A CaCO3/nanocellulose-based bioinspired nacre-like material

Department of Chemistry, Physical Chemist 10, Box 714, D-78457 Konstanz, Germany. E Department of Materials and Environmen 10691 Stockholm, Sweden Wallenberg Wood Science Center, Teknikrin Departament de F́ısica, Universitat Autonom Institucio Catalana de Recerca i Estudis Ava 08010 Barcelona, Spain † Electronic supplementary information description of wettability tests of NC kinetic controls of mineralizations. Fig. S 10.1039/c6ta09524k Cite this: J. Mater. Chem. A, 2017, 5, 16128


Introduction
In the quest for the development of materials with exceptional properties, biominerals fabricated by living organisms provide a vast source of inspiration. The properties of biominerals are diverse and relate to the combination of functions they serve in different organisms. The complex structures that have evolved to attain these specialized properties are genetically controlled and are formed under stringently controlled physiological conditions. Bone is a biomineral with exceptional mechanical properties conferred by the multilevel hierarchical structure of collagen and hydroxyapatite. 1 Another example of a material with intriguing mechanical properties is nacre. This iridescent and tough material protects the so body of mollusks from predators and irritants and also serves as an outer layer of pearls. 2 Nacre is composed of 95% aragonite and 5% of mostly chitin, 3 where the remarkable fracture resistance stems from the 'brick and mortar' arrangement of the mineral and organic components. 4 In biological nacre, the hard mineral provides structural rigidity, whereas the so organics dissipate fracture energy. 5 The biomineralization of calcium carbonate generally takes the advantage of amorphous calcium carbonate (ACC) as an intermediate to achieve the structurally complex combination of organic and inorganic constituents with a high degree of delity. 6,7 In bioinspired approaches, the great usefulness of droplets of liquid ACC stabilized by the presence of low amounts of polyanions, such as poly(aspartic acid), has been thoroughly demonstrated. 8,9 These so-called polymer-induced liquid precursors (PILPs; note that the polymer stabilizes the liquid precursors, rather than inducing them) 10-12 can be molded into any shape and thus enable the construction of tailor-made hybrids. 8,9,13,14 For instance, the successful in vitro re-mineralization of the biogenic insoluble matrix of nacre via PILPs suggests that this is also a viable pathway in vivo. 15 More recent work has demonstrated that polymer-mediated mineral growth in combination with a layer-by-layer deposition of porous organic lms allowed the rst replication of truly articial nacre using CaCO 3 . 16 In this study, we combined the most abundant biomineral, calcium carbonate, with the most abundant biopolymer, cellulose, and demonstrated that a bioinspired synthesis route can be used to produce nacre-like laminated materials with outstanding mechanical properties. The structure consists of inexpensive materials, which are green and truly sustainable.
More specically, we employed nanocellulose (NC) 17 that is a non-toxic, stiff, yet lightweight material with high tensile strength that is receiving rapidly increasing interest for the development of sustainable hybrid materials with excellent mechanical properties. [18][19][20][21][22] It has to be stressed that there are no previous examples of the successful combination of calcium carbonate and NC into a nacre-like structure as the bottom-up generation of an organic/inorganic-layered material with these compounds is highly challenging. This is due to the fact that none of the constituents provide an inherent 2D planar or scaffold basis, whereas a localized, controlled mineralization of the NC framework is required. Previous synthetic methods of other nacre-like materials involve predesigned matrix-directed mineralization, 23 electrophoretic deposition, 24,25 freeze casting, 26,27 templating, 15,27 layer-by-layer fabrication, 16,28,29 and self-assembly. [30][31][32] In this study, we showed that localized mineralization of deposited NC lms by controlled spreading and imbibition of liquid CaCO 3 precursors allowed the processing of nacre-like materials with alternating layers of mineralized and unmineralized NC. Controlling the wettability 33 of NC with hydroxyl and carboxyl surface functionalization towards liquid CaCO 3 precursors in the presence and absence of magnesium, respectively, leads to a very high degree of compositional and structural delity of the NC/CaCO 3 -based nacre-like material. Sequential assembly and inltration is a facile and scalable method that can be used for the generation of multi-layered organic-inorganic nacre-like hybrids. Fig. 1(a) illustrates the protocol for the generation of the nacrelike material. A detailed description of the syntheses of the NC samples, the lm preparations, and mineralization is provided in the ESI; Section S1-1-S1-5, including the choice of the suitable type of NC, control of lm thickness, and PILP wettability on the given lms. The morphology of the three different NC types used in this study is shown in the ESI, Fig. S1. † Films of NC functionalized with hydroxyl groups (NC-OH) and carboxyl groups (NC-COOH) are stable under the reaction conditions for CaCO 3 precipitation (high pH and initially 10 mM CaCl 2 , ESI, Section S1-3 and Fig. S2 †). Without Mg 2+ , the NC-OH lm shows very good wettability by CaCO 3 PILPs, whereas under the same conditions, NC-COOH cannot be wetted and thus remains unmineralized (ESI, Section S3-1-1 and Since the layer-by-layer technique requires a smooth base for further layering, obtaining a homogenous CaCO 3 mineralization is essential. In the kinetic setting of our mineralization procedure, the best mineralization results can be obtained from a NC dispersion with 0.11 wt% or less (ESI, Fig. S7-S9 †), providing a suitable lm thickness. With the established parameters, an initial mineralization of a single NC-OH layer was performed ( Fig. 1(a)-2). SEM images of the surface of the mineralized sample with 750 nm thickness suggest that the individual crystals are mutually aligned (ESI, Fig. S9 †), which is also apparent from the SAED pattern of the thin cuts (see below). Subsequently, a layer of NC-COOH followed by a layer of NC-OH was deposited, and then again mineralized ( Fig. 1(a)-3). The latter mineralization procedure was repeated several times until an iridescent composite lm was obtained ( Fig. 1(b)). The iridescence can be interpreted as interference colors caused by the existence of multiple uniform layers. 34 Eighteen mineralizations provide a sufficient basis to create this pattern. Again, note that the key for the generation of the nacre mimic is the wetting and inltration of the CaCO 3 PILPs on the NC-OH lms and their mineralization, as opposed to the inter-layers of lms of NC-COOH, which cannot be wetted in the absence of Mg 2+ and remain unmineralized.

Results and discussion
The obtained nacre-like material, based on the alternating organic and inorganic layers, was analyzed by scanning electron microscopy (SEM) techniques. The cross-section back-scattered electron (BSE) SEM image conrmed that the obtained material consists of layers of mineralized NC, which strongly backscatter the electrons and therefore appear much lighter than the unmineralized NC-COOH layers located between them (Fig. 1(c) and ESI, Fig. S10 †). The energy-dispersive X-ray spectroscopy (EDX) mapping of the cross-sections indicated that the distribution of Ca and C over the scanned area was concentrated within the NC-OH/CaCO 3 and NC-COOH layers, respectively ( Fig. 1(d) and ESI Fig. S11 †). Confocal laser scanning microscopy (CLSM) imaging of the NC component stained with Cal-couor White and combined with the collection of the light reected from the mineral phase also conrmed the layered arrangement of the mineral/organic phases (ESI, Fig. S12 †). To further analyze the mineralized parts, the samples were cut into sections with a microtome and analyzed by transmission electron microscopy (TEM) (Fig. 2(a and b), and ESI, Fig. S13 †). The TEM image shows a layer of brick-like inter-connected crystals. Note that imaging under higher magnication resulted in the burning of the samples and hence it was not possible to improve the image quality any further. The selected area electron diffraction (SAED) pattern of the thin sections shows arcs, indicating that the crystals are mutually oriented (Fig. 2(c) and ESI, Fig. S13 †). Evaluation of the SAED pattern reveals that these crystals are indeed calcite crystals, which are oriented in the [104] direction. Some of the reections, which cannot be assigned to this calcite pattern with the zone axis [À441], are likely randomly oriented in other directions or may arise from NC. Fig. 2 (a and b) TEM images of a sample cross-section obtained by microtome cuts of the layered composite structure with mineralized NC-OH layers after 60 layers. Note that the $300 nm thin cuts were very sensitive towards the electron beam, leading to sample deterioration, especially of the NC constituents. Thus, the length scales of the mineralized and unmineralized layers established by SEM and confocal microscopy ( Fig. 1c  and d)  In any case, this mutual orientation of individual calcite crystals suggests that the mineralized inter-layers exhibit at least partly a mesocrystalline structure. 35 To determine the composition of the obtained materials, thermogravimetric analysis (TGA) was performed (Fig. 2(d)). The TGA of a sample with 60 layers shows two main plateaus where the rst one occurs within the temperature range of 25-350 C. A close assessment reveals a smooth mass loss (ca. 2%) below 250 C, which was attributed to the loss of water. Note that the samples were incubated in a vacuum oven for 24 h before measuring the TGA curves. The existence of water in biogenic nacre was shown, 36 and herein, may be due to its trapping in the micropores resulting from the packing of the NC and is likely associated with the NC surface due to its highly hydrophilic character. Subtraction of the water content from the nal mass loss towards this rst plateau yields a fraction of organics of ca. 5%. The second major mass loss occurs between 650 and 830 C and was attributed to the calcination of the CaCO 3 phase (ca. 93%). Interestingly, the resulting composition is very similar to that of the biological nacre. 37 The TGA of a sample with 25 layers gives value of 2%, 7.5%, and 92.5% for water, organics, and CaCO 3 , respectively, and the minor differences between the 60-and 25-layers specimens illustrate that the bioinspired synthesis route yields a nacre-like material with a well-dened composition (Fig. 2(d)).
We also characterized the mechanical properties of the obtained nacre-like materials with different numbers of layers ( Fig. 2(e) and ESI, Table S2 †). The hardness of the composite increases with the increasing number of layers (Fig. 2(e)). The reduced Young's modulus of the composite with 90 layers was circa 14 GPa, which is similar to the value for human cortical bone 38 and exceeds the value of many nacre-like materials that are based on organic and inorganic constituents other than NC and CaCO 3 (reviewed and compiled in ref. 39). A quantitative comparison with the mechanical properties of different CaCO 3based articial nacre-like materials, also including biological examples of the nacreous layers of different abalone shells, is summarized in the ESI, Table S2. † Crack deection by the platelike crystals as well as crack trapping at the organic layers and the periodical variation of moduli are important for the impressive mechanical properties of biological nacre. 40 Because the NC/CaCO 3 -layered hybrids are not built up by individual crystalline platelets, it is expected that the hardness and fracture toughness is lower than those of natural nacre. Mao et al. recently presented a CaCO 3 -based nacre-like material with a platelet structure within the layers, 23 but note that also this material (ESI, Table S2 †) was inferior to natural nacre. In the case of the present nacre-like hybrid, the brillar NC-OH within the mineralized layers can serve as a ber-reinforcement, 41 where the tailored wetting of the liquid CaCO 3 precursors ensures the ber-mineral adhesion. Nano-interconnectivity was highlighted to play a crucial role for the toughening and stiffening of nacre-like hybrids, 42 whereas any further synergistic toughening effects arising from the interfacial interactions of the building blocks, as in graphene oxide/NC-based nacre-like materials, 43,44 do not likely play a role.
The nacre-like material fabricated herein does indeed exhibit a large plasticity index (dened as the plastic indentation energy divided by the total indentation energy). This parameter provides an estimate of how much energy can be absorbed by the material during, for example, an impact. It is relatively high for the NC/CaCO 3 nacre-like hybrid, and noteworthy, even higher than for the biological nacreous layer of Haliotis laevigata (ESI , Table S2 †).
It is also interesting to compare the material properties of the nacre-like material to another example of transparent hybrid lms composed of NC and nanoparticles of amorphous CaCO 3 (ACC). 18 While the hardness of the nacre-like hybrid and the transparent hybrid materials are similar, the reduced Young's modulus of the nacre-like structure was larger. This is likely due to the morphology of the hybrids, where the more organized and ordered structure of the nacre-like material was stiffer. However, calcite with a mesocrystalline structure comprises the nacre-like structure (see above), as opposed to the transparent hybrid lm that contained only ACC, which may also play a role in this context.
When the mineralizations were carried out under appropriate conditions in the presence of Mg 2+ , the wettability of the distinct lms with CaCO 3 PILPs is inverted, i.e., the NC-COOH lm can be mineralized as opposed to the NC-OH lms (ESI, Fig. S14 and S15 †). Moreover, the inorganic constituent is then not calcite, but ACC (ESI, Fig. S14; † see Section S3-3 for further discussions). 45

Conclusions
In conclusion, by controlling the reaction conditions and in particular, the wettability of NC-OH and NC-COOH towards PILPs in the absence and in the presence of magnesium ions, respectively, a layered hybrid material containing ca. 90% calcite exhibiting mesocrystalline features can be generated. The resulting nacre-like, iridescent structure shows mineralized parts with a thickness of ca. 20 mm separated by layers of unmineralized NC-COOH with a thickness of ca. 1 mm. The composition of the CaCO 3 -NC laminates is very similar to that of nacre, which suggests that the PILP-based mineralization pathway on organic matrices with a tunable wettability has a strong resemblance to the mineralization process occurring in living organisms. 46 The mechanical properties of the obtained structure reveal a relatively hard material with a reduced Young's modulus, similar to cortical bone, and a high plasticity-surpassing biological nacre-that is likely based upon NC-ber-reinforced, mesocrystalline CaCO 3 . The bio-inspired mineralization strategy is of interest to a wide range of materials also beyond NC with potential applications in e.g., packaging and building industries. The level of control over the localization of mineralization sites can be employed for the generation of even more complex patterns in hybrid materials, e.g., based on the phase behavior of NC 47 or self-assembled organic frameworks.