SHIM has emerged as a robust tool to capture high-resolution, high-content, 3D representations of fibrillar collagen in live and ex vivo tissue without the need for exogenous labeling18,19. In SHIM, a frequency doubling of the incident light occurs in molecular structures that are repetitive and non-centrosymmetric18,19,20,21,22,23. The resulting coherently forward propagated waveform is not accompanied by a loss of energy nor is subject to photobleaching, making it an ideal technique to examine and quantify endogenous collagen functional textiles. Simultaneously, two-photon excitation microscopy can be used to image autofluorescent elastin, through reflected fluorescence24,25.

Here, we first validate our combined SHIM-TPEM protocol on periosteum from sheep specimens prepared using standard protocols for undecalcified histology. Transverse sections were prepared from the ovine femoral diaphysis with intact surrounding musculature and vasculature stained in vivo using procion red per previous protocols26,27. Regions of interest (ROI) were imaged to highlight collagen and elastin fibers in adjacent musucloskeletal tissue compartments (bone, muscle, vasculature) and their respective microscopic structures along the major and minor axes of the diaphyseal shaft (Fig. 1a); these axes, calculated using an automated software, serve as objective indicators of tissue regions most and least able to resist bending forces in the axial plane28,29. For each ROI, a tiled image of the transverse (xy) plane, followed by a z-stack of one tile within the region, was captured to map in 3D space the composition and distribution of the collagen and elastin fibers as well as their higher order architectures.

The resulting merged tiled images demonstrate the high collagen content of bone (green) and high elastin content of muscle (brown) (Fig. 1). The thickness and composition of periosteum varies depending on proximity to nearby tissue compartments and structures such as muscle and vasculature. Where muscle or their fasciae adjoin to periosteum, the periosteum thickens, particularly in the posterior aspect. The periosteum and its neighboring muscle fasciae exhibit a varying composition and architecture of both collagen and elastin fibers. In some areas, fibers with a strong SHG signal, attributed to collagen, run along the bone’s circumference, though not spanning the entire ROI. These fibers represent components of periosteum’s fibrous layer, and their anisotropic arrangement likely underpins periosteum’s anisotropic mechanical reinforcement function and direction-dependent permeability2,3,15,16,17.

In other areas, collagen fibers insert at a slight angle directly into bone, indicative of Sharpey’s fiber structure and intrinsic function transducing force between muscle and bone (Fig. 1). The Sharpey’s collagen fiber bundles are thicker and emit a stronger signal than the collagen fibers within the periosteum sleeve composite. The thickness of the reflected fiber correlates to its contrast intensity, which varies as a function of the angle between the fiber and the laser beam20. Thus, for the first time to our knowledge, we were able to track in 3D space (in an approximately 40 μm thick z-stack) the angled collagen fibers making up single Sharpey’s fibers as they exit the periosteum and anchor into bone (Fig. 1b). Similarly, we also were able for the first time to visualize the interaction of the periosteum’s collagen fibers with neighboring muscle fascia and perimysium (Fig. 1c–g). Particularly in regions where muscle fascicles are in close association with the periosteum, collagen fibrils can be seen linking the periosteum to the perimysium, another indicator of direct force transfer (Fig. 1f). Though collagen is much less evident in the muscle per se, SHIM enables visualization of thin collagen fibers in the fasciae bordering muscle groups, perhaps providing mechanical support and stability during muscle distension. Based on these observations, we envision the muscle fascia as a continuous bounding layer of collagen fibers spanning superficial and deep muscle layers to the periosteum and finally, bone. In sum, the collagen fibers themselves exhibit different structures in different tissues, with loose and wavy collagen strands in muscle regions, to dense and straight bundles closer to the bone, and higher order architectures such as Sharpey’s fibers evident between tissue compartments.

Imaged in the transverse section of the long bone sample, the TPEM visualized elastin components of the periosteum resemble a dynamic layer enveloping bone. The elastin signal is also detected in muscle tissue structures, providing architectural information for fibers within muscle fascia, perimysium and blood vessels. With regards to muscle, distinct muscle groups can be seen connected via elastin coils resembling springs (Fig. 1d and Supplementary Animation 1 , 2 ) with other areas adhering directly to the periosteum (Fig. 1f and Supplementary Animation 3 ). In some ROIs multiple such coils formed a loosely woven webbed structure. These spatially varying features likely reflect the local mechanical environment of the tissue, e.g. elastin springs would maximize flexibility while providing elastic dampers during maximal muscle distension and spring back (Supplementary Fig. 1 and Supplementary Animation 3 ). Blood vessels, identified through the strong elastin signal of the vessel walls containing procion red filled channels, present abundantly in association with the periosteum (Fig. 1g and Supplementary Animation 4 ). Some blood vessels transect the periosteal layer to form the Volkmann canals, which insert into cortical bone and connect with the axially aligned Haversian channels. This multifunctional physiological tapestry comprises the fibrous weave of elastin and its higher order architecture into tissue fabric, bridging structures and vascular channels, highlighting the emergent structures which underlie the smart mechanical and permeability properties of the periosteum.

Imaging the periosteum using SHIM and TPEM enables high resolution mapping of elastin and collagen fibers and their higher order architectures in context of surrounding tissue compartments. As a next step in our bottom-up approach we used the z-stacks from our novel microscopy protocol to create scaled up 3D models, which accurately represent the composition and spatial architecture of the image sequences and the tissue itself (Fig. 2). To achieve this, each channel in the z-stack was imported separately as an image sequence into Mimics® for 3D rendering (Materialise, MIS v18.0 Beta) and then masked according to its signal intensity. The masks were converted to STL files and combined to create a composite 3D model comprising collagen, elastin and vascular components (Fig. 3). This model then served as a pattern template for a custom-configured jacquard-weaving algorithm (ArahWeave, arahne CAD/CAM for weaving) and for weaving of physical, scaled up prototypes (AVL Looms, Inc.).

In a first test of the scaled-up tissue weaving concept30,31, a series of textile swatch prototypes were woven, using specific combinations of collagen (nylon monofilament and braided silk suture) and elastin (elastane yarn), in a twill pattern designed to idealize periosteum’s intrinsic weave (Fig. 4a). As shown by the mechanical testing of the resulting textile swatches, the swatches’ capacity to resist tension are orthogonally anisotropic and highly dependent on the warp yarn composition of the weave. The scaled up textile swatches exhibit emergent properties suggestive of periosteum’s natural collagen and elastin weave. For example, although nylon monofilament (Nyl) suture weaves exhibit a lower elastic modulus (264.5 ± 73.1 MPa) than braided silk suture (Sil) warp weaves (461.7 ± 77.5 MPa), the observed strain distributions in the materials indicate that nylon warp weaves are much more resistant to tension than equivalent silk weaves (Fig. 4b–d). In contrast to both nylon and silk weaves, elastane (Ela) warp weaves are highly elastic, and exhibit little resistance to tension (2.106 ± 0.419 MPa), with a ubiquitously uniform strain profile (Fig. 4d). Interestingly, the weft yarns of the elastane warp weaves show the capacity to modulate strain distribution during axial loading, perhaps due to their low elastic modulus. Taken together, these first tests on a series of textile swatch prototypes demonstrate the feasibility of using SHIM in combination with TPEM to develop and prototype structurally relevant, scaled-up woven prototypes mimicking periosteum’s sophisticated, complex and composite tissue fabric of as well as its smart stress-strain properties. Such scaled up, mechanically functional textiles lend themselves for use in the safety and transport sector. Ongoing studies are implementing these approaches at the microscale using engineered collagen and elastin and other biological structural proteins, for rapid implementation in the medical sector30,31.

Figure 4 (a) Stereographs of woven swatches consisting of various warp and (weft) combinations (x12.5). (b,c) Individual value plot comparing elastic moduli of prototypes comprising different combinations, i.e. warp (weft), of nylon, elastane and silk (*p < 0.05). (d) Strain maps of prototypes at t 0 , t f/2 , t f , where t = time, and f = final. Full size image

The acquisition of high-resolution, architecturally accurate 3D models enables rapid prototyping using computer-controlled weaving and/or other rapid prototyping modalities, allowing for automated and spatially consistent fabrication at high throughput. Other contemporary rapid prototyping techniques have been applied to manufacture tissue engineering scaffolds including electrospinning of nanoscaffolds32,33, 3D organ printing34 and integrative weaving of porous cartilage scaffolds35. Developments in electrospun nanofiber scaffolds enable the creation and manipulation of scaffolds at the cellular length scale, although this manufacturing process is not yet amenable to customization of the architecture, geometry and mechanical attributes needed to mimic the composite and sophisticated material properties of the periosteum or other similarly complex tissues. 3D printing offers distinct advantages with regard to flexibility in customizing geometries but is not yet effective for prototyping pieces with seamless mechanical gradients or parts that can withstand dynamic tension and bending. To date, integrative weaving has not yet captured the detailed fiber arrangement of biological tissues. Hence, to our knowledge, this study is the first of its kind, where natural woven architectures are mapped and replicated in scaled-up models to develop novel advanced materials and functional textiles. The only other potential way to replicate this process recursively would be to ‘unravel’ an inverse representation of the tissue mechanics as a stiffness map (Fig. 5), providing a pattern for the anisotropic weave, as shown above. Both approaches have been reduced to practice and the intellectual property has been protected (patent pending)30,31. Tests are underway to optimize the degree to which emergent properties are compromised by using the latter technique.

Figure 5: Recursive weaving and spinning concept. (a–e) Example of a tensile testing of anisotropic sheep femur periosteum samples, analogous to testing of swatches in Fig. 4. (b,c) DIC imaging of displacements under load at time zero and 562 seconds. (d,e) Strain mapping overlay on (b,c). Used with permission after16. (f) Testing of the highly complex, multidimensional fabric of periosteum in situ and ex vivo, under stance shift load. Heterogenous strain map at one point in time is depicted in color using digital image correlation and high resolution imaging (using high definition television lens). The dashed line in this view is orthogonal to the middiaphyseal imaging carried out using second harmonic imaging of collagen and elastin per Fig. 1(a), on the anterior aspect (corresponds to side of bone with ROI indicated by dashed square 3. Used with permission after37. (g) Schematic representation of fabric complexity showing weave of spatially and temporally varying threads, where the color scale depicts either local strains analogous to the endogenous tissue or, from an inverse perspective, local fiber stiffness. (h) Schematic depiction of recursive step where program algorithm is used to spin anisotropic, viscoelastic threads and to weave fabric. Adapted with permission30,31. Full size image

In summary, here we have demonstrated our novel protocol using SHIM in conjunction with TPEM to elucidate the organization and distribution of the collagen and elastin fibers of the periosteal sheath and to replicate nature’s smart properties through creation of advanced materials via scale up of tissue architectures and multifunctional weaving. Inspired by nature’s paradigms, this disruptive technology (defined as an “innovation that creates a new market and value network and eventually disrupts and dispaces an existing market and value network”36) has significant implications for the development of next-generation advanced materials and mechanically functional textiles, including biomedical materials and even materials in transport and safety industries.