Collagen hydrogels are among ​the most well-studied platforms for drug delivery and in situ tissue engineering, thanks to their low cost, low immunogenicity, versatility, biocompatibility, and similarity to the natural extracellular matrix (ECM). Despite collagen being largely responsible for the tensile properties of native connective tissues, collagen hydrogels have relatively low mechanical properties in the absence of covalent cross-linking. This is particularly problematic when attempting to regenerate stiffer and stronger native tissues such as bone. Furthermore, in contrast to hydrogels based on ECM proteins such as fibronectin, collagen hydrogels do not have any growth factor (GF)-specific binding sites and often cannot sequester physiological (small) amounts of the protein. GF binding and in situ presentation are properties that can aid significantly in the tissue regeneration process by dictating cell fate without causing adverse effects such as malignant tumorigenic tissue growth. To alleviate these issues, researchers have developed several strategies to increase the mechanical properties of collagen hydrogels using physical or chemical modifications. This can expand the applicability of collagen hydrogels to tissues subject to a continuous load. GF delivery has also been explored, mathematically and experimentally, through the development of direct loading, chemical cross-linking, electrostatic interaction, and other carrier systems. This comprehensive article explores the ways in which these parameters, mechanical properties and GF delivery, have been optimized in collagen hydrogel systems ​and examines their in vitro or in vivo biological effect. This article can, therefore, be a useful tool to streamline future studies in the field, by pointing researchers into the appropriate direction according to their collagen hydrogel design requirements.

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices.

As cell function and phenotype can be directed by the mechanical characteristics of the surrounding matrix, hydrogels have become important platforms for cell culture systems, with properties that can be tuned by external stimuli, such as divalent cations, enzymatic treatment, and pH. However, many of these stimuli can directly affect cell behavior, making it difficult to distinguish purely mechanical signaling events. This study reports on the development of a hydrogel that incorporates photoswitchable cross-linkers, which can reversibly alter their stiffness upon irradiation with the appropriate wavelength of light. Furthermore, this study reports the response of bone-marrow-derived mesenchymal stem cells (MSCs) on these hydrogels that were stiffened systematically by irradiation with blue light. The substrates were shown to be noncytotoxic, and crucially MSCs were not affected by blue-light exposure. Time-resolved analysis of cell morphology showed characteristic cell spreading and increased aspect ratios in response to greater substrate stiffness. This hydrogel provides a platform to study mechanosignaling in cells responding to dynamic changes in stiffness, offering a new way to study mechanotransduction signaling pathways and biological processes, with implicit changes to tissue mechanics, such as development, ageing, and fibrosis.

Studies of cellular mechano-signaling have often utilized static models that do not fully replicate the dynamics of living tissues. Here, we examine the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 h, 4% CTS at 1 Hz), cell characteristics mimic responses to increased substrate stiffness. As the strain regime is intensified (frequency increased to 5 Hz), we characterize rapid establishment of a broad, structured and reversible protein-level response, even as transcription is apparently downregulated. Protein abundance is quantified coincident with changes to protein conformation and post-translational modification (PTM). Furthermore, we characterize changes to the linker of nucleoskeleton and cytoskeleton (LINC) complex that bridges the nuclear envelope, and specifically to levels and PTMs of Sad1/UNC-84 (SUN) domain-containing protein 2 (SUN2). The result of this regulation is to decouple mechano-transmission between the cytoskeleton and the nucleus, thus conferring protection to chromatin.

The mechanical environment can influence cell behaviour, including changes to transcriptional and proteomic regulation, morphology and, in the case of stem cells, commitment to lineage. However, current tools for characterizing substrates’ mechanical properties, such as atomic force microscopy (AFM), often do not fully recapitulate the length and time scales over which cells ‘feel’ substrates. Here, we show that an immortalised, clonal line of human mesenchymal stem cells (MSCs) maintains the responsiveness to substrate mechanics observed in primary cells, and can be used as a reporter of stiffness. MSCs were cultured on soft and stiff polyacrylamide hydrogels. In both primary and immortalised MSCs, stiffer substrates promoted increased cell spreading, expression of lamin-A/C and translocation of mechano-sensitive proteins YAP1 and MKL1 to the nucleus. Stiffness was also found to regulate transcriptional markers of lineage. A GFP-YAP/RFP-H2B reporter construct was designed and virally delivered to the immortalised MSCs for in situ detection of substrate stiffness. MSCs with stable expression of the reporter showed GFP-YAP to be colocalised with nuclear RFP-H2B on stiff substrates, enabling development of a cellular reporter of substrate stiffness. This will facilitate mechanical characterisation of new materials developed for applications in tissue engineering and regenerative medicine.

It has been established that the mechanical properties of hydrogels control the fate of (stem) cells. However, despite its importance, a one‐to‐one correspondence between gels' stiffness and cell behavior is still missing from literature. In this work, the viscoelastic properties of poly(ethylene‐glycol) (PEG)‐based hydrogels are investigated by means of rheological measurements performed at different length scales. The outcomes of this work reveal that PEG‐based hydrogels show significant stiffening when subjected to a compressional deformation, implying that conventional bulk rheology measurements may overestimate the stiffness of hydrogels by up to an order of magnitude. It is hypothesized that this apparent stiffening is caused by an induced “tensional state” of the gel network, due to the application of a compressional normal force during sample loading. Moreover, it is shown that the actual stiffness of the hydrogels is instead accurately determined by means of both passive‐video‐particle‐tracking (PVPT) microrheology and nanoindentation measurements, which are inherently performed at the cell's length scale and in absence of any externally applied force in the case of PVPT. These results underpin a methodology for measuring hydrogels' linear viscoelastic properties that are representative of the mechanical constraints perceived by cells in 3D hydrogel cultures.