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Medium chain-length polyhydroxyalkanoate copolymer modified by bacterial cellulose for medical devices Denis Mihaela Panaitescu, Irina Lupescu, Adriana Nicoleta Frone, Ioana Chiulan, Cristian Andi Nicolae, Vlad Tofan, Amalia Stefaniu, Raluca Somoghi, and Roxana Trusca Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00855 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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Biomacromolecules

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Medium Chain-Length Polyhydroxyalkanoate

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Copolymer Modified by Bacterial Cellulose for

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Medical Devices

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Denis Mihaela Panaitescu1*, Irina Lupescu2, Adriana Nicoleta Frone1, Ioana Chiulan1, Cristian

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Andi Nicolae1, Vlad Tofan3, Amalia Stefaniu2, Raluca Somoghi1, Roxana Trusca4

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1

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Independentei, 060021, Bucharest, Romania

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Romania

Polymer Department, National Institute for R&D in Chemistry and Petrochemistry, 202 Splaiul

National Institute for Chemical Pharmaceutical R&D, 112 Calea Vitan, 031299, Bucharest,

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Independentei, 050096, Bucharest, Romania

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Bucharest, 1-7 Gh. Polizu Street, 011061 Bucharest, Romania

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KEYWORDS: poly(3-hydroxyoctanoate), thermal stability, aging, mechanical properties, tubing,

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cytocompatibility

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ABSTRACT

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Medium chain-length PHAs (mPHAs) are flexible elastomeric biopolymers with valuable

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properties for biomedical applications like artificial arteries and other medical implants.

Cantacuzino National Institute of R&D for Microbiology and Immunology, 103 Splaiul

Science and Engineering of Oxide Materials and Nanomaterials, University Politehnica of

1 ACS Paragon Plus Environment

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However, an environmentally friendly and high productivity process together with the tuning of

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the mechanical and biological properties of mPHAs are mandatory for this purpose. Here, for the

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first time, a melt processing technique was applied for the preparation of bionanocomposites

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starting from poly(3-hydroxyoctanoate) (PHO) and bacterial cellulose nanofibers (BC). The

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incorporation of only 3 wt% BC in PHO improved its thermal stability with 25°C and reinforced

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it, increasing the Young’s modulus with 76% and the tensile strength with 44%. The percolation

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threshold calculated with the aspect ratio of the fibers after melt processing was very low and

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close to 3 wt%. We showed that this bionanocomposite is able to preserve the ductile behavior

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during storage, no important aging being noted between 3 h and one month after compression-

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molding. Moreover, this study is the first to investigate the melt processability of PHO

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nanocomposite for tube extrusion. In addition, biocompatibility study showed no pro-

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inflammatory immune response and better cell adhesion for PHO/BC nanocomposite with 3 wt%

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BC and demonstrated the high feasibility of this bionanocomposite for in vivo application of

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tissue-engineered blood vessels.

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INTRODUCTION

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Polyhydroxyalkanoates (PHAs) have been extensively studied over the past years due to their

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promising applications.1-4 Short-chain length PHAs (sPHAs) such as poly(3-hydroxybutyrate)

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(PHB)

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commercialized and tested for biomedical and packaging applications.2,4 However, sPHAs are

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extremely brittle and show poor melt processing behavior.4 Unlike sPHAs, elastomeric medium

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chain-length PHAs (mPHAs) are very promising for biomedical purposes due to their unique

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properties.2,5

and

poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

(PHBV)

have

been

already


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Poly(3-hydroxyoctanoate) (PHO) and its copolymers are from the most studied

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mPHAs.5,6 Cytocompatibility tests demonstrated the attachment, differentiation, and maturation

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of human keratinocytes cells on PHO (homopolymer) film obtained by solvent casting.6

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However, some biomedical applications require a specific range of mechanical and biological

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properties. For this purpose, PHO was modified by crosslinking or graft copolymerization or by

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the addition of other biopolymers and micro/nanofillers.7-11 PHO grafted with vinylimidazole

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showed higher hydrophilicity, biocompatibility and antimicrobial activity than the starting

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polymer.7 The nanocomposite from PHO copolymer (7.6 mol% of 3-hydroxyhexanoate (3HH))

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and 2.5 wt% organoclay showed a nanoindentation modulus five times higher than that of the

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pure matrix.9 PHO - diethylene glycol hybrid copolymer showed improved hydrophilicity and

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self-assembling ability forming microporous arrays.11

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PHB/PHO blends with adjustable biodegradability were studied for biodegradable stents.5

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These stents may be more advantageous than metal stents because they do not have to be

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removed after fulfilling their function to support the injured artery. The mPHAs are flexible

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elastomeric biopolymers with low crystallinity and glass transition temperature, moderate tensile

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strength and high elongation to break, which recommend them for heart valves and

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cardiovascular implants, skin and nerve tissue engineering.12,13 From aliphatic biopolyesters,

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only poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), which is available in larger quantities,

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has been studied for these purposes.5,13 Hence, PHAs are more hydrophobic than most of natural

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biopolymers, such as collagen14 and the use of hydrophilic agents is necessary to overcome this

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drawback and to enhance the biological properties of PHAs. It has been reported that both the

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hydrophilicity and mechanical properties of PHAs were improved by the addition of small

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amount of nanocellulose.15


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PHO/nanocellulose composites have been scarcely studied.10,12 In the pioneering work of

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Dubief et al. starch microcrystals from yellow pea and cellulose whiskers from tunicin were

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tested in a PHO latex to increase its hydrophilicity and mechanical properties.10 Higher storage

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modulus values were obtained in the case of PHO reinforced with high aspect ratio cellulose

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whiskers from a percolation threshold as low as 1.5 wt %.10 On the other hand, Basnett et al.

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were the first who addressed this issue in the perspective of medical applications.12 They

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obtained bacterial cellulose microcrystals by the weak acid hydrolysis of bacterial cellulose and

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modified their surface by acetylation. Composite films from PHO homopolymer and acetylated

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microcrystals in high amount (5, 15, and 25%), prepared by solvent casting using chloroform,

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showed enhanced in vitro degradability and increased cell growth and proliferation. However,

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some deterioration of the elastic properties was observed due to the high concentration of

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acetylated bacterial cellulose microcrystals in the films.12

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Great advantage could be brought to PHO by the nanodimension of the cellulosic filler,

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in agreement with the results obtained with cellulose whiskers from tunicates.10 Thus, the

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amount of bacterial cellulose modifier in PHO can be greatly decreased to a few percent by

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working with bacterial cellulose nanofibers instead of microfillers and the elastomeric properties

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of the matrix may be, thus, maintained. Moreover, for successful scale-up of these PHO

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nanocomposites to an industrially meaningful scale, free-solvent methods should be used. In the

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present work, we applied for the first time a melt processing technique for the preparation of

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PHO composites with bacterial cellulose nanofibers. Compared to the common method

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consisting in the solvent casting using chloroform, melt processing is environmentally friendly,

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cost effective and ensures high productivity, being a major step toward the fabrication of medical

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devices like stents.


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The main aim of this study was to obtain PHO/bacterial cellulose nanocomposites with

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enhanced mechanical properties and thermal stability, suitable for melting processing to obtain

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medical devices. For this purpose, bacterial cellulose nanofibers in dry form, that will be denoted

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from now on with BC for simplicity, were used in low amount (1.5 wt% and 3 wt%) in a PHO

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copolymer to obtain nanocomposites by melt processing. BC were obtained by the mechanical

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disintegration of bacterial cellulose pellicles; the size of BC and their dispersion in PHO were

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assessed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and

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by atomic force microscopy (AFM); calorimetric study was conducted to observe the effect of

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the nanofiller on the crystallization of PHO and the mechanical properties were determined for

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different aging times. Biocompatibility study was performed by assessing the pro-inflammatory

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immune response triggered by PHO/BC nanocomposite in an in vitro macrophage cell line

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model and by in vitro cytotoxicity tests through the analysis of L929 murine fibroblasts’

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behavior on the surface of neat PHO and PHO/BC nanocomposites. By this study we propose a

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green approach to obtain bionanocomposites for in vivo applications and we demonstrate that

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PHO nanomaterials are able to preserve their ductile behavior during storage, a step forward

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relative to PHB and other sPHAs. Moreover, this study is the first to investigate the melt

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processability of PHO/BC nanocomposites for tube extrusion as an important step for the

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manufacture of artificial arteries and other medical implants.

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EXPERIMENTAL PART

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Materials

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The wild-type Gram-negative bacteria Pseudomonas putida ICCF 391, from the Collection of

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Microorganisms of Industrial Interest of National Institute for Chemical-Pharmaceutical

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Research & Development (Bucharest, Romania) was used in this study. The strains have an 5 ACS Paragon Plus Environment

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optimum growth temperature range from 30°C to 34°C and were maintained in Cantacuzino

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Agar tubes at 4°C and used for all culture experiments. Bacterial cellulose pellicles were

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produced in static culture by the bacterial strain Gluconacetobacter Xylinus DSM 2004

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purchased from Leibniz Institute DSMZ (German Collection of Microorganisms and Cell

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Cultures) in the conditions mentioned in our previous work.16 All chemicals (of analytical grade)

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were supplied by Sigma-Aldrich and used as received.

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Biosynthesis of PHO copolymer

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PHO

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7.5:87.9:3.3) was produced by Pseudomonas putida using sodium octanoate as carbon source.

copolymer

(poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate)

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Further details about the biosynthesis of PHO are given in the Supporting Information.

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Preparation of bacterial cellulose nanofibers in dry form

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BC were obtained from fresh bacterial cellulose pellicles using a mechanical defibrillation

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process as described before16 with the differences: the gel-like sample collected from the blender

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was diluted to about 1% with distilled water and passed through a vertical colloid mill at about

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2000 min-1 for 180 min. From time to time, cold water was added to prevent the raising of the

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temperature above 40 ºC. The suspension containing BC was 5-fold concentrated in a rotavapor

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(Heidolph Instruments, Germany) at 40 °C and then freeze dried (FreeZone 2.5 L - Labconco

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USA) resulting powdered BC.

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Preparation of PHO/BC nanocomposites. PHO pellicles prepared in the conditions mentioned

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in the Supporting Information were introduced in the mixing chamber of a Brabender LabStation

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(Germany) heated to 45 ºC and melted. The calculated amount of BC powder was slowly poured

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into the melted polymer and, then, they were mixed for another 3 min at 40 min-1. The mixtures 6 ACS Paragon Plus Environment

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were shaped in foils on a laboratory two-roll mill (Polymix 110L, Germany) and compression

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molded in an electrically heated press (P200E Dr. Collin, Germany) at 46-48°C, 120 s preheating

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(5 bar), 30 s under pressure (50 bar) and cooling for another 120 s (5 bar) in a cooling cassette.

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Sheets (0.8-1 mm in thickness) of nanocomposites with 1.5 and 3 wt% BC (PHO-1.5BC and

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PHO-3BC) were thus obtained. A neat PHO reference was prepared in the same conditions.

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Before characterization, PHO and nanocomposite plates were kept for 30 days at room

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temperature.

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Characterization techniques

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Cryo-TEM analysis. The size and morphology of BC were investigated by TEM using a

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Tecnai™ G2 F20 TWIN Cryo-TEM instrument (FEI, Hillsboro, USA) at 200 kV accelerating

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voltage. The nanofibers were observed directly without any staining to improve contrast. A small

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droplet of diluted BC suspension in water was poured on a holey carbon grid and left to dry in

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vacuum at 25 °C. PHO-3BC nanocomposite sample was cut with a Leica EM FC7 cryo

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ultramicrotome at a temperature of -30 °C which is close to its Tg value. Slices of 100 nm were

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obtained at a cutting speed of 40 mm/s which were then carefully placed on holey carbon copper

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grids. Temperature and cutting speed were optimized for this sample. The dispersion of BC in

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the polymer matrix was then investigated using the Cryo-TEM instrument at an accelerating

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voltage of 200 kV in bright field mode. Images were analyzed with Digital Micrograph software

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(Gatan, USA) to obtain the width distribution of bacterial cellulose nanofibers.

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Thermal characterization. Thermo-gravimetric analysis (TGA) was carried out on a TA-Q5000

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V3.13 (TA Instruments Inc., USA) with nitrogen as the purge gas at a flow rate of 40 mL/min.

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The thermograms were acquired between 25 °C and 700 °C at a heating rate of 10 °C/min. The

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error on temperature determination was ±0.5 °C. Differential scanning calorimetry (DSC) was 7 ACS Paragon Plus Environment

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performed with a DSC Q2000 from TA Instruments under a helium flow (100 mL/min). Samples

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weighing around 7 mg were cooled to -10 ºC, heated to 80 ºC, held at that temperature for 3 min

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to delete the thermal history, then cooled to -80 ºC, kept isothermal for 2 min and heated again to

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80 oC at a constant heating/cooling rate of 10 oC/min. The melting temperature (Tm) was taken as

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the peak temperature of the melting endotherm. Two parallel samples were tested for the

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reference and for each PHO nanocomposite.

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Mechanical properties. Tensile properties of PHO/BC nanocomposites were measured at room

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temperature with a crosshead speed of 10 mm/min, using an Instron 3382 Universal Testing

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Machine equipped with a cell load of 1 kN. Five specimens, 5A type according to ISO 527, were

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tested for each sample and analyzed using the Bluehill 2 Software.

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Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. ATR-FTIR

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spectra were recorded on a FTIR spectrometer TENSOR 37 from Bruker (USA). Data were

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collected at room temperature from 4000 to 400 cm-1 with 16 scans.

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AFM investigation. AFM was employed to study the surface properties of PHO before and after

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BC addition. A MultiMode 8 from Bruker (USA) working in Peak Force (PF) Quantitative

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Nanomechanical Mapping (QNM) mode was used for this purpose. Silicon probe from Bruker

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with the following characteristics, stated by the specification - nominal tip radius of 8 nm,

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cantilever length of 225 µm and resonant frequency of 75 kHz - was used for the measurements.

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The tests were performed at room temperature, with a scan rate of 0.5 – 0.7 Hz. The images

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(256x256 pixels per line) were analyzed with NanoScope 1.20 software.

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SEM analysis. Surface morphology was assessed by scanning electron microscopy. A Quanta

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Inspect F50 scanning electron microscope (FEI, Hillsboro, USA), equipped with a field emission


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gun, with a resolution of 1.2 nm and working at an accelerating voltage of 30 kV was used for

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this purpose. The PHO and nanocomposite films were sputter-coated with gold before analysis.

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Cell line culture and differentiation into macrophage-like cells. Human monocytic cell line THP-

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1, purchased from Health Protection Agency Culture Collections, was cultured at 37 oC in a

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humidified atmosphere and 5% CO2 in a RPMI-1640 medium (Sigma-Aldrich Corporation,

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USA). This was supplemented with 10% fetal bovine serum (Biochrom AG, Germany), 1 mM L-

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glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Lonza, Belgium) and 5 mM 2-

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mercaptoethanol (complete culture medium). THP-1 cells were seeded at 2x105cells/mL, 200

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µL/well in a 96 -well flat bottom culture plate. They were treated with 30 ng/mL phorbol-12-

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myristate-13 acetate for 72h, resulting in macrophage-like cells.

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Evaluation of pro-inflammatory effect. Roughly equal amounts of each nanocomposite material

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were sterilized by overnight incubation in 70% ethanol solution followed by washing steps with

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sterile Phosphate Buffer Saline (PBS) to completely remove ethanol. Samples were incubated at

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37 oC in PBS for 20 days to allow elution of their constituents. PBS alone, incubated in a similar

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manner was used as a control. Following incubation, supernatants (elution samples) were

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collected and tested for endotoxin activity with LAL QCL-1000 kit (Lonza, Belgium).

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Differentiated macrophage-like cells were incubated in the presence of elution samples (diluted

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1/10 in complete culture medium) for 24h. Cells incubated with lipopolysaccharide (LPS 100

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ng/mL) or without stimuli were used as positive and negative controls. Supernatants were

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collected and stored at -80 oC until use. The influence of elution samples on differentiated

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macrophages was evaluated by measuring Tumor Necrosis Factor-α (TNF-α) and Interleukin-6

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(IL-6) concentrations in culture supernatants using ELISA (DuoSet kits from R&D Systems Inc.,

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USA). 9 ACS Paragon Plus Environment

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Evalutation of cytocompatibility. Cytotoxic effect of neat PHO, PHO-1.5BC and PHO-3BC on

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L929 cell line was evaluated using round disc-shaped samples, 10 mm in diameter. The samples

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were sterilized by incubation in 70% ethanol solution for 30 minutes and another 30 minutes in

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30% ethanol solution; washes with sterile Phosphate Buffer Saline (PBS) were performed to

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discard residual ethanol. Afterwards, the samples were equilibrated in DMEM medium (Sigma-

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Aldrich Co, USA) supplemented with 10% fetal bovine serum (Biochrom AG, Germany), 1 mM

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L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Lonza, Belgium). L929 murine

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fibroblast cells were cultured at 37ºC in a humidified atmosphere and 5% CO2 in complete

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DMEM culture medium. At passage 9, the cells were trypsinized and seeded on previously

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sterilized disc-shaped samples (neat PHO and nanocomposites) in ultra-low attachment flat-

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bottom 24 well plates (Greiner Bio-One, Austria) at a cell density of 20,000 cells/cm2. The

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medium was refreshed every two days. The disc-shaped samples were analyzed after 3 and 7

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days using fluorescence microscopy as described elsewhere.15 Images were acquired using a

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Eclipse TE2000 inverted fluorescence microscope (Nikon, Austria) and processed with Huygens

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software (SVI, Hilversum, Netherlands). Cellular morphology and cell density were assessed by

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using CellProfiler software; eight different images per well were analyzed. To observe the

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adhesion of L929 cells on the surface of the films, the cells were fixed with 4%

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paraformaldehyde and permeabilized with Triton X-100 before staining. Nuclei were stained

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with SYBR ® -Green I (Lonza, Belgium) and actin filaments with Texas-Red ® -X Phalloidin.

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RESULTS AND DISCUSSION

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TEM analysis

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TEM images of BC are shown in Figure 1. The pellicles were completely disintegrated in sparse

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network nanostructures (Figure 1a) and individual nanofibers (Figure 1b). The length of the 10 ACS Paragon Plus Environment

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nanofibers was larger than 5 µm and the width from a couple of nm to 100 nm. Therefore, the

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nanofibers have a high aspect ratio (AR), of more than 50. The detailed image from Figure 1

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shows a single crystalline microfibril of about 20 nm in width at the top right of the image and

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several protofibrils of about 4 nm. These protofibrils are the most elementary blocks in the

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formation of the bacterial cellulose network.17 Therefore, the morphology of BC nanofibers was

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preserved after the mechanical treatment, which succeeded in the release of the nanofibers from

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the bacterial pellicle.

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Figure 1. TEM images of BC as sparse network small piece (a) and individual nanofibers (b).

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Thermal stability

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The degradation of PHO is a single step process, similar to that observed in PHB (Figure 2a)

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with a maximum degradation temperature (Td) of 250 °C (Figure 2b). This Td value is close to

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that obtained for a PHO copolymer with 2 mol% 3HH,18 but lower than that obtained for a

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mPHA containing both saturated and unsaturated monomers (around 280 °C).19 It is worth

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mention that TGA experiments were made on melt processed samples, which were exposed to


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mechanical stress, temperature, oxygen and humidity influences; the original films showed a

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higher Td, of 277 ºC.

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The addition of BC greatly increased the thermal stability of PHO, regardless the amount

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of nanofibers; the Td value was around 275 °C for nanocomposites, with 25°C higher than that of

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PHO matrix and even better compared to that of a commercial PHB (fine powder, Biomer,

6

Germany) (Figure 2).

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The influence of BC nanofillers on the thermal stability of mPHAs was not studied so far;

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the previous works were focused on sPHAs composites with cellulose nanofillers and have

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reported different results.20,21 For example, nanofibrillated cellulose obtained from TEMPO

10

oxidized cellulose by mechanical defibrillation decreased the thermal stability of PHBV20 and

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cellulose nanocrystals (CNC) obtained by acid hydrolysis increased the thermal stability of

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PHBV.21 In this last case it was hypothesized that, in the early stages of PHBV decomposition,

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the formation of a six-membered ring ester was blocked due to the hydrogen bonds between

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CNC and PHBV, which hindered the degradation by chain scissions. A shift of Td to higher

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temperatures for PHBV with low amount of bacterial cellulose nanowhiskers (1 wt%) but a

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decrease of the thermal stability for higher loading (3 wt%) was also reported.22 Moreover, Sin et

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al have shown that the mechanism of mPHAs decomposition is different from that of sPHAs,19

18

which is associated with the ester cleavage by β-elimination reaction.23 It was hypothesized that

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the aliphatic side chain composed of more than three carbons in mPHAs could hinder the

20

formation of a stable six-membered ring ester intermediate.19 They proposed a random chain

21

scission reaction determined by the hydrolytic chain cleavage initiated at the ester bond at 160 -

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180 ºC, followed by a secondary process, the dehydration of hydroxyl-esters above 195 ºC,

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producing terminally unsaturated oligomers.19


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Figure 2. TGA (a) and DTG (b) diagrams for PHO and nanocomposites with PHB as a

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reference; inset – DTG curves (second degradation step) have been offset for clarity.

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The hydrolytic chain cleavage mechanism was considered as a possible mechanism in the

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case of PHO/BC nanocomposites. However, BC are highly hydrophilic and even after drying

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they contain a certain amount of bound water as reported before;24 their weight loss at 150 ºC 13 ACS Paragon Plus Environment

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determined by TGA in the same conditions with the nanocomposites was 2.0% (Figure 2a) and

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that of PHO and nanocomposites was less than 0.2%. Therefore, the higher thermal stability of

3

nanocomposites compared to that of the matrix can’t be explained by the influence of BC in

4

reducing the hydrolytic degradation. Indeed, BC should favor the hydrolytic degradation of

5

PHO, providing the necessary water for the chain cleavage. On the other hand, BC have better

6

thermal stability and much higher crystallinity than PHO. Their Td value (334 ºC) is 80 ºC higher

7

than that of PHO and a degree of crystallinity of 84% was reported for BC nanofibers16 while the

8

crystallinity of PHO do not, generally, exceed 30%.25 Therefore, BC may slow down the

9

absorption of water serving as a barrier and delaying the hydrolytic degradation. Likewise, the

10

interactions between BC and PHO by hydrogen bonding could also delay the degradation, as for

11

sPHAs.21 Therefore, the increase of BC concentration could have antagonistic effects on the

12

thermal stability of PHO, which may explain the small influence of BC concentration on the

13

thermal degradation of nanocomposites.

14

Unlike PHO, the thermal degradation of PHO-BC nanocomposites was a two step

15

process (Figure 2), with the main degradation caused by the chain scission of PHO and the

16

second due to BC degradation. The intensity of this second process depended on the amount of

17

BC, being less intense for the nanocomposite with 1.5 wt% BC. Interestingly, the Td value

18

corresponding to the degradation of BC in nanocomposites (346-348 ºC) was higher than that of

19

pure BC (334 ºC), before the melt compounding with PHO (Figure 2 - inset). A decrease of the

20

thermal stability of cellulose after melt processing would be expected because of the effects of

21

shear stress and temperature and not an increase. The increase of BC stability in nanocomposites

22

may result from the hydrogen bonds between the nanofibers and PHO matrix21 or from the

23

different thermal conductivity in pure BC and nanocomposites.26


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Biomacromolecules

1 2

DSC analysis

3

The thermal characteristics of PHO copolymer and the influence of BC on its glass

4

transition (Tg) and crystallinity were assessed by DSC (Figures S1 and S2). A very small

5

endothermic peak just above the glass transition temperature was detected in the thermograms of

6

PHO and composites (Figure S1). This event, characterized by a small enthalpy relaxation of

7

only 0.5 J/g, is commonly connected to the enthalpy recovered during the heating of an aged

8

sample.27 Since Tg was determined from the second melting, after a heating cycle which erased

9

the thermal history caused by the melt processing and ambient storage for 30 days, it can be

10

supposed that the physical aging was promoted in the previous cooling cycle, when the

11

temperature of the samples was lowered below Tg.

12

No change in the glass transition temperature was induced by the addition of BC; Tg

13

values between -37 and -38 °C were obtained for all the samples, likewise the temperature of the

14

relaxation peak was similar (about -34 °C). Therefore, the addition of BC did not influence the

15

mobility of PHO chains probably because the weight fraction of BC nanofibers was small and

16

their effect on the segmental dynamics of PHO was, thus, negligible. Moreover, previous works

17

on nanocomposites filled with cellulose whiskers reported no change of Tg value with the

18

increase of whiskers amount, regardless of the nature of the polymer matrix.28

19

Interestingly, a melting endotherm was obtained only in the first melting cycle for PHO

20

and nanocomposites; no crystallization was observed in the cooling step and no melting event in

21

the second melting cycle (Figure S2). This is a consequence of the very slow crystallization of

22

mPHAs, much slower than the cooling rate.29-31 The storage of the samples for 30 days in

23

ambient conditions, between the melting temperature and the glass transition of PHO, allowed


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Page 16 of 38

1

the crystallization before DSC analysis but the recrystallization could not take place during the

2

short time of the cooling cycle.

3

For a deeper insight into the crystallization process of PHO in PHO/BC nanocomposites,

4

the samples were annealed before DSC characterization in the following conditions, denoted as

5

“a”: PHO and nanocomposites were heated in an oven up to 80 °C with about 5 °C/min, kept

6

isothermally for 5 minutes, then stored in a fridge for 48 h at a temperature of 5 °C; thereafter

7

they were characterized by DSC in the same conditions as for the first test. The temperature of 5

8

°C was chosen because it was demonstrated that the crystallization rate of PHO reaches a

9

maximum at 0-5 °C.25 After annealing in “a” conditions, a melting endotherm was obtained for

10

all the samples only in the first heating cycle (Figure 3) but no crystallization appeared at

11

cooling, similar to the first test. Indeed, the annealing conditions “a” allowed the crystallization

12

of PHO but the short time of the cooling cycle was not enough for recrystallization.

13

14 15

Figure 3. DSC diagrams for un-annealed PHO (a), PHO-1.5BC (b), PHO-3BC (c) and annealed

16

samples (PHOa, PHO-1.5BCa and PHO-3BCa).

17 18

The DSC diagrams for un-annealed and annealed (“a”) samples are shown in Fig. 3 for

19

comparison. A melting peak around 53 ºC with a shoulder at lower temperature was obtained for


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1

PHO and nanocomposites in the first test. No variation of the melting point was caused by the

2

addition of BC up to 3 wt%. However, the shoulder was smaller and the main endotherm

3

narrower for the composite with 3 wt% BC compared to the pure matrix, which means increased

4

population of more perfect crystals due to BC addition. This may result from the nucleating

5

effect of BC, as previously reported for other PHAs modified by cellulose nanofibers.20-22

6

However, the degree of crystallinity remained almost unchanged as illustrated by the melting

7

enthalpy normalized by the proportion of PHO in the composite: 25.6 J/g for PHO, 25.4 J/g for

8

PHO-1.5BC and 26.2 J/g for PHO-3BC. The influence of BC on the degree of crystallinity of

9

PHO was verified by FTIR. The absorbance at 1165 cm-1, which is crystallinity-sensitive in

10

PHO,32 showed no change in intensity or position in nanocomposites compared to pure PHO;

11

therefore no variation in crystallinity was obtained from FTIR spectra, which is consistent with

12

the DSC results.

13

After annealing in conditions “a”, a lower melting temperature of 48 ºC and a lower

14

melting enthalpy of 20 J/g were obtained for all the samples, indicating smaller less perfect

15

crystals and lower crystallinity. Indeed, previous DSC study showed that about 7 weeks at 20 ºC

16

or 3 weeks at 5 ºC are required to reach the maximum level of crystallinity in PHO.25 As

17

expected, the annealing conditions “a”, in fact two days at 5 ºC, led to a lower degree of

18

crystallinity compared to the storage at room temperature for 30 days; this shows the influence of

19

the storage conditions on the properties of PHO. It has been reported that sPHAs undergo

20

physical ageing and secondary crystallization during storage, with dramatic effect on the

21

mechanical properties.33 Therefore, the mechanical properties of nanocomposites were

22

determined for different storage conditions.

23


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Page 18 of 38

Mechanical properties

2

The stress-strain curves of PHO and nanocomposites, determined after one month from

3

the compression-molding of the sheets, are shown in Figure 4a. A strong increase in both tensile

4

strength and modulus with the increase of BC concentration was noted although no chemical

5

modification of the filler or matrix was done: 3 wt % BC increased the Young’s modulus (YM)

6

with 76% and the tensile strength (TS) with 44%. A weaker increase of YM and TS was

7

observed for PHB or PHBV reinforced with similar amount of cellulose nanofillers.20,26 An

8

increase of the YM by 22% and almost no variation of TS were reported for PHBV

9

nanocomposite with 2.5 wt% nanofibrilated cellulose.20 Likewise, similar increase in YM (77%)

10

and TS (36%) were noted for a higher amount of cellulose nanowhiskers (5 wt%) in PHBV.26

11

The mechanical properties of PHO-3BC (YM of 25 MPa, TS greater than 12 MPa and

12

elongation at break (S) of 65%) promotes this nanocomposite as a suitable material for tissue

13

engineering; its properties fall in the characteristic limits for skin: 15-150 MPa for YM, 5-30

14

MPa for TS and 35-115 % for S, depending on the type of the tissue.34

15

The significant increase of TS in PHO-3BC is, probably, determined by the good

16

interfacial bonding between the carbonyl groups of PHO and the hydroxyl groups of cellulose

17

nanofibers.10,26 This was also suggested by the TGA results. The increase of YM in

18

nanocomposites compared to the matrix may be related to: (i) the restriction of the polymer chain

19

mobility; (ii) the increased crystallinity of the polymer or (iii) the high rigidity of the nanofibers

20

and their good dispersion in the polymer matrix.


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1 2

Figure 4. Stress-strain curves of PHO and nanocomposites (a); Young’s modulus (YM), tensile

3

strength – (TS) and strain at break (S) in function of the aging time (b).

4 5

DSC analysis showed no change in Tg value of PHO after BC addition, and hence chain

6

mobility was not restricted by the small amount of nanofibers. On the other hand, the increase in

7

polymer crystallinity and crystal perfection after nanofillers addition enhances the stiffness and is

8

considered the main cause of a higher YM in nanocomposites.5,29 However, no significant

9

increase of PHO crystallinity was noticed by DSC analysis after BC addition but more perfect

10

crystals in the case of PHO-3BC nanocomposite, which may contribute to some increase in YM.

11

Therefore, the high rigidity of BC nanofibers and their good dispersion in PHO must have

12

contributed to the improvement of YM; it should be kept in mind that the YM of cellulose

13

nanowhiskers was reported to be 143 GPa35, so more than 5000 times higher than that of PHO

14

matrix.

15

Besides polymer-fiber interactions which contribute to a good stress transfer from the

16

matrix to the nanofibers, the high aspect ratio of BC, illustrated by the TEM image (Figure 1), is

17

another factor that should be considered. A percolation model is commonly used to explain the 19 ACS Paragon Plus Environment

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Page 20 of 38

1

good reinforcing effect of cellulose nanofillers in polymers.10 The formation of a network is

2

possible from a low percolation threshold (P) for high aspect ratio nanofibers. In our system, a P

3

value as low as 1.4 vol% (1.8 wt%. considering the densities of BC and PHO as 1.5 and 1.2

4

g/cm3, respectively) was estimated on the bases of the aspect ratio (AR) of BC before processing:

5

P = 0.7/AR,22 with AR = 50. Therefore, the formation of a continuous network can be assumed

6

in PHO-3BC nanocomposite.

7

AFM – PF QNM technique was used to get more insight into the dispersion of BC in

8

PHO-3BC nanocomposite and the reinforcement mechanism (Figure 5). Long, thin nanofibers

9

(30-40 nm in width and less than 15 nm in height) were observed on the surface of the

10

nanocomposite (Figure 5b), at least some of them being in contact with each others. The larger

11

width of cellulose nanofibers compared to their height in AFM images could be an influence of

12

the tip widening effect.36

13 14

Figure 5. AFM topographic, peak force error and deformation images on the surface of PHO (a)

15

and PHO-3BC (b). 20 ACS Paragon Plus Environment

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Biomacromolecules

1

The length of nanofibers could not be determined in these images and, therefore, lower

2

magnification AFM scans were taken (Figure 6a and b). Many long nanofibers of more than 5

3

µm in length and 30 - 60 nm in width, besides a few short nanofibers, evenly spread on the

4

surface of PHO-3BC were observed. It should be noted that at a larger scan area the nanofibers

5

were not detected anymore. Interestingly, TEM image of the same magnification of cryo-

6

microtomed layer from the bulk material (Figure 6c) particularly showed well dispersed short

7

fibers; nanofibers of 60 - 80 nm in width and no more than 1 µm in length were observed by

8

TEM. This difference between the surface and bulk nanofibers is intriguing. The shear forces

9

during melt processing may influence both the fibers’ length and their dispersion in PHO, by

10

decreasing the length and improving the dispersion. However, the same size characteristics

11

should be obtained in bulk and on the surface. Moreover, scanning transmission electron

12

microscopy (STEM) image (Figure 6d) showed a thin, long and slightly curved nanofiber in

13

PHO-3BC nanocomposite; indeed, “zigzag” fibers (see Figure 6d – detail) were often observed

14

in TEM images. Hence, it can be hypothesized that the “zigzag” fibers may be cut during the

15

release of the slices, resulting fragments. Having in mind that the cryo-microtomed layer of

16

nanocomposite has a thickness of about 100 nm, which is close to the width of BC nanofibers, it

17

can be assumed that the cutting process may unfold the bundles of BC nanofibrils leading to

18

broaden fibers and may remove parts of the fibers leading to a “dash-dot” like structure in the

19

TEM image. SEM images (Figure 6e-g) confirm the high proportion of long nanofibers and their

20

good dispersion in PHO-3BC; a real network of BC nanofibers is obvious in these images. The

21

nanofibers’ width, determined after a systematic analysis of TEM, AFM and SEM images, was

22

usually between 40 to 110 nm (see histograms in Figure 6h), consistent with previously reported

23

data for BC nanofibers in the initial pellicle.16,17


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Page 22 of 38

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Biomacromolecules

1

Figure 6. Lower magnification AFM images of PHO-3BC nanocomposite - height (a) and peak

2

force error (b); Cryo-TEM image of PHO-3BC nanocomposite (c); STEM image of bacterial

3

cellulose nanofibril in PHO-3BC nanocomposite; curved nanofiber (detail) (d); SEM images of

4

PHO-3BC nanocomposite – different magnifications (e-g); histogram showing the width

5

distribution of BC nanofibers in PHO-3BC nanocomposite from TEM (154 counts), AFM (45

6

counts) and SEM (40 counts) analyses (h)

7 8

The mean width value was 79±22 nm when determined from TEM images analysis,

9

52±23 nm from AFM data and 56±29 nm from SEM analysis. The difference between the mean

10

values may come from the difficulty to “see” the entire fiber which is embedded in the polymer

11

in the case of surface analyses but also from the disruption of fibers as discussed above, in the

12

case of TEM analysis. In conclusion, the melt processing did not induce large changes in the

13

length and width of BC nanofibers in the composite; only a higher proportion of short fibers was

14

observed. Therefore, it can be assumed that the aspect ratio of nanofibers was only slightly

15

decreased and considering the bent and coiled nanofibers (see Fig. 6) as well as nanofiber

16

clusters, it may be supposed that 3 wt% BC is close to the limit from which the percolation is

17

occurring.

18

DSC analysis showed that the aging process influenced the degree of crystallinity of PHO

19

samples, which could affect the mechanical behavior. Therefore, the mechanical properties of

20

PHO-3BC nanocomposite and PHO matrix were determined at different intervals after

21

compression-molding: 3 h, 3days, one week and one month. During these times, the samples

22

were kept at room temperature and 50% relative humidity. The intervals were chosen based on

23

previously reported data regarding the crystallization of PHO.29 Thus, XRD study on a PHO 23 ACS Paragon Plus Environment

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Page 24 of 38

1

copolymer containing 5.7% 3HH showed that the first XRD peaks were observed after about 50

2

min for the sample isothermally crystallized at 21 °C and the maximum crystallization rate was

3

noted between 50 and 200 min (about 3 h). Likewise, in a long-term crystallization study by

4

means of DSC, the melting enthalpy showed only a slight growth between 2 to 4 weeks.29

5

The tensile test results (YM in MPa, TS in MPa and S in %) are given in Figure 4b;

6

higher values of tensile strength and modulus were obtained for the nanocomposite compared to

7

the pure matrix regardless the aging time. YM of PHO matrix slightly decreased after 3 days of

8

storage and finally recovered the initial value (measured after 3 h) (Figure 4b); similar variation

9

was noted for TS. Interestingly, only a slight decrease of S with about 10% was observed in the

10

case of PHO over one month of storage, the value of the elongation at break (77%) indicating a

11

ductile behavior. Indeed, PHO sample did not show significant aging during storage and it was

12

able to preserve the ductile behavior within the tested period. Similar conclusion was drawn by

13

Larrañaga et al on a PHO copolymer tested between 2 and 4 weeks of storage at 21 °C;

14

unfortunately, no initial measurement of the mechanical properties was done to observe the

15

variation of the properties between the first day and the 28th.29 Contrarily, a dramatic decrease of

16

the elongation at break (more than 75%) was reported for PHB after 2 weeks of ambient storage,

17

indicating a remarkable embrittlement.37 Similar results were obtained for a PHB copolymer

18

containing different amount, between 2.5 and 8.1 mol%, of 3HH.38 However, the copolymer with

19

3HH fraction higher than 10 mol% showed no significant changes in the mechanical properties

20

after 30 days room-temperature aging.39 The increased brittleness of PHAs was mainly explained

21

by the secondary crystallization and the constraints on the amorphous regions. Considering the

22

inherent low crystallinity of PHO,25 much lower than that of PHB and the higher crystallization

23

rate observed in the first 200 min (about 3 h) of storage close to the room temperature,29 the


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1

amorphous region can keep a high mobility regardless the storage time and the influence of the

2

small amount of tiny crystals formed during the storage is insignificant.

3

Several differences were noticed in the variation of the mechanical properties of PHO-

4

3BC with the aging time: almost no variation of the tensile strength but an increase with almost

5

15% of YM and a decrease with about 20% of S over one month of storage. Indeed, BC

6

nanofibers may put supplementary constraint on the amorphous region between crystallites, thus

7

increasing the rigidity. However, the elongation at break of PHO-3BC over one month of

8

storage, 65%, shows a ductile behavior.

9

The surface of PHO-3BC nanocomposite and PHO matrix was investigated by AFM

10

(Figure S3) to observe the crystalline structure, if any, after 3 h of storage. A lamellar structure

11

was visible on the surface of the nanocomposite but less clear on the surface of the matrix,

12

probably because of the nucleating effect of BC; this is consistent with DSC results. Indeed,

13

AFM investigation shows that some crystalline formations have already grown on the surface of

14

PHO samples after 3h of storage and confirms the previous observations for PHO-3HH

15

copolymer.29 The long spacing summing the thickness of crystalline and amorphous layers was

16

estimated to be 44 nm, which is a larger value than the previous estimate for PHB (below 14

17

nm40). This was expected because the crystallization of PHO was not completed and, unlike

18

PHB, the crystallinity in PHO is small. The tip widening effect could also influence the results.36

19

However, no comparison with previous results is possible since this is the first trial to estimate

20

the periodicity in mPHAS. No significant difference in AFM images after one week of storage

21

was noted for both samples, which support the observation that the aging process and

22

recrystallization are not important in the case of PHO matrix and PHO-3BC nanocomposite.

23


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Page 26 of 38

Melt processing of PHO/3BC nanocomposite for tube extrusion

2

A simulation of the melt processing of PHO-3BC nanocomposite was undertaken using a

3

homemade extrusion plastometer (Figure 7a) with a tube-shaped die shown as detail in Figure

4

7a. The tube-shaped die had an inner diameter of 3.5 mm, and an outer diameter of 5.5 mm. The

5

cylinder temperature was set to 50 °C. The nanocomposite was filled in the cylinder and kept at

6

the set temperature for about 4 min; then, a weight of 10 kg was applied on the piston and a

7

molded article having a tube-like shape was extruded through the die (Figure 7b). The tube was

8

highly flexible as can be seen in Figure 7c and recovered its form after bending (Figures 7d and

9

e).

10 11

Figure 7. Melt extrusion plastometer used in the tests (a) with the die shown in the detailed

12

image; the tube is flexible and recovered its form after bending (b-e).

13 14

Cell adhesion and cell proliferation studies were performed on PHO homopolymer6 but

15

no information on the pro-inflammatory immune response was found in the literature. Therefore,

16

an evaluation of pro-inflammatory effect was done on PHO and nanocomposites.

17 18

Evaluation of pro-inflammatory effect

19

Macrophages are known to engulf foreign material and to potentially elicit an immune signaling

20

cascade by means of secreted cytokines, chemokines and other immune effectors. TNF-α and IL26 ACS Paragon Plus Environment

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1

6 are potent pro-inflammatory effectors whose secretion by the macrophages can be triggered by

2

various danger signals, therefore evaluation of inflammatory susceptibility is of utmost

3

importance for biomedical applications.

4

Evaluation of the potential adverse immunological effects of PHO and PHO-3BC was

5

performed in an indirect manner: the eluted constituents of polyhydroxyalkanoate composite

6

were incubated with THP-1 derived macrophages. A PHB sample was tested in similar

7

conditions and served as a polymer reference. Prior to ELISA, all elutions were tested for

8

endotoxin activity to avoid inaccurate results due to bacterial contamination or residual LPS

9

from biosynthesis. All tested samples showed endotoxin activity under the 0.005 EU/mL

10

threshold. After incubation, TNF-α and IL-6 proteins were measured in culture supernatants. IL-

11

6 presence was undetectable in all the samples, except for the LPS treated sample. Likewise,

12

TNF-α concentration was very low (Figure 8), similar to PBS control or cell culture medium

13

control, which is less than 0.3 pg/mL.

14

15 16

Figure 8. TNF-α level in supernatants of THP-1 derived macrophage cells stimulated with

17

elution samples from polyhydroxyalkanoates (undetectable for PHO sample) 27 ACS Paragon Plus Environment

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Page 28 of 38

1 2

These results show that PHB, PHO and PHO-3BC nanocomposite will not release

3

components that can drive an acute pro-inflammatory response when used in-vivo, qualifying

4

them as feasible candidates for tissue engineering and biomedical devices.

5 6

Evaluation of cytotoxicity effect

7

The images acquired on the third day (Figure 9) showed the adhesion of L929 cells and

8

their characteristic spindle-shaped morphology for all the three samples, neat PHO, PHO-1.5BC

9

and PHO-3BC nanocomposites. In the seventh day, cells extensively proliferated and completely

10

cover the entire available surface of each tested samples (data not shown). These results prove

11

the ability of neat PHO and PHO nanocomposites to allow cell adherence and to promote cell

12

proliferation.

13

Images acquired on the third day were further analyzed with CellProfiler to yield

14

information about cell density and morphological features. Regarding cell density, the results

15

show small differences between samples, with a higher value for the nanocomposite with 1,5%

16

BC (Figure S4, Supporting Information). The evaluation of cell area revealed a higher tendency

17

of L929 cells to spread over the nanocomposite with 3 wt% BC (Figure S5). The result can be

18

explained by the presence of the more hydrophilic bacterial cellulose on the surface of PHO-

19

3BC, which may greatly promote cell anchoring and adhesion. This hypothesis is also sustained

20

by the form factor analysis (Figure S6). Although there are no statistical significant differences,

21

one may observe a lower form factor value for PHO-3BC sample, indicating a tendency to a

22

more elongated morphology corresponding to a heavily anchored fibroblast, as opposed to a

23

rounder one, characteristic to loosely adhered cells.

24 28 ACS Paragon Plus Environment

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Figure 9. Inverted fluorescence microscope images of L929 cells acquired in the third day on the

3

surface of neat PHO and nanocomposites 29 ACS Paragon Plus Environment

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Page 30 of 38

1 2

In conclusion, neither of the tested materials can be deemed cytotoxic. On the contrary,

3

both PHO and PHO/BC nanocomposites are highly biocompatible and favor cell attachment.

4

PHO-3BC, with a more hydrophilic surface due to the higher content of BC showed greater

5

compatibility with L929 cells allowing them to spread over larger areas and to adopt a more

6

natural morphology.

7

CONCLUSIONS

8

A melt processing technique was used for the first time to obtain nanocomposites from a

9

PHO copolymer and bacterial cellulose nanofibers with high aspect ratio. The addition of BC

10

had a benefic effect on the thermal stability of PHO which was increased with 25°C; this is very

11

important for the further melt processing of medical devices. A hydrolytic chain cleavage

12

mechanism was considered to explain the thermal behavior of the nanocomposites. No change of

13

chain mobility and glass transition was observed after BC addition in PHO; likewise, the melting

14

temperature was not influenced but the changes in the shape of the melting endotherm indicated

15

increased population of more perfect crystals in PHO-3BC nanocomposite. The slow

16

crystallization of pure PHO and PHO/BC nanocomposites was evidenced by the lack of

17

crystallization during the cooling cycle, regardless of the storage or annealing conditions. The

18

aging phenomena in PHO and nanocomposites were studied by testing the mechanical properties

19

at different intervals, between 3 h and one month after compression-molding. Crucially, PHO

20

and PHO-3BC nanocomposite were able to preserve their ductile behavior during storage and did

21

not show significant aging, totally opposite to PHB and other sPHAs which show secondary

22

crystallization and increased brittleness during storage. Higher values of tensile strength and

23

modulus were obtained for the nanocomposite compared to the pure matrix regardless the aging 30 ACS Paragon Plus Environment

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Biomacromolecules

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time. Only 3 wt % BC was enough to reinforce PHO, increasing the YM with 76% and TS with

2

44%. With a Young’s modulus of 25 MPa, a tensile strength of 12 MPa and an elongation at

3

break of 65%, PHO-3BC nanocomposite is a suitable material for tissue engineering. This

4

nanocomposite was successfully melt processed by tube extrusion and this is an important step

5

for the manufacture of artificial arteries and other medical implants. Biocompatibility study

6

showed no pro-inflammatory immune response of PHO and nanocomposites and no cytotoxic

7

effect on L929 cell line. PHO-3BC showed a greater compatibility and is considered a feasible

8

candidate for in vivo application of tissue-engineered blood vessels.

9

Supporting Information

10

Details on the biosynthesis of PHO copolymer; glass transition temperature from DSC

11

thermograms (second melting) (Figure S1); DSC thermograms (first melting, cooling, second

12

melting) of PHO and nanocomposites (Figure S2); AFM images after 3h of storage (Figure S3).

13

L929 cell density, cell area and factor form (Figures S4-S6).

14

Notes

15

The authors declare no competing financial interest.

16

AUTHOR INFORMATION

17

Corresponding Author

18

* E-mail: [email protected] (DMP)

19

Acknowledgement


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1

The financial support from the Romanian Ministry of Education-UEFISCDI, grant 122/2017

2

(PNIII PED-2016-0287 - CELLAB-SLP) and grant 158/2012 (POLYBAC) is greatly

3

acknowledged.

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