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The uterus is central to the establishment, maintenance, and delivery of a healthy pregnancy. Biomechanics is an important contributor to pregnancy success, and alterations to normal uterine biomechanical functions can contribute to an array of obstetric pathologies. Few studies have characterized the passive mechanical properties of the gravid human uterus, and ethical limitations have largely prevented the investigation of mid-gestation periods. To address this key knowledge gap, this study seeks to characterize the structural, compositional, and time-dependent micro-mechanical properties of the nonhuman primate (NHP) uterine layers in nonpregnancy and at three time-points in pregnancy: early 2 nd , early 3 rd , and late 3 rd trimesters. Distinct material and compositional properties were noted across the different tissue layers, with the endometrium-decidua being the least stiff, most viscous, least diffusible, and most hydrated layer of the NHP uterus. Pregnancy induced notable compositional and structural changes to the endometrium-decidua and myometrium, but no micro-mechanical property changes. Further comparison to published human data revealed notable similarities across species, with minor differences noted for the perimetrium and nonpregnant endometrium. This work provides insights into the material properties of the NHP uterus and demonstrates the validity of NHPs as a model for studying certain aspects of human uterine biomechanics.

Rising maternal mortality rates in the U.S. are a significant public health issue that must be addressed; however, much of the basic science information required to target pregnancy-related pathologies have not yet been defined. Placental and blastocyst implantation research are challenging to perform in humans because of the early time frame of these processes in pregnancy and limited access to first trimester tissues. As a result, there is a critical need to develop model systems capable of studying these processes in increasing mechanistic detail. With the recent passing of the FDA Modernization Act 2.0 and advances in tissue engineering methods, three-dimensional microphysiological model systems offer an exciting opportunity to model early stages of placentation. Here, we detail the synthesis, characterization, and application of gelatin methacryloyl (GelMA) hydrogel platforms for studying trophoblast behavior in three-dimensional hydrogel systems. Photopolymerization strategies to fabricate GelMA hydrogels render the hydrogels homogeneous in terms of structure and stable under physiological temperatures, allowing for rigorous fabrication of reproducible hydrogel variants. Unlike other natural polymers that have minimal opportunity to tune their properties, GelMA hydrogel properties can be tuned across many axes of variation, including polymer degree of functionalization, gelatin bloom strength, light exposure time and intensity, polymer weight percent, photoinitiator concentration, and physical geometry. In this work, we aim to inspire and instruct the field to utilize GelMA biomaterial strategies for future placental research. With enhanced microphysiological models of pregnancy, we can now generate the basic science information required to address problems in pregnancy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Samantha Zambuto reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This review considers fully three-dimensional biomaterial environments of varying complexity as these pertain to research on the placenta. The developments in placental cell sources are first considered, along with the corresponding maternal cells with which the trophoblast interact. We consider biomaterial sources, including hybrid and composite biomaterials. Properties and characterization of biomaterials are discussed in the context of material design for specific placental applications. The development of increasingly complicated three-dimensional structures includes examples of advanced fabrication methods such as microfluidic device fabrication and 3D bioprinting, as utilized in a placenta context. The review finishes with a discussion of the potential for in vitro, three-dimensional placenta research to address health disparities and sexual dimorphism, especially in light of the exciting recent changes in the regulatory environment for in vitro devices., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Samantha Zambuto and Adrienne Scott reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The vagina is a fibromuscular tube-shaped organ spanning from the hymenal ring to the cervix that plays critical roles in menstruation, pregnancy, and female sexual health. Vaginal tissue constituents, including cells and extracellular matrix components, contribute to tissue structure, function, and prevention of injury. However, much microstructural function remains unknown, including how the fiber-cell and cell-cell interactions influence macromechanical properties. A deeper understanding of these interactions will provide critical information needed to reduce and prevent vaginal injuries. Our objectives for this work herein are to first engineer a suite of biomaterials for vaginal tissue engineering and second to characterize the performance of these biomaterials in the vaginal microenvironment. We successfully created fiber-reinforced hydrogels of gelatin-elastin electrospun fibers infiltrated with gelatin methacryloyl hydrogels. These composites recapitulate vaginal material properties, including stiffness, and are compatible with the vaginal microenvironment: biocompatible with primary vaginal epithelial cells and in acidic conditions. This work significantly advances progress in vaginal tissue engineering by developing novel materials and developing a state-of-the-art tissue engineered vagina.

The leading cause of perinatal mortality is fetal growth restriction (FGR), defined as in utero fetal growth below the 10th percentile. Insufficient exchange of oxygen and nutrients at the maternal-fetal interface is associated with FGR. This transport occurs through the vasculature of the placenta, particularly in the terminal villi, where the vascular membranes have a large surface area and are the thinnest. Altered structure of the placenta villi is thought to contribute to decreased oxygen exchange efficiency, however, understanding how the three-dimensional microstructure and properties decrease this efficiency remains a challenge. Here, a novel, multiscale workflow is presented to quantify patient-specific biophysical properties, 3D structural features, and blood flow of the villous tissue. Namely, nanoindentation, optical coherence tomography, and ultrasound imaging were employed to measure the time-dependent material properties of placenta tissue, the 3D structure of villous tissue, and blood flow through the villi to characterize the microvasculature of the placenta at increasing length scales. Quantifying the biophysical properties, the 3D architecture, and blood flow in the villous tissue can be used to infer changes in maternal-fetal oxygen transport at the villous membrane. Overall, this multiscale understanding will advance knowledge of how microvascular changes in the placenta ultimately lead to FGR, opening opportunities for diagnosis and intervention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: We declare no competing interests. This work was supported by Wellcome Leap as part of the In Utero Program and the US National Institutes of Health T32 Postdoctoral Training Grant in Regenerative Medicine (T32 EB028092).

Gelatin methacryloyl (GelMA) hydrogels are widely used for a variety of tissue engineering applications. The properties of gelatin can affect the mechanical properties of gelatin gels; however, the role of gelatin properties such as bloom strength on GelMA hydrogels has not yet been explored. Bloom strength is a food industry standard for describing the quality of gelatin, where higher bloom strength is associated with higher gelatin molecular weight. Here, we evaluate the role of bloom strength on GelMA hydrogel mechanical properties. We determined that both bloom strength of gelatin and weight percent of GelMA influenced both stiffness and viscoelastic ratio; however, only bloom strength affected diffusivity, permeability, and pore size. With this library of GelMA hydrogels of varying properties, we then encapsulated Swan71 trophoblast spheroids in these hydrogel variants to assess how bloom strength affects trophoblast spheroid morphology. Overall, we observed a decreasing trend of spheroid area and Feret diameter as bloom strength increased. In identifying clear relationships between bloom strength, hydrogel mechanical properties, and trophoblast spheroid morphology, we demonstrate that bloom strength should considered when designing tissue engineered constructs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The uterus has critical biomechanical functions in pregnancy and undergoes dramatic material growth and remodeling from implantation to parturition. The intrinsic material properties of the human uterus and how they evolve in pregnancy are poorly understood. To address this knowledge gap and assess the heterogeneity of these tissues, the time-dependent material properties of all human uterine layers were measured with nanoindentation. The endometrium-decidua layer was found to be the least stiff, most viscous, and least permeable layer of the human uterus in nonpregnant and third-trimester pregnant tissues. In pregnancy, the endometrium-decidua becomes stiffer and less viscous with no material property changes observed in the myometrium or perimetrium. Additionally, uterine material properties did not significantly differ between third-trimester pregnant tissues with and without placenta accreta. The foundational data generated by this study will facilitate the development of physiologically accurate models of the human uterus to investigate gynecologic and obstetric disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The fetal membranes are an essential mechanical structure for pregnancy, protecting the developing fetus in an amniotic fluid environment and rupturing before birth. In cooperation with the cervix and the uterus, the fetal membranes support the mechanical loads of pregnancy. Structurally, the fetal membranes comprise two main layers: the amnion and the chorion. The mechanical characterization of each layer is crucial to understanding how each layer contributes to the structural performance of the whole membrane. The in-vivo mechanical loading of the fetal membranes and the amount of tissue stress generated in each layer throughout gestation remains poorly understood, as it is difficult to perform direct measurements on pregnant patients. Finite element analysis of pregnancy offers a computational method to explore how anatomical and tissue remodeling factors influence the load-sharing of the uterus, cervix, and fetal membranes. To aid in the formulation of such computational models of pregnancy, this work develops a fiber-based multilayer fetal membrane model that captures its response to previously published bulge inflation loading data. First, material models for the amnion, chorion, and maternal decidua are formulated, informed, and validated by published data. Then, the behavior of the fetal membrane as a layered structure was analyzed, focusing on the respective stress distribution and thickness variation in each layer. The layered computational model captures the overall behavior of the fetal membranes, with the amnion being the mechanically dominant layer. The inclusion of fibers in the amnion material model is an important factor in obtaining reliable fetal membrane behavior according to the experimental dataset. These results highlight the potential of this layered model to be integrated into larger biomechanical models of the gravid uterus and cervix to study the mechanical mechanisms of preterm birth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The endometrium undergoes rapid cycles of vascular growth, remodeling, and breakdown during the menstrual cycle and pregnancy. Decidualization is an endometrial differentiation process driven by steroidal sex hormones that is critical for blastocyst-uterine interfacing and blastocyst implantation. Certain pregnancy disorders may be linked to decidualization processes. However, much remains unknown regarding the role of decidualization and reciprocal trophoblast-endometrial interactions on endometrial angiogenesis and trophoblast invasion. Here, we report an engineered endometrial microvascular network embedded in gelatin hydrogels that displays morphological and functional patterns of decidualization. Vessel complexity and biomolecule secretion are sensitive to decidualization and affect trophoblast motility, but that signaling between endometrial and trophoblast cells was not bi-directional. Although endometrial microvascular network decidualization status influences trophoblast cells, trophoblast cells did not induce structural changes in the endometrial microvascular networks. These findings add to a growing literature that the endometrium has biological agency at the uterine-trophoblast interface during implantation. Finally, we form a stratified endometrial tri-culture model, combining engineered microvascular networks with epithelial cells. These endometrial microvascular networks provide a well-characterized platform to investigate dynamic changes in angiogenesis in response to pathological and physiological endometrial states., Competing Interests: Competing interests The authors declare no competing interests.

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Fetal membranes have important mechanical and antimicrobial roles in maintaining pregnancy. However, the small thickness (<800 µm) of fetal membranes places them outside the resolution limits of most ultrasound and magnetic resonance systems. Optical imaging methods like optical coherence tomography (OCT) have the potential to fill this resolution gap. Here, OCT and machine learning methods were developed to characterize the ex vivo properties of human fetal membranes under dynamic loading. A saline inflation test was incorporated into an OCT system, and tests were performed on n = 33 and n = 32 human samples obtained from labored and C-section donors, respectively. Fetal membranes were collected in near-cervical and near-placental locations. Histology, endogenous two photon fluorescence microscopy, and second harmonic generation microscopy were used to identify sources of contrast in OCT images of fetal membranes. A convolutional neural network was trained to automatically segment fetal membrane sub-layers with high accuracy (Dice coefficients >0.8). Intact amniochorion bilayer and separated amnion and chorion were individually loaded, and the amnion layer was identified as the load-bearing layer within intact fetal membranes for both labored and C-section samples, consistent with prior work. Additionally, the rupture pressure and thickness of the amniochorion bilayer from the near-placental region were greater than those of the near-cervical region for labored samples. This location-dependent change in fetal membrane thickness was not attributable to the load-bearing amnion layer. Finally, the initial phase of the loading curve indicates that amniochorion bilayer from the near-cervical region is strain-hardened compared to the near-placental region in labored samples. Overall, these studies fill a gap in our understanding of the structural and mechanical properties of human fetal membranes at high resolution under dynamic loading events., Competing Interests: The authors declare no conflicts of interest., (© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Recent engineering advances provide new tools and techniques to alleviate poor pregnancy outcomes that can lead to maternal and fetal death and long-term medical complications.

Major obstetrical syndromes related to preterm birth-including preterm pre-labor rupture of membranes, fetal growth restriction and pre-eclampsia-affect 10-15% of all pregnancies worldwide, resulting in substantial financial and human costs. Human pregnancy comprises a set of complex physiological processes, which involve most organ systems within the maternal body. There has been rapid recent growth of computational biomechanical approaches to the study of problems in pregnancy. These are particularly attractive for research that is logistically difficult and ethically challenging to execute in humans. Here, we present the history and current state-of-the-art in pregnancy bioengineering research, focusing on three case studies in which computational approaches have been used to explore the maternal-fetal dyad. First, fracture models are used to examine preterm pre-labor rupture of the fetal membranes, which is responsible for one-third of premature births. Next, models of the utero-placental interface are considered, focused on the trophoblast-the layer of fetal cells that directly contact the maternal uterus and thus form the immunological interface between two genetically different individuals. Finally, maternal cardiovascular function in pregnancy is examined in a multiscale framework considering interactions between hormonal and mechanical cues leading to heart growth. These three examples demonstrate the substantial potential for engineering approaches to pregnancy research, in which 'experiments' in silico can be deployed to examine complex systems that are otherwise not available for targeted research. (225 words)., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The characterization of human subcutaneous adipose tissue (SAT) under high-rate loading is valuable for development of biofidelic finite element human body models (FE-HBMs) to predict seat belt-pelvis interaction and injury risk in vehicle crash simulations. While material characterization of SAT has been performed at 25 °C or 37 °C, the effect of temperature on mechanical properties of SAT under high-rate and large-deformation loading has not been investigated. Similarly, while freezing is the most common preservation technique for cadaveric specimens, the effect of freeze-thaw on the mechanical properties of SAT is also absent from the literature. Therefore, the aim of this study was to determine the effect of freezing and temperature on mechanical properties of human SAT. Fresh and previously frozen human SAT specimens were obtained and tested at 25 °C and 37 °C. High-rate indentation and puncture tests were performed, and indentation-puncture force-depth responses were obtained. While the chance of material failure was found to be different between temperatures and between fresh and previously frozen tissue, statistical analyses revealed that temperature and freezing did not change the shear modulus and failure characteristics of SAT. Therefore, the results of the current study indicated that SAT material properties characterized from either fresh or frozen tissue at either 25 °C or 37 °C could be used for enhancing the biofidelity of FE-HBMs., (Copyright © 2022 by ASME.)

Medical conditions that primarily or disproportionately affect women have historically been poorly studied. In contrast to the musculoskeletal and cardiovascular systems, there is no lengthy record of biomaterials research addressing women's health needs. In this article, the historical reasons for this discrepancy are examined. The anatomy of both the nonpregnant and pregnant reproductive tissues is reviewed, including the ovaries, uterus, and (fetal) placenta. Examples of biomaterials-related women's health research are described, including tissue engineering, organoids, and microphysiological systems. The future of the field is considered with dual focuses. First, there is a significant need for novel approaches to advance women's health through materials and biomaterials, particularly in complex biomimetic hydrogels. Second, there is an exciting opportunity to enlarge the community of biomaterials scientists and engineers working in women's health to encourage more contributions to its rapidly emerging product development pipeline., (© The Author(s), under exclusive License to the Materials Research Society 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Research into the human placenta's complex functioning is complicated by a lack of suitable physiological in vivo models. Two complementary approaches have emerged recently to address these gaps in understanding, computational in silico techniques, including multi-scale modeling of placental blood flow and oxygen transport, and cellular in vitro approaches, including organoids, tissue engineering, and organ-on-a-chip models. Following a brief introduction to the placenta's structure and function and its influence on the substantial clinical problem of preterm birth, these different bioengineering approaches are reviewed. The cellular techniques allow for investigation of early first-trimester implantation and placental development, including critical biological processes such as trophoblast invasion and trophoblast fusion, that are otherwise very difficult to study. Similarly, computational models of the placenta and the pregnant pelvis at later-term gestation allow for investigations relevant to complications that occur when the placenta has fully developed. To fully understand clinical conditions associated with the placenta, including those with roots in early processes but that only manifest clinically at full-term, a holistic approach to the study of this fascinating, temporary but critical organ is required., (© 2021. Biomedical Engineering Society.)

Soft biological tissues demonstrate strong time-dependent mechanical behavior, arising from their intrinsic viscoelasticity and fluid flow-induced poroelasticity. It is increasingly recognized that time-dependent mechanical properties of soft tissues influence their physiological functions and are linked to several pathological processes. Nevertheless, soft tissue time-dependent characteristics, especially their micromechanical variation with tissue composition and location, remain poorly understood. Nanoindentation is a well-established technique to measure local elastic properties but has not been fully explored to determine micro-scale time-dependent properties of soft tissues. Here, a nanoindentation-based experimental strategy is implemented to characterize the micro-scale poroelastic and viscoelastic behavior of mouse heart, kidney, and liver tissues. It is demonstrated that heart tissue exhibits substantial mechanical heterogeneity where the elastic modulus varies spatially from 1 to 30 kPa. In contrast, both kidney and liver tissues show relatively homogeneous response with elastic modulus 0.5-3 kPa. All three tissues demonstrate marked load relaxation under constant indentation, where the relaxation behavior is observed to be largely dominated by tissue viscoelasticity. Intrinsic permeability varies among different tissues, where heart tissue is found to be less permeable compared to kidney and liver tissues. Overall, the results presented herein provide key insights into the time-dependent micromechanical behavior of different tissues and can therefore contribute to studies of tissue pathology and tissue engineering applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

There has long been anecdotal evidence of early labor and delivery in severe weather events leading to preterm birth. In particular, significant barometric pressure changes are associated with hurricanes and bomb cyclones. Some authors have related these low pressure weather events to premature rupture of fetal membranes, hypothesizing that the membranes act as an inflated balloon and respond directly to pressure changes. In this article, the key literature including data supporting this hypothesis is reviewed. A simple numerical model, based on a competition between the driving and resisting forces for fetal membrane rupture, is presented. This model provides a quantitative mechanism for membrane failure in the context of storms with low atmospheric pressure. Other sequelae of severe storms that are unrelated to fetal membrane rupture are also discussed. Labor and delivery in the context of major weather events should be understood in a holistic framework that includes both exogenous and endogenous factors relevant to the pregnant patient., (Copyright © 2020 Wheeler and Oyen.)

Amnion is a membrane that surrounds and structurally protects the developing fetus during pregnancy. The rupture of amniotic membranes prior to both normal and preterm deliveries involves stretch forces acting on a biochemically triggered weak zone of the membranes. Fracture toughness is an important mechanical property describing how the membranes containing a defect resist fracture, but this property has never been investigated in amniotic membranes. In this work, the fracture toughness of many samples cut from four pieces of amniotic membrane from different mothers was examined by uniaxial and pure shear (mode I) fracture tests. The measurement was checked for dependence on the sample geometry and notch length. Results from the uniaxial tensile test show J-shaped stress-strain curves and confirm that the amniotic membrane is a nonlinear material. The measured fracture toughness of four amniotic membranes ranged from 0.96 ± 0.11 to 1.83 ± 0.18 kJ m -2 . Despite considering the effect of the presence of the defect on mechanical property measurement, similar fracture behaviour was observed for pre-notched and unnotched specimens, indicating that the membranes were extremely tolerant to defects. This defect-tolerant characteristic provides insight into the understanding of fetal membrane rupture., Competing Interests: We declare we have no competing interests.

Study Question: What is the stiffness (elastic modulus) of human nonpregnant secretory phase endometrium, first trimester decidua, and placenta?, Summary Answer: The stiffness of decidua basalis, the site of placental invasion, was an order of magnitude higher at 103 Pa compared to 102 Pa for decidua parietalis, nonpregnant endometrium and placenta., What Is Known Already: Mechanical forces have profound effects on cell behavior, regulating both cell differentiation and migration. Despite their importance, very little is known about their effects on blastocyst implantation and trophoblast migration during placental development because of the lack of mechanical characterization at the human maternal-fetal interface., Study Design, Size, Duration: An observational study was conducted to measure the stiffness of ex vivo samples of human nonpregnant secretory endometrium (N = 5) and first trimester decidua basalis (N = 6), decidua parietalis (N = 5), and placenta (N = 5). The stiffness of the artificial extracellular matrix (ECM), Matrigel®, commonly used to study migration of extravillous trophoblast (EVT) in three dimensions and to culture endometrial and placental organoids, was also determined (N = 5)., Participants/materials, Setting, Methods: Atomic force microscopy was used to perform ex vivo direct measurements to determine the stiffness of fresh tissue samples. Decidua was stained by immunohistochemistry (IHC) for HLA-G+ EVT to confirm whether samples were decidua basalis or decidua parietalis. Endometrium was stained with hematoxylin and eosin to confirm the presence of luminal epithelium. Single-cell RNA sequencing data were analyzed to determine expression of ECM transcripts by decidual and placental cells. Fibrillin 1, a protein identified by these data, was stained by IHC in decidua basalis., Main Results and the Role of Chance: We observed that decidua basalis was significantly stiffer than decidua parietalis, at 1250 and 171 Pa, respectively (P < 0.05). The stiffness of decidua parietalis was similar to nonpregnant endometrium and placental tissue (250 and 232 Pa, respectively). These findings suggest that it is the presence of invading EVT that is driving the increase in stiffness in decidua basalis. The stiffness of Matrigel® was found to be 331 Pa, significantly lower than decidua basalis (P < 0.05)., Large Scale Data: N/A., Limitations, Reasons for Caution: Tissue stiffness was derived by ex vivo measurements on blocks of fresh tissue in the absence of blood flow. The nonpregnant endometrium samples were obtained from women undergoing treatment for infertility. These may not reflect the stiffness of endometrium from normal fertile women., Wider Implications of the Findings: These results provide direct measurements of tissue stiffness during the window of implantation and first trimester of human pregnancy. They serve as a basis of future studies exploring the impact of mechanics on embryo implantation and development of the placenta. The findings provide important baseline data to inform matrix stiffness requirements when developing in vitro models of trophoblast stem cell development and migration that more closely resemble the decidua in vivo., Study Funding/competing Interest(s): This work was supported by the Centre for Trophoblast Research, the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), the Medical Research Council (MR/P001092/1), the European Research Council (772426), an Engineering and Physical Sciences Research Council Doctoral Training Award (1354760), a UK Medical Research Council and Sackler Foundation Doctoral Training Grant (RG70550) and a Wellcome Trust Doctoral Studentship (215226/Z/19/Z)., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Collagen scaffolds are widely used in a range of tissue engineering applications, both in vitro and in vivo, where their permeability to fluid flow greatly affects their mechanical and biological functionality. This paper reports new insights into the interrelationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds, focussing in particular on the degree of closure and the alignment of the pores. Isotropic and aligned scaffolds of different occlusivity were produced by ice templating, and were characterised in terms of their structure and mechanical properties. Permeability studies were conducted using two experimental set-ups to cover a wide range of applied fluid pressures. The permeability was found to be constant at low pressures for a given scaffold with more open structures and aligned structures being more permeable. The deformation of scaffolds under high pressure led to a decrease in permeability. The aligned structures were more responsive to deformation than their isotropic equivalents with their permeability falling more quickly at low strain. For isotropic samples, a broad (1 - ɛ) 2 dependence for permeability was observed with the constant of proportionality varying with collagen fraction as the starting structures became more occluded. Aligned scaffolds did not follow the same behaviour, with the pores apparently closing more quickly in response to early deformation. These results highlight the importance of scaffold structure in determining permeability to interstitial fluid, and provide an understanding of scaffold behaviour within the complex mechanical environment of the body. STATEMENT OF SIGNIFICANCE: Collagen scaffolds are widely used in tissue engineering applications, for instance to contribute with wound healing. Their permeability to fluid flow, such as water and blood, is important to ensure they perform efficiently when inside the body. The present study reports new insights into the relationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds. It presents in particular the experimental setups used to measure these properties and the result of comparisons between collagen scaffolds with different structures: aligned and isotropic (non-aligned). It indicates quantitative differences in terms of permeability, and the effects of compression on such permeability. The results contribute to the development and understanding of collagen scaffolds and their applications., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cartilage wounds result in chronic pain and degradation of the quality of life for millions of people. A synthetic cellular scaffold able to heal the damage by substituting the natural tissue is of great potential value. Here, it is shown for the first time that the unique interplay between the molecular components of cartilage can be reproduced in composite materials made of a polyelectrolyte hydrogel embedding a collagen scaffold. These composites possess a mechanical response determined by osmotic and electrostatic effects, comparable to articular cartilage in terms of elastic modulus, time-dependent response, and permeability to interstitial fluid flow. Made entirely from biocompatible materials, the cartilage-like composite materials developed permit 3D culture of chondrocyte-like cells through their microporosity. The biomimetic materials presented here constitute an entirely new class of osmotically stiffened composites, which may find use outside of biomedical applications., (© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The transport properties of hydrogels largely affect their performance in biomedical applications ranging from cell culture scaffolds to drug delivery systems. Solutes can move through the polymer mesh as a result of concentration gradients in the interstitial fluid or pressure gradients that move the fluid and solutes simultaneously. The relationship between the two modalities of transport in hydrogels can provide insight for the design of materials that can function effectively in the dynamic conditions experienced in vitro and in vivo , yet this correlation has not been previously elucidated. Here, fluorescence recovery after photobleaching (FRAP) is used to measure the diffusivity of dextran molecules of different size within polyethylene glycol hydrogels. Spherical indentation analyzed in a poroelastic framework is used to measure the permeability to fluid flow of the same hydrogels. It is found that while the diffusivity varies with exp( ξ -2 ), where ξ is the mesh size of the hydrogels, it also varies with exp( k -1 ), where k is the intrinsic permeability. For the same hydrogel structure, diffusive transport is affected by the solute size, while convective transport is unaffected. As spherical indentation is a reliable, quick and non-destructive testing method for hydrated soft materials, the relationship provides the means to faster assessment of the transport properties of hydrogels and, ultimately, of their effective use in biomedical applications.

Native biological tissues are viscoelastic materials that undergo time-dependent loading in vivo. It is therefore crucial to ensure that biomedical materials have a suitable viscoelastic response for a given application. In this study, the viscoelastic properties of electrospun poly(vinyl alcohol) are investigated using tensile load relaxation testing. A five-parameter generalised Maxwell constitutive model is found to characterise the experimental response. The effect of polymer concentration and electrospinning voltage on model parameters is investigated in detail. The stiffness coefficients for the relaxation process appear to be dependent on the electrospinning conditions used whereas the time constants remain relatively unchanged. It is also observed that the stiffness parameters are linearly correlated with the equilibrium modulus, indicating that a single underlying material property dictates the relaxation moduli. Lastly, it is found that the viscoelastic model parameters are not predicted by the fibre diameter. These results provide an important understanding in designing electrospun mats with desired time-dependent properties., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The incidence of congenital spine deformities, including congenital scoliosis, kyphosis, and lordosis, may be influenced by the in utero mechanical environment, and particularly by fetal movements at critical time-points. There is a limited understanding of the influence of fetal movements on spinal development, despite the fact that mechanical forces have been shown to play an essential role in skeletal development of the limb. This study investigates the effects of muscle forces on spinal curvature, vertebral segmentation, and vertebral shape by inducing rigid or flaccid paralysis in the embryonic chick. The critical time-points for the influence of fetal movements on spinal development were identified by varying the time of onset of paralysis. Prolonged rigid paralysis induced severe defects in the spine, including curvature abnormalities, posterior and anterior vertebral fusions, and altered vertebral shape, while flaccid paralysis did not affect spinal curvature or vertebral segmentation. Early rigid paralysis resulted in more severe abnormalities in the spine than later rigid paralysis. The findings of this study support the hypothesis that the timing and nature of fetal muscle activity are critical influences on the normal development of the spine, with implications for the understanding of congenital spine deformities. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2135-2144, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The remarkable mechanical performance of biological materials is based on intricate structure-function relationships. Nanoindentation has become the primary tool for characterising biological materials, as it allows to relate structural changes to variations in mechanical properties on small scales. However, the respective theoretical background and associated interpretation of the parameters measured via indentation derives largely from research on 'traditional' engineering materials such as metals or ceramics. Here, we discuss the functional relevance of indentation hardness in biological materials by presenting a meta-analysis of its relationship with indentation modulus. Across seven orders of magnitude, indentation hardness was directly proportional to indentation modulus. Using a lumped parameter model to deconvolute indentation hardness into components arising from reversible and irreversible deformation, we establish criteria which allow to interpret differences in indentation hardness across or within biological materials. The ratio between hardness and modulus arises as a key parameter, which is related to the ratio between irreversible and reversible deformation during indentation, the material's yield strength, and the resistance to irreversible deformation, a material property which represents the energy required to create a unit volume of purely irreversible deformation. Indentation hardness generally increases upon material dehydration, however to a larger extent than expected from accompanying changes in indentation modulus, indicating that water acts as a 'plasticiser'. A detailed discussion of the role of indentation hardness, modulus and toughness in damage control during sharp or blunt indentation yields comprehensive guidelines for a performance-based ranking of biological materials, and suggests that quasi-plastic deformation is a frequent yet poorly understood damage mode, highlighting an important area of future research., Statement of Significance: Instrumented indentation is a widespread tool for characterising the mechanical properties of biological materials. Here, we show that the ratio between indentation hardness and modulus is approximately constant in biological materials. A simple elastic-plastic series deformation model is employed to rationalise part of this correlation, and criteria for a meaningful comparison of indentation hardness across biological materials are proposed. The ratio between indentation hardness and modulus emerges as the key parameter characterising the relative amount of irreversible deformation during indentation. Despite their comparatively high hardness to modulus ratio, biological materials are susceptible to quasiplastic deformation, due to their high toughness: quasi-plastic deformation is hence hypothesised to be a frequent yet poorly understood phenomenon, highlighting an important area of future research., (Copyright © 2017 Acta Materialia Inc. All rights reserved.)

Bone-tendon, bone-ligament and bone-cartilage junctions are multi-tissue interfaces that connect materials that differ by two orders of magnitude in mechanical properties, via gradual variations in mineral content and matrix composition. These sites mediate load transfer between highly dissimilar materials and are consequently a primary site of injury during orthopedic failure. Given the large incidence rate and the lack of suitable surgical solutions for their regeneration or repair, characterization of their natural structure and subsequent replication through tissue engineering is important. Here, we evaluate the ability and accuracy of instrumented indentation to characterize the mechanical properties of both biological tissues and engineered scaffolds with interfaces between materials that contain significant changes in mechanical properties. In this study, finite element simulations and reference samples are developed that characterize how accurately indentation measures the modulus of a material as it varies with distance across a continuous interface between dissimilar tissues with multiple orders of magnitude difference in properties. Finite element simulations accurately predicted discrepancies between the modulus function across an interface observed by indentation and the true modulus function of the material and hence allow us to understand the limits of instrumented indentation as a technique for quantifying gradual changes in material properties. It was found that in order to accurately investigate mechanical property variations in tissues with significant modulus heterogeneity the indenter size should be less than 10 percent of the expected length scale of the modulus variations., Statement of Significance: The interfaces between stiff and compliant orthopedic tissues such as bone-tendon, bone-ligament and bone-cartilage are frequent sites of failure during both acute and chronic orthopedic injury and as such their replication via tissue engineering is of importance. The characterization and understanding of these tissue interfaces on a mechanical basis is a key component of elucidating the structure-function relationships that allow them to function naturally and hence a core component of efforts to replicate them. This work uses finite element models and exeperiments to outline the ability of instrumented indentation to characterize the elastic modulus variations across tissue interfaces and provides guidelines for investigators seeking to use this method to understand any interface between dissimilar tissues., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Electrospinning is a simple and efficient process for producing sub-micron fibres. However, the process has many variables, and their effects on the non-woven mesh of fibres is complex. In particular, the effects on the mechanical properties of the fibre meshes are poorly understood. This paper conducts a parametric study, where the concentration and bloom strength of the gelatin solutions are varied, while all electrospinning process parameters are held constant. The effects on the fibrous meshes are monitored using scanning electron microscopy and mechanical testing under uniaxial tension. Mesh mechanical properties are relatively consistent, despite changes to the solutions, demonstrating the robustness of electrospinning. The gel strength of the solution is shown to have a statistically significant effect on the morphology, stiffness and strength of the meshes, while the fibre diameter has surprisingly little influence on the stiffness of the meshes. This experimental finding is supported by finite element analysis, demonstrating that the stiffness of the meshes is controlled by the volume fraction, rather than fibre diameter. Our results demonstrate the importance of understanding how electrospinning parameters influence the pore size of the meshes, as controlling fibre diameter alone is insufficient for consistent mechanical properties., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterized by inadequate invasion of extravillous placental trophoblast cells into the uterine arteries. How trophoblast invasion is controlled remains an unanswered question but is influenced by maternal uterine immune cells called decidual natural killer cells. Here, we describe an in vitro microfluidic invasion assay to study the migration of primary human trophoblast cells. Each experiment can be performed with a small number of cells making it possible to conduct research on human samples despite the challenges of isolating primary trophoblast cells. Cells are exposed to a chemical gradient and tracked in a three-dimensional microenvironment using real-time high-resolution imaging, so that dynamic readouts on cell migration such as directionality, motility and velocity are obtained. The microfluidic system was validated using isolated trophoblast and a gradient of granulocyte-macrophage colony-stimulating factor, a cytokine produced by activated decidual natural killer cells. This microfluidic model provides detailed analysis of the dynamics of trophoblast migration compared to previous assays and can be modified in future to study in vitro how human trophoblast behaves during placentation., (© 2017 The Authors.)

Background: In the human placenta, maternal and fetal bloods exchange substances through the surface of the villous trees: the fetal blood circulates in the villous trees, around which the maternal blood circulates. The blood flows directly influence fetal growth. Stem villi, the main supports of the villous tree, have contractile cells along the axes, whose contractions are expected to influence the blood circulations in the placenta. The displacement is neither measurable nor predictable while non-invasive measurements such as umbilical Doppler waveforms are helpful to predict the histological changes of the villous trees and vascularization in the placenta., Objective: The displacement caused by the contraction of the villous tree is necessary to predict the blood flows in the placenta. Hence, a computational villous tree model, which actively contracts, was developed in this study., Method: The villous tree model was based on the previous reports: shear moduli of the human placenta; branching patterns in the stem villi. The displacement pattern in the placenta was estimated by the computational model when the shear elastic moduli were changed., Results: The results show that the displacement caused by the contraction was influenced by the shear elastic moduli, but kept useful for the blood flows in the placenta. The characteristics agreed with the robustness of the blood flows in the placenta., Conclusion: The villous tree model, which actively contracts, was developed in this study. The combination of this computational model and non-invasive measurements will be useful to evaluate the condition of the placenta.

The fetal membrane surrounds the fetus during pregnancy and is a thin tissue composed of two layers, the chorion and the amnion. While rupture of this membrane normally occurs at term, preterm rupture can result in increased risk of fetal mortality and morbidity, as well as danger of infection in the mother. Although structural changes have been observed in the membrane in such cases, the mechanical behaviour of the human fetal membrane in vivo remains poorly understood and is challenging to investigate experimentally. Therefore, the objective of this study was to develop simplified finite element models to investigate the mechanical behaviour and rupture of the fetal membrane, particularly its constituent layers, under various physiological conditions. It was found that modelling the chorion and amnion as a single layer predicts remarkably different behaviour compared with a more anatomically-accurate bilayer, significantly underestimating stress in the amnion and under-predicting the risk of membrane rupture. Additionally, reductions in chorion-amnion interface lubrication and chorion thickness (reported in cases of preterm rupture) both resulted in increased membrane stress. Interestingly, the inclusion of a weak zone in the fetal membrane that has been observed to develop overlying the cervix would likely cause it to fail at term, during labour. Finally, these findings support the theory that the amnion is the dominant structural component of the fetal membrane and is required to maintain its integrity. The results provide a novel insight into the mechanical effect of structural changes in the chorion and amnion, in cases of both normal and preterm rupture.

Mechanically robust hydrogels are required for many tissue engineering applications to serve as cell-supporting structures. Unlike natural tissues, the majority of existing tough hydrogels lack ordered microstructures organized to withstand specific loading conditions. In this work, electrospun gelatin nanofibres, mimicking the collagen network in native tissues, are used to strengthen and resist crack propagation in brittle alginate hydrogels. Aligned nanofibre reinforcement enhances the tensile strength of the hydrogels by up to two orders of magnitude. The nanofibres can be arranged as multilayer laminates with varying orientations, which increases the toughness by two orders of magnitude compared with the unreinforced hydrogel. This work demonstrates a two-part strategy of fibre reinforcement and composite lamination in manufacturing strong and tough hydrogels with flexible microstructures to suit different mechanical and biomedical requirements., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Cartilage is a structural tissue with unique mechanical properties deriving from its electrically-charged porous structure. Traditional three-dimensional environments for the culture of cells fail to display the complex physical response displayed by the natural tissue. In this work, the reproduction of the charged environment found in cartilage is achieved using polyelectrolyte hydrogels based on polyvinyl alcohol and polyacrylic acid. The mechanical response and morphology of microporous physically-crosslinked cryogels are compared to those of heat-treated chemical gels made from the same polymers, as a result of pH-dependent swelling. In contrast to the heat-treated chemically-crosslinked gels, the elastic modulus of the physical cryogels was found to increase with charge activation and swelling, explained by the occurrence of electrostatic stiffening of the polymer chains at large charge densities. At the same time, the permeability of both materials to fluid flow was impaired by the presence of electric charges. This cartilage-like mechanical behavior displayed by responsive cryogels can be reproduced in other polyelectrolyte hydrogel systems to fabricate biomimetic cellular scaffolds for the repair of the tissue.

Severe degeneration of the intervertebral disc has an immensely debilitating effect on quality of life that has become a serious health and economic burden throughout the world. The disc plays an integral role in biomechanical movement and support within the spine. The emergence of tissue engineering endeavours to restore the structural characteristics and functionality of the native tissue. Hydrogels have been widely investigated as a candidate for regeneration of the gelatinous nucleus pulposus due to its architectural resemblance and fluid retention characteristics. However, hydrogels are often limited due to small compressive stiffness and tear resistance, leading to extrusion complications. Reinforcement of the hydrogel network using polymeric scaffolds may address these issues of inadequate mechanical properties and implant instability. This study investigates the potential of a carrageenan gel-infused polycaprolactone scaffold for nucleus pulposus tissue engineering. Mechanical properties were characterised using viscoelastic and poroelastic frameworks via microindentation. The incorporation of polymeric reinforcement within the gels increased material stiffness to that comparable to the native nucleus pulposus, however permeability was significantly greater than native values. A preliminary cell evaluation culturing NIH 3T3s over 21 days suggested the incorporation of polymeric networks also enhanced cellular proliferation compared to gels alone., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tissue architecture is intimately linked with its functions, and loss of tissue organization is often associated with pathologies. The intricate depth-dependent extracellular matrix (ECM) arrangement in articular cartilage is critical to its biomechanical functions. In this study, we developed a Raman spectroscopic imaging approach to gain new insight into the depth-dependent arrangement of native and tissue-engineered articular cartilage using bovine tissues and cells. Our results revealed previously unreported tissue complexity into at least six zones above the tidemark based on a principal component analysis and k -means clustering analysis of the distribution and orientation of the main ECM components. Correlation of nanoindentation and Raman spectroscopic data suggested that the biomechanics across the tissue depth are influenced by ECM microstructure rather than composition. Further, Raman spectroscopy together with multivariate analysis revealed changes in the collagen, glycosaminoglycan, and water distributions in tissue-engineered constructs over time. These changes were assessed using simple metrics that promise to instruct efforts toward the regeneration of a broad range of tissues with native zonal complexity and functional performance.

Placental oxygen transport takes place at the final branches of the villous tree and is dictated by the relative arrangement of the maternal and fetal circulations. Modeling techniques have failed to accurately assess the structure-function relationship in the terminal villi due to the geometrical complexity. Three-dimensional blood flow and oxygen transport was modeled in four terminal villi reconstructed from confocal image stacks. The blood flow was analyzed along the center lines of capillary segments and the effect of the variability in capillary diameter, tortuosity and branching was investigated. Additionally, a validation study was performed to corroborate the simulation results. The results show how capillary variations impact motion of the fetal blood, and how their bends and dilatations can decelerate the flow by up to 80%. Vortical flow is also demonstrated not to develop in the fetal capillaries. The different geometries are shown to dictate the transport of gases with differences of over 100% in the oxygen flux between samples. Capillary variations are key for efficient oxygen uptake by the fetus; they allow the blood to decelerate where the villous membrane is thinnest allowing for a better oxygenation, but also by reducing the vessel diameter they carry the oxygenated blood away fast. The methodology employed herein could become a platform to simulate complicated in-vivo and in-vitro scenarios of pregnancy complications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Three-dimensional (3D) bioprinting is on the cusp of permitting the direct fabrication of artificial living tissue. Multicellular building blocks (bioinks) are dispensed layer by layer and scaled for the target construct. However, only a few materials are able to fulfill the considerable requirements for suitable bioink formulation, a critical component of efficient 3D bioprinting. Alginate, a naturally occurring polysaccharide, is clearly the most commonly employed material in current bioinks. Here, we discuss the benefits and disadvantages of the use of alginate in 3D bioprinting by summarizing the most recent studies that used alginate for printing vascular tissue, bone and cartilage. In addition, other breakthroughs in the use of alginate in bioprinting are discussed, including strategies to improve its structural and degradation characteristics. In this review, we organize the available literature in order to inspire and accelerate novel alginate-based bioink formulations with enhanced properties for future applications in basic research, drug screening and regenerative medicine., Competing Interests: The authors declare no conflict of interest.