Tuneable hydrolytic degradation of poly(l -lactide) scaffolds triggered by ZnO nanoparticles

Accepted Manuscript Tuneable hydrolytic degradation of poly(l-lactide) scaffolds triggered by ZnO nanoparticles

E. Lizundia, P. Mateos, J.L. Vilas PII: DOI: Reference:

S0928-4931(16)31397-2 doi: 10.1016/j.msec.2017.02.104 MSC 7438

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

20 September 2016 14 December 2016 21 February 2017

Please cite this article as: E. Lizundia, P. Mateos, J.L. Vilas , Tuneable hydrolytic degradation of poly(l-lactide) scaffolds triggered by ZnO nanoparticles. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.02.104

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ACCEPTED MANUSCRIPT Tuneable hydrolytic degradation of Poly (L-lactide) scaffolds triggered by ZnO nanoparticles E. Lizundia1,2*, P. Mateos2, J. L. Vilas2 1

Dept. of Graphic Design and Engineering Projects, Bilbao Faculty of Engineering. University of the Basque Country (UPV/EHU) 2

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Macromolecular Chemistry Research Group (LABQUIMAC). Dept. of Physical Chemistry. Faculty of Science and Technology. University of the Basque Country (UPV/EHU), Spain.

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* Corresponding author:

University of the Basque Country (UPV/EHU)

48013 Bilbao - Spain Phone: +34 94 601 3965

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E-mail address: [email protected]

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Alameda Urquijo w/n

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Dept. of Graphic Design and Engineering Projects

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Abstract

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In this work we fabricate porous PLLA and PLLA/ZnO scaffolds with porosities ranging from 10 to 90% and average pore diameter of 125-250 µm by solvent casting/particulate leaching method. The structural evolution of PLLA/ZnO scaffolds during their in vitro degradation in phosphate buffered saline (PBS) at physiological pH (7.4) has been studied as a function of porosity and obtained results were compared to plain PLLA scaffolds. The changes induced upon the hydrolytic degradation of scaffolds have been explored by measuring the pH changes of the medium, the mass loss, thermal transitions, crystalline structure, thermal stability and the morphological changes. It is shown that the degradation profile of scaffolds could be successfully modified by tuning both the amount of ZnO nanoparticles and by varying the scaffold porosity. Results reveal that the water dissociated on ZnO nanoparticle surfaces initiate hydrolytic degradation reactions by reducing the strength of the chemical bonds of the adjacent PLLA chains, causing them to further divide into water-soluble oligomers. Obtained results may be useful towards the development of antibacterial porous structures with tuneable degradation rates to be used as a substrate for the growth of different kind of cells and tissues. Keywords: Poly(L-lactide); Zinc oxide; scaffold; hydrolytic degradation; tissue engineering; biomedical material.

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ACCEPTED MANUSCRIPT 1. Introduction

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Tissue engineering (TE) with porous three-dimensional (3D) biodegradable scaffolds has emerged as a promising method for tissue regeneration [1,2]. Porous scaffolds could recreate biological tissue substitutes that restore, maintain or improve tissue functions [3]. To obtain structurally and functionally developed tissues, these scaffolds used for TE should posses a three-dimensional highly porous structure with open and interconnected pore network that allows cell growth and at the same time permits the flow of nutrients and metabolic waste [4,5]. Scaffolds allow the development of less invasive and resorbable devices that would avoid the need for a second revision surgery to remove the implant, reducing the eventual infection risk for the patient [6,7]. Depending on the intended application, fast or slow degradation of scaffolds is desired since these structures should present a controllable degradation rate that matches the cell/tissue growth. For instance, a slow degradation could yield to stress shielding of the growing tissue and which limits the regeneration process [8], while a fast degraded scaffold does not allow the sufficient development of the neo-tissue [9]. This fact demonstrates the need of extensive works focused on adjusting the hydrolytic kinetics of scaffolds.

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Porous scaffolds composed of several biocompatible materials have been already proven to have potential uses for tissue engineering applications [10,11]. Currently, polymer-based scaffolds are the most commonly used materials for the fabrication of these three-dimensional porous architectures [12,13]. In comparison with natural biodegradable polymers, the use of synthetic polymers for TE presents the advantages of their mass production capability, high reproducibility, tailored structural features with custom-made mechanical behaviour and tuneable degradation rate to meet the intended biomedical application [14–16].

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Biocompatibility of the scaffold is an essential requirement and the materials used must not elicit any cytotoxic responses during their degradation into the human body. Besides of its already known biocompatibility and biodegradability, polylactides are an environmentally friendly polymer since it could be derived from completely renewable resources and could be ultimately degraded into metabolizable products by natural pathways via random-scission of their ester linkages [17,18].The semicrystalline poly (L-lactide) (PLLA) enantiomer emerges as the ideal candidate as it has shown a good in vivo track record [19,20]. This environmentally friendly polymer is currently being widely used as sutures, drug delivery devices and tissue engineering substrates as it’s possess favourable cell adhesion and proliferation properties. PLLA displays a tuneable degradation rate, good mechanical properties and easy of processing (they can be shaped into screws, scaffolds, pins, plates…) [21]. Moreover, its glass transition temperature (Tg) is located at about 55-60ºC, having therefore a high mechanical modulus at physiologic temperature [22]. However, its further application in tissue engineering has been largely limited owing to its slow degradation rate, which makes PLLA-based medical devices not optimal for short- and medium-term applications [21]. Indeed, several works have reported that high molecular weight PLLA lasts from 2 to 5.6 years for its total resorption in vivo [23,24]. In this sense, different copolymerization approaches have been investigated to tune its degrabalility [6]. To date, the composite strategy has been mainly used for fabricating polymeric scaffolds with desired mechanical properties [25] For example, natural-polymer based composites have been reported to present an increased mechanical stability and improved cell interaction [26,27]. Overall, traditional polymer-based composites require large filler volume fractions to reach the desired physical and mechanical properties. On the contrary, nanocomposites usually offer improved physico-mechanical performance at low loading fractions [28]. Therefore, an innovative approach could arise from the use of nanoparticles which would initiate hydrolytic degradation reactions of their hosting matrix and to thereby obtain scaffolds with tuneable degradation profiles. In a recent work, our group has shown that the hydrolytic degradation of PLLA could be markedly accelerated by adding zinc oxide nanoparticles (ZnO NPs) via H2O

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dissociation on ZnO oxygen vacancy sites and the attack of this new hydroxyl groups to the PLLA ester bonds [29]. Thus, it could be hypothesized that the selective addition of ZnO nanoparticles into PLLA matrix would yield scaffolds with larger degradation rates. More interestingly, it has been reported that these nanoparticles preferentially kill high proliferative cells, like cancer cells, versus normal cells and are able, as well, to act as effective antibacterial agents [30]. Additionally, zinc oxide nanoparticles are economically feasible and have been approved by the American Food and Drug Administration (FDA) [31]. In one of our previous works we have demonstrated that when ZnO nanoparticles are loaded into PLLA they activate the material surface and drive cell differentiation at the same time that the release of zinc within the culture medium remains negligible [32]. Consequently, it could be expected that in addition to modulating the degradation kinetic of PLLA scaffolds, ZnO nanoparticles may as well stimulate a faster regeneration of the healing tissue. Additionally, it has been proven that the presence of zinc oxide avoids biofilm formation and bacterial colonization on the implant devices, hindering bacterial an eventual bacterial infection caused by the medical implant [33].

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In this work we attempt to investigate the hydrolytic degradation mechanism of PLLA-based porous scaffolds that mimic the architecture of natural extracellular matrix (ECM). Since typically porosities of about 80-90% with a minimum average pore diameter of 60-100 µm are required for obtaining a successful cell penetration and vascularisation of the newly forming tissues [5], scaffolds having a pore diameter of 125-250 µm and porosities up to 90 % have been fabricated by solvent casting/particulate leaching method (SCPL). The structural evolution of PLLA/ZnO scaffolds during their hydrolytic degradation in phosphate buffered saline (PBS) has been studied as a function of porosity and obtained results were compared to plain PLLA scaffolds. The pH changes induced by the release of the acidic by-products, the mass loss, the thermal transitions and the thermal stability of the scaffolds were monitored during the in vitro degradation. Furthermore, the morphological evolution of scaffolds upon degradation has been explored by scanning electron microscopy (SEM). The combination of the inherent properties of zinc oxide make ZnO NPs great candidates to develop cheap antibacterial scaffolds with tuneable hydrolytic profiles which are expected to aid cells during their proliferation process. Obtained results may be useful towards the development of antibacterial scaffolds that cover a wide range of degradation rates for potential tissue engineering applications. 2. Experimental part

2.1. Starting materials

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PLLA with a number-average molecular weight (Mn) of 100.000g/mol and a polydispersity index (Mw/Mn) of 1.85 has been supplied by Purac Biochem. Phosphate buffered saline (PBS) tablets and NaCl was supplied by Sigma Aldrich, chloroform (reagent ≥ 99.8%) was purchased from LabScan. Zinc oxide (ZnO) nanoparticles have been kindly purchased by L´Urederra Technological Centre (Spain). 3D scaffolds were prepared via solvent casting/particulate leaching method using chloroform as a solvent and NaCl particles (sieved to be in between 120 and 250 µm) as a porogen. Firstly, ZnO nanoparticles were homogeneously dispersed in chloroform via mild sonication (20% output for 1 minute) in a Vibra-Cell™ CV 334 ultrasonic processor. These dispersed NPs were added to previously dissolved PLLA (in chloroform) to obtain a ZnO concentration of 1 wt% (with respect to polymer fraction). PLLA–ZnO dispersions were submitted to an additional sonication step for 5 minutes. Different amounts of NaCl particles were added them and the mixture was vigorously magnetically stirred for 1h. The resulting materials were transferred to circular Petri-dishes and were dried until constant weight. Square-shaped (1x1cm2) scaffolds were punched out and placed in distilled water for 48 h at room

ACCEPTED MANUSCRIPT temperature to leach out the salt particles. Non-porous samples were prepared in a hydraulic hot press by compression moulding at 200ºC for 4 minutes under a pressure of 150 MPa. In vitro degradation of PLLA and PLLA/ZnO scaffolds with a porosity ranging from 0 to 90% was carried out in an oven at 60ºC. Samples with a surface area to volume ratio of 0.2cm-1 were immersed in a PBS solution (PBS tablets dissolved in Milli-Q water; pH=7.4) and they were removed after different periods of time. The pH evolution of the medium has been monitored using a 691 pH Meter (Metrohm).

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Two samples from each composition and degradation time were removed from the PBS solution and weighed after wiping the surface with a filter paper to absorb the surface water. These samples were dried at 40ºC for 24h and were weighed again (dry weight, Wd). Remaining weight (%RW) was then calculated:

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where Wo represents the initial weight of scaffolds (~30mg). 2.2. Differential scanning calorimetry (DSC)

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Thermal transitions of degraded scaffolds were determined using a Mettler Toledo DSC 822e calorimeter under nitrogen atmosphere (30 ml/min). Samples having 6±1 mg were sealed in an aluminium pan and they were heated from 0 to 200ºC at a rate of 10ºC/min. The PLLA crystalline fraction Xc (%) has been determined as [34]:

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where ΔHf and ΔHc are respectively the enthalpy of fusion and cold crystallization of the samples determined on the DSC. ΔHf0 = 106 J/g was taken as the heat of fusion of an infinitely thick PLLA crystal [35].

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2.3. Thermogravimetric analysis (TGA)

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Thermal stability of scaffolds was studied in a TGA METTLER TOLEDO 822e Thermal Gravimetric Analysis instrument by heating the samples from room temperature to 500ºC at 10ºC/min with nitrogen flux of 50 ml/min. 2.4. Morphological characterization Scaffold morphology upon hydrolytic degradation has been analyzed in a Hitachi S-4800 field emission scanning electron microscope (FE-SEM) at an acceleration voltage of 5 kV. Surfaces were chromium-coated in a Quorum Q150T ES turbo-pumped sputter coater (5 nm thick coating). 3. Results and discussion 3.1. pH evolution and remaining weight (RW) Figure 1a shows the evolution of the medium pH as a function of hydrolysis time for PLLA/ZnO scaffolds in PBS at physiological pH (pH 7.4). It is observed that the pH of the

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incubation medium remains roughly constant during an initial period of ~5 days. Then, the pH gradually decays until a minimum value of 2.9 after 50 days due to the random-scission of ester linkages of the polymeric matrix, producing a continuous fragmentation of the PLLA macromolecules into oligomeric species which are then released as acidic by-products from the scaffold structure to the incubation medium [21,36]. Upon PLLA chain shortening, these newly formed oligomers become water- when their molar mass reaches about 1.000 g mol-1 (which corresponds to chains formed by 13 LA units) [29]. As a consequence, these short chains are expelled to the outside medium as acidic byproducts due to the presence of carboxylic end-groups. It is interesting to note that after being degraded in PBS for 20 days, the medium pH of PLLA/ZnO film has been measured to be 4.4 in comparison with 6.4 measured for the 88% porous scaffold. This slower pH decrease as the scaffold´s porosity increases suggests that the occurring accumulation of acidic degradation by-products within the dense samples, PLLA/ZnO film for instance, accelerate chain scission reactions of PLLA. Since an increased porosity facilitated the release of lactic acid oligomers to the surrounding medium, these chains do not autocatalyze hydrolytic degradation in PLLA/ZnO materials, leading to slower degradation profiles.

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Figure 1. pH evolution of the PBS degradation medium as a function of hydrolysis time (a) and remaining weight (b) of PLLA/ZnO scaffolds having different porosities.

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Moreover, Figure S1 depicts the pH values of the PBS medium when PLLA scaffolds are degraded. It is seen that the pH decay is slower when comparing with PLLA/ZnO nanocomposite scaffolds. This behaviour could a priori taken as counterintuitive since in a previous work we have shown that PLLA/ZnO materials are slightly more hydrophobic than neat PLLA (a water contact angle of 81º versus 88º for neat PLLA and PLLA having 1wt.% of ZnO nanoparticles) [37]. This behaviour could be attributed to the generation of new hydroxyl groups from the H2O dissociation on ZnO nanoparticle surfaces, which in turn accelerates the hydrolysis of PLLA ester bonds [29]. More interestingly, the pH decay barely depends on the porosity of the scaffolds, contrarily to that found for PLLA/ZnO scaffolds (after 20 days of degradation the pH difference is 0.5 in regard to 2 obtained for nanocomposites). Therefore, ZnO nanoparticles could serve to make the porosity a key variable on the resulting hydrolytic degradation of PLLA-based scaffolds. The fact that the degradation profile of PLLA/ZnO composite scaffolds could be selectively tuned by varying both the porosity and the ZnO concentration could be useful in the biomedical field because these two aspects could be easily modified during the scaffold fabrication by the common scaffold fabrication methods such as SCPL, electrospinning, solid–liquid phase separation, phase inversion or 3D printing [4]. The remaining weight (%RW) of PLLA/ZnO and PLLA scaffolds is shown in Figure 1b and Figure S1b respectively. It is seen that RW could be only computed during the first 27 days of degradation because longer times damage the structural integrity of the samples. RW values

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are found to decrease with degradation time in the same trend to the reported pH decay as a result of the polymer fragmentation and the subsequent release of lactide oligomers to the incubation medium. The fact that the RW of PLLA/ZnO film is larger than that corresponding to the scaffolds could be explained in terms of some differences on the crystalline structure of our samples. Since PLLA/ZnO film has a slightly larger crystalline fraction than that of scaffolds due to the different fabrication approaches employed, the early-stage hydrolytic degradation which mainly occurs within the amorphous regions is delayed (less material is able to undergo degradation reactions) [38]. Once the hydrolytic reactions begins, after being submerged in PBS for 10 days, the RW rapidly decreases, following the same porositydependence as reported for pH data. When compared to the RW of based PLLA scaffolds, it is also noticed that PLLA/ZnO film losses 29% of its weight after the first days of degradation, while PLLA film only losses 20% of its initial mass.

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The effect of the in vitro degradation on the thermal properties of scaffolds was followed by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Figure 2 shows the heating DSC traces of hydrolytically degraded 62 % porous PLLA/ZnO scaffolds as a function of degradation time, while Table 1 summarizes their corresponding main thermal parameters (glass transition temperature Tg, melting temperature Tm and crystallinity degree Xc). It could be observed that, for all the studied degradation periods, PLLA/ZnO structures present the characteristic thermograms corresponding to semicrystalline polyesters, with a second-order phase transition corresponding to the glass transition followed by a sharp melting endotherm located at temperatures above 150 ºC [39,40]. 0,0

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Overall, as degradation occurs, the Tg of scaffolds gradually drops from 69.7 ºC for nondegraded sample to 27.9 ºC at day 50. In the same way, Tm decreases from 173.3 to 154.2 ºC for the same period, while the crystallinity degree of scaffolds was found to slightly increase from 48.1 to 61.1%. These changes mainly occur during the initial stage of degradation, which, according to the pH changes displayed in Figure 1a, has been observed to be 27 days for 62 % porous scaffold. The occurring thermal transition changes are related to the increased molecular mobility of PLLA chains induced by the hydrolytic chain-cleavage, which upon degradation allow the development of well-ordered domains [21,41,42]. Therefore, the shorter the PLLA chains become, the lower Tg and Tm values are achieved, while Xc increases. The small exothermic peak occurring just before melting and centred at 161.1 ºC is ascribed to a phase transition of α´ to more stable α polymorph [43]. Therefore,

ACCEPTED MANUSCRIPT the initial structure of scaffolds, mainly composed by α´ crystals, is transformed to α form because of the occurring chain reorganization makes these crystals to undergo a phase transformation to a more stable polymorph, obliterating all traces of α´ crystal form. This fact suggest that the hydrolytic degradation not only occurs within the PLLA amorphous regions but also is able to modify the structure of the already existing polymorphs, at least the disordered α´ form, which has been reported to present a frustrated packing structure similar to α form [44]. Therefore, apparently water molecules are able to penetrate the disordered crystals (α´) and modify their chain conformation similarly to the effect that occurs when PLLA is heated during the DSC heating scans [40,45].

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Table 1. Main thermal properties of PLLA/ZnO scaffolds as determined by DSC.

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The thermal stability of scaffolds was recorded by thermogravimetric analysis (TGA). The TGA traces in Figure 3a reveal that the thermodegradation of PLLA/ZnO porous structures proceeds at lower temperatures as degradation time increases. For instance, the onset of thermal degradation (T5%, taken as the first 5% weight loss) is continuously lowered from 318.4 ºC for non-degraded sample to 222.2 ºC after 50 days of degradation in PBS (see Table S1 for the characteristic temperatures of thermal degradation). Similarly, the maximum degradation rate temperature (Tpeak) decreases from 347.8 to 313.2 ºC. Since it has been reported that the thermal stability of polyesters is strongly correlated to macromolecular length [46], the increased thermal susceptibility as scaffolds are submerged in PBS may be correlated to the presence of shorter PLLA chains within the scaffolds. These short chains, require less energy than the original long macromolecules (Mn=100.000g/mol) to undergo thermal diffusion processes, arise from the hydrolytically-induced chain scission [47]. As shown in Figure 3b, regardless of degradation time all samples display a bell-shaped derivative degradation curve, denoting a single-stage thermodegradation for all the samples occurring via intramolecular transesterification reactions [48]. In any case, it is also noted that the Full Width at Half Maximum (FWHM) of degradation peak is increased from 28.9 to 60.1 ºC after being the scaffolds immersed for 50 days in PBS as a result of increased polydispersity with increasing degradation time [21,49]. On the other side, and according to Figure 3c and 3d, for a given degradation time the thermal stability of scaffolds increases with porosity. In comparison to 79% porous PLLA/ZnO scaffold, the Tonset of the nanocomposite film occurs 35.8ºC below, while its Tpeak is lowered by 37.8 ºC (Table S2). This behaviour indicates that the hydrolytically-induced PLLA degradation is more pronounced in the case of dense films, which agrees well with reported pH, RW and DSC data. Moreover, plain PLLA film shows an enhanced stability in regard to nanocomposite film, indicating that during the degradation in PBS medium the polylactide backbone chain scission, which occurs through a random-scission of ester linkages, has not been as dramatic as in the case of the sample containing ZnO nanoparticles [21], highlighting the catalytic effect of zinc oxide on the hydrolytic degradation of PLLA [29].

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Figure 3. Thermogravimetric traces (a,c) and weight lost rates (b,d) of scaffolds corresponding having 50% porosity as a function of immersion time (a,b). The effect of composition after 8 days of degradation in PBS (c,d).

3.3. Morphological evolution of PLLA/ZnO scaffolds

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The morphological changes induced during the hydrolytic degradation of scaffolds were assessed by field emission scanning electron microscope. As shown by the FE-SEM crosssections in Figure 4, all the scaffolds show an isotropically distributed well interconnected porous structure with no significant differences in the pore size, shape and interconnectivity. This fact suggest that the scaffold structure is not affected by the presence of zinc oxide nanoparticles and it is mainly governed by the size and amount (concentration) of the NaCl porogen employed during the SCPL process. Non-degraded scaffolds present uniformly distributed pores of about 187 ± 54 and 184 ± 37 μm for 50% and 90% porous scaffolds respectively (statistics based on 100 counts; see Figure S2a and S2b in Supporting Information for pore size distribution). It could be observed that upon degradation in PBS medium PLLA/ZnO scaffolds are able to keep their highly porous structure. However, as a result of the occurring chain-scission, the average pore size decreases to 137 ± 44 and to 167 ± 39 μm after 20 days of degradation for 50% and 90% porous scaffolds (Figure S2c and S2d respectively). It should be pointed that this decrease results less marked as the degree of porosity increases, indicating that high porosities avoid the collapse of the three-dimensional structure because of the easier release of the acidic by-products to the incubating medium [21].

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Figure 4. FE-SEM micrographs of PLLA/ZnO scaffolds before (left column) and after being hydrolytically degraded for 20 days (right column) with different porosities: a) 0%; b) 50%; c) 70%; d) 75% and e) 90%.

It is interesting to note the good structural integrity of these scaffolds upon their immersion in PBS at 60 ºC in regard to other scaffolds composed by FDA-approved polymers such as PLGA, which have been reported to suffer notable shrinking and twisting after 2 weeks submerged in PBS medium [50]. More precisely, the dimensional stability of PLGA scaffolds was found to be inadequate because the lost of their initial pore structure at the early stages of hydrolysis. Since holding the original scaffold dimensions during the beginning of the degradation process, and thus, during the growth of the new tissue, results essential for TE applications, these PLLA/ZnO scaffolds may arise as suitable substrates for cell/tissue proliferation because of their tuneable degradability and structural integrity.

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Figure 5. FE-SEM micrographs of PLLA/ZnO film (a) and 90% porous scaffold after 20 days of hydrolytic degradation.

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Figure 5 shows high-magnification FE-SEM micrographs corresponding to hydrolytically degraded PLLA/ZnO film (a) and scaffold (b). It is observed that non-porous material evolves from a rather smooth surface (Figure 5a) to a highly rough surface as a result of the occurring molecular scissions, while the morphology of the porous scaffold remains virtually unchanged, indicating a stronger degradation of the PLLA/ZnO film when comparing with its porous counterpart. As highlighted by blue arrows, both samples show several pits on their structures as a result of the expelled ZnO nanoparticles [32]. This finding confirms that the PLLA degradation preferably occurs at the PLLA-ZnO interface [29]. Consequently, the chances for the PLLA molecular scission occurring as a result of the hydrolytic degradation are higher in the case of the PLLA/ZnO system in comparison with plain PLLA. Overall, these morphological findings suggest that lower porosities and higher ZnO concentrations induce faster degradation of scaffolds, fact that has been already proven by pH, DSC, TGA and FTIR experiments.

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In the light of obtained results we speculate that the water dissociated by the ZnO nanoparticles reduce the strength of the chemical bonds of the PLLA chains causing them to divide into water-soluble oligomers. Then, these short chains comprising carboxylic ends are able to autocatalyze ester hydrolysis reactions in the PLLA/ZnO system. These autocatalytic reactions could be finely tuned by simply modifying the amount of H2O-dissociation sites (or ZnO nanoparticles) or by fabricating structures with different porosities which allow oligomers and monomers to leach out from the polymeric structure, reducing their autocatalytic effect [51,52]. Conclusions

The aim of this work is to provide a novel and simple approach by which the hydrolytic degradation profile of PLLA-scaffolds could be tuned. To that end, PLLA/ZnO composite scaffolds having a pore diameter of 125-250 µm and porosities up to 90 % have been fabricated by solvent casting/particulate leaching method. The effect of the in vitro hydrolytic degradation of scaffolds in PBS medium on the physico-chemical and morphological has been investigated by commonly employed techniques. Results reveal and increased degradation rate in scaffolds having low porosities as a result of the accumulation of the acidic byproducts inside the composite materials. It is also observed that the addition of ZnO nanoparticles into PLLA matrix accelerates the hydrolytic degradation kinetics of scaffolds. DSC studies reveal that the hydrolytic degradation not only occurs within the PLLA amorphous regions but also is able to modify the structure of the already existing α´ disordered polymorph to a more compact α form. Morphological observations provide direct

ACCEPTED MANUSCRIPT evidence on the ZnO-initiated PLLA degradation, which is expected to occur via H2O dissociation onto zinc oxide nanoparticles, which initiate hydrolytic degradation reactions by reducing the strength of the chemical bonds of the adjacent PLLA chains. Although in vitro biological evaluation of these scaffolds are required for establishing pre-clinical application, the experimental findings shown here suggest that the development of PLLA/ZnO scaffolds may allow engineering antibacterial devices with tuneable degradation profiles which would match the tissue growth rate. Moreover, these dynamic structures would expose ZnO nanoparticles as the PLLA matrix degrades, which as has been recently proven by our group to drive cell differentiation [32].

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Acknowledgements

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E.L. thanks the University of the Basque Country (UPV/EHU) for a postdoctoral fellowship. In addition, we gratefully acknowledge Corbion-Purac for the kind donation of PLLA polymer. Authors thank the Basque Country Government for financial support (Ayudas para apoyar las actividades de los grupos de investigación del sistema universitario vasco, IT718-13).

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[1] D. Mohamad Yunos, O. Bretcanu, A.R. Boccaccini, Polymer-bioceramic composites for tissue engineering scaffolds, J. Mater. Sci. 43 (2008) 4433–4442. [2] B. Stevens, Y. Yang, A. Mohandas, B. Stucker, K.T. Nguyen, A review of materials, fabrication methods, and strategies used to enhance bone regeneration in engineered bone tissues, J. Biomed. Mater. Res. - Part B Appl. Biomater 85 (2008) 573–582. [3] R. Langer, J.P. Vacanti, Tissue engineering, Science 260 (1993) 920–926. [4] D.W. Hutmacher, Scaffolds in tissue engineering bone and cartilage., Biomaterials. 21 (2000) 2529–2543. [5] V. Karageorgiou, D. Kaplan, Porosity of 3D biomaterial scaffolds and osteogenesis, Biomaterials 26 (2005) 5474–5491. [6] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym. Sci. 32 (2007) 762–798. [7] J.-B. Zeng, K.-A. Li, A.-K. Du, Compatibilization strategies in poly(lactic acid)-based blends, RSC Adv. 5 (2015) 32546–32565. [8] E.A. Makris, P. Hadidi, K.A. Athanasiou, The knee meniscus: Structure-function, pathophysiology, current repair techniques, and prospects for regeneration, Biomaterials 32 (2011) 7411–7431. [9] Q.L. Loh, C. Choong, Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size., Tissue Eng. Part B. Rev. 19 (2013) 485–502. [10] M.M. Stevens, J.H. George, Exploring and engineering the cell surface interface, Science 310 (2005) 1135– 1138. [11] S. Zhang, Fabrication of novel biomaterials through molecular self-assembly, Nat. Biotechnol. 21 (2003) 1171–1178. [12] K.M. Woo, J.H. Jun, V.J. Chen, J. Seo, J.H. Baek, H.M. Ryoo, G.S. Kim, M.J. Somerman, P.X. Ma, Nanofibrous scaffolding promotes osteoblast differentiation and biomineralization, Biomaterials. 28 (2007) 335– 343. [13] J. Xie, X. Li, Y. Xia, Putting electrospun nanofibers to work for biomedical research, Macromol. Rapid Commun. 29 (2008) 1775–1792. [14] L. Almany, D. Seliktar, Biosynthetic hydrogel scaffolds made from fibrinogen and polyethylene glycol for 3D cell cultures, Biomaterials 26 (2005) 2467–2477. [15] M.P. Lutolf, J. a Hubbell, Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering., Nat. Biotechnol. 23 (2005) 47–55. [16] P.A. Gunatillake, R. Adhikari, N. Gadegaard, Biodegradable synthetic polymers for tissue engineering, Eur. Cells Mater. 5 (2003) 1–16. [17] U. Edlund, A.C. Albertsson, Polyesters based on diacid monomers, Adv. Drug Deliv. Rev. 55 (2003) 585– 609. [18] T. Maharana, B. Mohanty, Y.S. Negi, Melt-solid polycondensation of lactic acid and its biodegradability, Prog. Polym. Sci. 34 (2009) 99–124. [19] S.I. Jeong, B.S. Kim, S.W. Kang, J.H. Kwon, Y.M. Lee, S.H. Kim, Y.H. Kim, In vivo biocompatibilty and degradation behavior of elastic poly(L-lactide-co-epsilon-caprolactone) scaffolds, Biomaterials 25 (2004) 5939–5946.

ACCEPTED MANUSCRIPT

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[20] D.C. Surrao, S.D. Waldman, B.G. Amsden, Biomimetic poly(lactide) based fibrous scaffolds for ligament tissue engineering, Acta Biomater. 8 (2012) 3997–4006. [21] A. Larrañaga, P. Aldazabal, F.J. Martin, J.R. Sarasua, Hydrolytic degradation and bioactivity of lactide and caprolactone based sponge-like scaffolds loaded with bioactive glass particles, Polym. Degrad. Stab. 110 (2014) 121–128. [22] M. Obarzanek-Fojt, Y. Elbs-Glatz, E. Lizundia, L. Diener, J.-R. Sarasua, A. Bruinink, From implantation to degradation — are poly (L-lactide)/multiwall carbon nanotube composite materials really cytocompatible?, Nanomedicine 10 (2014) 1041–1051. [23] J.E. Bergsma, F.R. Rozema, R.R.M. Bos, G. Boering, W.C. de Bruijn, A.J. Pennings, In vivo degradation and biocompatibility study of in vitro pre-degraded as-polymerized polylactide particles, Biomaterials 16 (1995) 267–274. [24] J.C. Middleton, A.J. Tipton, Synthetic biodegradable polymers as orthopedic devices, Biomaterials 21 (2000) 2335–2346. [25] C. Liu, K.W. Chan, J. Shen, H.M. Wong, K.W. Kwok Yeung, S.C. Tjong, Melt-compounded polylactic acid composite hybrids with hydroxyapatite nanorods and silver nanoparticles: biodegradation, antibacterial ability, bioactivity and cytotoxicity, RSC Adv. 5 (2015) 72288–72299. [26] N. Kathuria, A. Tripathi, K.K. Kar, A. Kumar, Synthesis and characterization of elastic and macroporous chitosan-gelatin cryogels for tissue engineering, Acta Biomater. 5 (2009) 406–418. [27] A. Tripathi, N. Kathuria, A. Kumar, Elastic and macroporous agarose-gelatin cryogels with isotropic and anisotropic porosity for tissue engineering., J. Biomed. Mater. Res. A. 90 (2008) 680–694. [28] C. Sharma, A.K. Dinda, P.D. Potdar, C.F. Chou, N.C. Mishra, Fabrication and characterization of novel nano-biocomposite scaffold of chitosan-gelatin-alginate-hydroxyapatite for bone tissue engineering, Mater. Sci. Eng. C. 64 (2016) 416–427. [29] E. Lizundia, L. Ruiz-Rubio, L. Vilas J, M. León L, Towards the development of eco-friendly disposable polymers: ZnO-initiated thermal and hydrolytic degradation in Poly (L- lactide)/ZnO nanocomposites, RSC Adv. 6 (2016) 15660-15669. [30] M. Premanathan, K. Karthikeyan, K. Jeyasubramanian, G. Manivannan, Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation, Nanomedicine 7 (2011) 184–192. [31] D.R. Sambandan, D. Ratner, Sunscreens: An overview and update, J. Am. Acad. Dermatol. 64 (2011) 748– 758. [32] S. Trujillo, E. Lizundia, J.L. Vilas, M. Salmeron-Sanchez, PLLA/ZnO nanocomposites: dynamic surfaces to harness cell differentiation, Colloids Surf., B 144 (2016) 152-160. [33] P. Kanmani, J.W. Rhim, Properties and characterization of bionanocomposite films prepared with various biopolymers and ZnO nanoparticles, Carbohydr. Polym. 106 (2014) 190–199. [34] J. Del Rıó, A. Etxeberria, N.L. Opez-Rodrıuez, E. Lizundia, J.R. Sarasua, A PALS Contribution to the Supramolecular Structure of Poly(L-lactide), Macromolecules 43 (2010) 4698–4707. [35] J.-R. Sarasua, R.E. Prud’homme, M. Wisniewski, A. Le Borgne, N. Spassky, Crystallization and Melting Behavior of Polylactides, Macromolecules 31 (1998) 3895–3905. [36] E. Lizundia, A. Larrañaga, L. Vilas J, M. León L, Three-dimensional orientation of Poly(L-lactide) crystals under uniaxial drawing, RSC Adv. 6 (2016) 11943-11951. [37] E. Lizundia, L. Ruiz-Rubio, J. Luis Vilas, L. Manuel Leon, Poly(L-lactide)/ZnO nanocomposites as efficient UV-shielding coatings for packaging applications, J. Appl. Polym. Sci. 132 (2016) 42426. [38] R. Auras, B. Harte, S. Selke, An overview of polylactides as packaging materials, Macromol. Biosci. 4 (2004) 835–864. [39] E. Lizundia, A. Oleaga, A. Salazar, J.R. Sarasua, Nano- and microstructural effects on thermal properties of poly (l-lactide)/multi-wall carbon nanotube composites, Polymer 53 (2012) 2412–2421. [40] E. Lizundia, J.L. Vilas, L.M. León, Crystallization, structural relaxation and thermal degradation in Poly(llactide)/cellulose nanocrystal renewable nanocomposites, Carbohydr. Polym. 123 (2015) 256-265. [41] L. Lu, S.J. Peter, M.D. Lyman, H.L. Lai, S.M. Leite, J. A. Tamada, J.P. Vacanti, R. Langer, A.G. Mikos, In vitro degradation of porous poly(L-lactic acid) foams, Biomaterials 21 (2000) 1595–1605. [42] Y. Gong, Q. Zhou, C. Gao, J. Shen, In vitro and in vivo degradability and cytocompatibility of poly(l-lactic acid) scaffold fabricated by a gelatin particle leaching method, Acta Biomater. 3 (2007) 531–540. [43] E. Meaurio, I. Martinez De Arenaza, E. Lizundia, R. Sarasua J, Analysis of the C=O Stretching Band of the α-Crystal of Poly(L-lactide), Macromolecules 42 (2009) 5717–5727. [44] J. Puiggali, Y. Ikada, H. Tsuji, L. Cartier, T. Okihara, B. Lotz, The frustrated structure of poly(l-lactide), Polymer (Guildf). 41 (2000) 8921–8930. doi:10.1016/S0032-3861(00)00235-4. [45] E. Lizundia, S. Petisco, J.-R. Sarasua, Phase-structure and mechanical properties of isothermally melt-and cold-crystallized poly (L-lactide), J. Mech. Behav. Biomed. Mater. 17 (2012) 242–251. [46] H.L. Friedman, Kinetics of thermal degradation of char-forming plastics from thermogravimetry. Application to a phenolic plastic, J. Polym. Sci. Part C Polym. Symp. 6 (1964) 183–195. [47] E. Lizundia, E. Meaurio, J.M. Laza, J.L. Vilas, L.M. León Isidro, Study of the chain microstructure effects on the resulting thermal properties of poly(L-lactide)/poly(N-isopropylacrylamide) biomedical materials,

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Mater. Sci. Eng. C. 50 (2015) 97–106. [48] F. Kopinke, M. Remmler, K. Mackenzie, M. Milder, Thermal decomposition of biodegradable polyesters II. Poly (lactic acid), Polym. Degrad. Stab. 43 (1996) 329–342. [49] M. Partini, R. Pantani, FTIR analysis of hydrolysis in aliphatic polyesters, Polym. Degrad. Stab. 92 (2007) 1491–1497. [50] A.-M. Haaparanta, P. Uppstu, M. Hannula, V. Ellä, A. Rosling, M. Kellomäki, Improved dimensional stability with bioactive glass fibre skeleton in poly(lactide-co-glycolide) porous scaffolds for tissue engineering., Mater. Sci. Eng. C. Mater. Biol. Appl. 56 (2015) 457–66. [51] L. Wu, J. Ding, Effects of porosity and pore size on in vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering, J. Biomed. Mater. Res. - Part A. 75 (2005) 767–777. [52] I. Grizzi, H. Garreau, S. Li, M. Vert, Hydrolytic degradation of devices based on poly(DL-lactic acid) size dependence, Biomaterials. 16 (1995) 305–311.

ACCEPTED MANUSCRIPT Highlights Poly(L-lactide)/ZnO nanocomposite scaffolds are prepared

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The structural evolution of scaffolds during their in vitro degradation is studied

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The degradation profile of scaffolds could be successfully tailored

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The presence of ZnO initiate hydrolytic degradation reactions in PLLA matrix

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PLLA/ZnO is suitable for achieving porous structures with tuneable degradation rates

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