Figure - available from: Advanced Science
This content is subject to copyright. Terms and conditions apply.
Hydrolytically stable and nonswelling hydrogels are necessary to study cellular stiffness sensing in 3D. A) Scheme of DexVS hydrogel model. DexVS is reacted with the cell‐adhesive peptide cRGD and crosslinked through MMP‐cleavable peptides. B) Optical tweezers measurements of the storage (G') and loss (G'') modulus of hMSC encapsulated DexVS hydrogels crosslinked with 21.0 × 10⁻³ m di‐cysteine‐HD and 29.4 × 10⁻³ m mono‐cysteine‐HD from at least 50 different beads measured on n = 3 independent samples. All data are presented as a mean + s.d. C) Young's modulus of DexVS hydrogels as a function of crosslinker concentration, as measured by nanoindentation (n = 3 independent samples). D) Young's modulus of hMSC encapsulated hydrolytically labile DexMA hydrogels and stable DexVS with 21.0 × 10⁻³ m peptide crosslinker after 1, 7, and 21 days of culture (n = 3 independent samples). The orange X indicates that the hydrogel was fully hydrolyzed after 7 days in cell culture medium. E) Storage modulus of hMSC encapsulated DexVS hydrogels crosslinked with 21.0 × 10⁻³ m di‐cysteine‐HD and 29.4 × 10⁻³ m mono‐cysteine‐HD, measured by optical tweezers (at 22.7 Hz) within 1–8 µm near cells and >50 µm away from cells. The orange arrow indicates beads measured, while the green arrow indicates cells. F) Morphology of hMSCs encapsulated in nonswelling versus swelling soft (≈0.1 kPa) hydrogels (XZ plane shown, the red arrow indicates the swelling direction). G) Hydrogel swelling ratio of the nonswelling versus swelling soft (≈0.1 kPa) hydrogels. (n ≥ 3 independent samples). H) Cell shape index of hMSCs encapsulated in the nonswelling versus swelling soft (≈0.1 kPa) hydrogels. (n ≥ 10 cells). I) Cells elongate along the main axis of swelling (indicated by red arrows). Composite fluorescence images showing F‐actin (green) and nuclei (blue) (scale bar, 100 µm) (XZ plane shown). Hydrogel swelling in (F–I) was controlled by the hydrophilicity of the crosslinker peptide. All data are presented as a mean ± s.d. except for (E) as box‐and‐whisker plots (box, 25–75%; bar‐in‐box, median; whiskers, the largest or smallest point comprised within 1.5× of the interquartile range from both edges).
Source publication
While matrix stiffness regulates cell behavior on 2D substrates, recent studies using synthetic hydrogels have suggested that in 3D environments, cell behavior is primarily impacted by matrix degradability, independent of stiffness. However, these studies did not consider the potential impact of other confounding matrix parameters that typically co...
Citations
... The phosphopaxillin focal adhesion structures of osteoblasts in the 3D nanofibrous gelatin scaffolds were less than on 2D substrates [5], of which typical focal adhesion structure formations are rare in vivo [2,6]. Furthermore, cells within 3D hydrogelbased matrices did not exhibit large focal adhesions [7][8][9]. Third, for some cases, their functional contribution to the . Transverse cryostat craniofacial mesenchyme sections of an E13.5 mouse embryo (F-J). ...
In tissue engineering, the mechanical properties of the extracellular matrix (ECM) or scaffolds have increasingly been considered to impact therapeutic efficacy by regulating cell behaviors, including differentiation, proliferation, migration, and adhesion. However, the understanding of how cells sense, integrate, and convert the mechanical cues from the ECM cues into biochemical signals to control certain cell behaviors is still elusive, especially in 3D, which more closely mimics the natural microenvironment than 2D systems. This review highlights the key differences between 2 and 3D in the contexts of mechanoregulative cell behaviors such as cell adhesion, spreading, migration, and force transmission. Furthermore, critical designing factors that needs to be considered for the fabrication of 3D tissue engineering scaffolds is discussed: stiffness, viscoelasticity, degradability, and the immobilization of biomolecules. Although mechanotransduction in 3D is actively being studied, understanding cellular mechanotransduction in 3D and designing of mechanoregulative 3D scaffolds still presents several challenges, including varying mechanical properties depending on different tissues, dynamic mechanical environments, and integration of multimodal cues. Interdisciplinary methodologies encompassing material engineering, cell biology, and mechanical engineering would serve to mitigate these challenges and augment our understanding of mechanoregulation governing cellular behaviors, thus fostering advancements in biomedical applications in the future.
... 21−29 Indeed, hydrogel stiffness controls cell spreading and underlying biological function. 26,30,31 More recently, however, many groups are realizing that the native ECM is a viscoelastic material that continuously remodels according to cellular behavior, 32−35 which is reflected in the many artificial hydrogels that are being developed, which are dynamic and have remodeling capabilities. 4,27,36,37 The dynamic nature of the hydrogels can arise from matrix degradation 4,36 or from the dynamic nature of the bonds used. ...
... Hydrogels generated through thiol-ene coupling, the reaction of thiol residues with alkenes, are increasingly used for the encapsulation of cells within 3D hydrogels and the design of soft materials for drug release [1][2][3] . This enables to achieve a broad control of physico-chemical properties that, in turn, can regulate cell phenotype [4][5][6][7] , the delivery of therapeutics 8,9 and repair of soft tissues [10][11][12][13] . Indeed, the excellent control of crosslinking through Michael addition and radical mechanisms, even in the presence of air and in physiological conditions, allows the control of mechanical properties of resulting hydrogels whilst enabling the incorporation of a broad range of eukaryotic cells with excellent viabilities 4, 14-17 . ...
The formation of hybrid hydrogel-elastomer scaffolds is an attractive strategy for the formation of tissue engineering constructs and microfabricated platforms for advanced in vitro models. The emergence of thiol-ene coupling, in particular radical-based, for the engineering of cell-instructive hydrogels and the design of elastomers raises the possibility of mechanically integrating these structures, without relying on the introduction of additional chemical moieties. However, the bonding of hydrogels (thiol-ene radical or more classic acrylate/methacrylate radical-based) to thiol-ene elastomers and alkene-functional elastomers has not been characterised in detail. In this study, we quantify the tensile mechanical properties of hybrid hydrogel samples formed of two elastomers bonded to a hydrogel material. We examine the impact of radical thiol-ene coupling on the crosslinking of both elastomers (silicone or polyesters) and hydrogels (based on thiol-ene crosslinking or diacrylate chemistry), and on the mechanics and failure behaviour of resulting hybrids. This study demonstrates the strong bonding of thiol-ene hydrogels to alkene-presenting elastomers with a range of chemistries, including silicones and polyesters. Overall, thiol-ene coupling appears as an attractive tool for the generation of strong, mechanically integrated, hybrid structures for a broad range of applications.
Neural tissue engineering has emerged as a promising field that aims to create functional neural tissue for therapeutic applications, drug screening, and disease modelling. It is becoming evident in the...
Tissue homeostasis and disease states rely on the formation of new blood vessels through angiogenic sprouting, which is tightly regulated by the properties of the surrounding extracellular matrix. While physical cues, such as matrix stiffness or degradability, have evolved as major regulators of cell function in tissue microenvironments, it remains unknown whether and how physical cues regulate endothelial cell migration during angiogenesis. To investigate this, a biomimetic model of angiogenic sprouting inside a tunable synthetic hydrogel is created. It is shown that endothelial cells sense the resistance of the surrounding matrix toward proteolytic cleavage and respond by adjusting their migration phenotype. The resistance cells encounter is impacted by the number of covalent matrix crosslinks, crosslink degradability, and the proteolytic activity of cells. When matrix resistance is high, cells switch from a collective to an actomyosin contractility‐dependent single cellular migration mode. This switch in collectivity is accompanied by a major reorganization of the actin cytoskeleton, where stress fibers are no longer visible, and F‐actin aggregates in large punctate clusters. Matrix resistance is identified as a previously unknown regulator of angiogenic sprouting and, thus, provides a mechanism by which the physical properties of the matrix impact cell migration modes through cytoskeletal remodeling.
Effective tendon regeneration following injury is contingent on appropriate differentiation of recruited cells and deposition of mature, aligned, collagenous extracellular matrix that can withstand the extreme mechanical demands placed on the tissue. As such, myriad biomaterial approaches have been explored to provide biochemical and physical cues that encourage tenogenesis and template aligned matrix deposition in lieu of dysfunctional scar tissue formation. Fiber‐reinforced hydrogels present an ideal biomaterial system toward this end given their transdermal injectability, tunable stiffness over a range amenable to tenogenic differentiation of progenitors, and capacity for modular inclusion of biochemical cues. Here, tunable and modular, fiber‐reinforced, synthetic hydrogels are employed to elucidate salient microenvironmental determinants of tenogenesis and aligned collagen deposition by tendon progenitor cells. Transforming growth factor β3 drives a cell fate switch toward pro‐regenerative or pro‐fibrotic phenotypes, which can be biased toward the former by culture in softer microenvironments or inhibition of the RhoA/ROCK activity. Furthermore, studies demonstrate that topographical anisotropy in fiber‐reinforced hydrogels critically mediates the alignment of de novo collagen fibrils, reflecting native tendon architecture. These findings inform the design of cell‐free, injectable, synthetic hydrogels for tendon tissue regeneration and, likely, that of a range of load‐bearing connective tissues.
This article is protected by copyright. All rights reserved
Matrix remodeling plays central roles in a range of physiological and pathological processes and is driven predominantly by the activity of matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) proteins. How MMPs regulate cell and tissue dynamics is not well understood as in vivo approaches are lacking and many in vitro strategies cannot provide high‐resolution, quantitative measures of enzyme activity in situ within tissue‐like 3D microenvironments. Here, a Förster resonance energy transfer (FRET) sensor of MMP activity is incorporated into fully synthetic hydrogels that mimic many properties of the native ECM. Fluorescence lifetime imaging is then used to provide a real‐time, fluorophore concentration‐independent quantification of MMP activity, establishing a highly accurate, readily adaptable platform for studying MMP dynamics in situ. MCF7 human breast cancer cells encapsulated within hydrogels are then used to detect MMP activity both locally, at the sub‐micron level, and within the bulk hydrogel. This versatile platform may find use in a range of biological studies to explore questions in the dynamics of cancer metastasis, development, and tissue repair by providing high‐resolution, quantitative, and in situ readouts of local MMP activity within native tissue‐like environments.
Hydrogels with tailor‐made swelling‐shrinkable properties have aroused considerable interest in numerous biomedical domains. For example, as swelling is a key issue for blood and wound extrudates absorption, the transference of nutrients and metabolites, as well as drug diffusion and release, hydrogels with high swelling capacity have been widely applicated in full‐thickness skin wound healing and tissue regeneration, and drug delivery. Nevertheless, in the fields of tissue adhesives and internal soft‐tissue wound healing, and bioelectronics, non‐swelling hydrogels play very important functions owing to their stable macroscopic dimension and physical performance in physiological environment. Moreover, the negative swelling behavior (i.e., shrinkage) of hydrogels can be exploited to drive noninvasive wound closure, and achieve resolution enhancement of hydrogel scaffolds. In addition, it can help push out the entrapped drugs, thus promote drug release. However, there still has not been a general review of the constructions and biomedical applications of hydrogels from the viewpoint of swelling‐shrinkable properties. Therefore, this review summarizes the tactics employed so far in tailoring the swelling‐shrinkable properties of hydrogels and their biomedical applications. And a relatively comprehensive understanding of the current progress and future challenge of the hydrogels with different swelling‐shrinkable features is provided for potential clinical translations.
Mechanics, as a key physical factor which affects cell function and tissue regeneration, is attracting the attention of researchers in the fields of biomaterials, biomechanics, and tissue engineering. The macroscopic mechanical properties of tissue engineering scaffolds have been studied and optimized based on different applications. However, the mechanical properties of the overall scaffold materials are not enough to reveal the mechanical mechanism of the cell–matrix interaction. Hence, the mechanical detection of cell mechanics and cellular-scale microenvironments has become crucial for unraveling the mechanisms which underly cell activities and which are affected by physical factors. This review mainly focuses on the advanced technologies and applications of cell-scale mechanical detection. It summarizes the techniques used in micromechanical performance analysis, including atomic force microscope (AFM), optical tweezer (OT), magnetic tweezer (MT), and traction force microscope (TFM), and analyzes their testing mechanisms. In addition, the application of mechanical testing techniques to cell mechanics and tissue engineering scaffolds, such as hydrogels and porous scaffolds, is summarized and discussed. Finally, it highlights the challenges and prospects of this field. This review is believed to provide valuable insights into micromechanics in tissue engineering.
Matrix mechanics regulate essential cell behaviors through mechanotransduction, and as one of its most important elements, substrate stiffness was reported to regulate cell functions such as viability, communication, migration, and differentiation. Neutrophils (Neus) predominate the early inflammatory response and initiate regeneration. The activation of Neus can be regulated by physical cues; however, the functional alterations of Neus by substrate stiffness remain unknown, which is critical in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional (3D) culture system made of hydrogels was developed to explore the effects of varying stiffnesses (1.5, 2.6, and 5.7 kPa) on the states of Neus. Neus showed better cell integrity and viability in the 3D system. Moreover, it was shown that the stiffer matrix tended to induce Neus toward an anti-inflammatory phenotype (N2) with less adhesion molecule expression, less reactive oxygen species (ROS) production, and more anti-inflammatory cytokine secretion. Additionally, the aortic ring assay indicated that Neus cultured in a stiffer matrix significantly increased vascular sprouting. RNA sequencing showed that a stiffer matrix could significantly activate JAK1/STAT3 signaling in Neus and the inhibition of JAK1 ablated the stiffness-dependent increase in the expression of CD182 (an N2 marker). Taken together, these results demonstrate that a stiffer matrix promotes Neus to shift to the N2 phenotype, which was regulated by JAK1/STAT3 pathway. This study lays the groundwork for further research on fabricating engineered tissue mimics, which may provide more treatment options for ischemic diseases and bone defects. STATEMENT OF SIGNIFICANCE.
