Carbohydrate Polymers 124 (2015) 180–187

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Effect of crystallinity and plasticizer on mechanical properties and tissue integration of starch-based materials from two botanical origins Diego Velasquez a , Graciela Pavon-Djavid b , Laurent Chaunier a , Anne Meddahi-Pellé b,c , Denis Lourdin a,∗ a

INRA, UR1268 Biopolymères Interactions Assemblages, Rue de la Géraudière, 44316 Nantes, France INSERM, U1148, Université Paris 13, Université Paris7, Sorbonne Paris Cité LVTS, 46, rue Henri Huchard, 75877 Paris, France c Université Paris 13, Sorbonne Paris Cité, Institut Universitaire de Technologie de Saint-Denis, Place du 8 mai 1945, 93206 Saint-Denis, France b

a r t i c l e

i n f o

Article history: Received 13 September 2014 Received in revised form 2 February 2015 Accepted 3 February 2015 Available online 12 February 2015 Keywords: Starch Crystallization Mechanical properties Biocompatibility

a b s t r a c t The application of starch-based materials for biomedical purposes has attracted significant interest due to their biocompatibility. The physical properties and crystal structure of materials based on potato starch (PS) and amylomaize starch (AMS) were studied under physiological conditions. PS plasticized with 20% glycerol presented the best mechanical properties with an elastic modulus of 1.6 MPa and a weak swelling, remaining stable for 30 days. The in vitro cell viability of 3T3 cells after contact with extracts from PS and AMS with 20% glycerol is 72% and 80%, respectively. PS presented good tissue integration and no significant inflammation or foreign body response after 30 days intra-muscular implantation in a rat model, contrary to AMS. It was shown that glycerol plasticization favors a fast B-type crystallization of PS materials, enhancing their mechanical strength and durability, and making them a good candidate for bioresorbable and biocompatible materials for implantable medical devices. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Polysaccharides are widely used in biomaterials development (Khan & Ahmad, 2013). Among these, starch is commonly available since it can be obtained from maize, potato, wheat and rice, and is a low cost product because of its widespread availability. Starch is composed of two glucose homopolymers: amylose (a linear polymer) and amylopectin (a highly branched polymer), as well as minor components such as lipids and proteins. Depending on its botanical origin, the amylose/amylopectin ratio can vary: 20/80 for potato starch, 25/75 for normal maize starch, 70/30 for amylomaize (high amylose maize) starch and 34/66 for pea starch (Buléon, Colonna, Planchot, & Ball, 1998). Starch is degraded by alpha-amylase, an enzyme present in saliva during the first step

Abbreviations: PS-G0 , non-plasticized potato starch; PS-G10 , potato starch plasticized with 10% glycerol; PS-G20 , potato starch plasticized with 20% glycerol; AMS-G0 , non-plasticized amylomaize starch; AMS-G10 , amylomaize starch plasticized with 10% glycerol; AMS-G20 , amylomaize starch plasticized with 20% glycerol. ∗ Corresponding author. Tel.: +33 02 40 67 51 47; fax: +33 02 40 67 50 05. E-mail addresses: [email protected] (D. Velasquez), [email protected] (G. Pavon-Djavid), [email protected] (L. Chaunier), [email protected] (A. Meddahi-Pellé), [email protected] (D. Lourdin). http://dx.doi.org/10.1016/j.carbpol.2015.02.006 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

of human carbohydrate digestion (Pedersen, Bardow, Beier Jensen, & Nauntofte, 2002). Starch is a very versatile raw material that can be processed into thermoplastic materials by extrusion, injection molding and thermomolding to produce porous or dense materials. The mechanical properties and, particularly, the rigidity of starch-based materials can be modulated by the addition of plasticizers such as glycerol or sorbitol that decrease the glass transition of materials (Hulleman, Kalisvaart, Janssen, Feil, & Vliegenthart, 1999; Lourdin, Coignard, Bizot, & Colonna, 1997; van Soest & Knooren, 1997). However, a low glass transition can induce a slow crystallization that increases the stiffness and strength of materials (van Soest, Hulleman, de Wit, & Vliegenthart, 1996; Viguié, Molina-Boisseau, & Dufresne, 2007). In biomedical applications, the degradation profile, the swelling ratio and the controlled release capacity of materials containing modified starch (Björses et al., 2011; Dandekar et al., 2012; Echeverria, ˜ & Gurruchaga, 2005; Elvira, Mano, San Román, & Reis, Silva, Goni, 2002) such as hydroxypropyl starch-ethyl methacrylate, starchmethacrylate (Echeverria et al., 2005) and starch-ethylene vinyl alcohol have been studied, either as hydrogels or as thermoplastic materials (Alberta Araujo, Cunha, & Mota, 2004; Franco-Marquès, Méndez, Gironès, Ginebra, & Pèlach, 2009). Starch is also introduced into composite materials to improve the biocompatibility of the blended material (Rodrigues & Emeje, 2012). Recently, there has

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been a growing interest in starch as a biomaterial for tissue engineering applications. For example, starch added to poly(␧caprolactone) materials was used to improve cell adhesion and proliferation (Santos et al., 2008), enhance angiogenesis, and promote in vivo tissue integration (Santos et al., 2007; Santos, Unger, Sousa, Reis, & Kirkpatrick, 2009) and repair (Rodrigues, Gomes, Leonor, & Reis, 2012; Santos et al., 2010; Silva et al., 2012). In our previous work, we studied the structure-property relationships of starch materials (Bizot et al., 1997; Lourdin et al., 1997; Lourdin, Della Valle, & Colonna, 1995). We recently demonstrated their shape-memory effect (Véchambre, Chaunier, & Lourdin, 2010) and the possibility of triggering this effect in water at 37 ◦ C for the development of shape-memory stents (Beilvert, Chaubet, et al., 2014; Beilvert, Faure, et al., 2014). Despite a few studies (Shi et al., 2006), research focused on pure starch-based materials for biomedical purposes remains rare. In this study, we evaluated the influence of crystalline structure and plasticizer content on the physical properties of starch-based materials from two botanical origins (amylomaize and potato) immersed in physiological medium, as well as their in vitro cell effect and in vivo tissue response in a rat animal model. 2. Materials and methods 2.1. Production and characterization of starch materials Starches from two botanical origins were extruded: potato starch (PS) (Roquette, France) with a carbohydrate composition of approximately 20% amylose and 80% amylopectin, and amylomaize starch (AMS) (Eurylon7® , Roquette, France) with approximately 70% amylose and 30% amylopectin. Starch also contains minor components, i.e., lipids (1.1% in AMS and 0.1% in PS) and proteins (0.5% in AMS and 0.05% in PS). Prior to extrusion, the water content of starch flours was adjusted to 27%. For plasticized materials, 10% and 20% of glycerol, whose efficiency as a plasticizer was previously demonstrated (Lourdin et al., 1997), was added to the hydrated total mass of the preparation. Samples are referred to as PS-GX and AMS-GX for potato and amylomaize starch-based samples, respectively, where GX indicates the percentage of glycerol (0%, 10% and 20%). For extrusion, a Scamia single-screw device (Rheoscam Type 20.11d, France) was used. A flat die of 25 mm × 2.5 mm was adapted in order to obtain ribbon-shaped materials. The temperature profile for extrusion was [100 ◦ C, 110 ◦ C, 110 ◦ C] from the entry of the flour starch into the machine to the extrusion outlet. After extrusion and in order to have a normalized shape, samples were thermomolded and cut into squares (surface area of 1 cm2 ) to have controlled thickness, weight and surface state. In order to stabilize the water content, samples were kept in a closed vessel containing a saturated solution of NaBr, with a relative humidity of 57% at 20 ◦ C. After two weeks of moist stabilization, the glass transition temperature (Tg ) was determined by differential scanning calorimetry (DSC) with an automated T.A. Q100 instrument (T.A. Instruments, USA). The moisture content of stabilized samples was measured by thermogravimetry on a TGA2050 (T.A. Instruments, USA). The elastic modulus was measured with a dynamometer in compression mode (Instron Corporation, USA). The compression rate was 5 mm/min, with a force sensor of 500N and a surface contact of 78.5 mm2 . Samples with the normalized shape (area: 1 cm2 ; thickness: 1.5 mm) were used for in vitro and in vivo characterizations. 2.2. Characterization of materials under physiological conditions 2.2.1. Aspect and swelling The aspect and swelling evolution of starch materials (n = 3) at 5, 15, 60, 240 and 1440 min (1 day) and 43,200 min (30 days)

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was determined by immersion of samples weighing 0.21 ± 0.03 g in 2.5 mL 1× Phosphate Buffered Saline (PBS) at pH 7.4 and 37 ◦ C. After removing the PBS solution, samples were weighed and swelling was determined with the following equation: W=

Wf − W0 W0

where Wf is the mass at the time of measurement and W0 is the mass before immersion. Thickness was measured at the same end points of swelling for another series of samples with a sliding caliper. The physical appearance of materials immersed in 24well plates containing 2.5 mL of PBS at 37 ◦ C was photographically recorded over one month using a digital camera (Casio QV-5700, Japan). 2.2.2. Remaining glycerol The plasticizer release from samples (n = 3) containing glycerol in 2.5 mL of PBS at 37 ◦ C was determined by high-pressure liquid chromatography (HPLC). The chromatographic system consisted of a degasser (ERC-3310, ERMA Inc., Japan) a programmable pump (Waters 590 Programmable HPLC pump, Waters, USA), an auto sampler (Waters 717 plus, Waters, USA) with an HX 87H Aminex column (BioRad, USA) at 40 ◦ C, and a differential refractor (Waters 2414 Refractive Index Detector, Waters, USA). The glycerol was dosed from aliquots of 500 ␮L from the filtered supernatant using a syringe-filter of 0.47 ␮m. The glycerol peak appears at 10.8 min for a flow rate of 0.7 mL/min and a pressure of 7.6 MPa. 2.2.3. Mechanical properties The evolution of the elastic modulus during immersion of materials in physiological conditions (PBS, pH 7.4 at 37 ◦ C) was measured with a dynamometer in compression mode as described in Section 2.1. These measurements (n = 3) were taken after 5, 15, 60, 240, 1440 (1 day) and 43,200 min (30 days). 2.2.4. Crystallinity The evolution of crystallinity of starch materials in physiological conditions was measured by Wide Angle X-ray Scattering using a Bruker D8 X-ray diffractometer (Bruker, Germany). Following their removal from the PBS solution, samples were rapidly dried in order to stop the crystallization and to avoid the high X-ray absorbance of water. Samples were immersed in two baths of 95% ethanol, followed by two baths of absolute ethanol. Dehydrated samples were kept in a vacuum container at 375 mbar overnight and then re-stabilized in a closed environment at a relative humidity of 75% with a NaCl solution. The relative crystallinity of samples was determined using Wakelin’s method, with two standards: an amorphous spectrum of extruded potato starch and spherulites of recrystallized potato starch (Wakelin, Virgin, & Crystal, 1959). 2.3. In vitro and in vivo studies 2.3.1. Cell cytotoxicity of starch-based material extracts Samples (0.21 ± 0.03 g) were immersed in 3 mL of Minimum Essential Medium (MEM, Gibco, USA) supplemented with 10% fetal bovine serum, 1% l-glutamine and 1% of an antibiotic solution of penicillin-streptomycin (Gibco, USA) for 24 h at 37 ◦ C, and 5% CO2 . BALB/c 3T3 (CCL-163TM , ATCC, USA) were cultured and 3 × 104 cells were plated in 48-well plates until confluence. Then, 250 ␮L of the extract solution (pure at 100% and diluted at 25% in supplemented MEM) were added. Addition of 250 ␮L of dimethyl sulfoxide (DMSO) at 10% represented the positive control of cytotoxicity. The complete medium represented the negative control. After 24 h, an MTT assay was carried out and

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Fig. 1. In vitro characterization of extruded potato starch (PS) and amylomaize starch (AMS) samples: (A) swelling ratio, (B) thickness evolution, (C) kinetics of remaining glycerol, and (D) macroscopic appearance of samples (10 mm × 10 mm × 1.5 mm) after 30 days under physiological conditions (PBS, 37 ◦ C, pH 7.4). For all insets: PS-G0 (䊉), PS-G10 (), PS-G20 (), AMS-G0 (), AMS-G10 (), and AMS-G20 ().

the viability of cells was determined as previously described (Mosmann, 1983). 2.3.2. In vivo implantation of samples Both the procedure and the animal treatment complied with the Principles of Laboratory Animal Care formulated by the French National Society for Medical Research. The studies were carried out under Authorization N◦ 006235 of the French Ministry of Agriculture. Samples used for implantation were PS-G0 , PS-G10 , PS-G20 and AMS-G0 . Male Wistar rats, 8-weeks-old and weighing 300 ± 50 g (Janvier Labs, France), were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg of body weight). After anesthesia, the abdomen wall was shaved, disinfected, and then draped in a sterile fashion. A vertical incision was made on both the left and right side of the abdominal midline and the materials were implanted in an abdominal intramuscular position. The skin and the muscle layer were sutured (Vicryl® 4/0) after intramuscular implantation. Rats were euthanized by an intraperitoneal injection of sodium pentobarbital (60 mg/kg) at day 30 postsurgery. Implanted materials were dissected, and at least 0.5 cm of surrounding tissue was excised, gently rinsed in saline solution and fixed in 4% paraformaldehyde solution, dehydrated and

embedded in paraffin. Five micron-thick sections were obtained (Leitz Wetzlar microtome, France), stained with HematoxylinPhloxine-Saffron (HPS). Labeling of Reca-1 for endothelial cells, CD80 (type-1 macrophages, M1) and CD163 (type-2 macrophages, M2), was performed by immunostaining (working dilution: 1/30) using the EnVision kit (Dako, Denmark). Digital images were obtained with a NanoZoomer 2.0-RC C 10730 (Hamamatsu, Japan) and image analysis was performed using NDP.view 2 software (Hamamatsu, Japan). Fibrotic capsule thickness and granuloma size were analyzed using three visual fields from three different sections. 2.4. Statistical analysis Data were expressed as the mean ± standard deviation, and one-way analysis of variance (ANOVA) was performed (JMP software, JMP® 9.0.1). In order to be analyzed, data was subdivided into groups according to their glycerol content (0%, 10% and 20%) and physicochemical (water uptake, thickness evolution, glycerol release, crystalline evolution), mechanical (elastic modulus) and biological properties. Differences among the means of p < 0.05 were considered to be significant. 3. Results

Table 1 Physical properties of starch-based materials. Sample

Moisture content (%)

Tg (◦ C)

Initial elastic modulus (MPa)

PS-G0 PS-G10 PS-G20 AMS-G0 AMS-G10 AMS-G20

10.5 10.4 12.6 11.9 10.7 15

87 40 16 77 32 17

105 89.9 64.8 114 107 83

± ± ± ± ± ±

4 1.3 0.6 16 16 4

3.1. Production and characterization of starch-based materials Reproducible starch-based samples were obtained with stable thickness (1.5 ± 0.2 mm) and weight (0.21 ± 0.03 g). Moisture content and Tg of samples after extrusion and stabilization in a relative humidity of 57% are presented in Table 1. The water content of approximately 10–12% increases up to 15% for

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Fig. 3. In vitro cell viability on BALB/c 3T3 cells after exposure to AMS (A) or PS (B) extraction in supplemented MEM at 37 ◦ C. *Samples with viability values that are significantly different (p < 0.05).

Information) for AMS samples. PS-G0 , PS-G10 and AMS-G0 samples began to degrade at 90 min. AMS-G10 and AMS-G20 samples began to degrade rapidly as of 5 min after immersion. PS-G20 did not present an obvious degradation, and lasted for 30 days.

Fig. 2. In vitro characterization of mechanical properties and crystallinity of extruded potato starch (PS) and amylose-rich maize starch (AMS) samples under physiological conditions (PBS, 37 ◦ C, pH 7.4). Elastic modulus of (A) AMS samples and (B) PS samples, as well as crystallinity (C). Arrows in (A) indicate the last collected values of the elastic modulus. For all insets: PS-G0 (䊉), PS-G10 (), PS-G20 (), AMS-G0 (), AMS-G10 (), and AMS-G20 ().

compositions containing 20% glycerol. PS-G0 and AMS-G0 had a Tg of 87 and 77 ◦ C, respectively. Addition of glycerol decreased this Tg to below room temperature at 16 and 17 ◦ C for PS-G20 and AMS-G20 , respectively. This means that PS-G0 and AMS-G0 were glassy, rigid and weakly deformable materials with a high elastic modulus (105 ± 4 and 114 ± 16 MPa, respectively). Samples PS-G20 and AMS-G20 containing 20% glycerol were rubbery and less rigid, with a lower elastic modulus (64.8 ± 0.6 and 83 ± 4 MPa, respectively). 3.2. Characterization of materials under physiological conditions 3.2.1. Macroscopic aspect The macroscopic aspect of the sample during the follow-up in PBS at 37 ◦ C is presented in Fig. 1D and in Figure S1 (Supporting Information) for PS samples, and in Figure S2 (Supporting

3.2.2. Swelling and thickness The evolution of the swelling ratio under physiological conditions is shown in Fig. 1A. At day 1, PS-G0 presented a maximum swelling ratio that was significantly higher (4.5 ± 0.7) than PS-G10 (3.5 ± 0.2), with p < 0.05. The swelling ratio decreased for PS-G0 and PS-G10 (to 4.2 ± 0.6 and 3.2 ± 0.1, respectively) after 1 day, probably due to the dissolution of amylopectin. The swelling ratio of PS-G20 was 1.20 ± 0.04 and stabilized after 240 min. In the case of AMS materials, swelling moderately increased during the first day and stabilized at a ratio of 0.7. Thickness also evolved over time, notably for PS-G0 and PS-G10 , as shown in Fig. 1B, meaning that both water uptake and thickness considerably increased under physiological conditions. Due to the considerable erosion of the samples AMSG10 and AMS-G20 , no data were collected after 5 and 15 min of immersion. 3.2.3. Determination of remaining glycerol Fig. 1C represents the amount of glycerol remaining in the samples containing 10% and 20% glycerol in PBS at 37 ◦ C. Different time sequences were observed: there is a rapid release during the first 15 min that mainly depends on the initial concentration of glycerol. As of 240 min, there is a slow release of glycerol, and a low percentage of glycerol (1.1%) remained in AMS-G20 after 1 day. No significant difference was observed between PS and AMS starch (p < 0.05). 3.2.4. Mechanical properties and crystallinity Fig. 2A and B shows the decrease of the elastic modulus of all samples during the first minutes after immersion. This corresponds to the very efficient plasticizing effect of water on amorphous starch (Bizot et al., 1997). For AMS-G10 and AMS-G20 , the measurement of mechanical properties was not possible after 15 min due to their rapid disintegration (the last values are indicated by arrows in Fig. 2A). For the other samples, the mechanical properties remained measurable throughout the experiment. PSG0 presented a low elastic modulus of 0.2 MPa, whereas PS-G20

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Fig. 4. Non-plasticized starch materials from amylose-rich maize (AMS-G0 ) and potato starch (PS-G0 ) after 30 days in vivo intramuscular implantation in a rat model. (A) and (E): Macroscopic view after skin removal. A granuloma (white slanted line in (A)) was observed in the AMS-G0 sample. In AMS-G0 , the fibrotic capsule ((B), double arrow) surrounding a necrotic core (inner tilted black shape) was shown. (C) Magnification of (B) with black arrows indicating the remaining fragments of AMS-G0 . In PS-G0 , the sample was revealed between the slanted black lines (F). Cell infiltration and matrix deposition were observed at higher magnifications (G). (B, C, F, G) hematoxylin-phloxine-saffron staining. CD163 immunohistological analysis of AMS-G0 (D) and PS-G0 (H).

and AMS-G0 maintained an elastic modulus at 1.5 and 9.8 MPa, respectively, after 43,200 min (30 days) of immersion in physiological conditions. Stabilization of the geometry and the mechanical properties was probably due to the simultaneous evolution of crystallinity in PS samples, as shown in Fig. 2C. PS-G20 presented a rapid increase of B-type crystallinity from 15% after 1 h, to 25% after 30 days of immersion. Crystallization of PS-G10 and PS-G0 is observed after 1 day and 30 days, respectively. Crystallinity of PS-G0 and PS-G10 was statistically different from PS-G20 (p < 0.05). The evaluation of B-type crystallinity on AMS samples was disturbed by the presence of amylose-lipid complexes that lead to a superimposition of a V-type pattern. Patterns of X-ray

diffraction are shown in Figures S3 and S4 (Supporting Information).

3.3. In vitro and in vivo studies 3.3.1. Cell viability of starch material extracts The in vitro viability test is presented in Fig. 3. At 25% dilution, 100% cell viability was obtained. No significant difference was observed in cell viability at a 25% dilution of extracts. In pure extracts (100%), cell viability was 88.3 ± 1.7% (AMS-G0 ), 91 ± 17.8% (AMS-G10 ) and 80.3 ± 12.6% (AMS-G20 ). With pure extracts (100%)

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Fig. 5. Plasticized potato starch materials (PS-G20 ) after 30 days intramuscular implantation in a rat model. (A). Macroscopic view after skin removal revealed the remaining sample. Hematoxylin-phloxine-saffron staining shows the sample cell colonization (B) (black slanted lines) and the remaining starch material (black arrows in (C)). (D) CD163 immunohistological staining.

of PS, a slight decrease was observed with the presence of glycerol, with the lowest value of 71.8 ± 9.2% for PS-G20 . 3.3.2. In vivo response of starch materials During the in vivo implantation study, no animals died nor presented signs of infection. At 30 days post-implantation, only the AMS-G0 samples induced macroscopically visible tissue reactions (Fig. 4A). The histological and immunohistochemistry study of AMS-G0 samples (n = 3) confirmed the presence of a granuloma with a core diameter of 4.84 ± 0.8 mm, surrounded by a thick fibrotic capsule (1.6 ± 0.7 mm) composed of starch debris (black arrows, Fig. 4C). CD163 immunostaining revealed M2 macrophages within samples (Fig. 4D), whereas CD80 was negative for M1 macrophages (Figure S5, Supporting Information). Cell colonization was observed in PS samples (Figs. 4G, 5C and S7B, Supporting Information). This cell colonization was achieved within PS-G0 (Fig. 4G). Starch remained visible after the addition of 10% or 20% glycerol, as indicated with arrows in Figure S7B (Supporting Information) and Fig. 5C. CD163 immunolabeling showed the presence of M2 macrophages in PS samples (Figs. 4H, 5D and S7C, Supporting Information), whereas no M1 macrophages (CD80) were shown in Figure S5 (Supporting Information). For all samples, Reca-1 immunostaining did not show the presence of endothelial cells (Figure S5, Supporting Information). 4. Discussion In a previous study, we demonstrated the possibility of obtaining potato-starch devices that exhibit a small-size wire design, as well as shape-memory properties that make it possible to use a stent at 37 ◦ C in water (Beilvert, Chaubet, et al., 2014). This device had short-term durability in an alpha-amylase solution and did not induce a pathological foreign-body reaction when it was implanted in vivo during a short period of time (8 days) (Beilvert, Chaubet, et al., 2014). In the present study, we focused on the effect of crystallinity and plasticizer content on the physical properties of starch materials immersed in a physiological solution at 37 ◦ C and then studied the in vivo response over a longer period (30 days).

Without plasticizer, the immersion of amylomaize starch (AMS-G0 ) and potato starch materials (PS-G0 ) in PBS at 37 ◦ C led to elastic moduli at equilibriums that were approximately 50 times higher for AMS-G0 than for PS-G0 , with 9.8 and 0.2 MPa, respectively. It is well known that the increase of the amylose/amylopectin ratio promotes mechanical properties of starch materials in the “dry” state (Lourdin et al., 1995; van Soest & Essers, 1997). In this study, differences in mechanical properties are instead attributable to the plasticizing effect of water uptake whose level is almost six times higher for PS-G0 (450%) than for AMS-G0 (80%). Such a behavior is probably due to the higher hydrophilic capacity and solubility of amylopectin in comparison to amylose (Leloup, Colonna, & Buleon, 1991). The immersion of materials in PBS also has a significant impact on crystallization. In the case of potato starch, it led to B-type crystallization whose kinetic is strongly promoted by the initial plasticizer concentration. This could be due to the presence of crystal germs induced by glycerol during the fabrication and the storage of samples that exhibit a crystallinity of 5% before immersion for PS-G20 , whereas other compositions present quasi-amorphous initial structures. The rapid crystallization of PS-G20 that takes place during the first few hours of immersion simultaneously with glycerol release preserves the material from swelling and stabilizes its elastic modulus at between 1 and 2 MPa. These results are in accordance with the previous study carried out in water and characterized at small deformations by dynamic mechanical analysis, where the dynamic modulus E decreased from 1 GPa to 2.4 MPa after 6 h (Beilvert, Chaubet, et al., 2014). Such a modulus in a rubbery state can be compared to polydimethylsiloxane (PDMS) used for biomedical applications (Nunes, 2011; Pinto et al., 2010). In contrast, without glycerol, the rapid swelling due to water uptake appears well before crystallization. With a low glycerol content of 10%, the delay in crystallization is slightly shortened but is still too long to avoid swelling. The crystallization behavior of AMS containing glycerol is more complicated than potato starch since B and V types of crystalline structures are present, which is consistent with previously published results (Zabar, Shimoni, & Bianco-Peled, 2008). They showed

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a significant delamination and erosion during immersion, in comparison to PS-G20 . This behavior could be due to heterogeneities present in the initial material, leading to a water uptake gradient. Concerning in vitro studies, PS-G0 and AMS-G0 extracts show 100% cell viability, regardless of the botanical origin. Increasing the concentration of plasticizer in samples induced a cellular effect on viability with a decrease to 71% for PS-G20 and to 80% for AMS-G20 , although these percentages remain within the ranges imposed by the standard, which is up to 70% (Sundback et al., 2005). However, glycerol can be used as a component of medical devices (Gal & Nussinovitch, 2009; Hermans et al., 2014; Sundback et al., 2005). It has been used in the preparation of freezing medium for organ transplantation preservation solutions (Miyata, Hayakawa, Kajiwara, & Kanno, 2012) and has been found to be non-cytotoxic (Yang et al., 2010). In vivo implantation of materials leads to a tissue response that follows the general principles of healing, such as acute inflammation, tissue granulation formation and tissue remodeling (Bartneck et al., 2012). At this stage, a successful repair depends on the material composition, material crystallinity, surface structure and rate of degradation as well as the interactions between the materials and the biological environment (Helmus, Gibbons, & Cebon, 2008). At day 30 post-implantation, amylomaize starch materials induced a pathological foreign-body reaction revealed by granuloma formation, the presence of giant cells and macrophages going towards AMS degradation (Bartneck et al., 2012). Potato starch materials were integrated into the tissue and a limited inflammatory reaction was observed, essentially due to the presence of M2 that is generally associated with implant healing (Badylak, Valentin, Ravindra, McCabe, & Stewart-Akers, 2008). These differences in host-tissue response between AMS and PS are probably linked to the material composition. Although both AMS and PS materials are mainly composed of amylose and amylopectin, minor components such as proteins and lipids are present in a ten-fold higher concentration in AMS than in PS, and play a role in the modulation of body immune response. The addition of glycerol to PS materials did not have an effect on the immune response. These results are consistent with those obtained with the starch-based stent material (Beilvert, Chaubet, et al., 2014). The presence of the plasticizer, primarily 20% glycerol, increases material resistance to degradation, which was also observed in vivo where fragments of PS-G10 and PS-G20 starch remained observable after 30 days. Resorption times for PS-G10 or PS-G20 material were similar to those observed with starch-blended materials such as starch-polyvinylidene fluoridetrifluoroethylene membrane (Marques et al., 2013), or longer than those observed with biological matrices such as non-cellularized extracellular matrices (Abed et al., 2011). 5. Conclusion The crystallinity induced by the physiological environment is a key parameter for the design of new degradable materials with a controlled shelf life. Due to its ability to crystallize, which improves its durability, as well as to its good tissue integration, plasticized potato starch could be a new candidate for biomedical applications. This study opens perspectives in the field of biomaterial design, targeting tissue engineering or the development of biodegradable surgical implants. Acknowledgements Mr. Velasquez would like to thank the Pays de la Loire Region for providing a partial PhD scholarship. The authors are grateful to A. Buléon for his helpful discussions and B. Quemener, B. Pontoire,

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