**2. Dynamic nature of the cell microenvironment**

**Normal tissues** The micro-environment within which cells reside in natural tissues under‐ goes constant synthesis and degradation [16, 17], and has long been recognized as dynamic and changing [25, 28]. Although the composition of tissues generally remain tightly control‐ led in maturation, ECM remodeling constantly takes place [25, 29, 30], particularly when un‐ der hormonal stimulation or stress responses (e.g., [31]). Cells actively participate in tissue remodeling by secreting and mobilizing ECM molecules [32]. Alterations in ECM composi‐ tion may result in changes in cell adhesion and /or tissue stiffness [33, 34] which further stimulates cellular responses. For instance, laminin component involved in cell adhesion to ECM is variant due to dynamics in exogenous factors [35]; normal cartilage shows elevated stiffness [36]; and vocal fold tissue exhibits dynamic viscoelastic properties [37]. Some speci‐ alized cell types can experience fast adhesion and detachment from ECM [38]. Changing ECM can also modify cell-cell interactions, further affecting cell behavior [39].

**Pathological tissues** Diseased tissue may possess properties such as mechanical stiffness different from those of the normal tissue [40, 41]. As a typical example, it has been found that tumor cells display enhanced movement towards ECM with lower mechanical rigidity, which is interesting considering the general stiffening phenomenon of tumor tissues [42], and biomechanical characteristics of tissues play a crucial role in tumor development [41]. It has also been shown that during the surgical procedures such as radio-frequency (RF) abla‐ tion, tissue properties can be modified [43]. Moreover, changes in ECM composition and rel‐ ative quantity of ECM molecules can be correlated to pathology. For instance, ECM composition change that occurs during sub-epithelial tissue remodeling proved associated with asthma [33]. ECM remodeling in diseased heart valves is correlated to myofibroblast contractility [44], and certain cell types such as valvular interstitial cells can be activated and contribute to further tissue remodeling [45]. Additionally, ECM remodeling affects tissue mechanical properties in addition to inflammatory responses [46]. Moreover, mechanical forces, as experienced in traumatic brain injury or even under normal conditions, could po‐ tentially cause protein aggregation, giving rise to various diseases including neurodegenera‐ tive diseases [47]. Furthermore, early investigation of properties of central nervous (CNS) tissue under impact yielded modulus values with considerable variation. As an example, Fallenstein and coworker reported storage modulus of human brain tissue of 0.6 ~ 1.1 kPa under sinusoidal shear stress input mimicking head impact [48].

especially for load-bearing and mechanically active tissues (e.g., heart, cartilage, lung) [15]. Not only do these changes naturally occur, but there are also benefits associated with them from a tissue engineering viewpoint, as highlighted in the series of discussions in the March 2005 issue of MRS (Materials Research Society) Bulletin [16, 17] and later studies. Whitesides [18] and Mrksich [19] and their coworkers among a number of investigators pioneered the work on engineering cell growth by using dynamic substrates. Their work and later reports on differential cell responses to materials with different properties suggest that it is benefi‐ cial for biomaterials to have controlled changing properties [20]. These facts make it very de‐ sirable for the bio-mimetic materials to have the capability of undergoing controlled remodeling with respect to time. They also raised caution in interpretation of the observa‐ tions made from the majority of the biological studies, where properties of the substrates (e.g., culture flask, Petri dish, and hydrogels) remain unchanged throughout the process. **The scope of this work** A significant number of reviews are available on the changes in soluble factors of ECM that may affect cellular behavior (e.g., [21]) and particularly on the changing environment in bioreactors [22] (e.g., nutrients concentration, oxygen level, tem‐ perature). Thus, they are not covered in this review. Moreover, flow conditions and the re‐ sulting traction forces, and their effect on certain cell types including blood cells (i.e., endothelial cells and red blood cells) have also been intensively reviewed and hence are not discussed here. This is also the case for mechanical forces, strain and stress applied directly to the cells (e.g., [23, 24]) in load-bearing tissues such as bone, cartilage and lung (for refer‐

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 proper‐ ties, design and conditions to trigger and achieve designed dynamics, and the impact of insitu change of these properties on cell behavior, which provides guideline for design of biomaterials for their applications in medical and healthcare applications. Note that me‐

**Normal tissues** The micro-environment within which cells reside in natural tissues under‐ goes constant synthesis and degradation [16, 17], and has long been recognized as dynamic and changing [25, 28]. Although the composition of tissues generally remain tightly control‐ led in maturation, ECM remodeling constantly takes place [25, 29, 30], particularly when un‐ der hormonal stimulation or stress responses (e.g., [31]). Cells actively participate in tissue remodeling by secreting and mobilizing ECM molecules [32]. Alterations in ECM composi‐ tion may result in changes in cell adhesion and /or tissue stiffness [33, 34] which further stimulates cellular responses. For instance, laminin component involved in cell adhesion to ECM is variant due to dynamics in exogenous factors [35]; normal cartilage shows elevated stiffness [36]; and vocal fold tissue exhibits dynamic viscoelastic properties [37]. Some speci‐ alized cell types can experience fast adhesion and detachment from ECM [38]. Changing

chanical stiffness and elastic modulus were used interchangeably in this work.

ECM can also modify cell-cell interactions, further affecting cell behavior [39].

**2. Dynamic nature of the cell microenvironment**

ence, see, e.g., [25-27] ).

84 Advances in Biomaterials Science and Biomedical Applications

**Development and aging** During development, synthesis and degradation of ECM is a con‐ trolled process (e.g., [8, 49]), and mis-regulation contributes to many forms of diseases [30] Particularly, the microenvironment for embryonic and adult stem cells is regulated both temporally and spatially [2, 34], and is involved in various developmental processes includ‐ ing responses to soluble factors, cell differentiation, and morphogenesis [12]. ECM in mus‐ culoskeletal and other tissues adapts to increasing mechanical requirements by altering the size of tissue components [50] during development. Structural dynamics of ECM compo‐ nents such as collagen, laminin, and fibronectin coincides with estrous cycle and develop‐ mental progression [51]. Besides development, aging is also accompanied by changing ECM composition and structures. For example, in connective tissues, aging has been reported to be associated with increase in type I collagen content and decrease in both type III collagen and proteoglycans content, and with collagen fiber disruption and unraveling [50].

**Tissue-implant interfaces** With the growing interest in developing biomimetic materials for tissue engineering applications, tissue-implant interfaces have been subject to considerable research effort. Previous reports showed that cells can actively modify ECM at the interfa‐ ces, and cause drastic changes in tissue or construct mechanics using fibroblast-populated construct and other biomaterials [52, 53]. The study by Lee and co-workers suggested that dynamic moduli of an alginate material may be due to the bioactivities of the chondrocytes encapsulated in the scaffold [54]. In a similar study, different substrate composition and ar‐ chitecture gave rise to distinct levels of modulus increase owing to chondrocytes responses [55]. To take another example, smooth muscle cells (SMCs) in contact with engineered arteri‐ al construct displayed distinctive responses in protein synthesis and consequently the me‐ chanical properties of ECM were significantly different [56]. Additionally, biodegradable materials used in various tissue engineering applications possess changing properties asso‐ ciated with specific degradation profiles.

**Engineering advantages** It has been suggested that temporal control over substrate or scaf‐ fold properties may entail great benefits in engineering cell growth. Among the notable ex‐ amples is the stem cell differentiation and proliferation. A recent work showed enhanced hepatic functions from differentiated stem cells on softer substrates and improved expan‐ sion of undifferentiated cells on stiffer ones [57]. Therefore, it is promising to use stiffer sub‐ strates for optimal proliferations and subsequently soften them to gain better hepatic functions once differentiation completes. Langrana group also found that different neurite properties (e.g., axonal length and primary dendrite number) show differential preference towards substrate stiffness [58], suggesting the strategy of promoting nerve regeneration with scaffold of varying properties. Similar approach can be adopted to take advantage of differential cell responses (e.g., migration and functions) to adhesive properties.

The recurring indication from the above discussions is that *in vivo* ECM interacts with cells in many ways, and that the alteration in ECM composition or structures leads to changes in adhesive properties (hence cell adhesion) and/or mechanical properties. This potentially af‐ fects a variety of cell types and their properties and functioning, at different developmental stages, under normal or pathological conditions, or upon impact or injury. It also holds promises in offering novel approaches to tissue engineering applications. As a result, it is imperative to understand cellular responses to *changing substrate properties* for basic biology and biomimetic material (including biodegradable materials) design.

### **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 [59] and do not involve changes that mimic dy‐ namic 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 [60], and have emerged as an important candidate of materials in studying cellular responses to dynamic substrates [60, 61] where modifica‐ tions could be made *in situ*. One of the major research focuses in this direction is to exam‐ ine the effect of dynamic adhesive property of the substrate on cells, particularly by leveraging the ability to selectively capture or release cells upon application of a variety of stimuli (Table 1).


materials used in various tissue engineering applications possess changing properties asso‐

**Engineering advantages** It has been suggested that temporal control over substrate or scaf‐ fold properties may entail great benefits in engineering cell growth. Among the notable ex‐ amples is the stem cell differentiation and proliferation. A recent work showed enhanced hepatic functions from differentiated stem cells on softer substrates and improved expan‐ sion of undifferentiated cells on stiffer ones [57]. Therefore, it is promising to use stiffer sub‐ strates for optimal proliferations and subsequently soften them to gain better hepatic functions once differentiation completes. Langrana group also found that different neurite properties (e.g., axonal length and primary dendrite number) show differential preference towards substrate stiffness [58], suggesting the strategy of promoting nerve regeneration with scaffold of varying properties. Similar approach can be adopted to take advantage of

The recurring indication from the above discussions is that *in vivo* ECM interacts with cells in many ways, and that the alteration in ECM composition or structures leads to changes in adhesive properties (hence cell adhesion) and/or mechanical properties. This potentially af‐ fects a variety of cell types and their properties and functioning, at different developmental stages, under normal or pathological conditions, or upon impact or injury. It also holds promises in offering novel approaches to tissue engineering applications. As a result, it is imperative to understand cellular responses to *changing substrate properties* for basic biology

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 [59] and do not involve changes that mimic dy‐

SAMs are formed by adsorption of molecules in solution or gas phase onto substrates in a spontaneous and organized fashion [60], and have emerged as an important candidate of materials in studying cellular responses to dynamic substrates [60, 61] where modifica‐ tions could be made *in situ*. One of the major research focuses in this direction is to exam‐ ine the effect of dynamic adhesive property of the substrate on cells, particularly by leveraging the ability to selectively capture or release cells upon application of a variety of

differential cell responses (e.g., migration and functions) to adhesive properties.

and biomimetic material (including biodegradable materials) design.

**3. Types of dynamic substrates and stimuli**

namic ECM are beyond the scope of the review.

**3.1. Self-assembled monolayer (SAM)**

stimuli (Table 1).

ciated with specific degradation profiles.

86 Advances in Biomaterials Science and Biomedical Applications



Note: ssDNA: single-stranded DNA; ↑ increase; ↓ decrease. \*For 'Promising materials', most provides in vitro cyto-tox‐ icity study, and cellular responses to dynamic properties remain to be investigated. N/A: not available

**Table 1.** A partial list dynamic substrates currently used in studying cell responses.

**Type Substrates Properties changed**

Methacrylated HA hydrogel

Hydrogel based on PAM crosslinked by photosensitive reagent

PEG based hydrogel with photosensitive crosslinker

Piezo-controlled substrate and AFM cantilever

Photo-active glass substrate with modifications

Substrates with photo-responsive caged peptides

PEG-modified ITO microelectrodes on glass substrates

Photo-crosslinked alginate hydrogel

Gellan Gum hydrogel with both ionic crosslinking

and

**Other types of substrates**

**Promising materials\***

**and stimuli**

88 Advances in Biomaterials Science and Biomedical Applications

*for*

UV exposure è stiffness ↑ *Stimulus: UV radiation and addition of reactive groups*

Mechanical Stiffness (global or local)↓ *Stimulus: UV radiation*

Mechanical Stiffness↓ Adhesive property change *Stimulus: UV radiation*

Mechanical stiffness with cycling changes *Stimulus: stiffness clamp*

*on AFM*

Adhesion on/off *Stimulus: UV radiation & pro- adhesive molecules*

Adhesion on/off *Stimulus: UV*

Adhesion on/off *Stimulus: electric potential*

Stiffness change; *Stimulus: light or hydrolysis*

Stiffness, swelling, and degradation change *Stimulus: light or ion exchange*

3T3 fibroblasts

hMSCs and Valvular inter-stitial cells (VICs)

HEK293, COS, NIH 3T3

3T3 fibroblasts

HepG2 (hepatic) and 3T3 cells, coculture

Primary bovine chondrocyte

s

**Cell model Observations and notes Ref.**

Adipogenic differentiation favored when cells is on the softer substrate long (stiffening at later times) Osteogenic differentiation when cells are on the stiffer

[79]

[76]

[77]

[84]

[62]

[63]

[92]

[88]

hMSCs Fate of hMSCs differentiate depends on the dynamics of

stiffness change of substrates

substrate (stiffening at early times).

to differential responses

inhibited by softening Good viability of hMSCs

contraction velocity↓

Single cell control

responses

ones

radiation

Stiffness decrease of 20-30% upon propose UV

Global stiffness decrease results in less spreading Localized softening to anterior and posterior area gives

Valvular cell differentiation into myo-fibroblasts is

NIH 3T3 Apparent stiffness↑ leads to cells contraction rate↑ and

Spatio-temporal control over cell adhesion

Changes took place instantaneously, and so did

Responses were reversible, and consistent for same cell.

Modifications of non-adhesive surfaces to adhesive

High survival rate for primary bovine chondrocytes Cellular responses to dynamic changes to be studied

NIH 3T3 Swelling and hydrolytic degradation vary with respect

Stiffness may be changed quickly during photo-

to crosslinking mechanism

crosslinking process

Micro-patterned co-culture made possible [85]

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 photo‐ chemical 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 sub‐ strates by integrating surface chemistry, micro-patterning, and cell microenvironment engi‐ neering [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 re‐ lease 3T3 fibroblasts from designed areas on the substrate, and subsequently encouraged mi‐ gration of neighboring cells to those areas with newly added adhesion molecules [65]. Refining this design by adding responsiveness to both negative and positive electric potentials, they demonstrated selective control over cell release [67] (Fig. 1C). Other groups have also engaged in the effort along this direction. By employing a hydroquinone terminated SAM based on re‐ dox 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 [68], and selectively re‐ leased 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 cel‐ lular responses and cell-ECM interactions [60]. 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[69] (Fig. 1B). While the find‐ ing is interesting, the long exposure of cells to UV may be problematic despite the reported neg‐ ligible impact of light with wavelength over 320 nm on cells [63].

**Figure 1.** SAM-based dynamic substrates. (A) Schematic of the approach based on redox reaction (A1) by adjusting elec‐ trochemical potential, and cell detachment upon application of electric potential (A2). Extracted from [68]. (B) Schematic of altering configuration of azobenzene group under light of different ranges of wave length (B1) [69] and application to cell culture (B2) where NIH 3T3 fibroblasts initially adhere to adhesive surface (a) which was inhibited upon surface modifi‐ cation (b) followed by recovery of adhesion due to azobenzene configuration change (c). Extracted from [69]. (C) Illustra‐ tion of a SAM that allows different modifications with positive and negative electric potential (C1) and its application in selective release of Swiss 3T3 cells (C2). Extracted from [67]. All with permission from publishers.

The above surveys part of the key advancement using SAM in modifying adhesion proper‐ ties 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 mo‐ lecular 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., pro‐ tein conformation) cues whose applicability under *in vivo* conditions is problematic. Next, the resulting changes in these studies are mostly of surface biochemical properties or of the presentation and biological activities of the surface ligands. Nevertheless, SAMs have great‐ ly facilitated the probe and understanding of cell-ECM interactions and particular interplay between cells and ECM with dynamics in adhesive properties.

#### **3.2. Polymeric hydrogels**

dox 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 [68], and selectively re‐ leased 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 cel‐ lular responses and cell-ECM interactions [60]. 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[69] (Fig. 1B). While the find‐ ing is interesting, the long exposure of cells to UV may be problematic despite the reported neg‐

**Figure 1.** SAM-based dynamic substrates. (A) Schematic of the approach based on redox reaction (A1) by adjusting elec‐ trochemical potential, and cell detachment upon application of electric potential (A2). Extracted from [68]. (B) Schematic of altering configuration of azobenzene group under light of different ranges of wave length (B1) [69] and application to cell culture (B2) where NIH 3T3 fibroblasts initially adhere to adhesive surface (a) which was inhibited upon surface modifi‐ cation (b) followed by recovery of adhesion due to azobenzene configuration change (c). Extracted from [69]. (C) Illustra‐ tion of a SAM that allows different modifications with positive and negative electric potential (C1) and its application in

selective release of Swiss 3T3 cells (C2). Extracted from [67]. All with permission from publishers.

ligible impact of light with wavelength over 320 nm on cells [63].

90 Advances in Biomaterials Science and Biomedical Applications

Hydrogel materials are gaining popularity in the development of biomimetic materials, pri‐ marily due to the hydrated nature of natural ECM [14, 71]. Implantable hydrogel materials are increasingly being used in cardiovascular disease, nerve regeneration, and other condi‐ tions [59]. 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 [72]. Kim and colleagues took advantages of the thermo-responsive swelling behavior of copolymer between NIPAM and sodium acryl‐ ate, and created a hydrogel film that can be used to control cell encapsulation with surface topography [73]. Moreover, biomaterials responsive to the natural stimuli such as those ex‐ perienced by biodegradable materials were found useful in mimicking biological events un‐ der physiological conditions, as illustrated in cell invasion to a MMP-responsive hydrogel scaffold [74]. This finding, among others, exemplifies the strategy of triggering material dy‐ namic 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 [75]. 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 [76] showed that upon UV induced substrates softening, spreading of 3T3 fibroblasts was hindered in contrast to that under static condi‐ tions (Fig. 2A). More interestingly, localized softening at anterior and posterior of cells yield‐ ed differential cellular morphology and migration responses [76]. Meanwhile, a PEG based polymer (PEGA) crosslinked by photosensitive crosslinker (PEGdiPDA) has been developed by Kloxin et al. [77], 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 mi‐ croenvironment, and potentially allow creation of dynamic stiffness gradient.

**Figure 2.** Photosensitive hydrogels and the study of cellular responses. (A) On a polyacrylamide hydrogel with photo‐ sensitive crosslinker, NIH 3T3 cells contract as indicated by projection area in response to UV-induced substrate soften‐ ing. Extracted from [76]. (B) Valvular interstitial cells (VICs) on a PEG based hydrogel with photosensitive crosslinker displayed de-activation when UV radiation triggered substrate stiffness decrease. Extracted from [77]. Both with per‐ mission from publishers.

Similar observations were made by Burdick group for human mesenchymal stem cells (hMSCs) by using hyaluronic acid (HA) hydrogel degradable from hydrolytic and enzy‐ matic reactions [78] (Fig. 3A). Very recently, a new material platform has been con‐ structed by this group [79] and others [80] where the stiffness of a methacrylated hyaluronic acid hydrogel is increased via addition of photo-initiator and UV light expo‐ sure. 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 [81], 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.

these methods made possible high precision in applying changes of cellular mechanical mi‐

**Figure 2.** Photosensitive hydrogels and the study of cellular responses. (A) On a polyacrylamide hydrogel with photo‐ sensitive crosslinker, NIH 3T3 cells contract as indicated by projection area in response to UV-induced substrate soften‐ ing. Extracted from [76]. (B) Valvular interstitial cells (VICs) on a PEG based hydrogel with photosensitive crosslinker displayed de-activation when UV radiation triggered substrate stiffness decrease. Extracted from [77]. Both with per‐

mission from publishers.

croenvironment, and potentially allow creation of dynamic stiffness gradient.

92 Advances in Biomaterials Science and Biomedical Applications

Factors other than environmental conditions (e.g., light, pH, temperature) can also be delivered to stimulate dynamics in substrate properties. Incorporating DNA as cross‐ linker, Jiang and colleagues have developed a hydrogel system for cell attachment where mechanical properties of the substrates can be altered *in situ* in a controlled fash‐ ion when the cell culture is present [20, 81]. These DNA crosslinked hydrogels may al‐ so be designed to be potentially responsive to bio-stimuli, such as temperature or enzymes. Two representative cell types were chosen for the study of cell responses to dynamic substrate: fibroblasts whose sensitivity to mechanical cues is well documented, and neurons whose mechanosensing capability has recently just started being appreciat‐ ed. The reports [20, 81] offered evidence that both cell types do respond to dynamic al‐ ternations in the mechanical characteristics of ECM, and suggest that the *alternations* in the mechanical stiffness may be involved in disease progression (Fig. 3B). It has been shown that the stiffness change resulting from pathological processes, may also aid in further progression of diseases [82].

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 dy‐ namic processes, and secondly contrasted cell behavior to stiffness decrease with that for hardening of the substrates [83]. 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 prop‐ erties (e.g., projection area vs. aspect ratio and protrusion for fibroblasts). The stress generation may also be involved in affecting cell behavior [83].

**Figure 3.** Substrates with dynamic mechanical stiffness and their application in cell culture studies. (A) Live/deal cell staining of human mesenchymal stem cells (hMSCs) in a hyaluronic acid hydrogel (A1) and the analysis of gene expres‐ sion of type I/II collagen and aggrecan revealed that aside from type I collagen, both type II collagen and aggrecan exhibited an elevated level of expression on dynamic gels from the static ones. Extracted from [78]. With permission from publisher. (B) L929 fibroblast growth in a DNA crosslinked hydrogel with dynamic stiffness from crosslinking den‐ sity change (B1) and the quantification of spreading area and aspect ratio (B2) showed that dynamic gels are signifi‐ cantly different from their static counterparts. Extracted from [81].

#### **3.3. Other types of materials**

The approach of employing polymeric hydrogel to study dynamic changes has certain limi‐ tations, one of which is the coupling of mechanical stiffness and forces (e.g., [83]). To ad‐ dress this concern and others, different from the approach by using SAM or polymeric hydrogel, AFM based method put forth by Webster and co-workers [84] probed cellular re‐ sponse to instant step change in stiffness excluding influence from stress or strain in the sub‐ strates (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 [84]. However, this approach is most likely with inherent limitation in mimicking natural cell environment while remains an interesting tool in probing cellular re‐ sponses 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-cleava‐ ble agents (NPE-TCSP) were shown to be useful for controlling cell adhesion selectively and temporally [62]. 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, fol‐ lowed by cell seeding. It is useful to study dynamics in interactions between single cells. Pe‐ tersen et al. [63] 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 re‐ versible. With a sequential activation of adhesive sites upon application of electric potential, a recent study [85] demonstrated the utility of substrates with ITO (indium tin oxide) micro‐ electrodes modified with poly(ethylene glycol), or PEG, in co-culture of two cell types (hep‐ atic cells and fibroblasts) in a controlled manner (Fig. 4B).

**Figure 3.** Substrates with dynamic mechanical stiffness and their application in cell culture studies. (A) Live/deal cell staining of human mesenchymal stem cells (hMSCs) in a hyaluronic acid hydrogel (A1) and the analysis of gene expres‐ sion of type I/II collagen and aggrecan revealed that aside from type I collagen, both type II collagen and aggrecan exhibited an elevated level of expression on dynamic gels from the static ones. Extracted from [78]. With permission from publisher. (B) L929 fibroblast growth in a DNA crosslinked hydrogel with dynamic stiffness from crosslinking den‐ sity change (B1) and the quantification of spreading area and aspect ratio (B2) showed that dynamic gels are signifi‐

The approach of employing polymeric hydrogel to study dynamic changes has certain limi‐ tations, one of which is the coupling of mechanical stiffness and forces (e.g., [83]). To ad‐ dress this concern and others, different from the approach by using SAM or polymeric hydrogel, AFM based method put forth by Webster and co-workers [84] probed cellular re‐ sponse to instant step change in stiffness excluding influence from stress or strain in the sub‐ strates (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 [84]. However, this approach is most likely with inherent limitation in mimicking natural cell environment while remains an interesting tool in probing cellular re‐ sponses to instantaneously change in stiffness. Additionally, this approach is applicable

Common cell culture substrates (e.g., glass coverslip) modified with common photo-cleava‐ ble agents (NPE-TCSP) were shown to be useful for controlling cell adhesion selectively and temporally [62]. In this method, target areas were first irradiated to remove BSA known to

cantly different from their static counterparts. Extracted from [81].

94 Advances in Biomaterials Science and Biomedical Applications

mostly to cells with dynamic morphology.

**3.3. Other types of materials**

**Figure 4.** Cellular behavior in responses to other substrates with dynamic properties. (A) 3T3 fibroblasts grown in areas of patterned stripes (A1) generated from UV radiation based on the surface chemistry involving a caging group (A2). Extract‐ ed from [63]. (B) With PEG-modified ITO microelectrodes on glass substrates (B1), co-culture of two different cell types, HepG2 (hepatic) and 3T3 cells, was made possible. Extracted from [85]. Both with permission from publishers.

#### **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 frequen‐ cy mimicking the load from human vocal tissues [86] 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 [87]. Ion concen‐ tration may be another stimulus to allow for temporal modification of hydrogel properties as exchange of ions between monovalant and divalant cations [88], and further work is needed to confirm it. Temperature-dependent substrate softening has been demonstrated by Krekhova et al. [89] and 3D complex with temperature-mediated crosslinking hence me‐ chanical properties has been proposed by Stahl et al. [90], while applicability of these sys‐ tems in mimicking cellular microenvironment remains to be seen. Zustiak et al. [91] 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 evi‐ dence 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-cross‐ linked alginate hydrogels based on alginate methacrylation were presented by Jeon et al [92], and cyto-toxicity has been found to be low. In summary, these substrates holds prom‐ ises as substrates with modifiable properties *in situ*, and need to be carefully tuned and eval‐ uated for use as substrates with dynamic properties (Table 1). Other materials responsive to various stimuli including pH, temperature, and biochemical factors for a variety of applica‐ tions, including can be found in the earlier reviews [64, 70, 93], and thus are not discussed in detail here due to the focus of the current analysis.

#### **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 *per se* in light of the observations in normal and pathological tissues, development and aging, and potential engineering bene‐ fits. Towards this end, rate of change (e.g., gradual vs. abrupt), range of change (e.g., small perturbation vs. drastic modifications), and change profile (e.g., monotonic increase vs. fluc‐ tuation) characterizing the nature of changes and their impact on cellular processes are sub‐ ject to research effort, apparently adding to the complexity of the problem (Table 1). Take biodegradable material (e.g., [94]) as an example. It would be relevant to understand how mechanical and adhesive characteristics evolve with degradation and how the degradation profile affects the changes in the cellular micro-environment. Experimental design along this line may include, for example, different rates of release of RGD motif decreasing adhesive‐ ness while keeping the same range of change (e.g., half of the total RGD presenting sites), or altering the range of change while maintaining the same rate of change. Furthermore, it is not clear at this point whether cellular responses to opposite changes (e.g., increase vs. de‐ crease in adhesiveness or rigidity) of substrate properties are symmetric, thus their behavior to one direction of dynamic alterations may not be a reliable predictor of that to the opposite changes.

#### **4.2. Potential effect on cell-cell interactions**

**3.4. Promising substrates**

96 Advances in Biomaterials Science and Biomedical Applications

detail here due to the focus of the current analysis.

**4. Design considerations and outlook**

**4.1. Dynamic properties of the 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 frequen‐ cy mimicking the load from human vocal tissues [86] 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 [87]. Ion concen‐ tration may be another stimulus to allow for temporal modification of hydrogel properties as exchange of ions between monovalant and divalant cations [88], and further work is needed to confirm it. Temperature-dependent substrate softening has been demonstrated by Krekhova et al. [89] and 3D complex with temperature-mediated crosslinking hence me‐ chanical properties has been proposed by Stahl et al. [90], while applicability of these sys‐ tems in mimicking cellular microenvironment remains to be seen. Zustiak et al. [91] 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 evi‐ dence 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-cross‐ linked alginate hydrogels based on alginate methacrylation were presented by Jeon et al [92], and cyto-toxicity has been found to be low. In summary, these substrates holds prom‐ ises as substrates with modifiable properties *in situ*, and need to be carefully tuned and eval‐ uated for use as substrates with dynamic properties (Table 1). Other materials responsive to various stimuli including pH, temperature, and biochemical factors for a variety of applica‐ tions, including can be found in the earlier reviews [64, 70, 93], and thus are not discussed in

As indicated in the discussions in Background and Motivation, the progression in changes of ECM properties is also critical in addition to changes *per se* in light of the observations in normal and pathological tissues, development and aging, and potential engineering bene‐ fits. Towards this end, rate of change (e.g., gradual vs. abrupt), range of change (e.g., small perturbation vs. drastic modifications), and change profile (e.g., monotonic increase vs. fluc‐ tuation) characterizing the nature of changes and their impact on cellular processes are sub‐ ject to research effort, apparently adding to the complexity of the problem (Table 1). Take biodegradable material (e.g., [94]) as an example. It would be relevant to understand how mechanical and adhesive characteristics evolve with degradation and how the degradation profile affects the changes in the cellular micro-environment. Experimental design along this line may include, for example, different rates of release of RGD motif decreasing adhesive‐ ness while keeping the same range of change (e.g., half of the total RGD presenting sites), or

Changes in adhesive or mechanical properties of ECM can stimulate cells, which, in re‐ sponse, 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 [95], 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 [33].

#### **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 polymer‐ ic 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 applica‐ tions 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 attrac‐ tive [84] 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., [88]) 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 envi‐ ronmental 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/cool‐ ing 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.



**Table 2.** Design considerations in constructing dynamic substrates mimicking extra-cellular matrix (ECM).

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 *in situ* changes to cells will be desired.
