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Microgel Film Dynamics Modulate Cell Adhesion Behavior
Abstract
A material's mechanical properties greatly control cell behavior at the cell-substrate interface. In this work, we demonstrate that microgel multilayers have unique elastic and viscoelastic-like properties that can be modulated to produce morphological changes in fibroblasts cultured on the film. Protein adsorption is also examined and the data are contrasted with the number of cells adhered. The dynamic interaction of cell and substrate is only partially explained by conventional understanding of surface-receptor interactions and substrate elasticity. Viscoelasticity, a mechanical property not often considered, plays a significant role at cellular length and time scales for microgel films.
... 1,2 Matrix mechanics have been proven to be an important regulator for cellular behavior and function, including cell adhesion, proliferation, migration, and differentiation. 3−7 Engler et al. 8 have demonstrated that human mesenchymal stem cells (MSCs) can differentiate into neurons, myoblasts, or osteoblasts when they are cultured on collagencoated polyacrylamide (PA) hydrogels with stiffness similar to that of the brain (0.1−1 kPa), muscle (8)(9)(10)(11)(12)(13)(14)(15)(16)(17), or collagenous bone (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Recently, Yang et al. 9 also found that a one-week culture of MSCs on rigid surfaces (10 kPa) promotes nuclear runx2 expression that remains active after the surrounding matrix is softened. ...
... The proportion of collagen plays an important role in determining the mechanical property of the matrix. 33 To increase the matrix stiffness, the proportion of collagen in the collagen/HA mixture was increased in our experiment. As shown in Table 1, the compressive elastic modulus of collagen/HA mixtures were 6.74 ± 1.16 kPa, 8.82 ± 2.12 kPa, and 23.61 ± 8.06 kPa. ...
... Above this approximate plateau, NIH3T3 fibroblasts show little variation in adhesion characteristic regardless of E. However, below this threshold, fibroblast cells have extraordinary sensitivity to the E value. 33 Similarly, the characteristic of cell differentiation is perhaps to have a plateau for the scaffold stiffness. In our study, the E of the control group (decellularized bone scaffold without coating) is about 1.46 ± 0.44 GPa measured by nanoindentation testing (Agilent Technologies, NANO Indenter G200, USA), which is far beyond the elasticity threshold that MSCs can sense. ...
A growing body of evidence has shown that extracellular matrix (ECM) stiffness can modulate stem cell adhesion, proliferation, migration, differentiation and signaling. Stem cells can feel and respond sensitively to the mechanical microenvironment of the ECM. However, most studies have focused on classical two-dimensional (2D) or quasi-three-dimensional environment, which cannot represent the real situation in vivo. Furthermore, most the current methods used to generate different mechanical properties invariably change the fundamental structural properties of the scaffolds (such as morphology, porosity, pore size, and pore interconnectivity). In this study, we have developed a novel three-dimensional (3D) scaffolds with different degrees of stiffness, but the same 3D microstructure, that was maintained by using decellularized cancellous bone. Mixtures of collagen and hydroxyapatite [HA: Ca10(PO4)6(OH)2] with different proportions were coated on decellularized cancellous bone to vary the stiffness (local stiffness: 13.00±5.55 kPa, 13.87±1.51 kPa, and 37.7±19.6 kPa; bulk stiffness: 6.74±1.16 kPa, 8.82±2.12 kPa, and 23.61±8.06 kPa). Micro computed tomography (μ-CT) assay proved that there was no statistically significant difference in the architecture of the scaffolds before or after coating. Cell viability, osteogenic differentiation, cell recruitment and angiogenesis were determined to characterize the scaffolds and evaluate their biological responses in vitro and in vivo. The in vitro results indicate that the scaffolds developed in this study could sustain adhesion and growth of rat mesenchymal stem cells (MSCs) and promote their osteogenic differentiation. The in vivo results further demonstrated that these scaffolds could help to recruit MSCs from subcutaneous tissue, induce them to differentiate into osteoblasts, and provide the 3D environment for angiogenesis. These findings showed that the method we developed can build scaffolds with tunable mechanical properties almost without variation in 3D microstructure. These preparations not only can provide a cell-free scaffold with optimal matrix stiffness to enhance osteogenic differentiation, cell recruitment and angiogenesis in bone tissue engineering, but also have significant implications for studies on the effects of matrix stiffness on stem cells differentiation in 3D environment.
... They have useful features such as temperature-, pH-, salinityand concentration-dependent 1 swelling behavior as well as elasticity and exibility. This renders them as good candidates for various applications such as drug delivery agents, 2-4 the engineering of tissues, [5][6][7][8] and the modication of rheological properties. 9 The elasticity of microgels can be controlled, e.g., by the amount of crosslinker, and the length of polymer chains used in the synthetization process. ...
... The accuracy of the analytic scaling formula in eqn (26) in combination with eqn (27) for the short-time self-diffusion coefficient of spherical annulus particle systems is established in Fig. 6 by the comparison with high-precision benchmark simulation data for the spherical annulus 21 and uniformly permeable particle models. 17 Regarding the latter model, the conversion of l x to the related reduced hydrodynamic radius parameter g in the spherical annulus model was done using eqn (8). This corresponds to the inversion of the curve for l t x in Fig. 4 in terms of g. ...
... From Fig. 10, the good agreement of the semi-empirical formula for K(f, g) in eqn (32) with the spherical annulus simulation data 21 is noticed for g $ 0.8. The gure depicts also simulation data for permeable spheres where l x has been converted to the respective g using eqn (8). Moreover, results for 9 Concentration dependence of the scaling function u K (f, g) associated with the sedimentation coefficient of spherical annulus particles, for values of g as indicated. ...
We present an easy-to-use analytic toolbox for the calculation of short-time transport properties of concentrated suspensions of spherical colloidal particles with internal hydrodynamic structure, and direct interactions described by a hard-core or soft Hertz pair potential. The considered dynamic properties include self-diffusion and sedimentation coefficients, the wavenumber-dependent diffusion function determined in dynamic scattering experiments, and the high-frequency shear viscosity. The toolbox is based on the hydrodynamic radius model (HRM) wherein the internal particle structure is mapped on a hydrodynamic radius parameter for unchanged direct interactions, and on an existing simulation data base for solvent-permeable and spherical annulus particles. Useful scaling relations for the diffusion function and self-diffusion coefficient, known to be valid for hard-core interaction, are shown to apply also for soft pair potentials. We further discuss extensions of the toolbox to long-time transport properties including the low-shear zero-frequency viscosity and the long-time self-diffusion coefficient. The versatility of the toolbox is demonstrated by the analysis of a previous light scattering study of suspensions of non-ionic PNiPAM microgels [Eckert et al., J. Chem. Phys., 2008, 129, 124902] in which a detailed theoretical analysis of the dynamic data was left as an open task. By the comparison with Hertz potential based calculations, we show that the experimental data are consistently and accurately described using the Verlet-Weis corrected Percus-Yevick structure factor as input, and for a solvent penetration length equal to three percent of the excluded volume radius. This small amount of solvent permeability of the microgel particles has a significant dynamic effect at larger concentrations.
... They have useful features such as temperature-, pH-, salinity-and concentrationdependent [1] swelling behavior as well as elasticity and flexibility. This renders them as good candidates for various applications such as drug delivery agents [2][3][4], the engineering of tissues [5][6][7][8], and the modification of rheological properties [9]. The elasticity of microgels can * j.riest@fz-juelich.de ...
... Eqs. (8)(9)(10)(11) can be inverted to obtain the materialspecific parameters λ x and l * s in terms of the ratio ...
... The figure depicts also simulation data for permeable spheres where λ x has been converted to the respective γ using Eq. (8). Moreover, results for K(φ, γ) are displayed as predicted by the self-part corrected δγ scheme with VW-PY structure factor input where the self-part contribution to K was calculated according to Eq. (26). ...
We present an easy-to-use analytic toolbox for the calculation of short-time
transport properties of concentrated suspensions of spherical colloidal
particles with internal hydrodynamic structure, and direct interactions
described by a hard-core or soft Hertz pair potential. The considered dynamic
properties include self-diffusion and sedimentation coefficients, the
wavenumber-dependent diffusion function determined in dynamic scattering
experiments, and the high-frequency shear viscosity. The toolbox is based on
the hydrodynamic radius model (HRM) wherein the internal particle structure is
mapped on a hydrodynamic radius parameter for unchanged direct interactions,
and on an existing simulation data base for solvent-permeable and spherical
annulus particles. Useful scaling relations for the diffusion function and
self-diffusion coefficient, known to be valid for hard-core interaction, are
shown to apply also for soft pair potentials. We further discuss extensions of
the toolbox to long-time transport properties including the low-shear
zero-frequency viscosity and the long-time self-diffusion coefficient. The
versatility of the toolbox is demonstrated by the analysis of a previous light
scattering study of suspensions of non-ionic PNiPAM microgels [Eckert et al.,
J. Chem. Phys., 2008, 129, 124902] in which a detailed theoretical analysis of
the dynamic data was left as an open task. By the comparison with Hertz
potential based calculations, we show that the experimental data are
consistently and accurately described using the Verlet-Weis corrected
Percus-Yevick structure factor as input, and for a solvent penetration length
equal to three percent of the excluded volume radius. This small solvent
permeability of the microgel particles has a significant dynamic effect at
larger concentrations.
... Saxena et al. investigated self-healing microgel multilayers atop PDMS substrates coated with aminopropyltrimethoxysilane (APTMS). [94] The deformability of the PDMS substrate allowed exploring the effects of stretching on the recovery of multilayers with different thicknesses and crosslinking methods. ...
... Saxena et al. employed a combined approach to microgel adhesion to investigate the self-healing behavior of multilayered microgel system: the first layer of P(NIPAm-co-AA) microgels was covalently anchored to the surface of PDMS functionalized with APTMS, while subsequent strata were deposited via electrostatic LbL assembly, by alternating anionic and cationic polymers PEI and PDADMAC. [94] Deformation of the flexible PDMS substrate disrupted the multilayer without removing the basal covalently bound monolayer, allowing the team to probe the self-healing behavior of the rearrangeable upper layers at various values of strain (0-60%) and pH (3-7.4). Ramesh et al. repaired damaged polypropylene (PP) nonwoven fibermats with P(NIPAm-co-AA) microgels by co-depositing the microgels with a P(NIPAm-co-benzophenone acrylamide) copolymer. ...
This study presents a comprehensive survey of microgel-coated materials and their functional behavior, describing the complex interplay between the physicochemical and mechanical properties of the microgels and the chemical and morphological features of substrates. The cited literature is articulated in four main sections: i) properties of 2D and 3D substrates, ii) synthesis, modification, and characterization of the microgels, iii) deposition techniques and surface patterning, and iv) application of microgel-coated surfaces focusing on separations, sensing, and biomedical applications. Each section discusses – by way of principles and examples – how the various design parameters work in concert to deliver functionality to the composite systems. The case studies presented herein are viewed through a multi-scale lens. At the molecular level, the surface chemistry and the monomer make-up of the microgels endow responsiveness to environmental and artificial physical and chemical cues. At the micro-scale, the response effects shifts in size, mechanical, and optical properties, and affinity towards species in the surrounding liquid medium, ranging from small molecules to cells. These phenomena culminate at the macro-scale in measurable, reversible, and reproducible effects, aiming in a myriad of directions, from lab-scale to industrial applications.
... These are produced by deposition, usually layer-by-layer, of discrete hydrogel particles (microgels) with sizes ranging from the nanometers to micrometers. [105,106] An earlier report showed that their viscoelastic behavior, beside granting them the ability to self-heal after stretching in the absence of covalent crosslinking of the film, affected cell behavior. [105] This was further addressed by Chester and co-workers, who designed a family of layer-by-layer microgel thin films with similar elastic modulus (≈107 kPa) but decreasing loss tangent (from 1.8 to 0.8) by tuning the internal crosslinking of the particles. ...
... [105,106] An earlier report showed that their viscoelastic behavior, beside granting them the ability to self-heal after stretching in the absence of covalent crosslinking of the film, affected cell behavior. [105] This was further addressed by Chester and co-workers, who designed a family of layer-by-layer microgel thin films with similar elastic modulus (≈107 kPa) but decreasing loss tangent (from 1.8 to 0.8) by tuning the internal crosslinking of the particles. [106] The loss tangent of the films was found to be the main regulator in fibroblast migration on these viscoelastic films: on more dissipative substrates, an increase in ROCK activity prompted amoeboid migration, with ellipsoid cells loosely attached to the substrate. ...
Cell mechanotransduction is an area of intense research focus. Until now, very limited tools have existed to study how cells respond to changes in the extracellular matrix beyond, for example, mechanical deformation studies and twisting cytometry. However, emerging are a range of elastic, viscoelastic and even purely viscous materials that deform and dissipate on cellular length and timescales. This article reviews developments in these materials, typically translating from 2D model surfaces to 3D microenvironments and explores how cells interact with them. Specifically, it focuses on emerging concepts such as the molecular clutch model, how different extracellular matrix proteins engage the clutch under viscoelastic‐stress relaxation conditions, and how mechanotransduction can drive transcriptional control through regulators such as YAP/TAZ.
... Although stiffness (or elastic modulus) 66,69,70 has been well explored, loss modulus (also called viscous modulus) 64 Outside-in and inside-out pathway have been studied as two common features for the mutual interaction between cells and their microenvironment. 39 In other words, the substrate mechanics influence cell mechanics, and simultaneously cell mechanics can in turn remodel the substrate and tune its mechanics. ...
In order to achieve successful tissue engineering, biomaterials need to possess some essential properties such as porosity, mechanical strength, biocompatibility and immune acceptance. Freeze casting has been proved as a good approach for making porous scaffolds due to its high processing biocompatibility and the resultant high porosity (Chapter 1). In Chapter 2, we applied freeze casting to reverse engineer silk matrix with separated silk fibroin and silk sericin in order to obtain antibacterial dermal patches. Freeze casting also enables encapsulation (or cryopreservation) of cells and tissues. In Chapter 3, a combination of directional freezing under confocal microscope and differential scanning calorimetry analysis were performed to reveal surrounding environment of Saccharomyces cerevisiae during freezing and in situ interactions between cells and freezing front. This technique was also proved useful for red blood cell cryopreservation in presence of albumin (Chapter 4). In conclusion, freeze casting/directional freezing enabled antibacterial matrix fabrication and also cryopreservation of yeast and red blood cells.
... Under this scenario, Langmuir monolayers are a versatile and well-established methodology to elucidate the structural and mechanical properties of biomolecules interacting with different systems [33,34]. In this sense, microgel particles at interfaces are currently being intensively studied for their potential use as emulsion stabilizers [35][36][37][38] as well as for cell adhesion and for haptics as tactile feedback devices [39][40][41]. However, as far as we know, there are no studies in which Langmuir monolayers have been used to study the complexation of DNA by microgels at the air-water interface. ...
Micro- and nanogels are being increasingly used to encapsulate bioactive compounds. Their soft structure allows large loading capacity while their stimuli responsiveness makes them extremely versatile. In this work, the complexation of DNA with thermoresponsive microgels is presented. To this end, PEGylated charged microgels based on poly-N-isopropylacrylamide have been synthesized, allowing one to explore the electrostatics of the complexation. Cationic microgels complexate spontaneously by electrostatic attraction to oppositely charged DNA as demonstrated by electrophoretic mobility of the complexes. Then, Langmuir monolayers reveal an increased interaction of DNA with swollen microgels (20 °C). Anionic microgels require the presence of multivalent cations (Ca2+) to promote the complexation, overcoming the electrostatic repulsion with negatively charged DNA. Then again, Langmuir monolayers evidence their complexation at the surface. However, the presence of Ca2+ seems to induce profound changes in the interaction and surface conformation of anionic microgels. These alterations are further explored by measuring adsorbed films with the pendant drop technique. Conformational changes induced by Ca2+ on the structure of the microgel can ultimately affect the complexation with DNA and should be considered in the design. The combination of microstructural and surface properties for microgels offers a new perspective into complexation of DNA with soft particles with biomedical applications.
... However, external crosslinking can also be performed by utilizing EDC and NHS coupling chemistry to crosslink the carboxyl groups found in the AAc within the particle to the amine groups found in the PEI that diffuses in and out of the microgel particles and between layers. 26,27 In the absence of external crosslinking, changing the internal crosslinking density by altering the amount of BIS used during particle polymeriza- Figure 1C and equations are presented in Table S3. ...
Cells maintain tensional homeostasis by monitoring the mechanics of their microenvironment. In order to understand this mechanotransduction phenomenon, hydrogel materials have been developed with either controllable linear elastic or viscoelastic properties. Native biological tissues, and biomaterials used for medical purposes, often have complex mechanical properties. However, due to the difficulty in completely decoupling the elastic and viscous components of hydrogel materials, the effect of complex composite materials on cellular responses has largely gone unreported. Here, we characterize a novel composite hydrogel system capable of decoupling and individually controlling both the bulk stiffness and surface viscoelasticity of the material by combining polyacrylamide (PA) gels with microgel thin films. By taking advantage of the high degree of control over stiffness offered by PA gels and viscoelasticity, in terms of surface loss tangent, of microgel thin films, it is possible to study the influence that bulk substrate stiffness and surface loss tangent have on complex fibroblast responses, including cellular and nuclear morphology and gene expression. This material system provides a facile method for investigating cellular responses to complex material mechanics with great precision and allows for a greater understanding of cellular mechanotransduction mechanisms than previously possible through current model material platforms. A novel composite hydrogel system capable of decoupling and individually controlling both the bulk stiffness and surface viscoelasticity of the material by combining polyacrylamide gels with microgel thin films is described. Fibroblast cellular and nuclear morphological changes and changes in gene expression in response to a wide range of these parameters are characterized.
... However, these coatings were found to be unstable under physiological conditions since the contact point between the particle and surface is limited [270]. From this point of view, nanogels, as being soft deformable colloidal particles, can stick to the surface firmly and create remarkably homogenous coatings [271,272]. Nanogel-based surface coatings can be easily prepared by physical adsorption without requiring any harsh chemical procedures such as grafting or covalent binding [268]. Besides, the characteristics of the nanogel-coated surface can be simply modified by just modulating the chemical functionalities of the adsorbed nanogel particles. ...
The implementation of nanotechnology to develop efficient antimicrobial systems has a significant impact on the prospects of the biomedical field. Nanogels are soft polymeric particles with an internally cross-linked structure, which behave as hydrogels and can be reversibly hydrated/dehydrated (swollen/shrunken) by the dispersing solvent and external stimuli. Their excellent properties, such as biocompatibility, colloidal stability, high water content, desirable mechanical properties, tunable chemical functionalities, and interior gel-like network for the incorporation of biomolecules, make them fascinating in the field of biological/biomedical applications. In this review, various approaches will be discussed and compared to the newly developed nanogel technology in terms of efficiency and applicability for determining their potential role in combating infections in the biomedical area including implant-associated infections.
... Even though PEGDA possesses great properties, such as good biocompatibility, low immunogenicity, ideal 3D porous structure, and great water absorption, it is widely known to resist cell adhesion due to its highly hydrophilic nature. 93−96 Multiple methods have been adopted to improve PEGDA hydrogels' adhesion properties, for example, by adding positively charged monomer 2-(methacryloyloxy)ethyl-trimethylammonium chloride (MAE-TAC), 97 arginine-glycine-aspartic (RGD) peptide, 98,99 and sodium methallyl sulfonate (SMAS). 57 PEGDA has also been blended with other materials such as PCL due to its predominant viscoelastic properties, exceptional cytocompatibility, and relatively inexpensive production costs to improve the scaffold's properties. ...
The fabrication of scaffolds that precisely mimic the natural structure and physiochemical properties of bone is still one of the most challenging tasks in bone tissue engineering. 3D printing techniques have drawn increasing attention due to their ability to fabricate scaffolds with complex structures and multiple bioinks. For bone tissue engineering, lithography-based 3D bioprinting is frequently utilized due to its printing speed, mild printing process, and cost-effective benefits. In this review, lithography-based 3D bioprinting technologies including SLA and DLP are introduced; their typical applications in biological system and bioinks are also explored and summarized. Furthermore, we discussed possible evolvement of the hardware/software systems and bioinks of lithography-based 3D bioprinting, as well as their future applications.
... Generally, the basic biological functionality of the 3D matrix can be achieved using natural biopolymers (e.g., collagen and chitosan) and synthetic biodegradable polymers (e.g., PEG and fibroin), which ensure the biocompatibility and adhesivity of the system. The 3D substrate should be porous with sufficient support strength for ensuring the exchange of nutrients and waste between the system and external matrix 126 . In previous studies, electrospun cross-linked LPEI was used to synthesize 3D scaffold for investigating the growth behavior of normal human fibroblast (NHF) cells 127 . ...
Cationic polymers, known for their highly positive charges, have historically dominated the materials used in bioengineering. However, the demand for intelligent systems with high efficiency, bio-mimetic and tunable features is increasing. Artificial composites that mimic the biorecognition and periodic structures may propel the development of advanced materials with outstanding properties. Polyethyleneimines (PEIs) constitute a valuable class of polycations because they have repetitive structural units, a wide molecular weight range and flexible polymeric chains, which facilitate customization of functional composites. Specific advantageous features could be introduced by purposeful modification or functionalization, such as the specificity and sensitivity, distinct geometry, biocompatibility, and long service life. Thus, PEIs have been rapidly used in a wide range of applications in the fields of biomedicine, biotechnology and biomaterial science. This article provides an overview of recent advancements in the fabrication of PEI-based materials and corresponding applications in gene and drug delivery, bioinhibitors, bio-separation, bioimaging, cell culture, and production of antibacterial and self-healing materials. The effects of molecular weight, topological structure, positive charges and hydrophilic properties on the performance of PEIs have been illustrated in detail. Finally, current technological limitations, research challenges, and future aspects are also discussed.
... While natural hydrogels are broadly implemented in cell culture, the biological impacts of viscoelasticity have just recently gained attention, and important and unexpected biological impacts have been reported [178,179] . Many cellular behaviors, including focal adhesion formation, cell spreading, MSC differentiation, cell migration and ECM synthesis, have been found to be regulated by matrix viscoelasticity rather than the initial elastic modulus as discussed in detail below ( Fig. 4 ). ...
Viscoelasticity of living tissues plays a critical role in tissue homeostasis and regeneration, and its implication in disease development and progression is being recognized recently. In this review, we first explored the state of knowledge regarding the potential application of tissue viscoelasticity in disease diagnosis. In order to better characterize viscoelasticity with local resolution and non-invasiveness, emerging characterization methods have been developed with the potential to be supplemented to existing facilities. To understand cellular responses to matrix viscoelastic behaviors in vitro, hydrogels made of natural polymers have been developed and the relationships between their molecular structure and viscoelastic behaviors, are elucidated. Moreover, how cells perceive the viscoelastic microenvironment and cellular responses including cell attachment, spreading, proliferation, differentiation and matrix production, have been discussed. Finally, some future perspective on an integrated mechanobiological comprehension of the viscoelastic behaviors involved in tissue homeostasis, cellular responses and biomaterial design are highlighted.
Statement of Significance
Tissue- or organ-scale viscoelastic behavior is critical for homeostasis, and the molecular basis and cellular responses of viscoelastic materials at micro- or nano-scale are being recognized recently. We summarized the potential applications of viscoelasticity in disease diagnosis enabled by emerging non-invasive characterization technologies, and discussed the underlying mechanism of viscoelasticity of hydrogels and current understandings of cell regulatory functions of them. With a growing understanding of the molecular basis of hydrogel viscoelasticity and recognition of its regulatory functions on cell behaviors, it is important to bring the clinical insights on how these characterization technologies and engineered materials may contribute to disease diagnosis and treatment. This review explains the basics in characterizing viscoelasticity with our hope to bridge the gap between basic research and clinical applications.
... Their ability to react to external stimuli like pH value, solvent composition, ionic strength or temperature by rapidly changing their particle size granted microgels the attribute ''smart''. 1 Considering the numerous potential applications for microgels, 2 e.g. for drug delivery, 3-6 intelligent coatings 7,8 and membranes, 9,10 as emulsifiers, [11][12][13] sensors [14][15][16][17] or as carriers of nanoparticles, [18][19][20][21] tailoring the microgel properties has become important since the early 2000's. ...
Thermo- and pH-responsive copolymer microgels were obtained by surfactant-assisted precipitation poylmerization of N-isopropylacrylamide (NiPAM) and acrylic acid (AAc). The surfactants used were sodium dodecylsulfate (SDS), dodecyltrimethylammonium bromide (DTAB) and the nonionic n-octyl-b-D-glucopyranoside (C⁸G¹). We investigate the influence of the surfactants on the acrylic acid incorporation rate, the particle size, particle morphology, and the swelling behaviour at pH 4 and pH 7, at which AAc is neutral or charged, respectively. It is shown that each surfactant has a specific influence, which is connected to its role in the polymerization mechanism and its charge. A combined FTIR and PCS study reveals that the particles undergo a temperature-induced change in microstructure, even if the particle hydrodynamic radius does not change significantly.
... In our study, there was almost no expression of OC and OPN in MSCs on the decellularized bone scaffold (indicated in Fig. 5). The elastic modulus of the decellularized bone scaffold selected in this study was (230.93 ± 72.65) MPa, which far exceeded the matrix stiffness threshold which MSCs can sense [46]. ...
... LbL films with different surface chemistries have shown diverse influences on cell adhesion. LbL films can be made to exhibit cytophobicity by the use of nonfouling groups or molecules such as PEG [237,238], phosphorylcholine (PC) groups [239,240], and zwitterionic groups [241,242]. In contrast, by using building blocks with particular chemical components, cell adhesion could be effectively increased [243,244]. ...
Cellular behavior is crucially dependent on the biophysical and biochemical properties of the extracellular matrix (ECM), in which the properties of biochemistry, topography, and mechanics are critically important and dominantly studied. Since its introduction by Decher and Lvov in the early 1990s, layer-by-layer (LbL) assembly technology has received considerable interest for constructing polymeric thin films in both academic and industrial studies. The technology has been especially important in applications involving biomedical materials, tissue engineering, and regenerative medicine. In recent years, because of outstanding flexibility and multipotency, polymeric LbL thin films have been extensively studied to create a biomimetic cellular microenvironment with one or more biophysical and biochemical properties. The field has moved from simple mimicking to active control of various cellular behaviors. This review first introduces the basic background of the natural cellular microenvironment, the LbL assembly, and progress in polymeric LbL thin films. Next, biomimetic films constructed using the LbL technique are introduced. The biochemical components, topographical features, and mechanical properties of the films are detailed. Furthermore, progress in thin LbL films for controlling cell behavior, such as cell adhesion, stem cell differentiation, and cell-cell interactions, are highlighted. Finally, the review closes with a summary and a brief outlook of the opportunities and challenges associated with polymeric LbL thin films for advancing promising future developments.
... Various modifications have been applied to improve cell attachment onto PEGDA. For example, arginine-glycine-aspartic (RGDS) immobilization into PEGDA could promote cell attachment on the hydrogel [7]. However, the process of bone development is inherently dependent on the interaction between osteoblasts and extracellular matrix (ECM) proteins. ...
To improve the biological performance of poly(ethylene glycol)-diacrylate (PEGDA) hydrogel as an injectable bone grafting scaffold, sodium methallyl sulphonate (SMAS) was incorporated into PEGDA hydrogel. The physiochemical properties of the resultant polymers were assessed via Fourier transform infrared spectroscopy (FTIR), swelling ratio, zeta potential, surface morphology, and protein adsorption analysis. MC3T3-E1 cells were seeded on the hydrogel to evaluate the effect of the sulphonated modification on their attachment, proliferation, and differentiation. The results of FTIR and zeta potential evaluations revealed that SMAS was successfully incorporated into PEGDA. With increasing concentrations of SMAS, the swelling ratio of the hydrogels increased in deionized water but stayed constant in phosphate buffered saline. The protein adsorption also increased with increasing concentration of SMAS. Moreover, the sulphonated modification of PEGDA hydrogel not only enhanced the attachment and proliferation of osteoblast-like MC3T3-E1 cells but also up-regulated alkaline phosphatase activity as well as gene expression of osteogenic markers and related growth factors, including collagen type I, osteocalcin, runt related transcription factor 2, bone morphogenetic protein 2, and transforming growth factor beta 1. These findings indicate that the sulphonated modification could significantly improve the biological performance of PEGDA hydrogel. Thus, the sulphonated PEGDA is a promising scaffold candidate for bone grafting.
... To elaborate, with each amine addition, a trifunctional acrylate molecule became a difunctional acrylate molecule by reacting with the carbon double bond on the TMPeTA monomer [28]. This finding is consistent with the results from swelling ratio study (supplementary 3) as well as other studies wherein the higher the average functionality of a polymer, the higher the overall crosslinking density of the network [29][30][31] which explains a decrease in Young's modulus by increasing the DEA concentration in both TMPeTA (692) and TMPeTA (912) samples. Stiffness does not seem to play a significant role in cell attachment in this study. ...
Thiol-acrylate polymers have therapeutic potential as biocompatible scaffolds for bone tissue regeneration. Synthesis of a novel cyto-compatible and biodegradable polymer composed of trimethylolpropane ethoxylate triacrylate-trimethylolpropane tris (3-mercaptopropionate) (TMPeTA-TMPTMP) using a simple amine-catalyzed Michael addition reaction is reported in this study. This study explores the impact of molecular weight and crosslink density on the cyto-compatibility of human adipose derived mesenchymal stem cells. Eight groups were prepared with two different average molecular weights of trimethylolpropane ethoxylate triacrylate (TMPeTA 692 and 912) and four different concentrations of diethylamine (DEA) as catalyst. The materials were physically characterized by mechanical testing, wettability, mass loss, protein adsorption and surface topography. Cyto-compatibility of the polymeric substrates was evaluated by LIVE/DEAD staining® and DNA content assay of cultured human adipose derived stem cells (hASCs) on the samples over over days. Surface topography studies revealed that TMPeTA (692) samples have island pattern features whereas TMPeTA (912) polymers showed pitted surfaces. Water contact angle results showed a significant difference between TMPeTA (692) and TMPeTA (912) monomers with the same DEA concentration. Decreased protein adsorption was observed on TMPeTA (912) -16% DEA compared to other groups. Fluorescent microscopy also showed distinct hASCs attachment behavior between TMPeTA (692) and TMPeTA (912), which is due to their different surface topography, protein adsorption and wettability. Our finding suggested that this thiol-acrylate based polymer is a versatile, cyto-compatible material for tissue engineering applications with tunable cell attachment property based on surface characteristics.
... 15 Furthermore, microgels have been used alone to fabricate thin film substrates or as constitutive units combined with other polyelectrolytes. 16 For practical in vivo applications, particularly long-time ones, the approval of any of the reagents employed in the synthesis due to their possible toxicity is a comprehensible limitation. ...
The modulation of cell adhesion via biologically inspired materials plays a key role in the development of realistic platforms to envisage not only mechanistic descriptions of many physiological and pathological processes but also new biointerfacial designs compatible with the requirements of biomedical devices. In this work, we show that the cell adhesion and proliferation of three different cell lines can be easily manipulated by using a novel biologically inspired supramolecular coating generated via dip coating of the working substrates in an aqueous solution of polyallylamine in the presence of phosphate anions−a simple one-step modification procedure. Our results reveal that selective cell adhesion can be controlled by varying the deposition time of the coating. Cell proliferation experiments showed a cell type-dependent quasi-exponential growth demonstrating the nontoxic properties of the supramolecular platform. After reaching a certain surface coverage, the supramolecular films based on phosphate-polyamine networks displayed antiadhesive activity towards cells, irrespective of the cell type. However and most interestingly, these antiadherent substrates developed strong adhesive properties after thermal annealing at 37 ºC for 2-3 days. These results were interpreted based on the changes in the coating hydrophilicity, topography and stiffness, with the latter being assessed by atomic force microscopy imaging and indentation experiments. The reported approach is simple, robust and flexible, and offers opportunities for the development of tunable, biocompatible interfacial architectures to control cell attachment for various biomedical applications.
... This information is difficult to obtain by other means and is relevant for applications that rely on functional species encapsulated in the cavity of hollow microgels, for example, in sensors or functional coatings. 18,57,58 In addition, it should be noted that the kink is not observed above the VPTT. This indicates that the penetration is limited by the dense network at high temperatures and the probe mostly compresses the collapsed microgels. ...
Microgels composed of thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAM) are interfacial active. Their adsorption leads to deformation causing conformational changes that have profound effects on the macroscopic properties of these films. Yet, methods to quantitatively probe the local density are lacking. We introduced scanning force microscopy (SFM) to quantitatively probe the internal structure of microgels physically adsorbed on a solid (SiO2) / water interface. Using a sharp SFM tip we investigated the two types of microgels, (i) core-shell microgels featuring a hard silica core and a PNIPAM shell, and (ii) hollow microgels obtained by dissolution of the silica core. Thus, both systems have the same polymer network as peripheral structure but a distinctly different internal structure i.e. a rigid core vs. a void. Evaluation of the force-distance curves, the force profile during insertion of the tip into the polymer network enables to determine a depth dependent contact resistance which closely correlates with the density profiles determined in solution by small-angle neutron scattering (SANS). We found that the cavity of the swollen hollow microgels is still present when adsorbed to the solid substrate. Remarkably, while currently used techniques such as colloidal probe or reflectometry only provide an average of the z-profile, the methodology introduced herein actually probes the real three dimensional density profile, which is ultimately important to understand the macroscopic behavior of microgel films. This will bridge the gap between the colloidal probe experiments that deform the microgel globally and the insertion in which the disturbance is located near the tip.
... For example, microgel particles have been designed as drug delivery vehicles [3][4][5], enzyme supports [6,7], biosensors [8,9], and hemostatic agents [10]. Recently, microgels have also been used with great success as building blocks in a bottom-up approach to develop responsive surface coatings [11][12][13], self-healing materials [14], and tissue engineering scaffolds [15,16]. ...
We present a systematic study of self-cross-linked microgels formed by precipitation polymerization of oligo ethylene glycol methacrylates. The cross-linking density of these microgels and, thus, the network flexibility can be easily tuned through the modulation of the reaction temperature during polymerization. Microgels prepared in absence of any difunctional monomer, i.e. cross-linker, show enhanced deformability and particle spreading on solid surfaces as compared to microgels cross-linked with varying amounts of poly(ethylene glycol diacrylate) (PEG-DA) in addition to self-crosslinking. Particles prepared at low reaction temperatures exhibit the highest degree of spreading due to the lightly cross-linked and flexible polymer network. Moreover, AFM force spectroscopy studies suggest that cross-linker-free microgels constitute of a more homogeneous polymer network than PEG-DA cross-linked particles and have elastic moduli at the particle apex that are ~5 times smaller than the moduli of 5 mol-% PEG-DA cross-linked microgels. Resistive pulse sensing experiments demonstrate that microgels prepared at 75 and 80°C without PEG-DA are able to deform significantly to pass through nanopores that are smaller than the microgel size. Additionally, we found that polymer network flexibility of microgels is a useful tool to control the formation of particle dewetting patterns. This offers a promising new avenue for build-up of 2D self-assembled particle structures with patterned chemical and mechanical properties.
... Cell adhesion to the surface of a synthetic biomaterial or a natural matrix is a complex process that involves integrin receptors in the cell membrane binding to surface-localized ECM proteins [12]. Because cellular adhesion is critical to most cell functions and tissue development, artificial biointerfaces are often functionalized with ECM proteins or related peptides to improve cell attachment. ...
The interaction between cancer cells and their microenvironment is an indispensable link in cancer progression that occurs on the interfaces between them and presents typical biointerfacial behavior. Recently, the cancer cell/microenvironment interface has begun to attract more attention because of its fundamental roles in cancer growth and metastasis, which is promising for the efficacy of anti-cancer drugs and other important effects. In this review, we focused on mechanical coupling of the biointerfaces and their application in cancer early diagnosis, the pharmacology of anticancer agents and the design of the anticancer drug carriers. Newly developed strategies for cancer therapy based on mechanical coupling, such as correcting cell mechanics defects, tunable rigidity for drug delivery and topography-coupled-mechanical drug design, and drug screening, provide a proof of concept that cell mechanics offer a rich drug target space, allowing for the possible corrective modulation of tumor cell behavior. Biomechanopharmacology is therefore important to recognize the biomechanical factors and to control them not only for improvement in our knowledge of cancer but also for the development of new drugs and new uses of old drugs.
... Nevertheless, the experimental procedures for PEG-RGDS conjugation is tedious and time-consuming. Other researchers have proposed an interesting method to fabricate maleic chitosan-PEGDA hybrid hydrogel through inserting acrylate functional groups on a polysaccharide by use of the well-known Michael-type reaction between the amines of chitosan and the double bonds of the diacrylate (Saxena et al. 2014). Another PEGDA surface modification method involves the hydroxyl carboxyl reaction. ...
Poly(ethylene glycol) diacrylate (PEGDA) is a common hydrogel that has been actively investigated for various tissue engineering applications owing to its biocompatibility and excellent mechanical properties. However, the native PEGDA films are known for their bio-inertness which can hinder cell adhesion, thereby limiting their applications in tissue engineering and biomedicine. Recently, nano composite technology has become a particularly hot topic, and has led to the development of new methods for delivering desired properties to nanomaterials. In this study, we added polystyrene nano-spheres (PS) into a PEGDA solution to synthesize a nano-composite film and evaluated its characteristics. The experimental results showed that addition of the nanospheres to the PEGDA film not only resulted in modification of the mechanical properties and surface morphology but further improved the adhesion of cells on the film. The tensile modulus showed clear dependence on the addition of PS, which enhanced the mechanical properties of the PEGDA-PS film. We attribute the high stiffness of the hybrid hydrogel to the formation of additional cross-links between polymeric chains and the nano-sphere surface in the network. The effect of PS on cell adhesion and proliferation was evaluated in L929 mouse fibroblast cells that were seeded on the surface of various PEGDA-PS films. Cells density increased with a larger PS concentration, and the cells displayed a spreading morphology on the hybrid films, which promoted cell proliferation. Impressively, cellular stiffness could also be modulated simply by tuning the concentration of nano-spheres. Our results indicate that the addition of PS can effectively tailor the physical and biological properties of PEGDA as well as the mechanical properties of cells, with benefits for biomedical and biotechnological applications.
The porous structure of microgels significantly influences their properties and, thus, their suitability for various applications, in particular as building blocks for tissue scaffolds. Porosity is one of the crucial features for microgel‐cell interactions and significantly increases the cells' accumulation and proliferation. Consequently, tailoring the porosity of microgels in an effortless way is important but still challenging, especially for non‐spherical microgels. This work presents a straightforward procedure to fabricate complex‐shaped poly(N‐isopropyl acrylamide) (PNIPAM) microgels with tuned porous structures using the so‐called cononsolvency effect during microgel polymerization. Therefore, the classical solvent in the reaction solution is exchanged from water to water‐methanol mixtures in a stop‐flow lithography process. For cylindrical microgels with a higher methanol content during fabrication, a greater degree of collapsing is observed, and their aspect ratio increases. Furthermore, the collapsing and swelling velocities change with the methanol content, indicating a modified porous structure, which is confirmed by electron microscopy micrographs. Furthermore, swelling patterns of the microgel variants occur during cooling, revealing their thermal response as a highly heterogeneous process. These results show a novel procedure to fabricate PNIPAM microgels of any elongated 2D shape with tailored porous structure and thermo‐responsiveness by introducing the cononsolvency effect during stop‐flow lithography polymerization.
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Although many investigations of thermoresponsive microgels have been reported, their surface properties, which are crucial in colloid science, are still not fully understood. In this study, microgels with surface-localized charged groups were synthesized by precipitation polymerization, and their electrophoretic behaviors were analyzed using a modified version of Ohshima's equation to obtain two surface properties of the soft particles: the softness parameter and the surface charge density. This systematic evaluation allows us to discuss the thermoresponsiveness of the overall microgels and their surfaces separately. Furthermore, the validity of the surface properties obtained from electrophoresis was verified by comparing them with the results of seeded emulsion polymerization in the presence of the microgels and the force-indentation curves obtained via high-speed atomic force microscopy (HS-AFM).
Herein, a new non-vinylic type of cationic microgels (MG) was readily prepared from ethylene glycol diglycidyl ether and Jeffamine T-403 in water. The MG was responsive to both temperature and pH, and oxidatively stable as demonstrated by the hydrogen peroxide study. Using glass as a model substrate, its surface was easily imparted with a heterogeneous morphology by simply adsorbing MG dispersed in basic solution. Specifically, the morphology features patches made of a monolayer of connected yet individually recognizable MG. Through in vitro cell studies, we show that a mere change of the extent of surface coverage by such a patchy morphology can strike a balance in promoting adhesion and differentiation of neuron-like PC-12 cells and primary cortical neurons of chick embryo, without soliciting proliferative response from non-neuronal cells of NIH3T3 fibroblast and CTX astrocyte. This simple yet unconventional surface morphology created by MG could be leveraged in the future as an alternative strategy for neural interface engineering.
Multifunctional nanogel coatings provide a promising antimicrobial strategy against biomedical implant-associated infections. Nanogels can create a hydrated surface layer to promote antifouling properties effectively. Further modification of nanogels with quaternary ammonium compounds (QACs) potentiates antimicrobial activity owing to their positive charges along with the presence of a membrane-intercalating alkyl chain. This study effectively demonstrates that poly(N-isopropylacrylamide-co-N-[3(dimethylamino)propyl]methacrylamide) (P(NIPAM-co-DMAPMA)-based nanogel coatings possess antifouling behavior against S. aureus ATCC 12600, a Gram-positive bacterium. Through the tertiary amine in the DMAPMA comonomer, nanogels are quaternized with a 1-bromo-dodecane chain via an N-alkylation reaction. The alkylation introduces the antibacterial activity due to the bacterial membrane binding and the intercalating ability of the aliphatic QAC. Subsequently, the quaternized nanogels enable the formation of intraparticle hydrophobic domains because of intraparticle hydrophobic interactions of the aliphatic chains allowing for Triclosan incorporation. The coating with Triclosan-loaded nanogels shows a killing efficacy of up to 99.99% of adhering bacteria on the surface compared to nonquaternized nanogel coatings while still possessing an antifouling activity. This powerful multifunctional coating for combating biomaterial-associated infection is envisioned to greatly impact the design approaches for future clinically applied coatings.
Material scaffolds that mimic the structure, function, and bioactivity of native biological tissues are in constant development. Recently, material scaffolds composed of microgel particles have shown promise for applications ranging from bone regeneration to spheroid cell growth. Previous studies with poly N-isopropylacrylamide microgel scaffolds utilized a layer-by-layer (LBL) technique where individual, uniform microgel layers are built on top of each other resulting in a multilayer scaffold. However, this technique is limited in its applications due to the inability to control microscale deposition or patterning of multiple particle types within a microgel layer. In this study, an ultrasonic microplotting technique is used to address the limitations of LBL fabrication to create patterned microgel films. Printing parameters, such as bioink formulation, surface contact angle, and print head diameter, are optimized to identify the ideal parameters needed to successfully print microgel films. It was found that bioinks composed of 2 mg/mL of microgels and 20% polyethylene glycol by volume (v/v), on bovine serum albumin-coated glass, with a print head diameter of 50 μm resulted in the highest quality prints. Patterned films were created with a maximum resolution of 50 μm with the potential for finer resolutions to be achieved with alternative bioink compositions and printing parameters. Overall, ultrasonic microplotting can be used to create more complex microgel films than is possible with LBL techniques and offers the possibility of greater printing resolution in 3D with further technology development.
Matrix stiffness has been demonstrated in many studies to adjust the biological behaviors of mesenchymal stem cells (MSCs). However, in the initial phase of bone restoration, MSCs will encounter a hypoxic microenvironment. Studying the connection existing between the matrix stiffness and biological behavior of MSCs under hypoxic condition can better simulate the microenvironment at the prime period of bone repairment. In this work, three-dimensional (3D) decalcified bone scaffolds with diverse stiffness [high stiffness (66.06 ± 27.83) MPa, medium stiffness (26.90 ± 13.16) MPa, and low stiffness (0.67 ± 0.14) MPa] but same microstructure have been prepared by controlling decalcification time. In addition, the decellularized bone scaffold was regard as control group and its stiffness was (230.93 ± 72.65) MPa. The viability, proliferation, infiltration, and osteogenic differentiation of MSCs seeded into these 3D demineralized bone scaffolds were systematically investigated under 100 μM CoCl2-simulated hypoxic and normoxic environments. The results showed that the viability, proliferation, and extracellular matrix (ECM) secretion of MSCs had no significant difference on scaffolds with diverse stiffness, but the degree of collagen deposition of MSCs gradually increased with the increase of scaffold stiffness both under normoxia and hypoxia. Compared to normoxia, the viability, proliferation, ECM secretion, vascular endothelial growth factor (VEGF) expression, and osteogenesis of MSCs on the scaffolds with the same stiffness were evidently inhibited by hypoxia. Additionally, under hypoxic condition, the expression of VEGF and hypoxia inducible factor 1α (HIF-1α) in MSCs on the low stiffness scaffold was markedly increased comparing to those on other groups. In summary, we found that the low-stiffness scaffold can improved the proliferation and osteogenic differentiation of MSCs under hypoxic environment, which may help to explore efficient methods for bone defect repairing.
Bacterial infection is a severe problem especially when associated with biomedical applications. This study effectively demonstrates that poly-N-isopropylmethacrylamide based microgel coatings prevent bacterial adhesion. The coating preparation via a spraying approach proved to be simple, and both cost and time efficient creating a homogeneous dense microgel monolayer. In particular, the influence of cross-linking density, microgel size, and coating thickness was investigated on the initial bacterial adhesion. Adhesion of Staphylococcus aureus ATCC 12600 was imaged using a parallel plate flow chamber setup, which gave insights in the number of the total bacteria adhering per unit area onto the surface and the initial bacterial deposition rates. All microgel coatings successfully yielded more than 98% reduction in bacterial adhesion. Bacterial adhesion depends both on the cross-linking density/stiffness of the microgels and on the thickness of the microgel coating. Bacterial adhesion decreased when a lower cross-linking density was used at equal coating thickness and at equal cross-linking density with a thicker microgel coating. The highest reduction in the number of bacterial adhesion was achieved with the microgel that produced the thickest coating (h = 602 nm) and had the lowest cross-linking density. The results provided in this paper indicate that microgel coatings serve as an interesting and easy applicable approach and that it can be fine-tuned by manipulating the microgel layer thickness and stiffness.
Cell behavior is influenced by the biophysical properties of their microenvironments, and the linear elastic properties of substrates strongly influences adhesion, migration, and differentiation responses. Because most biological tissues exhibit non-linear elastic properties, there is a growing interest in understanding how the viscous component of materials and tissues influences cell fate. Here we describe the use of microgel thin films with controllable non-linear elastic properties for investigating the role of material loss tangent on cell adhesion, migration, and myofibroblastic differentiation, which have implications in fibrotic responses. Fibroblast modes of migration are dictated by film loss tangent; high loss tangent induced ROCK-mediated amoeboid migration while low loss tangent induced Rac-mediated mesenchymal cell migration. Low loss tangent films were also associated with higher levels of myofibroblastic differentiation. These findings have implications in fibrosis and indicate that slight changes in tissue viscoelasticity following injury could contribute to early initiation of fibrotic related responses.
Hydrogels are promising 3D materials that have demonstrated increasing applications in the encapsulation and delivery of drugs and cells. Herein we report an injectable colloidal hydrogel that directly assembled by graphene oxide (GO) and thermo-sensitive nanogels (tNG). The pH dependent hydrogen bonding interactions between the carboxyl and oxethyl groups induce the reversible assembly of GO and nanogels. The hydrogel is mouldable and can be shaped into different macroscopic objects, and the mechanical strengths are tunable with pH and temperature adjustment. The hybrid hydrogel by its own possesses high antibacterial activity, and demonstrates responsive drug release behaviour and high viability of 3D encapsulated cells. We expect this hybrid colloidal hydrogel can serve as an interesting scaffold for active cargo delivery and cell culture.
Poly(ethylene glycol) diacrylate (PEGDA) are being investigated for various tissue engineering applications for its biocompatibility and excellent mechanical properties. However, the native PEGDA films can hinder cell adhesion which limits applications in the field of tissue engineering and biomedicine. Recently, nano composite technology has been a particularly hot topic because of it can be used to modify deliver to the materials properties. In this paper, we have added polystyrene nano-spheres into the PEGDA solution to synthesize nanocomposites film. The experimental results show that the adhesion of cell on PEGDA film can be regulated by the concentration of polystyrene nano-spheres. The cell adhesion and spread can be improved through increasing concentration of polystyrene nano-spheres. Furthermore, we find that the spheres can also change the mechanical properties of PEGDA films and further affect the cell growth status.
Development of materials with fine spatial control over topographical, mechanical, or chemical features has been investigated for a variety of applications. Here we present a method to fabricate an array of polyelectrolyte constructs including two-dimensionally and three-dimensionally patterned assemblies using both compressible and incompressible colloidal building blocks. This method eliminates prior constraints associated with specific chemistries, and can be used to develop modular, multi-component, patterned assemblies. In particular, development of constructs were investigated using microgels, which are colloidally stable hydrogel microparticles, polystyrene (PS) beads, and PS-microgel core-shell building blocks in conjunction with the polycation poly(ethyleneimine) (PEI). The topography, mechanical properties, and microstructure of these materials were characterized via bright field microscopy, laser scanning confocal microscopy (LSCM), atomic force microscopy (AFM), and AFM nanoindentation. Cellular studies demonstrate that such patterned film constructs can be used as model systems to investigate and direct cellular adhesion and spreading. Finally, this fabrication method is expanded to develop bulk polyelectrolyte gels that can be used to develop cell-laden gels.
Polyelectrolyte multilayers (PEMs) have many potential applications in tissue engineering and regenerative medicine. However, the softness of biocompatible PEMs results in limited cell adhesion. A novel strategy for the enhancement of cell adhesion on PEMs based on thermal annealing is presented here. The impact of thermal annealing at 37 oC of poly-l-lysine (PLL) and alginate (Alg) polyelectrolyte multilayers on the adhesion of human lung cancer A549 and myoblast C2C12 cell lines is studied. The properties of the PEMs after annealing are characterized by means of the quartz crystal microbalance with dissipation, atomic force microscopy, atomic force spectroscopy, zeta potential, and contact angle measurements. After annealing, PLL/Alg PEMs become smoother displaying an increase in stiffness. Furthermore, PEMs become more hydrophobic, with an increase in contact angle from 36° to 90°. Additionally, the surface charge decreases and protein deposition on PEMs significantly diminishes after annealing. Cell adhesion, measured by the projected average cell spreading and focal contact formation, is remarkably improved for the annealed PEMs.
Polyelectrolyte multilayers (PEMs) with different polycation/polyanion pairs are fabricated by the layer-by-layer technique employing synthetic, natural, and both types of polyelectrolytes. The impact of the chemical composition of PEMs on cell adhesion is assessed by studying cell shape, spreading area, focal contacts, and cell proliferation for the A549 cell line. Cells exhibit good adhesion on PEMs containing natural polycations and poly(sodium 4-styrenesulfonate) (PSS) as polyanion, but limited adhesion is observed on PEMs fabricated from both natural polyelectrolytes. PEMs are then assembled, depositing a block of natural polyelectrolytes on top of a stiffer block with PSS as polyanion. Cell adhesion is enhanced on top of the diblock PEMs compared to purely natural PEMs. This fact could be explained by the interdigitation between polyelectrolytes from the two blocks. Diblock PEM assembly provides a simple means to tune cell adhesion on biocompatible PEMs.
Stimuli-responsive gels are known as smart gels. Such gels respond adaptively to a variety of external stimulation. Different classes of smart gels, according to the name of the stimulus, are thermoresponsive, pH-responsive, photoresponsive, electroresponsive, magnetoresponsive gels, chemically activated gels, and others. Such smart gel materials are discussed in this article along with their synthesis, characterization, properties, and various applications such as drug delivery systems, thermoresponsive cell culture dishes, microfluidic gel photovoltaics, etc. The advantages and disadvantages of electroactive gels have been discussed for their application as actuators. Microporous and superporous gels, microgels/nanogels, shape-memory and self-healing gels, and fullerene (C60)-containing nanostructured smart polymers/gels are also depicted in this article. The state-of-the-art research and development of smart gels along with the opportunities and challenges of this emerging field of research are highlighted in this article.Keywords: Smart gels;thermoresponsive gels;electroresponsive gels;magnetoresponsive gels;photoresponsive gels;pH-responsive gels;chemically activated gels;microgels and nanogels;microporous and superporous gels;shape-memory and self-healing gels;fullerene (C 60)-containing nanostructured smart polymers/gels;drug delivery systems;thermoresponsive cell culture dishes;gel photovoltaics;gel actuators
Thin films composed entirely of microgel building blocks were fabricated using two kinds of self-cross-linked, oppositely charged microgels, via centrifugal deposition. Atomic force microscopy studies revealed that both microgels form very thin monolayer films due to a large degree of microgel deformation during deposition. Meanwhile, centrifugal deposition from a mixture of these two kinds of microgels resulted in the formation of microgel bilayers with a total thickness of around 20 nm. The film thickness increased linearly with the deposition time. Additionally, isotropic stretching/release by heating/cooling of the dried microgel films generated complicated buckling patterns, while anisotropic (uniaxial) stretching/release resulted in parallel buckling perpendicular to the stretching direction. The damage caused by anisotropic stretching and 100 °C treatment can be healed by addition of water, while damage caused via treatment at 150 °C cannot be healed due to the occurrence of polymer cross-linking, which inhibits the mobility of the microgel building blocks.
Microgels are colloidally stable, hydrogel microparticles that have previously been used in a range of (soft) material applications due to their tunable mechanical and chemical properties. Most commonly, thermo and pH-responsive poly(N-isopropylacrylamide) (pNIPAm) microgels can be fabricated by precipitation polymerization in the presence of the co-monomer acrylic acid (AAc). Traditionally pNIPAm microgels are synthesized in the presence of a crosslinking agent, such as N,N’-methylenebisacrylamide (BIS), however, microgels can also be synthesized under ‘crosslinker free’ conditions. The resulting particles have extremely low (<0.5%), core-localized crosslinking resulting from rare chain transfer reactions. AFM nanoindentation of these ultralow crosslinked (ULC) particles indicate that they are soft relative to crosslinked microgels, with a Young’s modulus of ~10 kPa. Furthermore, ULC microgels are highly deformable as indicated by a high degree of spreading on glass surfaces and the ability to translocate through nanopores significantly smaller than the hydrodynamic diameter of the particles. The size and charge of ULCs can be easily modulated by altering reaction conditions, such as temperature, monomer, surfactant and initiator concentrations, and through the addition of co-monomers. Microgels based on the widely utilized, biocompatible polymer polyethylene glycol (PEG) can also be synthesized under crosslinker free conditions. Due to their softness and deformability, ULC microgels are a unique base material for a wide variety of biomedical applications including biomaterials for drug delivery and regenerative medicine.
The compartmentalization of microgels is a challenging task for synthetic polymer chemistry. Although the complexation with low molecular weight compounds or the use of microfluidic techniques offer attractive possibilities for other length scales, it is difficult to implement compartments in the mesoscale range of 10-100 nm. Herein we show how simple blending of reactive prepolymers is suitable to design new microgel morphologies with tailored compartments. We use poly(EEGE)-block-poly(AGE) as crosslinkable, pro-hydrophilic prepolymer in blends with varying amounts of crosslinkable, yet hydrophobic poly(THF-stat-AllylEHO) or inert and hydrophobic polystyrene, and crosslink the allyl functional prepolymer(s) in a thiol-ene click-type reaction after miniemulsification. Our strategy shows how arrested versus free nanophase separation can be used to control easily the morphology and polarity of microgel particles.
Studies focused on understanding the role of matrix biophysical signals on cells, especially those when cells are encapsulated in hydrogels that are locally remodelled, are often complicated by appropriate methods to measure differences between the bulk and local material properties. From this perspective, stress-relaxing materials that allow long-term culture of embedded cells provide an opportunity to elucidate aspects of this biophysical signalling. In particular, rheological characterization of the stress relaxation properties allows one to link a bulk material measurement to local aspects of cellular functions by quantifying the corresponding cellular forces that must be applied locally. Here, embryonic stem cell-derived motor neurons were encapsulated in a well-characterized covalently adaptable bis-aliphatic hydrazone crosslinked PEG hydrogel, and neurite outgrowth was observed over time. Using fundamental physical relationships describing classical mechanics and viscoelastic materials, we calculated the forces and energies involved in neurite extension, the results of which provide insight to the role of biophysical cues on this process.
The field of polymeric biomaterials has received much attention in recent years due to its potential for enhancing the biocompatibility of systems and devices applied to drug delivery and tissue engineering. Such applications continually push the definition of biocompatibility from relatively straightforward issues such as cytotoxicity to significantly more complex processes such as reducing foreign body responses or even promoting/recapitulating natural body functions. Hydrogels and their colloidal analogues, microgels, have been and continue to be heavily investigated as viable materials for biological applications because they offer numerous, facile avenues in tailoring chemical and physical properties to approach biologically harmonious integration. Mechanical properties in particular are recently coming into focus as an important manner in which biological responses can be altered.
Poly(N-isopropylacrylamide) (PNIPAM) microgel is perhaps the most well-known intelligent soft nanomaterial. Combining the strengths of hydrogel and nanoparticles, with unique stimuli-responsivity, PNIPAM microgels have found numerous biomedical applications, such as drug delivery, biosensing, and so on. Usually they were used as dispersed particles, however, they can also be used as building blocks to fabricate 2D films and 3D aggregates. These nanostructured assemblies exhibit new properties which the dispersed particles do not have, and new biomedical applications have been found for these assemblies. In this paper, the biomedical applications of PNIPAM microgels in the form of dispersed particles, 2D films and 3D aggregates were reviewed and some recent progress in this area was highlighted.
In the past two decades, layer-by-layer (LbL) assembly has been proven to be a convenient and versatile method to fabricate functional films. However, using traditional dipping LbL assembly to fabricate micrometer-thick films is time consuming. Compared with ultrathin films, micrometer-thick films prepared by LbL assembly possess enhanced mechanical stability, and allow deposition of a significantly increased amount of materials and the integration of multiple functions. These merits of thick films produced by LbL assembly can result in new functions and allow the functions of ultrathin films fabricated by LbL assembly to be optimized. In this tutorial review, the methods for rapid fabrication of thick polymeric films involving LbL assembly are reviewed. The functions of such films that are relevant to their micrometer thickness are discussed.
Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.
To study cell biology, cells are typically removed from their host organism and cultured on an entirely different environment. Tools taken from traditional material engineering are adopted to create spatially and structurally defined biological microenvironments. While cell spreading influences a variety of cellular behaviors, like migration, proliferation, and differentiation, micropatterning techniques have demonstrated that cellular architecture is an integral mechanism by which cells regulate their behavior. Microfabrication is used to determine the cell response to the adhesive cues in their microenvironment. The biomaterials can be used in future to integrate biochemical cues with structural cues to generate highly defined microenvironments.
Recent progress in the design and application of artificial cellular microenvironments and nanoenvironments has revealed the extraordinary ability of cells to adjust their cytoskeletal organization, and hence their shape and motility, to minute changes in their immediate surroundings. Integrin-based adhesion complexes, which are tightly associated with the actin cytoskeleton, comprise the cellular machinery that recognizes not only the biochemical diversity of the extracellular neighbourhood, but also its physical and topographical characteristics, such as pliability, dimensionality and ligand spacing. Here, we discuss the mechanisms of such environmental sensing, based on the finely tuned crosstalk between the assembly of one type of integrin-based adhesion complex, namely focal adhesions, and the forces that are at work in the associated cytoskeletal network owing to actin polymerization and actomyosin contraction.
We have measured the traction forces generated by fibroblasts using a novel micromachined device that is capable of determining the subcellular forces generated by individual adhesive contacts. The front of migrating fibroblasts produced intermittent rearward forces whereas the tail produced larger forward directed forces. None of the forces were steady; they all had periodic fluctuations. The transition between forward and rearward traction forces occurred at the nucleus, not at the rear of the cell or the border between the endoplasm and the ectoplasm. We propose that the coupling of lamella extensions to fluctuating rearward tractions in front of the nuclear region move the front of a fibroblast forward, while force-facilitated release of rear adhesive contacts and anterior-directed tractions allow the region behind the nucleus to advance.
Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH- and NH(2)-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH(3) groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against beta(1) integrin inhibited matrix mineralization on the OH and NH(2) surfaces, whereas function-perturbing antibodies specific for beta(3) integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.
Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such
cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells
on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify
cancer cells. However, an understanding of how tissue cells—including fibroblasts, myocytes, neurons, and other cell types—sense
matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity
can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin
cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness
on cell state likely has important implications for development, differentiation, disease, and regeneration.
Since RGD peptides (R: arginine; G: glycine; D: aspartic acid) have been found to promote cell adhesion in 1984 (Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule, Nature 309 (1984) 30), numerous materials have been RGD functionalized for academic studies or medical applications. This review gives an overview of RGD modified polymers, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on polymers. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed.
Microgel multi-layer films assembled from anionic particles and linear polycation were prepared on elastomeric substrates and their self-healing properties studied. Dried films were imaged in situ during mechanical deformation and were determined to undergo plastic deformation in response to linear strain, leading to film buckling upon strain relaxation. Hydration leads to rapid reorganization of the film building blocks, permitting recovery of the film to the undamaged state. Additionally, films were determined to heal in the presence of high relative humidity environments, suggesting that film swelling and hydration is a major factor in the restoration of film integrity, and that full immersion in solvent is not required for healing. Films prepared from microgels with lower levels of acid content and/or polycation length, factors strongly connected to the charge density and presumably the connectivity of the film, also display self-healing characteristics.
Nanoparticle (NP)-bioconjugates hold great promise for more sensitive disease diagnosis and more effective anticancer drug delivery compared with existing approaches. A critical aspect in both applications is cellular internalization of NPs, which is influenced by NP properties and cell surface mechanics. Despite considerable progress in the optimization of the NP-bioconjugates for improved targeting, the role of substrate stiffness on cellular uptake has not been investigated. Using polyacrylamide (PA) hydrogels as model substrates with tunable stiffness, we quantified the relationship between substrate stiffness and cellular uptake of fluorescent NPs by bovine aortic endothelial cells (BAECs). We found that a stiffer substrate results in a higher total cellular uptake on a per cell basis, but a lower uptake per unit membrane area. To obtain a mechanistic understanding of the cellular uptake behavior, we developed a thermodynamic model which predicts that membrane spreading area and cell membrane tension are two key factors controlling cellular uptake of NPs, both of which are modulated by substrate stiffness. Our experimental and modeling results not only open up new avenues for engineering NP-based cancer cell targets for more effective in vivo delivery, but also contribute a remarkable example of how the physical environment dictates cellular behavior and function.
Millions of people lose or damage their organs or tissues due to disease, birth defects, or accidents each year. Stem cells, such as embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), adult stem cells, and fetal stem cells are an attractive prospect for regenerative medicine and tissue engineering. The pluripotent nature of ESCs and iPSCs opens many avenues for potential stem cell-based regenerative therapies and the development of drug discovery platforms. Nowadays, it is difficult to find novel biomolecules for differentiation of stem cells and to find much higher efficiency of stem cell differentiation into desired lineages solely by combination of these biomolecules in culture medium. Biomaterials for stem cell culture are focused as a tool for finetuning of stem cell differentiation, because it is quite recent for researchers to realize biomaterials can guide stem cell fate of differentiation.
Natural extracellular matrix (ECM) proteins possess critical biological characteristics that provide a platform for cellular adhesion and activation of highly regulated signaling pathways. However, ECM based biomaterials can have several limitations including poor mechanical properties and risk of immunogenicity. Synthetic biomaterials alleviate the risks associated with natural biomaterials but often lack the robust biological activity necessary to direct cell function beyond initial adhesion. A thorough understanding of receptor mediated cellular adhesion to the ECM and subsequent signaling activation has facilitated development of techniques that functionalize inert biomaterials providing a biologically active surface. Here we review a range of approaches used to modify biomaterial surfaces for optimal receptor mediated cell interactions as well as provide insights into specific mechanisms of downstream signaling activation. In addition to a brief overview of integrin receptor-mediated cell function, so-called "biomimetic" techniques reviewed here include (1) surface modification of biomaterials with bioadhesive ECM macromolecules or specific binding motifs, (2) nanoscale patterning of the materials, and (3) use of "natural-like" biomaterials.
We describe the surface modification of magnetic nanoparticles (MNPs), the coverage of poly(N-isopropylacrylamide) (PNiPAM) microgel with the MNPs and the inductive heating of these carriers. PNiPAM surface itself was modified using the layer-by-layer (LbL) assembly of polyelectrolytes to facilitate the deposition of surface-modified MNPs. One advantage of this concept is it allows the tuning of the magnetic and thermoresponsive properties of individual components (nanoparticles and microgels) separately before assembling them. Characterisations of the hybrid core–shell are discussed. In particular, it is shown that (i) each layer is successfully deposited and, more importantly, (ii) the coated microgel retains its thermoresponsive and magnetic behaviour.
In the realm of soft nanotechnology, hydrogel micro- and nanoparticles represent a versatile class of responsive materials. Over the last decade, our group has investigated the synthesis and physicochemical properties of a variety of synthetic hydrogel particles. From these efforts, several particle types have emerged with potentially enabling features for biological applications, including nanogels for targeted drug delivery, microlenses for biosensing, and coatings for biomedical devices. For example, core/shell nanogels have been used to encapsulate and deliver small interfering RNA to ovarian cancer cells; nanogels used in this fashion may improve therapeutic outcomes for a variety of macromolecular therapeutics. Microgels arranged as multilayers on implantable biomaterials greatly minimize the host inflammatory response to the material. Furthermore, the triggered release of drugs (i.e., insulin) has been demonstrated from similar assemblies. The goal of this feature article is to highlight developments in the design of responsive microgels and nanogels in the context of our recent efforts and in relation to the community that has grown up around this fascinating class of materials.
The elastic properties of thin gelatin films were investigated with the atomic force microscope (AFM). The degree of swelling and thus the softness of the gelatin can be tuned by immersing it in mixtures of propanol and water. Therefore, we have chosen gelatin films as a model system to characterize the measurement of elasticity of thin and soft samples. The major aim of this study was to investigate the influence of the film thickness on the apparent elastic (Young's) modulus. Thus, we prepared wedge-shaped samples with a well-defined thickness of up to 1 mu m. The Young's modulus of our samples was between 1 MPa and 20 kPa depending on the degree of swelling. The elasticity was calculated by analyzing the recorded force curves with the help of the Hertz model. We show that the calculated Young's modulus is dependent on the local film thickness and the applied loading force of the AFM tip. Thus, the influence of the hard substrate on the calculated softness of the film can be characterized as a function of indentation. It was possible to determine the elastic properties of gelatin films with a thickness down to 50 nm and a Young's modulus of similar to 20 kPa.
Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) microgel films are shown to allow controlled detachment of adsorbed cells via temperature stimuli. Cell response occurs on the timescale of several minutes, is reversible, and allows for harvesting of cells in a mild fashion. The fact that microgels are attached non-covalently allows using them on a broad variety of (charged) surfaces and is a major advantage as compared to approaches relying on covalent attachment of active films. In the following, the microgels’ physico-chemical parameters in the adsorbed state and their changes upon temperature variation are studied in order to gain a deeper understanding of the involved phenomena. By means of atomic force microscopy (AFM), the water content, mechanical properties, and adhesion forces of the microgel films are studied as a function of temperature. The analysis shows that these properties change drastically when crossing the critical temperature of the polymer film, which is the basis of the fast cell response upon temperature changes. Furthermore, nanoscale mechanical analysis shows that the films posses a nanoscopic gradient in mechanical properties.
We report on the finding that absorption of citrate-stabilized Au nanoparticles into microgel/polyelectrolye multilayer thin
films results in an increase in the resistance of those films to strain-induced damage in the dry state while maintaining
the remarkable self-healing properties of the films following rehydration. Films were fabricated atop elastomeric poly(dimethylsiloxane)
substrates by a centrifuge-assisted layer-by-layer technique using anionic hydrogel microparticles (microgels) and cationic
linear polymers as the building blocks. Gold nanoparticles were embedded into swollen hydrogel films by a simple immersion
method wherein the Coulombic interactions between the anionic Au particles and the polycation are likely important. After
drying, the mechanical properties of films were inferred from the observation of cracks/wrinkles formed during stretching
of the elastomeric substrate. As-prepared (no Au) hydrogel films revealed the presence of damage perpendicular to the stretching
direction (10% strain), as observed previously. However, Au nanoparticle-doped films displayed significantly reduced damage
under identical stretching conditions while forming cracks and wrinkles under higher strains (20–30%). Importantly, all films
displayed excellent self-healing behavior upon rehydration regardless of Au content, suggesting that the nanoparticle toughening
effect does not interfere with the film mobility required to achieve autonomic repair.
KeywordsLayer-by-layer–Microgel–Gold nanoparticle–Self-healing
The successful incorporation of biomaterials implanted in the human body depends on many factors, amongst. which are the surface properties of the solid implant, the surrounding liquid with its dissolved proteins and the surface characteristics of cells.Under physiological conditions, biomaterials are immediately covered with proteins before cells can adhere to the material, thereby changing the original characteristics of the substratum surface. Cell spreading was significantly better on high surface free energy substrata than on low energy substrata, both in the absence and in the presence of preadsorbed proteins, despite the fact that a small convergence of the surface free energies of the various substrata occurred upon adsorption. Adsorbed proteins thus seem to transfer substratum characteristics towards adhering and spreading cells. Fourier transform infrared (FTIR) spectra of proteins adsorbed on various substrata indeed demonstrated the appearance of the amide I and amide II bands, with minor shifts in the wave numbers and small changes in the shape of the absorption bands. These band shifts might indicate conformational changes of the adsorbed protein layers, which are probably responsible for the transfer of substratum properties to the interface with adhering and spreading cells.
The binding of cytochrome c to pH and thermoresponsive colloidal hydrogels was investigated using multiangle light scattering, measuring loading through changes in particle molar mass and root mean square radius. Loosely cross-linked microgels [composed of a random copolymer of N-isopropylacrylamide (NIPAm) and acrylic acid (AAc)] demonstrated a high loading capacity for protein. Encapsulation was dependent on both the charge characteristics of the network and the salinity of the medium. Under favorable binding conditions (neutral pH, low ionic strength), microgels containing the highest studied charge density (30 mol% AAc) were capable of encapsulating greater than 9.7 × 10(5) cytochrome c molecules per particle. Binding resulted in the formation of a polymer-protein complex and condensation of the polymer. Anionic microgels demonstrated a change in density ~20-fold in the presence of oppositely charged proteins. These studies of cytochrome c encapsulation represent a significant step towards direct measurement of encapsulation efficiency in complex media as we pursue responsive nanogels and microgels for the delivery of macromolecular therapeutic agents.
Bioactive glasses and ceramics enhance bone formation and bond directly to bone, and have emerged as promising substrates for bone tissue engineering applications. Bone bioactivity involves physicochemical surface reactions and cellular events, including cell attachment to adsorbed extracellular matrix proteins. The effects of fibronectin (Fn) adsorption and glass surface reaction stage on the attachment of osteoblast-like cells (ROS 17/2.8) to bioactive glass were analyzed. Bioactive glass disks were pretreated in a simulated physiologic solution to produce three reaction layers: unreacted glass (BG0), amorphous calcium phosphate (BG1d), and carbonated hydroxyapatite (BG7d). Synthetic hydroxyapatite (sHA) and nonreactive borosilicate glass (CG) were used as controls. A spinning disk device which applied a linear range of forces to attached cells while maintaining uniform chemical conditions at the interface was used to quantify cell adhesion. The number of adherent cells decreased in a sigmoidal fashion with applied force, and the resulting detachment profile provided measurements of adhesion strength. For the same amount of adsorbed Fn, cell adhesion was higher on surface-reacted bioactive glasses (BG1d and BG7d) than on BG0, CG, and sHA. For all substrates, cell attachment was primarily mediated by the RGD binding site of Fn, as demonstrated by blocking experiments with antibodies and RGD peptides. Cell adhesion strength increased linearly with adsorbed Fn surface density. Analysis of this fundamental relationship revealed that improved adhesion to reacted bioactive glasses resulted from enhanced cell receptor-Fn interactions, suggesting substrate-dependent conformational changes in the adsorbed Fn.
Here we describe a simple approach to create various sizes of protein nanoarrays for the investigation of cell adhesion. Using a combination of nanosphere lithography, oxygen plasma treatment, deep etching and nanomolding processes, well-ordered polymeric nanopillar arrays have been fabricated with diameters in the range of 50-600 nm. These nanopillar arrays were used as stamps for nanocontact printing to create fibronectin nanoarrays, which were used to study the size dependent formation of focal adhesion. It was found that cells can adhere and spread on fibronectin nanoarrays with a fibronectin pattern as small as 50 nm. It was also found that the average size of focal adhesion decreased as the size of the fibronectin pattern was reduced.
This paper reports the development of a novel force measurement device based on polymeric nanopillar arrays. The device was fabricated by a process combining nanosphere lithography, oxygen plasma treatment, deep etching and nano-molding. Well-ordered polymeric nanopillar arrays with various diameters and aspect ratios were fabricated and used as cell culture substrates. Cell traction forces were measured by the deflection of the nanopillars. Since the location of the nanopillars can be monitored at all times, this device allows for the measurement of the evolution of adhesion forces over time.
Many biological materials are hierarchically structured which means that they are designed from the nano- to the macro-scale in a sometimes self-similar way. There are lots of papers published including very detailed descriptions of these structures at all length scales--however, investigations of mechanical properties are most often focused on either nano-indentation or bulk mechanical testing characterizing properties at the smallest or largest size scale. Interestingly, there are hardly any investigations that systematically interconnect mechanical properties of different length scales. Nevertheless there are often conclusions drawn like the one that "biological materials exhibit their excellent mechanical properties due to their hierarchical structuring". Thus, we think there is a gap and discrepancy between the detection and description of biological structures and the correlated determination and interpretation of their mechanical properties. Hence, in this paper we order hierarchically structured biological materials with high mineral content according to their hierarchical levels and attribute measured mechanical properties to them. This offers the possibility to gain insight into the mechanical properties on different hierarchical levels even though the entire biological materials were tested. On the other hand we use data of one material, namely enamel, where mechanical properties were measured on every length scale. This kind of data analysis allows to show how a theoretical model developed by Huajian Gao and co-workers can be used to get closer insights into experimental data of hierarchically structured materials.
Thin films assembled from microgel building blocks have been constructed using a simple, high-throughput, and reproducible centrifugation (or "active") deposition technique. When compared to a common passive adsorption method (e.g., dip coating), microgels that are actively deposited onto a surface have smaller footprints and are more closely packed. Under both active and passive deposition conditions, the microgel footprint areas decrease during deposition. However, under active deposition, the microgel footprint appears to decrease continually and to a greater degree over the course of the deposition, forming a tightly packed, homogeneous film. Taking advantage of the rapid and uniform assembly of these films, we demonstrate the use of active deposition toward the fabrication of polyelectrolyte multilayers containing anionic microgels and a cationic linear polymer. Microgel multilayers successfully demonstrated effective blocking of the underlying substrate toward macrophage adhesion, which is a highly sought-after property for modulating the inflammatory response to an implanted biomaterial.
(Figure Equation Presentation) Soft yet strong: Colloidal hydrogel films, which are constructed using a layer-by-layer polyelectrolyte approach, are easily damaged by mechanical disruption, but can also autonomically heal (see picture). The healing event occurs within seconds once the film has been resolvated. The lability of the coulombic interactions between hydrogel particle and linear polymer plays a direct role in its ability to self-heal.
Implantation of synthetic materials into the body elicits inflammatory host responses that limit medical device integration and biological performance. This inflammatory cascade involves protein adsorption, leukocyte recruitment and activation, cytokine release, and fibrous encapsulation of the implant. We present a coating strategy based on thin films of poly(N-isopropylacrylamide) hydrogel microparticles (i.e. microgels) cross-linked with poly(ethylene glycol) diacrylate. These particles were grafted onto a clinically relevant polymeric material to generate conformal coatings that significantly reduced in vitro fibrinogen adsorption and primary human monocyte/macrophage adhesion and spreading. These microgel coatings also reduced leukocyte adhesion and expression of pro-inflammatory cytokines (TNF-alpha, IL-1beta, MCP-1) in response to materials implanted acutely in the murine intraperitoneal space. These microgel coatings can be applied to biomedical implants as a protective coating to attenuate biofouling, leukocyte adhesion and activation, and adverse host responses for biomedical and biotechnological applications.
Tissue engineering in vitro and in vivo involves the interaction of cells with a material surface. The nature of the surface can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation. Initial events at the surface include the orientated adsorption of molecules from the surrounding fluid, creating a conditioned interface to which the cell responds. The gross morphology, as well as the microtopography and chemistry of the surface, determine which molecules can adsorb and how cells will attach and align themselves. The focal attachments made by the cells with their substrate determine cell shape which, when transduced via the cytoskeleton to the nucleus, result in expression of specific phenotypes. Osteoblasts and chondrocytes are sensitive to subtle differences in surface roughness and surface chemistry. Studies comparing chondrocyte response to TiO2 of differing crystallinities show that cells can discriminate between surfaces at this level as well. Cellular response also depends on the local environmental and state of maturation of the responding cells. Optimizing surface structure for site-specific tissue engineering is one option; modifying surfaces with biologicals is another.
Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.
Mechanical interactions between cell and substrate are involved in vital cellular functions from migration to signal transduction. A newly developed technique, traction force microscopy, makes it possible to visualize the dynamic characteristics of mechanical forces exerted by fibroblasts, including the magnitude, direction, and shear. In the present study such analysis is applied to migrating normal and transformed 3T3 cells. For normal cells, the lamellipodium provides almost all the forces for forward locomotion. A zone of high shear separates the lamellipodium from the cell body, suggesting that they are mechanically distinct entities. Timing and distribution of tractions at the leading edge bear no apparent relationship to local protrusive activities. However, changes in the pattern of traction forces often precede changes in the direction of migration. These observations suggest a frontal towing mechanism for cell migration, where dynamic traction forces at the leading edge actively pull the cell body forward. For H-ras transformed cells, pockets of weak, transient traction scatter among small pseudopods and appear to act against one another. The shear pattern suggests multiple disorganized mechanical domains. The weak, poorly coordinated traction forces, coupled with weak cell-substrate adhesions, are likely responsible for the abnormal motile behavior of H-ras transformed cells.
This study investigated whether a nanometer scale of surface roughness could improve the adhesion and growth of human endothelial cells on a biomaterial surface. Different molecular weights or chain lengths of polyethylene glycol (PEG) were mixed and then grafted to a polyurethane (PU) surface, a model smooth surface, to form a nanometer (nm) scale of roughness for PU-PEG surfaces (PU-PEG(mix)) while PEG with a molecular weight of 2000 was also grafted to PU to form PU-PEG(2000) for comparison. In addition, the concept was tested on cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) that was photochemically grafted to PU-PEG(mix) and PU-PEG(2000) surfaces (e.g., PU-PEG(mix)-GRGD and PU-PEG(2000)-GRGD surfaces, respectively). To prepare GRGD-grafted PU-PEG(mix) and PU-PEG(2000) surface, 0.025M of GRGD-SANPAH (N-Succinimidyl-6-[4'-azido-2'-nitrophenylamino]-hexanoate) solutions was grafted to PU-PEG(mix) and PU-PEG(2000) by surface adsorption of the peptide and subsequent ultraviolet (UV) irradiation for photoreaction. The grafting efficiencies for GRGD to PU-PEG(mix) and PU-PEG(2000) surfaces were about 67% for both surfaces, semi-quantitatively analyzed by an HPLC. The surface roughness, presented with a roughness parameter, R(a), and the topography of the tested surfaces were both measured and imaged by an atomic force microscope (AFM). Among the R(a) values of the films, PU was the smoothest (e.g., R(a)=1.53+/-0.20 nm, n=3) while PU-PEG(mix) was the roughest (e.g., R(a)=39.79+/-10.48 nm, n=4). Moreover, R(a) values for PU-PEG(mix) and PU-PEG(mix)-GRGD surfaces were about 20 nm larger than those for PU-PEG(2000) and PU-PEG(2000)-GRGD, respectively, which were consistent with the topographies of the films. Human umbilical vein endothelial cells (HUVECs) were adhered and grown on the tested surfaces after 36 h of incubation. Among the films, HUVEC's adhesion on the surface of PU-PEG(mix)-GRGD was the densest while that on the surface of PU-PEG(2000) was the sparsest. Also, the adhesion and growth of HUVECs for the roughness surfaces were statistically significantly better than that of smooth surface for both GRGD grafted and un-grafted surfaces, respectively. The viability for the growth of HUVECs on the tested surfaces analyzed by MTT assay also confirmed the efficacy of the increased surface roughness. In conclusion, increased surface roughness of biomaterial surfaces even at 10-10(2) nm scale could enhance the adhesion and growth of HUVECs on roughness surfaces that could be useful for applications of tissue engineering.
The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands.
New generations of synthetic biomaterials are being developed at a rapid pace for use as three-dimensional extracellular microenvironments to mimic the regulatory characteristics of natural extracellular matrices (ECMs) and ECM-bound growth factors, both for therapeutic applications and basic biological studies. Recent advances include nanofibrillar networks formed by self-assembly of small building blocks, artificial ECM networks from protein polymers or peptide-conjugated synthetic polymers that present bioactive ligands and respond to cell-secreted signals to enable proteolytic remodeling. These materials have already found application in differentiating stem cells into neurons, repairing bone and inducing angiogenesis. Although modern synthetic biomaterials represent oversimplified mimics of natural ECMs lacking the essential natural temporal and spatial complexity, a growing symbiosis of materials engineering and cell biology may ultimately result in synthetic materials that contain the necessary signals to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis.
Polyelectrolyte multilayer films were employed to support attachment of cultured rat aortic smooth muscle A7r5 cells. Like smooth muscle cells in vivo, cultured A7r5 cells are capable of converting between a nonmotile "contractile" phenotype and a motile "synthetic" phenotype. Polyelectrolyte films were designed to examine the effect of surface charge and hydrophobicity on cell adhesion, morphology, and motility. The hydrophobic nature and surface charge of different polyelectrolyte films significantly affected A7r5 cell attachment and spreading. In general, hydrophobic polyelectrolyte film surfaces, regardless of formal charge, were found to be more cytophilic than hydrophilic surfaces. On the most hydrophobic surfaces, the A7r5 cells adhered, spread, and exhibited little indication of motility, whereas on the most hydrophilic surfaces, the cells adhered poorly if at all and when present on the surface displayed characteristics of being highly motile. The two surfaces that minimized cell adhesion consisted of two varieties of a diblock copolymer containing hydrophilic poly(ethylene oxide) and a copolymer bearing a zwitterionic group AEDAPS, (3-[2-(acrylamido)-ethyldimethyl ammonio] propane sulfonate). Increasing the proportion of AEDAPS in the copolymer decreased the adhesion of cells to the surface. Cells presented with micropatterns of cytophilic and cytophobic surfaces generated by polymer-on-polymer stamping displayed a surface-dependent cytoskeletal organization and a dramatic preference for adhesion to, and spreading on, the cytophilic surface, demonstrating the utility of polyelectrolyte films in manipulating smooth muscle cell adhesion and behavior.
It is well known that mechanical stimuli induce cellular responses ranging from morphological reorganization to mineral secretion, and that mechanical stimulation through modulation of the mechanical properties of cell substrata affects cell function in vitro and in vivo. However, there are few approaches by which the mechanical compliance of the substrata to which cells adhere and grow can be determined quantitatively and varied independent of substrata chemical composition. General methods by which mechanical state can be quantified and modulated at the cell population level are critical to understanding and engineering materials that promote and maintain cell phenotype for applications such as vascular tissue constructs. Here, we apply contact mechanics of nanoindentation to measure the mechanical compliance of weak polyelectrolyte multilayers (PEMs) of nanoscale thickness, and explore the effects of this tunable compliance for cell substrata applications. We show that the nominal elastic moduli E(s) of these substrata depend directly on the pH at which the PEMs are assembled, and can be varied over several orders of magnitude for given polycation/polyanion pairs. Further, we demonstrate that the attachment and proliferation of human microvascular endothelial cells (MVECs) can be regulated through independent changes in the compliance and terminal polyion layer of these PEM substrata. These data indicate that substrate mechanical compliance is a strong determinant of cell fate, and that PEMs of nanoscale thickness provide a valuable tool to vary the external mechanical environment of cells independently of chemical stimuli.
Thermoresponsive poly(N-isopropylacrylamide) (pNIPAm) microgel particles cross-linked with various concentrations of PEG diacrylates of 3 different PEG chain lengths were synthesized via free-radical precipitation polymerization in order to investigate the phase transition and protein adsorption behavior as the hydrophilicity of the network is increased. Photon correlation spectroscopy (PCS) reveals that, as the concentration of PEG cross-linker incorporated into the particles is increased, an increase in the temperature and breadth of the phase transition occurs. Qualitative differences in particle density using isopycnic centrifugation confirm that higher PEG concentrations result in denser networks. The efficient incorporation of PEG cross-linker was confirmed with (1)H NMR, and variable temperature NMR studies suggest that, in the deswollen state, the longer PEG cross-links protrude from the dense globular network. This behavior apparently manifests itself as a decrease in nonspecific protein adsorption with increasing PEG length and content. Furthermore, when electrostatically attached to a glass surface, the particles containing the longer chain lengths exhibited enhanced nonfouling behavior and were resistant to cell adhesion in serum-containing media. The excellent performance of these particulate films and the simplicity with which they are assembled suggests that they may be applicable in a wide range of applications where nonfouling coatings are required.
Mechanical properties of model and natural gels have recently been demonstrated to play an important role in various cellular processes such as adhesion, proliferation, and differentiation, besides events triggered by chemical ligands. Understanding the biomaterial/cell interface is particularly important in many tissue engineering applications and in implant surgery. One of the final goals would be to control cellular processes precisely at the biomaterial surface and to guide tissue regeneration. In this work, we investigate the substrate mechanical effect on cell adhesion for thin polyelectrolyte multilayer (PEM) films, which can be easily deposited on any type of material. The films were cross linked by means of a water-soluble carbodiimide (EDC), and the film elastic modulus was determined using the AFM nanoindentation technique with a colloidal probe. The Young's modulus could be varied over 2 orders of magnitude (from 3 to 400 kPa) for wet poly(L-lysine)/hyaluronan (PLL/HA) films by changing the EDC concentration. The chemical changes upon cross linking were characterized by means of Fourier transform infrared spectroscopy (FTIR). We demonstrated that the adhesion and spreading of human chondrosarcoma cells directly depend on the Young's modulus. These data indicate that, besides the chemical properties of the polyelectrolytes, the substrate mechanics of PEM films is an important parameter influencing cell adhesion and that PEM offer a new way to prepare thin films of tunable mechanical properties with large potential biomedical applications including drug release.
Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.
Cell adhesion to the extracellular matrix is a dynamic process involving numerous focal adhesion components, which act in coordination to strengthen and optimize the mechanical anchorage of cells over time. A method for systematically analyzing the cell adhesion strengthening process and the components involved in this process is described here. The method combines an adhesion strength assay based on applying fluid shearing to a population of cells and quantitative biochemical analyses.
Soft-tissue cells are surprisingly sensitive to the elasticity of their microenvironment, suggesting that traditional culture plastic and glass are less relevant to tissue regeneration and chemotherapeutics than might be achieved. Cells grown on gels that mimic the elasticity of tissue reveal a significant influence of matrix elasticity on adhesion, cytoskeletal organization, and even the differentiation of human adult derived stem cells. Cellular forces and feedback are keys to how cells feel their mechanical microenvironment, but detailed molecular mechanisms are still being elucidated. This review summarizes our initial findings for multipotent stem cells and also the elasticity-coupled effects of drugs on cancer cells and smooth muscle cells. The drugs include the contractility inhibitor blebbistatin, the proliferation inhibitor mitomycin C, an apoptotis-inducing antibody against CD47, and the translation inhibitor cycloheximide. The differential effects not only lend insight into mechano-sensing of the substrate by cells, but also have important implications for regeneration and molecular therapies.