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The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicators that make use of a fluorene-based molecular wire as a voltage-sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2), readily reports on action potentials in mammalian neurons, detects perturbations to the cardiac action potential waveform in human induced pluripotent stem cell-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlight the utility of fVF 2 for interrogating membrane potential dynamics.

The creation of human induced pluripotent stem cells (hiPSCs) has provided an unprecedented opportunity to study tissue morphogenesis and organ development through 'organogenesis-in-a-dish'. Current approaches to cardiac organoid engineering rely on either direct cardiac differentiation from embryoid bodies (EBs) or generation of aligned cardiac tissues from predifferentiated cardiomyocytes from monolayer hiPSCs. To experimentally model early cardiac organogenesis in vitro, our protocol combines biomaterials-based cell patterning with stem cell organoid engineering. 3D cardiac microchambers are created from 2D hiPSC colonies; these microchambers approximate an early-development heart with distinct spatial organization and self-assembly. With proper training in photolithography microfabrication, maintenance of human pluripotent stem cells, and cardiac differentiation, a graduate student with guidance will likely be able to carry out this experimental protocol, which requires ∼3 weeks. We envisage that this in vitro model of human early heart development could serve as an embryotoxicity screening assay in drug discovery, regulation, and prescription for healthy fetal development. We anticipate that, when applied to hiPSC lines derived from patients with inherited diseases, this protocol can be used to study the disease mechanisms of cardiac malformations at an early stage of embryogenesis.

Despite their preclinical promise, few recombinant growth factors have been fully developed into effective therapies, in part, due to the short interval of therapeutic activity after administration. To address this problem, we developed nanoscale polymer conjugates for multivalent presentation of therapeutic proteins that enhance the activation of targeted cellular responses. As an example of this technology, we conjugated multiple Sonic hedgehog (Shh) proteins onto individual hyaluronic acid biopolymers to generate multivalent protein clusters at defined ratios (i.e., valencies) that yield enhanced Shh pathway activation at equivalent concentrations relative to unconjugated Shh. In this study, we investigated whether these multivalent conjugates (mvShh) could be used to improve the therapeutic function of Shh. We found that a single treatment with mvShh significantly accelerated the closure of full-thickness wounds in diabetic (db/db) mice compared to either an equivalent dose of unconjugated Shh or the vehicle control. Furthermore, we identified specific indicators of wound healing in fibroblasts and endothelial cells (i.e., transcriptional activation and cell migration) that were activated by mvShh in vitroand at concentrations approximately an order of magnitude lower than the unconjugated Shh. Taken together, our findings suggest that mvShh conjugates exhibit greater potency to activate the Shh pathway, and this multivalency advantage improves its therapeutic effect to accelerate wound closure in a diabetic animal model. Our strategy of multivalent protein presentation using nanoscale polymer conjugates has the potential to make a significant impact on the development of protein-based therapies by improving their in vivoperformance.

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering.

Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term ‘stem cell therapy’ is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call “design for optimal functionality.” Third, we describe an alternative approach to sECM design and implementation, called “design for simplicity,” in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ.We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.

Enzymatically degradable semi-interpenetrating polymer networks (edsIPNs) were explored for their biocompatibility and ability to promote new scleral tissue growth, as a means of reinforcing the posterior wall of the eye. The edsIPNs comprised thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid), customizable peptide crosslinkers cleavable by matrix metalloproteinases, and interpenetrating linear poly(acrylic acid)-graft-peptide chains to engage with cell surface receptors. Rheological studies revealed an increase in stiffness at body temperature; the complex shear modulus |G*| was 14.13 ± 6.13 Pa at 22°C and 63.18 ± 12.24 Pa at 37°C, compatible with injection at room temperature. Primary chick scleral fibroblasts and chondrocytes cultured on edsIPN increased by 15.1- and 11.1-fold, respectively, over 11 days; both exhibited delayed onset of exponential growth compared with the cells plated on tissue culture polystyrene. The edsIPN was delivered by retrobulbar injection (100 μL) to nine 2-week-old chicks to assess biocompatibility in vivo.Ocular axial dimensions were assessed using A-scan ultrasonography over 28 days, after which eyes were processed for histological analysis. Although edsIPN injections did not affect the rate of ocular elongation, the outer fibrous sclera showed significant thickening. The demonstration that injectable biomimetic edsIPNs stimulate scleral fibrous tissue growth represents proof-of-principle for a novel approach for scleral reinforcement and a potential therapy for high myopia.

Stem cells are defined by their ability to self‐renew and to differentiate into one or more mature lineages, and they reside within natural niches in many types of adult and embryonic tissues that present them with complex signals to regulate these two hallmark properties. The diverse nature of these in vivomicroenvironments raises important questions about the microenvironmental cues regulating stem cell plasticity, and the stem cell field has built a strong foundation of knowledge on the biochemical identities and regulatory effects of the soluble, cellular, and extracellular matrix factors surrounding stem cells through the isolation and culture of stem cells in vitrowithin microenvironments that, in effect, emulate the properties of the natural niche. Recent work, however, has expanded the field's perspective to include biophysical and dynamic characteristics of the microenvironment. These include biomechanical characteristics such as elastic modulus, shear force, and cyclic strain; architectural properties such as geometry, topography, and dimensionality; and dynamic structures and ligand profiles. We will review how these microenvironmental characteristics have been shown to regulate stem cell fate and discuss future research directions that may help expand our current understanding of stem cell biology and aid its application to regenerative medicine. Copyright © 2009 John Wiley & Sons, Inc.

Although biochemical signals that modulate stem cell self-renewal and differentiation were extensively studied, only recently were the mechanical properties of a stem cell's microenvironment shown to regulate its behavior. It would be desirable to have independent control over biochemical and mechanical cues, to analyze their relative and combined effects on stem-cell function. We developed a synthetic, interfacial hydrogel culture system, termed variable moduli interpenetrating polymer networks (vmIPNs), to assess the effects of soluble signals, adhesion ligand presentation, and material moduli from 10–10,000Pa on adult neural stem-cell (aNSC) behavior. The aNSCs proliferated when cultured in serum-free growth media on peptide-modified vmIPNs with moduli of ≥100Pa. In serum-free neuronal differentiation media, a peak level of the neuronal marker, β-tubulin III, was observed on vmIPNs of 500Pa, near the physiological stiffness of brain tissue. Furthermore, under mixed differentiation conditions with serum, softer gels (∼100–500Pa) greatly favored neurons, whereas harder gels (∼1,000–10,000Pa) promoted glial cultures. In contrast, cell spreading, self-renewal, and differentiation were inhibited on substrata with moduli of ∼10Pa. This work demonstrates that the mechanical and biochemical properties of an aNSC microenvironment can be tuned to regulate the self-renewal and differentiation of aNSCs.

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, Vpeel, was measured as a continuously increasing function of the imposed tension, Tpeel, which ranges from ∼0 to 50nN/μm. For each cell, peeling proved highly heterogeneous, with Vpeel fluctuating between 0μm/s (∼80% of time) and ∼10μm/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.

Interpenetrating polymer networks IPNs of polyacrylamidecoethylene glycolacrylic acid pAAmcoEGAAc applied to model surfaces prevent protein adsorption and cell adhesion. Subsequently, IPN surfaces functionalized with the RGD cellbinding domain from rat bone sialoprotein BSP modulated bone cell adhesion, proliferation, and matrix mineralization. The objective of this study was to utilize the same biomimetic modification strategy to produce functionally similar pAAmcoEGAAc IPNs on clinically relevant titanium surfaces. Contact angle goniometry and Xray photoelectron spectroscopy XPS data were consistent with the presence of the intended surface modifications. Cellular response was gauged by challenging the surfaces with primary rat calvarial osteoblast RCO surfaces in serumcontaining media. IPN modified titanium and negative control RGEIPN surfaces inhibit cell adhesion and proliferation, while RGDmodified IPNs on titanium supported osteoblast attachment and spreading. Furthermore, the latter surfaces supported significant mineralization despite exhibiting lower levels of proliferation than positive control surfaces. These results suggest that with the appropriate optimization, this approach may be practical for surface engineering of osseous implants. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 38–47, 2003

Sequential Robust Design experiments and Xray photoelectron spectroscopic XPS studies were performed to examine the immobilization of hyaluronic acid HA on glass substrates chemisorbed with N2aminoethyl3aminopropyltrimethoxysilane EDS. Numerous reaction conditions were investigated, including the concentrations of 1ethyl33dimethylaminopropyl carbodiimide hydrochloride EDC, Nhydroxysulfosuccinimide SulfoNHS, and HA, and the reaction buffer type, concentration, and pH. The elemental surface compositions of carbon and silicon CSi ratio were used to assess the extent of HA immobilization, leading to the identification of critical HAbinding reaction conditions and the determination of an optimum surface chemistry. The optimum chemistry consisted of 200 mMEDC, 50 mMSulfoNHS, 10 mMN2hydroxyethylpiperazineN′2ethanesulfonic acid HEPES buffer at a pH of 7.0, and 3 mgmL HA. This work emphasizes the advantages of using Robust Design methods over traditional statistical experimental design, particularly when large numbers of variables are examined and costly analytical techniques are employed. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 61: 391–398, 2002

There is presently significant interest in cellular responses to physical forces, and numerous devices have been developed to apply stretch to cultured cells. Many of the early devices were limited by the heterogeneity of deformation of cells in different locations and by the high degree of anisotropy at a particular location. We have therefore developed a system to impose cyclic, large-strain, homogeneous stretch on a multiwell surface-treated silicone elastomer substrate plated with pulmonary epithelial cells. The pneumatically driven mechanism consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle. We present the design of the device and membrane shape, the surface modifications of the membrane to promote cell adhesion, predicted and experimental measurements of the strain field, and new data using cultured airway epithelial cells. We present for the first time the relationship between the magnitude of cyclic mechanical strain and the extent of wound closure and cell spreading.

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivoimplants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitroand in vivo.A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d,7 were identified. The corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A−4] (6.9 kDa), were produced. Both peptides enhanced in vitrosulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivoimplant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, [A+4] and [A−4], may have a role in preodontoblast maturation. The [A+4] and [A−4] may thus be tissue-specific epithelial mesenchymal signaling molecules.

The density of ArgGlyAspcontaining peptides covalently grafted to solid materials has been shown to affect adhesion, spreading, and focal contact formation. The objective of this study was to examine the effect of ligand density on mineralization of the extracellular matrix deposited by osteoblasts. In particular, RGDmodified quartz surfaces with ligand densities varying over two orders 0.01–3.6 pmolcm2 of magnitude were prepared to assess the longterm function of osteoblasts on peptidederivatized surfaces. After 3 weeks in culture, surfaces modified with a 15 amino acid peptide AcCysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyrNH2 at a density ≥0.62 pmolcm2significantly p< 0.05 enhanced mineralization compared with a RGD surface density of 0.01 pmolcm2, RGE surfaces, or clean surfaces adsorbed with serum proteins. These results suggest that regulation of the surface density of adhesive ligands on biomaterial surfaces is a critical determinant in a strategy to alter the degree of extracellular matrix maturation in contact with solid surfaces e.g., implants. Further studies are required to elucidate the intracellular signal transduction pathways that mediate longterm matrix mineralization through the initial engagement of these adhesive ligands. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 52, 595–600, 2000.

Fluorescence microscopy can offer unique advantages for biomaterials characterization. Like spectroscopy or radioactivity, it can be used to quantify specific binding to surfaces, but it can also assess surface homogeneity at the micron scale or detect protein aggregation. To fully utilize the potential of this technique, there must be a way to calibrate the microscope in terms of the moles of a fluorophore per unit area. The method we propose involves the following steps: fluorescent labeling of erythrocytes and quantification of the label by flow cytometry; flattening of fluorescent erythrocytes for microscopic observation; imaging and digital analysis to relate the gray level intensities to the fluorophore density; and using this procedure to characterize a different, more easily obtainable, standard. The latter can be a 50 solution of Na fluorescein that yields a highly reproducible and uniform fluorescence. Concentrated fluorescein solution can also be used to correct images for the spatial nonuniformity of illumination and detection shading correction. By applying this method to study the binding of IgG and fibrinogen to glass or amidated glass, we showed that protein adsorption to glass may result in protein aggregation that may affect the biological activity of the adsorbed protein. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 50, 90–96, 2000.

To better understand the events involved in the generation of defined tissue architectures on biomaterials, we have examined the mechanism of attachment of human bonederived cells HBDC to surfaces with patterned surface chemistry in vitro. Photolithography was used to generate alternating domains of N2aminoethyl3aminopropyltrimethoxysilane EDS and dimethyldichlorosilane DMS. At 90 min after seeding, HBDC were localized preferentially to the EDS regions of the pattern. Using sera specifically depleted of adhesive glycoproteins, this spatial organization was found to be mediated by adsorption of vitronectin Vn from serum onto the EDS domains. In contrast, fibronectin Fn was unable to adsorb in the face of competition from other serum components. These results were confirmed by immunostaining, which also revealed that both Vn and Fn were able to adsorb to EDS and DMS regions when coated from pure solution, i.e., in the absence of competition. In this situation, each protein was able to mediate cell adhesion across a range of surface densities. Cell spreading was constrained on the EDS domains, as indicated by cell morphology and the lack of integrin receptor clustering and focal adhesion formation. This spatial constraint may have implications for the subsequent expression of differentiated function. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 49, 200–210, 2000.

No abstract.

In an effort to regulate mammalian cell behavior in contact with solid material surfaces, we have functionalized surfaces with different ratios of both the putative cell binding (‐Arg‐Gly‐Asp‐) domain and a consensus heparan‐binding domain. The peptide sequences ‐Arg‐Gly‐Asp‐ (‐RGD‐) and ‐Phe‐His‐Arg‐Arg‐Ile‐Lys‐Ala‐ (‐FHRRIKA‐) or mixtures of the two in the ratios of 75:25 (mimetic peptide surface I), 25:75 (mimetic peptide surface II), and 50:50 (mimetic peptide surface III) were immobilized on model surfaces using a heterobifunctional cross‐linker to link the peptide(s) to amine‐functionalized quartz surfaces. Contact angle measurements, spectroscopic ellipsometry, and X‐ray photoelectron spectroscopy were used to confirm the chemistry, thickness of the overlayers, and surface density of immobilized peptides (∼4−6 pmol/cm2). The degree of rat calvaria osteoblast‐like cell spreading, focal contact formation, cytoskeletal organization, proliferation, and mineralization of the extracellular matrix (ECM) on model biomaterial surfaces was examined. Mimetic peptide surface II (MPS II) and MPS III supported the highest degree of cell spreading (p< 0.05), following 4 h of incubation, compared to MPS I, homogeneous ‐RGD‐, and homogeneous ‐FHRRIKA‐ grafted surfaces. Furthermore, MPS I, MPS II, MPS III, and homogeneous ‐RGD‐ surfaces promoted the formation of focal contacts and stress fibers by attached bone cells. The strength of bone cell detachment following 30 min of incubation was significantly higher (p< 0.05) on MPS II surfaces compared to homogeneous ‐RGD‐ and ‐FHRRIKA‐. However, the degree of cell proliferation on the peptide surfaces were not significantly different from each other (p> 0.1). Following 24 d in culture, the areas of mineralized ECM formed on MPS II and MPS III surfaces were significantly (p< 0.05) larger than those of other surfaces. These results demonstrate that utilizing peptide sequences incorporating both cell‐ and heparin‐adhesive motifs can enhance the degree of cell surface interactions and influence the long‐term formation of mineralized ECM in vitro.

We have identified the integrin subunits responsible for the initial adhesion of human osteoblast‐like cells to peptide‐modified surfaces. Biomimetic peptide surfaces containing homogenous RGD (Arg‐Gly‐Asp), homogenous FHRRIKA (Phe‐His‐Arg‐Arg‐Ile‐Lys‐Ala), and a mixed ratio of FHRRIKA:RGD (25:75) were used to assess integrin‐mediated adhesion. The RGD and FHRRIKA peptides were selected from the cellbinding and putative heparin‐binding domains of bone sialoprotein. A panel of monoclonal antibodies against human α1, α2, α3, α4, α5, β1, αv, and αβ3was used to identify the subunits most dominant in mediating short‐term (10 or 30 minutes) and long‐term (4 hours) cell adhesion to the peptide surfaces. Anti‐α2, anti‐β1, and anti‐αvsignificantly (p < 0.05) diminished cell attachment to homogenous RGD surfaces following 30 minutes of incubation. After 4 hours of incubation on RGD‐grafted surfaces, immunostaining of these integrin subunits revealed discrete localization of the αvsubunit at the periphery of the cell (similar to focal contact points), whereas the α2and β1subunits stained very diffusely throughout the cell. A radial‐flow apparatus was used to determine the effect of anti‐integrin antibodies on strength of cell detachment following 10 minutes of incubation on peptide‐grafted surfaces. The strength of detachment from surfaces containing RGD was significantly reduced (p < 0.05) in the presence of anti‐α2, anti‐αv, or anti‐β1compared with controls (presence of preimmune mouse IgG). None of the antibodies significantly influenced cell attachment to homogenous FHRRIKA‐grafted surfaces. These results demonstrate that initial (30 minutes) attachment of human osteoblast‐like cells to homogenous RGD surfaces was mediated by the collagen receptor α2β1and the vitronectin receptor αvβ3, whereas only the vitronectin receptor governed longer term (longer than 30 minutes) adhesion (localization to focal contacts). The importance of distinct integrins in mediating the attachment of bone cells to RGD‐immobilized surfaces indicates a strategy for engineering orthopaedic implants with a built‐in surface specificity for cell adhesion.

ABSTRACT: We have set forth a design strategy for creating biomimetic materials that direct the formation of tissue surrounding implants or regeneration within porous scaffolds. Our studies have established that heterogeneous mimetic peptide surfaces (MPS) containing both the ‐RGD‐ (cell‐binding) and ‐FHRRIKA‐(putative heparin‐binding) peptides, unique to BSP, in the ratio of 75:25 (MPS II) or 50:50 (MPS III) proved to be more biologically relevant and specific for RCO cell function. The initial response of human osteoblast‐like cells to these surfaces was mediated by the collagen (α2β1) and vitronectin receptors (αvβ3), whereas the vitronectin receptor alone dominated longer‐term events (> 30 min). MPS II and III surfaces enhanced cell spreading and long‐term events such as mineralization of the extracellular matrix compared to homogenous peptide surfaces and controls. Furthermore, extensive mineralization of the ECM deposited by RCOs occurred when the peptide was coupled to an interfacial interpenetrating polymer network (IPN) that resisted protein deposition (i.e., non‐specific adsorption) and fouling. Work on thermo‐reversible P(NIPAAm‐co‐AAc) hydrogels demonstrated the ability to create materials that can be delivered to the body in a minimally invasive manner and support tissue regeneration. These hydrogels can be modified to incorporate biofunctional components such as the biomimetic peptides, theoretically enhancing their ability to foster tissue regeneration. These results suggest that biomaterials can be engineered to mimic ECM components of bone (e.g., various organs) by grafting peptides in the appropriate ratios of the cell and heparin‐binding domains, and ultimately modulate the expression of the osteoblast cell phenotype. Approaches similar to the one presented in this work can be used to design materials for hybrid artificial organs and other tissues.

In detecting receptor antagonists or enzyme inhibitors, there are three parameters that often affect the outcome in a predictable quantitative manner: concentrations of the receptors (enzyme), labeled ligand (substrate), and antagonist (inhibitor). The usual goal of assay optimization is to maximize the ability of the assay to detect low concentrations of the analyte. Another question of practical importance, especially in screening of large numbers of samples, would be minimization of the reagent cost. Although the mathematical theory of optimization of the receptor binding assay was developed a long time ago, the resulting formulas (in the general case of unequal affinities of ligand and competitor) were not well suited for practical use. The current availability of computational programs, such as Mathematica, makes possible an efficient solution, both for receptor- and enzyme-based assays. We use a graphical approach to assay optimization and apply it to the following problems: (1) optimization of assay sensitivity, (2) optimization of the reagent cost, and (3) analysis of the entire range of the parameter values since the mathematically optimal values may sometimes be impractical. The computation is extremely simple and the problem can sometimes be solved in several minutes.

In recent years a central objective of tissue engineering has been understanding the interaction of cells with biomaterial surfaces. In this study we examined the protein adsorption events necessary to control the attachment and the subsequent spatial distribution of bone-derived cells exposed to chemically modified surfaces. Silane chemistry and photolithography techniques were used to create substrates with alternating regions of an aminosilane, N-(2-amino-ethyl)-3-aminopropyl-trimethoxysilane (EDS), alongside an alkylsilane, dimethyldichlorosilane (DMS), on quartz surfaces. Sera depleted of fibronectin (Fn), vitronectin (Vn), or both were used to determine if these proteins were necessary for the initial attachment and spatial distribution of bone-derived cells exposed to modified surfaces in vitro. The kinetics and mechanisms of the spatial distribution of cells were examined using light microscopy and digital image acquisition and subsequently were analyzed. Compared to complete serum, the use of serum depleted of fibronectin with vitronectin included had minimal effect on the cell attachment, spreading, and spatial distribution on the EDS regions of the surface. However, the use of serum depleted of vitronectin with or without fibronectin included resulted in greatly reduced cell attachment and spreading. Thus the presence of vitronectin was required for the attachment, spreading, and spatial distribution of bone-derived cells exposed to EDS/DMS-patterned surfaces. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 81–93, 1997.

Adhesion, spreading, and focal contact formation of primary bone-derived cells on quartz surfaces grafted with a 15 amino acid peptide that contained a -RGD-(-Arg-Gly-Asp-) sequence unique to bone sialoprotein was investigated. The peptide surfaces were fabricated by using a heterbifunctional crosslinker, sulfosuccinimidyal 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, to link the peptide to amine functionalized quartz surfaces. Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to confirm the chemistry and thickness of the overlayers. A radial flow apparatus was used to characterize cell detachment from peptide-grafted surfaces. After 20 min of cell incubation, the strength of cell adhesion was significantly (p< 0.05) higher on the -RGD- compared to -RGE- (control) surfaces. Furthermore, the mean area of cells contacting the -RGD- was significantly (p< 0.05) higher than -RGE- surfaces. Vinculin staining showed formation of small focal contact patches on the periphery of bone cells incubated for 2 h on the -RGD- surfaces; however, few or no focal contacts were formed by cells seeded on the -RGE- grafted surfaces. The methods of peptide immobilization utilized in this study can be applied to implants, biosensors, and diagnostic devices that require specificity in cell adhesion. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 9–19, 1997.

Patterned surfaces with alternating regions of amino silanes [N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS)] and alkyl silanes [dimethyldichlorosilane (DMS)] have been used to alter the kinetics of spatial distribution of cellsin vitro. In particular, we have previously observed the preferential spatial distribution of bone cells on the EDS regions of EDS/DMS patterned surfaces (10). In this study, we examined whether the mechanism of spatial distribution of cells on the EDS regions was adhesion mediated. Homogeneous layers of EDS and DMS were immobilized on quartz substrates and characterized by contact angle, X-ray photoelectron spectroscopy, and spectroscopic ellipsometry. The strength of bone cell attachment to the modified substrates was examined using a radial flow apparatus, within either 20 min or 2 hr of cell incubation in the presence of serum. A Weibull distribution was chosen to characterize the strength of cell-substratum adhesion. Within 20 min of cell exposure, the strength of adhesion was significantly larger on EDS and clean surfaces, compared with DMS surfaces (p<0.0001). Within 2 hr of cell incubation, there was no statistical difference between the strength of cell adhesion to EDS, DMS, and clean surfaces. The results of this study suggest that the surface chemistry mediates adhesion-based spatial cell arrangement through a layer of adsorbed serum proteins.

Human pluripotent stem cells harbor an unlimited capacity to generate therapeutically relevant cells for applications in regenerative medicine. However, to utilize these cells in the clinic, scalable culture systems that activate defined receptors and signaling pathways to sustain stem cell self-renewal are required; and synthetic materials offer considerable promise to meet these needs. De novodevelopment of materials that target novel pathways has been stymied by a limited understanding of critical receptor interactions maintaining pluripotency. Here, we identify peptide agonists for the human pluripotent stem cell (hPSC) laminin receptor and pluripotency regulator, α6-integrin, through unbiased, library-based panning strategies. Biophysical characterization of adhesion suggests that identified peptides bind hPSCs through α6-integrin with sub-μM dissociation constants similar to laminin. By harnessing a high-throughput microculture platform, we developed predictive guidelines for presenting these integrin-targeting peptides alongside canonical binding motifs at optimal stoichiometries to generate nascent culture surfaces. Finally, when presented as self-assembled monolayers, predicted peptide combinations supported hPSC expansion, highlighting how unbiased screens can accelerate the discovery of targeted biomaterials.

Volumetric muscle loss (VML) injuries are characterized by a degree of tissue loss that exceeds the endogenous regenerative capacity of muscle, resulting in permanent structural and functional deficits. Such injuries are a consequence of trauma, as well as a host of congenital and acquired diseases and disorders. Despite significant preclinical research with diverse biomaterials, as well as early clinical studies with implantation of decellularized extracellular matrices, there are still significant barriers to more complete restoration of muscle form and function following repair of VML injuries. In fact, identification of novel biomaterials with more advantageous regenerative profiles is a critical limitation to the development of improved therapeutics. As a first step in this direction, we evaluated a novel semisynthetic hyaluronic acid-based (HyA) hydrogel that embodies material features more favorable for robust muscle regeneration. This HyA-based hydrogel is composed of an acrylate-modified HyA (AcHyA) macromer, an AcHyA macromer conjugated with the bsp-RGD(15) peptide sequence to enhance cell adhesion, a high-molecular-weight heparin to sequester growth factors, and a matrix metalloproteinase-cleavable cross-linker to allow for cell-dependent remodeling. In a well-established, clinically relevant rat tibialis anterior VML injury model, we report observations of robust functional recovery, accompanied by volume reconstitution, muscle regeneration, and native-like vascularization following implantation of the HyA-based hydrogel at the site of injury. These findings have important implications for the development and clinical application of the improved biomaterials that will be required for stable and complete functional recovery from diverse VML injuries.

Efforts to understand and engineer cell behavior in mechanically soft environments frequently employ two-dimensional cell culture substrates consisting of thin hydrogel layers with low elastic modulus supported on rigid substrates to facilitate culturing, imaging, and analysis. Here we characterize how an elastic creasing instability of the gel surface may occur for the most widely used soft cell culture substrate, polyacrylamide hydrogels, and show that stem cells respond to and change their behavior due to these surface features. The regions of stability and corresponding achievable ranges of modulus are elucidated in terms of the monomer and cross-linker concentrations, providing guidance for the synthesis of both smooth and creased soft cell substrates for basic and applied cell engineering efforts.

Cardiac tissues are able to adjust their contractile behavior to adapt to the local mechanical environment. Nonuniformity of the native tissue mechanical properties contributes to the development of heart dysfunctions, yet the current in vitro cardiac tissue models often fail to recapitulate the mechanical nonuniformity. To address this issue, a 3D cardiac microtissue model is developed with engineered mechanical nonuniformity, enabled by 3D‐printed hybrid matrices composed of fibers with different diameters. When escalating the complexity of tissue mechanical environments, cardiac microtissues start to develop maladaptive hypercontractile phenotypes, demonstrated in both contractile motion analysis and force‐power analysis. This novel hybrid system could potentially facilitate the establishment of “pathologically‐inspired” cardiac microtissue models for deeper understanding of heart pathology due to nonuniformity of the tissue mechanical environment. Native heart tissues adjust their function when experiencing nonuniform mechanical environments caused by heart diseases (e.g., partial fibrosis). A 3D cardiac microtissue model under a nonuniform mechanical environment is developed by combining fibers with high or low mechanical resistance. This “pathologically‐inspired” 3D in vitro cardiac microtissue model allows for the probing of the adaptivity of cardiac microtissues to nonuniform tissue mechanical environments.

In article number 1901373by Kevin E. Healy, Zhen Ma, and co‐workers, a “pathologically‐inspired” 3D cardiac microtissue model is established based on human induced pluripotent stem cells and laser‐based 3D printing technology. This engineered in vitro model system can facilitate better understanding of the correlation between cardiac contractile dysfunctions and nonuniform tissue mechanical environments.

The survival of a biomaterial or tissue engineered construct is mainly hampered by the deficient microcirculation in its core, and limited nutrients and oxygen availability to the implanted or colonizing host cells. Aiming to address these issues, we herein propose bioresponsive gellan gum (GG) hydrogels that are biodegradable by metalloproteinase 1 (MMP‐1) and enable endothelial cells adhesion and proliferation. GG is chemically functionalized with divinyl sulfone (DVS) and then biofunctionalized with thiol cell‐adhesive peptides (T1 or C16) to confer GG endothelial cell biorecognition cues. Biodegradable hydrogels are then formed by Michael type addition of GGDVS or/and peptide‐functionalized GGDVS with a dithiol peptide crosslinker sensitive to MMP‐1. The mechanical properties (6 to 5580 Pa), swelling (17 to 11), MMP‐1‐driven degradation (up to 70%), and molecules diffusion coefficients of hydrogels are tuned by increasing the polymer amount and crosslinking density. Human umbilical cord vein endothelial cells depict a polarized elongated morphology when encapsulated within T1‐containing hydrogels, in contrast to the round morphology observed in C16‐containing hydrogels. Cell organization is favored as early as 1 d of cell culture within the T1‐modified hydrogels with higher concentration of peptide, while cell proliferation is higher in T1‐modified hydrogels with higher modulus. In conclusion, biodegradable and bioresponsive GGDVS hydrogels are promising endothelial cell responsive materials that can be used for vascularization strategies. Gellan gum (GG) hydrogelsare extensively studied in the tissue engineering and regenerative medicine field but their lack of cell adhesion sites and cell‐mediated degradation hinders their application in a cell‐responsive environment. Herein, for the first time, a one‐step chemical modification of GG is proposed, which allows the attachment of any thiol peptide and crosslinking with a biscisteine peptide.

The long-awaited translation of stem cell–based therapies to the clinic requires engineering approaches.

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