Cell size and shape affect cellular processes such as cell survival, growth and differentiation, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

We report the thermodynamically controlled growth of solution-processable and free-standing nanosheets via peptide assembly in two dimensions. By taking advantage of self-sorting between peptide β-strands and hydrocarbon chains, we have demonstrated the formation of Janus 2D structures with single-layer thickness, which enable a predetermined surface heterofunctionalization. A controlled 2D-to-1D morphological transition was achieved by subtly adjusting the intermolecular forces. These nanosheets provide an ideal substrate for the engineering of guest components (e.g., proteins and nanoparticles), where enhanced enzyme activity was observed. We anticipate that sequence-specific programmed peptides will offer promise as design elements for 2D assemblies with face-selective functionalization.

Extracellular vesicles (EVs) have recently gained significant attention as important mediators of intercellular communication, potential drug carriers, and disease biomarkers. These natural cell-derived nanoparticles are postulated to be biocompatible, stable under physiological conditions, and to show reduced immunogenicity as compared to other synthetic nanoparticles. Although initial clinical trials are ongoing, the use of EVs for therapeutic applications may be limited due to undesired off-target activity and potential “dilution effects” upon systemic administration which may affect their ability to reach their target tissues. To fully exploit their therapeutic potential, EVs are embedded into implantable biomaterials designed to achieve local delivery of therapeutics taking advantage of enzyme prodrug therapy (EPT). In this first application of EVs for an EPT approach, EVs are used as smart carriers for stabilizing enzymes in a hydrogel for local controlled conversion of benign prodrugs to active antiinflammatory compounds. It is shown that the natural EVs’ antiinflammatory potential is comparable or superior to synthetic carriers, in particular upon repeated long-term incubations and in different macrophage models of inflammation. Moreover, density-dependent color scanning electron microscopy imaging of EVs in a hydrogel is presented herein, an impactful tool for further understanding EVs in biological settings.

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Cells in the body use a variety of mechanisms to ensure the specificity and efficacy of signal transduction. One way that this is achieved is through tight spatial control over the position of different proteins, signaling sequences, and biomolecules within and around cells. For instance, the extracellular matrix protein fibronectin presents RGDS and PHSRN sequences that synergistically bind the αβ integrin when separated by 3.2 nm but are unable to bind when this distance is >5.5 nm.1 Building biomaterials to controllably space different epitopes with subnanometer accuracy in a three-dimensional (3D) hydrogel is challenging. Here, we synthesized peptides that self-assemble into nanofiber hydrogels utilizing the β-sheet motif, which has a known regular spacing along the peptide backbone. By modifying specific locations along the peptide, we are able to controllably space different epitopes with subnanometer accuracy at distances from 0.7 nm to over 6 nm, which is within the size range of many protein clusters. Endothelial cells encapsulated within hydrogels displaying RGDS and PHSRN in the native 3.2 nm spacing showed a significant upregulation in the expression of the alpha 5 integrin subunit compared to those in hydrogels with a 6.2 nm spacing, demonstrating the physiological relevance of the spacing. Furthermore, after 24 h the cells in hydrogels with the 3.2 nm spacing appeared to be more spread with increased staining for the αβ integrin. This self-assembling peptide system can controllably space multiple epitopes with subnanometer accuracy, demonstrating an exciting platform to study the effects of ligand density and location on cells within a synthetic 3D environment.

Acoustic patterning using ultrasound standing waves has recently emerged as a potent biotechnology enabling the remote generation of ordered cell systems. This capability has opened up exciting opportunities, for example, in guiding the development of organoid cultures or the organization of complex tissues. The success of these studies is often contingent on the formation of tightly-packed and uniform cell arrays; however, a number of factors can act to disrupt or prevent acoustic patterning. Yet, to the best of our knowledge, there has been no comprehensive assessment of the quality of acoustically-patterned cell populations. In this report we use a mathematical approach, known as Voronoï tessellation, to generate a series of metrics that can be used to measure the effect of cell concentration, pressure amplitude, ultrasound frequency and biomaterial viscosity upon the quality of acoustically-patterned cell systems. Moreover, we extend this approach towards the characterization of spatiotemporal processes, namely, the acoustic patterning of cell suspensions and the migration of patterned, adherent cell clusters. This strategy is simple, unbiased and highly informative, and we anticipate that the methods described here will provide a systematic framework for all stages of acoustic patterning, including the robust quality control of devices, statistical comparison of patterning conditions, the quantitative exploration of parameter limits and the ability to track patterned tissue formation over time.

SummaryObjectiveTo investigate and validate digital X-ray microradiography as a novel, high-throughput and cost-effective screening approach to identify abnormal joint phenotypes in mice.MethodDigital X-ray microradiography was used to quantify the subchondral bone mineral content (BMC) in the medial tibial plateau. Accuracy and reproducibility of the method were determined in 22 samples from C57BL/6(B6Brd;B6Dnk;B6N-Tyrc-Brd) wild-type mice. The method was then validated in wild-type mice that had undergone surgical destabilisation of medial meniscus (DMM) and in a genetically modified mouse strain with an established increase in trabecular bone mass.ResultsThe measurement of subchondral BMC by digital X-ray microradiography had a coefficient of variation of 3.6%. Digital X-ray microradiography was able to demonstrate significantly increased subchondral BMC in the medial tibial plateau of male mice 4 and 8 weeks after DMM surgery and in female mice 8 weeks after surgery. Furthermore, digital X-ray microradiography also detected the increase in subchondral BMC in a genetically modified mouse strain with high trabecular bone mass.ConclusionQuantitation of subchondral BMC by digital X-ray microradiography is a rapid, sensitive and cost-effective method to identify abnormal joint phenotypes in mice of both genders at several ages.