A partial list dynamic substrates currently used in studying cell responses.
Therefore, this review is focused on the latest studies and current knowledge of two- or three- dimensional substrates with changing or dynamic mechanical and adhesive properties, design and conditions to trigger and achieve designed dynamics, and the impact of in-situ change of these properties on cell behavior, which provides guideline for design of biomaterials for their applications in medical and healthcare applications. Note that mechanical stiffness and elastic modulus were used interchangeably in this work.
2. Dynamic nature of the cell microenvironment
The recurring indication from the above discussions is that
3. Types of dynamic substrates and stimuli
Here we consider two major classes of dynamic substrates that are based on self-assembled monolayer, or SAM, and hydrogels, as well as other types of substrates with surface or structural modifications. Since the focus of this work is on mimicking dynamic nature of ECM to examine cellular responses, those dynamic materials that are developed for other specific applications such as drug delivery  and do not involve changes that mimic dynamic ECM are beyond the scope of the review.
3.1. Self-assembled monolayer (SAM)
SAMs are formed by adsorption of molecules in solution or gas phase onto substrates in a spontaneous and organized fashion , and have emerged as an important candidate of materials in studying cellular responses to dynamic substrates [60, 61] where modifications could be made
|SAM incorporating O-silyl hydroquinone moiety||Adhesion on/off||3T3 fibroblasts||Modulation of cell adhesion and migration||[17, 61, 65]|
|Electro-active quinine monolayer on Au||Adhesion on/off||3T3 fibroblasts||Selective release of adherent cells|||
|Azobenzene containing SAM on Au||Adhesion on/off||3T3 fibroblasts||Attachment and release of adherent cells|
Potential to control part of a single cell or groups of cells
|MMP responsive polymer hydrogel network||Degradation of hydrogel||Human foreskin fibroblasts (HFFs)||Cell infiltration into the gel network with time|||
|Thermo-responsive polymer with photosensitive surface||Adhesion on/off||CHO-K1 cells||Reversible control over cell adhesion|
Ability to control a population of cells
|poly(NIPAM-co-sodium acrylate) hydrogel films on rigid substrates.||Topographic change (swelling/de-swelling of gels)||Porcine epithelial cells||Dynamic patterned substrates|
Reversible encapsulation of adherent cells
|DNA crosslinked PAM gels||Crosslinking density↑|
Mechanical stiffness ↑, vice versa
|L929 & GFP fibroblasts||On dynamic substrate, L929 cells spread more than those on static stiff substrates (~23 kPa), while GFP fibroblasts respond differently to stiffening and softening of substrates|
Cell spreading and polarity (aspect ratio) respond differently to stiffness dynamics
The range, starting point, and end point of change matter
|DNA crosslinked PAM gels||Crosslinking density↓||Primary spinal cord cells||Neurite outgrowth respond to dynamic stiffness|
The trend in the response match that to the static stiffness except for primary dendrite length
|HA hydrogel||Crosslinking density change and ECM deposition|
Mechanical stiffness change
|human mesenchymal stem cells (hMSCs)||Mechanical properties can be engineered with degradation|
Stiffness ↑ when degradation equals ECM deposition, and Stiffness ↓ at rapid degradtion
Cellular responses to dynamic stiffness are different from static gels with the same initial or ending conditions
|Methacrylated HA hydrogel||UV exposure||hMSCs||Fate of hMSCs differentiate depends on the dynamics of stiffness change of substrates|
Adipogenic differentiation favored when cells is on the softer substrate long (stiffening at later times)
Osteogenic differentiation when cells are on the stiffer substrate (stiffening at early times).
|Hydrogel based on PAM crosslinked by photosensitive reagent||Mechanical Stiffness (global or local)↓||3T3 fibroblasts||Stiffness decrease of 20-30% upon propose UV radiation|
Global stiffness decrease results in less spreading
Localized softening to anterior and posterior area gives to differential responses
|PEG based hydrogel with photosensitive crosslinker||Mechanical Stiffness↓|
Adhesive property change
Valvular inter-stitial cells (VICs)
|Valvular cell differentiation into myo-fibroblasts is inhibited by softening|
Good viability of hMSCs
|Piezo-controlled substrate and AFM cantilever||Mechanical stiffness with cycling changes||NIH 3T3||Apparent stiffness↑ leads to cells contraction rate↑ and contraction velocity↓|
Changes took place instantaneously, and so did responses
Responses were reversible, and consistent for same cell.
|Photo-active glass substrate with modifications||Adhesion on/off||HEK293, COS, NIH 3T3||Spatio-temporal control over cell adhesion|
Single cell control
|Substrates with photo-responsive caged peptides||Adhesion on/off||3T3 fibroblasts||Modifications of non-adhesive surfaces to adhesive ones|||
|PEG-modified ITO microelectrodes on glass substrates||Adhesion on/off||HepG2 (hepatic) and 3T3 cells, co-culture||Micro-patterned co-culture made possible|||
|Photo-crosslinked alginate hydrogel||Stiffness change;||Primary bovine chondrocytes||High survival rate for primary bovine chondrocytes|
Cellular responses to dynamic changes to be studied
|Gellan Gum hydrogel with both ionic crosslinking and||Stiffness, swelling, and degradation change||NIH 3T3||Swelling and hydrolytic degradation vary with respect to crosslinking mechanism|
Stiffness may be changed quickly during photo-crosslinking process
|Methacrylated HA hydrogel with photo-crosslinker||Stiffening||NIH3T3L|
|Good cell viability|
Cellular responses to dynamic changes to be studied
|PEG-based hydrogel incorporating CMP*||Softening||N/A||Cellular responses to dynamic changes to be studied|||
|PEG hydrogel (PEG vinyl sulfone crosslinked with PEG-diester-dithiol)||Softening||3T3 balb fibroblasts||Good cell viability in 3D gels|
Cellular responses to dynamic changes to be studied
|Resilin-like polypeptide (RLP) network crosslinked by THPP||Dynamic stiffness||N/A||Cellular responses to dynamic changes to be studied|||
|Thermo-reversible hydro-ferrogels (FGs)||Mechanical stiffness change||N/A||Cellular responses to dynamic changes to be studied|||
These stimuli, applied to initiate substrate dynamic, include light [62, 63], electricity [16, 17], pH, temperature, and others [16, 64] (Fig. 1). These approaches generally involve photochemical or electrochemical conversion, redox reactions, or stimulated configuration change of surface proteins, which leads to the attachment, detachment, shielding, or exposing of cell adhesion molecules, among which a popular choice is RGD peptide.
Mrksich group has been actively engaged in the development of SAM-based dynamic substrates by integrating surface chemistry, micro-patterning, and cell microenvironment engineering [17, 19, 61, 65, 66]. Based on an elegant design of SAM with electrochemically responsive group on a micro-patterned substrate, they first applied electrical stimulation to release 3T3 fibroblasts from designed areas on the substrate, and subsequently encouraged migration of neighboring cells to those areas with newly added adhesion molecules . Refining this design by adding responsiveness to both negative and positive electric potentials, they demonstrated selective control over cell release  (Fig. 1C). Other groups have also engaged in the effort along this direction. By employing a hydroquinone terminated SAM based on redox reactions, Chan and colleagues proposed a SAM on gold surface that enables attachment and release of cell adhesion molecules such as those with RGD motif , and selectively released adherent 3T3 fibroblasts bound through RGD motifs but not those adherent based on hydro-phobic interactions (Fig. 1A). Reversibility of cell adhesion is attractive in studying cellular responses and cell-ECM interactions . As an example, a surface chemistry involving azobenzene capable of switching between two configurations was utilized to expose or hide adhesion sites (e.g., RGD motif) upon photochemical stimulation (Fig. 1B). While the finding is interesting, the long exposure of cells to UV may be problematic despite the reported negligible impact of light with wavelength over 320 nm on cells .
The above surveys part of the key advancement using SAM in modifying adhesion properties of the substrates mimicking those of natural cellular microenvironment. For a complete analysis of SAMs and their various applications, readers are referred to other reviews (e.g., [60, 70]). It suffices to point out that SAMs possess advantages in the precision (down to molecular level) of the control that can be applied in mechanistic studies [60, 66] of cell-ECM interactions, and are potentially useful for cell-based diagnostics among many applications. However, this approach has certain limitations. First, it mostly relies on coupling between electrical, chemical (including pH), mechanical, thermal, optical and biochemical (e.g., protein conformation) cues whose applicability under
3.2. Polymeric hydrogels
Hydrogel materials are gaining popularity in the development of biomimetic materials, primarily due to the hydrated nature of natural ECM [14, 71]. Implantable hydrogel materials are increasingly being used in cardiovascular disease, nerve regeneration, and other conditions . With careful design, hydrogel materials can have tunable materials properties, which have been demonstrated in a myriad of examples (Table 1). For instance, different than SAM-based approach, a polymer with both thermo- and photo-sensitivity was used to reversibly control adhesion of a group of cells . Kim and colleagues took advantages of the thermo-responsive swelling behavior of copolymer between NIPAM and sodium acrylate, and created a hydrogel film that can be used to control cell encapsulation with surface topography . Moreover, biomaterials responsive to the natural stimuli such as those experienced by biodegradable materials were found useful in mimicking biological events under physiological conditions, as illustrated in cell invasion to a MMP-responsive hydrogel scaffold . This finding, among others, exemplifies the strategy of triggering material dynamic from bio-responsiveness to potential site- or disease-specific cues. The information from these studies is instrumental to the design of biodegradable materials in optimizing degradation profile for target cellular responses . Naturally, in order to achieve desired outcome in adopting these strategies, it is important to gain thorough understanding of the natural environment, and minimize risks associate with biodegradable materials such as premature degradation, and potential toxicity of intermediate products from degradation.
Using a popular polyacrylamide hydrogel culture system with modifications that impart it with photo-sensitivity, Wang and colleagues  showed that upon UV induced substrates softening, spreading of 3T3 fibroblasts was hindered in contrast to that under static conditions (Fig. 2A). More interestingly, localized softening at anterior and posterior of cells yielded differential cellular morphology and migration responses . Meanwhile, a PEG based polymer (PEGA) crosslinked by photosensitive crosslinker (PEGdiPDA) has been developed by Kloxin et al. , and used to lower gel stiffness upon UV exposure, which resulted in de-activation of myofibroblasts (Fig. 2B). Although UV radiation is preferentially avoided, these methods made possible high precision in applying changes of cellular mechanical microenvironment, and potentially allow creation of dynamic stiffness gradient.
Similar observations were made by Burdick group for human mesenchymal stem cells (hMSCs) by using hyaluronic acid (HA) hydrogel degradable from hydrolytic and enzymatic reactions  (Fig. 3A). Very recently, a new material platform has been constructed by this group  and others  where the stiffness of a methacrylated hyaluronic acid hydrogel is increased via addition of photo-initiator and UV light exposure. In response to elevated stiffness, human mesenchymal stem cells (hMSCs) spread more and exert greater traction forces in hours (almost one magnitude of difference), and the rate of stiffness elevation dictates fate of cell differentiation towards adipogenic (slower) or osteogenic (faster) lineage. Their work highlighted that cellular behavior on dynamic gels is not the same as that on static gels with same initial or final properties, underlining the significance of dynamics in gel properties. This has been echoed in the concurrent work , where, for instance, the fibroblasts on 100% crosslinked hydrogels demonstrated different morphology from that on 100% crosslinked gels modified from 50% gels. Therefore, it is conceivable that the previous state of the cells and their ECM is also among the determinants of their current state, and that time dimension of ECM is of great importance.
Factors other than environmental conditions (e.g., light, pH, temperature) can also be delivered to stimulate dynamics in substrate properties. Incorporating DNA as crosslinker, Jiang and colleagues have developed a hydrogel system for cell attachment where mechanical properties of the substrates can be altered
The same material system was employed by Previera and co-workers, and they firstly proved the dual mechanical stimuli, namely strain and stiffness drop, during the dynamic processes, and secondly contrasted cell behavior to stiffness decrease with that for hardening of the substrates . On hardening gels (from 12 kPa to 22 kPa), cells spread more than those on static substrates of higher stiffness (22 kPa), whereas on softening ones, they have greater spreading area than that on either starting or ending stiffnesses. In these studies, cell responses are determined by the range of rigidity change (due to crosslinking density), starting and ending rigidity, and specific cell properties (e.g., projection area vs. aspect ratio and protrusion for fibroblasts). The stress generation may also be involved in affecting cell behavior .
3.3. Other types of materials
The approach of employing polymeric hydrogel to study dynamic changes has certain limitations, one of which is the coupling of mechanical stiffness and forces (e.g., ). To address this concern and others, different from the approach by using SAM or polymeric hydrogel, AFM based method put forth by Webster and co-workers  probed cellular response to instant step change in stiffness excluding influence from stress or strain in the substrates (Table 1). It has been confirmed that indeed individual 3T3 fibroblasts are able to sense and respond to the stiffness in a scale of seconds as demonstrated in traction rate and contraction velocity . However, this approach is most likely with inherent limitation in mimicking natural cell environment while remains an interesting tool in probing cellular responses to instantaneously change in stiffness. Additionally, this approach is applicable mostly to cells with dynamic morphology.
Common cell culture substrates (e.g., glass coverslip) modified with common photo-cleavable agents (NPE-TCSP) were shown to be useful for controlling cell adhesion selectively and temporally . In this method, target areas were first irradiated to remove BSA known to prevent cell adhesion, and then pro-adhesive molecules (e.g., fibronectin) were added, followed by cell seeding. It is useful to study dynamics in interactions between single cells. Petersen et al.  used light to stimulate photosensitive surface modification resulting in uncovering of the RGD motif upon release of a caging group (Fig. 4A). In doing so, adhesion of 3T3 fibroblasts was first inhibited and then encouraged, although this process is not reversible. With a sequential activation of adhesive sites upon application of electric potential, a recent study  demonstrated the utility of substrates with ITO (indium tin oxide) microelectrodes modified with poly(ethylene glycol), or PEG, in co-culture of two cell types (hepatic cells and fibroblasts) in a controlled manner (Fig. 4B).
3.4. Promising substrates
By applying an oscillation to a resilin-like polypeptide network crosslinked by THPP (β-[Tris(hydroxymethyl) phosphino] propionic acid (betaine), Li and colleagues were able to observe dynamic mechanical stiffness of the gels varying with regard to oscillatory frequency mimicking the load from human vocal tissues  Oscillatory shear induced stiffening and softening of the collagen network might also serve as good substrates for mimicking cellular microenvironment particularly that in mechanically active tissues . Ion concentration may be another stimulus to allow for temporal modification of hydrogel properties as exchange of ions between monovalant and divalant cations , and further work is needed to confirm it. Temperature-dependent substrate softening has been demonstrated by Krekhova et al.  and 3D complex with temperature-mediated crosslinking hence mechanical properties has been proposed by Stahl et al. , while applicability of these systems in mimicking cellular microenvironment remains to be seen. Zustiak et al.  reported mechanical stiffness drop, from approximately 1 kPa and at different rate, along with degradation of a poly(ethylene glycol), or PEG, hydrogel which might serve as not only drug delivery vehicle but also biomaterial construct, and they have offered preliminary evidence of good viability of 3T3 balb fibroblasts on the hydrogel substrate. Similarly, rigidity change from ~180 kPa to tens of kPa in 3-week period of degradation from a photo-crosslinked alginate hydrogels based on alginate methacrylation were presented by Jeon et al , and cyto-toxicity has been found to be low. In summary, these substrates holds promises as substrates with modifiable properties
4. Design considerations and outlook
4.1. Dynamic properties of the substrates
As indicated in the discussions in Background and Motivation, the progression in changes of ECM properties is also critical in addition to changes
4.2. Potential effect on cell-cell interactions
Changes in adhesive or mechanical properties of ECM can stimulate cells, which, in response, secrete soluble factors and ECM molecules, and this further impacts neighboring cell types. Additionally, some cell types such as neurons may use other cells (e.g., astroglia) as substrate , and stiffness change of ‘underlying’ cells per se due to ECM stimulation may give rise to further alternations thanks to cell-cell interactions. For instance, during asthma, ECM stiffening contributes to stiffness increase of airway smooth muscle (ASM) cells, which potentially affects other cell types in the close proximity .
4.3. Design parameters for biomaterials and outlook
The design parameters of dynamic substrates from current studies are summarized in Table 2, which includes, but are not limited to, material system to consider (e.g., SAM or polymeric hydrogel materials), nature of change (mechanical stiffness or adhesion), rate of change (e.g., transient or gradual change, controllability of the rate of change), range of change (e.g., at different stiffness range) as well as potential issues in further investigations and applications to medical and healthcare applications. If the interest is in understanding the cellular behavior to mechanical stiffness alone, then an AFM based approach might be more attractive  as others will involve stress or strain as part of the stiffening or softening process. If precise control over stiffness range is desired, the DNA crosslinked PAM hydrogel system will serve the purpose better [20, 58, 81]. Polymeric hydrogel materials with controllable degradation profile and hence mechanical stiffness dynamics during degradation (e.g., ) will serve the purpose best when biodegradable materials are applied. Some of the material systems do offer unique benefits such as reversible property change or without using environmental factors (by applying oscillation, crosslinker, or ssDNA).
Meanwhile, there are inherent limitations to each of the material system under discussion (Table 2). UV exposure generally causes concern to its impact on cellular activities despite the findings of little impact from a number of studies based on a range of biological assays. Under physiological conditions, application of certain cues (e.g., ssDNA, light, or ion) might be too difficult or it might be greatly limited (e.g., temperature triggered changes). However, it is still possible to find ways to apply these cues with careful design. For instance, ssDNA design based on pre-screening using BLAST search against targeted specie or tissue type may minimize the chance of interfering with normal biological activities. Local heating/cooling may be carefully applied to induce dynamic changes to achieve cellular responses. Three dimensional system may better mimic natural cellular micro-environment than their 2D counter parts.
|Ion||Ion-crosslinked GC hydrogel||Stiffness||~22 to ~17 kPa (with chemical crosslinking)||N/A||Under physiological conditions, divalent ions exchanged by mono-valent ones|||
|Light||Hydrogels based on PAM crosslinked by photosensitive agents||Softening||Stiffness: 5.5~7.2 kPa||Approximately 0.5~0.6 kPa/ min||UV exposure for 3 min|
UV radiation with low energy density
Depth of penetration and limit on dose
|Methacrylated HA hydrogel||Stiffening|
|Stiffness: ~3 to ~30 kPa||Approximately 9 kPa/hr (short term); 2 kPa/day (long-term)||UV exposure for a few min|
Potential toxicity of photoinitiator
Depth of penetration and limit on dose
|Photo-crosslinked methacrylated Gellan Gum hydrogel||Stiffness; Swelling|
|Stiffness: a few kPa to 22 kPa (by physical crosslinking)||Approximately 20 kPa/ min||UV exposure for one min|
Depth of penetration and limit on dose
|Methacrylated HA hydrogel with photo-crosslinker||Stiffness;|
|Stiffness: 1.6 to 3.8 kPa; 3-12 kPa||Approximately 0.1 or 0.3 kPa/min (during gelation)||UV exposure for a few min|
Potential toxicity of photo-initiator
Depth of penetration and limit on dose
|PEG based hydrogel with photosensitive crosslinker||Stiffness↓|
|N/A||N/A||Depth of penetration and limit on dose|||
|DNA||DNA crosslinked PAM system||Stiffening & softening, potentially coupled with strain/stress|
|Stiffness: ~5.9 to 22.9 kPa|
Stress > 0.5 Pa
|Up to 8.5 kPa/ day||No differentiation in cellular responses between forces, stress, and stiffness|
Potentially interference from DNA with bio-activity (e.g., as anti-sense DNAs), and potential issue with DNase
BLAST search against target specie & tissue type
|[20, 81, 83]|
|AFM/ stiffness clamp||Piezo-controlled substrates and AFM stiffness clamp||Instantaneous change in stiffness|
|Stiffness: 3.6 to 90 nN/µm||Step change (instantane-|
|Applicable only to cells with dynamic morphology|||
|Hydro-lysis||Photo-crosslinked alginate hydrogel||Softening due to degradation||Stiffness: ~25 to ~180 kPa||7-8 kPa/ day||In sample preparation (with cells), UV exposure for 10 mins|||
|HA hydrogel||Stiffening & structure change||Stiffness: e.g., ~5 to 30 kPa for one case||0.7 kPa/ day||Dense crosslinking may impede cellular growth limited by diffusion& concentration of radicals|||
|PEG hydrogel (PEG vinyl sulfone crosslinked with PEG-diester-dithiol)||Softening due to degradation||Stiffness: from ~1 kPa-3 kPa to very low||From ∼900 Pa/day to 500 Pa/day||Good cell viability|
Hydrogel degraded in 16 hours
|Temp-erature||Thermo-reversible hydro-ferrogels (FGs)||Stiffening due to structural transition||Stiffness: ~28-24 kPa for 2ºC change at 37ºC||A few kPa for 1ºC of temperature change||Temperature change needs to be defined to be relevant to cell culture|||
|PEG-based hydrogel incorporating CMP*||Stiffness change|
Due to temperature and free CMP
|Stiffness (indirect measurement)||N/A||Temperature change needs to be defined to be relevant to cell culture|
Bio-compatibility of free CMP
|Oscilla-tion||Resilin-like polypeptide based elastomer||Stiffness change due to oscillation||Storage modulus between 0.5 and 10 kPa||Highly dynamic||Strain/stress that is associated with oscillation|||
A few new material system have been identified with the potential as dynamic cell culture platform as well as choice of biomaterials (Table 1). Many of them have demonstrated good cyto-compatibility, and investigation of impact of
5. Concluding remarks
There is an increasing recognition of the discrepancy between static nature of the current cell culture substrates or scaffolds and the dynamics in ECM in natural or diseased tissue, during development and aging, or at tissue-scaffold interfaces. This has motivated the development of materials with controlled changing properties that mimic those of ECM. An array of stimuli, including environmental factors (temperature, pH, light, electrical potential) and non-environmental cues including enzyme and DNA, have been implemented to trigger dynamics in a number of material platform such as SAMs, polymeric hydrogels and other substrates with surface chemistry and modifications.
To date, most of the effort along this line has been devoted to
Understanding the interplay between cells and the extracellular matrix (ECM) including its dynamic aspect is fundamental to biology, development, aging and pathology, and can aid in the design of biomaterials. Ultimately, the system enabling both spatial and temporal control  of cells would be most relevant in terms of bio-mimicry and tissue engineering applications. Some of the potential directions include creating dynamic adhesive gradient to guide cell migration or neurite outgrowth at desired time point, constructing scaffolds with suitable mechanical rigidity to inhibit glia cell growth (thus hinder scar formation) while promoting nerve regeneration with compliance gradient, and developing dynamic platform for stem cell harvesting and differentiation for cell-based therapies.
The helpful discussions and advice from Langrana group at Rutgers University, New Jersey, USA as well as previous collaborators are greatly appreciated.
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