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Collagen is a major structural protein in the human body. It not only provides connective tissues such as ligaments and tendons with toughness and strength but it also constitutes the biomechanical scaffold for cell attachment in the extracellular matrix. Collagen molecules aggregate to form collagen fibrils, which are fibers with a diameter of 10 nm to 500 nm and a length of up to a centimeter. Tensile tests on individual collagen fibrils reveal a strongly non-linear stress-response to deformation with the tensile modulus reaching the gigapascal range. But not only that, collagen fibrils have been found to be viscoelastic which means collagen fibrils have both elastic and viscous characteristics. However, direct measurement of viscoelastic material properties in tension is only possible with force-controlled tensile tests, which can not be conducted with state-of-the-art methods. In this paper, we report the first force-controlled tensile tests of individual collagen fibrils. To account for the non-linear material characteristics, high responsiveness in the force control and robustness about a property change of the collagen fibril are needed. Therefore, a two-degrees-of-freedom (2-DOF) controller is applied for force control with high responsiveness. The 2-DOF controller is composed of feedforward (FF) and feedback (FB) controllers. In addition, a modeling error compensation is implemented for robustness. The modeling error is calculated from the difference between the actual force response measured by the sensor and the ideal force response calculated from the plant model. The validity of the proposed control method is confirmed from simulation and experimental results.

Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates (“2D”) or embedded in fibrin hydrogels (“3D”) with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels’ fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7–30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.

Achieving strong adhesion in wet environments remains a technological challenge in biomedical applications demanding biocompatibility. Attention for adhesive motifs meeting such demands has largely been focused on marine organisms. However, bioadhesion to inorganic surfaces is also present in the human body, in the hard tissues of teeth and bones, and is mediated through serines (S). The specific amino acid sequence DpSpSEEKC has been previously suggested to be responsible for the strong binding abilities of the protein statherin to hydroxyapatite, where pS denotes phosphorylated serine. Notably, similar sequences are present in the non-collagenous bone protein osteopontin (OPN) and the mussel foot protein 5 (Mefp5). OPN has previously been shown to promote fracture toughness and physiological damage formation. Here, we investigated the adhesion strength of the motif D(pS)(pS)EEKC on substrates of hydroxyapatite, TiO2, and mica using atomic force microscopy (AFM) single-molecule force spectroscopy (SMFS). Specifically, we investigated the dependence of adhesion force on phosphorylation of serines by comparing findings with the unphosphorylated variant DSSEEKC. Our results show that high adhesion forces of over 1 nN on hydroxyapatite and on TiO2 are only present for the phosphorylated variant D(pS)(pS)EEKC. This warrants further exploitation of this motif or similar residues in technological applications. Further, the dependence of adhesion force on phosphorylation suggests that biological systems potentially employ an adhesion-by-demand mechanism via expression of enzymes that up- or down-regulate phosphorylation, to increase or decrease adhesion forces, respectively.

Collagen fibrils are the fundamental structural elements in vertebrate animals and compose a structural framework that provides mechanical support to load-bearing tissues. Understanding how these fibrils initially form and mechanically function has been the focus of a myriad of detailed investigations over the last few decades. From these studies a great amount of knowledge has been acquired as well as a number of new questions to consider. In this review, we examine the current state of our knowledge of the mechanical properties of extant fibrils. We emphasize on the mechanical response and related deformation of collagen fibrils upon tension, which is the predominant load imposed in most collagen-rich tissues. We also illuminate the gaps in knowledge originating from the intriguing results that the field is still trying to interpret. STATEMENT OF SIGNIFICANCE: Collagen is the result of millions of years of biological evolution and is a unique family of proteins, the majority of which provide mechanical support to biological tissues. Cells produce collagen molecules that self-assemble into larger structures, known as collagen fibrils. As simple as they appear under an optical microscope, collagen fibrils display a complex ultrastructural architecture tuned to the external forces that are imposed upon them. Even more complex is the way collagen fibrils deform under loading, and the nature of the mechanisms that drive their formation in the first place. Here, we present a cogent synthesis of the state-of-knowledge of collagen fibril mechanics. We focus on the information we have from in vitro experiments on individual, isolated from tissues, collagen fibrils and the knowledge available from in silico tests.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFβ increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Description, The architecture of the tendon-to-bone attachment is designed for toughness., Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and previously unexplored imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we show that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of oxygen status (pseudohypoxia). Whilst TGFβ increased rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting ‘bone-type’ cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knock down of Factor Inhibiting HIF (FIH) or oxidative stress caused pseudohypoxic HIF activation in normal fibroblasts. In contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of IPF lung mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Collagen is the major structural protein in human bodies constituting about 30% of the entire protein mass. Through a self-assembly process, triple helical collagen molecules assemble into high aspect-ratio fibers of tens to hundreds of nanometer diameter, known as collagen fibrils (CFs). In the last decade, several methods for tensile testing these CFs emerged. However, these methods are either overly time-consuming or offer low data acquisition bandwidth, rendering dynamic investigation of tensile properties impossible. Here, we describe a novel instrument for tensile testing of individual CFs. CFs are furnished with magnetic beads using a custom magnetic tweezer. Subsequently, CFs are lifted by magnetic force, allowing them to be picked-up by a microgripper structure, which is mounted on a cantilever-based interferometric force probe. A piezo-lever actuator is used to apply tensile displacements and to perform tensile tests of tethered CFs, after alignment. Once the mechanical tests are finished, CFs are removed from the microgripper by application of a magnetic field. Our novel instrument enables tensile tests with at least 25-fold increased throughput compared to tensile testing with an atomic force microscope while achieving force resolution (p–p) of 10 nN at a strain resolution better than 0.1%.

Osteoporosis is the most common bone disease and is conventionally classified as a decrease of total bone mass. Current diagnosis of osteoporosis is based on clinical risk factors and dual energy X‐ray absorptiometry (DEXA) scans, but changes in bone quantity (bone mass) and quality (trabecular structure, material properties, and tissue composition) are not distinguished. Yet, osteoporosis is known to cause a deterioration of the trabecular network, which might be related to changes at the tissue scale—the material properties. The goal of the current study was to use a previously established test method to perform a thorough characterization of the material properties of individual human trabeculae from femoral heads in cyclic tensile tests in a close to physiologic, wet environment. A previously developed rheological model was used to extract elastic, viscous, and plastic aspects of material behavior. Bone morphometry and tissue mineralization were determined with a density calibrated micro‐computed tomography (μCT) set‐up. Osteoporotic trabeculae neither showed a significantly changed material or mechanical behavior nor changes in tissue mineralization, compared with age‐matched healthy controls. However, donors with osteopenia indicated significantly reduced apparent yield strain and elastic work with respect to osteoporosis, suggesting possible initial differences at disease onset. Bone morphometry indicated a lower bone volume to total volume for osteoporotic donors, caused by a smaller trabecular number and a larger trabecular separation. A correlation of age with tissue properties and bone morphometry revealed a similar behavior as in osteoporotic bone. In the range studied, age does affect morphometry but not material properties, except for moderately increased tissue strength in healthy donors and moderately increased hardening exponent in osteoporotic donors. Taken together, the distinct changes of trabecular bone quality in the femoral head caused by osteoporosis and aging could not be linked to suspected relevant changes in material properties or tissue mineralization. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., Individual trabeculae of human femoral heads were tested cyclically in tensile mode. Tensile modulus was determined in each loading and unloading cycle and significantly larger in the unloading phase. Determination of material properties with a rheological model revealed no significant difference between control and osteoporotic trabeculae. In contrast, bone morphometry was significantly deteriorated in osteoporosis and with increased ageing.

Background In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. Methods Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. Findings Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. Interpretation Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. Funding Austrian Research Promotion Agency FFG (“CartiScaff” #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08), Graphical abstract Image, graphical abstract

Ageing, disease and osteoporosis treatment have been linked to accumulation of microdamage, which is caused by repetitive loading and may eventually causes fatigue failure of bones. Post-hoc investigations for in vivo loading and in vitro experiments have been developed to better understand microdamage formation. In this context, previous studies were not able to discriminate the effects caused by structural changes of the trabecular network from differences of tissue/material properties on microdamage formation. In the present study a fatigue test protocol was established to induce microdamage at a defined tensile stress state of individual trabeculae. Further, a thorough analysis of microdamage analysis was presented for 2D and 3D confocal images, enabling a comparison between the tissue and the meso-scale. Eight individual trabeculae were tested for 1500 cycles, six for 2100 cycles and seven for 3000 cycles (close to failure). Microdamage increased slowly from 1500 to 2100 cycles and showed a rapid increase at 3000 cycles. Diffuse damage was mainly present, although also linear microcracks were visible at 2100 and 3000 cycles. Average microcrack length was 93 µm and diffuse damage density was 4.4% for samples tested for 3000 cycles, comparable to previous studies on trabecular bone cores. Only one to three large microdamage sites were observed in the central region, connected to the trabecular surface with small straight cracks. The presented procedure is a first step to better understand how microdamage formation is influenced by material properties in aged and diseased bone, independently of deteriorated trabecular microarchitecture.

Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by testing agarose samples with indentation moduli ranging over three orders of magnitude via AFM and compared these results with bulk compression tests. Our results show that quantitative measurements of indentation modulus is achieved over three orders of magnitude ranging from 1 kPa to 1000 kPa via AFM cantilever-based microindentation experiments. Therefore, our approach could be used for quantitative micromechanical measurements without the need to perform further validation via bulk compression experiments.

Cortical bone is permeated by a system of pores, occupied by the blood supply and osteocytes. With ageing, bone mass reduction and disruption of the microstructure are associated with reduced vascular supply. Insight into the regulation of the blood supply to the bone could enhance the understanding of bone strength determinants and fracture healing. Using synchrotron radiation-based computed tomography, the distribution of vascular canals and osteocyte lacunae was assessed in murine cortical bone and the influence of age on these parameters was investigated. The tibiofibular junction from 15-week- and 10-month-old female C57BL/6J mice were imaged post-mortem. Vascular canals and three-dimensional spatial relationships between osteocyte lacunae and bone surfaces were computed for both age groups. At 15 weeks, the posterior region of the tibiofibular junction had a higher vascular canal volume density than the anterior, lateral and medial regions. Intracortical vascular networks in anterior and posterior regions were also different, with connectedness in the posterior higher than the anterior at 15 weeks. By 10 months, cortices were thinner, with cortical area fraction and vascular density reduced, but only in the posterior cortex. This provided the first evidence of age-related effects on murine bone porosity due to the location of the intracortical vasculature. Targeting the vasculature to modulate bone porosity could provide an effective way to treat degenerative bone diseases, such as osteoporosis.

Tissue hydration is well known to influence tissue mechanics and can be tuned via osmotic pressure. Collagen fibrils are nature's nanoscale building blocks to achieve biomechanical function in a broad range of biological tissues and across many species. Intrafibrillar covalent cross-links have long been thought to play a pivotal role in collagen fibril elasticity, but predominantly at large, far from physiological, strains. Performing nanotensile experiments of collagen fibrils at varying hydration levels by adjusting osmotic pressure in situ during atomic force microscopy experiments, we show the power the intrafibrillar noncovalent interactions have for defining collagen fibril tensile elasticity at low fibril strains. Nanomechanical tensile tests reveal that osmotic pressure increases collagen fibril stiffness up to 24-fold in transverse (nanoindentation) and up to 6-fold in the longitudinal direction (tension), compared to physiological saline in a reversible fashion. We attribute the stiffening to the density and strength of weak intermolecular forces tuned by hydration and hence collagen packing density. This reversible mechanism may be employed by cells to alter their mechanical microenvironment in a reversible manner. The mechanism could also be translated to tissue engineering approaches for customizing scaffold mechanics in spatially resolved fashion, and it may help explain local mechanical changes during development of diseases and inflammation.

Bone is a biological tissue with unique mechanical properties, owing to a complex hierarchical structure ranging from the nanoscale up to the macroscale. To better understand bone mechanics, investigation of mechanical properties of all structural elements at every hierarchical level and how they interact is necessary. Testing of bone structures at the lower microscale, e.g. bone lamellae, has been least performed and remains a challenge. Focused ion beam (FIB) milling is an attractive technique for machining microscopic samples from bone material and performing mechanical testing at the microscale using atomic force microscopy (AFM) and nanoindentation setups. So far, reported studies at this length scale have been performed on bone samples of animal origin, mostly in a dehydrated state, except for one study. Here we present an AFM-based microbeam bending method for performing bending measurements in both dehydrated and rehydrated conditions at the microscale. Single lamella bone microbeams of four human donors, aged 65–94 yrs, were machined via FIB and tested both in air and fully submerged in Hank's Balanced Salt Solution (HBSS) to investigate the effect of (de)hydration and to a certain extent, of age, on bone mechanics. Bending moduli were found to reduce up to 5 times after 2 h of rehydration and no trend of change in bending moduli with respect to age could be observed. Mechanical behavior changed from almost purely elastic to viscoelastic upon rehydration and a trend of lower dissipated energy in samples from older donors could be observed in the rehydrated state. These results confirm directly the importance of water for the mechanical properties of bone tissue at the microscale. Moreover, the trend of lowered capability of energy dissipation in older donors may contribute to a decrease of fracture toughness and thus an increase in bone fragility with age.

Osteoporosis is defined as a decrease of bone mass and strength, as well as an increase in fracture risk. It is conventionally treated with antiresorptive drugs, such as bisphosphonates (BPs) and selective estrogen receptor modulators (SERMs). Although both drug types successfully decrease the risk of bone fractures, their effect on bone mass and strength is different. For instance, BP treatment causes an increase of bone mass, stiffness and strength of whole bones, whereas SERM treatment causes only small (4%) increases of bone mass, but increased bone toughness. Such improved mechanical behavior of whole bones can be potentially related to the bone mass, bone structure or material changes. While bone mass and architecture have already been investigated previously, little is known about the mechanical behavior at the tissue/material level, especially of trabecular bone. As such, the goal of the work presented here was to fill this gap by performing cyclic tensile tests in a wet, close to physiologic environment of individual trabeculae retrieved from the vertebrae of beagle dogs treated with alendronate (a BP), raloxifene (a SERM) or without treatments. Identification of material properties was performed with a previously developed rheological model and of mechanical properties via fitting of envelope curves. Additionally, tissue mineral density (TMD) and microdamage formation were analyzed. Alendronate treatment resulted in a higher trabecular tissue stiffness and strength, associated with higher levels of TMD. In contrast, raloxifene treatment caused a higher trabecular toughness, pre-dominantly in the post-yield region. Microdamage formation during testing was not affected by either anti-resorptive treatment regimens. These findings highlight that the improved mechanical behavior of whole bones after anti-resorptive treatment is at least partly caused by improved material properties, with different mechanisms for alendronate and raloxifene. This study further shows the power of performing a mechanical characterization of trabecular bone at the level of individual trabeculae for better understanding of clinically relevant mechanical behavior of bone.

3D imaging of the bone vasculature is of key importance in the understanding of skeletal disease. As blood vessels in bone are deeply encased in the calcified matrix, imaging techniques that are applicable to soft tissues are generally difficult or impossible to apply to the skeleton. While canals in cortical bone can readily be identified and characterised in X-ray computed tomographic data in 3D, the soft tissue comprising blood vessels that are putatively contained within the canal structures does not provide sufficient image contrast necessary for image segmentation. Here, we report an approach that allows for rapid, simultaneous visualisation of calcified bone tissue and the vasculature within the calcified bone matrix. Using synchrotron X-ray phase contrast-enhanced tomography we show exemplar data with intracortical capillaries uncovered at sub-micrometre level without the need for any staining or contrast agent. Using the tibiofibular junction of 15 week-old C57BL/6 mice post mortem, we show the bone cortical porosity simultaneously along with the soft tissue comprising the vasculature. Validation with histology confirms that we can resolve individual capillaries. This imaging approach could be easily applied to other skeletal sites and transgenic models, and could improve our understanding of the role the vasculature plays in bone disease.

The ability to measure bone tissue material properties plays a major role in diagnosis of diseases and material modeling. Bone’s response to loading is complex and shows a viscous contribution to stiffness, yield and failure. It is also ductile and damaging and exhibits plastic hardening until failure. When performing mechanical tests on bone tissue, these constitutive effects are difficult to quantify, as only their combination is visible in resulting stress–strain data. In this study, a methodology for the identification of stiffness, damping, yield stress and hardening coefficients of bone from a single cyclic tensile test is proposed. The method is based on a two-layer elasto-visco-plastic rheological model that is capable of reproducing the specimens’ pre- and postyield response. The model’s structure enables for capturing the viscously induced increase in stiffness, yield, and ultimate stress and for a direct computation of the loss tangent. Material parameters are obtained in an inverse approach by optimizing the model response to fit the experimental data. The proposed approach is demonstrated by identifying material properties of individual bone trabeculae that were tested under wet conditions. The mechanical tests were conducted according to an already published methodology for tensile experiments on single trabeculae. As a result, long-term and instantaneous Young’s moduli were obtained, which were on average 3.64 GPa and 5.61 GPa, respectively. The found yield stress of 16.89 MPa was lower than previous studies suggest, while the loss tangent of 0.04 is in good agreement. In general, the two-layer model was able to reproduce the cyclic mechanical test data of single trabeculae with an root-mean-square error of 2.91 ± 1.77 MPa. The results show that inverse rheological modeling can be of great advantage when multiple constitutive contributions shall be quantified based on a single mechanical measurement.

Two-photon polymerization (2PP) is a lithography-based 3D printing method allowing the fabrication of 3D structures with sub-micrometer resolution. This work focuses on the characterization of gelatin-norbornene (Gel-NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol-ene system allows 2PP processing of cell-containing materials at remarkably high scanning speeds (1000 mm s

Bacterial infections of central venous catheters (CVCs) cause much morbidity and mortality, and are usually diagnosed by concordant culture of blood and catheter tip. However, studies suggest that culture often fails to detect biofilm bacteria. This study optimises X-ray micro computed tomography (X-ray µCT) for the quantification and determination of distribution and heterogeneity of biofilms in in vitro central venous catheter (CVC) model systems.Bacterial culture and scanning electron microscopy (SEM) were used to detect Staphylococcus epidermidis ATCC 35984 biofilms grown on catheters in vitro in both flow and static biofilm models. Alongside this, X-ray µCT techniques were developed in order to detect biofilms inside CVCs. Various contrast agent stains were evaluated using energy dispersive X-ray spectroscopy (EDS) to further optimise these methods. Catheter material and biofilm were segmented using a semi-automated MATLAB script and quantified using the Avizo Fire software package.X-ray µCT was capable of distinguishing between the degree of biofilm formation across different segments of a CVC flow model. EDS screening of single and dual compound contrast stains identified 10 nm gold and silver nitrate as the optimum contrast agent for X-ray µCT. This optimised method was then demonstrated to be capable of quantifying biofilms in an in vitro static biofilm formation model, with a strong correlation between biofilm detection via SEM and culture.X-ray µCT has good potential as a direct, non-invasive, non-destructive technology to image biofilms in CVCs, as well as other in vivo medical components in which biofilms accumulate in concealed areas.

Adhesion processes play a decisive role in the animal and human body and have been studied in great detail. Phosphorylation of serine as adhesion strategy is found in different species and serves different purposes, e.g. under water surface adhesion and protection strategies. Based on these biological adhesion applications, we present a biomimetic phosphonate-containing block copolymer approach to study surface adhesion. We synthesized two block copolymers (28 kDa and 39 kDa), which differed in their phosphonate-containing block dimethyl(2-methacryloyloxyethyl phosphonate) (DMMEP), using reversible addition fragmentation-chain transfer (RAFT) polymerization and tethered these polymers onto atomic force microscopy (AFM) probes. After performing AFM in single molecule force spectroscopy (SMFS) mode under physiological-like conditions (phosphate buffered saline - PBS, pH 7.2) on different substrates (mica, calcium deficient hydroxyapatite, TiO2 coated Si-wafer) we determined adhesion forces of 1610 ± 76 pN and 2257 ± 48 pN for the 28 kDa and the 39 kDa block copolymer, respectively. Our results show higher adhesion on hydroxyapatite, TiO2 and mica using polymers with a longer phosphonate block. This phosphonate containing block copolymer could serve as adhesion motif in several applications, and is very promising in the biomedical field, especially for tissue engineering applications due to its excellent adhesion on hydroxyapatite and titanium under physiological-like conditions.

The ability to measure bone tissue material properties plays a major role in diagnosis of diseases and material modeling. Bone’s response to loading is complex and shows a viscous contribution to stiffness, yield and failure. It is also ductile and damaging and exhibits plastic hardening until failure. When performing mechanical tests on bone tissue, these constitutive effects are difficult to quantify, as only their combination is visible in resulting stress–strain data. In this study, a methodology for the identification of stiffness, damping, yield stress and hardening coefficients of bone from a single cyclic tensile test is proposed. The method is based on a two-layer elasto-visco-plastic rheological model that is capable of reproducing the specimens’ pre- and postyield response. The model’s structure enables for capturing the viscously induced increase in stiffness, yield, and ultimate stress and for a direct computation of the loss tangent. Material parameters are obtained in an inverse approach by optimizing the model response to fit the experimental data. The proposed approach is demonstrated by identifying material properties of individual bone trabeculae that were tested under wet conditions. The mechanical tests were conducted according to an already published methodology for tensile experiments on single trabeculae. As a result, long-term and instantaneous Young’s moduli were obtained, which were on average 3.64 GPa and 5.61 GPa, respectively. The found yield stress of 16.89 MPa was lower than previous studies suggest, while the loss tangent of 0.04 is in good agreement. In general, the two-layer model was able to reproduce the cyclic mechanical test data of single trabeculae with an root-mean-square error of 2.91 ± 1.77 MPa. The results show that inverse rheological modeling can be of great advantage when multiple constitutive contributions shall be quantified based on a single mechanical measurement.

Continuing technological advancement of mechanical characterization at the microscale has enabled the isolation of micron-sized specimens and their direct mechanical characterization. Such techniques, initially developed for engineering materials and MEMS, can also be applied on hard biological materials. Bone is a material with a complex hierarchical structure ranging from the macro- all the way down to the nanoscale. To fully understand bone tissue mechanics, knowledge of the mechanics of all structural elements i.e. at every length scale is necessary. Particularly, the mechanical properties of microstructural elements, such as bone lamellae are still largely unknown. In the last decade, testing protocols have been devised to close this gap including bending and compression of micrometer-sized bone specimens. However, the precision and accuracy of results obtained have not been discussed. In this study, we aim to do exactly this: we validate microbeam bending by testing silicon microbeams with known mechanical constants, and evaluate the precision and sources of errors in both microbeam bending and micropillar compression by means of finite element (FE) modeling. Bending of Si-microbeams reproduced the expected value for the bending modulus within 17% accuracy, although the effect of geometrical uncertainties was estimated to result in relative errors of up to 50%. The deformation of constraining bulk material had a smaller influence, with relative errors of 11%, for microbeam bending and 25% for micropillar compression. For the latter this error could be sufficiently eliminated by the Sneddon correction. The tapering of micropillars had a negligible effect on overall apparent stiffness, but induced inhomogeneous stress state within micropillars may lead to superposed structural deformation mechanisms and be responsible for failure patterns observed in past studies.

Interest in bone as a functional material, for tools, weapons, etc., dates over 1.5 million years. Much more recently, researchers have sought to determine how the combination of collagen, calcium phosphate, and other organic compounds results in a material with considerable stiffness, strength, and toughness. The major challenge is that bone is a hierarchical material such that changes in the properties at any hierarchical level, including the nano- and microscale, impact macroscale bone mechanics. With improved imaging modalities, sample preparation, and mechanical characterization techniques, smaller and smaller length scales have become accessible. This article summarizes techniques used to assess bone micro- and nanomechanics in the context of bone hierarchy to elucidate mechanisms and structure–function relationships.

Osteons are the end product of cortical bone remodeling. Changes in their mechanical properties may be an indicator of overall bone health. Aszenci and Bonucci first evaluated the compressive properties of individual osteons in 1968. However, these results have never been independently validated and technological advances allow for further investigation of osteon mechanics. In the present study, an experimental protocol was established such that osteons ~250 µm in diameter were successfully extracted from human cortical bone (males, ages 93 and 64) and loaded in compression. Both micro-computed tomography and second harmonic generation (SHG) microscopy imaging methods were incorporated into the protocol to improve the structural and compositional assessment of each osteon. Further, a high-resolution videography system was used to monitor the osteons during mechanical testing, permitting insight into deformation mechanisms in situ. The results show that the older donor osteons (male, age 93 (n = 13)) were stiffer (p = 0.04) and more highly mineralized (p = 0.03), while the younger donor osteons (male, 64 (n = 14)) exhibited more heterogeneity in the measured mechanical properties with the presence of both stiff and more compliant osteons. SHG intensity indicated predominantly longitudinal fiber alignment for all osteons while the videography data revealed three distinct failure modes and confirmed that whitening observed during yielding of macroscale bone specimens represents failure at the local level. The stiffness and strength of the younger donor osteons were heavily dependent on the gross structure of the osteon, while these properties were dominated by collagen orientation in the older donor osteons.

Accumulation of advanced glycation end-products (AGEs) in biological tissues occurs as a consequence of normal ageing and pathology. Most biological tissues are composed of considerable amounts of collagen, with collagen fibrils being the most abundant form. Collagen fibrils are the smallest discernible structural elements of load-bearing tissues and as such, they are of high biomechanical importance. The low turnover of collagen cause AGEs to accumulate within the collagen fibrils with normal ageing as well as in pathologies. We hypothesized that collagen fibrils bearing AGEs have altered hydration and mechanical properties. To this end, we employed atomic force and Brillouin light scattering microscopy to measure the extent of hydration as well as the transverse elastic properties of collagen fibrils treated with ribose. We find that hydration is different in collagen fibrils bearing AGEs and this is directly related to their mechanical properties. Collagen fibrils treated with ribose showed increased hydration levels and decreased transverse stiffness compared to controlled samples. Our results show that BLS and AFM yield complementary evidence on the effect of hydration on the nanomechanical properties of collagen fibrils.

Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo‐induced degradation of such hydrogel systems is commonly based on the integration of photolabile o‐nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one‐ and two‐photon absorption. Herein we describe a cytocompatible click‐based hydrogel containing o‐nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone‐based small molecule as photosensitizer the efficiency of the two‐photon degradation process can be improved significantly. Biocompatibility of both the improved two‐photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (

Trabecular bone is located inside flat bones as well as in the epi- and metaphysis of long bones and plays a key role with respect to load transfer. Disorders, such as osteoporosis, weaken the structural integrity and may also cause changes in the mechanical properties of individual trabeculae, such as Young's modulus. Knowledge of mechanical tissue properties are necessary to assess risk of bone fracture with finite element analysis (FEA). However, such parameters are most often obtained from experiments on air-dried specimens which do not reflect the physiological conditions. In this study, micro-tensile tests of individual bovine trabeculae were performed until fracture to evaluate the influence of hydration state on the elastic and post-yield behavior. Dehydration resulted in significantly (p 0.001) lower post yield work and ultimate strain, whereas stiffness, yield stress and ultimate stress were significantly (p 0.001) larger. Further, inelastic strain of dehydrated samples was confined to a small region, whereas it was distributed over a larger area in wet samples. Similarly, microdamage accumulation was confined to a significantly smaller region (p 0.05) in dry samples, compared to wet ones. Thus, damage localization resulted in a quasi-brittle failure in dry samples. In contrast, hydrated samples showed a much larger area of microdamage accumulation, resulting in a ductile failure. These results emphasize the need to keep bone samples hydrated during mechanical testing. Sequentially, the findings may help to improve clinical applications like FEA-based bone strength predictions.

Extracellular matrix stiffness has a profound effect on the behaviour of many cell types. Adherent cells apply contractile forces to the material on which they adhere, and sense the resistance of the material to deformation – its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (

Graphical abstract, Structure-function relationships in tendons are directly influenced by the arrangement of collagen fibres. However, the details of such arrangements in functionally distinct tendons remain obscure. This study demonstrates the use of quantitative polarised light microscopy (qPLM) to identify structural differences in two major tendon compartments at the mesoscale: fascicles and interfascicular matrix (IFM). It contrasts functionally distinct positional and energy storing tendons, and considers changes with age. Of particular note, the technique facilitates the analysis of crimp parameters, in which cutting direction artefact can be accounted for and eliminated, enabling the first detailed analysis of crimp parameters across functionally distinct tendons. IFM shows lower birefringence (0.0013 ± 0.0001 [−]), as compared to fascicles (0.0044 ± 0.0005 [−]), indicating that the volume fraction of fibres must be substantially lower in the IFM. Interestingly, no evidence of distinct fibre directional dispersions between equine energy storing superficial digital flexor tendons (SDFTs) and positional common digital extensor tendons (CDETs) were noted, suggesting either more subtle structural differences between tendon types or changes focused in the non-collagenous components. By contrast, collagen crimp characteristics are strongly tendon type specific, indicating crimp specialisation is crucial in the respective mechanical function. SDFTs showed much finer crimp (21.1 ± 5.5 µm) than positional CDETs (135.4 ± 20.1 µm). Further, tendon crimp was finer in injured tendon, as compared to its healthy equivalents. Crimp angle differed strongly between tendon types as well, with average of 6.5 ± 1.4° in SDFTs and 13.1 ± 2.0° in CDETs, highlighting a substantially tighter crimp in the SDFT, likely contributing to its effective recoil capacity. Statement of Significance This is the first study to quantify birefringence in fascicles and interfascicular matrix of functionally distinct energy storing and positional tendons. It adopts a novel method – quantitative polarised light microscopy (qPLM) to measure collagen crimp angle, avoiding artefacts related to the direction of histological sectioning, and provides the first direct comparison of crimp characteristics of functionally distinct tendons of various ages. A comparison of matched picrosirius red stained and unstained tendons sections identified non-homogenous staining effects, and leads us to recommend that only unstained sections are analysed in the quantitative manner. qPLM is successfully used to assess birefringence in soft tissue sections, offering a promising tool for investigating the structural arrangements of fibres in (soft) tissues and other composite materials.

Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular d -Ala- d -Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

Strength is the most widely reported parameter with regards to bone failure. However, bone contains pre-existing damage and stress concentration sites, perhaps making measures of fracture toughness more indicative of the resistance of the tissue to withstand fracture. Several toughening mechanisms have been identified in bone, prominently, at the microscale. More recently, nanoscale toughness mechanisms, such as sacrificial-bonds and hidden-length or dilatational band formation, mediated by noncollagenous proteins, have been reported. Absence of specific noncollagenous proteins results in lowered fracture toughness in animal models. Further, roles of several other, putative influencing, factors such as closely bound water, collagen cross-linking and citrate bonds in bone mineral have also been proposed. Yet, it is still not clear if and which mechanisms are hallmarks of osteoporosis disease and how they influence fracture risk. Further insights on the workings of such influencing factors are of high importance for developing complementary diagnostics and therapeutics strategies.

Skin wound healing is a vital process that is important for re-establishing the epithelial barrier following disease or injury. Aberrant or delayed skin wound healing increases the risk of infection, causes patient morbidity, and may lead to the formation of scar tissue. One of the most important events in wound healing is coverage of the wound with a new epithelial layer. This occurs when keratinocytes at the wound periphery divide and migrate to re-populate the wound bed. Many approaches are under investigation to promote and expedite this process, including the topical application of growth factors and the addition of autologous and allogeneic tissue or cell grafts. The mechanical environment of the wound site is also of fundamental importance for the rate and quality of wound healing. It is known that mechanical stress can influence wound healing by affecting the behaviour of cells within the dermis, but it remains unclear how mechanical forces affect the healing epidermis. Tensile forces are known to affect the behaviour of cells within epithelia, however, and the material properties of extracellular matrices, such as substrate stiffness, have been shown to affect the morphology, proliferation, differentiation and migration of many different cell types. In this review we will introduce the structure of the skin and the process of wound healing. We will then discuss the evidence for the effect of tissue mechanics in re-epithelialisation and, in particular, on stem cell behaviour in the wound microenvironment and in intact skin. We will discuss how the elasticity, mechanical heterogeneity and topography of the wound extracellular matrix impact the rate and quality of wound healing, and how we may exploit this knowledge to expedite wound healing and mitigate scarring.