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Making microporous nanometre-scale fibrous PLA aerogels with clean and reliable supercritical CO2 based approaches
Microporous and Mesoporous Materials 184 (2014) 162–168
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Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Making microporous nanometre-scale fibrous PLA aerogels with clean and reliable supercritical CO2 based approaches Aurelio Salerno ⇑, Concepción Domingo Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus de la UAB s/n, Bellaterra 08193, Spain
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Article history: Received 23 July 2013 Received in revised form 12 October 2013 Accepted 16 October 2013 Available online 24 October 2013 Keywords: Aerogel Nanofibre Polylactic acid Pore structure Supercritical CO2
a b s t r a c t Polylactic acid (PLA) aerogels, with a multiscale structure consisting of nanometre-scale fibres and interconnected micropores, were here fabricated by a novel thermal induced phase separation (TIPS) approach. The developed process is based on a biocompatible route combining ethyl lactate (EL) as a non-toxic solvent for PLA and supercritical CO2 (scCO2) as a clean drying agent. First, PLA was dissolved in EL to prepare homogeneous solutions with a polymer concentration ranging from 3 to 5.5 wt%. Subsequently, TIPS was generated by the controlled decrease of the temperature down to a temperature lower than the solution gelation point. Finally, solvent exchange, alcogel formation and scCO2 drying allowed the manufacture of the desired nanometre-scale fibrous PLA aerogels. In particular, PLA aerogels with homogeneous morphology and constituted by an overall porosity in the range of 90–95% and a specific surface area in the range of 70–95 m2/g were manufactured by modulating polymer concentration in the starting EL solution, gelation temperature and EL extraction conditions. The obtained aerogels possessed a bimodal structure of fibres with a mean length of 100–200 nm coupled with nanopores of a mean diameter down to 2 nm. Finally, the combination of TIPS with gas foaming and porogen leaching techniques was explored as a suitable strategy to obtain multifunctional micro- and nano-sized fibrous PLA materials, suitable of providing biomimetic three-dimensional platforms for tissue engineering scaffolds. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction The pursuit of nanoscale porous aerogels characterised by a three dimensional fibre structure represents a new realm of matter of current research in functional materials design and fabrication. Porous aerogels with fibre diameter scales of the order of tens to few hundred nanometres possess unique transport, structural and biophysical properties for technological and biomedical applications [1–3]. Owning a high porosity and a large surface area, nanometre-scale fibrous materials offers outstanding properties in terms of the flexibility of surface functionalities and the control of transport properties [1]. Nanometre-scale fibres of various biocompatible materials have been deeply studied and are currently applied in tissue engineering as scaffolds for cell culture and tissue development [3,4]. Indeed, nanometre-scale fibrous scaffolds are able to mimic the collagen structure of the extracellular matrix, enhancing cell/material cross-talking at the interface and promoting cell adhesion, proliferation and differentiation [4]. However, the true potential to create functional nanometre-scale fibrous materials depends on the control of the fibres structure. In addition, for tissue engineering applications the materials should be prepared preferably by using non toxic large-scale ⇑ Corresponding author. Tel.: +34 935801853. E-mail address: [email protected] (A. Salerno). 1387-1811/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.micromeso.2013.10.019
manufacturing processes. Electrospinning, molecular self assembly and phase separation are common bottom-up nanofabrication techniques used to create three dimensional scaffolds composed of interwoven nanometre-scale fibres [4–9]. Phase separation is one of the most versatile approach designed to large-scale fabrication of nanofibrous materials with controlled morphology and structure [8,9]. Phase separation from a polymer–solvent solution is based on the thermodynamic de-mixing of the system into a polymer-rich and a polymer-poor phases, which can be caused by antisolvent addition, using either conventional liquids or supercritical fluids [10,11], or by cooling down the solution below a binodal solubility curve [8,9]. This last approach, named thermally induced phase separation (TIPS), allows for the large-scale formation of nanometre-scale fibrous structures with characteristic diameters in the 50–500 nm interval, controlled by selecting the appropriate polymer/solvent combination, the cooling temperature and the process kinetics [12]. Nanometre-scale fibrous polylactic acid (PLA) [12], polyhydroxyalkanoate (PHA) [13], chitosan [14] and gelatin [15] materials have been successfully manufactured by means of the TIPS technique. However, the solvent choice for polymers processing is still an opening question, as solvents are present not only in the production route, but also in the final product as a residue. In this work, the fabrication of nanometre-scale fibrous aerogels by using ethyl lactate (EL) to process PLA was investigated to overcome the limitations related to the use of toxic
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organic solvents, such as dioxane or tetrahydrofuran, for polymers processing. EL is a member of the lactate esters family. It does not show any potential health risk and it has been approved by FDA as additive in food products [16–20]. Therefore, the use of EL may allow for the green and sustainable large-scale fabrication of nanometre-scale fibrous PLA materials. Another critical step when preparing nanometre-scale fibrous aerogels by phase separation is the drying of the gel. In general, a polymeric gel formed upon drying in air provides a white and dense collapsed xerogel, in which the fibre network does not retain the original three-dimensional structure [21]. Supercritical CO2 (scCO2) solvent extraction is a process that allows the drying of the gel through the formation of a supercritical mixture of the CO2 and the liquid solvent, which is typically ethanol. The supercritical mixture shows no surface tension and can be easily eliminated in a single step by venting the vessel [21,22]. Herein, we report a comprehensive study of a novel and clean approach, based on the combination of TIPS route for phase separation and scCO2 for drying, for the design and fabrication of nanometre-scale fibrous PLA aerogels with controlled morphology and structural properties. The effect of important operating conditions, such as polymer concentration in the initial solution, gelation temperature and solvent choice for EL exchange, was investigated in terms of produced aerogels morphology, porosity, specific surface area, pore size distribution, mean fibre diameter and fibre diameter size distribution. 2. Experimental 2.1. Materials PLA with an 80/20 L/DL ratio, a molecular weight of 200 kDa and an inherent viscosity at midpoint of 3.8 DL/g, was provided by Purac Biochem (Gorinchem, The Netherlands). EL (photoresist grade; purity P 99.0%) and ethanol (99.5%) were provided by Sigma–Aldrich (Madrid, Spain) and used without further purification. Gelatin particles (Merck, Darmstadt, Germany) were used as a particulate porogen. CO2 (99.95 w%, Carburos Metálicos) was used as the drying agent.
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procedure. EL and water were completely miscible at the temperature used to prepare the polymeric solution. In the step (2), aA definite amount of the starting solutions was added to moulds prepared by folding an aluminium foil and transferred into a thermostatic bath (D8, Haake, Karlsruhe, Germany), pre-cooled to a temperature of either 15, 0 or 15 °C, for gelation during 3 h. In the step (3), gelled samples were removed from the mould by unfolding the aluminium foil and immersed into either distilled water or ethanol for solvent exchange. This step allows extracting the EL leading to polymer precipitation and aerogel structure stabilisation. The EL extraction was carried out by two different ways. In the first approach, the gels were soaked in excess of water and the medium changed 5 times up to the almost complete elimination of the EL. The obtained hydrogels were then soaked in ethanol to prepare PLA alcogels. In the second approach, the gels were directly immersed in ethanol to remove the EL. (4) In both cases, obtained alcogel samples were finally dried using scCO2. No evidence of significant shrinking was observed for the samples after the gelation and drying steps. Drying was carried out in a high-pressure autoclave (TharDesign, Pittsburgh, USA) with a cylindrical section and a total volume of 114 mL. The autoclave is provided of two sapphire windows, which enable the on-time visual monitoring of the process, and 4 electric resistances placed inside the wall of the vessel, which ensure a homogeneous distribution of the temperature inside the chamber. Samples were placed inside of the autoclave on the top of a metallic support to allow for the addition of a magnetic stirrer at the bottom of the vessel. This set-up improved fluids mixing and reduced equilibrium time. After the reactor was charged with the gelled sample and sealed, the temperature was raised up to 39 °C. Liquid CO2 was subsequently pumped inside the vessel to raise the pressure up to 19 MPa, thus, ensuring the achievement of supercritical conditions. The operative temperature was selected to avoid crossing PLA glass transition temperature. These pressure/temperature conditions ensured the supercritical/gaseous state for the used scCO2/ ethanol mixture, without passing through the liquid state. Samples
2.2. Measurement of the cloud point and the gelation point curves The cloud-point for binary PLA/EL solutions was assessed by turbidimetry. PLA was dissolved in EL at 70 °C under magnetic stirring for 8 h in order to prepare a homogeneous solution. The concentration of PLA in EL was selected in the 3–5.5 wt% range, based on the study of the viscosity-concentration curve performed by Levato et al. on a similar system [23]. Subsequently, 1 mL of this solution was sealed in a 2 mL glass tube and placed in a water bath pre-heated to a temperature ca. 10 °C higher than the expected cloud-point. The cloud-point temperature, defined as the temperature at which the clear solution became turbid, was visually determined by slowly cooling the solution in steps of 1 °C giving an equilibrium time of 10 min. The samples were then further cooled again in steps of 1 °C to achieve the gelation point, defined as the temperature at which the solution ceased to flow. The gelation point was determined by inverting the vial vertically? 2.3. Nanometre-scale fibrous PLA aerogels fabrication via TIPS/scCO2 drying combined processes The nanometre-scale fibrous aerogels were manufactured in four steps (Fig. 1). In the step (1), PLA/EL solutions, with a polymer concentration in the 3–5.5 wt% range, were prepared at 70 °C under magnetic stirring for 8 h. Moreover, a 3 wt% PLA solution in a 95/5 v% of EL/water mixture was prepared following the same
Fig. 1. Scheme of the TIPS and scCO2 drying combined process used for the design and fabrication of nanoscale fibrous PLA aerogels: (A) PLA solutions with a polymer concentration in the 3–5.5 wt% range were prepared at 70 °C during 8 h under magnetic stirring; (B) TIPS was induced by cooling the solution down to 15, 0 or 15 °C; (C) EL was extracted by soaking the samples in water or ethanol; (D) the materials were further soaked in ethanol and dryied with scCO2 at 19 MPa, 39 °C for 1.5 h.
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were held at these conditions for 1.5 h. Finally, the vessel was depressurised, lasting the venting ca. 1 h. In order to exploit the possibility of further improving the control of aerogels morphology and structure, the combination of TIPS with either gas foaming (GF) and porogen leaching (PL) techniques was assessed following two different approaches. In the first approach, in the step (4) the pressure was slowly released to an intermediate pressure of 6 MPa. Subsequently, the vessel was vented very fast (5 s) to induce foaming. In the second approach, in the step (1) the polymeric solution was mixed with gelatin particles in a 1.2 v/wt%. The mixing was performed manually mixing with the aid of a spatula. The obtained mixture was placed in a mould and maintained at 0 °C for 3 h for gelation and, subsequently, the EL was extracted by soaking in ethanol. The obtained material was further soaked in water at 40 °C for 2 days to selectively extract the gelatin particles. The final PLA product was obtained by exchanging the water with ethanol and drying the alcogel following the previously described protocol in step (4). Prepared PLA aerogels evidenced excellent stability in air and no additional treatment was necessary to stabilise them before characterisation.
CPA225D, Sartorius, Gottingen, Germany), while the volume was determined by geometrical calculation using a high precision calliper. The BET specific surface area of the aerogels was determined by N2 adsorption–desorption measurements at 196 °C using an ASAP 2000 Micromeritics Inc. Instrument (Aachen, Germany). Prior to measurements, samples were dried at 35 °C under a reduced pressure for 3 days. The mean fibres diameter and diameter size distribution were assessed by Image analysis (Image JÒ). A minimum of one hundred fibres was analysed for each sample. 3. Results and discussion 3.1. Phase diagram of PLA/EL solution The TIPS technique is based on the thermodynamic separation of a homogeneous polymer solution into a polymer-rich and a polymer-poor phases as a consequence of a temperature variation.
2.4. Aerogels characterisation Differential scanning calorimetric (DSC) analysis was used to assess the thermal properties of the aerogels. The samples were tested on a DSC8500 apparatus (Perkin Elmer, Massachusetts, USA) equipped with a liquid N2 controller CRYOFILL. Samples were first equilibrated at 20 °C for 5 min and then heated up to 200 °C at a scanning rate of 10 °C/min under inert atmosphere. The morphology of the aerogels was assessed by scanning electron microscopy (SEM). Samples were cross-sectioned and gold sputtered prior to be analysed by SEM (QUANTA 200F FEG-ESEM, FEI, The Netherlands). The porosity of the aerogels was determined by geometrical calculation from the mass and volume measurements [24]. The mass was measured by using a high precision balance (10 4 g,
Fig. 2. Cloud point and gelation point of PLA/EL solution as a function of polymer concentration.
Fig. 3. Low and high magnification SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels as a function of polymer concentration in the initial solution: (A, B) 3 and (C, D) 5.5 w%. Samples were obtained at 0 °C gelation temperature and by extracting the EL in water.
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To optimise the experimental protocol necessary to design the desired nanometre-scale fibrous materials, it is important to determine the phase diagram of the starting polymeric solution by assessing its cloud and gelation points. The cloud point and gelation temperatures of solutions with different concentrations of PLA in EL were measured and are presented in Fig. 2. The temperature at which both thermal transitions occurred increased with the increase of PLA concentration in the starting solution [25]. The gelation curve was about 10 °C lower than that of the cloud point curve. In particular, the gelation and cloud temperatures increased from 24 and 34 °C up to 33 and 40 °C when the PLA concentration was increased from 3 to 5.5 wt%, respectively. The observed results are in agreement to those reported for binary solutions of PLA in tetrahydrofuran [25] or in dioxane/water binary solvent [26]. 3.2. Properties of nanometre-scale fibrous PLA aerogels prepared via TIPS A series of porous nanometre-scale fibrous PLA aerogels was fabricated by varying the composition of the polymeric solution, the gelation temperature and the extraction medium. Fig. 3 shows the SEM micrographs of samples prepared at 0 °C gelation temperature and by extracting the EL in water as a function of the concentration of polymer in solution. The aerogels were characterised by a fibrous structure, with fibre diameters in the submicron range, and high porosity. Furthermore, the low magnification SEM images of Fig. 3A–D indicated that fibres density significantly increased with the increase of the concentration of PLA in the starting solution. Fig. 4 reports the porosity, specific surface area, pore size distribution and mean fibre diameter of the PLA samples in Fig. 3. As shown in Fig. 4A, the aerogels were characterised by porosity degrees higher than 90%. The porosity progressively decreased from 95.2 ± 0.5% to 90.3 ± 1.0% by increasing the PLA concentration in the starting solution from 3 to 5.5 wt%. The opposite trend was observed for the specific surface area, which increased from 69.4 ± 1.3 to 94.9 ± 0.5 m2/g when the concentration of PLA increased from 3 to 5.5 wt%. These results were in accordance with the information supplied by the images in Fig. 3 and demonstrated the possibility of controlling the density of the fibrous network by modulating polymer concentration in the starting solution. The pore volume measured using the adsorption data in the BET analysis was in the order of 0.15 cm3/g. The pore volume distribution plot showed in Fig. 4B shows that the three dimensional fibrous network of the prepared samples had both meso and macropores. The pore volume fraction corresponding to pores of 2 nm mean diameter increased progressively from 0.044 up to 0.055 cm3/g by rising the PLA concentration from 3 to 5.5 wt%. The pore volume fraction corresponding to pores of 10–100 nm mean diameter also increased with the increase of PLA concentration (Fig. 4). This nanometre-scale porosity is coupled concordant to the macroscopic porosity observed in the SEM images of Fig. 3 formed by randomly oriented pores passing throughout the entire sample volume. The average fibres diameter vs. PLA concentration is represented in Fig. 4C. The fibres diameter was in the range of 25–400 nm. The mean values slightly decreased from 196 ± 101 to 122 ± 63 nm by increasing PLA solution concentration from 3 to 5.5 wt%. Although the decrease of the average diameter of PLA fibres with the increase of polymer concentration could not be considered as statistically significant, this still observed effect can be ascribable to the effect of polymer concentration on the phase separation and polymer crystallisation processes. In particular, it was reported that, depending on the initial polymer concentration, the crystallisation of such polymers from solution can lead to different morphologies [9]. For dilute solutions, the formation of chain folded lamellae is usually observed. Conversely, at high polymer
Fig. 4. (A) Porosity and specific surface; (B) pore volume vs pore diameter plot and (C) mean fibres diameter of PLA aerogels as a function of polymer concentration in the initial solution. Samples were obtained at 0 °C gelation temperature and by extracting the EL in water.
concentration, suspensions of supramolecular architectures of these lamellae, such as axialites and spherulites, are obtained The values obtained in this work of average porosity and surface area are significantly higher than those reported for PLA scaffolds prepared by the standard phase separation approach (ca. 1 m2/g) and in accordance to those detailed by Chen and Ma for PLA aerogels prepared from a polymer solution in a dioxane/water mixture [27]. Further experiments were carried out in order to assess the effect of the used gelation temperature and the extraction medium on the morphology, mean fibres diameter and diameter size distribution of PLA aerogels prepared by TIPS. In particular, we investigated the effect of two alternatives gelation temperatures, 15 and 15 °C, as well as two different EL extraction media, water and ethanol, on samples morphology and the fibrous structure. The selected gelation temperature was higher than the EL freezing temperature, which is 26 °C, therefore ensuring that phase separation occurred without solvent crystallisation. Regarding the extraction
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Fig. 5. SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels as a function of polymer concentration in the initial solution, gelation temperature and extraction solvent: (A, C, E, G) 3 w% and (B, D, F, H) 5 w% PLA; (A, B, E, F) 15 °C and (C, D, G, H) 15 °C gelation temperature; (A–D) water and (E–H) ethanol extraction media. (K) The mean fibres diameter is also showed for each sample.
medium, in the first approach the gels were soaked in water to extract the EL and the obtained hydrogels were further dehydrated by soaking in ethanol to obtain alcogels suitable for sCO2 drying. In the second approach, the gels were directly soaked in ethanol to extract the EL and to obtain the alcogel and further dried by means of scCO2. The results of these tests are reported in Fig. 5. As shown in Fig. 5A–H, nanoscale fibrous PLA aerogels were also achieved at the gelation temperatures of 15 and 15 °C and by performing extraction in either water or ethanol. In particular, as previously observed for the samples prepared at 0 °C, the porosity and fibres diameter decreased with the increase of polymer concentration in the starting solution from 3 to 5.5 w% (Fig. 5K) and, concomitantly, the fibre diameter size distribution shifted to lower values with the increase of PLA concentration (Fig. 5I and L). The evolution of the structure and nanometre-scale properties of PLA fibres prepared by the TIPS process strongly depends on the route of polymer crystallisation during solution gelation [12]. It has been recently demonstrated that nanofibres formation via TIPS involves the initial condensation of amorphous nanoparticles with a gradual crystallisation from the gel. Extending the gelation step, a porous structure, characterised by an interconnected network of fibres, is formed. The final morphological, structural and thermal properties of the samples are temperature dependent in regard of the initial quenching period [12]. Table 1 reports the glass transition temperature (Tg), melting peak temperature (Tp), melting temperature range (DTp) and degree of crystallinity (vC) of PLA samples prepared in this work by the TIPS method as a function of polymer concentration and gelation temperature. For the prepared fibrous PLA, the thermal properties were not significantly affected by the different operating conditions investigated. For all of the precipitated samples, Tg values close to 66–67 °C were measured. Furthermore, vC and Tp values were in the order of 22–23% and 135–136 °C, respectively. These values are in accordance with those reported by other works for PLA membranes prepared by means of the TIPS process and using different solvents [12,28]. The use of binary mixtures of polymer solvent may be also a powerful approach to improve the control over the morphological and structural properties of PLA scaffolds prepared via TIPS technique. Phase separation for binary solvent mixtures depends not only of the temperature drop, but it is also strongly related to the effect of the composition of the binary solvent mixture on solution gelation and polymer crystallisation. For instance, solvent mixtures with a relatively high freezing point would crystallise and freeze when quenching the system to a low temperature. As a direct consequence, multi-scaled nanofibrous materials characterised by the
Table 1 Effect of concentration of polymer and gelation temperature on the thermal properties of nanoscale fibrous PLA prepared by TIPS and scCO2 drying. Sample
Tg (°C)
Tp (°C)
DTp (°C)
vC (%)
3% PLA/15 °C 4% PLA/15 °C 5% PLA/15 °C 5.5% PLA/15 °C 3% PLA/ 15 °C 5.5% PLA/ 15 °C
66.5 66.4 66.3 66.2 66.7 66.8
136.1 135.9 136.0 136.5 135.1 136.0
116–142 114–143 115–142 115–144 114–141 115–143
22.9 23.2 23.1 23.2 23.2 22.2
presence of larger pores can be fabricated for application such as tissue engineering scaffolds [29]. In this work, the possibility to fabricate nanometre-scale fibrous PLA aerogels has been further explored by adding water to the initial polymer solution as the antisolvent. In Fig. 6, the morphology and porosity of PLA aerogels obtained from a 3 wt% polymer solution in a 95/5 v/v% EL/water mixture are reported as a function of the gelation temperature. PLA polymeric networks, with a homogeneous distribution of uniform nanometre-scale fibres, were achieved at the gelation temperature of 15 °C (Fig. 6A). Conversely, when the gelation temperature decreased down to 0 or 15 °C, the miscibility of EL and water decreased and ice crystals could precipitate out from the solution during the phase separation step. This effect could be responsible for the formation of a porous structure characterised by large pores and an irregular fibrous structure (Fig. 6B, C). The same effect could explain the porosity trend observed in Fig. 6D as a function of the gelation temperature, because the formation of ice crystals could increase the porosity of the samples prepared at 15 °C. The porosity data reported in Fig. 6D as a function of the gelation temperature indicated that samples porosity slightly decreased from 94.3 ± 1.1% at 15 °C to 92.4 ± 0.6% at 0 °C, and subsequently increased up to 94.0 ± 0.8% at 15 °C. 3.3. Nanometre-scale fibrous PLA aerogels prepared via TIPS combined with GF or PL techniques The possibility to design and manufacture porous PLA aerogels characterised by multi-scaled pore structures is of great importance for applications related to tissue engineering, were scaffolds with pores larger than 100 lm are necessaries to allow for the three dimensional adhesion and colonisation with cells, as well as for new tissue development in vitro and in vivo [24,30].
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Fig. 6. SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels prepared starting from a 3 w% polymer in a 95/5 v/v EL/water solution and gelified at (A) 15, (B) 0 and (C) 15 °C. (D) Porosity of nanoscale fibrous PLA aerogels prepared starting from a 3 w% solution of polymer in a 95/5 v/v EL/water as a function of the gelation temperature.
Fig. 7. (A, B) SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels prepared by combining the TIPS method with the GF process. (C) Porosity of nanoscale fibrous PLA aerogels prepared by combining TIPS and GF techniques as a function of polymer concentration in the starting solution. Samples were obtained at 0 °C gelation temperature and by extracting EL in water.
Fig. 8. SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels prepared by combining the TIPS method with the PL process. Samples were obtained at 0 °C gelation temperature and by extracting EL in water.
Therefore, in the last part of this study the developed TIPS method has been improved in this regard by combining it with either GF or PL processes. A two-step depressurization approach was developed
in the case of the TIPS/GF combined process, aiming to concomitantly achieve gel drying and foaming, avoiding the use of an additional processing step. In Fig. 7A–C the morphology and the
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porosity of the TIPS/GF prepared PLA samples is reported as a function of polymer concentration in the initial solution. The PLA samples were characterised by a nanometre-scale fibrous morphology, while there was no evidence of multi-scaled pore structures (Fig. 7A, B). The evaluation of the porosity demonstrated that the combined approach was able to increase the overall porosity of PLA by 0.5% in the case of using a 3 wt% PLA polymer solution and by 1.9% for a 5.5 wt% solution. This result indicated that the method involving two depressurisation steps, characterised by a final fast depressurisation during vessel venting, induced partial foaming, but did not allow for the formation of the large pores required for tissue engineering. To overcome the previous limitation, micrometric gelatin particles were used in a combined TIPS/PL process. The gelatin particles acted as a templating agent for large pores formation. The morphological characterisation of obtained PLA sample prepared from a 5 wt% solution of PLA in EL and gelatin particles is shown in Fig. 8A–C. The addition of the micrometric particulate porogen effectively allowed for the fabrication of multi-scaled porous PLA aerogels with large pores, in the order of 300 lm, replicating the size of the starting gelatin particles. Furthermore, by optimising the TIPS steps, it was possible to induce high pore interconnectivity (Fig. 8B) as well as to recreate a nanometre-scale fibrous architecture on the pore walls (Fig. 8C). Previous works have shown that a similar pore structure is very helpful to improve the biological response of PLA scaffolds [29]. The as prepared PLA multi-scaled porous scaffolds were characterised by a 95% overall porosity, which is an optimal value for several tissue engineering applications. 4. Conclusions In this work, a clean and sustainable approach to manufacture multimodal nanometre-scale fibrous PLA aerogels with controlled morphology and architecture is reported. The aerogels were fabricated by means of TIPS process by using the green solvents EL and scCO2 to dissolve the polymer and to dry the aerogel, respectively. By controlling the concentration of polymer in solution, the gelation temperature and the extraction media, nanometrescale fibrous PLA aerogels, with homogeneous distribution of fibres and controlled structural features, were obtained. In particular, by increasing polymer concentration from 3 to 5.5 wt%, the porosity was modulated from 90% to 95% and the specific surface area from 70 to 95 m2/g, with a mean fibres diameter in the range of 100–200 nm. Finally, the combination of TIPS with either GF or PL processing techniques was explored to improve the control on pore structure features and to fabricate multi-scaled porous PLA aerogels with potential applications in tissue engineering as scaffolds.
Acknowledgements The authors gratefully acknowledge Julio Fraile for his support during the experimental activity. Aurelio Salerno gratefully acknowledges the CSIC for the financial support through a JAE-DOC contract cofinancied by the FSE. The authors also gratefully acknowledge the financial support of the Ministerio de Economía y Competitividad through the research project BIOREG (MAT2012-35161) and POLREMED (MAT2010-18155). References [1] Z. Huang, Y.-Z. Zhang, M. Kotaki, S. Ramakrishna, Comp. Sci. Tech. 63 (2003) 2223–2253. [2] G. Hayase, K. Kanamori, K. Nakanishi, Micropor. Mesopor. Mater. 158 (2012) 247–252. [3] Y. Zhang, C.T. Lim, S. Ramakrishna, Z. Huang, J. Mater. Sci. Mater. Med. 16 (2005) 933–946. [4] T. Dvir, B.P. Timko, D.S. Kohane, R. Langer, Nat. Nanotech. 6 (2011) 13–22. [5] H. Li, J.D. Carter, T.H. LaBean, Mater. Today 12 (2009) 24–32. [6] K.H. Smith, E. Tejeda-Montes, M. Poch, A. Mata, Chem. Soc. Rev. 40 (2011) 4563–4577. [7] V. Guarino, V. Cirillo, P. Taddei, M.A. Alvarez-Perez, L. Ambrosio, Macromol. Biosci. 11 (2011) 1694–1705. [8] H. Tanaka, Adv. Mater. 21 (2009) 1872–1880. [9] P. van de Witte, P.J. Dijkstra, J.W.A. van den Berg, J. Feijen, J. Membr. Sci. 117 (1996) 1–31. [10] A. Vega-Gonzalez, P. Subra-Paternault, A.M. Lopez-Periago, C.A. GarciaGonzalez, C. Domingo, Eur. Poly. J. 44 (2008) 1081–1094. [11] C.A. Garcia-Gonzalez, A. Vega-Gonzalez, A.M. Lopez-Periago, P. SubraPaternault, C. Domingo, Acta Biomater. 5 (2009) 1094–1103. [12] J. Shao, C. Chen, Y. Wang, X. Chen, C. Du, Reac. Func. Polym. 72 (2012) 765–772. [13] X. Li, Y. Zhang, G. Chen, Biomaterials 29 (2008) 3720–3728. [14] J. Zhao, W. Han, H. Chen, M. Tu, R. Zeng, Y. Shi, Z. Cha, C. Zhou, Carbohydr. Polym. 83 (2011) 1541–1546. [15] X. Liu, P.X. Ma, Biomaterials 30 (2009) 4094–4103. [16] S. Aparicio, L. Alcalde, Green Chem. 11 (2009) 65–78. [17] C.S.M. Pereira, V.M.T.M. Silva, A.E. Rodrigues, Green Chem. 13 (2011) 2658– 2671. [18] A. Salerno, R. Levato, M.A. Mateos-Timoneda, E. Engel, P.A. Netti, J.A. Planell, J. Biomed. Mater. Res. Part A 101A (2013) 720–732. [19] X. Ye, C.M. Wai, J. Chem. Ed. 80 (2003) 198. [20] A. Salerno, C. Domingo, RSC Adv. 3 (2013) 17355. [21] F. Placin, J.P. Desvergne, F. Cansell, J. Mater. Chem. 10 (2000) 2147–2149. [22] E. Reverchon, S. Cardea, C. Rapuano, J. Supercr. Fluids 45 (2008) 365–373. [23] R. Levato, M. Mateos-Timoneda, J.A. Planell, Macromol. Biosci. 12 (2012) 557. [24] A. Salerno, S. Zeppetelli, E. Di Maio, S. Iannace, P.A. Netti, Biotech. Bioeng. 108 (2011) 963–976. [25] F. Yang, R. Murugan, S. Ramakrishna, X. Wang, Y.-X. Ma, Biomaterials 25 (2004) 1891–1900. [26] L. Budyanto, Y.Q. Goh, C.P. Ooi, J. Mater. Sci. Mater. Med. 20 (2009) 105–111. [27] V.J. Chen, P.X. Ma, Biomaterials 27 (2006) 3708–3715. [28] P.X. Ma, R. Zhang, J. Biomed. Mater. Res. Part A 46 (1999) 60. [29] L. He, Y. Zhang, X. Zeng, D. Quan, S. Liao, Y. Zeng, J. Lu, S. Ramakrishna, Polymer 50 (2009) 4128–4138. [30] A. Salerno, S. Zeppetelli, E. Di Maio, S. Iannace, P.A. Netti, Macromol. Rapid Comm. 32 (2011) 1150–1156.
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Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Making microporous nanometre-scale fibrous PLA aerogels with clean and reliable supercritical CO2 based approaches Aurelio Salerno ⇑, Concepción Domingo Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus de la UAB s/n, Bellaterra 08193, Spain
a r t i c l e
i n f o
Article history: Received 23 July 2013 Received in revised form 12 October 2013 Accepted 16 October 2013 Available online 24 October 2013 Keywords: Aerogel Nanofibre Polylactic acid Pore structure Supercritical CO2
a b s t r a c t Polylactic acid (PLA) aerogels, with a multiscale structure consisting of nanometre-scale fibres and interconnected micropores, were here fabricated by a novel thermal induced phase separation (TIPS) approach. The developed process is based on a biocompatible route combining ethyl lactate (EL) as a non-toxic solvent for PLA and supercritical CO2 (scCO2) as a clean drying agent. First, PLA was dissolved in EL to prepare homogeneous solutions with a polymer concentration ranging from 3 to 5.5 wt%. Subsequently, TIPS was generated by the controlled decrease of the temperature down to a temperature lower than the solution gelation point. Finally, solvent exchange, alcogel formation and scCO2 drying allowed the manufacture of the desired nanometre-scale fibrous PLA aerogels. In particular, PLA aerogels with homogeneous morphology and constituted by an overall porosity in the range of 90–95% and a specific surface area in the range of 70–95 m2/g were manufactured by modulating polymer concentration in the starting EL solution, gelation temperature and EL extraction conditions. The obtained aerogels possessed a bimodal structure of fibres with a mean length of 100–200 nm coupled with nanopores of a mean diameter down to 2 nm. Finally, the combination of TIPS with gas foaming and porogen leaching techniques was explored as a suitable strategy to obtain multifunctional micro- and nano-sized fibrous PLA materials, suitable of providing biomimetic three-dimensional platforms for tissue engineering scaffolds. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction The pursuit of nanoscale porous aerogels characterised by a three dimensional fibre structure represents a new realm of matter of current research in functional materials design and fabrication. Porous aerogels with fibre diameter scales of the order of tens to few hundred nanometres possess unique transport, structural and biophysical properties for technological and biomedical applications [1–3]. Owning a high porosity and a large surface area, nanometre-scale fibrous materials offers outstanding properties in terms of the flexibility of surface functionalities and the control of transport properties [1]. Nanometre-scale fibres of various biocompatible materials have been deeply studied and are currently applied in tissue engineering as scaffolds for cell culture and tissue development [3,4]. Indeed, nanometre-scale fibrous scaffolds are able to mimic the collagen structure of the extracellular matrix, enhancing cell/material cross-talking at the interface and promoting cell adhesion, proliferation and differentiation [4]. However, the true potential to create functional nanometre-scale fibrous materials depends on the control of the fibres structure. In addition, for tissue engineering applications the materials should be prepared preferably by using non toxic large-scale ⇑ Corresponding author. Tel.: +34 935801853. E-mail address: [email protected] (A. Salerno). 1387-1811/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.micromeso.2013.10.019
manufacturing processes. Electrospinning, molecular self assembly and phase separation are common bottom-up nanofabrication techniques used to create three dimensional scaffolds composed of interwoven nanometre-scale fibres [4–9]. Phase separation is one of the most versatile approach designed to large-scale fabrication of nanofibrous materials with controlled morphology and structure [8,9]. Phase separation from a polymer–solvent solution is based on the thermodynamic de-mixing of the system into a polymer-rich and a polymer-poor phases, which can be caused by antisolvent addition, using either conventional liquids or supercritical fluids [10,11], or by cooling down the solution below a binodal solubility curve [8,9]. This last approach, named thermally induced phase separation (TIPS), allows for the large-scale formation of nanometre-scale fibrous structures with characteristic diameters in the 50–500 nm interval, controlled by selecting the appropriate polymer/solvent combination, the cooling temperature and the process kinetics [12]. Nanometre-scale fibrous polylactic acid (PLA) [12], polyhydroxyalkanoate (PHA) [13], chitosan [14] and gelatin [15] materials have been successfully manufactured by means of the TIPS technique. However, the solvent choice for polymers processing is still an opening question, as solvents are present not only in the production route, but also in the final product as a residue. In this work, the fabrication of nanometre-scale fibrous aerogels by using ethyl lactate (EL) to process PLA was investigated to overcome the limitations related to the use of toxic
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organic solvents, such as dioxane or tetrahydrofuran, for polymers processing. EL is a member of the lactate esters family. It does not show any potential health risk and it has been approved by FDA as additive in food products [16–20]. Therefore, the use of EL may allow for the green and sustainable large-scale fabrication of nanometre-scale fibrous PLA materials. Another critical step when preparing nanometre-scale fibrous aerogels by phase separation is the drying of the gel. In general, a polymeric gel formed upon drying in air provides a white and dense collapsed xerogel, in which the fibre network does not retain the original three-dimensional structure [21]. Supercritical CO2 (scCO2) solvent extraction is a process that allows the drying of the gel through the formation of a supercritical mixture of the CO2 and the liquid solvent, which is typically ethanol. The supercritical mixture shows no surface tension and can be easily eliminated in a single step by venting the vessel [21,22]. Herein, we report a comprehensive study of a novel and clean approach, based on the combination of TIPS route for phase separation and scCO2 for drying, for the design and fabrication of nanometre-scale fibrous PLA aerogels with controlled morphology and structural properties. The effect of important operating conditions, such as polymer concentration in the initial solution, gelation temperature and solvent choice for EL exchange, was investigated in terms of produced aerogels morphology, porosity, specific surface area, pore size distribution, mean fibre diameter and fibre diameter size distribution. 2. Experimental 2.1. Materials PLA with an 80/20 L/DL ratio, a molecular weight of 200 kDa and an inherent viscosity at midpoint of 3.8 DL/g, was provided by Purac Biochem (Gorinchem, The Netherlands). EL (photoresist grade; purity P 99.0%) and ethanol (99.5%) were provided by Sigma–Aldrich (Madrid, Spain) and used without further purification. Gelatin particles (Merck, Darmstadt, Germany) were used as a particulate porogen. CO2 (99.95 w%, Carburos Metálicos) was used as the drying agent.
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procedure. EL and water were completely miscible at the temperature used to prepare the polymeric solution. In the step (2), aA definite amount of the starting solutions was added to moulds prepared by folding an aluminium foil and transferred into a thermostatic bath (D8, Haake, Karlsruhe, Germany), pre-cooled to a temperature of either 15, 0 or 15 °C, for gelation during 3 h. In the step (3), gelled samples were removed from the mould by unfolding the aluminium foil and immersed into either distilled water or ethanol for solvent exchange. This step allows extracting the EL leading to polymer precipitation and aerogel structure stabilisation. The EL extraction was carried out by two different ways. In the first approach, the gels were soaked in excess of water and the medium changed 5 times up to the almost complete elimination of the EL. The obtained hydrogels were then soaked in ethanol to prepare PLA alcogels. In the second approach, the gels were directly immersed in ethanol to remove the EL. (4) In both cases, obtained alcogel samples were finally dried using scCO2. No evidence of significant shrinking was observed for the samples after the gelation and drying steps. Drying was carried out in a high-pressure autoclave (TharDesign, Pittsburgh, USA) with a cylindrical section and a total volume of 114 mL. The autoclave is provided of two sapphire windows, which enable the on-time visual monitoring of the process, and 4 electric resistances placed inside the wall of the vessel, which ensure a homogeneous distribution of the temperature inside the chamber. Samples were placed inside of the autoclave on the top of a metallic support to allow for the addition of a magnetic stirrer at the bottom of the vessel. This set-up improved fluids mixing and reduced equilibrium time. After the reactor was charged with the gelled sample and sealed, the temperature was raised up to 39 °C. Liquid CO2 was subsequently pumped inside the vessel to raise the pressure up to 19 MPa, thus, ensuring the achievement of supercritical conditions. The operative temperature was selected to avoid crossing PLA glass transition temperature. These pressure/temperature conditions ensured the supercritical/gaseous state for the used scCO2/ ethanol mixture, without passing through the liquid state. Samples
2.2. Measurement of the cloud point and the gelation point curves The cloud-point for binary PLA/EL solutions was assessed by turbidimetry. PLA was dissolved in EL at 70 °C under magnetic stirring for 8 h in order to prepare a homogeneous solution. The concentration of PLA in EL was selected in the 3–5.5 wt% range, based on the study of the viscosity-concentration curve performed by Levato et al. on a similar system [23]. Subsequently, 1 mL of this solution was sealed in a 2 mL glass tube and placed in a water bath pre-heated to a temperature ca. 10 °C higher than the expected cloud-point. The cloud-point temperature, defined as the temperature at which the clear solution became turbid, was visually determined by slowly cooling the solution in steps of 1 °C giving an equilibrium time of 10 min. The samples were then further cooled again in steps of 1 °C to achieve the gelation point, defined as the temperature at which the solution ceased to flow. The gelation point was determined by inverting the vial vertically? 2.3. Nanometre-scale fibrous PLA aerogels fabrication via TIPS/scCO2 drying combined processes The nanometre-scale fibrous aerogels were manufactured in four steps (Fig. 1). In the step (1), PLA/EL solutions, with a polymer concentration in the 3–5.5 wt% range, were prepared at 70 °C under magnetic stirring for 8 h. Moreover, a 3 wt% PLA solution in a 95/5 v% of EL/water mixture was prepared following the same
Fig. 1. Scheme of the TIPS and scCO2 drying combined process used for the design and fabrication of nanoscale fibrous PLA aerogels: (A) PLA solutions with a polymer concentration in the 3–5.5 wt% range were prepared at 70 °C during 8 h under magnetic stirring; (B) TIPS was induced by cooling the solution down to 15, 0 or 15 °C; (C) EL was extracted by soaking the samples in water or ethanol; (D) the materials were further soaked in ethanol and dryied with scCO2 at 19 MPa, 39 °C for 1.5 h.
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were held at these conditions for 1.5 h. Finally, the vessel was depressurised, lasting the venting ca. 1 h. In order to exploit the possibility of further improving the control of aerogels morphology and structure, the combination of TIPS with either gas foaming (GF) and porogen leaching (PL) techniques was assessed following two different approaches. In the first approach, in the step (4) the pressure was slowly released to an intermediate pressure of 6 MPa. Subsequently, the vessel was vented very fast (5 s) to induce foaming. In the second approach, in the step (1) the polymeric solution was mixed with gelatin particles in a 1.2 v/wt%. The mixing was performed manually mixing with the aid of a spatula. The obtained mixture was placed in a mould and maintained at 0 °C for 3 h for gelation and, subsequently, the EL was extracted by soaking in ethanol. The obtained material was further soaked in water at 40 °C for 2 days to selectively extract the gelatin particles. The final PLA product was obtained by exchanging the water with ethanol and drying the alcogel following the previously described protocol in step (4). Prepared PLA aerogels evidenced excellent stability in air and no additional treatment was necessary to stabilise them before characterisation.
CPA225D, Sartorius, Gottingen, Germany), while the volume was determined by geometrical calculation using a high precision calliper. The BET specific surface area of the aerogels was determined by N2 adsorption–desorption measurements at 196 °C using an ASAP 2000 Micromeritics Inc. Instrument (Aachen, Germany). Prior to measurements, samples were dried at 35 °C under a reduced pressure for 3 days. The mean fibres diameter and diameter size distribution were assessed by Image analysis (Image JÒ). A minimum of one hundred fibres was analysed for each sample. 3. Results and discussion 3.1. Phase diagram of PLA/EL solution The TIPS technique is based on the thermodynamic separation of a homogeneous polymer solution into a polymer-rich and a polymer-poor phases as a consequence of a temperature variation.
2.4. Aerogels characterisation Differential scanning calorimetric (DSC) analysis was used to assess the thermal properties of the aerogels. The samples were tested on a DSC8500 apparatus (Perkin Elmer, Massachusetts, USA) equipped with a liquid N2 controller CRYOFILL. Samples were first equilibrated at 20 °C for 5 min and then heated up to 200 °C at a scanning rate of 10 °C/min under inert atmosphere. The morphology of the aerogels was assessed by scanning electron microscopy (SEM). Samples were cross-sectioned and gold sputtered prior to be analysed by SEM (QUANTA 200F FEG-ESEM, FEI, The Netherlands). The porosity of the aerogels was determined by geometrical calculation from the mass and volume measurements [24]. The mass was measured by using a high precision balance (10 4 g,
Fig. 2. Cloud point and gelation point of PLA/EL solution as a function of polymer concentration.
Fig. 3. Low and high magnification SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels as a function of polymer concentration in the initial solution: (A, B) 3 and (C, D) 5.5 w%. Samples were obtained at 0 °C gelation temperature and by extracting the EL in water.
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To optimise the experimental protocol necessary to design the desired nanometre-scale fibrous materials, it is important to determine the phase diagram of the starting polymeric solution by assessing its cloud and gelation points. The cloud point and gelation temperatures of solutions with different concentrations of PLA in EL were measured and are presented in Fig. 2. The temperature at which both thermal transitions occurred increased with the increase of PLA concentration in the starting solution [25]. The gelation curve was about 10 °C lower than that of the cloud point curve. In particular, the gelation and cloud temperatures increased from 24 and 34 °C up to 33 and 40 °C when the PLA concentration was increased from 3 to 5.5 wt%, respectively. The observed results are in agreement to those reported for binary solutions of PLA in tetrahydrofuran [25] or in dioxane/water binary solvent [26]. 3.2. Properties of nanometre-scale fibrous PLA aerogels prepared via TIPS A series of porous nanometre-scale fibrous PLA aerogels was fabricated by varying the composition of the polymeric solution, the gelation temperature and the extraction medium. Fig. 3 shows the SEM micrographs of samples prepared at 0 °C gelation temperature and by extracting the EL in water as a function of the concentration of polymer in solution. The aerogels were characterised by a fibrous structure, with fibre diameters in the submicron range, and high porosity. Furthermore, the low magnification SEM images of Fig. 3A–D indicated that fibres density significantly increased with the increase of the concentration of PLA in the starting solution. Fig. 4 reports the porosity, specific surface area, pore size distribution and mean fibre diameter of the PLA samples in Fig. 3. As shown in Fig. 4A, the aerogels were characterised by porosity degrees higher than 90%. The porosity progressively decreased from 95.2 ± 0.5% to 90.3 ± 1.0% by increasing the PLA concentration in the starting solution from 3 to 5.5 wt%. The opposite trend was observed for the specific surface area, which increased from 69.4 ± 1.3 to 94.9 ± 0.5 m2/g when the concentration of PLA increased from 3 to 5.5 wt%. These results were in accordance with the information supplied by the images in Fig. 3 and demonstrated the possibility of controlling the density of the fibrous network by modulating polymer concentration in the starting solution. The pore volume measured using the adsorption data in the BET analysis was in the order of 0.15 cm3/g. The pore volume distribution plot showed in Fig. 4B shows that the three dimensional fibrous network of the prepared samples had both meso and macropores. The pore volume fraction corresponding to pores of 2 nm mean diameter increased progressively from 0.044 up to 0.055 cm3/g by rising the PLA concentration from 3 to 5.5 wt%. The pore volume fraction corresponding to pores of 10–100 nm mean diameter also increased with the increase of PLA concentration (Fig. 4). This nanometre-scale porosity is coupled concordant to the macroscopic porosity observed in the SEM images of Fig. 3 formed by randomly oriented pores passing throughout the entire sample volume. The average fibres diameter vs. PLA concentration is represented in Fig. 4C. The fibres diameter was in the range of 25–400 nm. The mean values slightly decreased from 196 ± 101 to 122 ± 63 nm by increasing PLA solution concentration from 3 to 5.5 wt%. Although the decrease of the average diameter of PLA fibres with the increase of polymer concentration could not be considered as statistically significant, this still observed effect can be ascribable to the effect of polymer concentration on the phase separation and polymer crystallisation processes. In particular, it was reported that, depending on the initial polymer concentration, the crystallisation of such polymers from solution can lead to different morphologies [9]. For dilute solutions, the formation of chain folded lamellae is usually observed. Conversely, at high polymer
Fig. 4. (A) Porosity and specific surface; (B) pore volume vs pore diameter plot and (C) mean fibres diameter of PLA aerogels as a function of polymer concentration in the initial solution. Samples were obtained at 0 °C gelation temperature and by extracting the EL in water.
concentration, suspensions of supramolecular architectures of these lamellae, such as axialites and spherulites, are obtained The values obtained in this work of average porosity and surface area are significantly higher than those reported for PLA scaffolds prepared by the standard phase separation approach (ca. 1 m2/g) and in accordance to those detailed by Chen and Ma for PLA aerogels prepared from a polymer solution in a dioxane/water mixture [27]. Further experiments were carried out in order to assess the effect of the used gelation temperature and the extraction medium on the morphology, mean fibres diameter and diameter size distribution of PLA aerogels prepared by TIPS. In particular, we investigated the effect of two alternatives gelation temperatures, 15 and 15 °C, as well as two different EL extraction media, water and ethanol, on samples morphology and the fibrous structure. The selected gelation temperature was higher than the EL freezing temperature, which is 26 °C, therefore ensuring that phase separation occurred without solvent crystallisation. Regarding the extraction
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Fig. 5. SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels as a function of polymer concentration in the initial solution, gelation temperature and extraction solvent: (A, C, E, G) 3 w% and (B, D, F, H) 5 w% PLA; (A, B, E, F) 15 °C and (C, D, G, H) 15 °C gelation temperature; (A–D) water and (E–H) ethanol extraction media. (K) The mean fibres diameter is also showed for each sample.
medium, in the first approach the gels were soaked in water to extract the EL and the obtained hydrogels were further dehydrated by soaking in ethanol to obtain alcogels suitable for sCO2 drying. In the second approach, the gels were directly soaked in ethanol to extract the EL and to obtain the alcogel and further dried by means of scCO2. The results of these tests are reported in Fig. 5. As shown in Fig. 5A–H, nanoscale fibrous PLA aerogels were also achieved at the gelation temperatures of 15 and 15 °C and by performing extraction in either water or ethanol. In particular, as previously observed for the samples prepared at 0 °C, the porosity and fibres diameter decreased with the increase of polymer concentration in the starting solution from 3 to 5.5 w% (Fig. 5K) and, concomitantly, the fibre diameter size distribution shifted to lower values with the increase of PLA concentration (Fig. 5I and L). The evolution of the structure and nanometre-scale properties of PLA fibres prepared by the TIPS process strongly depends on the route of polymer crystallisation during solution gelation [12]. It has been recently demonstrated that nanofibres formation via TIPS involves the initial condensation of amorphous nanoparticles with a gradual crystallisation from the gel. Extending the gelation step, a porous structure, characterised by an interconnected network of fibres, is formed. The final morphological, structural and thermal properties of the samples are temperature dependent in regard of the initial quenching period [12]. Table 1 reports the glass transition temperature (Tg), melting peak temperature (Tp), melting temperature range (DTp) and degree of crystallinity (vC) of PLA samples prepared in this work by the TIPS method as a function of polymer concentration and gelation temperature. For the prepared fibrous PLA, the thermal properties were not significantly affected by the different operating conditions investigated. For all of the precipitated samples, Tg values close to 66–67 °C were measured. Furthermore, vC and Tp values were in the order of 22–23% and 135–136 °C, respectively. These values are in accordance with those reported by other works for PLA membranes prepared by means of the TIPS process and using different solvents [12,28]. The use of binary mixtures of polymer solvent may be also a powerful approach to improve the control over the morphological and structural properties of PLA scaffolds prepared via TIPS technique. Phase separation for binary solvent mixtures depends not only of the temperature drop, but it is also strongly related to the effect of the composition of the binary solvent mixture on solution gelation and polymer crystallisation. For instance, solvent mixtures with a relatively high freezing point would crystallise and freeze when quenching the system to a low temperature. As a direct consequence, multi-scaled nanofibrous materials characterised by the
Table 1 Effect of concentration of polymer and gelation temperature on the thermal properties of nanoscale fibrous PLA prepared by TIPS and scCO2 drying. Sample
Tg (°C)
Tp (°C)
DTp (°C)
vC (%)
3% PLA/15 °C 4% PLA/15 °C 5% PLA/15 °C 5.5% PLA/15 °C 3% PLA/ 15 °C 5.5% PLA/ 15 °C
66.5 66.4 66.3 66.2 66.7 66.8
136.1 135.9 136.0 136.5 135.1 136.0
116–142 114–143 115–142 115–144 114–141 115–143
22.9 23.2 23.1 23.2 23.2 22.2
presence of larger pores can be fabricated for application such as tissue engineering scaffolds [29]. In this work, the possibility to fabricate nanometre-scale fibrous PLA aerogels has been further explored by adding water to the initial polymer solution as the antisolvent. In Fig. 6, the morphology and porosity of PLA aerogels obtained from a 3 wt% polymer solution in a 95/5 v/v% EL/water mixture are reported as a function of the gelation temperature. PLA polymeric networks, with a homogeneous distribution of uniform nanometre-scale fibres, were achieved at the gelation temperature of 15 °C (Fig. 6A). Conversely, when the gelation temperature decreased down to 0 or 15 °C, the miscibility of EL and water decreased and ice crystals could precipitate out from the solution during the phase separation step. This effect could be responsible for the formation of a porous structure characterised by large pores and an irregular fibrous structure (Fig. 6B, C). The same effect could explain the porosity trend observed in Fig. 6D as a function of the gelation temperature, because the formation of ice crystals could increase the porosity of the samples prepared at 15 °C. The porosity data reported in Fig. 6D as a function of the gelation temperature indicated that samples porosity slightly decreased from 94.3 ± 1.1% at 15 °C to 92.4 ± 0.6% at 0 °C, and subsequently increased up to 94.0 ± 0.8% at 15 °C. 3.3. Nanometre-scale fibrous PLA aerogels prepared via TIPS combined with GF or PL techniques The possibility to design and manufacture porous PLA aerogels characterised by multi-scaled pore structures is of great importance for applications related to tissue engineering, were scaffolds with pores larger than 100 lm are necessaries to allow for the three dimensional adhesion and colonisation with cells, as well as for new tissue development in vitro and in vivo [24,30].
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Fig. 6. SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels prepared starting from a 3 w% polymer in a 95/5 v/v EL/water solution and gelified at (A) 15, (B) 0 and (C) 15 °C. (D) Porosity of nanoscale fibrous PLA aerogels prepared starting from a 3 w% solution of polymer in a 95/5 v/v EL/water as a function of the gelation temperature.
Fig. 7. (A, B) SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels prepared by combining the TIPS method with the GF process. (C) Porosity of nanoscale fibrous PLA aerogels prepared by combining TIPS and GF techniques as a function of polymer concentration in the starting solution. Samples were obtained at 0 °C gelation temperature and by extracting EL in water.
Fig. 8. SEM micrographs showing the morphology of nanoscale fibrous PLA aerogels prepared by combining the TIPS method with the PL process. Samples were obtained at 0 °C gelation temperature and by extracting EL in water.
Therefore, in the last part of this study the developed TIPS method has been improved in this regard by combining it with either GF or PL processes. A two-step depressurization approach was developed
in the case of the TIPS/GF combined process, aiming to concomitantly achieve gel drying and foaming, avoiding the use of an additional processing step. In Fig. 7A–C the morphology and the
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porosity of the TIPS/GF prepared PLA samples is reported as a function of polymer concentration in the initial solution. The PLA samples were characterised by a nanometre-scale fibrous morphology, while there was no evidence of multi-scaled pore structures (Fig. 7A, B). The evaluation of the porosity demonstrated that the combined approach was able to increase the overall porosity of PLA by 0.5% in the case of using a 3 wt% PLA polymer solution and by 1.9% for a 5.5 wt% solution. This result indicated that the method involving two depressurisation steps, characterised by a final fast depressurisation during vessel venting, induced partial foaming, but did not allow for the formation of the large pores required for tissue engineering. To overcome the previous limitation, micrometric gelatin particles were used in a combined TIPS/PL process. The gelatin particles acted as a templating agent for large pores formation. The morphological characterisation of obtained PLA sample prepared from a 5 wt% solution of PLA in EL and gelatin particles is shown in Fig. 8A–C. The addition of the micrometric particulate porogen effectively allowed for the fabrication of multi-scaled porous PLA aerogels with large pores, in the order of 300 lm, replicating the size of the starting gelatin particles. Furthermore, by optimising the TIPS steps, it was possible to induce high pore interconnectivity (Fig. 8B) as well as to recreate a nanometre-scale fibrous architecture on the pore walls (Fig. 8C). Previous works have shown that a similar pore structure is very helpful to improve the biological response of PLA scaffolds [29]. The as prepared PLA multi-scaled porous scaffolds were characterised by a 95% overall porosity, which is an optimal value for several tissue engineering applications. 4. Conclusions In this work, a clean and sustainable approach to manufacture multimodal nanometre-scale fibrous PLA aerogels with controlled morphology and architecture is reported. The aerogels were fabricated by means of TIPS process by using the green solvents EL and scCO2 to dissolve the polymer and to dry the aerogel, respectively. By controlling the concentration of polymer in solution, the gelation temperature and the extraction media, nanometrescale fibrous PLA aerogels, with homogeneous distribution of fibres and controlled structural features, were obtained. In particular, by increasing polymer concentration from 3 to 5.5 wt%, the porosity was modulated from 90% to 95% and the specific surface area from 70 to 95 m2/g, with a mean fibres diameter in the range of 100–200 nm. Finally, the combination of TIPS with either GF or PL processing techniques was explored to improve the control on pore structure features and to fabricate multi-scaled porous PLA aerogels with potential applications in tissue engineering as scaffolds.
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