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Feb 1, 2013 - For these measurements, three liquids (water, diiodomethane, and ethylene glycol) with known surface energy were used. The total surface ...
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Effects of Ar–N2–O2 Microwave Plasma on Poly-L-Lactic Acid Thin Films Designed for Tissue Engineering Emna Chichti, Gerard Henrion, Franck Cleymand, Majid Jamshidian, Michel Linder, Elmira Arab-Tehrany*

Poly-lactic acid (PLA) is the most used biopolymer in both biomedical and food packaging fields to replace petrochemical plastics. The surface properties of PLA thin films were studied before and after plasma treatment to enhance its wettability and its adhesive properties. Based on the experimental design, the most significant parameters of the plasma process were specified. The effect of the cold plasma treatment on the mechanical, topographic composition, thermal and barrier properties of the PLA was carried out using different Ar–N2–O2 gas mixture. Results show that the discharge gas can have a significant influence on the chemical composition and the wettability of the PLA surfaces. As the plasma processing is a surface treatment without affecting the bulk properties, it did not change the PLA properties.

1. Introduction Tissue engineering (TE) is a multidisciplinary field focused on the development and application of knowledge in chemistry, physics, engineering, life, and clinical sciences to the solution of critical medical problems, as tissue loss and

E. Chichti, M. Jamshidian, M. Linder, Dr. E. Arab-Tehrany Universite´ de Lorraine, Laboratoire d’inge´nierie des Biomolecules, 2 avenue de la Foreˆt de Haye, TSA 40602, 54518 – Vandoeuvre Cedex, France E-mail: [email protected] G. Henrion, F. Cleymand CNRS, Institut Jean Lamour, UMR 7198, 54042 Nancy, France G. Henrion, F. Cleymand Universite´ de Lorraine, Institut Jean Lamour UMR 7198 CNRS – Ecole des Mines – Parc de Saurupt - CS 14234, 54042 Nancy, France Plasma Process. Polym. 2013, 10, 535–543 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

organ failure.[1] The development of biomaterials (biodegradable and bioabsorbable) with the required characteristics to aid in the recovery of tissues damaged by accident or human disease is one of the greatest research challenges involving areas such as medicine and engineering. Biopolymers offer an alternative to traditional biocompatible materials (metallic and ceramic) and non-biodegradable polymers for a large number of applications. Synthetic biodegradable poly-lactones such as poly-lactic acid (PLA), poly-glycolic acid (PGA), and poly-caprolactone (PCL) as well as their copolymers are a family of linear aliphatic polyesters, which are most frequently used in TE because of their excellent biocompatibility.[2–5] Poly (L-lactic acid) (PLLA) (–[CH(CH3)COO]n–) is a wellknown biodegradable and biocompatible semi-crystalline aliphatic polyester with a wide range of medical, textile, and packaging applications.

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DOI: 10.1002/ppap.201200124

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E. Chichti, G. Henrion, F. Cleymand, M. Jamshidian, M. Linder, E. Arab-Tehrany

However, the hydrophobicity and low surface energy of these polymers lead to inefficient cell attachment, spreading, and proliferation.[6] Biologic response to biomaterials implantation is determined by their surface characteristics rather than bulk properties, therefore a wide range of surface modification methodologies have been used to modify their surface chemistry, wettability, surface energy, and topography in order to modulate cellular responses such as cell adhesion or foreign body reaction.[7–10] Many methods can be used to overcome this low hydrophilicity such as chemical treatment or physical ones like UV irradiation or plasma treatment. Non-thermal plasmas are commonly used to modify the surface properties of polymers.[11–13] Plasma treatments have been successfully applied to popular polymers, only more recently they have been used to modify the surface properties of uncommon biodegradable polymers such as, for example, polylactic acid (PLA).[14–23] Regarding the plasma treatment that could be used to improve the PLA film properties, the most critical parameters are the injected power, gas mixture composition, and process duration. To avoid a too strong degradation of temperature sensitive materials, polymer samples can be positioned in the afterglow, downstream the plasma source. In that case, the distance between the plasma core and the sample is a supplemental parameter. In the present study, Ar/O2/N2 gas mixture was used to improve their hydrophilicity and surface energy. Doehlert experimental design was used to obtain a number of distinct levels and the response surface methodology (RSM) was applied as an effective tool to get an optimal response.

chloroform was evaporated in a dark place, under hood, during 48 h at 25 8C. Then films were placed in incubator (Memmert, Germany) at 30 8C for 7 d for complete solvent evaporation. The Petri dishes were kept in a hermetic container containing P2O5 powder before each analysis. The final film thickness was 50  2 mm measured (at least 8 points) by a mechanical micrometer (Messmer, UK) according to ASTM D374.

2.2. Plasma Treatment The plasma set-up is a home-made system whose details have been reported previously.[24] The plasma reactor consists of a 5 mm inner diameter cylindrical quartz tube in which flows a O2/N2/Ar gas mixture. The quartz tube crosses a 2.45 GHz microwave surfaguide[25] wave launcher. Downstream the plasma, the postdischarge enters a PyrexTM tube (28 mm inner diameter) 365 mm far away from the plasma gap. Such a long distance is necessary to prevent any effect of the pink afterglow in pure nitrogen. PLA samples (2 cm  1 cm) were fixed on a metallic sample holder, which was inserted in the post-discharge tube. This allowed us to precisely place the sample in the process chamber and avoided displacement of the light sample within the gas flow during the process. Total Ar flow rate was 1 L min1 and reaction gas mixture was 100 cm3 min1. The total pressure was fixed at 6 MPa and the treatment time varied in the range of 50 to 300 s. The microwave power ranged from 150 to 450 W. The gas purities are 99.995%. After treatment, samples are kept at 4 8C to reduce the mobility of molecular chains under the surface before assessing the water contact angle (WCA) and the weight before and after plasma treatment.

2.3. Experimental Design

2. Experimental Section 2.1. Film Preparation Poly (lactic acid) (PLA), 2002 D, was purchased in pellet form from Natureworks1 Co., Minnetonka (USA); Chloroform D (99.8% CDCL3) from Euriso-Top (France), and phosphorus pentoxide from Sigma– Aldrich (France). A solution of 3.5% w/w of PLA was prepared in chloroform and mixed during 1 h. Then 10 g of solution was casted in Teflon 90  110 mm Petri dishes (Welch, USA) the extra

A Doehlert experimental design was adopted.[26] The total number of points (N) for four factors (k) was 21 (N  k2 þ k þ 1), and 25 experiments were carried out. The last five assays were performed at the center of the experimental domain in order to estimate the residual variance. The processing variables of plasma treatment investigated were microwave power (X1, five levels), N2/O2 mixture gas ratio (X2, seven levels), treatment time (X3, seven levels), and distance between transmitting terminal and sample (X4, three levels). The independent variables (Xi) and their levels are presented in Table 1.

Table 1. Experimental domain and level distribution variables used for minimization the WCA.

Independent variables

Symbol

Experimental values

Levels

Microwave power (W) N2/O2 mixture gas ratio

X1

150, 225, 300, 375, 450

5

X2

0, 17, 33, 50, 67, 83, 100

7

Treatment time (s)

X3

50, 81, 144, 175, 206, 269, 300

7

Distance between transmitting

X4

50, 175, 300

3

terminal and sample (mm)

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Plasma Process. Polym. 2013, 10, 535–543 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

DOI: 10.1002/ppap.201200124

Effects of Ar–N2–O2 Microwave Plasma. . .

2.4. Data Analysis For data analysis, analysis of variance (ANOVA) and multiple linear regressions were performed using the NEMROD1software.[27] A quadratic model containing 15 coefficients, including interaction terms, was assumed to describe relationships between the response Y and the experimental factors Xi:

Y ¼ b0 þ

4 X i¼1

bi Xi þ

4 X

bii Xii2 þ

i¼1

3 X 4 X

bij Xi Xj

The degree of crystallinity was evaluated according to Equation (2): XC ¼

DHm  DHc  100 DHf

(2)

where: DHm ¼ fusion enthalpy, DHc ¼ crystallization enthalpy, DHf ¼ melting enthalpy of 100% crystalline PLA (93.6 J g1).[34]

(1)

i¼1 j¼1þ1

2.8. Water Contact Angle (WCA) where b0 is the constant coefficient (intercept), bi the linear coefficient, bii the quadratic coefficient, and bij the second-order interaction coefficient. Response surfaces and contour plots were developed using the fitted quadratic polynomial equations obtained from the response surface regression analysis. The level of significance for all tests was set at 95%. Numerous experimental and model prediction results of independent assays[28–31] carried out to confirm the adequacy of the model predicted values at the optimal point were very close.

2.5. Water Vapor Permeability (WVP) Measurement The water vapor transition rate (WVTR) measurements were done using a Permatran W3/31 (Mocon, Inc., Minneapolis, MN, USA) according to ASTM standard F1249.[32] The calibration of the instrument for WVTR measurements was performed using polyester standard films provided by Mocon. Water vapor concentration within the nitrogen stream is measured by an infrared detector contained within the instrument. Samples were exposed to 90% relative humidity (RH) and tested at 38  1 8C. WVP (g m1 s1 bar1) was calculated by multiplying the WVTR by film thickness. The tests were done in triplicate and the mean values were reported.

2.6. Mechanical Properties Tensile tests were done using a Lloyd instruments testing machine (AMETEK, UK) according to ASTM D 882[33] to determine different mechanical properties including tensile strength, Young’s modulus and % of elongation at break. Before testing, all samples were equilibrated for 48 h at 50  2% RH in a container using magnesium nitrate saturated solution at 20  1 8C. Equilibrated film specimens were mounted in the film-extending grips of the testing machine and stretched at a rate of 25 mm min1 until breaking. The RH and temperature of the testing environment was held at 52  2% and 20  2 8C, respectively.

Contact angle measurements of PLA thin films were performed by following the sessile drop method with a contact angle instrument (Digidrop Contact AngleMeter) equipped with an image analysis attachment (Windrop). The probe liquids used were milli-Q1 water. Uniform drops of liquids (2.5 mL) were carefully deposited on the film surface using micrometer syringe. The volume of the drops was kept constant since variations in the volume of the drops can lead to inconsistent contact angle measurements. Measurements were consistently conducted under the constant conditions of RH (39%) and temperature (23 8C). Contact angle measurements were recorded three times on three different locations of PLA surface within 5 s for a given thin film.

2.9. Surface Energy Measurement The total surface energy of the films was determined graphically by the Owens–Wendt method, which is usually applied for solids with low surface energy like polymers. The Owens–Wendt theory divides the surface energy into two components: one due to dispersive interactions and one due to polar interactions.[35] Owens and Wendt proved that the total surface energy of a solid, g Ts , can be expressed as the sum of contributions from dispersive g ds and polar p g s force components. These can be determined from the contact angle (u) data of polar and non-polar liquids with known dispersive p g dL and polar g L parts of their surface energy, via the following relations: pffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffi g L ð1 þ cosuÞ ¼ 2 g Lp g Sp þ 2 g Ld g Sd

(3)

For these measurements, three liquids (water, diiodomethane, and ethylene glycol) with known surface energy were used. The total surface energy, polar and dispersive components of PLA thin films before and after plasma treatment were determined according to the water diiodomethane and ethylene glycol measured contact angle.

2.10. Atomic Force Microscopy 2.7. Thermal Properties Modulated differential scanning calorimetry (2920 Modulated DSC, TA instruments) is used to assess glass transition (Tg), crystalline (Tc) and melting (Tm) temperatures and their corresponding enthalpy (DHc, DHm). A single cycle is programmed from 25 to 250 8C at 10 8C min1 to avoid creating the thermal history and thus losing plasma effect. Plasma Process. Polym. 2013, 10, 535–543 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The surface morphology was analyzed by using a D3100 AFM equipped with a Nanoscope 5 electronic from Bruker instruments (Madisson, USA). The images were recorded at ambient conditions (20 8C and 30% RH) and in soft intermittent contact mode (IC-AFM or TappingTM AFM). Tap150 tapping mode cantilevers (Veeco model No MPP-12100) with a typical spring constant of 5 N m1 and a resonance frequency of 165 kHz was used for scanning. Tapping

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force was controlled by the ratio between set point amplitude (Asp) and free-air amplitude (A0). The scan rate was adjusted in the range of 0.5–1 Hz depending on the image quality. Each scan line contains 512 pixels and a whole image is composed of 512 scan lines. For acquisition of the surface morphology amplitude error, phase and height images were recorded on several areas of film surface. Only the representative height images (10  10 mm2) were presented here for discussion. All offline image flattening and analyses of the images were conducted at the software environment provided by the AFM manufacturer. The statistical parameters related with sample roughness[36] were estimated by the software equipment in a 10 mm2 area of AFM image, so average roughness (Ra) and root mean square roughness (Rq) were only presented.

2.11. X-Ray Photoelectron Spectroscopy (XPS) The XPS analyses were carried out with a Kratos Axis Ultra spectrometer (Kratos Analytical, Manchester, UK) using a monochromatic Al–Ka source. All spectra were recorded at 908 take-off angle and analyzed area was about 700  300 mm2. Survey spectra were recorded with 1.0 eV step and 160 eV analyzer pass energy and the high resolution regions with 0.1 eV step and 20 eV pass energy, with an exception for the carbon with 0.05 eV step. In both cases, the hybrid lens mode was employed (magnetic and electrostatic). During the data acquisition the Kratos charge neutralizer system was used on all specimens with the following settings: filament current 2 A, charge balances 3.5 V, filament bias 1.0 V and magnetic lens trim coil 0.34 A. The C–(C, H) carbon was set to 284.60 eV and therefore used as an internal energy reference. Spectra were analyzed using the Vision software from Kratos (Vision 2.2.6). A Shirely base line was used for the subtraction of the background from each peak. Quantification was performed using the photoemission cross-sections and transmission coefficients given in the Vision package.

3. Results and Discussion 3.1. Experimental Design The experimental design is a good tool to determine the optimal parameters of plasma process. Contact angle and weight loss of the PLA films were measured before and after plasma treatment to observe the effect of each parameter (microwave power (W), gas mixture (Ar–(100–x) N2–O2) (%), time of treatment and distance between the discharge and the film (mm)) on surface and bulk properties. The results presented in Table 2 are very significant for WCA and weight loss before and after plasma treatment with 99.9% confidence level and consequently a great repeatability of plasma process. When focusing on the impact of each parameter and their influence, the result of the experimental design showed that gas mixture had negative effect on the weight loss response, which means that the PLA surface was engraved

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Table 2. Impact of each parameter and their interactions.

WCA difference Coefficient b0

27.625

b1

1.215

b2

0.240

b3

1.423

Weight loss

Signif.%