Acta Biomaterialia

BackgroundAortic microstructure-function relationships and the pathophysiology of how medial degeneration leads to aortic dissection remain poorly defined. We aimed to determine how degeneration of individual components of the extracellular matrix (ECM), namely elastin, collagen, and proteoglycans, influence biomechanical properties of aortic tissue through an improved, disease-motivated enzymatic digestion framework. MethodsPorcine aortic tissue was sectioned into 200 {micro}m thick samples in the media, and progressively digested with elastase or collagenase for selective degradation of these ECM components. Full thickness human aortic tissues were treated with chondroitinase, hyaluronidase, and heparinase to completely remove proteoglycans. Biomechanical characterization was performed using planar biaxial tensile testing, from which low- and high-strain modulus, transition-zone behaviour, strain-energy density, and energy loss were derived. Degree of elastin fiber degradation was analyzed using two photon excitation fluorescence imaging. Analysis of collagen degradation was performed using picrosirius red staining under brightfield and polarized light. Alcian blue staining was used to evaluate proteoglycan content. ResultsInduced fragmentation and disorganization of elastin fibers reduced low-strain load bearing capacity, evidenced by reduced low-strain modulus, strain-energy density, and transition zone stress, along with reduced energy loss. Targeted collagen disorganization similarly reduced strain-energy density and decreased strain at the onset of transition, consistent with premature collagen recruitment, and was accompanied by reductions in high strain modulus and energy loss with increasing collagen degradation. Proteoglycan removal decreased energy loss and was found to modulate low- and high-strain behaviour, including reduced strain-energy density and strain at onset of transition, and increased high strain modulus. ConclusionsThrough targeted modelling of ECM degenerative features on aortic tissue mechanics, we have identified distinct disease-associated biomechanical roles for major matrix constituents, with overlapping effects. These findings delineate mechanical consequences of component-specific matrix degeneration while underscoring the complex, multifactorial nature of structure-function relationships in aortic disease.

Tendon/enthesis injuries are a worldwide clinical problem. Along the enthesis, collagen fibrils show a progressive loss of anisotropy and an increase in mineralization reaching the bone. This causes gradients of mechanical properties. The design of scaffolds to regenerate these load-bearing tissues requires of being validated in vivo in relevant large animal models. The sheep tendon of triceps surae muscle is an optimal animal model for this scope with limited knowledge about its structure and mechanics. We decided to understand in-depth its structure and full-field mechanics. Collagen fibrils morphology was investigated via scanning electron microscopy revealing a marked change in orientation/dimensions passing from tendon to enthesis. Backscatter electron images and nanoindentation at the enthesis/bone marked small gradients of mineralization at the mineralized fibrocartilage reaching 27%wt and indentation modulus around 17-30 GPa. The trabecular bone instead had indentation modulus around 15-22 GPa. Mechanical tensile tests with digital image correlation confirmed the typical non-linear behavior of tendons (failure strain = 8.2{+/-}1.0%; failure force = 1369{+/-}187 N) with maximum principal strains reaching mean values of {varepsilon}p1[~]7%. The typical auxetic behavior of tendon was highlighted by the minimum principal strains ({varepsilon}p2[~]5%), progressively dampened at the enthesis. Histology revealed that this behavior was caused by a local thickening of the epitenon. Cyclic tests showed a force loss of 21{+/-}7 % at the last cycle. These findings will be fundamental for biofabrication and clinicians interested in designing the new generation of scaffolds for enthesis regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/630234v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@143f0a8org.highwire.dtl.DTLVardef@16cb394org.highwire.dtl.DTLVardef@181be61org.highwire.dtl.DTLVardef@f9f2d9_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceTendon and enthesis lesions are a clinical problem. To validate scaffolds for these applications large animal models are needed. Sheep tendon of triceps surae muscle is an optimal site for this scope. However, little is known about its extracellular matrix structure and mechanical properties. This work investigates the structure and mechanics of this tissue from different points of view. Scanning electron microscopy and histology studied its extracellular matrix morphology and composition. Backscattered electron images and nanoindentation assessed gradients of mineralization and stiffness at the enthesis. Mechanical tensile and cyclic tests coupled with digital image correlation elucidated its mechanics and superficial strain distribution. These findings will be fundamental for biofabrication and clinician experts to design innovative scaffolds to regenerate the enthesis.

Collagen is the most abundant protein in the extracellular matrix, crucial for wound healing and cell proliferation. While it holds promise as a scaffold for tendon, skin, and ligament reconstruction, collagens mechanical strength, particularly under stretch, is poor. Previous attempts to improve collagen strength involved blending it with silkworm or recombinant spider silk. In this study, for the first time, we evaluated whether collagen gel from fish skin could be strengthened by infusing it with native spider silk, specifically the major ampullate (MA) silk of Nephila pilipes, known for superior mechanical properties. MA silk was woven onto a frame, pressed into a PDMS platform, and then used to create a collagen scaffold. Youngs modulus of the infused collagen scaffold, subjected to either stretching or non-stretching treatments, was measured using AFM. After 24 hours of cyclic stretching, collagen infused with silk showed less fragility, higher Youngs modulus, and no bacterial growth. Immunohistochemical staining showed that after stretching, the thickness and architecture of the collagen gel infused with silk were maintained, and the fibers were reorganized in a more compact, aligned, and denser manner. Overall, collagen infused with native spider silk exhibited improved mechanical stability and stiffness under cyclic stretching, suggesting that this combination could serve as a robust matrix for bioengineering applications while preventing bacterial infiltration.

Tendons hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer, however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy (SBF-SEM) to investigate the threedimensional microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite element modeling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.

Many animals rely on specialised mouthparts to process food. Because this is a mechanically demanding task, mouthparts often wear, with potentially serious consequences for feeding performance and thus fitness. The biomechanics of wear are therefore of clear biological relevance, but remain poorly understood, especially in insects, where conventional engineering wear tests are hard to implement. Here, we present a nanomechanical characterisation of the mandibular epicuticle of three insect species: two leaf-cutting specialists, one with and one without transition-metal inclusions, and an omnivore. Contrary to predictions from simple engineering wear theory, wear resistance was neither directly proportional to indentation hardness nor inversely proportional to wear load. We suggest that this discrepancy arises in part from the high hardness-to-modulus ratio of mandibular epicuticle, which renders indentation hardness a poor proxy for resistance to plastic deformation. A simple elasto-plastic wear model qualitatively captures the main discrepancies between experiment and theory, and points to a revised set of wear proxies that may allow at least a qualitative ranking of biological materials via iso-performance lines on Ashby plots. Yet, as with most wear models, the wear coefficient remains unpredictable, a limitation strikingly illustrated by the increase in epicuticular wear resistance upon hydration despite a decrease in both hardness and modulus. Together, these observations suggest that purely plastic wear models may often be inadequate for biological materials with a high hardness-to-modulus ratio, and that even elasto-plastic models require careful validation against experimental wear assays. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/731969v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@13e958org.highwire.dtl.DTLVardef@13ca5fdorg.highwire.dtl.DTLVardef@b585adorg.highwire.dtl.DTLVardef@15b3be3_HPS_FORMAT_FIGEXP M_FIG C_FIG

In pulmonary fibrosis, excessive scar tissue accumulates in the alveolar interstitial space, impairing gas exchange and compromising lung function. This fibrotic remodeling results in tissue stiffening, but more complex lung mechanical properties critical to tissue function, such as viscoelasticity and stress relaxation, remain poorly defined. To address this gap, we use the bleomycin aged mouse model to characterize both bulk and spatially-resolved viscoelastic mechanical properties of normal and fibrotic lungs. Our analysis reveals that while bleomycin-induced fibrosis leads to heterogeneously increased lung stiffness, viscoelasticity as measured by tan delta (ratio of loss to storage modulus) and stress relaxation timescales remains remarkably consistent as a function of both age and bleomycin treatment. This unexpected preservation of viscoelasticity despite fibrotic stiffening highlights a previously underappreciated mechanical phenotype of fibrotic lungs. To model these distinct mechanical features in vitro, we utilize a hyaluronic acid-based hydrogel system that largely recapitulates the viscoelastic mechanical properties observed in both normal and fibrotic lungs. These findings provide new insight into the mechanical consequences of fibrosis and establish a tunable in vitro hydrogel platform mimicking key tissue viscoelastic properties.

Collagen scaffolds seeded with human chondrocytes have shown great potential for cartilage repair and regeneration. However, these porous scaffolds buckle under low compressive forces, creating regions of highly localized deformations that can cause cell death and deteriorate the integrity of the engineered tissue. We perform three-dimensional (3D) tomography-based characterization to track the evolution of collagen scaffolds microstructure under large deformation. The results illustrate how instabilities produce a spatially varying compaction across the specimens, with more pronounced collapse near the free boundaries. We discover that, independent of differences in pore-size distributions, all collagen scaffolds examined displayed strong auxetic behavior i.e., their transverse area contracts under compression, as a result of the instability cascade. This feature, typically characteristic of engineered metamaterials, is of critical importance for the performance of collagen scaffolds in tissue engineering, especially regarding the persistent challenge of lateral integration in cartilage constructs.

The human femoral neck is particularly vulnerable to fracture, with failure most often initiating in the superior region. While age-related microstructural changes such as cortical thinning and increased porosity are well established, the contribution of material properties at the lamellar and mineralised collagen fibril (MCF) levels remains poorly understood. Here, regional differences in nanostructural properties of cortical bone from 78 femoral necks obtained from 44 donors aged 54-96 are investigated using a combined 2D and 3D X-ray scattering imaging approach. This approach quantifies MCF orientation and structure averaged over multiple lamellae in large fields of view, capturing tissue heterogeneity through the hierarchical scales. We identified misalignment between the scattering signals arising from the MCF bundles -- specifically those associated with mineral inclusions in the collagen fibril gap regions, the mineral nanostructure, and the mineral crystal lattice -- suggesting the presence of distinct mineral phases within and around the collagen fibers. Despite substantial intra-sample variability, the superior region displays on average more oblique MCF orientations, larger and thicker mineral platelets arranged in a less-ordered structure, greater misalignment between mineral and collagen at the MCF level, and possibly stiffer collagen fibres, with no significant trends observed with donor age or sex. The cumulative effect of these material property differences may contribute to the increased susceptibility of the superior cortex to compressive failure.

This study investigates the evolution with strain of the material volume fraction (i.e., porosity) and geometry in porous scaffolds to obtain a more accurate description of their stress-strain behavior. Single bundles and hierarchical structures (8 bundles enveloped by a membrane) were produced by electrospinning as tendon/ligament scaffolds. They underwent a micro-tomography in situ tensile test. Apparent and net stress were obtained using the initial sample cross-section and material volume fraction to normalize axial force. Micro-tomography revealed sample morphology change with strain to calculate the actual stress-strain. Moreover, nanofibers arrangement was revealed by scanning electron microscopy on both bundles and membranes. The description of the mechanical response significantly changed using evolving morphometry (actual stress-strain) instead of initial static one (apparent stress-strain), for both single bundle and hierarchical structure. The actual elastic modulus of the single bundles (583{+/-}97 MPa) was statistically higher than that of the hierarchical structures (163{+/-}107 MPa). This is related to the membrane, membrane-bundle and inter-bundle interactions. In the hierarchical structure, portions of the material resisting traction are constituted by nanofibers not aligned with the load. The different definitions for the stress-strain behavior allow different accuracy levels depending on the experimental complexity. The evolution of morphology with deformation can significantly affect the description of the mechanical response of porous scaffolds. This has a double impact in practical applications: at the body scale, it allows a better comparison between the scaffold behavior and the target tissue; at the cellular scale, it predicts the actual substrate stiffness that cells will face. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/630537v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5d5337org.highwire.dtl.DTLVardef@1027047org.highwire.dtl.DTLVardef@11966a9org.highwire.dtl.DTLVardef@a2e4e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1+/D menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1+/D meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

Mechanical deformation applied to tendon at the tissue-scale is transferred to the microscale -- including the extracellular matrix (ECM), the pericellular matrix (PCM), the cell and the nucleus -- through a process known as strain transfer. Microscale strains, in turn, trigger biological activity that plays an important role in the maintenance of tendon phenotype and homeostasis. Although tendon predominantly experiences longitudinal tensile forces, transverse forces due to bony impingement have been implicated in both physiological (e.g., maintenance of the tendon insertion) and pathophysiological (e.g. insertional Achilles tendinopathy) processes. However, to our knowledge, prior studies have not characterized the micromechanical strain environment in the context of tendon impingement. Therefore, the objective of this study was to characterize the micromechanical strain environment in the impinged Achilles tendon insertion using a novel mouse hindlimb explant model in combination with finite element (FE) modeling. We hypothesized that impingement would generate large magnitudes of transverse compressive strain at the local matrix, PCM, and cell scales. Mouse hindlimb explants were imaged on a multiphoton microscope, and image stacks of the same population of tendon cells were obtained at the Achilles tendon insertion before and after dorsiflexion-induced impingement. Using an innovative multiphoton elastography approach, three-dimensional Green-Lagrange and principal strains were measured at the matrix scale, while longitudinal strain and aspect ratio were measured at the PCM and cell scales. Our results demonstrate that impingement generated substantial transverse compression at the matrix-scale, which led to longitudinal stretching of cells, an increase in cell aspect ratio, and -- surprisingly -- longitudinal compression of the tendon PCM. These experimental results were corroborated by an FE model developed to simulate the micromechanical environment in impinged regions of the Achilles tendon. Moreover, in both experiments and simulations, impingement-generated microscale stresses and strains were highly dependent on initial cell-cell gap spacing. Understanding the factors that influence the microscale strain environment generated by impingement could contribute to a more mechanistic understanding of impingement-induced tendinopathies and inform the development of approaches that disrupt the progression of pathology.

Engineered living materials (ELMs) at the multicelluar level represent an innovation that promises programmable properties for biomedical, environmental, and consumer applications. However, the rational tuning of the mechanical properties of such ELMs from first principles remains a challenge. Here we use synthetic cell-cell adhesins to systematically characterize how rheological and viscoelastic properties of multicellular materials made from living bacteria can be tuned via adhesin strength, cell size and shape, and adhesion logic. We confirmed that the previous results obtained for non-living materials also apply to bacterial ELMs. Additionally, the incorporation of synthetic adhesins, combined with the adaptability of bacterial cells in modifying various cellular parameters, now enables novel and precise control over material properties. Furthermore, we demonstrate that rheology is a powerful tool for actively shaping the microscopic structure of ELMs, enabling control over cell aggregation and particle rearrangement, a key feature for complex material design. These results deepen our understanding of tuning the viscoelastic properties and fine structure of ELMs for applications like bioprinting and microbial consortia design including natural systems.

Enthesis tissue engineering aims to develop scaffolds that replicate the mechanical and structural gradients of the tendon/ligament-bone interface. Among the different biofabrication techniques, electrospinning is surely one of the most promising to fabricate morpho-mechanically relevant enthesis fascicle-inspired scaffolds. An interesting and totally unexplored characteristic of these nanofibrous scaffolds is their ability, when mechanically tested, to produce/transmit strain rate and nanofiber fracture-dependent mechanical vibrations, which can potentially influence surrounding tissues and cells. This study develops a method to investigate how scaffold geometry and material affect vibrational behavior under mechanical stimulation. Electrospun bundles of poly(L-lactic) acid/collagen type I (PLLA/Coll) were fabricated to mimic the fibrocartilage, the enthesis junction, and the tendon/ligament regions, while block copolymer poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT-PBT) bundles represented only the tendon/ligament. Scaffolds were morphologically and mechanically characterized, including strain rate-dependent vibrational response. Scanning electron microscopy confirmed distinct fiber architectures. Under monotonic tensile tests to failure, scaffolds exhibited strain rate-dependent mechanical behavior, with PLLA/Coll bundles showing dominant vibrational frequencies up to 4.2 {+/-} 0.9 Hz with a scaffolds geometry-dependent manner. PEOT-PBT scaffolds instead, displayed higher vibration attenuation, with dominant frequencies peaking at 0.539 {+/-} 0.063 Hz. They also showed lower tensile properties, reflecting a different mechanical and vibrational profile respect to PLLA/Coll bundles. Integrating vibrational characterization with mechanical testing offers a novel framework for designing scaffolds that more accurately reproduce the gradient mechanical environment of fibrous musculoskeletal tissues such as tendons/ligaments and their entheses. These findings highlight the potential of this combined approach to further increase the mechanical comprehension of electrospun scaffolds. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/699441v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1e4f38dorg.highwire.dtl.DTLVardef@31e658org.highwire.dtl.DTLVardef@9c590org.highwire.dtl.DTLVardef@18996e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tendon is critical for musculoskeletal function as it transfers forces generated by muscle to bone and stores energy during movement. Impaired mechanical function in tendon limits mobility and results from fatigue-induced damage progression that outpaces the restorative processes maintaning tissue health, a phenomenom we term mechanopathology. Early and non-invasive detection of tendon mechanopathologies is vital to prevent further damage, but is lacking in the clinical space. Here, we evaluate the ability of diffusion tensor magnetic resonance imaging (DT-MRI) to detect mechanical fatigue damage in tendon, and validate our findings using histologic assessments of collagen fiber microstructure and molecular structure. We found that fatigue-induced changes in DT-MRI metrics of tendon are spatially heterogeneous, and correspond to regions with damaged collagen fiber microstructure. While secondary structures of collagen molecules were damaged by fatigue loading, they do not spatially correspond to fatigue-induced changes in DT-MRI metrics. Fatigueinduced changes in DT-MRI metrics can be partially explained by quantitative metrics of post-fatigue collagen fiber microstructure, estimating the limit of detection of DT-MRI metrics to fatigue-induced damage in tendon. Our findings indicate that DT-MRI metrics are sensitive to fatigue-induced local damage in tendon, supporting the potential of DT-MRI as a non-invasive and translatable tool to clinically detect mechanopathologies in tendon.

Understanding the multiscale mechanics of the colorectum is essential for uncovering the mechanotransductive pathways underlying visceral nociception. Intraluminal distension of the large intestine reliably evokes pain in disorders of gut-brain interaction (DGBIs), yet the tissue-level and nerve fiber-level responses to mechanical loading remain poorly defined. Here, we present results from a novel biomechanical testing framework that integrates uniaxial circumferential extension with high-resolution optical imaging to quantify deformation in both bulk colorectal tissue and embedded sensory nerve fibers. We tested intact, cylindrical colorectal segments from mice using a custom 3-D-printed chamber with intraluminal stainless-steel rods to apply circumferential stretch while maintaining a planar imaging field. We measured bulk-tissue deformation via Digital Image Correlation (DIC), while we assessed stretch in nerve fibers through fluorescence imaging of VGLUT2-labeled afferents analyzed using a custom fiber-network analyses. Across specimens, we observed a consistent auxetic response-characterized by positive axial strain during circumferential extension-at both the macroscale and microscale. Five out of six colorectal specimens exhibited positive axial Green-Lagrange strain (Exx), with an average median Exx of 0.0177, during circumferential extension generating an average median Eyy of 0.1273. Nerve fiber analysis across nine specimens revealed an average median stretch ratio of 1.0631, indicating 6.31% elongation, with substantial heterogeneity driven by fiber orientation. These findings demonstrate that the colorectum and its embedded network of nerve fibers exhibit auxetic behavior, a property that may amplify mechanical signaling and influence nociceptive signaling. Our methods and results provide foundational insight into structure-function relationships of colorectum and inform design of bioinspired auxetic materials.

Successful tissue engineering requires biomedical devices that initially stabilize wounds, then degrade as tissue is regenerated. However, the material degradation rates reported in literature are often conflicting. Incorporation of in situ monitoring functionality into implanted devices would allow real time assessment of degradation and potential failure. This necessitates introduction of contrast agent as most biomedical devices are composed of polymeric materials with no inherent contrast in medical imaging modalities. In the present study, computed tomography (CT)-visible radiopaque composites were created by adding 5-20wt% tantalum oxide (TaOx) nanoparticles into polymers with distinct degradation profiles: polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15 and PLGA 50:50, representing slow, medium and fast degrading materials respectively. Radiopaque phantoms, mimicking porous tissue engineering devices, were implanted into mice intramuscularly or intraperitoneally, and monitored via CT over 20 weeks. Changes in phantom volume, including collapse and swelling, were visualized over time. Phantom degradation profile was determined by polymer matrix, regardless of nanoparticle addition and foreign body response was dictated by the implant site. In addition, degradation kinetics were significantly affected in mid-degrading materials, transitioning from linear degradation intramuscularly to exponential degradation intraperitoneally, due to differences in inflammatory responses and fluid flow. Nanoparticle excretion from degraded phantoms lagged behind polymer, and future studies will modulate nanoparticle clearance. Utilizing in situ monitoring, this study seeks to unify literature and facilitate better tissue engineering devices, by highlighting the relative effect of composition and implant site on important materials properties.

AO_SCPLOWBSTRACTC_SCPLOWLiving organisms have developed design principles, such as functional gradients (FGs), to interface hard materials with soft ones (e.g., bone and tendon). Mimicking such design principles can address the challenges faced when developing engineered constructs with soft-hard interfaces. To date, implementing these FG design principles has been primarily performed by varying the ratio of the hard phase to that of the soft phase. Such design approaches, however, lead to inaccurate mechanical properties within the transition zone. That is due to the highly nonlinear relationship between the material distribution at the microscale and the macroscale mechanical properties. Here, we 3D print micro-bricks from either a soft or a hard phase and study the nonlinear relationship between their arrangements within the transition zone and the resulting macroscale properties. We carry out experiments at the micro- and macroscales as well as finite element simulations at both scales. Based on the obtained results, we develop a co-continuous power-law model relating the arrangement of the micro-bricks to the local mechanical properties of the micro-brick composites. We then use this model to rationally design FGs at the individual micro-brick level and create two types of biomimetic soft-hard constructs, including a specimen modeling bone-ligament junctions in the knee and another modeling the nucleus pulposus-annulus fibrosus interface in intervertebral discs. We show that the implemented FGs drastically enhance the stiffness, strength, and toughness of both types of specimens as compared to non-graded designs. Furthermore, we hypothesize that our soft-hard FGs regulate the behavior of murine preosteoblasts and primary human bone marrow-derived mesenchymal stromal cells (hBMSCc). We culture those cells to confirm the effects of soft-hard interfaces on cell morphology as well as on regulating the expression of focal adhesion kinase, subcellular localization, and YAP nuclear translocation of hBMSCs. Taken together, our results pave the way for the rational design of soft-hard interfaces at the micro-brick level and (biomedical) applications of such designs.

Cellular agriculture, as an emerging food production system, holds potential to address sustainability, food security, and agricultural resilience. Within the cell-based meat supply chain, one of the key steps is scaffolding. In this study, we assessed decellularized banana leaves, various coating materials, and different cell seeding strategies to determine their effects on cell viability, cell growth, cell alignment, and the response of the materials to thermal processing. The efficiency of decellularization was verified through DNA quantification, which decreased from 445 ng/mg in fresh banana leaves to non-detectable levels in the decellularized samples. This was further confirmed by FTIR and PCA modeling. Cell viability exceeded 98% on uncoated, soy-coated, and gelatin-coated samples of the decellularized banana leaves. Alignment of cells on gelatin-coated samples was the highest among the samples, with a dominant orientation of 65.8{degrees}, compared to soy-coated and uncoated samples with dominant orientations of 9.2{degrees} and -6.3{degrees}, respectively. In terms of quality attributes, the kinetics of shrinkage indicated that coating with soy and the presence of cells increased the activation energy due to the higher energy required for protein denaturation. Moreover, the kinetics of area changes in plain scaffolds without cells followed a first-order pattern, while with seeded cells a second-order pattern was followed. In summary, decellularized banana leaves present a sustainable and suitable biomaterial to support cells towards future needs related to meat production.

Dissection of the thoracic aorta includes delamination of medial lamellae and permeation of blood within the media. Quantifying how biaxial loading of a vulnerable wall and fluid mechanics interact to drive dissection remains a central challenge. Here we combine controlled distension-extension testing of intact porcine descending thoracic aortas with forced intramural fluid injection to investigate how axial stretch, injection rate, and needle gauge modulate the initiation and propagation of intramural delamination. Across experiments, injection pressure-volume curves exhibited nonlinear responses characterized by pressure peaks followed by stepwise pressure drops, suggesting progressive micro-delamination events within the medial lamellar networks. Increasing axial stretch significantly elevated peak injection pressure and promoted preferential axial propagation of the permeation / delamination front. Higher injection rates induced abrupt lamellar separation and larger dissected areas, whereas smaller needle gauges generated higher upstream pressures due to increased hydraulic resistance. Synchrotron imaging revealed the microstructural transition from intralamellar fluid permeation and wall swelling to the formation of a large fluid-filled delamination cavity. These results support a mechanistic framework in which the introduction of pressurized fluid within the aortic media behaves as a hydraulic fracture process in a layered poroelastic tissue, governed by balance across fluid pressurization, wall loading, and interlamellar strength. The findings provide quantitative insight into the biomechanical conditions that contribute to the initiation and propagation of aortic dissection.

The intervertebral disc (IVD) is a key contributor to the spines biomechanical functions. It consists of a gelatinous core (nucleus pulposus, NP) surrounded by a fibrous ring (annulus fibrosus, AF). Accumulation of microcracks and tears in the AF can lead to NP herniation outside the disc space, compressing the nerve roots and causing pain. Herniation is a leading cause of low back pain and represents a major socioeconomic burden, with no effective regenerative treatment currently available. Previously, we demonstrated the potential of a poly({varepsilon}-caprolactone) (PCL)-based implant for the closure of an annulus fibrosus (AF) defect. However, the slow in vivo degradation of PCL may hinder timely AF regeneration. Here, we hypothesized that accelerating PCL degradation kinetics by incorporating a lactide component, known for its rapid degradation, could enhance AF repair. To that aim, PCLA polymers of {varepsilon}-caprolactone and lactide were synthesised as a copolymer (C-CL90%LA10%) or a blend (B-PCL80%PLA20%). These polymers were electrospun into aligned nanofibrous sheets, which were then assembled into a biomimetic multi-lamellar 3D implant. Cytocompatibility was assessed in vitro using ovine AF cells and ex vivo using a bovine tail disc model. In vitro, PCLA sheets promoted ovine AF cell alignment, proliferation, and expression of type I and II collagen. Ex vivo, the multi-lamellar implant remained in place within a full-thickness (4 mm) annular defect in bovine tail discs for 4 weeks, with initial cell infiltration. Degradation and regenerative potential were then evaluated in an ovine lumbar full-thickness annular defect model at 1 month (n=4) and 6 months (n=10) after implantation. Histological and immunohistochemical analyses revealed substantial cellular infiltration and neo-tissue ingrowth within implant layers, despite implant displacement in some cases. At 1 month, all implants retained their multi-lamellar structure with no visible degradation. By 6 months, the copolymer implants exhibited marked degradation, whereas PCL and blend implants remained structurally intact. Furthermore, ex vivo biomechanical testing revealed comparable flexibility to intact but much lower than non-implanted controls in axial rotation. This study confirms the accelerated degradation kinetics of PCLA copolymers within the disc microenvironment and demonstrates that the multi-lamellar PCLA implant can guide AF tissue repair. Further optimization is needed to enhance implant retention and ensure long-term functional integration.