with the structural features highlighted by SEM analyses and by the experiments carried out to measure pore size. The lowest number of viable cells was found out both onto samples with the highest and heterogeneous pore size values (scaffolds 3D-1, pore size 100–800 μm) and the prototypes with the smallest pore size and dense and compacted texture (scaffolds 3D-4, pore size b 100 μm). Very large pores prevent cell attachment due to an area reduction and, to a limited available density, while very small pores prevent cells to penetrate and diffuse into the scaffold. Considering all these preliminary biological data, cell-seeding surface was selected as the most suitable technique to seed cells on LMP-3055 3D scaffolds. Moreover, 3D-2 and 3D-3 were chosen as the most appropriate and useful substrates to study and investigate the in vitro cell proliferation and degradation properties. 2D film presented a number of cells

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Fig. 3. SEM images of cross section of 3D scaffold composed of LMP-3055: a, b and c 3D-2 (113, 500× and 2.64 k× magnification) and d, e and f 3D-3 (507×, 3.58 and 21.10 k× magnification). Scale bars are shown in the micrographs.

lower with respect to 3D scaffold as a result of the structural features that characterized the 2D-prototypes: reduced porosity and limited surface area. Cell proliferation studies were carried out on 3D scaffolds and 2D film (Fig. 6). Differences were highlighted in cell behavior for 3D scaffolds and 2D film. In vitro 3-D cell culture conditions more accurately reproduce in vivo biological responses, as these conditions more strictly match the in vivo tissue structure and functions [26]. In vivo environments can be reproduced using 3D scaffolds that act as templates allowing cells to attach, proliferate and eventually deposit extracellular matrix (ECM). 2D films do not allow cells to move and interact freely as

it occurs in vivo. Thus, cells are not able to take advantage of the inherent features of 3D templates such as the exchange of nutrients, oxygen molecules and degradation by-products and the propagation of biological signals. The in vitro proliferation study of cells cultured onto/into 3D prototypes allows one to investigate deeply on the complex events which should take place in vivo, such as the mechanical and chemical signals arising from both bordering and even isolated cells. The data plotted in Fig. 6 showed a three-phase profile: for 3D scaffolds a gradual growth of cells till day 7th then a plateau phase till day 21st followed by a further increase of cells at day 28th was detected. 3D prototypes presented an equal number of viable cells onto/into scaffold (Fig. 6a). At day 28th, the cell numbers detected into the 3D-2 and 3D-3 scaffolds were 1.68 × 106/ and 1.83 × 106/ scaffold, respectively. 2D film presented a specific proliferation profile: a lag-time till day 7, for 2F-2 the cell number detected onto the surface of 2D-2 was 2.6 × 105 cells/film while 1.9 cells/film were calculated for 2D-3. The initial lag phase was followed by a gradual increment of cells till it reaches a plateau phase at day 21st, when the number of cells recovered on the 2F-2 and 2F-3 surfaces was almost equal (6.7 × 105 cells/film). No further increase was measured after day 21st (Fig. 6b). The results of the in vitro biological studies showed a positive result in terms of cell growth. Knowing that scaffold thickness is a critical parameter controlling the ability of cell to proliferate in the central part of the scaffold, these results are related to the experimental conditions here reported. Since this is a preliminary evaluation, the positive in vitro results will be deepened and further evaluated. 3.6. In vitro degradation study

Fig. 4. DSC traces recorded on 3D scaffolds (3D-1, 3D-2, 3D-3 and 3D-4), polymer raw material and paraffin porogen; data were collected between −80 and 200 °C.

Table 1 reported the GPC data referred to freeze-dried 3D scaffolds. The analyses were performed to assess how the preparation protocol affected the chemical properties of LMP-3055 graft copolymer. On the basis of the GPC data, it is possible to identify the use of organic solvents (1,4-dioxane to dissolve polymer, hexane and cyclohexan to remove paraffin porogen particles) as the most critical steps. The results demonstrated that organic solvents led to a statistically significant reduction of average Mw and Mn that was dependent on the LMP-3055 solution concentration. The Mw reduction of 3D-1 and 3D-2, calculated as percentage of the polymer in bulk Mw (~500 kDa), was 13.5 and 5.76%. Moreover, the Mn reduction was detected to be much higher with

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Fig. 5. Effect of seeding methods on cell seeding in 3D scaffolds. Total cell results determined 6 h after seeding and after 7 days of culture in complete DMEM with serum, at 37 °C, 5% CO2 using MTT assay. Equivalent cell number (5 × 105 cells) cultured on the well plates was used as control.

respect to Mw changes, ranging between about 45 and 21%. These results suggested an unzipping degradation mechanism which led to the release of small oligomers characterized by low Mw and good

Fig. 6. Cell proliferation study. Total cell results determined after 6 h, 7, 14, 21 and 28 days of culture in DMEM with serum, at 37 °C, 5% CO2. Equivalent cell number (5 × 105 cells for 3D scaffold and 2 × 105 for 2D film) cultured on the well plates was used as control (CRT).

water solubility, resulting in a higher decrement of the Mn with respect to the Mw [27]. The results of the Mw and Mn of 3D-1 and 3D-2 were stable at storage conditions; after two months of storage at 5 °C ± 2 °C the scaffolds' average Mw and Mn were not considerably changed (unreported data). The in vitro degradation study was assessed on 2F-2 and 3F-2 and it was set up following the approach used to make the in vitro proliferation study and the degradation performances of LMP 3055 as raw material was considered as control reference. 3D scaffolds and 2D film were incubated in KRH at 37 °C, for 35 days in static conditions. The in vitro degradation of LMP-3055 graft copolymer, as raw material, was also performed and considered as a control. Data demonstrated that the degradation rate of 3D scaffold was much higher with respect to the 2D prototype and to the LMP-3055 raw material, Fig. 7a. LMP-3055 graft copolymer presented a biphasic profile characterized by an initial lag-time of 14 days, followed by a slow increase of the Mw reduction percentage till 8% is achieved at day 28th. No further evidences of degradation reactions were detected for all incubation time (35 days). 2D film profile presented a short lag-time (7 days), followed by a gradual increase of the Mw reduction percentage reaching 16% after 21 days of incubation. 3D scaffold showed a profile characterized by a rapid Mw reduction (20%) in the first three days, followed by a plateau phase between day 3rd and day 14th. Further Mw reductions were detected at day 14th and day 28th, till a percentage reduction of 25% at day 35th is reached. The GPC data were supported by mass loss experiments (Fig. 7b). The mass loss profiles of 3D prototypes and LMP-3055 polymer (raw material) presented a similar trend of Mw reduction profiles, while the mass loss profile of 2D film reflected Mn reduction manner (data are included as Supplementary Fig. 7). The water uptake (WU) profiles are shown in Fig. 8. As expected, the trabecular structure and the internal pore volume of 3D scaffold were more appropriate to entrap and contain a much higher water amount with respect to 2D film and LMP-3055 polymer which had a dense and solid structure with no internal pore volume. For 3D scaffold, a gradual increase WU percentage was observed till an uptake of 250%

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through physiological buffer systems preventing and buffering inflammation reactions. The pH data did not highlight any statistically significant variations during the in vitro degradation time and at day 35th, the buffer pHs of 3D-, 2D-prototypes and LMP-3055 polymer were 7.8, 7.36 and 7.3, respectively. 3.7. Mechanical testing The results of compression testing on 3D prototype are summarized in Table 2. The compressive and tensile properties of 3D prototypes appeared to be sensitive to the pore volume and, consequently, to the porosity and pore size. Samples with high pore volume (3D-2) presented the lowest value of Young's compressive modulus (0.13 MPa), whereas 3D-3 which was characterized by pores that ranged from 100 to 200 μm had a Young's modulus of 0.27 MPa. Tensile Young's modulus, yield stress and ultimate tensile stress also critically depend on the structural features of scaffolds. The stiffness of 3D-3 was dictated by pore size. Initial mechanical stiffness increased or decreased varying the initial porosity: it was proved that the small pore size produced the highest stiffness but the lowest value of cell growth (Fig. 8). The increased mechanical stiffness was related to the higher polymer concentration used for the scaffold preparation, while the reduced number of cells could be correlated to small pore size and probably the restricted space available for proliferation. The tensile results obtained with 2D film exhibited stress whitening, neck formation and cold drawing with high elongation percentage (150 ± 27%). 4. Conclusion Fig. 7. In vitro degradation study. Mw reduction (a) and mass loss (b) percentages of LMP3055 raw material (bulk), 2F-2 film and 3D-2 scaffold (10% w/v) incubated in KRH (pH 7.4), at 37 °C for 35 days.

after 35 days of incubation is obtained. On the contrary, for 2D film and LMP-3055 polymer a more static situation was highlighted with respect to the 3D samples. These samples were able to entrap small percentages of buffer (50%) during all the incubation times. KRH pHs were measured by pH meter for all tested samples in order to complete and support the mass loss experiments. The release of the acidic oligomers from the polymer matrix should induce the reduction of buffer pH to acidic values when the buffer capabilities were not adequate to contrast pH changes. The buffer was selected to simulate the in vivo conditions: the acidic degradation by-products formed during the hydrolytic degradation process of polymer matrix are buffered

LMP-3055 could be considered a good candidate for tissue engineering applications. The cytotoxicity studies, performed on polymer demonstrated that the LMP-3055 was not toxic and no toxic leachable substance was released during the incubation in DMEM. Moreover, the study carried out on LMP-3055 polymer samples sterilized by gamma irradiation at 25 kGy suggested that the sterilization treatment did not affect the biofunctionality of cells. 3D scaffold and 2D film prepared with LMP-3055 could be considered a suitable substrate for the in vitro and in vivo tissue regeneration. SEM analysis revealed the porous structure of 3D-2 and 3D-3 scaffolds with well interconnected pores promoting nutrients and waste product exchanges. Degradation test revealed that the degradation of 3D scaffold polymer matrix (ΔMw) and water uptake exceed those of LMP-3055 polymer (as raw material) and 2D film. Nevertheless, the mass loss was controlled, resulting in good stability of the construct and capacity to maintain the physical integrity. Mechanical analysis showed that the compressive and tensile strength were related to the structural features of scaffolds. Indeed, the trabecular structure of 3D scaffold led to a system with more limited tensile strength with respect to the 2D film. The study performed on LMP-3055 demonstrated the capability of using the polymer as a biodegradable and biocompatible substrate for TE, in particular it will be used to formulate graft to repair urethral strictures or defects.

Table 2 Mechanical properties of LMP-3055 3D scaffolds. Polymer type

Compression Tensile LMP-3055 concentration Ec Et (%, w/v) (Mpa)a (Mpa)a

LMP-3055 10 12.5 a

Fig. 8. In vitro degradation study: water uptake behavior of LMP-3055 as raw material (bulk), 2F-2 and 3D-2 scaffold (10% w/v) incubated in KRH (pH 7.4), at 37 °C for 35 days.

b c

0.13 ± 0.06 0.27 ± 0.09

Yield stress (Mpa)b

0.25 ± 0.16 0.04 ± 0.06 0.36 ± 0.24 0.02 ± 0.01

Modulus of elasticity or Young's modulus. Yield strength point or yield stress. Ultimate tensile strength (UTS) or failure stress.

UTS (Mpa)c 0.04 ± 0.07 0.02 ± 0.01

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.08.037.

Acknowledgment

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Rossella Dorati is an assistant professor at the Department of Drug Sciences, College of Pharmacy, University of Pavia. She received her PhD in Pharmaceutical Chemistry & Technology in 2005. The research activity of Dr. Rossella Dorati develops in the Pharmaceutical Technology area. Her current research interest revolves around formulation and characterization of scaffolds as implantable and temporal devices in tissue engineering, based on synthetic and natural polymers.

The authors would like to thank the Marc Hillmyer research group (University of Minnesota) for donating samples of graft copolymer of polylactic acid (PLA), LMP-3055.

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Claudia Colonna received her PhDs in Pharmaceutical Chemistry & Technology in 2007 and in Biomolecular Sciences and Biotechologies at the Istituto Universitario di Studi Superiori (IUSS) of Pavia in 2011. Most recent research interests involve: formulation, characterization and study of micro- and nanoparticulate drug delivery systems made of biodegradable natural polymers such as chitosan, its salts and derivatives and loaded with macromolecular drugs, biomolecules, proteins and peptides.

Corrado Tomasi is a member of the I.E.N.I. C.N.R. (National Research Council) in Pavia He received his PhD in Chemical Sciences in 2003. His current research interests are dealing with the physico-chemical characterization of polymers and sol–gel compounds applied on different fields such as pharmacy, biology and energy storage and production. His research activity is documented by more than 75 scientific publications on international journals.

Ida Genta is Associate Professor at the Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Pavia, Italy. Professor Genta's research focus on the areas of Pharmaceutical Technology and Applied Pharmaceutical Chemistry: micro- and nano-particles based on biodegradable poly-alfa-hydroxyacids such as poly-lactide, poly-glycolide and their copolymers and intended for parenteral, intraarticular and local drug administration. More recently block copolymers made of polyethylenglicole and lactic acid (PEG-PLLA, PEG-PDLA) have been used because of their usefulness in parenteral administration.

Dr. Giovanna Bruni is Researcher at the School of Pharmacy of the University of Pavia. She teaches the courses Physical Chemistry and Physical Chemistry of Dispersions. Her main research interests are in the field of physico-chemical characterization of compounds of pharmaceutical interest. The long experience in this field and the expertise gained in the use of thermal techniques, FT-IR spectroscopy and electron microscopy are the basis for an activity of problem solving in the pharmaceutical industry. The research activity is documented by about seventy scientific publications on peer-reviewed international journals (ISI).

Bice Conti is a full professor of Applied Pharmaceutical Chemistry at the Department of Drug Sciences of the University of Pavia, Italy and is a coordinator of the undergraduate Pharmacy and Pharmaceutical Chemistry and Technology Courses of the University of Pavia, Italy. She teaches undergraduate, graduate and PhD students either Pharmacy or Biotechnology courses. She is a professor in the project of “Continuing Education in Medicine”. Researches in these years involved characterization of new biopolymers and their behavior to gamma-irradiation, formulation of drug delivery systems such as micro and nanoparticles and films. Her current research interest revolves around formulation and characterization of scaffolds as implantable and temporal devices in tissue engineering.