Aerogel Microspheres from Natural Cellulose Nanofibrils and Their

Jun 4, 2014 - College of Quartermaster Technology, Jilin University, Changchun, Jilin ... at Georgia Tech, Georgia Institute of Technology, Atlanta, G...
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Aerogel Microspheres from Natural Cellulose Nanofibrils and Their Application as Cell Culture Scaffold Hongli Cai,†,‡ Sudhir Sharma,‡ Wenying Liu,‡ Wei Mu,‡ Wei Liu,‡ Xiaodan Zhang,‡ and Yulin Deng*,‡ †

College of Quartermaster Technology, Jilin University, Changchun, Jilin Province 130062, China School of Chemical and Biomolecular Engineering and IPST at Georgia Tech, Georgia Institute of Technology, Atlanta, Georgia 30318, United States

‡

S Supporting Information *

ABSTRACT: We demonstrated that ultralight pure natural aerogel microspheres can be fabricated using cellulose nanofibrials (CNF) directly. Experimentally, the CNF aqueous gel droplets, produced by spraying and atomizing through a steel nozzle, were collected into liquid nitrogen for instant freezing followed by freeze-drying. The aerogel microspheres are highly porous with bulk density as low as 0.0018 g cm−3. The pore size of the cellulose aeogel microspheres ranges from nano- to macrometers. The unique ultralight and high porous structure ensured high moisture (∼90 g g−1) and water uptake capacity (∼100 g g−1) of the aerogel microspheres. Covalent crosslinking between the native nanofibrils and cross-linkers made the aerogel microspheres very stable even in a harsh environment. The present study also confirmed this kind of aerogel microspheres from native cellulose fibers can be used as cell culture scaffold.

1. INTRODUCTION Cellulose has received increasing interest in research because it is safe, stable, nontoxic, biodegradable, renewable, low cost, and abundant in nature. The polysaccharide could be modified in different chemical or physical ways to obtain derivatives with specific properties. Polysaccharide polymers can be used as thickeners, stabilizers, suspending agents, gelling agents, film formers, emulsifiers, lubricants, and over recent years especially in medicine as scaffold materials in tissue engineering and as carriers for drug delivery.1,2 Aerogel is a type of intriguing material prepared by replacing the liquid solvent in a gel with air without substantially altering the network structure or the volume of the gel body.3 Since they could be synthesized with a unique topological porous structure with porosity up to 99%, aerogels have many exciting properties, including low density, high specific surface area, and low dielectric permittivity as well as extraordinarily low thermal conductivity, holding 15 entries in Guinness World Records for material properties.3−6 As the new generation of materials, cellulose aerogel possesses features of a traditional aerogel7,8 and, at the same time, has its own excellent specificity. It promises not only a very low density as typical for aerogels but also a comparatively high strength and ductility compared with inorganic or polymeric aerogels.9−11 Therefore, cellulose aerogels have received great attention.12−16 Different cellulose sources and preparation methods could make the aerogel materials with totally different microstructures and properties. Usually, cellulose aerogels were prepared from regenerated cellulose that was made by dissolving native cellulose in a solvent followed by regeneration using a nonsolvent.12,14,17−20 More recently, aerogels from regenerated cellulose were prepared © XXXX American Chemical Society

using a freeze-drying process or a supercritical carbon dioxide process as subsequent drying to prevent pore collapse. With these drying methods, the resulting cellulose aerogels could have a high specific surface area as desired.12,21−23 However, the limitation of these methods was obvious. The conventional cellulose solvents are toxic and have only limited dissolution capability for high molecular weight cellulose.17 The required processing steps of dissolution, gelation, and solvent exchange are very slow and can take several days.24 Different from most aerogels reported in the literature, microsphere structured aerogel is a unique material that can be used as a template for drug delivery, superabsorbent, synthesizing porous inorganic catalysts or functional composite particle, cell culture growth template, targeted release system, etc.25−29 Microspheres can be principally produced using various wellknown techniques. However, because of aerogel’s mechanical properties, it is difficult, rather impossible, to obtain spherical microparticles by milling or crushing of the monolithic aerogels. Previous studies have shown that most synthesis routes to produce different kinds of inorganic, polysaccharide, and hybrid aerogel microspheres were based on the various combinations of sol−gel formation, emulsion process, ambient pressure drying, and supercritical extraction technique.30−33 Chaichanawong et al. successfully prepared carbon aerogel microspheres using sol−gel polycondensation of a resorcinol-formaldehyde solution containing sodium carbonate as a catalyst and emulsification method, followed by solvent exchange, supeŕ critical drying, and carbonization.34 Garcia-Gonzá lez syntheReceived: March 14, 2014 Revised: May 15, 2014

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mechanical blending for 10 min to get the consistent fiber suspensions. After that, the obtained cellulose fiber suspensions were diluted to the desired concentration through adding deionized water and then subjected to the mechanical defibrillation process using a high speed shear homogenizer (T18 basic, Ultra Turrax, IKA Works Inc., USA) at 20 000 rpm for 3 h. A weighted amount of cross-linker, Kymene, was added to the cellulose suspensions with mechanical stirring and finally treated in an ultrasonic bath for 10 min to obtain the native cellulose nanofibril aqueous gels. The direct evidence of cross-linking between cellulose and epichlorohydrin resin has been previously studied.41,42 After being transferred into a 500 mL steel feed tank, the cellulose aqueous gel was sprayed and atomized at 40 MPa constant pressure through a 1.0 mm inner diameter steel nozzle. The spray droplets of the suspension were collected in a cooled beaker full of liquid nitrogen. Then the frozen samples were freeze-dried in a lyophilizer (VirTis Freezemobile 25EL Sentry 2.0, USA) at a condenser temperature of −84.5 °C. The samples were kept frozen during the drying under a vacuum of 80 mTorr for 24 h. The cross-linking of aerogel microspheres was achieved by curing the dried samples containing the cross-linker in a vacuum oven at 120 °C for 3 h. In order to make a comparison of the properties, cross-linked aerogel microspheres with different starting pulp fibers (cellulose content in the suspension) and cross-linker concentrations were prepared following the same procedure as mentioned above. 2.3. Characterization. 2.3.1. SEM. Scanning electron microscopy (the Zeiss LEO 1530 microscopy) was carried out to study their morphological characteristics. The samples were attached to the holders with conductive double side carbon tape and sputter coated with gold to avoid charging during the tests. 2.3.2. Bulk Density. Bulk density was determined by transferring a known quantity of microspheres to a 100 mL measuring cylinder and tapping it 3 times at 2 s intervals. The bulk density was obtained by dividing the weight of the sample by the final volume of the sample.43 2.3.3. Particle Size Analysis. The samples of prepared microspheres were randomly selected, and their size was determined by averaging more than 100 particles using an optical microscope (Olympus, India). 2.3.4. TGA. Thermal stability of the aerogel microspheres was tested on a PerkinElmer STA 6000 thermal analyzer. The experiment was done at a heating rate of 10 °C min−1 under a nitrogen flow of 20 mL min−1. 2.3.5. Moisture Rate and Water Uptake Capacity. The aerogel microspheres were placed in 51% relative humidity conditions at 23 °C for 1 week and then weighed before and after drying in a vacuum oven at 105 °C for 24 h to measure their moisture rate. The water uptake capacity of the aerogel microspheres was calculated by setting the weight difference between the dried and the fully hydrated samples. The aerogel microspheres were dried at 105 °C for several days to obtain unchanged weight. About 1 g of dried microspheres were weighed and then soaked in water until the weight remained constant. The wetted samples were wiped with a blotting paper and immediately weighed to determine their wet mass weighed again. The water uptake of aerogel microspheres was calculated. 2.3.6. BET. The specific surface area was determined by N2 adsorption/desorption measurements (Gemini, Micromeritics, USA). Both adsorption and desorption isotherms were measured, and the surface area was determined from the adsorption results using the Brunauer−Emmet−Teller (BET) method. All samples were first heated at 95 °C in an oven and then sealed in 9 mm BET tubes and weighed. The samples were degassed for 24 h at 100 °C under vacuum using a Quantachrome FloVac degassing unit. After degassing the sealed tubes were weighed again, and the dry weight of the samples was calculated. The BET isotherms were then measured using a Quantachrome Quadrasorb SI BET instrument. High purity N2 was used as the adsorbate; the isotherms were measured at 77 K. Upon obtaining the isotherms, the BET surface area and pore size distribution were determined using DFT model analysis. 2.3.7. Cell Culture Study. The model selected for cell culture experiment is 3T3 NIH cell. 3T3 NIH cells were grown in a 75 cm flask in DMEM with 10% FBS and 1% P/S. The flask was trypsinized with 0.25% trypsin solution for 3 min at 37 °C upon confluence. The

sized corn starch aerogel microspheres by the combination of emulsion gelation and supercritical drying, and the starch aerogel microspheres have specific surface areas in the range of 34−120 m2 g−1 and particle sizes in the range of 215−1226 μm diameter.35 Yun et al. employed a water in-oil (W/O) emulsion method with a subsequent ambient pressure drying step to prepare silica aerogel microspheres using methyltrimethoxysilane (MTMS) as the silica precursor. The obtained silica aerogel microspheres have a very low bulk density (0.08 g cm−3, approaching 96% porosity) and are superhydrophobic with a contact angle as high as 172°.36 All these aerogel microspheres showed excellent properties and their properties were found to be influenced by agitation rate, precursor concentration, surfactant concentration, phase ratios, and drying parameters. Although many different methods have been reported for preparation of functional cellulose aerogels microspheres, all these are made from regenerated cellulose, the microsphere structured nanofibrous aerogel from nature cellulose directly has not been reported. As we all know, 3D cell cultures are emerging tools in cell biology, regenerative medicine, cell therapy, chemical testing, and drug discovery. The main principle requirements of the 3D cell culture scaffolds include the following: (a) the materials should be nontoxic and biocompatible and in conductive to cell attachment, differentiation, and proliferation;37,38 (b) the morphologies should resemble the micro/nanoscale architectures of the native extracellular matrix and consist of a highly interconnected porous network, which can encourage cellular infiltration and allow the proper exchange of nutrients and metabolic waste throughout the cell culture scaffold.39,40 In this study, we reported a facile approach for preparation of ultralight cellulose aerogel microspheres by spray-freeze-drying. With this preparation route, natural cellulose nanofibrils rather than regenerated cellulose were used as the basic building blocks. As a result, the method for cellulose aerogel microsphere synthesis completely avoided the use of toxic solvent so no dissolution and purification processes are required. The obtained materials are highly lightweight (bulk density is only one tenth of the silica aerogel microspheres mentioned above) with high porous and have excellent water uptake capacity. The cellulose nanofibril aerogel microspheres reported in this study are native, nontoxic, biocompatible, interconnected nanofibrous with high porous structure, which could be suitable for cell culture scaffold application. The result of cell culture experiment is promising. We believe our research not only will help the development of the fabrication and the application of cellulose aerogel microspheres but also contribute to the synthesis of other materials with desirable porous structure.

2. EXPERIMENTAL SECTION 2.1. Materials. Kymene (polyamide-epichlorohydrin resin) was purchased from Ashland Hercules Inc., USA. Commercial bleached softwood kraft pulp was used as the raw material. 3T3 NIH cell was purchased from ATCC and cultured following the instructions on its information sheet. DMEM (Dulbecco’s modified eagle medium), FBS (Fetal bovine serum), P/S (penicillin-streptomysin), and MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Invitrogen and used as received. Trypsin and isopropyl alcohol were purchased from Gibco and Sigma-Aldrich, respectively. 2.2. Preparation of Cross-Linked Aerogel Microspheres. Commercial bleached softwood kraft pulp was used as the starting material for preparing cellulose nanofibrils. The dry cellulose pulp board was soaked in deionized water for 24 h and then dispersed by B

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Figure 1. Schematic diagram of the atomization and spray-freeze-drying process for fabricating the porous nanofibril aerogel microsphere using liquid nitrogen as the cryogenic medium. The feed solution was transported by pressurized air, and frozen droplets were collected in a liquid nitrogen cooled beaker underneath the spray nozzle. cells were then transferred to a 15 mL conical tube and centrifuged for 10 min at 250 g. The supernatant was removed, and the cells were resuspended in fresh media. Four flasks of cells were combined to give a total volume of 20 mL and a cell count of 8 × 106 cells. Around 8 × 103 cellulose microspheres were added, and the solution was placed on an orbital shaker set to 40 rpm, in an incubator at 37 °C. The spheres were seeded for 24 h, after which the mixture was gently centrifuged for 5 min at 47 g. The supernatant was carefully removed, and the spheres were resuspended in 1 mL of fresh media. The seeded spheres were transferred to a 12-well plate, 60 μL per well. Three mL of fresh media was then added to each well. The plates were placed in a 37 °C incubator for 2 h. After that the media was transferred to a new plate in order to remove unseeded cells. To prepare the seeded microspheres for MTT, the contents of a well were transferred to a 15 mL conical tube and gently centrifuged for 5 min. The supernatant was removed, and the spheres were resuspended in 500 μL of fresh media and transferred to a 1.5 mL tube. Background was measured as the media supplemented with pristine microspheres. The solution containing 20 μL of 5 mg mL−1 MTT was added to each tube, which was then incubated for 3 h at 37 °C. After incubation, 500 μL of isopropyl alcohol was added to each tube. The tubes were shaken thoroughly to dissolve MTT crystals. The solutions were then transferred to a 96well plate, 300 μL per well, with each sample taking up 3 wells. The data was acquired using a plate reader (Infinite 200 Pro, Tecan).

3. RESULTS AND DISCUSSION Figure 1 shows a schematic diagram of the spray-freeze-drying apparatus fabricated to produce ultralight porous cellulose aerogel microspheres. In a typical demonstration, to make cross-linked cellulose nanofibril aerogel microspheres, softwood pulp cellulose fibers were used as the original material which were about 20−50 μm in diameter. The defibrillation started at the surface of the cellulose fibers, and small-sized fibers were peeled off from the cellulose fibers under high speed mechanical shearing. After high speed homogenizer mechanical shearing, the softwood fibers almost lost their pristine structure, and thinner fibrils with diameters ranging from nano- to microsize were found. Further defibrillation resulted in highly viscous gel-like nanofibril cellulose suspension in water, which was stable for several months under the ambient environment. As demonstrated in Figure 2a, the final products were mainly fibril bundles of several micrometers. Most fibrils had a diameter below 50 nm. These suspensions with different concentrations of nanofibrils and cross-linker were sprayed and atomized at constant pressure through a steel nozzle directly into liquid nitrogen for instant freeze. It was found that the cellulose nanofibril solution could not form microdroplets if the pressure was too low.

Figure 2. SEM of the aerogel microspheres with a different cellulose nanofibril concentration or cross-linker ratio (cellulose nanofibrils to cross-linker, w/w): (a) image of native nanofibrils; (b) image of aerogel microspheres with a nanofibril concentration of 1.5% and a cross-linker ratio of 10:1; (c) image of aerogel microspheres with a nanofibril concentration of 0.6% and a cross-linker ratio of 10:1 at higher magnification; (d) image of aerogel microspheres with a nanofibril concentration of 2.0% and a cross-linker ratio of 10:1 at higher magnification; (e) image of aerogel microspheres with a nanofibril concentration of 0.6% and a cross-linker ratio of 10:2; (f) low magnification image shows multiple cellulose nanofibril aerogel microspheres.

However, when the pressure was too high, the water in the nanofibril solution was atomized to liquid droplets alone, leading to the nanofibrils left behind. As a result, the water droplets were separated from nanofibrils. In other words, the droplets had more water and less nanofibrils compared to its mother solution. Based on experimental experience, 40 MPa was a suitable pressure that could produce uniform droplets with a consistent concentration of cellulose nanofibrils in the C

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Figure 3. Bulk density of the aerogel microspheres with a different cellulose nanofibril concentration or cross-linker ratio (cellulose nanofibrils to cross-linker, w/w): (a) same nanofibril concentration (0.6%) with a different cross-linker ratio and (b) different nanofibril concentration with the same cross-linker ratio (10:1).

The samples of prepared microspheres were randomly selected, and their size was determined using an optical microscope. A typical example of microsphere distribution is shown in Figure 4. The detail size distributions can be found

droplets. It was noted that small cellulose aqueous gel droplets maintained their spherical shape without agglomeration in the process. The frozen droplets were subsequently freeze-dried to obtain aerogel microspheres. In order to improve the stability of the samples in solvent, the dried aerogel microspheres with different amounts of cross-linkers were further cured in a vacuum oven at 120 °C for 3 h to achieve sufficient covalent cross-linking. Kymene can form both self-cross-linking through its epichlorohydrin groups and external cross-linking with cellulose, which can increase the mechanical strength, particularly the wet strength, of the aerogels.41,42 SEM images of typical aerogel microspheres are shown in Figure 2 b-e. It was clear that the aerogel microspheres were composed of individual nanofibrils. Though some nanofibril bundles still could be found, they are much smaller compared with original materials. Under higher magnification, it was confirmed that the bundles are composed of nanofibrils with diameters ranging from several tens to hundreds of nanometers. Also, from the SEM images, it was found that pore size varied widely from a few nanometers to micrometers throughout the aerogel microspheres. Comparing the SEM images, it is obvious that when the cellulose concentration or cross-linker concentration increased, the pore size became smaller. This observation could be attributed to two reasons. When the nanofibrils concentration in cellulose aqueous gels increases, the density of cellulose in the droplet also increases, resulting in denser aerogels after freeze-dry. On the other hand, more crosslinker was added in the samples, and more covalent crosslinking occurred resulting in denser aerogel microspheres. The low magnification image of Figure 2f shows that the sizes of cellulose nanofibrils aerogel microspheres are in the range of a few tens to hundreds of micrometers. Bulk density was determined by transferring a known quantity of microspheres to a measuring cylinder and tapping it 3 times at 2 s intervals.43 Through dividing the weight by the final volume of the sample, the bulk density was calculated. The results are shown in Figure 3. As the cellulose concentration was higher, the bulk density increased. This was in accordance with the previous observation by Aulin et al., who reported that the cellulose aerogel density was almost linearly proportional to the dispersion concentrations.44 With more cross-linkers, the bulk density of the final resulting microspheres became higher, too.

Figure 4. Particle size distribution of the aerogel microspheres with a cellulose nanofibril concentration of 0.6% and a cross-linker ratio of 10:1 (cellulose nanofibrils to cross-linker, w/w).

from Supporting Information S-1. The diameter of most cellulose aerogel microspheres was around 60−120 μm, and higher concentration cellulose solution resulted in bigger sized aerogel microspheres. All together, these results indicate that the diameter, porosity, and pore size of cellulose aerogel microspheres can be tuned by varying the concentration of CNF aqueous suspensions. The thermal stability of the nanofibril aerogel microspheres was studied from the TGA curves as shown in Figure 5. Since the cellulose aerogel microspheres have very low bulk density, it is very difficult to get an accurate weight in small TGA crucible volume, and we could not find the actual trend of the effect of cellulose nanofibrils or cross-linker concentration on the decomposing temperature of aerogel microspheres. It was indicated that the starting decomposing temperature and the maximal weight loss rate of the aerogel microspheres trended toward a lower temperature than that of the large sized aerogel made from the same nanofibrils but without using the spray method. A possible reason for the lowered thermal stability of D

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Figure 5. Typical TGA curves of the aerogel microspheres: (a) TGA curves and (b) DTG curves. In the pictures, 0.2, 0.6, 1.0, and 1.5 represent the cellulose nanofibril concentration (%), and 10:1 and 10:2 represent the cross-linker ratio (cellulose nanofibrils to cross-linker, w/w).

Figure 6. Moisture rate and water uptake capacity curves of the aerogel microspheres with different cellulose concentrations or cross-linker ratios (cellulose nanofibrils to cross-linker, w/w): (a) moisture rate of a different nanofibril concentration with the same cross-linker ratio (10:1); (b) moisture of the same nanofibril concentration (0.6%) with a different cross-linker ratio; (c) water uptake of a different nanofibril concentration with the same cross-linker ratio (10:1); (d) water uptake of the same nanofibril concentration (0.6%) with a different cross-linker ratio.

more than 100 g of water, which was a typical superabsorbent property. It was also found that the nanofibril concentration and cross-linker dosage had an effect on the water uptake properties. The water absorption of aerogel increased when the cross-linker dosage increased. This phenomenon was presumably a result of the pore structure difference under different nanofibril concentration or cross-linking degree. The superabsorbent properties of cellulose aerogel microspheres depend

the aerogel microspheres is that they have a higher specific surface area than the larger sized aerogel, making them easier for thermal degradation from the cellulose surface. The aerogel microspheres made from cellulose nanofibrils by the spray-freeze-drying method showed a high moisture rate (Figure 6 a-b) because of their high surface area and porous structure.45 The water uptake capacity tests (shown in Figure 6 c-d) indicated that 1 g of the cellulose aerogel could absorb E

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Figure 7. Nitrogen adsorption−desorption isotherms curve and pore distribution. The specific surface area is 389.9 m2 g−1 for the aerogel microspheres made from a solution with a cellulose nanofibril concentration of 0.6% and a cross-linker ratio of 10:1 (cellulose nanofibrils to crosslinker, w/w).

Figure 8. MTT data of 3T3 NIH cells seeded on cellulose aerogel microspheres: (a) standard curve and (b) quantitative data for cells seeded on cellulose microspheres.

three-dimensional cell cultures could provide quite similar conditions with real physiological situations, which is important to avoid erroneous conclusions in the experiments. Porous structure materials could provide a suitable environment for cell culture by enhancing the supply of nutrients, diffusion of gases, and removal of metabolic wastes. The openness of the pores is one of the critical factors to be carefully considered because entry of cells into porous materials is only possible through pores at the perimeter. By considering this issue, the nontoxic green ultralight high porous aerogel microspheres of native cellulose nanofibrils produced by spray-freeze-drying methods could be suitable in the fields. To keep the good enough mechanical properties in culture environment, covalent crosslinking between the native cellulose nanofibrils were taken in this study. The resulting aerogel microspheres made using the method reported in this paper were very robust even in a harsh environment. The cellulose aerogel microspheres without cross-linking were broken in water after agitation for a few minutes, but for these cellulose aerogel nanofibril microspheres after cross-linking, they could still keep their three-dimensional shape after 6 h of agitation in water with a magnetic stir bar. 3T3 NIH cells were seeded onto the aerogel microspheres made from a solution with a cellulose nanofibril concentration of 0.6% and a cross-linker ratio of 10:1 (cellulose nanofibrils to cross-linker, w/w) to evaluate the potential use of the gelatin

not only on the hydrophilic nature of cellulose but also largely on the storability of those nano- and microsized pores inside. The specific surface area and pore distribution of the aerogel microspheres were measured by BET absorption, and a typical curve is shown in Figure 7. The results showed that the aerogel microspheres, made from a solution with a cellulose nanofibril concentration of 0.6% and a cross-linker ratio of 10:1 (cellulose nanofibrils to cross-linker, w/w), had a specific surface area about 389 m2 g−1. However, no clear dependence of specific surface area on the cross-linking degree and nanofibrils concentration was found when the different aerogel microspheres were measured. It should be noted that poor reproducibility in the specific surface area measurement was observed. The possible reason was that these aerogel microspheres were so light. In order to add enough mass of the aerogel microspheres in the BET test tube for measurement, the aerogel microspheres had to be compressed and densely filled in the test tube. Since it is hard to control the degree of the compression when the ultralight microsphere samples were filled in the test tube, the specific surface area of these aerogel microspheres greatly varied from sample to sample. Three-dimensional cell cultures are emerging tools in cell biology, regenerative medicine, cell therapy, chemical testing, and drug discovery. Compared to two-dimensional cultures, F

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ACKNOWLEDGMENTS The authors would like to acknowledge Jilin University and thte Chinese Scholarship Council of China for the financial support of this study.

porous beads for cell culture. Through the cell culture operation, the staining showed the cells could be seeded on the aerogel microspheres and accumulated successfully. The cell proliferation was also assessed at 1, 7, and 14 days, suggesting a high proliferation rate of the cells on the porous aerogel microspheres. For further studies the differentiation state of the cell spheroids, MTT, was used. According to the cell number standard curve (shown in Figure 8(a)), the quantity of the cell number was calculated. As time passed, the number of cells seeded on the aerogel microspheres increased during 2 weeks of culture implicating the process of differentiation (shown in Figure 8(b)). The results clearly showed that the cellulose nanofibril aerogel microspheres were nontoxic and biocompatible and that their interconnected high porous nanofibrous structure could facilitate cell attachment, penetration, differentiation, and proliferation and allow proper transfer of nutrient/oxygen and metabolic wastes. The results demonstrated that the nonhuman and nonanimal derived single component cellulose aerogel microspheres with porous structure were able to support cell growth and differentiation. Although the overall dimensions of the aerogel microspheres might not be suitable for in vivo cell delivery by injection, we believe that these aerogel microspheres have great potential for the tissue engineering area.

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ASSOCIATED CONTENT

S Supporting Information *

Diagrams of the detail particle size distribution of the aerogel microspheres with different cellulose nanofibrils concentration or cross-linker ratio. This material is available free of charge via the Internet at http://pubs.acs.org.

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4. CONCLUSION Herein, we successfully fabricated plant-derived native cellulose nanofibrils aerogel microspheres prepared by facile sprayfreeze-drying methods. These aerogel microspheres had a porous structure with pore sizes ranging from nano- to macroscale. This unique porous structure combining with nanosized water swellable cellulose nanofibrils allowed the aerogel microspheres to have low bulk density, high moisture rate, and high water absorption capacity. Moreover, to some extent, the porous system and properties could be further controlled by changing the beginning concentration of cellulose aqueous gel or cross-linker ratio. Through covalently crosslinking, the aerogel microspheres could be very stable even in a harsh environment. These features are important for some applications, especially in various biomedical applications, which was proved by the 3T3 NIH cell culture experiments. For the first time, the present study reported that aerogel microspheres could be made directly from native cellulose fibers and could be used as cell culture scaffold. In addition, this simple spray-freeze-drying direct synthesis technique could be easily extended to a variety of other systems with desired high porous structure.

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