Control Cell Adhesion Research Articles

Identification and Analysis of Key Parameters for the Ossification on Particle Functionalized Composites Hydrogel Materials.

Developing materials for tissue engineering and studying the mechanisms of cell adhesion is a complex and multifactor process that needs analysis using physical chemistry and biology. The major challenge is the labor-intensive data mining as well as requirements of the number of advanced techniques. For example, hydrogel-based biomaterials with cell-binding sites, tunable mechanical properties, and complex architectures have emerged as a powerful tool to control cell adhesion and proliferation for tissue engineering. Composite hydrogels could be used for bone tissue regeneration, but they exhibit poor ossification properties. In current work, we have designed new osteoinductive gellan gum hydrogels by a thermal annealing approach and consequently functionalized them with Ca/Mg carbonate submicron particles. Determination of key parameters, which influence a successful hydroxyapatite generation, was done via the principal component analysis of 18 parameters (Young's modulus of the hydrogel and particles, particle size, and mass) and cell behavior at various time points (like viability, numbers of the cells, rate of alkaline phosphatase production, and cells area) obtained by characterizing such composite hydrogel. It is determined that the particles size and concentration of calcium ions have a dominant effect on the hydroxyapatite formation, because of providing local areas with a high Young's modulus in a hydrogel, a desirable property for cell adhesion. The detailed analysis presented here allows identifying hydrogels for cell growth applications, while on the other hand, material properties can be predicted, and their overall number can be minimized leading to efficient optimization of bone reconstruction and other cell growth applications.

Myoblast adhesion and proliferation on biodegradable polymer films with femtosecond laser-fabricated micro through-holes.

Controlling cell adhesion and cell differentiation is necessary to fabricate a tissue with arbitrary properties for tissue engineering applications. A substrate with a porous structure as a cell scaffold allows the diffusion of the cell culture medium through the scaffold. In this work, we show that the femtosecond laser fabricated micro through-holes in biodegradable polymer films, enhance myoblast adhesion, and accelerates proliferation and differentiation. ChR2-C2C12 and UT-C2C12 cells were seeded on the films with micro through-holes each fabricated by a single femtosecond laser pulse. Cell adhesion was enhanced on films with holes fabricated by laser irradiation. In addition, cell proliferation was accelerated on films with micro through-holes that penetrate the film, compared to on films with micro craters that do not penetrate the film. On films with arrays consisting of micro through-holes, cells aligned along the arrays and cell fusion was enhanced, indicating the acceleration of cell differentiation.

Cells-Grab-on Particles: A Novel Approach to Control Cell Focal Adhesion on Hybrid Thermally Annealed Hydrogels.

Biomaterials engineered with specific cell binding sites, tunable mechanical properties, and complex architectures are essential to control cell adhesion and proliferation. The influence of the local properties, such as the local hardness and stability on the interaction with cells, has not been yet fully understood and exploited. This is particularly relevant for hydrogels, very promising materials with, unfortunately, poor cell adhesion properties, attributed mostly to their softness. Here, we propose a new approach for producing hybrid hydrogels by functionalizing them with particles and performing a thermal treatment. Exploring the interaction of cells with these materials we introduce a new concept, cells-grabbing-onto-particles, a facilitation of the cell adhesion through modulation of local properties. The approach is implemented on alginate hydrogels typically unsuitable for cell growth by turning them into a very effective cell culture growth platform. Specifically, alginate hydrogels are bio-mineralized with calcium carbonate (CaCO3) particles, where an additional thermal annealing (T-A) process has been applied. The local Young's modulus of new T-A treated hybrid hydrogels has increased to over 3 MPa on areas of hydrogels containing particles and to around 1 MPa on areas without particles, which is drastically different from 130 to 180 kPa values for unmodified hydrogels. Intriguingly, our results show that enhancement of local mechanical properties alone is a necessary, but insufficient, condition; the particles must be stably fixed in gels for cell growth and proliferation. Extended for hydrogels functionalized with silica particles too, the cells-grab-on-particles concept is shown applicable to different materials and cells for cell biology and tissue engineering.

A practical guide to microfabrication and patterning of hydrogels for biomimetic cell culture scaffolds

This review article describes microfabrication techniques to define chemical, mechanical and structural patterns in hydrogels and how these can be used to prepare in vivo like, i.e. biomimetic, cell culture scaffolds. Hydrogels are attractive materials for 3D cell cultures as they provide ideal culture conditions and they are becoming more prominently used. Single material gels without any modifications do however have their limitation in use and much can be gained by in improving the in vivo resemblance of simple hydrogel cell culture scaffolds. This review article discusses the most commonly used cross-linking strategies used for hydrogel-based culture scaffolds and gives a brief introduction to microfabrication methods that can be used to define chemical, mechanical and structural patterns in hydrogels with micrometre resolution. The review article also describes a selection of literature references using these microfabrication techniques to prepare organ and disease models with controlled cell adhesion, proliferation and migration. It is intended to serve as an introduction to microfabrication of hydrogels and an inspiration for novel interdisciplinary research projects.

Author Correction: Photoinduced self-initiated graft polymerization of methacrylate monomers on poly(ether ether ketone) substrates and surface parameters for controlling cell adhesion

One of the super engineering plastics, poly(ether ether ketone) (PEEK), was functionalized by photoinduced self-initiated graft polymerization of various methacrylate monomers. Anionic, cationic, zwitterionic, nonionic hydrophilic, and nonionic hydrophobic polymer layers were formed onto PEEK substrates. Physical and chemical surface characterizations of the resultant polymer-grafted PEEK substrates were performed through measurements of their surface free energies and ζ potentials. The values of these parameters varied in the ranges of 39–71 mJ/m2 and −69 to 46 mV, respectively. These parameters reflected the chemical structures of the grafted polymers. To understand the effects of these surface parameters on cell adhesion behavior at the substrate surface, the amount of fibronectin adsorbed on the plasma-contacting surface and the density of fibroblast cells adhered to the surface were determined. The adherent cell density showed a good linear correlation with the amount of fibronectin adsorbed on the plasma-contacting surface. The polymer surface with zero ζ potential showed a lower adsorbed fibronectin density. Both anionic and cationic polymer layers had increased cell adhesion compared with that on the original PEEK substrate, whereas the zwitterionic polymer layers significantly prevented cell adhesion. In conclusion, grafting zwitterionic polymers onto a PEEK substrate is anticipated to be useful in the development of a biomedical PEEK substrate. To obtain functionalization of super-engineering plastic, poly(ether ether ketone) (PEEK) substrate, photoinduced self-initiated graft polymerization of several kinds of functional methacrylate was carried out. Graft polymerization proceeded well, and 50–250 nm in thickness graft polymer layers were generated on the PEEK substrate. Surface characteristics such as surface free energy and surface ζ potential on the polymer-grafted PEEK substrate were corresponded to the chemical structure of the graft polymers. Cell attachment on the polymer-grafted PEEK substrate was examined. The surface ζ potentials of the polymer-grafted PEEK substrates governed the cell adhesion density.

Supramolecular Hydrogels with Tunable Chirality for Promising Biomedical Applications.

Chirality exits from molecular-level, supramolecular, and nanoscaled helical structures to the macroscopic level in biological life. Among these various levels, as the central structural motifs in living systems (e.g., double helix in DNA, α-helix, β-sheet in proteins), supramolecular helical systems arising from the asymmetrical spatial stacking of molecular units play a crucial role in a wide diversity of biochemical reactions (e.g., gene replication, molecular recognition, ion transport, enzyme catalysis, and so on). However, the importance of supramolecular chirality and its potential biofunctions has not yet been fully explored. Thus, generating chiral assembly to transfer nature's chiral code to artificial biomaterials is expected to be utilized for developing novel functional biomaterials. As one of the most commonly used biomaterials, supramolecular hydrogels have attracted considerable research interest due to their resemblance to the structure and function of the native extracellular matrix (ECM). Therefore, the performance and manipulation of chiral assembled nanoarchitectures in supramolecular hydrogels may provide useful insights into understanding the role of supramolecular chirality in biology.In this Account, recent progress on chiral supramolecular hydrogels is presented, including how to construct and regulate assembled chiral nanostructures in hydrogels with controllable handedness and then use them to develop chiral hydrogels that could be applied in biology, biochemistry, and medicine. First, a brief introduction is provided to present the basic concept related to supramolecular chirality and the importance of supramolecular chirality in living systems. The chiral assemblies in supramolecular hydrogels are strongly driven by noncovalent interactions between molecular building blocks (such as hydrogen bonding, π-π stacking, hydrophobic, and van der Waals interactions). Consequently, the handedness of these chiral assemblies can be regulated by many extra stimuli including solvents, temperature, pH, metal ions, enzymes, and photoirradiation, which is presented in the second section. This manipulation of the chirality of nanoarchitectures in supramolecular hydrogels can result in the development of potential biofunctions. For example, specific supramolecular chirality-induced biological phenomena (such as controlled cell adhesion, proliferation, differentiation, apoptosis, protein adsorption, drug delivery, and antibacterial adhesion) are presented in detail in the third section. Finally, the outlook of open challenges and future developments of this rapidly evolving field is provided. This account that highlights the diverse chirality-dependent biological phenomena not only helps us to understand the importance of chirality in life but also provides new ideas for designing and preparing chiral materials for more bioapplications.

Photoinduced self-initiated graft polymerization of methacrylate monomers on poly(ether ether ketone) substrates and surface parameters for controlling cell adhesion

Photoinduced self-initiated graft polymerization of methacrylate monomers on poly(ether ether ketone) substrates and surface parameters for controlling cell adhesion

Surface Patterning of Self-healing P(MMA/nBA) Copolymer for Dynamic Control Cell Behaviors

Cell behaviors are regulated by a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties. However, most works regulate cell behaviors under static conditions or by external factors. To control cell adhesion and proliferation with a dynamic and mechanical environment, we pattern the surface on self-healing copolymer P(MMA/nBA). The copolymer P(MMA/nBA) with the composition of 48/52 (MMA/nBA) recovers nearly 100% of its original tensile strains after 86 h of recovery from deformation. The physical patterns on P(MMA/nBA) film are obtained over large areas and the size of the hole and the width of connecting bar are in line with the copper grid specifications. The patterned surface tends to be flat after 12 h with almost 75%-80% recovery. Compared with cell incubation on polystyrene flat and patterned surface of P(MMA/nBA) film without self-healing capability, the number and morphology of cells are well manipulated on the patterned surface of self-healing P(MMA/nBA) film. This approach provides a convenient method for dynamically regulating the cell behaviors on the surface of self-healing materials without chemical or biological modifications.

C-Src promotes tumor progression through downregulation of microRNA-129-1-3p.

MicroRNAs (miRNAs) fine‐tune cellular signaling by regulating expression of signaling proteins, and aberrant expression of miRNAs is observed in many cancers. The tyrosine kinase c‐Src is upregulated in various human cancers, but the molecular mechanisms underlying c‐Src‐mediated tumor progression remain unclear. In previous investigations of miRNA‐mediated control of c‐Src‐related oncogenic pathways, we identified miRNAs that were downregulated in association with c‐Src transformation and uncovered the signaling networks by predicting their target genes, which might act cooperatively to control tumor progression. Here, to further elucidate the process of cell transformation driven by c‐Src, we analyzed the expression profiles of miRNAs in a doxycycline‐inducible Src expression system. We found that miRNA (miR)‐129‐1‐3p was downregulated in the early phase of c‐Src‐induced cell transformation, and that reexpression of miR‐129‐1‐3p disrupted c‐Src‐induced cell transformation. In addition, miR‐129‐1‐3p downregulation was tightly associated with tumor progression in human colon cancer cells/tissues. Expression of miR‐129‐1‐3p in human colon cancer cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasion. We also identified c‐Src and its critical substrate Fer, and c‐Yes, a member of the Src family of kinases, as novel targets of miR‐129‐1‐3p. Furthermore, we found that miR‐129‐1‐3p‐mediated regulation of c‐Src/Fer and c‐Yes is important for controlling cell adhesion and invasion. Downregulation of miR‐129‐1‐3p by early activation of c‐Src increases expression of these target genes and synergistically promotes c‐Src‐related oncogenic signaling. Thus, c‐Src‐miR‐129‐1‐3p circuits serve as critical triggers for tumor progression in many human cancers that harbor upregulation of c‐Src.

The influence of structural gradients in large pore organosilica materials on the capabilities for hosting cellular communities.

Cells exist in the so-called extracellular matrix (ECM) in their native state, and numerous future applications require reliable and potent ECM-mimics. A perspective, which goes beyond ECM emulation, is the design of a host-material with features which are not accessible in the biological portfolio. Such a feature would, for instance, be the creation of a structural or chemical gradient, and to explore how this special property influences the biological processes. First, we wanted to test if macroporous organosilica materials with appropriate surface modification can act as a host for the implementation of human cells like HeLa or LUHMES. It was possible to use a commercially available polymeric foam as a scaffold and coat it with a thiophenol-containing organosilica layer, followed by biofunctionalization with biotin using click chemistry and the subsequent coupling of streptavidin–fibronectin to it. More importantly, deformation of the scaffold allowed the generation of a permanent structural gradient. In this work, we show that the structural gradient has a tremendous influence on the capability of the described material for the accommodation of living cells. The introduction of a bi-directional gradient enabled the establishment of a cellular community comprising different cell types in spatially distinct regions of the material. An interesting perspective is to study communication between cell types or to create cellular communities, which can never exist in a natural environment.

Polymorphic calcium alginate microfibers assembled using a programmable microfluidic field for cell regulation.

Effectively guiding and accurately controlling cell adhesion and growth on the surfaces of specific morphological materials are key issues and hot research topics for optimizing biomaterials. Herein, novel polymorphic alginate microfibers formed through microfluidic spinning technology in a single microchip are presented. Through programming the flow and reaction kinetics in microchannels, other than self-modified micromorphic channel geometry, polymorphic microfibers with precisely tuned curvature-adjustable morphology can be obtained. Finite element (FE) simulations of the flow field (unidirectional fluid-solid coupling) proved the efficacy of the proposed control strategy. Moreover, the specific disordered-ordered cell arrangements showed a linear relationship between bioinspired alginate microfibers with different curvatures and the orientation angle of L929 cells, and diversified growth morphologies, including oblate ellipse, star, tree and strip shapes, occurred on the customizable interface curvature of the calcium alginate microfibers, providing a paradigm for using specific structured natural biomedical materials for cell regulation. This work represents a new design concept for manufacturing polymorphic fibrous biomedical materials through a unique marriage of the fields of green chemistry, hydromechanics, and biomaterials, which should be very useful for guiding the controllable construction of alginate materials for use in structural materials for biomedical and engineering purposes.

C-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

SummaryStreptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

EpCAM associates with integrin and regulates cell adhesion in cancer cells

The epithelial cell adhesion molecule (EpCAM) is one of the most frequently and intensely expressed of tumor-associated antigens, but the role that EpCAM plays in the proliferation, adhesion and migration properties of cancer cells remains unclear. In the present study, we screened several tumor cell lines and found that colorectal cancer CW-2 and epidermoid carcinoma A431 cells expressed relatively higher levels of EpCAM. In order to assess the biological functions of EpCAM expression in cell adhesion and migration, we established a knock out (KO) of EpCAM genes in both of these types of cancer cells via a CRISPR/Cas9 system. The elongated cell morphology was converted to a rounded morphology in the EpCAM-KO cells. These cells showed decreases in cell proliferation and migration into extracellular matrix proteins, as well as decreases in cellular signaling elements such as phosphorylated focal adhesion kinase (FAK), AKT and ERK. Moreover, the cell growth and the colony formation abilities were significantly decreased in EpCAM-KO cells. Importantly, co-immunoprecipitation analysis revealed that EpCAM associated with integrin β1. Also, the expression levels of integrin α5 were decreased in EpCAM-KO cells, compared with that in the wild-type cells. Taken together, these data clearly demonstrate that EpCAM associates with integrin β1 to regulate FAK/ERK signaling pathways in controlling cell adhesion, migration and proliferation via extracellular matrix adhesion, which provides novel mechanisms for EpCAM-mediated biological functions and cancer phenotypes.

Ultrashort Pulsed Laser Surface Patterning of Titanium to Improve Osseointegration of Dental Implants

Ultrashort pulsed direct laser interference patterning (DLIP) is used to generate hierarchical line‐like patterns on titanium surfaces to control cell adhesion and spreading on dental implants, thereby improving osseointegration. The DLIP structures have spatial periods of 3, 5, 10, and 17 μm. They are produced using a laser source with a pulse duration of 10 ps and cumulated energy densities between 0.1 and 78.9 J cm−2. Laser‐induced periodic surface structures (LIPSS) and submicron features are obtained on the treated samples. The DLIP treatment leads to the development of a thick titanium oxide layer, which is imaged and quantified using time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). Several days (30–56) after the laser treatment, specimens with larger spatial periods are hydrophilic, whereas samples with spatial periods of 3 μm are hydrophobic. Seeded human osteoblasts on the laser‐structured samples show 2.5 times higher cell numbers after 7 days in vitro culture compared with osteoblasts on a grit‐blasted and etched reference sample. Finally, cell adhesion to a structured 3D dental implant is demonstrated.

Macrophage Adhesion and Phenotypic Shifts on Hydrophobically Modified Hydrogels

A balance of macrophage phenotypes is essential in successful implantation. Therefore, biomaterials should be carefully designed to better regulate the response of macrophages. We demonstrate macrophage adhesion and phenotypic shifts can be modulated with hydrophobically modified hydrogels. This study represents a fundamental concept in controlling cell adhesion and differentiation with substrate hydrophobicity. Gel synthesis was carried out using methacrylated gellan gum (MGG), which was polymerized and crosslinked through a photo-initiated thiol-ene reaction in situ. The degree of hydrophobicity was controlled by attaching hydrophobic branches of different lengths to the MGG. The attachment was carefully controlled so that the hydrogel compressive modulus was not affected by the hydrophobic modification. Compared to unmodified MGG, the water contact angle increased and the swelling ratio decreased for modified MGG hydrogels. Naive and activated macrophages were subsequently seeded on the hydrogels with different surface wettability. Cell proliferation and spreading increased with increasing surface hydrophobicity. Hydrogel surface adhesion to a hydrophobic probe was quantified and showed an increasing trend with increasing substrate hydrophobicity. This suggests that the hydrophobic surface may increase protein adsorption, which, in turn, increases cell spreading. Cytokine secretion by M1 and M2 macrophages were also altered by substrate hydrophobicity. Both nitrite production, a marker of M1, and urea production, a marker of M2, increased with substrate hydrophobicity, with nitrate production being more pronounced. RAW 264.7 macrophages seeded on these substrates shifted to a more pro-inflammatory phenotype with increasing substrate hydrophobicity.

Proteome-level assessment of origin, prevalence and function of leucine-aspartic acid (LD) motifs.

MotivationLeucine-aspartic acid (LD) motifs are short linear interaction motifs (SLiMs) that link paxillin family proteins to factors controlling cell adhesion, motility and survival. The existence and importance of LD motifs beyond the paxillin family is poorly understood.ResultsTo enable a proteome-wide assessment of LD motifs, we developed an active learning based framework (LD motif finder; LDMF) that iteratively integrates computational predictions with experimental validation. Our analysis of the human proteome revealed a dozen new proteins containing LD motifs. We found that LD motif signalling evolved in unicellular eukaryotes more than 800 Myr ago, with paxillin and vinculin as core constituents, and nuclear export signal as a likely source of de novo LD motifs. We show that LD motif proteins form a functionally homogenous group, all being involved in cell morphogenesis and adhesion. This functional focus is recapitulated in cells by GFP-fused LD motifs, suggesting that it is intrinsic to the LD motif sequence, possibly through their effect on binding partners. Our approach elucidated the origin and dynamic adaptations of an ancestral SLiM, and can serve as a guide for the identification of other SLiMs for which only few representatives are known.Availability and implementation LDMF is freely available online at www.cbrc.kaust.edu.sa/ldmf; Source code is available at https://github.com/tanviralambd/LD/.Supplementary information Supplementary data are available at Bioinformatics online.

Impact of UV- and carbodiimide-based crosslinking on the integrin-binding properties of collagen-based materials

Collagen constructs are widely used for tissue engineering. These are frequently chemically crosslinked, using EDC, to improve their stability and tailor their physical properties. Although generally biocompatible, chemical crosslinking can modify crucial amino acid side chains, such as glutamic acid, that are involved in integrin-mediated cell adhesion. Instead UV crosslinking modifies aromatic side chains. Here we elucidate the impact that EDC, in combination with UV, exerts on the activity of integrin-binding motifs. By employing a model cell line that exclusively utilises integrin α2β1, we found that whilst EDC crosslinking modulated cell binding, from cation-dependent to cation-independent, UV-mediated crosslinking preserved native-like cell binding, proliferation and surface colonisation. Similar results were observed using a purified recombinant I-domain from integrin α1. Conversely, binding of the I-domain from integrin α2 was sensitive to UV, particularly at low EDC concentrations. Therefore, from this in vitro study, it appears that UV can be used to augment EDC whist retaining a specific subset of integrin-binding motifs in the native collagen molecule. These findings, delineating the EDC- and UV-susceptibility of cell-binding motifs, permit controlled cell adhesion to collagen-based materials through specific integrin ligation in vitro. However, in vivo, further consideration of the potential response to UV wavelength and dose is required in the light of literature reports that UV initiated collagen scission may lead to an adverse inflammatory response. Statement of significanceRecently, there has been rapid growth in the use of extracellular matrix-derived molecules, and in particular collagen, to fabricate biomaterials that replicate the cellular micro-environment. Often chemical or physical crosslinkers are required to enhance the biophysical properties of these materials. Despite extensive use of these crosslinkers, the cell-biological consequences have not been ascertained. To address this, we have investigated the integrin-binding properties of collagen after chemically crosslinking with EDC and physically crosslinking with UV-irradiation. We have established that whilst EDC crosslinking abates all of the integrin binding sites in collagen, UV selectively inhibits interaction with integrin-α2 but not -α1. By providing a mechanistic model for this behaviour, we have, for the first time, defined a series of crosslinking parameters to systematically control the interaction of collagen-based materials with defined cellular receptors.

Potential-Responsive Surfaces for Manipulation of Cell Adhesion, Release, and Differentiation.

In living systems, interfacial molecular interactions control many biological processes. New stimuli-responsive strategies are desired to provide versatile model systems that can regulate cell behavior in vitro. Described here are potential-responsive surfaces that control cell adhesion and release as well as stem cell differentiation. Cell adhesion can be modulated dynamically by applying negative and positive potentials to surfaces functionalized with tailored monolayers. This process alters cell morphology and ultimately controls behavior and the fate of the cells. Cells can be detached from the electrode surface as intact clusters with different geometries using electrochemical potentials. Importantly, morphological changes during adhesion guide stem cell differentiation. The higher accessibility of the peptide under a positive applied potential causes phenotypic changes in the cells that are hallmarks of osteogenesis, whereas lower accessibility of the peptide promoted by negative potentials leads to adipogenesis.

Potential‐Responsive Surfaces for Manipulation of Cell Adhesion, Release, and Differentiation

AbstractIn living systems, interfacial molecular interactions control many biological processes. New stimuli‐responsive strategies are desired to provide versatile model systems that can regulate cell behavior in vitro. Described here are potential‐responsive surfaces that control cell adhesion and release as well as stem cell differentiation. Cell adhesion can be modulated dynamically by applying negative and positive potentials to surfaces functionalized with tailored monolayers. This process alters cell morphology and ultimately controls behavior and the fate of the cells. Cells can be detached from the electrode surface as intact clusters with different geometries using electrochemical potentials. Importantly, morphological changes during adhesion guide stem cell differentiation. The higher accessibility of the peptide under a positive applied potential causes phenotypic changes in the cells that are hallmarks of osteogenesis, whereas lower accessibility of the peptide promoted by negative potentials leads to adipogenesis.

Subtle Regulation of Scaffold Stiffness for the Optimized Control of Cell Behavior.

The rigidity of extracellular matrices can impact cell fate, guide tissue development, and initiate tumor formation. Scaffolds such as hydrogels with tunable levels of stiffness have been developed to control cell adhesion, migration, and differentiation, providing suitable microenvironments for different tissue outcomes. However, studies of cell-material interactions are largely confined to biomaterials with stiffness values that are coarsely regulated, so refinements in sensitive cellular responses and optimal stiffness values that determine cell fate remain elusive. Here, a freezing temperature, as a tunable regulating factor, was introduced to freeze-drying processes to form silk fibroin (SF) scaffolds with refined control of stiffness values. Due to this control of intermediate structural conformations of SF, the scaffolds exhibited differences in stiffness values to permit refined assessments of impact on cell behavior on cell-friendly surfaces. Both in vitro and in vivo results with these scaffolds exhibited gradually changeable cell migration and differentiation outcomes, as well as differences in tissue ingrowth, demonstrating the sensitivity of cellular responses to such refined mechanical cues. The optimal vascularization capacity of these SF scaffolds was in the 3-7.4 kPa range, suggesting a key range to develop bioactive biomaterials. Systematic fine regulation of scaffold rigidity based on the present strategy provides a platform for an improved understanding of cell-material interactions and also for generating optimized microenvironments for tissue regeneration.