Stimuli-responsive hydrogels for manipulation of cell microenvironment: From chemistry to biofabrication technology
Graphical abstract
Introduction
Cellular microenvironment plays a crucial role in the regulation of cell behavior. Cells reside within intricate microenvironments consisting of extracellular matrix (ECM), soluble factors, and neighboring cells [1]. They interact dynamically with and reconstitute the surrounding ECM during development, tissue regeneration post-injury or disease, or to maintain tissue homeostasis [[2], [3], [4], [5], [6], [7]]. A variation of composition and structure of ECM by cell-mediated remodeling alters the presentation of signaling molecules and changes biophysical properties spatially and temporally [8]. Cell-mediated cleavage of proteolytic domains of ECM proteins by secretion of matrix metalloproteinases (MMPs), collagenases, plasmin, and elastases is critical for cell migration. For example, endothelial and tumor cell invasion in collagen is governed by activation of MMP-1 and MMP-8 collagenases [9,10]. Moreover, following injury, cells degrade the provisional fibrin matrix by MMP enzymes to allow deposition of new ECM proteins [7]. At the same time, cells also secret MMP inhibitors to regulate ECM degradation and remodeling in order to promote regeneration [11]. Under certain conditions such as chronic wounds, aging or certain diseases, the balance between ECM production and degradation is lost, resulting in overproduction of ECM, excessive ECM crosslinking, and cell contraction, ultimately leading to fibrosis and loss of tissue function [[12], [13], [14]]. For instance, excessive deposition of ECM by the fibroblasts of the heart may result in myocardial fibrosis, the hallmark of hypertrophic cardiomyopathy, a well-known disease that causes arrhythmias and heart failure [15]. Also, matrix overproduction and stiffening during breast cancer progression promote integrin clustering and tumor invasion [16,17].
Besides temporal modulation of ECM stiffness, the spatial physical gradients of ECM properties (e.g. stiffness, porosity, and topography) also play a significant role during tissue development, disease progression, and wound healing [13]. For instance, stiffness gradients at the injured sites guide directional cell migration (durotaxis) to promote wound healing [18]. Bone matrix is characterized by inherent porosity gradients, spanning from compact (5–30% porosity) to spongy (30–90% porosity) structure [19]. In addition, biophysical forces such as shear stress and strain experienced by blood and vascular cells due to blood flow in vasculature; cyclic tensile stress and strain experienced by cardiac and lung cells; and dynamic compressive stress acting on bone and cartilage cells due to body movement are critical determinants of cell behavior [13]. For example, the application of tensile stress and strain promotes migration of fibroblast and endothelial cells [[20], [21], [22], [23]], differentiation of myoblast into myotubes [24,25], maturation of cardiomyocytes [26], and differentiation of mesenchymal stem cells into smooth muscle cell lineages, [27] while compressive stress induces mesenchymal stem cell differentiation into chondrocytes [28].
Similar to physical cues, biochemical cues such as soluble (e.g., chemokines, cytokines, and growth factors) or insoluble signals (e.g., binding domains of ECM) are constantly regulated spatially and temporally by cells to enable specific cell behavior such as migration, proliferation, and differentiation [7]. For example, in vivo differentiation of human mesenchymal stem cells toward chondrocytes is associated with downregulation of fibronectin (FN) at day 7–12 of differentiation by upregulation of FN-cleavage enzyme, MMP-13 [29]. In addition, the concentration gradient of soluble factors is well-regulated in space and time by on-demand release and sequestration [30]. For instance, vascular endothelial growth factor (VEGF) has been demonstrated to promote endothelial cell proliferation, while VEGF concentration gradient guides the directional growth of vessels toward hypoxic sites [31]. Moreover, chemical gradients of morphogens such as hedgehog, bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β), and fibroblast growth factors (FGFs) control differentiation during embryogenesis [19,32,33]. Similarly, chemical gradients regulate the direction of axonal growth [34], as well as the migration of leukocytes and fibroblasts to sites of injury [19,35]. Collectively, a plethora of biological studies suggest that, in order to mimic the cell microenvironment and engineer tissues that recapitulate the structure and function of their native counterparts, it is necessary to engineer biomaterials whose biophysical and biochemical cues can be modulated in space and time.
The progress toward understanding cell-ECM interaction has long been devoted to studying the cellular behavior in preprogrammed static three-dimensional (3D) scaffolds, which are designed to mimic certain biophysical and biochemical features of native ECMs. However, native ECMs are inherently heterogeneous and constantly undergo dynamic remodeling mediated by cells to enable specific cell event [8,[36], [37], [38]]. Therefore, traditionally developed 3D scaffolds with temporally homogeneous cues are unable to provide sustainable guidance to cells. Indeed, most of the reported 3D scaffolds direct cell fate only at specific time points, and thereafter the scaffolds lose their instructive features because the presented signals are no longer effective in guiding the cells at new time points.
Benefited from the advances in biorthogonal chemistries and the growing library of stimuli-responsive functionalities, there has been a substantial paradigm shift in the design criteria of 3D scaffolds. Accordingly, development of stimuli-responsive scaffolds has attracted increasing attention, because they can emulate to a great extent the dynamic nature of native ECMs by undergoing unidirectional or cyclical structural changes with physiologically benign stimuli. Moreover, stimuli-responsive scaffolds can permit user-defined spatiotemporal modulation of biophysical and biochemical cues to direct cell behavior, paving the way to understand complex interdependent cell signaling. In addition, integration of stimuli-responsive materials with the state-of-art biofabrication technologies has enabled engineering of complexity of cell microenvironment over multiple length scales, and opened new avenues toward the development of functional tissue-like replacements with clinically relevant importance. Furthermore, scaffolds with tunable sensitivity to pH, temperature, light, mechanical, and electrical stimuli have been widely exploited for sustainable and on-demand delivery of bioactive cues to cells, as well as the generation of biochemical gradients to spatially regulate cell growth. Finally, reversible modulation of biophysical cues, such as mechanical stiffness and stress/strain behavior, has also been achieved using stimuli-responsive 3D platforms.
Stimuli-responsive hydrogels have received great attention in cell biology and tissue engineering (TE) fields because of their capability to change their physical and chemical properties in response to user-defined stimuli, allowing modulation of cell microenvironment. Moreover, hydrophilicity and unique physical properties of hydrogels permit diffusion of oxygen, nutrients, and bioactive molecules. In addition, hydrogels are crosslinked macromolecules that retain large amounts of water, thereby acting as reservoirs of signaling molecules that influence cell fate [39,40]. Hydrogels can be classified into two major categories: chemically crosslinked hydrogels in which polymer chains are crosslinked through covalent bonds; and physically crosslinked hydrogels in which 3D structures are formed via weak forces like hydrogen bonding, hydrophobic and ionic interactions, metal-ligand coordination, host-guest intercalation, and stereocomplexation [41]. The materials used in stimuli-responsive hydrogel formation can be natural, synthetic or hybrids of natural and synthetic. The most common natural materials used for hydrogel formation include polysaccharides (such as chitosan, alginate, hyaluronic acid (HA), dextran, and agarose) and proteins (such as collagen, fibrin, elastin, and gelatin) [42]. Natural materials are usually biocompatible and biodegradable, containing biological signals that affect cell behaviors; however, they typically possess poor mechanical properties, with batch-to-batch variability. Importantly, protein-based materials exhibit a complex myriad of bioactive functionalities, resulting in difficulties in defining which bioactive signal elicits a specific cell response [43]. In contrast, synthetic materials offer reproducible and tunable physical, chemical and mechanical properties, but lack biological cues that promote tissue formation and regeneration [44]. Therefore, combining natural and synthetic materials is an intriguing research area that may open avenues toward fabrication of biomimetic scaffolds with tunable mechanical and biological properties [45].
Integration of stimuli-responsive materials with current biofabrication techniques including lithography, micromolding, microcontact printing, 3D bioprinting modalities, and textile fabrication methods have emerged as promising tools for the construction of 3D scaffolds that mimic the architecture of native tissues [[46], [47], [48], [49]]. Thus, such integration provides an opportunity to manipulate the structural and organizational features of scaffolds to direct cell behavior. Some of these techniques can be linked to imaging modalities to reveal recreated tissue architecture [50,51]. Recent studies have demonstrated the development of 3D constructs with organizational features similar to aortic valve, ear, skin, and blood vessels [52]. However, significant efforts are still needed to improve the spatial resolution and vertical buildup of the scaffolds. Furthermore, the development of new biomaterials that meet the requirement of biofabrication techniques, such as proper viscosity and rapid crosslinking process, is also important.
The main goal of this review is to summarize the state-of-art stimuli-responsive hydrogels that recapitulate critical features of native ECMs and can be modulated with external and/or internal stimuli to alter cell microenvironment, thereby enabling real-time manipulation of cellular response as well as revealing cell-directing cues. The review highlights the multifaceted perspectives of the stimuli-responsive hydrogel, spanning from biomaterials chemistry to biofabrication technology. Specifically, the Introduction section highlights ECM dynamics and the importance of stimuli-responsive hydrogels in studies to understand complex cellular processes. Then, the next section summarizes the chemistry toolsets employed in fabricating chemically and physically crosslinked scaffolds, as well as imparting bioactivity to the design of the scaffolds. Subsequently, the third section discusses various categories of stimuli-responsive hydrogels including pH-, thermo-, photo-, electro, mechano-responsive platforms, with particular emphasis on the stimuli-responsive functionalities and structure-property relationship. Afterward, the fourth section highlights the vital role of stimuli-responsive hydrogels in the modulation of biochemical and biophysical cell-directing cues. Manipulation of cell attachment using stimuli-responsive two-dimensional (2D) substrates, and controlled modulation of structural, mechanical, and biochemical characteristics in stimuli-sensitive 3D platforms, are reviewed in this section. The fifth section highlights the integration of stimuli-responsive hydrogels with recent biofabrication technologies including microcontact printing, micromolding, lithography, textile fabrication technologies and bioprinting modalities, for building stimuli-responsive constructs in vitro with geometrical and dynamic features similar to native tissue. Given the multidisciplinary nature of the research involving stimuli-responsive scaffolds, it is impossible to include a discussion with specific details on every aspect of such stimuli-responsive platforms in this review.
Section snippets
Hydrogels
Native ECMs are mainly composed of two components: 1) proteins, such as collagen, laminin, fibronectin, and elastin; and 2) glycosaminoglycans (GAGs), such as sulfated heparin, chondroitin, and keratin, which bind to the protein backbone to form proteoglycan [53]. The negatively charged GAGs fill the interstitial spaces within ECMs and sequester soluble signaling molecules (e.g., growth factors) via non-covalent ionic interaction and hydrogen bonding [54]. In addition, ECMs incorporate cell
Stimuli-responsive hydrogels
Stimuli-responsive scaffolds are defined as scaffolds that undergo significant physical or chemical changes upon small alteration of external stimuli or changes within their environment. Depending on the magnitude of the stimulus and the response sensitivity, several kinds of physical, chemical, and biological stimuli have been employed to trigger the changes within scaffolds [139]. Bond cleavage, bond formation, swelling/deswelling, and conformational changes are the most common responses. In
Modulation of cell microenvironment using stimuli-responsive scaffolds
Thus far, we have provided an overview of chemical and physical strategies employed in hydrogels formation, as well as the chemical toolset of stimuli-responsive functionalities that brings up dynamicity to the scaffold structures. In this section, how stimuli-responsive scaffolds are being utilized to modulate the biophysical and biochemical cell-directing cues in space and time will be elucidated.
Integration of stimuli-responsive scaffolds with biofabrication technologies
Native ECMs composed of highly organized multi-cellular structures over multiscale sizes, spanning from nanoscale and mesoscale to macroscale dimensions [311]. Thus, engineering of cell microenvironment necessitates not only endowing dynamicity to the scaffolds, but also emulating the structure organization of native tissues. Unlike the random distribution of cells within scaffolds that lack structural arrangement, coupling the structural organization capacity of biofabrication technology with
Conclusions and future perspectives
Hydrogel-based scaffolds with stimuli-responsive behavior have attracted significant interest in the development of dynamic 3D constructs, which allow spatiotemporal modulation of physical and biological cues, as well as real-time monitoring of cell behavior. This review highlights the intriguing role of stimuli-responsive hydrogels in on-demand manipulation of cell-directing cues reversibly and irreversibly. It also emphasizes the integration of stimuli-responsive biomaterials with advanced
Acknowledgements
Mohamed Alaa Mohamed acknowledges the Egyptian Ministry of Higher Education and Scientific Research for supporting him during his PhD study in University at Buffalo.
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