Acta Biomaterialia

This paper concerns the tear properties and behaviour of Bombyx mori silk cocoons. The tear resistance of cocoon layers is found to increase progressively from the innermost layer to the outermost layer. Importantly, the increase in tear strength correlates with increased porosity, which itself affects fibre mobility. We propose a microstructural mechanism for tear failure, which begins with fibre stretching and sliding, leading to fibre piling, and eventuating in fibre fracture. The direction of fracture is then deemed to be a function of the orientation of piled fibres, which is influenced by the presence of junctions where fibres cross at different angles and which may then acts as nucleating sites for fibre piling. The interfaces between cocoon wall layers in Bombyx mori cocoon walls account for 38% of the total wall tear strength. When comparing the tear energies and densities of Bombyx mori cocoon walls against other materials, we find that the Bombyx mori cocoon walls exhibit a balanced trade-off between tear resistance and lightweightness.

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.

Electrospun fiber meshes have long served as biomaterials in a wide range of biomedical applications due to their functional similarities to extracellular matrix and highly tunable properties. Altering the mechanical behaviors of individual fibers and their microarchitecture (e.g.; diameter, crimp, orientation, density) can in principle be used to control bulk level behaviors. Moreover, electrospun meshes are often combined with softer coatings and hydrogels to control surface interactions with body tissues. Yet, fully optimizing their behaviors for specific applications remains an elusive target due to a continued lack of understanding of the micromechanical mechanisms and their relation to bulk mechanical behaviors. Our goal herein was to understand how actual nanoCT-generated 3D microfiber geometry can be used to predict bulk mechanical properties of hydrogel-mesh composites. Electrospun polyurethane meshes were fabricated with a random fiber orientation and coated with a PEG-based hydrogel. The fiber-hydrogel composite was then imaged with a nanoCT scanner at a voxel resolution of 180 nm. From these images, custom Python programs were written to segment, refine, and tesselate a high-resolution finite element of the fiber mesh and hydrogel volumes into a single integrated bi-material finite element model. The resulting mesh was used to run simulations of the planar biaxial mechanical tests used to characterize the bulk mechanical behaviors. Our framework thus enabled systematic investigations of both the macroscopic bulk mechanical response of the overall fiber mesh and the microscopic localized mechanical response of fibers under various stages of loading. The resultant simulations were accurate and predictive of the bulk mechanical responses. It is interesting to note that the fiber-hydrogel composite material experienced the largest stresses within the fiber phase and the largest strains within the hydrogel. This key result underscores that while the previous analytical model assumed local affine deformations, at the microscale this assumption does not hold. We also found very different effective fiber stress-strain responses in each model. It is likely these differences are due to the substantial heterogeneous non-affine local deformations present in the actual fiber-hydrogel composite. This finding further reveals the need for more rigorous approaches to better understand how electrospun-based materials function in order to improve their use in modern medical devices and implants.

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

Human hair is a keratin-based fiber with mechanical properties relevant to load-bearing biomaterials; however, its smooth cuticle limits fiber-fiber cohesion during textile-style processing. This study examines how controlled chemical decuticularization influences surface morphology and tensile behavior of intact human hair assembled into continuous one-dimensional (1D) strands. Hair was treated with oxidative bleach, sodium hydroxide (NaOH), or formic acid (FA), carded, and spun using a standardized protocol. SEM imaging showed treatment-dependent surface disruption, from minimal cuticle modification (bleach) to partial scale lifting (NaOH) and extensive cuticle removal (FA). Tensile testing revealed significant differences in Youngs modulus, ultimate tensile strength (UTS), and elongation at break (EAB) across treatments (ANOVA, p < 0.05). NaOH-treated strands exhibited the highest modulus (207 MPa), UTS (34 MPa), and moderate extensibility (28%), whereas bleach- and FA-treated strands showed reduced stiffness and strength. Compared with reference yarns, NaOH-treated strands approached the stiffness of wool and retained greater extensibility than cotton. These findings support a processing window in which partial decuticularization enhances fiber cohesion while preserving mechanical integrity. The resulting 1D strands provide a potential building block for woven biomesh structures, motivating further evaluation of durability, cyclic behavior, multi-ply configurations, and computational modeling.

Mechanical properties of the extracellular matrix (ECM) modulate cell-substrate interactions and influence cellular behaviors such as contractility, migrations, and proliferation. Although the effects of substrate stiffness on mechanobiology have been well studied, the role of ECM viscoelasticity in fibrotic progression remains less understood. To examine how viscoelasticity affects the biophysical properties and regulates the signaling of human mammary fibroblasts, we engineered elastic (E) and viscoelastic (VE) polyacrylamide hydrogels with comparable storage moduli ([~]14.52 {+/-} 1.03 kPa) but distinctly different loss moduli. Fibroblasts cultured on E hydrogels spread extensively (2428.93 {+/-} 864.71 m{superscript 2}), developed prominent stress fibers with higher zyxin intensity, and generated higher traction stresses (2931.57 {+/-} 1732.61 Pa). In contrast, fibroblasts on VE substrates formed smaller focal adhesion areas (54.2% reduction), exhibited lower critical adhesion strengths (51.8%), and generated 21% lower traction stresses (p < 0.001), indicating weaker adhesions. These substrates also promoted migrations and showed enhanced proliferation accompanied by reduced YAP activity, suggesting a mechanotransduction shift that may involve alternative signaling pathways. In contrast, E substrates showed YAP nuclear translocation, consistent with greater cytoskeletal tension and contractility. These findings highlight the importance of energy dissipation mechanisms in regulating fibroblast function on substrates mimicking the fibrotic milieu. Our results demonstrate that tuning the ECM viscoelasticity is a useful strategy to regulate cell behaviors in tissue engineered scaffolds, and develop better disease modeling for regenerative medicine.

Biomaterials can influence the coordinated efforts required to achieve tissue rehabilitation. Sponge-like silk fibroin scaffolds that include bioactive molecules have been shown to influence tissue repair. However, the mechanisms by which scaffold formulations elicit desired in vivo responses is unclear. Here, acellular silk scaffolds consisting of type I collagen, heparin, and/or vascular endothelial growth factor (VEGF) were used to investigate material fabrication and composition parameters that drive scaffold degradation, cell infiltration, and adipose tissue deposition in vivo. In subcutaneous implants, scaffold degradation was assessed, and results show that the percentage of cells infiltrating the scaffold increased when scaffold formulations contained bioactive molecules. To gain further insight, calculated in vitro enzymatic degradation rates increased with higher enzyme concentrations and theoretical cleavage sites. However, the addition of type I collagen and heparin to the scaffold at relevant concentrations did not change degradation rates, compared to silk alone. These in vitro results are contrary to observations in vivo, where bioactive molecules influence local protein deposition, immune cell infiltration rates, and vascularization. Thus, quantitative in vitro and in vivo evaluations aid in determining the mechanisms by which biomaterials influence tissue repair and support intentional biomaterial design for clinical applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/493207v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@7b403forg.highwire.dtl.DTLVardef@1b6ed79org.highwire.dtl.DTLVardef@a0b439org.highwire.dtl.DTLVardef@9843f3_HPS_FORMAT_FIGEXP M_FIG C_FIG This work examines the role of scaffold fabrication and bioactive molecule inclusion on the enzymatic degradation of silk fibroin-based lyophilized sponges. Specifically, the roles of collagen I, heparin, and vascular endothelial growth factor are analyzed to determine the impact of formulation on rate of degradation. In addition, scaffolds are either pre-fabricated, where these bioactive molecules are included in the polymer solution prior to casting the scaffold or the bioactive molecules are introduced following scaffold formation through passive adsorption to the silk fibroin scaffold surface. Scaffolds are enzymatically degraded in vitro, and kinetic rate constants are calculated for the different formulations. In vivo, cellularity, adipose tissue accumulation, and scaffold area are assessed over time. Additionally, immunohistochemistry is used to visualize VEGF Receptor 2, CD 68, and -smooth muscle actin over time.

Soft tissues exhibit predominantly time-dependent mechanical behavior critical for their biological function in organs like the lungs and aorta, as they can deform and stretch at varying rates depending on their function. Collagen type I serves as the primary structural component in these tissues. The viscoelastic characteristics of such tissues, stemming from diverse energy dissipation mechanisms across various length scales, remains poorly characterized at the nanoscale. Prior experimental investigations have predominantly centered on analyzing tissue responses largely attributed to interactions between cells and fibers. Despite many studies on tissue viscoelasticity from scaffolds to single collagen fibrils, the time-dependent mechanics of collagen fibrils at the sub-fibrillar level remain poorly understood. This pioneering study employs atomic force microscopy (AFM) nano-rheometry and indentation testing to examine the viscoelastic characteristics of individual collagen type I fibrils at the ultrastructural level within distinct topographical zones, specifically focusing on gap and overlap regions. Our investigation has unveiled that collagen fibrils display a viscoelastic response that replicates the mechanical behavior of the tissue at the macroscale. Further, our findings suggest a distinct viscoelastic behavior between the gap and overlap regions, likely stemming from variances in molecular organization and cross-linking modalities within these specific sites. The results of our investigation provide unequivocal proof of the temporal dependence of mechanical properties and provides unique data to be compared to atomistic models, laying a foundation for refining the precision of macroscale models that strive to capture tissue viscoelasticity across varying length scales.

This paper considers the mechanical response of Bombyx mori silk cocoons to knife stabbing, a simple but controlled way of simulating predaceous penetration. Here, we stab test both entire cocoons (EC) and cocoon wall segments (CWS) statically and dynamically, and note that the process can be broken down in three stages. The first stage involves material deflection, the second is knife penetration, and the third is knife perforation. We find that ca. 95 % of the kinetic energy is lost during the penetration stage. There are noticeable differences in strain between the equatorial [Formula] and meridional [Formula] directions before and after the stabbing of EC specimens (p < 0.001). The apparent area of the cocoon is noted to be on average 7 % lower after stabbing than it is prior to being stabbed (p < 0.01). We find that while compression of the cocoon from stabbing results in equatorial expansion (with a Poissons ratio,{nu} = 0.25), in the meridional direction the cocoon contracts ({nu} = -0.05) thus showing auxetic behaviour. Force-deflection curves are different in CWS specimens as compared to EC specimens, and this is attributable to natural curvatures in CWS specimens remaining even after a being flattened for mounting and testing. Differences between EC and CWS specimens are also noticeable in the sizes of the stab footprints, with EC samples exhibiting 33 % smaller footprints than CWS samples (p < 0.001). We conclude that testing whole cocoon structures provides a more accurate understanding of their properties as compared to cut and flattened structures. This is because flattening cocoon wall specimens induces delamination and multiple failure zones, reducing the natural stab resistance of the material.

Soft hydrated materials, including biological tissues and hydrogels, exhibit complex time-dependent mechanical behaviors due to their poroelastic and viscoelastic properties. These properties often manifest on overlapping time scales, making it challenging to isolate the individual contributions of poroelasticity and viscoelasticity to the overall mechanical response. This study presents a novel semi-analytical model for characterizing these properties through sequential microscale load relaxation indentation testing. By extending existing theories, we developed a poroviscoelastic framework that enables the deconvolution of poroelastic and viscoelastic effects. Using this model to fit sequential microscale indentation data, we characterized porcine heart and liver tissues, as well as collagen and GelMA hydrogels, revealing distinct differences in their poroelastic and viscoelastic parameters. Our findings demonstrate that this approach not only provides rapid and detailed insights into the mechanical properties at the microscale but also offers significant advantages over traditional methods in terms of speed, computational efficiency, and practicality. This methodology has broad implications for advancing the understanding of tissue mechanics and the design of biomimetic materials for tissue engineering applications. Statement of SignificanceThis study introduces a novel approach to understanding the mechanical behavior of soft hydrated materials, like tissues and hydrogels. This study introduces a semi-analytical model to describe the time dependent behavior and a practical approach to distinguish between poroelasticity and viscoelasticity at the microscale. By providing this model along with a rapid and efficient characterization method, our approach enhances understanding of time-dependent mechanical behaviors critical for soft tissue mechanics and biomaterials design.

Cell fate in tissue engineering (TE) strategies is paramount to regenerate healthy, functional organs. The mechanical loads experienced by cells play an important role in cell fate. However, in TE scaffolds with a cell-laden hydrogel matrix, it is prohibitively complex to prescribe and measure this cellular micromechanical environment (CME). Accordingly, this study aimed to develop a finite element (FE) model of a TE scaffold unit cell that can be subsequently implemented to predict the CME and cell fates under prescribed loading. The compressible hyperelastic mechanics of a fibrin hydrogel were characterized by fitting unconfined compression and confined compression experimental data. This material model was implemented in a unit cell FE model of a TE scaffold. The FE mesh and boundary conditions were evaluated with respect to the mechanical response of a region of interest (ROI). A compressible second-order reduced polynomial hyperelastic model gave the best fit to the experimental data (C10 = 1.72×10-4, C20 = 3.83×10-4, D1 = 3.41, D2 = 8.06×10-2). A mesh with seed sizes of 40 μm and 60 μm in the ROI and non-ROI regions, respectively, yielded a converged model in 54 minutes. The in-plane boundary conditions demonstrated minimal influence on ROI mechanics for a 2-by-2 unit cell. However, the out-of-plane boundary conditions did exhibit an appreciable influence on ROI mechanics for a two bilayer unit cell. Overall, the developed unit cell model facilitates the modeling of the mechanical state of a cell-laden hydrogel within a TE scaffold under prescribed loading. This model will be utilized to characterize the CME in future studies, and 3D micromechanical criteria may be applied to predict cell fate in these scaffolds.View Full Text

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.

Fibrin is a fibrous protein network that entraps blood cells and platelets to form blood clots following vascular injury. As a biomaterial, fibrin acts a biochemical scaffold as well as a viscoelastic patch that resists mechanical insults. The biomechanics and biochemistry of fibrin have been well characterized independently, showing that fibrin is a hierarchical material with numerous binding partners. However, comparatively little is known about how fibrin biomechanics and biochemistry are coupled: how does fibrin deformation influence its biochemistry at the molecular level? In this study, we show how mechanically-induced molecular structural changes in fibrin affect fibrin biochemistry and fibrin-platelet interaction. We found that tensile deformation of fibrin lead to molecular structural transitions of -helices to {beta}-sheets, which reduced binding of tissue plasminogen activator (tPA), an enzyme that initiates fibrinolysis, at the network and single fiber level. Moreover, binding of tPA and Thioflavin T (ThT), a commonly used {beta}-sheet marker, was primarily mutually exclusive such that tPA bound to native (helical) fibrin whereas ThT bound to strained fibrin. Finally, we demonstrate that conformational changes in fibrin suppressed the biological activity of platelets on mechanically strained fibrin due to attenuated IIb{beta}3 integrin binding. Our work shows that mechanical strain regulates fibrin molecular structure and fibrin biological activity in an elegant mechano-chemical feedback loop, which likely influences fibrinolysis and wound healing kinetics.

Electrospun (ESP) scaffolds are a promising type of tissue engineering constructs for large defects with limited depth. To form new functional tissue, the scaffolds need to be infiltrated with cells, which will deposit extracellular matrix. However, due to dense fiber packing and small pores, cell and tissue infiltration of ESP scaffolds is limited. Here, we combine two established methods, increasing fiber diameter and co-spinning sacrificial fibers, to create a porous ESP scaffold that allows robust tissue infiltration. Full cell infiltration across 2 mm thick scaffolds is seen 3 weeks after subcutaneous implantation in rats. After 6 weeks, the ESP scaffolds are almost fully filled with de novo tissue. Cell infiltration and tissue formation in vivo in this thickness has not been previously achieved. In addition, we propose a novel method for in vitro cell seeding to improve cell infiltration and a model to study 3D migration through a fibrous mesh. This easy approach to facilitate cell infiltration further improves previous efforts and could greatly aid tissue engineering approaches utilizing ESP scaffolds. Statement of significanceElectrospinning creates highly porous scaffolds with nano- to micrometer sized fibers and are a promising candidate for a variety of tissue engineering applications. However, smaller fibers also create small pores which are difficult for cells to penetrate, restricting cells to the top layers of the scaffolds. Here, we have improved the cell infiltration by optimizing fiber diameter and by co-spinning a sacrificial polymer. We developed novel culture technique that can be used to improve cell seeding and to study cytokine driven 3D migration through fibrous meshes. After subcutaneous implantation, infiltration of tissue and cells was observed up to throughout up to 2 mm thick scaffolds. This depth of infiltration in vivo had not yet been reported for electrospun scaffolds. The scaffolds we present here can be used for in vitro studies of migration, and for tissue engineering in defects with a large surface area and limited depth.

This study aimed at developing a formulation to link microscopic structure and macroscopic mechanics of a fibrous scaffold filled with a hydrogel for use as a tissue-engineered patch for local epicardial support of the infarcted heart. Mori-Tanaka mean field homogenisation, closed-cell foam mechanics and finite element (FE) methods were used to represent the macroscopic elastic modulus of the filled fibrous scaffold. The homogenised constitutive description of the scaffold was implemented for an epicardial patch in a FE model of a human cardiac left ventricle (LV) to assess effects of patching on myocardial mechanics and ventricular function in presences of an infarct. The macroscopic elastic modulus of the scaffold was predicted to be 0.287 MPa with the FE method and 0.290 MPa with the closed-cell model for the realistic fibre structure of the scaffold, and 0.108 and 0.540 MPa with mean field homogenization for randomly oriented and completely aligned fibres. Epicardial patching was predicted to reduce maximum myocardial stress in the infarcted LV from 19 kPa (no patch) to 9.5 kPa (patch), and to increase the ventricular ejection fraction from 40% (no patch) to 43% (patch). The predictions of the macroscopic elastic modulus of the realistic scaffold with the FE and the closed-cell model agreed well, and were bound by the mean field homogenisation prediction for random and fully aligned fibre orientation of the scaffold. This study demonstrates the feasibility of homogenization techniques to represent complex multiscale structural features in an simplified but meaningful manner.

The Achilles tendon is the strongest tendon in the human body, but the basis of its high tensile strength has not been elucidated in detail. Here we have loaded healthy, human, Achilles tendons to failure in an anatomically authentic fashion while studying the local three-dimensional deformation and strains in real time, with very high precision, using digital image correlation (DIC). These studies identified a remarkable degree of anisotropic, medio-lateral auxetic behavior, with Poissons ratios not exceeding minus 1 in any part of the tendon at any time; under certain loads, discrete areas within the tendon had a Poissons ratio below minus 6. Early in the loading cycle, the proximal region of the tendon accumulated high lateral strains while longitudinal strains remained low. This behavior shielded the mid-substance of the tendon, its weakest part, from high longitudinal strains until immediately before rupture. These new insights are of great relevance to understanding the material basis of tendon injuries, designing improved prosthetic replacements, and developing regenerative strategies.

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.

PurposeFibrosis is the pathological remodeling of the extracellular matrix (ECM) that is largely orchestrated by activated fibroblasts. The mechanical properties of the ECM change drastically during fibrosis, and fibroblasts become increasingly activated by mechanical environments that mimic the properties of fibrotic tissues. While the effects of increased elastic modulus (stiffness) on fibroblast activation have been well-studied, the impact of changes in viscoelasticity are less clear. Here, we sought to determine how fibroblast activation is altered by changes in viscoelasticity in a three-dimensional, fibrillar microenvironment. MethodsWe employed 3D alginate collagen I hydrogels with independently tunable stiffness and stress relaxation rates. Dermal fibroblasts were encapsulated in hydrogels with four distinct mechanical profiles (soft: 3 kPa or stiff: 10 kPa, fast stress relaxing: {tau}1/2 {approx} 160 s or slow stress relaxing: {tau}1/2 {approx} 1600 s). We assessed fibroblast activation by changes in cell morphology, expression of key activation markers, and evidence of ECM remodeling. ResultsFibrillar alginate collagen networks enhanced fibroblast spreading, -smooth muscle actin stress fiber formation, and fibroblast activation protein- expression in matrices that were slow relaxing or stiff. The presence of the fibrillar network further enhanced fibroblast activation, independent of the changes driven by matrix viscoelasticity. ECM remodeling was also promoted by slow relaxing matrices, with increased fibronectin deposition and more remodeling of the local collagen fiber network. ConclusionsOur results demonstrate that fibroblast activation is highly responsive to matrix stress relaxation rate, and that models incorporating fibrillar, viscoelastic networks can provide new insights into the role of ECM mechanics driving fibroblast activation.

Extracellular matrix (ECM) proteins assemble to form a heterogeneous connective scaffold that supports cells. Physical interactions between cells and the matrix regulate cellular behaviors and influence subsequent tissue construction. However, there is a lack of fundamental understanding regarding the contributions of individual native ECM proteins to the matrix. This gap arises from the need for nanoscopic characterization, which operates on a much smaller length scale than typical assessments in cell and tissue cultures, as well as in tissue reconstruction and clinical implantation. This study aims to systematically investigate how individual ECM proteins affect lipid membranes structurally and mechanically, and how these influences regulate cell migration. Results from Langmuir isotherm analysis, X-ray reflectivity measurements, and cell scratch assays demonstrate that strong collagen adsorption on the membrane surface disrupts lipid packing. However, its rigid network provides a sturdy scaffold for cell adhesion, thereby enhancing cell attachment and promoting cell migration. In contrast, elastin has a minimal structural or mechanical impact on the membrane during both adsorption and compression, but it benefits cells by facilitating migration and reducing the risk of infection. Fibronectin, on the other hand, exhibits complex mechanical responses to compression, characterized by significant structural rearrangements that occur during adsorption. This strong interaction with the membrane can result in excessively high adhesion forces, ultimately limiting cell motility. These findings lay the foundation for the design of artificial scaffolds that can manipulate cellular responses, a critical step toward advancing regenerative medicine and tissue engineering. SignificanceFabricating extracellular matrix (ECM) scaffolds from cells offers advantages over traditional approaches, such as decellularized tissues, which face donor limitations, and artificial scaffolds, which may hinder cellular communication. However, the slow harvesting process of cell-derived ECM has limited its clinical applications. This research is part of a larger mission to engineer ECM prescaffolds on lipid carriers tailored to cell requirements, enhancing ECM production and regulating cell behavior. The first step involves systematically analyzing the structural and mechanical effects of ECM on lipid membranes and how these effects regulate cellular behavior. This work confirms distinct characteristics of ECM proteins, advancing fundamental understanding of cell-matrix interactions and paving the way for scaffold engineering.