Tough and Strong: Cross-Lamella Design Imparts Multifunctionality to Biomimetic Nacre

: The creation of structural composites with combined strength, toughness, low density, and biocompatibility remains a long-standing challenge. On the other hand, bivalve marine shells – Clinocardium exhibit strength, stiffness and toughness that surpass even that of the nacre which is the most widely mimicked model for structural composites. The superior mechanical properties of Clinocardium shells originate from their cross-lamella design, comprising CaCO 3 mineral platelets arranged in an ‘interlocked’ herringbone fashion. Reproduction of such hierarchical designs could offer multifunctionality, potentially combing strength and toughness at low densities and capability for seamless integration with biological systems. Here, we demonstrate manufacturing of the cross-lamella design by biomineralizing aragonite films with saw-tooth patterns and assembling them in a chitosan/fibroin matrix to generate a composite with interlocked mineral layers. The resultant composite —with a similar constitution to the biological counterpart, nearly doubles the strength of previous nacre-mimetic composites while improving the tensile toughness and simultaneously exhibiting stiffness and biocompatibility.

Bioinspired manufacturing -in the sense of replicating the way nature fabricates-holds great potential for supporting technological transformations in a variety of fields, from general manufacturing to medicine. 1 Bioinspired materials are produced by linking the structural components of life with the hierarchical designs with which they have evolved . 2 By doing so, biological molecules not only become relevant for engineering applications due to their abundance and/or integration with biological systems (e.g., human host or ecological cycle) but also become necessary for reproducing the synergies behind the extraordinary mechanical properties of structural biomaterials.
One key property of structural bio-composites is their multifunctionality, which enables a single material to perform satisfactorily in the many multidimensional loading scenarios that an organism may face during its life. 3 Mollusk shells, fish scales, arthropod exoskeletons, mammalian bones, and sponge spicules are examples of natural composites that exhibit such remarkably rich and isotropic combination of mechanical properties. 4,5 All these materials, despite their evolutionary disconnection and different compositions, are made of hard but brittle inorganic minerals and soft organic phases. However, the strength and toughness of these composites are orders of magnitude higher than those of their constituents, 6 and they result from the arrangement of the hard and soft phases in the composite matrix. 7 In the paradigmatic example of nacre seashells, the hard-mineral platelets are sandwiched between soft, sub-micron layers of organic macromolecules, which is one of the most followed models for the synthesis of high mechanical performance artificial materials. 7 Current approaches to producing biomimetic nacre using its native components focus on the reproduction of a "planar lamellar" model, where the mineral platelets organize parallel to each other across different levels. 8,9 Even this simplified model of nacre, which can be reproduced by freeze-casting, 10,11 slip/tape casting, 12 layer-by-layer bonding, 13,14 and vacuum-assisted filtration, 15 has demonstrated outstanding results in the reproduction of nacre's toughness.
However, strength of nacre-mimetic composites along the platelets remains lower than that of the biological counterpart. 16 Although, introduction of nanoscale bridges between inorganic platelets [17][18][19] laterally interlocked platelets 20 and 3-D printed platelet geometries 21,22 have yielded improvements in mechanical properties in these composites, the micro-level design of mineral platelets have not been fully considered yet. In particular, the planar lamellar models assume that the mineral platelets of nacre arrange in a brick wall structure where the mineral "bricks" have flat interfaces. By contrast, in native nacre, mineral platelets have a wavy cross-section, which causes transverse interlocking between adjacent platelets to increase the composite's resistance to deformation and prevent localized strain. 23,24 An extreme case of this mineral-interlocking can be observed in the inner crossed-lamellar design of the shells of some mollusks, where mineral interlocking occurs at the first-order domain; 25 instead of the mineral plates being "stacked" in a parallel manner, they are slanted toward the adjacent domain, resulting in intricate herringbone patterns ( Figure 1a). This interlocking strategy in structural materials is shown across different species in different forms. For instance, the shells of cockles (Clinocardium) have periodic inner crossed-lamellar structures, 26 while the conch shell (Strombus gigas) have a pattern, where the orientation of the crossed-lamellar changes (from 45°-90°) in different regions of the shell, resulting in one of the toughest known materials. 27 Notably, this interlocking strategy in structural bio-composites goes beyond mollusks; it is found in other phyla, such as the mineralized microridges in the exoskeleton of sea urchins (Phormosoma placenta), 28,29 and even in other kingdoms, such as the teeth-like interlocked mineral shells of diatoms, 30 which is a perfect example of convergent evolution.

Results and discussion:
Here, we report combined strength, stiffness and toughness in a biomimetic composite by reproducing the internal structure and composition of the shells of cockles by a distinctive approach of biomineralizing microscopically patterned aragonite platelets and assembling them hierarchically into an "interlocked" architecture across the composite. In the shells of cockles, the mineral platelets (constituting the first-order lamella) are stacked against the next domain at an angle of 30°-40° (Figure 1a-b). We replicated the localized biomineralization in natural systems using polyacrylic acid (PAA)-induced aragonite biomineralization and micro-structured chitosan films ( Figure S1). [31][32][33] This resulted in larger mineral platelets as the PAA−Ca 2+ complex being adsorbed and subsequently covering the entire chitosan surface (Figure 1c). With the increasing packing density, the adsorbed layer eventually underwent an amorphous-to-crystalline transition, leading to the formation of aragonite, as confirmed by X-ray diffraction ( Figure S2a). Areas of several square centimeters (3 × 1 cm 2 ) of chitosan films with micrometric topography were mineralized by this approach (Figure 1c and Figure S3). Notably, the only factor that limited the size of the patterned aragonite was the size of the reusable mold used for patterning the chitosan films used as template; by using polymer casting, chitosan films with nanometric features have been produced over large areas, 34 which is the same low-cost, high-throughput technology that is used here, highlighting the readiness of this approach to scale up to an industrially relevant level.
Layer by layer, using chitosan films with grating-like patterns, we built the crossed-lamellar structure analogous to the shells of cockles (Figure 1d). While we focused on the reproduction of the inner structures of the shell model, the ability to control the geometry of the aragonite platelets using patterned chitosan was observed to be quite general. Without aiming to explore the global limits of the approach but rather those relevant for this study, we found no apparent constraints to producing mineralized platelets with features ranging from 5-2µm and multiple geometries.  Figure S2b,c). In the specific case of the saw-tooth-like patterned features, the aragonite conforming to the chitosan patterned films was grown to a thickness of ~5µm ( Figure   1f). Interestingly, a more detailed study of the mineral platelets revealed that the patterned aragonite layers had a hierarchical sub-structure, resembling the meso-crystal morphology of the mineral component of natural nacre 23,24 (Figure 1g). Formation of such hierarchical mineralized structures has been attributed to the non-classical pathways of crystallization that involve an amorphous-to-crystalline transformation, such as that used here. 35 The main challenge to fabricating a hierarchical composite using freestanding films of a few microns thick is the process of handling and stacking each layer individually. For this purpose, we developed a strategy to exfoliate the patterned chitosan-aragonite (CA) films from the backing of a supporting polymethyl methacrylate (PMMA) substrate. By optimizing the thickness of the PMMA and CA films, the latter spontaneously delaminated from the support due to differential shrinking during dehydration ( Figure S4). This approach enabled the production of structured films of chitosan on PMMA substrates (Figure 2a The CA films were successively stacked with a fixed volume of silk fibroin solution, which was dispensed between each layer ( Figure 3a)-an approach that we have successfully used in the past to produce chitosan-fibroin laminates (i.e., Shrilk). 36 In total, nearly 300 CA films were stacked ( Figure 3b) with fibroin bonding the films together ( Figure S5). Importantly, the fabrication approach enabled the patterned side of adjacent CA films to face each other, thereby forming the "interlocked" arrangement ( Figure 3c) of CA films. Finally, the composite was pressed together to avoid air entrapment and relatively flatten the layers ( Figure S6), and dried in ambient conditions, which consolidated it to ~ 1.5 mm thick composite with a 70.4±1.8% mineral content ( Figure S7). Objects that are manufactured with chitinous polymers tend to shrink after manufacturing, which causes warping distortion and variation from the intended geometry ( Figure   S8) and thus limits their capability to be cast using standard molds. 36 The inclusion of the mineral phase in the matrix reported here resulted in a sturdy composite that conformed to the intended geometry after drying ( Figure 3d). As we demonstrated in the past, 37 freestanding chitosan films can be dried on a macroscopic mold to retain their geometry. As expected, the mineralized composites show a higher stiffness (Er = 20.4 ± 5.4 GPa) than that of the non-mineralized counterpart (evident from the slope of the unloading part of the indentation curves of both composites in Figure 4a). The stiffness of the mineralized composite is albeit lower than natural nacre due to relatively lower mineral content of ~ 70% vol. than that of nacre's ~ 95% vol.
Nevertheless, the layer-by-layer bonding approach presented here enabled fabrication of nacreous composites diverging from typical flat shapes. Tubular shapes (Figure 3e) were produced by drying the mineralized layers on cylindrical molds. We believe this demonstration of manufacturing of a non-planar biomimetic nacre would help the general use of these composites in engineering applications.
The interlocked mineral composite was observed to have simultaneously high ultimate tensile strength (~ 48 MPa) and nearly 4 times higher tensile toughness (area under the tensile stressstrain) in comparison to that of the planar mineral composite, while matching the stiffness (~ 25.8 GPa) and density (~ 1.51 ± 0.74 × 10 3 kg/m 3 ) of previous biomimetic nacres (Figure 4a and 4b). 10 A summary of all the mechanical properties of the interlocked mineral and planar mineral composites is provided in supporting information, Table S1. The tensile strength of the interlocked mineral composite (43.59 ± 4.5 MPa) is ~ 85% higher that of the planar-mineral composites that were produced using the same methodology (23 ± 2.8 MPa) (Figure 4b) and is also close to that of the nacre species Anodonta woodiana reported in literature. 18 The reason for the improvement in tensile strength of the interlocked mineral composite can be understood from finite element analysis of the composites (Figure 4c-e). In the interlocked-mineral composites, the mineral interlocking enables stress transfer through the stiffer mineral phase as opposed to stress transfer through the softer matrix in case of planar mineral composite. This helps increase the tensile strength of the interlocked mineral composites in comparison to that of the planar count erpart. 38 This is evident from Figure 4e, showing the maximum principal stress field of composites under same tensile load and shows that more load can be transferred directly through the interlocked stiffer mineral phase than that of the planar mineral composite. In fact, in previous works on nacremimetic composites, the load transfer through the soft organic layer between planar platelets is implicated for the eventual failure of the composite (platelet pull-out is observed when platelets' aspect ratio is less than a critical value); Hence, ways to arrest the sliding of the platelets (e.g., by incorporating nano-scale bridges between platelets) is emerging as a proven strategy to improve their tensile strength. 18,21 Interestingly, our interlocked mineral composite nearly doubled the strength that was reported for the best nacre replicas to date ( Figure 5), 10 which follow a planar lamella model and are produced using an "assembly-and-mineralization" approach. Additionally, the similar mechanical strength of our planar mineral composite presented here and those produced previously ( Figure 5), when considered independently of the technique used for its production, might be indicative of the full potential of the planar model, and highlights the limitations of a simplified laminated design and the need to move beyond it by including more complex features, such as mineral interlocking, to achieve full reproduction of the mechanical properties of natural nacre.
The improved tensile strength of the interlocked mineral composite also showed improved bending strength over the planar counterpart ( Figure S9a). This enhanced bending strength has been reported in natural systems of similar characteristics and is a result of an interface that is strategically designed to guide the propagation and dissipation of cracks. 22 In particular, the angled interfaces (with an interfacial angle between 30°-40°) that were chosen here as a biological model have been reported to be effective in deflecting fissures. 20 This effect is clearly observed in the optical examination of the fractured regions of crossed-lamellar specimens, where the topography of the layers results in extended and tortuous deflections of the cracks, resulting in dissipation of fracture energy in the composite before causing complete fracture ( Figure S9b). This effect can also be observed in the absence of surface cracks in the composite (Figure 4g) during indentation, which is a characteristic of monolithic calcite but is absent in the biological organic-inorganic composites comprising the same mineral. 10 The flexural work-of-fracture (determined by integrating the area under the flexural stress-strain curve 39,40 ) showed the interlocked-mineral composite having a 36% higher value than that of the plan-mineral composites (Table ST1).
The approach of linking natural molecules with a design they evolve to follow is a complex task when compared to the reproduction of a biological design with materials of known manufacture.
However, this additional complexity enables the exploration of enhanced synergies between components as well as seamless integration with biological systems, from the human body to entire ecosystems. This biocompatibility was demonstrated for the composite through the culturing and growth of a mammalian embryonic stem cell population for a week on the surface of the interlocked mineral composite reported here. The use of components of known biocompatibility resulted in a composite that supports the growth of these cells without any sign of cytotoxicity, suggesting the suitability of the composite for medical uses (Figure S10a,b). Interestingly, the structured minerals showed significant enhancement of cell proliferation compared to that of flat mineral platelets ( Figure S10c), which was attributed to the additional anchor points on the surface of the former. Additionally, the structured platelets showed an ability to drive cell growth along the direction of the pattern, an effect previously reported for other structured surfaces of similar dimensions. 41,42 Conclusion: In conclusion, we have demonstrated the reproduction of a nacre mimic using native components (namely chitin and fibroin as the organic phase and calcium carbonate as the inorganic phase) by following a crossed-lamellar model. This result not only achieved the characteristic toughness of the planar lamellar model but also realized the multifunctionality of the native material by nearly duplicating the tensile strength of the composite without altering its low density and biocompatibility ( Figure 5). This result was achieved by reorganizing the components rather than the composition, which demonstrated the interlocking phenomenon between the different mineral phases as a generalizable strategy for the development of multifunctional composites.

Fabrication of patterned chitosan surface
Chitosan (medium molecular weight, high degree of deacetylation; Sigma Aldrich) was dissolved at 2 and 3% w/v in 1% v/v acetic acid. A few drops of the chitosan solution were dispensed on PMMA sheets and pressed with a PDMS mold comprising the topographical pattern. The softimprinting was carried out until the chitosan film was dried after which the PDMS mold was peeled off. Subsequently, the patterned chitosan film was submerged in NaOH 4% (w/v) for 10 min to neutralize the protonated amino groups and avoid further dissolution. 36 Finally, the films were extensively washed in deionized water to remove the remaining NaOH and dried at 37°C.

Biomineralization of the patterned chitosan films
Patterned aragonite was grown on the patterned chitosan substrates by using a PAA-based mineralization precursor comprising 5mM CaCl2 solution and 10 µg/ml PAA and by implementing the traditional ammonia diffusion technique. The patterned chitosan films were submerged in the mineralization precursor held in a petri dish and the latter was covered with an Al foil with one perforation and then was placed inside a closed desiccator along with ~ 2 g of NH4HCO3. The mineralization was performed for different durations (12 h, 18 h, 24 h) to determine the uniformity of mineral growth on the chitosan substrate.

Synthesis of silk fibroin
A 5% w/v silk fibroin aquous solution was prepared from Degummed silk from Bombyx Mori (Mielke's Fiber Arts USA) by implementing the protocol described in ref. 36 .

Structural and chemical characterization
The morphology of the aragonite laminates and the composite specimen were investigated using a JEOL JSM-7600F field-emission scanning electron microscope (FE-SEM) operating at an accelerating voltage of 5 kV. The XRD measurements of the mineralized CaCO3 crystals was carried out with a Bruker D8 Discover high-resolution XRD (HRXRD) with a Cu Kα1 source. The

Mechanical characterization
An Instron ® 5940 single-column universal testing machine (UTM) equipped with a 500 N load cell was used for the tensile testing of the composite specimens. Dog-bone-shaped specimens with size of the reduced section of ~ 1 mm width and 0.5 mm thickness were used. Minimum 10 samples of each composite type were tested. The tensile toughness is obtained from the area under the tensile stress-strain curve between zero and ultimate strain. 36 Three-point bending tests were also performed on the Instron ® 5940 universal testing machine.
Beam-shaped composite specimens (of each type) of length 15 mm, width 2 mm and thickness 2 mm, were prepared. The bending test was conducted at a loading rate of 0.05 mm/min. Minimum 5 samples of each composite type were tested . The flexural work-of-fracture is obtained by integrating the area under the flexural stress-strain curve. 39,40 The observation of crack propagation was performed on a composite beam-shaped specimen of the same dimensions, with a notch of 300 µm in the middle.
Indentation was performed using NanoTest NTX (Micro Materials Ltd., UK)) equipped with a with a Berkovich indenter. For increased precision, indentation was repeated five times at ten different sites (total 50 indents per sample). The final depth of each indent never exceeded ten percent of the sample thickness and was at least 4 times larger than the thickness of individual mineral layer present in the composite. A loading rate of 80mN/sec was used so that the loading and unloading time segments fell between 20 and 50 seconds. The reduced modulus was calculated (by the Oliver-Pharr method) 49 and an average value was determined from all the tests for each sample.

Finite Element Analysis
Numerical simulations were conducted using the commercial FE package ABAQUS/Standard to capture the mechanical response of the tensile specimens. The stiff and soft materials were both Media was reconstituted every alternative day. Upon 80% confluency the cells were detached off the petri dish (Sigma Aldrich,USA) by treating with 0.25% of trypsin-EDTA solution (Nacalai Tesque, Japan) and 1×10 5 cells were taken for the biocompatibility experiment.

3D cell culturing and characterization
The viability assay was carried out by using mouse embryonic stem cells (E14TG2A). These cells upon 80% confluence were trypsin treated to dissociate the cells from the petri dish prior to seeding them on the sterilized composite specimen. The cells were then centrifuged and re-suspended in the supplemented media before counting the cell number with a cell counter. The calcium carbonate bio-composite was sterilized with 70% ethanol for 15 min followed by UV exposure for another 15 min before inoculating the stem cells. JGF, HKR and CR wrote the manuscript, with input of all the other authors.
Author Information: Correspondence and requests for materials should be addressed to CR (caross@mit.edu) or JGF (javier.fernandez@sutd.edu.sg)