Research Article pubs.acs.org/journal/ascecg
Fabrication of Nanocomposite Bioelastomer Porous Scaffold Based on Chitin Nanocrystal Supported Emulsion-Freeze-Casting Yaling Tian,† Kai Liang,‡ Xin Wang,† and Yali Ji*,† †
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State Key Laboratory for Modification of Chemical Fibers and Polymer Materials & College of Materials Science and Engineering, Donghua University, 2999 North Renmin Road, Songjiang, Shanghai 201620, China ‡ College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Songjiang, Shanghai 201620, China ABSTRACT: Degradable bioelastomers as porous scaffold materials have great potentials in tissue regeneration due to their compatible mechanical properties to the body’s soft tissues. A citrate-based elastomer is a recently developed bioelastomer possessing good surface affinities toward many cell types, but its insoluble and unmeltable characteristics caused difficulties in fabricating porous scaffolds. Herein, a new method where chitin nanocrystal (ChiNC) supported emulsion-freeze-casting was utilized to fabricate porous scaffolds of thermoset elastomer. ChiNC was initially used as a Pickering emulsifier to stabilize prepolymer latex spheres, then as a supporting agent to prevent ice-templated pores from collapse during thermocuring, and last as a biobased nanofiller to reinforce the porous scaffold. When ChiNC loading was at least 15% or greater, the directional porous structure were facilely obtained via use of a thick-walled Teflon container in a freezer at −40 °C. The nanocomposite elastomer porous scaffold exhibited good elastic resilience and improved mechanical properties with increasing ChiNC content. The entire process was easily performed and eco-friendly without use of a toxic agent. KEYWORDS: Bioelastomer, Poly(1,8-octanediol citrate), Pluronic F127, Chitin nanocrystal, Nanocomposite
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induced phase separation (TIPS),15,16 solvent casting/particulate leaching,17−19 sintering,20 gas foaming,21 melt-molding,22 solid free-form fabrication (SFF)23 and electrospinning.24 Unlike other common biopolymers, POC thermoset elastomer reveals certain limitations and challenges in using those methods to manufacture porous scaffolds due to the harsh curing condition, i.e., a long curing time (several days) in a vacuum environment, with the exception of solvent casting/ particulate leaching. Ameer et al.25,26 used particulate-leaching technique to prepare porous POC scaffolds for tissue regeneration with salt as porogen. They also tried to use poly(ethylene glycol) dimethyl ether (PEGDM) as porogen and obtained nanoporous POC scaffolds with porosity of 86.8% and average pore diameter of 151 nm, suitable for nerve soft tissue engineering applications.27 Although the particulate leaching method is easy to perform without the need of specialized equipment, it is a tedious process involving repeated washing, and not easy to prepare a large-volume scaffold due to the difficulty in leaching out the particles from the inside. Another processing method that has been utilized to fabricate interconnected porous POC scaffolds is the use of inverse 3D hydroxyapatite (HA) porous molds, constructed by a 3D
INTRODUCTION Poly(1,8-octanediol citrate) (POC), a cross-linked polyester elastomer, has great potential in the tissue engineering field due to the compatible mechanical properties to soft tissues and controllable and linear degradation profiles.1−3 POC was synthesized by a facile low-temperature melt polycondensation of citric acid and 1,8-octanediol and subsequent thermo-crosslinking without using any additives.1 To tailor the functionality and physicochemical property of POC, various POC-based copolymeric systems were developed, including urethanedoped POC (CUPE),4,5 fumaric, maleic or acrylate-doped POC,6−8 and our newly synthesized poly(1,8-octanediol-coPluronic F127 citrate) (POFC).9 POC-based bioelastomers have been explored for numerous tissue engineering applications because of the surface affinities toward many cell types,4,10−13 such as endothelial cells, mesenchymal stem cells and chondrocytes, but the cross-linked structure makes them insoluble and unmeltable, which restricts processing techniques that can be used to fabricate porous scaffolds for tissue regeneration. As a cell scaffold, an interconnected pore network structure is required in order to support the attachment, migration, proliferation and differentiation of cells in a well controlled manner, facilitate the transport of nutrients and metabolites to and from the cells, and thus, direct the regeneration process.14 Commonly used conventional techniques for fabrication of porous scaffolds include thermally © 2017 American Chemical Society
Received: December 24, 2016 Revised: February 24, 2017 Published: February 26, 2017 3305
DOI: 10.1021/acssuschemeng.6b03146 ACS Sustainable Chem. Eng. 2017, 5, 3305−3313
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printing method.28,29 The complex 3D HA molds were immersed in the POC prepolymer solution, followed by curing POC prepolymer and subsequently dissolving HA to obtained 3D POC scaffolds. But these 3D POC scaffolds exhibited a large average pore size of 0.75−0.9 mm and a low porosity of 50−62% due to the limitations of 3D printing resolution. Additionally, a new method referred to as low-pressure foaming (LPF) was proposed for the fabrication of POC porous scaffolds.30 During LPF, air bubbles, nucleated within the prepolymer matrix during a mixing step, expanded under vacuum, coalesced, and ultimately produced an interconnected pore network. LPF was easily performed without the use of organic solvents, supercritical fluid, or particle leaching, and the reported average pore size was in the range of 85−1000 μm, but the porosity had a range of only 24.8−47.2%. The electrospinning process was also used to produce POC-based porous nonwoven meshes.31,32 The meshes exhibited large surface areas, nonuniform pore structures and small pore sizes (several micrometers). But only blend fibers were produced because the POC prepolymer cannot be electrospun. Thus, it is indispensable to select a spinnable carrier polymer to be well mixed with POC prepolymer for smooth electospinning, such as poly(lactic acid-co-caprolactone)31 and collagen.32 Chitin, the second most abundant biopolymer following cellulose, has become an attractive biomaterial for applications in tissue engineering and biomedicine.33 Compared with its chitosan derivative, chitin possesses a more stable structure and improved compatibility,34 but the difficulties in dissolving and processing constrained its wide development in biomedicine field. Recently, Zhang et al.34,35 used a special solvent system to dissolve chitin and prepare hydrogel/fibrous chitin scaffolds for biomedical applications. Different from Zhang et al., Ehrlich and colleagues36−39 initiatively isolated three-dimensional nanofibrous chitin scaffolds from the skeleton of marine sponges for potential applications in tissue engineering. It is an environmentally sustainable route to generate chitin as a biomaterial. Here, we introduce a new processing technique described as chitin nanocrystals (ChiNCs) supported emulsion-freezecasting to fabricate thermoset nanocomposite porous scaffolds. ChiNC, a product of acid-treatment of chitin, is an emerging, novel nanofiller that is nontoxic, biocompatible and biodegradable.40−42 It can be used to regulate the mechanical properties of POC elastomer,43 and neutralize the acidic physiological environment resulting from the degradation products of POC. Moreover, it has already been reported that ChiNC particles are able to stabilize oil/water interfaces, forming so-called Pickering emulsions.44,45 In our previous study,9 Pluronic F127 was incorporated into a POC network to generate a new bioelastomer referred to as POFC, of which the prepolymer could disperse in water without the help of organic solvent and emulsifier. Thus, in this study we used ChiNC and POFC prepolymer to construct a Pickering-like emulsion, and then used freeze-casting method to fabricate a ChiNC/prepolymer porous scaffold, subsequently thermo-cross-linked the prepolymer, and ultimately obtained the ChiNC reinforced nanocomposite elastomer porous scaffold. In this process, ChiNC was first used as an emulsifier to stabilize prepolymer latex spheres, then as a supporting agent to prevent pores’ collapse during thermo-cross-linking, and last as a biobased nanofiller to reinforce elastomer porous scaffold. The entire process was easily performed and eco-friendly without using any toxic agent.
Research Article
EXPERIMENTAL SECTION
Materials. Pluronic F127 (PEO99−PPO65−PEO99) was purchased from Sigma-Aldrich Co. Ltd., USA. Citric acid (AR grade) and 1,8octanediol (AR grade) were supplied by Sinopharm Chemical Reagent Co. Ltd., China. Chitin flakes (from snow-crab shell) were provided by Kehai Chitin Co. Ltd., Shangdong, China. All chemicals were used as received without further purification. Preparation of ChiNC. Briefly, chitin flakes were hydrolyzed in 3 M HCl under stirring and refluxing for 6 h. The ratio of HCl solution to chitin was 30 mL/g. The residue was collected by centrifugation (9000 rpm, 10 min), and then repeated the above operations two more times. Then, the residue was washed with deionized (DI) water for three times via centrifugation (9000 rpm, 10−30 min). The obtained suspension was further dialyzed using regenerated cellulose dialysis tubes (25 mm in width, MWCO 8000−14 000, Sinopharm Chemical Reagent Co., China) in DI water at room temperature for 3 d, followed by ultrasonic treatment for 20 min and subsequent filtration to remove residual aggregates. Finally, the clear suspension was lyophilized to obtain light brown powders. Transmission electron microscopy (TEM) was used to estimate the average length and width of ChiNC, about 300 and 20 nm, respectively. This size is within the normal size of ChiNC that was extracted from shrimp/crab shell, i.e., 200−600 nm in length and 10−50 nm in width.41 Before use, the ChiNC were redispersed in DI water by sonification. Preparation of POFC Prepolymer. POFC prepolymer was synthesized according to Yang’s method but with different monomers.1 Briefly, citric acid, 1,8-octanediol (molar ratio 1.1:1) and F127 (20% with respect to total mass of citric acid and 1,8octanediol) were added to a 250 mL round-bottom flask and exposed to a constant flow of nitrogen gas. The mixture was fully melted at 160 °C under vigorous stirring and then kept stirring for 2 h at 140 °C to create POFC prepolymer. The 1H NMR spectroscopy (400 MHz, DMSO-d6) was used to confirm the chemical structures of the prepolymer. The peak assignments were listed below: 1.24−1.55 ppm, from −(CH2)6− of 1,8-octanediol; ∼2.79 ppm, from −CH2CO2− of citric acid; ∼4.0 ppm, from −OH of citric acid; ∼1.05 ppm, from −CH3− of F127; ∼3.5 ppm, from −OCH2− of F127 and 1,8octanediol. The molecular weight (Mn) and molecular weight distribution (d) of the prepolymer measured by gel permeation chromatograph-light scattering (GPC-LS, BI-MwA, THF) were Mn = 9.4 × 104 and d = 1.12. Preparation of ChiNC/POFC Porous Scaffold. POFC prepolymer was dissolved in DI water, and to it a desired amount of ChiNC aqueous suspension was added. The mixture was sonicated for 1 h, poured into a cylindrical thick-walled (∼10 mm in thickness, ∼50 mm in inner diameter, ∼20 mm in depth) Teflon mold without lid, frozen at −40 °C in a freezer for over 4 h, and lyophilized at −50 °C, followed by thermo-cross-linking at 80 °C in a vacuum for 3 d. For convenient comparison, the total mass of POFC and ChiNC (i.e., solid content) was controlled at 16 wt %. The mass percentages of ChiNCs filler were controlled at 15%, 20%, 25%, 30%, 35% and 40%. Because the upper surface of the emulsion closely contacted the cryogen, there existed a uniaxial temperature gradient, which caused the emulsion to undergo unidirectional freezing from the top to the bottom. In addition, an aluminum mold (∼2 mm in thickness, ∼45 mm in inner diameter, ∼25 mm in depth) with an aluminum lid was also used to freeze homogeneously the sample at −40 °C. Moreover, a simple experimental setup was designed to investigate directional-freezecasting using liquid nitrogen as cryogen. It consisted of an aluminum finger, of which one end was immersed in a liquid nitrogen bath and the other end supported a tube (∼2 mm in thickness, ∼30 mm in inner diameter, ∼30 mm in depth) filled with suspension. The tube was made of ABS resin and insulated using polystyrene foam to prevent horizontal heat transfer to the suspension. A copper sheet was attached to the bottom of the tube in favor of vertical heat conduction. Thus, a uniaxial temperature gradient was formed and the suspension underwent directional freeze from the bottom to the top. Measurement and Characterization. Dynamic light scattering (DLS) measurements were performed using a Zetasizer Nano ZS 3306
DOI: 10.1021/acssuschemeng.6b03146 ACS Sustainable Chem. Eng. 2017, 5, 3305−3313
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Figure 1. Illustration of preparation of ChiNC/POFC porous scaffolds. (a) reaction scheme for POFC prepolymer; (b) photograph of POFC prepolymer aqueous suspension; (c) latex size from DLS measurement; (d) FESEM micrograph from ChiNC/POFC emulsion; (e) schematic presentation of ChiNC supported emulsion-freeze-casting process. instrument (Malvern Instruments Ltd., UK) to obtain the latex’ size and distribution in the dispersion containing POFC prepolymer and ChiNC. The dispersions were diluted to 0.1% (w/w) with DI water. The Pickering-like emulsion structure was explored via field emission scanning electron microscopy (FESEM, SU8010, Hitachi Ltd., Japan). The diluted emulsion was dropped onto an aluminum foil, evaporated and observed. The morphologies of porous scaffolds were examined on a FESEM (SU8010, Hitachi Ltd., Japan). The samples were cryo-fractured in liquid nitrogen and sputter coated with platinum. The porosity was examined using the ethanol displacement method.46 In brief, a pycnometer was filled with ethanol to the brim (weighed as W1), and the scaffold (weighed as Ws) was placed into it. Subsequently, the pycnometer was vacuumized to force ethanol into the internal pores of the scaffold and again filled with ethanol to the brim (weighted as W2). Then, the scaffold was taken out and the pycnometer was weighed again as W3. The porosity is calculated as
porosity (%) = (W2 − W3 − Ws)/(W1 − W3)
The cubic samples (10 mm in side length) underwent 20 compressive loading−unloading cycles to test the compressive recovery property and assess the ability of scaffold to recover from deformation. The compression modulus (Young’s) was calculated from the initial slope of the stress−strain curve. The compression stress at 60% strain was also recorded at first compression cycle. Degradation was conducted in PBS medium (pH = 7.4). The specimens were weighed (Wc), placed into 50 mL of PBS, and then incubated at 37 °C for a period of time. At predetermined time intervals from week 1 to week 9, the sample was removed and thoroughly washed with DI water, lyophilized and weighed (Wd). The weight loss was calculated using the equation as
weight loss = [(Wc − Wd)/Wc] × 100%
Three individual tests were performed and data were averaged. Data are expressed as means and standard deviations (SD), with at least three samples. One-way analysis of variance (ANOVA) with Bonferroni analysis was performed for comparing means between two or multiple groups, and a p value of 0.05 was considered statistically significant.
(1)
Three individual tests were performed and data were averaged. Liquid absorption was examined in phosphate buffered saline medium (PBS, pH = 7.4). Cubic samples (10 mm × 10 mm × 10 mm) of the known mass (Wa) were incubated in PBS medium for 72 h, removed and gently wiped with filter paper to remove excess PBS on the surface, and weighed (Wb). The PBS uptake was calculated using the expression as PBS uptake = [(Wb − Wa)/Wa] × 100%
(3)
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RESULTS AND DISCUSSION Preparation of ChiNC/POFC Porous Scaffold. The strategy of fabricating ChiNC/POFC porous scaffold is schematically illustrated in Figure 1. First, the POFC prepolymer was prepared by a facile one-pot additive-free melt polycondensation of 1,8-octanediol, F127 and citric acid (see Figure 1a), and its structure was confirmed by 1H NMR and GPC-LS analyses. The prepolymer containing 20 wt % F127 could disperse in water to form emulsion without using any organic solvent and emulsifier because of its amphiphilic
(2)
Three individual experiments were performed and data were averaged. The mechanical behaviors were examined on an Instron-5969 material testing machine (Instron Ltd., USA) under compression mode at a crosshead speed of 10 mm/min and a force of 100 N at room temperature. The ultimate compression strain was set to 60%. 3307
DOI: 10.1021/acssuschemeng.6b03146 ACS Sustainable Chem. Eng. 2017, 5, 3305−3313
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cold source, the temperature gradient direction was in a sense vertically downward and resulted in the preferential growth of ice crystals vertically downward as well. Eventually, a long-range irregular lamellae-channeled architecture is formed as shown in Figure 2a,b,c,g,h,i. Furthermore, the interlamellae space was
nature endowed by F127 (see Figure 1b). The ChiNC is also well dispersed in water. It was assumed that once the prepolymer emulsion was mixed with ChiNC aqueous suspension, the hydrophilic superficial layer of the prepolymer latex sphere would attract ChiNC nanoparticles and be coated with ChiNC layer to construct so-called nanoparticle stabilized Pickering emulsion. The DLS measurement results (see Figure 1c) presented that the size of latex sphere in pure POFC dispersion was ∼190 nm; however, the measured size gradually increased to ∼340 nm under 10% ChiNCs loading, 1100 nm under 20% loading and 1200 nm 30% loading, indirectly confirming our assumption that a Pickering-like emulsion composed of ChiNCs coated POFC latex particles. This point was further proved by FESEM observation of the diluted emulsion containing 30% ChiNCs, as shown in Figure 1d. In the FESEM micrograph, there appeared a large number of irregular spherical particles. Several typical particles were deliberately highlighted using red circles in the image. The particle size was estimated ∼1 μm, which was in accordance with the results of DLS measurement. Similarly, using ChiNC nanoparticle as stabilizer to form Pickering emulsion was also reported in other works,44,45 in which ChiNCs were always used to stabilize micromolecular oil droplets. In our case, ChiNCs were used to stabilize macromolecular micelles, but the nature was the same. Subsequently, the ice-templated assembly was utilized to create porous structure via directly freeze-drying the Pickering-like emulsion (see Figure 1e). At the stage of freezing, the growth of ice crystals repelled latex spheres and solidified water; and at the next stage of lyophilization, the sublimation of the solidified water left porous frame. It was found though the freeze-dried POFC prepolymer possessed porous structure, when it underwent thermo-cross-linking, the formed porous structure inevitably collapsed and ultimately formed nonporous elastomer due to the melt flow of the prepolymer. Contrarily, as for the ChiNC/ POFC prepolymer system, with the growth of ice crystals, the ChiNC coated latex spheres were squeezed, causing the ChiNCs to locate selectively at the interstitial space between prepolymer latex spheres and form an interconnected network structure, into which the prepolymer latex spheres were embedded. Such ChiNC-based network structure firmly supported the prepolymer domains and prevented pores from distortion and collapse during thermosetting. Eventually, the porous structure formed during freeze-drying was retained after thermocuring as long as the ChiNCs loading was 15% or greater. When there is insufficient ChiNC to form a continuously interconnected structure, only nonporous elastomer was obtained. Morphologies of ChiNC/POFC Porous Scaffold. In the emulsion-freeze-casting method, the porous structure can be tailored by the growth of ice crystals including growth direction, growth rate, water content, etc.47,48 Ice crystals grow preferentially along temperature gradient, and thus controlling temperature gradient can realize regulation of scaffold pore direction. In addition, a fast freezing rate favors supercooling and, hence, impedes the formation of large ice crystals, resulting in smaller-sized pores.48 The volume of water directly decides porosity as long as the framework does not collapse. Thus, the freeze-drying ice-templated assembly is a promising technique to fabricate porous scaffolds with tuned pore architecture. In our case, a lidless thick-walled Teflon container was used to hold the suspensions and then put into a −40 °C freezer. Because the upper surface of the suspension directly contacted
Figure 2. Representative SEM images of ChiNC/POFC porous scaffolds with different ChiNC loading. (a, d) 15% ChiNC; (b, e) 20% ChiNC; (c, f) 25% ChiNC; (g, j) 30% ChiNC; (h, k) 35% ChiNC; (i, l) 40% ChiNC. The top 2 rows of images show the morphologies of perpendicular (cross section) planes to ice-growth direction, and bottom 2 rows of images show the morphologies of parallel (side view) planes to ice-growth direction, as shown in the corresponding cartoons (scale bar = 200 μm, freezing temperature = −40 °C).
bridged by many protrusions or pillars to form irregular cells, meaning smaller pores were constructed simultaneously (see Figure 2d,e,f,j,k,l). It was found that increasing ChiNC loading did not take much effect on the interlamellae distance, all in the range of 60−120 μm (see Figure 2a,b,c,g,h,i); however, the surface morphologies of lamellae varied from the irregular dendritic-like features (see Figure 2d−f) to the denser “fishbone” features49 (see Figure 2j−l), meaning the size of irregular cells between layers was evidently scaled down. Zhou et al.49 once observed when the ChiNC/PVA aqueous system underwent directional freeze-casting by liquid nitrogen, the interlamellae spaces varied from 180 to 30 μm with increasing PVA content from 0−2.0 wt %. Thus, the interlamellae distance is related to the solid content, and high solid content results in short interlamellae distance. In our case, solid content and freezing velocity for all samples were the same, thus we supposed that ice crystal growth suffered the equivalent resistance generated by excluding solid substance from its growth front, leading to nearly similar interlamellae distance. Whereas, at a constant solid content, increasing ChiNCs resulted in smaller-sized cells between lamellas, in that largersized latex particles, formed from higher content of ChiNCs, moved relatively slowly and did not have enough time to travel to the gaps before ice crystal grew forward. As a result, more particles were enveloped by the ice crystals (tip splitting) instead of being entrapped between ice crystals. After that, subsequent tip healing occurred and formed additional bridge structures.50 We also explored the effects of solid content on the pore structure. The ChiNC/POFC (30:70) suspensions with 5, 10 and 20 wt % solid contents underwent freeze-casting and 3308
DOI: 10.1021/acssuschemeng.6b03146 ACS Sustainable Chem. Eng. 2017, 5, 3305−3313
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ACS Sustainable Chemistry & Engineering subsequent thermocuring. The results are shown in Figure 3. At a lower solid content, i.e., 5 wt % (see Figure 3a,d), there
Figure 4. Representative SEM images of ChiNC/POFC (30:70) porous scaffolds. (a) Cross section, low magnification; (b) cross section, high magnification; (c) side view, low magnification; (d) side view, high magnification. The cross section shows the morphology of perpendicular plane to ice-growth direction, and side view shows the morphology of parallel plane to ice-growth direction, as shown in the corresponding cartoons (16% solid content, the freezing temperature = −40 °C, freezing in a lidded aluminum container).
Figure 3. Representative SEM images of ChiNC/POFC (30:70) porous scaffolds with different solid contents. (a, d) 5% solid content; (b, e) 10% solid content; (c, f) 20% solid content. The top row of images show the morphologies of perpendicular (cross section) planes to ice-growth direction, and bottom row of images show the morphologies of parallel (side view) planes to ice-growth direction, as shown in the corresponding cartoons (scale bar = 200 μm, freezing temperature = −40 °C).
that lamellae-channeled structure is scaled down. In addition, a denser irregular dendritic-like morphology was observed on the lamellar walls, indicating smaller-sized interlayer pores (see Figure 5c,d). Porosity and PBS Uptake of ChiNC/POFC Porous Scaffold. The porosity and PBS uptake for the porous scaffolds with various ChiNC loading levels are depicted in Figure 6a,b. The porosity of all scaffolds was in the range of 78.4 ± 5.1− 79.1 ± 4.6%, which was comparable to that obtained from solvent casting/particulate leaching technique,25,26 and much higher than that obtained from 3D printing technique28,29 and LPF technique.30 The porosity is related to the solid content of suspension in freeze-casting technique, and here, the solid content for all the suspensions remained constant, i.e., 16%, thus the porosity was basically unchanged with ChiNC content. The PBS uptake for all the scaffolds was in the range of 490.9 ± 27.5−510.2 ± 40.5%. This value was much larger than that of corresponding nonporous elastomers,51 whose uptake ratio was influenced by ChiNC content. Unlike that, here, the PBS uptake ratio of porous scaffolds was irrelevant to ChiNC content, but mainly dependent on the porosity. Mechanical Properties of ChiNC/POFC Porous Scaffold. Because the directional freeze-casting process endowed the porous scaffolds with anisotropic lamellae pore structures, we hypothesized that the mechanical properties in longitudinal direction and transverse direction were different. However, the compression tests (see Figure 7a,b) showed there was no pronounced difference in Young’s modulus and stress (at 60% strain) along the two orthogonal directions, indicating anisotropic pore structure did not induce anisotropic mechanical behavior in this system. This result was different from the study of Chau et al.,52 in which the Young’s moduli from longitudinal direction were much larger than that from transverse direction in the cellulose nanocrystal/POEGMA system. Generally, for the lamellar channel structures, compression in the longitudinal direction led to buckling of the lamellar walls, and compression in the transverse direction, at low strain, led to the collapse of the lamellar pores, and then, the stressing of the pore walls.52 Because there existed many cells formed from protrusions or pillars between the layers in our system, which supported lamellar channels, the mechanical properties in the transverse direction was strengthened and caused no difference in the two orthogonal directions. This point needs to be further explored in the future study.
appeared random and nondirectional pore structure instead of lamellar channel structure; with increasing solid content to 10 wt % (see Figure 3b,e), the lamellar channel structure started to be distinct. However, the significant structural defects and large size cracks were found on the lamellar walls; further increasing solid content to 20 wt % (see Figure 3c,f) produced more distinct lamellar channels, and the lamellar walls presented “fish-bone” morphology,49 meaning smaller-sized cells formed. Lee et al.50 also used directional-freeze-casting technique to fabricate cellulose microfibril foam and observed that increasing the concentration of cellulose microfibril suspension led to a gradual transition from a disordered network structure to a lamellar channel structure, which is very similar to our results. But the potential mechanism is still unknown. In addition, the interlamellae distance decreased with increasing solid content, which was consistent with the results of Zhou et al.49 In brief, in our system the solid content is a key factor that affected both the regularity of lamellar channels and the interlamellae distance. For contrast, a lidded aluminum container containing the suspension (30% ChiNC loading percentage) was also put into −40 °C freezer to perform nondirectional-freeze-casting. As expected, after lyophilization and thermosetting, an isotropic porous structure occurred as shown in Figure 4a−d. Whether the cross section view or side view, both exhibited irregular rounded pores with tens of micrometer size, very different from that obtained using a thick-walled Teflon mold, further confirming that using a lidless thick-walled Teflon mold as container can facilely realize directional-freeze-casting and construct anisotropic porous structure. The pore morphology is also dependent on the freeze-casting temperature and thus the rate of solvent crystallizes.47 To explore the effects of temperature on the pore morphology of ChiNC/POFC scaffold, a self-made directional-freeze-casting setup consisted of an aluminum finger was applied, of which one end of the finger was immersed in liquid nitrogen and the other end was used to support a container filled with the suspension (see animated setup in Figure 5). As expected, a lamellar channel morphology also appeared, but the interlamellae distance became much smaller than that obtained from −40 °C freezer (see Figure 5a,b). Such result is relatively rational because a lower temperature results in a faster freezing rate, and, hence, impedes the formation of larger ice crystals, so 3309
DOI: 10.1021/acssuschemeng.6b03146 ACS Sustainable Chem. Eng. 2017, 5, 3305−3313
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Figure 5. Representative SEM images of ChiNC/POFC (30:70) porous scaffolds. (a) Cross section, low magnification; (b) cross section, high magnification; (c) side view, low magnification; (d) side view, high magnification. The cross section shows the morphology of perpendicular plane to ice-growth direction, and side view shows the morphology of parallel plane to ice-growth direction, as shown in the corresponding cartoons (freezing at liquid nitrogen temperature).
Figure 6. (a) Porosity and (b) PBS uptake for ChiNC/POFC porous scaffolds with different ChiNC content (porosity, p > 0.05; PBS uptake, p > 0.05).
degradation rate. Moreover, the ChiNC loading ratio did not have significant effects on degradation rate, either. However, compared with POFC, the presence of ChiNC highly prolonged in vitro degradation whether in porous scaffold form or in nonporous film form.
Furthermore, it could be confirmed that with increasing ChiNC loading the Young’s moduli and stresses (at 60% strain) highly increased in both directions, which endowed the scaffolds with the ability to sustain the structural integrity under a certain amount of compression. The 20 compressive cycle tests were performed both in longitudinal direction and transverse direction. It was found the stress−strain curves for all the porous scaffolds exhibited similar behavior. Representative stress−strain curves for the scaffold containing 20% ChiNC are as an example shown in Figure 7c,d. The loading stress−strain curve for the first compressive (virgin) cycle both in longitudinal direction (see Figure 7c) and transverse direction (see Figure 7d) did not well overlap with other 19 compressive cycles, due to preconditioning.53 Essentially, the successive compressive cycles seemed to nearly overlap in the loading−unloading compressive curves, indicating all the porous scaffolds exhibited cyclable compressibility and elastic resilience, especially in the transverse direction. In Vitro Degradability of ChiNC/POFC Porous Scaffold. In vitro degradation behavior of ChiNC/POFC porous scaffolds in PBS medium is depicted in Figure 8. The pure POFC elastomer film exhibited a quick degradation rate in comparison to ChiNC containing elastomers, in that the incorporation of ChiNCs provided additional cross-links due to the chemical bonding between ChiNC and POFC,51 and those extra crosslinks resulted in a denser network structure, and thus delayed in vitro degradation of ChiNC/POFC. However, taking the sample containing 30% ChiNC for example, comparing its porous scaffold form with nonporous film form, it was evident that the porous structure did not exert much influence on the
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CONCLUSION The ChiNC reinforced nanocomposite elastomer porous scaffolds have been produced by a facile and eco-friendly process of ChiNC supported emulsion-freeze-casting as long as ChiNC loading percentage was 15% or greater. The ChiNC was initially used as a Pickering emulsifier to stabilize POFC prepolymer latex spheres, then as a supporting agent to prevent ice-templated pores from collapse during thermocuring, and last as a biobased nanofiller to reinforce porous scaffolds. Furthermore, by using a thick-walled Teflon container as mold, the lamellae-channeled pore architecture was produced in a freezer at −40 °C resulting from the unidirectional growth of ice crystals. The nanocomposite elastomer porous scaffold exhibited cyclable compressibility and elastic resilience. The Young’s moduli and compression stresses both in longitudinal direction and transverse direction increased with increasing ChiNC content. The presence of ChiNC highly prolonged in vitro degradation for both the porous scaffold form and the nonporous film form. This preliminary work provided a facile method to fabricate ChiNC/POFC nanocomposite elastomer porous scaffold for potential applications in tissue regeneration; however, more detailed studies on regulation of pore size and pore interconnectivity, etc. need to be carried out in future. 3310
DOI: 10.1021/acssuschemeng.6b03146 ACS Sustainable Chem. Eng. 2017, 5, 3305−3313
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Figure 7. Mechanical properties of ChiNC/POFC porous scaffolds. (a) Young’s modulus and (b) compression stress at 60% strain at the first compression cycle in the longitudinal direction and transverse direction. (c) Compressive stress−strain curves for a representative sample containing 20% ChiNC over 20 compression cycles in the dry state (at 25 °C) along (c) longitudinal direction (ice-growth direction) and (d) transverse direction (perpendicular to ice-growth direction) (*p < 0.05).
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ACKNOWLEDGMENTS This work was supported by the program from the National Natural Science Foundation of China (Grant No. 51303024), and the Fundamental Research Funds for the Central Universities (Grant No. 2232014D3-10).
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Figure 8. In vitro degradation of ChiNC/POFC porous scaffolds and elastomer films in PBS medium.
Moreover, as a potential scaffold material, the good surface affinity toward cells is very essential for cell adhesion and proliferation on it. On this basis, our future work will focus on the study of scaffold’s affinity toward chondrocytes in order to explore its application in cartilage reconstruction.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Y. Ji. E-mail:
[email protected]. ORCID
Yali Ji: 0000-0003-2316-5388 Notes
The authors declare no competing financial interest. 3311
DOI: 10.1021/acssuschemeng.6b03146 ACS Sustainable Chem. Eng. 2017, 5, 3305−3313
Research Article
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