Morphological, compositional, and mechanical properties

The chiton Rhyssoplax canariensis (Chitonidae: Chitoninae) was chosen as a representative model system to conduct a detailed investigation of chiton girdle scale armors. Like other chitons, this species (ca. 20 mm in length) is covered by eight bilaterally symmetrical overlapping mineralized shell plates, which are arranged dorsally along the longitudinal axis (in an anterior-posterior direction, Fig. 1a, b). In addition to the eight primary shell plates, protection is provided by the scaled girdle, which runs along the perimeter of the entire body (Fig. 1a, b). The girdle scales (ranging in size from ca. 100 μm to ca. 500 μm) are tightly packed and extend from the primary shell plates to the girdle edge (Fig. 1c). These tiny scales cover the girdle completely, and no gaps are observed between the primary plates and girdle scales when viewed dorsally. In this work, a pseudo-cylindrical coordinate system was used for denoting the sample orientations, where N, R, and C refer, respectively, to the normal (dorsal), radial, and circumferential directions at a given point along the girdle.

When viewed in cross-section, the girdle has three main structural components: the layer of large dorsal girdle scales (upper), a fibrous layer (middle), and a layer of smaller ventral scales (lower) (Fig. 1d, e). The dorsal scales exhibit a hook-like geometry with curvature towards the body, and while gaps between adjacent dorsal scales can be easily seen in sectioned or torn samples (red arrow, Fig. 1d), which are not externally visible due to the overlapping of individual scales (Fig. 1c). The bases of these dorsal scales are directly attached to the underlying fibrous layer (Fig. 1d). The white dashed lines in Fig. 1d, located close to the height at which the overlapping dorsal hook begins to form, suggest possible locations up to which the scales were covered by inter-scale soft tissues (also see Fig. 2c). The presence of this inter-scale organic matrix was also supported by the fact that after mechanically removing the underlying fibrous layer, the integrity of the dorsal girdle scales assembly was maintained (Supplementary Fig. 1). In contrast to the larger dorsal scales, the ventral scales are smaller and characterized by a comparatively simpler rod-like morphology (diameter, ca. 20 μm; length, 100–300 μm), with their longitudinal axes oriented in the radial direction from the animal’s geometric center (Fig. 1f).

Fig. 2 Compositional and mechanical characteristics of the girdle scales in the chiton R. canariensis. Backscattered SEM images of polished cross sections of the chiton’s scaled girdles in two orientations: a radial and b circumferential. Note that the middle organic fibrous layer can be clearly distinguished from the dorsal and ventral scales due to its electron density difference. c Energy-dispersive X-ray (EDX) spectroscopic elemental maps (O, C, Ca, and N) of the scale assembly taken from the region shown in a. The white arrow indicates the presence of nitrogen between adjacent scales above the fibrous middle layer. SEM image of the d fibrous middle layer after dorsal scale removal, and e the rod-like crystals observed on the fracture surface of an individual dorsal scale. f Indentation moduli, E r , and hardness, H, maps of the chiton scales taken in the region shown in b obtained from instrumented indentation measurements. Full size image

Synchrotron X-ray micro-computed tomography (μ-CT) was used to further investigate the three-dimensional geometry of the girdle scale assembly (Fig. 1g–j). The highly X-ray absorbent mineralized scales were easily segmented from the non-mineralized fibrous layer and inter-scale organic matrix. The dorsal girdle scales of R. canariensis are tightly packed despite a gradual decrease in scale size from the proximal to distal margins (Fig. 1g), and the overlap between adjacent scales can be visualized by using the transparent mode of 3D reconstructions (Fig. 1h). The rod-like ventral scales, by contrast, align their longitudinal directions along the radial (proximal-distal) direction, providing a complete and uniform coverage of the ventral surface of the girdle (Fig. 1i). The tight packing of the dorsal scales results in their diamond-shaped bases forming a diamond mosaic pattern when viewed ventrally, as revealed by the reconstructions with their ventral scales removed (Fig. 1j).

Compositional and nanomechanical measurements of the individual components of the girdle armor were then conducted to gain additional insight into its mechanical performance. Backscattered SEM (BSEM) images acquired from polished cross sections of the girdle armor in both radial and circumferential orientations demonstrate the high and uniform electron density of scales relative to the middle fibrous layer (Fig. 2a, b). The spacing between adjacent dorsal scales measures ca. 5 μm throughout the assembly. The circumferential cross-section shows that the thickness of the middle fibrous layer gradually decreases from the chiton body (ca. 200 μm) to the distal margin of the girdle (ca. 10 μm), and accompanies a similar corresponding decrease in dorsal scale sizes. Energy-dispersive X-ray spectroscopy (EDS) measurements confirmed that calcium carbonate is the main constituent in the dorsal and ventral scales, while the fibrous middle layer is not mineralized (Fig. 2c). Nitrogen was detected in the middle layer and in the regions between the scales (white arrow in Fig. 2c, N map), but not in the surrounding epoxy, suggesting the presence of a N-containing inter-scale organic matrix. Higher magnification SEM studies of fractured girdle samples revealed that the constituent fibers (measuring several hundred nm in diameter and >μm in length) of the middle fibrous layer exhibit distinctive band-like striations (Fig. 2d and Supplementary Fig. 2). High-resolution SEM image of fractured dorsal scales revealed an internal microstructure of tightly packed rod-like building blocks (Fig. 2e).

Instrumented nanoindentation measurements showed that the mechanical properties within individual scales were uniform due to the lack of significant sub-layering or structural gradients. The mineralized scales had a reduced modulus of 79.7 ± 4.6 GPa and hardness of 3.8 ± 0.6 GPa (n = 96), values which are comparable to those obtained from aragonite-based mineralized structures found in other mollusks52,53 (Fig. 2f).

Three-dimensional geometry

The 3D geometry and surface morphology of individual girdle scales from R. canariensis based on synchrotron μ-CT measurements are summarized in Fig. 3. Three-dimensional reconstructions of individual dorsal scales in different views highlight their two main geometrical features: a diamond-shaped prismatic ventral base and a shallow, cup-like dorsal hook that overlaps with adjacent scales (Fig. 3a–f). Fine structural features include a dimple at the scale base (white arrow in Fig. 3c) and a roughening of the surface of the posterior margin of each scale (white arrows in Fig. 3d, e). Small depressions were observed on the dorsal surface of scales (Fig. 3b), typically extending to a depth of ca. 100 μm, as shown in the transparent reconstruction in Fig. 3f.

Fig. 3 Three-dimensional geometry and surface morphology of individual dorsal scales of the chiton R. canariensis. a–f μ-CT data-based 3D rendering of individual girdle scales in different view angles and modes: a front view, b top view (yellow arrows indicate pore openings), c bottom view (white arrow shows a depression at the base of the scale), d two side-views (white arrows shows the surface roughness at the lower surface of backside), e back view, and f transparent mode (the yellow arrows show holes in the dorsal surface of scales and the white arrow indicates depression in base). g Projection contours along two orientations (transverse and bottom) are used to describe the geometries of chiton scales. Geometrical parameters are defined, including lengths (w, l, h 1 , h 2 , h (=h 1 + h 2 )), angles (α and β), areas (A 1 , A 2 , and A total (=A 1 + A 2 )), and volumes (V). h Top view of a μ-CT data-based reconstruction of the girdle scale assembly of R. canariensis. Three columns of scales used in the geometrical measurement are highlighted in pink color and their positions are indicated. i Variations of geometrical parameters as a function of scale position. The solid line represents the average and the shaded area shows the standard deviation (N = 3 for each measurement). j SEM image of a scale’s back surface. k Magnified-view of scale surface with microscopic bumps at the underside of the back surfaces of chiton scales, as indicated by the white box in j. l SEM-derived stereographic reconstruction of microscopic bumps in a similar region shown in k. m Backscattered SEM image of a polished cross-section in the region of microscopic bumps of a scale, highlighting the difference in morphology between the anterior (front) and posterior (back) surfaces of the dorsal scales. Full size image

In order to successfully mimic scale morphology for the production of a 3D-printed structural analogue (as discussed later), quantitative measurements of the scale geometry were conducted by defining several morphometric parameters, including W (width of base), L (length of base), h 1 (the vertical height from the base to the inflection point), and h 2 (the vertical height from the inflection point to the top of the scale), imbrication angle α (describing the medial lean of the hook), inclination angle β (describing the lateral pitch of the base), and two projection areas A 1 and A 2 (Fig. 3g). Morphometric measurements from three rows of dorsal scales within the girdle scale assembly were conducted (Fig. 3h), and the results are summarized in Fig. 3i. The scale size gradually decreases from the proximal to distal margins, as indicated by the decrease of scale volume from ca. 16 × 10−3 mm3 to ca. 5 × 10−3 mm3. Despite variations in size, all scales are roughly geometrically similar, as evidenced by the consistency of all parametric ratios (H/L, ca. 0.5–0.6; W/L, ca. 0.4–0.5; and h 1 /H, ca. 0.3–0.4), angles (α, ca. 110–120° and β, ca. 20–30°), and overlapping ratio (A 1 /A total , ca. 0.3–0.4), where H = h 1 + h 2 and A total = A 1 + A 2 .

SEM imaging of isolated individual dorsal scales revealed the presence of evenly spaced ridges (spacing: ca. 40 μm) running along the dorsal surface of the scales proximodistally toward the hook tip (Fig. 3j). The rear view of isolated scales shown in Fig. 3j illustrates the gradual increase in surface roughness towards the base of the scales, consistent with our μ-CT measurements (Fig. 3d, e). Figure 3k shows that the roughness of the posterior surface is caused by evenly distributed cone-shaped protrusions, with the corresponding stereographic reconstruction of the surface revealing that both their height and wavelength are ca. 5 μm (Fig. 3l). The surface rugosity of posterior side is in stark contrast to the surface smoothness of the anterior side of scales, when viewed in cross-section (Fig. 3m). Similar surface features were described by Bullock from the genus Chiton (also Chitoninae)54.

Interspecific comparison

We extended our 3D morphometric measurements of girdle dorsal scales to multiple chiton species from two families, the Ischnochitonidae and the Chitonidae (Fig. 4a). Scales were selected from the middle region of the girdles (approximately equidistant from the proximal and distal margins) for consistent geometrical and size comparisons. Top-, bottom-, front-, and side-view images of the different scales are shown in Fig. 4e. Five scales were segmented from the μ-CT data obtained from each of the ten species, and their morphometric measurements were recorded (Figs. 3g and 4b–d). The scales exhibited large variations in volume from ca. 0.005 mm3 (Lepidozona mertensii) to 0.1 mm3 (Rhyssoplax polita) (Fig. 4b). Despite size differences, a majority of the scales exhibited the same simple hook-like form, except L. mertensii (Ischnochitonidae), which displayed a unique geometry with two inflection points (Figs. 4e and 5d). While in most species the prismatic base accounted for ca. 30–40% of the scale height (i.e., h 1 /H: ca. 0.3–0.4), the prismatic base was ca. 50% of the total height in Chiton cumingsii and Ischnochiton contractus. The imbrication angle, α, ranged from ca. 90° in I. australis and L. mertensii to ca. 130° in I. lentiginosus. The inclination angle, β, ranged broadly from ca. 60° in I. australis to ca. 10° in C. cumingsii. In all species, the sum of the imbrication and inclination angles was <180°, signifying that all scales were capable of overlapping with their neighbors. β was usually larger in the Ischnochitonidae than in the Chitonidae, and as such, scales from the Ischnochitonidae exhibit a greater degree of inclination along the posterior direction for the diamond-shaped prismatic ventral base. A general morphological difference between the two examined groups was that Ischnochitonid scales exhibited more pronounced ridge structures on their dorsal surfaces. The H/L, W/L, and A 1 /A total ratios for all scales ranged from ca. 0.4–0.8, ca. 0.4–0.8, and ca. 0.1–0.6, respectively.

Fig. 4 Interspecific comparison of the dorsal scale geometries among ten chiton species. a The phylogenetic distribution of the chiton species used in this study. Variations of morphological parameters, b volume, c length and area ratios, and d angles for different species. The solid line represents the average and the shaded area shows the standard deviations (N = 5 for each measurement). e μ-CT data-based 3D reconstructions of individual dorsal scales from the ten chiton species in four different views: top, bottom, front, and side. All the reconstructions are at the same length scale except those from Rhyssoplax polita. Full size image

Fig. 5 Three-dimensional parametric modeling of chiton scale geometries. a Top, the 3D scale model with three principal scaffolding curves, XZ, YZ, and BASE. Bottom, 3D scale model highlighted with the central spine for generating the surface meshes. b Three principal curves with geometrical landmarks indicated. c, d Comparison of the original chiton scales with corresponding mimicked scale models for two species: c a single-curved scale from chiton Rhyssoplax canariensis and d a double-curved scale from chiton Lepidozona mertensii. Full size image

Parametric modeling of chiton girdle scales

We developed a parametric geometrical model to reproduce the observed morphometrics of chiton scales to facilitate the subsequent modeling of chiton-inspired scaled arrays. First, three principal sections were made through the scale: one horizontal section through the base (denoted as BASE) and two vertical sections running transversely and longitudinally in the (YZ) and (XZ) planes, respectively, both passing through the geometric center of the scale (Fig. 5a, top). We then selected 20 spatial markers on these principal sections. Using third-order polynomial interpolation, we generated a spline through each set of points, creating parametric versions of the three principal curves (Fig. 5b; see Supplementary Fig. 3 and Supplementary Table 1 for more details). We then generated a central spine running medially within the YZ cross-section (Fig. 5a, bottom, red line). Constructing planes normal to the spine along its length permitted the reconstruction of the complete scale surface. Parametrically controlling the relative positions of spatial markers along the principle curves enabled variation of scale geometries across a large design space (see Supplementary Video). Figure 5c illustrates the modeled geometry for R. canariensis, which agrees well with the geometry obtained from the native μ-CT data. The doubly curved geometry that characterizes the L. mertensii scales could also be replicated using the same parametric model (Fig. 5d), highlighting the morphological diversity that can be accommodated with this approach.

The successful 3D modeling of individual scales allowed us to design a composite scale armor assembly similar to that of chitons. The bio-inspired armor system included rigid scales embedded in an underlying soft substrate (Fig. 6a). The surrounding substrate matrix was modeled with a thickness equal to the height of the inflection point of the scale (h 1 ), as in R. canariensis (Fig. 6b). The ratio between the inter-scale spacing d and scale length L (d/L, ca. 5%) was also similar to that in the native girdle (Fig. 6c). To create a uniform pattern, scales with the same size and geometry were arranged into a diamond grid shown in Fig. 6c.

Fig. 6 Design and fabrication of a bio-inspired flexible scaled armor. a Schematic diagram showing the basic components of the bio-inspired scaled armor, i.e., (1) scales, (2) matrix, and (3) soft underlying layer. b Side- and c bottom view of the scaled armor. d, the inter-scale spacing. d Flat panel with uniform scales fabricated through additive manufacturing. e A bent panel showing its excellent flexibility. f, g Design of scale pattern with size gradients. h Scale assembly in flat (top) and curved (bottom) substrate. i Scale assembly on double-curved surfaces. j X-ray projection images of a kneepad based on the bio-inspired scaled protective panel in j extended and k bent positions, demonstrating its conformability and flexibility. l Demonstration of the protection capability of the chiton scale-inspired kneepad on broken glass. Full size image

Prototypes of bio-inspired scale armor were fabricated using multi-material 3D printing (Fig. 6d–l). To mimic the interaction of scales and soft girdle tissue, materials with moduli of ca. 2 GPa and ca. 0.7 MPa were used for the scales and surrounding matrix, respectively. Owing to the manufacturability and material choice limitations, the design was simplified to include only the large dorsal scales and a homogeneous flexible basal layer. The resultant fabricated scale assembly exhibited excellent flexibility, with ranges of motion similar to its biological analogue (Fig. 6e). The parametric nature of our model allowed us to quickly and efficiently explore arrangements beyond uniform patterns on a flat substrate. For example, we first explored varying the scale size in a fashion similar to that of the chiton’s native girdle scale geometry. Rather than using two sets of intersecting parallel lines as was done for the construction of the uniform scale tiling shown in Fig. 6a–d, we instead used the intersection points of two expanding fan-like grids to dictate the scale positions and sizes needed to create a gradual change in scale size (Fig. 6f, g), which closely resembled the geometry of the native girdle scales (Fig. 2b). In addition to flat substrates, substrates of varying curvature were also explored. Figure 6h shows a comparison between flat and low-curvature assemblies of scales with uniform sizes. Figure 6i shows a high-curvature assembly, where local surface curvature dictates both the orientation and size of each individual scale.

Finally, to demonstrate the utility of the chiton-inspired system for applications requiring both combined flexibility and protection, a scaled kneepad prototype was developed (Fig. 6j–l). Current kneepad designs often fall in one of two extremes: hard and rigid plates that create heavy protection but limit flexibility, or elastomeric rubbers/foams that provide high flexibility but limited protection (especially against sharp objects). The chiton scale-inspired knee protection pad offers a unique solution to this dilemma. The printed scale assemblies were easily attached on or inserted into standard knee sleeves and exhibited good shape conforming capabilities in both bent and extended configurations (Fig. 6j, k). The system also provides much higher puncture resistance to hard sharp objects compared to typical kneepads with single-material rubber- or foam-based inserts, as illustrated in Fig. 6l and insets.

Anisotropic flexibility

Owing to the anisotropic geometry of the scales, we hypothesized that the scale assembly would exhibit orientation-dependent bending stiffness. Using the scales of R. canariensis as a model system, our parametric prototypes allowed us to systematically study the mechanical performance of the chiton scale assembly. Using multi-material 3D-printed models, we were able to conduct direct mechanical tests on these prototypes using a pinned post-buckling bending test (see section Mechanical testing of 3D-printed protypes in Methods). Each prototype was printed with rigid rods attached to either side, which were inserted into two supporting bases that were connected to the mechanical testing rig (Fig. 7a). The pinned boundary conditions defined the axis of displacement and allowed the pins to rotate freely about their central axes during the bending tests. For these measurements, we only considered concave bending modes (scales facing inwards), so that we could directly investigate the mechanical consequences of scale jamming and other scale–scale interactions. This is in contrast to the convex bending mode, which was not constrained due to the lack of scale–scale interactions and therefore any mechanical properties would be largely dependent on the flexibility of the inter-scale flexible phase. We defined the scale orientation angle, φ, as the angle between the loading axis of the prototype and the overlapping (hook) direction of the scales (Fig. 7b). Three prototypes were fabricated (φ = 0°, 60°, and 90°), where the 0° sample corresponds to a bending of the scale array towards the chiton’s body, and the 90° sample models anterior-posterior bending of the girdle.

Fig. 7 Analysis of anisotropic flexibility of the bio-inspired scale armor prototypes. a Schematic diagram of the post-buckling bending experiments. b Left, schematic diagrams of specimens tested in three orientations by varying the angle, φ, which is defined between the loading direction and the width direction of scales. Right, the modeled three φ angles indicated on the chiton’s body. c Experimentally measured and corresponding FE predictions of the force–displacement curves for prototypes with φ of 0°, 60°, and 90°. Photos of the φ = 90° sample at displacement of d 0 and e 60 mm, and f, g corresponding configurations from the FE modeling. The left and right photos in d, e show the front and side views, respectively. The distributions of von Mises stress of the φ = 90° sample from h the front and i the back view. Magnified views of the interaction between adjacent scales at displacement of j, m, p 0 and k, n, q 60 mm for φ = (j, k) 0°, (m, n) 60°, and (p, q) 90° sample, respectively. The red dots indicate the interlocking (contact) among adjacent scales. The von Mises stress distributions within individual scales at displacement of 60 mm due to interscale contact for φ = (l) 0°, (o) 60°, and (r) 90° sample, respectively. s A magnified view of the interlocked scales (indicated by yellow arrows) and t corresponding stress distributions from FE analysis for the φ = 0° sample at displacement of 60 mm. Full size image

The force–displacement curves from the bending tests are summarized in Fig. 7c, and finite element (FE) analysis based on models with same number of scales under the same loading conditions was also conducted to investigate the deformation process at both the local scale and the global assembly level (Fig. 7d–g). Figure 7d, e depict a 90° sample at the initial and later stage of the test, whose deformation behavior was also successfully captured in the simulations (Fig. 7f, g). The stress distribution maps indicate that the central region of the test samples has an overall high stress level due to its smaller local radius of curvature compared to the two edge regions (Fig. 7h, i). The predicted orientation-dependent mechanical behavior was observed from the force–displacement curves (Fig. 7c). In general, all three orientations demonstrate a certain degree of strain stiffening behavior, although the onset and rate of stiffening varied significantly. The initial soft regime with low bending resistance corresponds to the scales rotating relative to one another. The subsequent strain stiffening is a function of adjacent scales coming into contact with one another, with the differing strain stiffening behaviors the result of the different scale interactions modeled. Since the stiffening rate is governed by post-contact friction behavior, the lack of measured surface friction properties for the 3D-printed models inadvertently leads to the discrepancy between experimental and computational load-displacement curves in the post-contact regimes (Fig. 7c). The system is highly flexible in the 0° orientation (bending toward the chiton’s body) as exhibited by its lower initial bending load and larger soft regime compared to the other two orientations. In this orientation, and when viewed dorsally, the hook of each scale contacts the back of the scale immediately in front of it (Fig. 7j–l), which was confirmed in sectioned profile views both experimentally (Fig. 7s) and computationally (Fig. 7t). In the cases of the 60° and 90° samples, each scale eventually made contact with neighboring scales in the vertical and adjacent diagonal rows, respectively (Fig. 7m–o and Fig. 7p–r, respectively). For the 90° sample, despite early contact and four contact points per scale (Fig. 7q, red dots), the scales are able to slide past each other with relative ease as indicated by the lower stress levels within scales (Fig. 7r), resulting in a low stiffening rate. In contrast, for the 60° samples, the scales make contact with adjacent scales in a single row (Fig. 7n); however, a much higher local contact stress due to significant interlocking among adjacent scales (Fig. 7o) leads to a higher stiffening rate.