Generation of Solid Foams with Controlled Polydispersity Using

Article Cite This: Langmuir XXXX, XXX, XXX−XXX

pubs.acs.org/Langmuir

Generation of Solid Foams with Controlled Polydispersity Using Microfluidics Sébastien Andrieux,† Wiebke Drenckhan,‡ and Cosima Stubenrauch*,† †

Institute of Physical Chemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany Institut Charles Sadron, CNRS, 23 Rue du Loess, 67200 Strasbourg, France

‡

ABSTRACT: Many properties of solid foams depend on the distribution of the pore sizes and their organization in space. However, these two parameters are very difficult to control with most traditional foaming techniques. Here we show how microfluidics can be used to tune the polydispersity of the foams (mono- vs different polydispersities) and the spatial organization of the pores (ordered vs disordered). For this purpose, the microfluidic flow-focusing technique was modified such that the gas pressure oscillates periodically, which translates into periodically oscillating bubble sizes in the liquid foam template. The liquid foams were generated from chitosan solutions and then gelled via cross-linking with genipin before we freeze-dried them to obtain a solid foam with a specific structure. The study at hand fills two existing scientific gaps. On the one hand, we present a novel approach for the generation of foams with controlled polydispersity. On the other hand, we obtained a solid foam with a new structure for foam templating consisting of rhombic dodecahedra. The controlled variation of the foam’s structure will allow studying systematically structure−property relations. Moreover, being fully biobased, this type of solid foam is a suitable candidate for applications in tissue engineering.

1. INTRODUCTION Polymer foams are materials consisting of a gas phase dispersed in a polymer matrix. The gas bubbles within the polymer matrix do not affect the intrinsic properties of the polymer such as its chemical reactivity or wettability. However, the presence of the gas bubbles results in materials that are lightweight and have a large specific surface area. The extent to which the disperse gas phase affects the properties of a porous polymer depends on the morphology of the foam, i.e., on its relative density, the average pore size, the interconnectivity between the pores, the thickness of the pore walls, etc. Twenty years ago, Gibson and Ashby1 reported on the effect of the relative density of porous polymers on their response to compression and the interdependency between architecture and mechanical properties. However, it is not yet well understood how the pore size distribution affects the mechanical properties. The reason for this lack of knowledge is the fact that the pore size distribution is a parameter that is very difficultif not even impossibleto control by traditional techniques. In recent years liquid templating has proved to be a promising method toward a better control over the morphologies of polymer foams, and this holds true for the use of both emulsion templates2−4 and foam templates.5−7 Liquid templating consists in producing a liquid template, typically an emulsion or a foam, composed of either a monomer or a diluted polymer solution. The template is subsequently solidified either via polymerization (monomer) or cross-linking © XXXX American Chemical Society

(polymer), which, in turn, results in the desired polymer foam after simple purification and/or drying steps. In conjunction with microfluidics, liquid templating allows for the synthesis of solid monodisperse polymer foams via the solidification of liquid monodisperse emulsions8,9 or liquid monodisperse foams.9−14 To the best of our knowledge, the first study in which specific properties of monodisperse and polydisperse foams were compared was carried out by Colosi et al.13 The authors investigated the morphological differences between monodisperse and polydisperse poly(vinyl alcohol) scaffolds. The monodisperse foams were produced via microfluidic bubbling, while the polydisperse foams were produced via gas foaming. It is true that this study presents some interesting results regarding the morphological differences between monodisperse and polydisperse foams. The authors show for example that monodisperse foams had not only a better homogeneity in pore sizes but also a better homogeneity in pore wall thickness. This may lead to interesting results regarding the mechanical properties of the monodisperse and polydisperse foams, but these were not investigated. Moreover, the monodisperse and polydisperse foams had porosities of 63% and 78%, respectively. This large difference in porosities makes a comparison between both materials on a deeper level Received: October 17, 2017 Revised: November 30, 2017

A

DOI: 10.1021/acs.langmuir.7b03602 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 1. Generation of monodisperse and polydisperse chitosan foams using a microfluidic device.

than just morphology unreliable. The main problem lies in the liquid fraction of polydisperse foams which can hardly be controlled. Indeed, different foaming methods may result in different bubble size distributions, different average bubble sizes, and liquid fractions, but these parameters are difficult to fine-tune.15 Simple foaming techniques such as mechanical frothing provide littlemost of the time even nocontrol over the foam’s morphology. In a later work, Costantini et al.16 also produced monodisperse and polydisperse foams by means of microfluidics and gas foaming, respectively, with the aim of studying how the polydispersity affects the cell seeding efficiency of polymeric scaffolds. The authors managed to reach closer porosities (66% for the polydisperse foam and 70% for the monodisperse foam) than in Colosi et al.13 The mechanical properties of microfluidic-generated solid foams were investigated for different porosities, but these results were not compared with polydisperse solid foams. Moreover, the liquid phase was not identical for the two foaming methods, as the surfactant concentration was 2% and 0.2% w/v for the polydisperse and monodisperse foams, respectively. For an accurate comparison between monodisperse and polydisperse systems, the chemical composition of the liquid templates needs to be identical. This holds also true for the gas phase. Indeed, in simple foaming methods such as mechanical frothing, the gas phase is often air and cannot be modified. In the case of closed-cell foams, the gas phase significantly affects the mechanical properties of the foam. In this context, it is important to know that often perfluorohexane is added to the gas phase to stabilize the liquid foam against coarsening. Perfluorohexane can easily be added in microfluidic setups but not with foaming methods such as mechanical frothing. The lack of knowledge about how perfluorohexane affects the mechanical properties of the resulting polymer foams makes it difficult to compare polymer foams generated either with or without added perfluorohexane. Thus, for a direct comparison monodisperse and polydisperse polymer foams need to be produced by means of the same technique and with the same composition. Since microfluidics is considered to be the best technique to produce monodisperse foams, we used microfluidics to produce both monodisperse and polydisperse foams. This enables us to produce foams with comparable average pore sizes and densities but different polydispersities so that the

pore size distribution becomes a parameter that can be studied independently. For the study at hand, we made use of our knowledge and know-how about monodisperse foam templating with chitosan solutions.17,18 Chitosan is a polysaccharide derived from chitin, which is itself extracted from the shells of crustaceans and can thus be produced from food waste.19 Chitosan is slightly soluble in acid conditions under which the amino groups are protonated, thus making chitosan a polycation.20 The presence of these same amino groups allows chitosan to form a hydrogel via chemical cross-linking, for example with genipin, which we chose as it is biobased and biocompatible. Having previously managed to synthesize monodisperse chitosan solid foams with the help of microfluidics,17,18 we now used microfluidics to synthesize polydisperse liquid foams with a controlled average bubble size and polydispersity. We will show the potential of microfluidics for the production of both monodisperse and polydisperse liquid templates. In the latter case, we can adjust different polydispersities, thus enabling us to study polydispersity as an independent parameter (section 3.1). The ground principle of foam templating is the ability to set the morphology of the solid foam by setting the morphology of the liquid foam, making solidification a critical step. We will thus detail the solidification of the liquid templates (section 3.2). This is the first study of its kind in which monodisperse and polydisperse foams with a controlled polydispersity are compared. We will discuss the morphological similarities and differences of both systems (section 3.3).

2. EXPERIMENTAL SECTION Chemicals. Chitosan was purchased from Glentham Life Sciences Ltd. and has a molecular weight of 30 000 g mol−1 and a deacetylation degree of 90.56% (data from supplier). Glacial acetic acid was purchased from VWR. Bidistilled water was used. Cognis Deutschland GmbH & Co (now BASF) donated the surfactant Plantacare 2000 UP, which is an alkyl polyglycoside with a headgroup composed of 1.5 glycoside units and alkyl chains between 8 and 16 carbons. The crosslinker genipin was purchased from Challenge Bioproducts Co., Ltd. Perfluorohexane was purchased from Alfa Aesar. All chemicals were used without further purification. Preparation of Chitosan Solutions. 4 wt % chitosan was dissolved in a solution containing 1 vol % acetic acid via magnetic stirring for 2 h. The few remaining undissolved particles were dissolved via ultrasonication for 5 min using an ultrasonic homogenizer B


Article

Langmuir

fraction of the initially generated foam. In order to obtain φ, we determined the total volume of liquid Vl in the cylinder from the sum of the flow rates Qtotal of the syringe pumps as Vl = tQtotal, while the volume Vl,drain of the drained liquid and the foam volume Vfoam were measured from the graduation of the cylinder. The volume of liquid contained Vl,foam in the foam was calculated as the difference between the total volume and the volume of the drained phase, i.e., Vl,foam = Vl − Vl,drain. The liquid fraction φ was calculated as the ratio of the liquid volume in the foam and the foam volume, i.e., φ = Vl,foam/Vfoam. The liquid foam stability was assessed by collecting the foams in test tubes. We took pictures of the foams right after their formation and at different time intervals. We measured the foam height at different time intervals, and the foam stability was assessed by studying the decay of the liquid foam height h(t) with time. We used the normalized value h(t)/h0, h0 being the initial foam height, as a measurement for the foam decay and plotted it versus the time t. Gel Point Measurements. The gel point of chitosan cross-linked with genipinin bulk and foamedwas determined by oscillatory rheometry using a Physica MCR 501 rheometer from Anton Paar. The geometry used was plate−plate with a plate radius of 25 mm. The gap was 1 mm, and a 1% deformation was applied at a frequency of 1 Hz. The temperature was set at 23 °C with a Peltier system with a temperature accuracy of 0.1 K. All parameters were identical for the gel point measurements of the bulk chitosan solution and the chitosan foam. Cross-Linking and Freeze-Drying. The Petri dishes containing the foams were sealed with Parafilm and left to cross-link at room temperature for 18 h. This time was determined to be long after the gel time, as is discussed in section 3.2. The foams became blue during cross-linking, which is the result of the reaction of genipin with the amino groups of chitosan in the presence of oxygen.25 Once crosslinked, the foams were frozen in liquid nitrogen and freeze-dried in an Alpha 1-4 LSC freeze-dryer from CHRIST. Density of Chitosan Cross-Linked with Genipin. The density of the material constituting the solid foams, namely chitosan crosslinked with genipin, was measured with a helium porosimeter AccuPycII 1340 from Micrometrics. We conducted the density measurements on chitosan solid foams which were ground into a powder. Characterization of Bubble/Pore Size Distribution. The bubble size distributions were determined from microscopy images taken with the Nikon SMZ-800 N optical microscope with an Optronis CL600X2 high-speed camera using the image analysis software ImageJ. We measured the bubble diameters in two ways. First, we measured the bubble size directly in the microfluidic channel on images like the ones shown in Figure 2C,D. At high gas pressures, the bubbling frequencies were so high that the bubbles did not appear individually on the image, but they traced a continuous line as shown in Figure 2D. The height of this line was taken as the bubble diameter. The second set of measurements was carried out with foams as those shown in Figure 2A,B, i.e., with bubbles that had left the microfluidic device. We determined the diameter dbubble of at least 60 bubbles, and the polydispersity index (PDI) was calculated according to

SONOPULS HD2200 from BANDELIN at a power of 40%. The surfactant was added such that a concentration of 0.1 wt % with regard to the solvent was obtained, which corresponds to 10 cmc. Genipin was dissolved in a 1 vol % acetic acid in water at a concentration of 1 wt % by magnetic stirring. The surfactant was added such that a concentration of 0.1 wt % with regard to the solvent was obtained. The chitosan and genipin solutions mixed after bubble formation via microfluidics. The flow rate of the genipin solution was set as one-sixth of the flow rate of the chitosan solution to keep the relative concentrations of chitosan and genipin constant. Thus, the resulting solution contained 3.43 wt % chitosan, 0.14 wt % genipin, and 0.1 wt % Plantacre 2000 UP with regard to the solvent. Microfluidic Foam Generation. Microfluidic foam generation was carried out using a commercial glass chip purchased from Dolomite Microfluidics whose X-junction has a depth of 190 μm and a width of 195 μm, and main channels have a depth of 190 μm and a width of 390 μm, and which was described in our previous work.18 The general setup for the generation of monodisperse and polydisperse liquid foam templates is shown in Figure 1. The gas flow was pressure-driven at a pressure pgas using an OB1Mk2 pressure controller from Elveflow connected to a nitrogen tap. Using a pressure-drivenrather than a flow-rate-drivengas flow ensures rapid stabilization of the viscous two-phase flow in the microfluidic chip since the gas is compressible at the characteristic pressures encountered in the device (order 1−2 bar). The gas phase was nitrogen with traces of perfluorohexane in order to hinder Ostwald ripening, which was used in many studies about liquid foams.21−24 For this purpose, a small amount of liquidyet highly volatileperfluorohexane was put in a glass bottle sealed with a GL45 cap from Vaplock allowing the plugging of the tubings from the pump and to the chip; i.e., the nitrogen passed through this bottle, carrying away traces of perfluorohexane before entering the chip. An aqueous solution containing 4 wt % chitosan and 0.1 wt % surfactant Plantacare 2000 UP was injected in the microfluidic chip with a syringe pump Pump 11 Elite from Harvard Apparatus at a controlled flow rate Qchitosan. The chitosan flow was separated with a Tjunction into two flowswith the same flow rate Qchitosan/2which met at the X-junction of the microfluidic chip with the gas flow. The generated foam moves through the microfluidic channels up until it gets in contact with an aqueous solution containing 1 wt % genipin and 0.1 wt % surfactant to initiate cross-linking. The genipin flow was controlled by a syringe pump and had a flow rate Qgenipin which was set to 1/6 of the chitosan flow rate Qchitosan. This ratio Qchitosan/Qgenipin = 6 was kept constant for all experiments (even when we changed the chitosan flow rate) in order to maintain the same genipin ratio and thus cross-linking density. Having explicit control over the genipin ratio is also the reason why we chose to control the flow rate rather than the pressure of the liquids. By adding genipin in a separate flow to the chitosan solution, one dilutes the chitosan and the genipin solution. At a flow rate ratio of 1/6 and at initial concentrations of 4 wt % chitosan and 1 wt % genipin, one obtains concentrations of 3.43 wt % chitosan and 0.14 wt % genipin, respectively, in the resulting foam. The bubble size and bubbling frequency can be adjusted by varying the liquid flow rates and the gas pressure. Here, we chose to work at a constant chitosan flow rate of Qchitosan = 180 μL min−1 and to vary the gas pressure pgas. The chitosan flow rate value of 180 μL min−1 was chosen because the corresponding genipin flow rate was Qgenipin = 30 μL min−1, which was the lowest flow rate for the bubbly flow not to enter the tubing carrying the genipin solution. Bubbling was monitored by means of a Nikon SMZ-800N optical microscope coupled with an Optronis CL600X2 high-speed camera. The foams were collected in polystyrene Petri dishes of 3.5 cm diameter and 1 cm height. Liquid Foam Characterization. The liquid fractions of the foams were determined by collecting the foams in a graduated cylinder for a given period of time t. After letting the foam settle for 30 min, part of the liquid has drained from the foam under the influence of gravity and fills the bottom of the cylinder. The final liquid fraction φ of the drained foam in the cylinder is therefore different from the liquid

PDI = 100 ×

⟨dbubble 2⟩ − ⟨dbubble⟩2 ⟨dbubble⟩

(1)

A foam can be called monodisperse if its PDI lies below 5%, while for PDI > 5% the foam is polydisperse, and the PDI quantifies the degree of polydispersity.26 For polydisperse foams, the diameters were calculated from the area of the bubbles. In the case of monodisperse ordered foams where the bubbles are in close contact, we calculated the diameters as the center-to-center distances between two neighboring bubbles. The coordinates of the bubble center were calculated as the average x and y positions of all the pixels contained in the bubble. In the case of polydisperse foams, the bubble diameters were calculated from the bubble areas. Note that neighboring bubbles in a foam are touching each other and that the black rings visible in the liquid foam templates (see Figure 4a−c) are optical effects and do not correspond to the outer boundaries of the bubbles.27 C


Article

Langmuir

estimation is to some extent due to optical effects and light refraction in the channel, as described by van der Net et al.27 Moreover, the bubbles are not always separated but are sometimesat high bubbling frequenciesseen as a continuous train of bubbles, as observed in Figure 2D. Thus, we had to measure the bubble diameters in the direction perpendicular to the flow. By doing so, we did not take into account the bubble deformation along the direction of the flow due to viscous forces and the confinement between the walls. But if the determination of bubble sizes within the channel is so inaccurate, why do we measure it this way? First, to point out how strongly the diameter of a flowing bubble constricted within a channel and the diameter of a bubble at rest can differ and attract the attention of our colleagues on the necessity of characterizing microfluidic-generated foams outside of the channels, whenever possible. Second, because one sometimes has no choice, e.g., during periodic bubbling (as discussed in the next paragraph) where the gas pressure varies with time and one cannot trace back from outside the chip the pressure at which a given bubble was formed. Polydisperse Bubbling. Applying periodically varying gas pressures around a constant value according to pgas(t) = ⟨pgas⟩ + [pgas,max − ⟨pgas⟩] sin(2πt/τ) mbar, we can produce foams with specific polydispersities. Figure 3 (bottom) shows how the bubble size (measured in the microfluidic channel) varies with time t for two different gas pressure protocols which are plotted in Figure 3 (top). The chosen protocols are pgas(t) = 1250 + 150 sin(πt/2) mbar and pgas(t) = 1350 + 250 sin(πt/4) mbar. The main parameters for polydisperse bubbling are thus the average gas pressure ⟨pgas⟩, the gas pressure amplitude pgas,max − ⟨pgas⟩, and the period τ. For clarity, we will denote these gas pressure protocols according to their average pressures ⟨pgas⟩, which are 1250 and 1350 mbar, respectively. One sees that the periodic pressure variation leads to a periodic variation of the bubble size and that despite an average gas pressure difference of 100 mbar between the two protocols, the average bubble size in the microfluidic channel lies around 210 μm in both cases. Moreover, for ⟨pgas⟩ = 1250 mbar (filled triangles) the maximum bubble size is 250 μm, and the minimum bubble size is 180 μm for pgas = 1100 mbar. The bubble size shows a sinusoidal response to a sinusoidal variation of the pressure. However, one observes a different behavior for the pressure protocol with ⟨pgas⟩ = 1350 mbar. The bubble size is of 290 μm at the pressure maximum of 1600 mbar, but for the pressure minimum of 1100 mbar, bubbling stops. Both pressure protocols share the same minimum pressure, i.e., 1100 mbar, but the microfluidic bubbling differs between both protocols, even when compared to monodisperse bubbling, for which a gas pressure of 1100 mbar results in bubbles with a diameter equal to 250 μm. At the pressure maxima of both pressure protocols, i.e., at 1400 and 1600 mbar, the maximum bubble sizes are 250 and 290 μm, respectively. The bubble size measured in the channel for monodisperse bubbling at pressures of 1400 and 1600 mbar are displayed in Figure 2 and are equal to ca. 260 μm in both cases, which corresponds to the upper limit of the bubble size observed in the channel. Thus, varying the gas pressure allows for larger bubble sizes than in the monodisperse case. The observation of different bubble sizes for the same gas pressure but a different pressure protocol entails the presence of dynamics effects: the speed at which the gas pressure variesor whether it varies at all affects the size of the bubbles. This relation illustrates the

Figure 2. Bubble diameter dbubble measured in the chip and from a foam monolayer as a function of the gas pressure pgas. The chitosan flow rate was set to Qchitosan = 180 μL min−1. The insets show pictures of the microfluidic bubbling (C, D) and the corresponding foam monolayers (A, B) from which the bubble sizes were determined at pgas = 500 mbar (A, C) and pgas = 1300 mbar (B, D). The error bars correspond to the standard deviations. All scale bars are 500 μm. The pore size distributions of the solid foams were calculated from scanning electron microscopy (SEM) images taken with a CamScan CS 44 microscope at a voltage of 5 kV. At least 40 pores were measured using ImageJ for each pore size distribution. The pore diameters in monodisperse foams were calculated as the center-tocenter distances such as in the liquid state. The pore diameters in polydisperse foams were calculated from the pore areas such as in the liquid state. We concede, however, that this method is to be seen as an approximation.

3. RESULTS AND DISCUSSION 3.1. Generation of Monodisperse and Polydisperse Liquid Chitosan Foams. Monodisperse Bubbling. Figure 2 shows how the bubble size can be tuned by changing the gas pressure pgas at constant liquid flow rate Qchitosan in our microfluidic geometry. If one works with a fixed gas pressure and a constant liquid flow rate in the correct pressure/flow rate range, one obtains perfectly monodisperse foams whose bubble sizes depend on the pressure/flow rate ratio as reported in previous studies.10,14,18,28 The overall trend is an increase of the bubble size with gas pressure, with a steep increase at low pressuresuntil ca. pgas = 600 mbarand a slower increase at higher pressures. The range of accessible bubble sizes lies between 180 and 400 μm for the used chip. In Figure 2 we made a clear distinction between the bubble sizes measured from the collected foams (circles) and the bubble sizes measured during bubbling in the microfluidic channel (triangles). One sees that the measurement of the bubble sizes in the channel systematically underestimates the bubble sizes of the produced foams and that an upper limit equal to 260 μm is reached. Note that this value is lower than the channel width, which is 390 μm, and larger than the channel height, which is 190 μm. When measured outside the microfluidic chip, the bubbles reach a diameter of 400 μm which is larger than the channel heightindicating that from a threshold diameter of 190 μm the bubbles are squeezed within the channel. However, even below 190 μm, the bubble sizes measured in the channel are underestimated. This underD


Article

Langmuir

foam produced with ⟨pgas⟩ = 1250 mbar had a PDI of 14.2%, and the polydisperse foam produced with ⟨pgas⟩ = 1350 mbar had a PDI of 26.2%. Increasing the amplitude of the pressure variation therefore led clearly to an increase of the polydispersity index, which was predicted by the on-chip measurements of the bubble sizes presented in Figure 3. Despite the fact that all average gas pressures were different, the three bubble size distributions are centered between 300 and 400 μm. Microfluidics with varying flow conditions thus allows producing liquid foams with comparable average bubble sizes but different PDIs. In other words, we are able to control independently the average bubble size and the polydispersity by setting the appropriate pressure parameters. Note that the bubble size distribution of the foam produced with ⟨pgas⟩ = 1350 mbar is slightly shifted toward higher bubble sizes, which corresponds to what was already observed in Figure 3, namely, the absence of bubbling at the lowest pressures. Moreover, the bubbling frequency also decreases with the gas pressure, yielding fewer small bubbles and more big bubbles. Analysis of Figure 4a shows that the monodisperse foam is ordered whereas both polydisperse foams are disordered. It has already been shown that monodisperse foams do self-order under gravity and confinement.26 The organizational difference between monodisperse and polydisperse templates needs to be taken into account and may play a role in the mechanical properties of the resulting solid foams. Changing the dimensions of the microfluidic chip and, more specifically, increasing the width of the microfluidic channel, one may have access to larger pressure amplitudes and thus larger PDIs. Future work should thus focus on the design of microfluidic chips with large channels, in order to reach the largest range of bubble sizes possible. 3.2. Cross-Linking of the Liquid Chitosan Foam Templates. All following experiments were carried out with two different types of foams: the monodisperse foams produced at pgas = 1200 mbar and the polydisperse foams produced at ⟨pgas⟩ = 1250 mbar. The corresponding liquid templates are shown in Figures 4a and 4b, respectively. For clarity and reading fluency, we will denote the two systems simply as “monodisperse foams” and “polydisperse foams” in the following. In order to obtain the desired monodisperse and polydisperse solid foams, the liquid templates need to be crosslinked and dried. Figure 5 shows the evolution of the elastic modulus G′ and the loss modulus G″ as a function of time for a foamed chitosan solution during cross-linking with genipin. The values of G′ are nonzero before cross-linking, which arises from the elastic deformation of the bubbles and can be approximated by G′ ∼ γ/R which is known for liquid foams and viscous liquids in general.29 Both G′ and G″ increase significantly during cross-linking with a well-defined crossover which defines the transition from a liquid-like to a solid-like state, i.e., the gel point. The gel point of the studied sample is reached after 47 min. For comparison, the inset in Figure 5 shows the evolution of G′ and G″ with time for the unfoamed chitosan solution during cross-linking with genipin. The initial value of G″ is several orders of magnitude lower than what is measured for the foam, while the value of G′ is negligibly small. This is expected for a highly viscous liquid which does not contain bubbles. As in the case of the foam, G′ and G″ increase during cross-linking with a well-defined crossover. One sees that the gel point in the absence of bubbles is reached after ca. 4 h (247 min), which is significantly longer when compared to the time of the

Figure 3. (top) Time dependence of the gas pressure pgas over a single period τ for two different pressure protocols. Note that the pressures are the ones set with the microfluidic software and thus do not account for experimental fluctuations. (bottom) Variation of the bubble size dbubble measured in the microfluidic channel over a single period τ for the two different pressure protocols. A bubble size of 0 corresponds to the absence of bubbling. The insets are pictures of microfluidic bubblings from which the bubble sizes were measured. All scale bars are 500 μm.

importance of the period τ on the bubbles size for a given gas pressure protocol. The period τ was different in the two gas pressure protocols and was equal to 4 and 8 s for the ⟨pgas⟩ = 1250 mbar and ⟨pgas⟩ = 1350 mbar protocols, respectively, because the period needs to be increased when the amplitude increases. This constraint comes from the fact that quick and significant pressure variations induce perturbations which prevent bubbling periodicity and may induce an arrest of bubbling. In order to reach a periodic flow, one needs to set the wished average pressure, a low amplitude, and a long period. One then increases the amplitude stepwise up until the desired amplitude is reached and adjust the period. Because of the huge influence the gas pressure protocol has on the bubble size, the calibration presented in Figure 2 is only valid for steady state bubbling, i.e., for the production of monodisperse foams. One cannot predict the bubble size distribution from pressure variations in polydisperse bubbling, but one needs to measure the bubble size distribution for each combination of average pressure, amplitude, and period. Characterization of Liquid Foams. Figure 4 shows typical photographs of the liquid foams which were produced from the different bubbling protocols presented in Figure 3. The monodisperse foam had a PDI of 3.7%, the polydisperse E


Article

Langmuir

Figure 4. Photographs of (a) a monodisperse liquid foam produced with pgas = 1200 mbar, (b) a polydisperse liquid foam produced with an average pressure of ⟨pgas⟩ = 1250 mbar and a pressure amplitude of 150 mbar, (c) a polydisperse liquid foam produced with an average pressure of ⟨pgas⟩ = 1350 mbar and a pressure amplitude of 300 mbar, and (d) their corresponding bubble size distributions. Qchitosan = 180 μL min−1.

However, if the gelation is too quick, the chitosan solution can solidify in the microfluidic channel and disturb the generation of bubbles. Indeed, microfluidics is a very sensitive technique and responds instantaneously to any pressure variation. Any increase of viscosity due to cross-linking, even locally, will lead to a local pressure change, which will induce a response at the constriction of the chip and thus a modification of the bubble size. In order to ensure that microfluidic bubbling is reproducible, one needs to make sure that the rheological properties of the bubbling solution do not change within the microfluidic setup. One can define the residence time as the duration between the time at which the bubble is formed and the time at which the bubble is collected in the Petri dish. The solution should not solidify within this time in order to avoid brutal pressure changes in the microfluidic chip. Once the foam is collected into the Petri dish, the bubbles of the monodisperse foam arrange in ordered layers of close-packed bubbles.18 This reorganization takes timetypically up to 10 minand should not be disturbed by an ill-timed solidification of the continuous phase. All the time constraints presented above lead us to estimate a minimum acceptable gel point of around 20 min. As already discussed, gelation needs to occur quickly enough so that the foam template does not have time to collapse before it is stabilized by gelation. The stability of the liquid foam templates in the absence ofcross-linker is assessed in Figure 6 by looking at the evolution of the relative foam height h/h0 with time. The foams drain during the time necessary to collect them, i.e., about 20 min. The initial time for the foam stability tests is the time at which we stop collecting the foam. One sees that at after 50 min the foams have at least 80% of their initial height even without cross-linking. Moreover, the larger part of the decrease of the foam height is due to drainage and thus

Figure 5. Gel point measurement at 23 °C via oscillatory rheology for a monodisperse chitosan foam cross-linked with genipin. The gel point is defined as the intersection of the storage and loss moduli and was determined to be at 47 min. The inset shows the gel point measurement for a bulk chitosan solution cross-linked with genipin. The bulk gel point reads 247 min.

corresponding foam. The acceleration of cross-linking in the foam in comparison with cross-linking in the bulk may be due to the confinement of the chitosan molecules in the foam. Once a threshold value of the Gibb’s elastic modulus is reached, the foam no longer follows typical destabilization mechanisms, and one can call it stable.30 The earlier this threshold is reached, the more the solid foam will resemble its liquid template. F


Article

Langmuir

nitrogen, are shown in Figure 7. The samples observed are typical examples of the foams before they are freeze-dried. The

Figure 7. Pictures of (a) monodisperse and (b) polydisperse chitosan foams cross-linked with genipin. The chitosan foams were cross-linked for 18 h at room temperature to produce foamed hydrogels which were frozen in liquid nitrogen to render the sample brittle enough to be broken. The scale bars are 2 mm.

blue color comes from the reaction of genipin with chitosan.25 The whiter areas visible on both samples come from the formation of ice crystals on the hydrogels which were frozen in liquid nitrogen in order to be cut. Notice the polyhedral shape of the monodisperse pores and the ordered pore layers present over almost the total height of the sample. The polydisperse foam, however, does not show any regularity, neither in the shape of the pores nor in its architecture. Since the monodisperse and polydisperse foams are handled the same way after their formation, i.e., cross-linked at room temperature for the same amount of time and frozen, one can conclude that monodispersity is the sole parameter responsible for the architectural ordering of the bubbles/pores and their shape. Moreover, the cross-linked foams do not show signs of coarsening, meaning that C6F14 does indeed prevent coarsening in the liquid template. 3.3. Structure of Monodisperse and Polydisperse Solid Chitosan Foams. Once cross-linked, the chitosan foams are freeze-dried. During this process water, which constitutes more than 96% of the mass of the foam, is removed. The resulting cross-linked, freeze-dried chitosan foams are shown in Figure 8. Looking at Figure 8, one sees that the

Figure 6. Pictures of (a) monodisperse and (b) polydisperse foam produced via microfluidics different times after their generation without genipin. The scales bars are 1 cm. (c) Plot of the foam height at time t over the initial foam height against time for the monodisperse and polydisperse foams presented in (a) and (b). For clarity, not all the pictures used to draw (c) are displayed in (a) and (b). The dotted line in (c) is a guide to the eye and marks the gel point of a monodisperse chitosan foam cross-linked with genipin as determined in Figure 5.

does not affect the size of the bubbles but their shape. Given that the gel point of the foams is at ca. 50 min, one can conclude that the cross-linking kinetics is rapid enough and fits well with the stability of the foam. Moreover, even after 6 h, the foams do not collapse below 75% of their initial height, which is a sign of good stability despite the absence of cross-linker. Figure 6c also shows that the polydispersity of the foam does not significantly affect the stability of the liquid foam. Comparing the residence time, gel point, and the lifetime of the foam template, one sees that the proposed formulation does not block the microfluidic device and leaves enough time for the foam to organize but solidifies before ageing sets in. The structure of the liquid template is thus maintained during crosslinking. Macroscopic images of the obtained monodisperse and polydisperse foamed hydrogels, which were frozen in liquid

Figure 8. Photographs of (a) monodisperse and (b) polydisperse solid chitosan foams. The scale bars are 1 mm.

crystallinity of the monodisperse foams already observed in the cross-linked foams (see Figure 7) is preserved throughout freeze-drying. The polydisperse cross-linked foam is neither crystalline nor does it have pores with a regular shape. Figure 9 provides a closer look at the structures of the monodisperse and polydisperse solid foams. The average pore sizes and PDIs are listed in Table 1. The monodisperse pores are polyhedral, and their structure is that of a rhombic dodecahedron, as sketched in Figure 10b.31 The spatial organization of the cells observed in Figures 8a and 9a corresponds to an FCC ordering. This is surprising for a G


Article

Langmuir

From this we can determine the pore diameter dpore = 2(3Vpore/ 4π)1/3 defined as the diameter the pore would have if it were spherical. This calculation allows determining a reasonably accurate pore dimension from SEM pictures which is important for a comparison with the bubble size of the liquid template. Looking at the pore size distributions of the monodisperse and polydisperse foams, one sees that the pores in the monodisperse solid foam remain narrowly distributed with an average pore size of 237 ± 9 μm and a PDI of 5.8%. The average pore size of the polydisperse solid foam is 304 ± 61 μm, yielding a PDI of 20.1%. When working with foam templating, one needs to compare the solid foams with the liquid templates they are made out of. The relevant parameters of the solid foams and their liquid counterparts are listed in Table 1. One sees that the monodisperse foam has an average bubble size of 364 ± 14 μm in the liquid state and an average pore size of 237 ± 14 μm in the solid state corresponding to a size reduction of 35%. On the other hand, the polydisperse foam has an average bubble size of 322 ± 48 μm in the liquid state and an average pore size of 304 ± 61 μm in the solid state; i.e., we observe a size reduction of 5.6% only. The densities of the two foams also strongly differ. The density of the monodisperse chitosan foam is ρ = 0.0113 ± 0.0017 g cm−3, while that of the polydisperse foam is ρ = 0.0083 ± 0.0009 g cm−3. The relative densities of the foams are calculated by dividing the densities of the foams by the density of cross-linked chitosan, which is ρc = 1.4207 ± 0.0031 g cm−3. We speculate that the different shrinkage and density originate from the liquid templates themselves: the monodisperse liquid template has a liquid fraction of φ = 0.11, while the polydisperse liquid template has a liquid fraction of φ = 0.06, i.e., a difference of almost a factor 2. The lower liquid fraction of the polydisperse foam template is consistent with the physics of packing, since in a polydisperse foam smaller bubbles can fill the voids between the bigger ones. Note that the relative densities of both systems are very low and yield porositiesthe porosity being defined as one minus the relative densityabove 99%. This high porosity comes from the combination of (i) the macroporous nature of the chitosan foams, much like any other porous polymer, and (ii) the low amount of polymer in the solution constituting the liquid foam template, namely only 3.43 wt % of chitosan. As a result, the struts of the solid foams must be porous, although the pores are too small to be visible with the used SEM.

Figure 9. SEM pictures of (a) monodisperse and (b) polydisperse solid chitosan foams.

low-density solid foam originating from a liquid foam template, since below a liquid fraction of φ = 0.06323 the FCC structure is energetically less favorable than the Kelvin structure (which has a BCC ordering).12,18 The reason for this observation is the fact that the liquid foam template has a liquid fraction which is high enough (φ > 0.11) for the formation of a stable FCC configuration in the liquid state. Since the template is gelled and frozen before the removal of water, the bubble positions remain fixed with respect to each other during the drying process. Thus, one obtains regions with low-density, polyhedral foam structures with an FCC ordering, which, in turn, results in rhombic dodecahedra cells. Studying the system with micro computed tomography (μ-CT) one would be able to determine the range over which the pores show this FCC ordering. In order to calculate the pore volume of the monodisperse foam, we measure the center-to-center distance between two neighboring pores, as sketched in Figure 10a. All edges of a rhombic dodecahedron are of equal length L, which one can calculate from the center-to-center distance. The pore volume Vpore is then given by31 Vpore =

16L3 3 3

4. CONCLUSIONS We describe a novel microfluidic foam templating approach which makes it possible to produce polymer foams with tunable pore size distributions (mono- vs polydisperse) and different pore organizations (ordered vs disordered). The initially liquid, chitosan-containing foams are cross-linked by genipin at a rate which allows the foams to retain their structures during solidification. After an additional freeze-drying step, solid foams are obtained which show tremendous differences in their

(2)

Table 1. Comparison of Liquid and Solid Foam Structures for Monodisperse and Polydisperse Foams liquid foam

monodisperse polydisperse

solid foam

av bubble size

PDI

liquid

av pore size

PDI

av window diam

density

(μm)

(%)

fraction φ

(μm)

(%)

(μm)

(g cm−3)

rel density

364 ± 14 322 ± 48

3.7 14.2

0.11 0.06

237 ± 14 304 ± 61

5.8 20.1

35 ± 9 77 ± 30

0.0113 ± 0.0017 0.0083 ± 0.0009

0.0080 ± 0.0012 0.0058 ± 0.0006

H


Article

Langmuir

Figure 10. (a) 2D representation of the structure observed in monodisperse solid foams showing four pores. The hexagons drawn with dotted lines correspond to pores belonging to the layer below the observed pore. The grey shapes represent the regions where the windows between pores occur. dcc is the center-to-center distance between two neighboring pores and corresponds to the distance separating the two parallel faces oriented upward of a polyhedral pore. (b) 3D representation of a rhombic dodecahedron. α is the characteristic angle used to calculate L from dcc and is equal to 35.265°. L is the edge length from which one can measure the volume of the rhombic polyhedron. All edges have the same length L. Adapted from ref 31.

■

morphologies, for example, in how much they shrink, in the degree of order of the foams, or in the structure of the pores themselves. These differences can be attributed to properties of the liquid templates, particularly to the ordering of the bubbles and the liquid fraction. The bubble size distribution comes into play when the liquid foam settles and dictates how the bubbles self-organize, which, in turn, determines the liquid fraction. Interestingly, gelling the monodisperse chitosan liquid foam leads to the formation of a solid foam with rhombic dodecahedron-shaped pores. It will be interesting to investigate the mechanical properties of this structure and compare it to already existing computer simulations.31 Future work should explore a wider range of polydispersities and compare polydisperse foams with different polydispersities but the same average pore size. In conclusion, the method presented in the paper at hand lends itself to studying the influence of the pore size distribution on the mechanical properties of solid foams and is thus a promising tool for gaining a deeper understanding of structure−property relations of solid foams. Moreover, open-cell, biobased scaffolds with controllable pore sizes and pore size distributions are currently discussed as scaffolds for tissue engineering applications.

■

REFERENCES

(1) Gibson, L. J.; Ashby, M. F. Cellular Solids: Structure and Properties; Cambridge University Press: Cambridge, 1997. (2) Barby, D.; Haq, Z. Low density porous cross-linked polymeric materials and their preparation. EP0060138A1, 1982. (3) Cameron, N. R. High internal phase emulsion templating as a route to well-defined porous polymers. Polymer 2005, 46, 1439−1449. (4) Carnachan, R. J.; Bokhari, M.; Przyborski, S. A.; Cameron, N. R. Tailoring the morphology of emulsion-templated porous polymers. Soft Matter 2006, 2, 608−616. (5) Friberg, S. E.; Fang, J.-H. Foams from aqueous systems of polymerizable surfactants. J. Colloid Interface Sci. 1987, 118, 543−552. (6) Raj, W. R. P.; Sasthav, M.; Cheung, H. M. Formation of polymeric foams from aqueous foams stabilized using a polymerizable surfactant. J. Appl. Polym. Sci. 1993, 49, 1453−1470. (7) Barbetta, A.; Gumiero, A.; Pecci, R.; Bedini, R.; Dentini, M. Gasin-liquid foam templating as a method for the production of highly porous scaffolds. Biomacromolecules 2009, 10, 3188−3192. (8) Costantini, M.; Colosi, C.; Guzowski, J.; Barbetta, A.; Jaroszewicz, ́ J.; Swięszkowski, W.; Dentini, M.; Garstecki, P. Highly ordered and tunable polyHIPEs by using microfluidics. J. Mater. Chem. B 2014, 2, 2290−2300. (9) Quell, A.; Elsing, J.; Drenckhan, W.; Stubenrauch, C. Monodisperse polystyrene foams via microfluidics - A novel templating route. Adv. Eng. Mater. 2015, 17, 604−609. (10) Chung, K.-Y.; Mishra, N. C.; Wang, C.-C.; Lin, F.-H.; Lin, K.-H. Fabricating scaffolds by microfluidics. Biomicrofluidics 2009, 3, 022403. (11) van der Net, A.; Gryson, A.; Ranft, M.; Elias, F.; Stubenrauch, C.; Drenckhan, W. Highly structured porous solids from liquid foam templates. Colloids Surf., A 2009, 346, 5−10. (12) Testouri, A.; Arriaga, L.; Honorez, C.; Ranft, M.; Rodrigues, J.; van der Net, A.; Lecchi, A.; Salonen, A.; Rio, E.; Guillermic, R.-M.; Langevin, D.; Drenckhan, W. Generation of porous solids with wellcontrolled morphologies by combining foaming and flow chemistry on a lab-on-a-chip. Colloids Surf., A 2012, 413, 17−24. (13) Colosi, C.; Costantini, M.; Barbetta, A.; Pecci, R.; Bedini, R.; Dentini, M. Morphological comparison of PVA scaffolds obtained by gas foaming and microfluidic foaming techniques. Langmuir 2013, 29, 82−91. (14) Costantini, M.; Colosi, C.; Mozetic, P.; Jaroszewicz, J.; Tosato, A.; Święszkowski, W.; Dentini, M.; Garstecki, P.; Barbetta, A. Microfluidic foaming: A powerful tool for tailoring the morphological and permeability properties of sponge-like biopolymeric scaffolds. ACS Appl. Mater. Interfaces 2015, 7, 23660−23671. (15) Drenckhan, W.; Saint-Jalmes, A. The science of foaming. Adv. Colloid Interface Sci. 2015, 222, 228−259.

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (C.S.). ORCID

Cosima Stubenrauch: 0000-0002-1247-4006 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Dr. Alexander Fels for his support with the SEM measurements. We thank Dr. Angelika Menner for useful discussions and for providing the helium porosimeter. We acknowledge funding from the German Research Foundation (STU 287/4-1) and from the European Research Council (ERC, FP7/2007-2013, 307280-POMCAPS). Part of this work has been published within the IdEx Unistra framework and has benefited from funding from the state, managed by the French National Research Agency as part of the “Investments for the future” program. I


Article

Langmuir (16) Costantini, M.; Colosi, C.; Jaroszewicz, J.; Tosato, A.; Rainer, A.; Trombetta, M.; Święszkowski, W.; Dentini, M.; Barbetta, A. Correlation between porous texture and cell seeding efficiency of gas foaming and microfluidic foaming scaffolds. Mater. Sci. Eng., C 2016, 62, 668−677. (17) Testouri, A.; Honorez, C.; Barillec, A.; Langevin, D.; Drenckhan, W. Highly structured foams from chitosan gels. Macromolecules 2010, 43, 6166−6173. (18) Andrieux, S.; Drenckhan, W.; Stubenrauch, C. Highly ordered biobased scaffolds: From liquid to solid foams. Polymer 2017, 126, 425−431. (19) Muzzarelli, R. A. A. Natural Chelating Polymers; Alginic Acid, Chitin and Chitosan; Pergamon Press: Oxford, 1973. (20) Berger, J.; Reist, M.; Mayer, J. M.; Felt, O.; Gurny, R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. Eur. J. Pharm. Biopharm. 2004, 57, 35−52. (21) Weaire, D.; Pageron, V. Frustrated froth: Evolution of foam inhibited by an insoluble gaseous component. Philos. Mag. Lett. 1990, 62, 417−421. (22) Gandolfo, F. G.; Rosano, H. L. Interbubble gas diffusion and the stability of foams. J. Colloid Interface Sci. 1997, 194, 31−36. (23) Höhler, R.; Yip Cheung Sang, Y.; Lorenceau, E.; Cohen-Addad, S. Osmotic pressure and structures of monodisperse ordered foam. Langmuir 2008, 24, 418−425. (24) Meagher, A. J.; Whyte, D.; Banhart, J.; Hutzler, S.; Weaire, D.; García-Moreno, F. Slow crystallisation of a monodisperse foam stabilised against coarsening. Soft Matter 2015, 11, 4710−4716. (25) Butler, M. F.; Ng, Y.-F.; Pudney, P. D. A. Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 3941−3953. (26) Drenckhan, W.; Langevin, D. Monodisperse foams in one to three dimensions. Curr. Opin. Colloid Interface Sci. 2010, 15, 341−358. (27) van der Net, A.; Blondel, L.; Saugey, A.; Drenckhan, W. Simulating and interpretating images of foams with computational raytracing techniques. Colloids Surf., A 2007, 309, 159−176. (28) Raven, J.-P.; Marmottant, P.; Graner, F. Dry microfoams: formation and flow in a confined channel. Eur. Phys. J. B 2006, 51, 137−143. (29) Cantat, I.; Cohen-Addad, I.; Elias, F.; Graner, F.; Höhler, R.; Pitois, O.; Rouyer, F.; Saint-Jalmes, A. Foams - Structure and Dynamics; Oxford University Press: Oxford, 2013. (30) Kloek, W.; van Vliet, T.; Meinders, M. Effect of bulk and interfacial rheological properties on bubble dissolution. J. Colloid Interface Sci. 2001, 237, 158−166. (31) Babaee, S.; Jahromi, B. H.; Ajdari, A.; Nayeb-Hashemi, H.; Vaziri, A. Mechanical properties of open-cell rhombic dodecahedron cellular structures. Acta Mater. 2012, 60, 2873−2885.

J


Generation of Solid Foams with Controlled Polydispersity Using

Recommend Documents