Cell Adhesion Domain Research Articles

Hydrogels with Independently Controlled Adhesion Ligand Mobility and Viscoelasticity Increase Cell Adhesion and Spreading.

A primary objective in designing hydrogels for cell culture is recreating the cell-matrix interactions found within human tissues. Identifying the most important biomaterial features for these interactions is challenging because it is difficult to independently adjust variables such as matrix stiffness, stress relaxation, the mobility of adhesion ligands and the ability of these ligands to support cellular forces. In this work we designed a hydrogel platform consisting of interpenetrating polymer networks of covalently crosslinked poly(ethylene glycol) (PEG) and self-assembled peptide amphiphiles (PA). We can tailor the storage modulus of the hydrogel by altering the concentration and composition of each network, and we can tune the stress relaxation half-life through the non-covalent bonding in the PA network. Ligand mobility can be adjusted independently of the matrix mechanical properties by attaching the RGD cell adhesion ligand to either the covalent PEG network, the dynamic PA network, or both networks at once. Interestingly, our findings show that endothelial cell adhesion formation and spreading is maximized in soft, viscoelastic gels in which RGD adhesion ligands are present on both the covalent PEG and non-covalent PA networks. The dynamic nature of cell adhesion domains, coupled with their ability to exert substantial forces on the matrix, suggests that having different presentations of RGD ligands which are either mobile or are capable of withstanding significant forces are needed mimic different aspects of complex cell-matrix adhesions. By demonstrating how different presentations of RGD ligands affect cell behavior independently of viscoelastic properties, these results contribute to the rational design of hydrogels that facilitate desired cell-matrix interactions, with the potential of improving in vitro models and regenerative therapies.

Tunable Light-Actuated Interpenetrating Networks of Silk Fibroin and Gelatin for Tissue Engineering and Flexible Biodevices.

Soft materials with tunable properties are valuable for applications such as tissue engineering, electronic skins, and human-machine interfaces. Materials that are nature-derived offer additional advantages such as biocompatibility, biodegradability, low-cost sourcing, and sustainability. However, these materials often have contrasting properties that limit their use. For example, silk fibroin (SF) has high mechanical strength but lacks processability and cell-adhesive domains. Gelatin, derived from collagen, has excellent biological properties, but is fragile and lacks stability. To overcome these limitations, composites of gelatin and SF have been explored. However, mechanically robust self-supported matrices and electrochemically active or micropatterned substrates were not demonstrated. In this study, we present a composite of photopolymerizable SF and photogelatin, termed photofibrogel (PFG). By incorporating photoreactive properties in both SF and gelatin, control over material properties can be achieved. The PFG composite can be easily and rapidly formed into free-standing, high-resolution architectures with tunable properties. By optimizing the ratio of SF to gelatin, properties such as swelling, mechanical behavior, enzymatic degradation, and patternability are tailored. The PFG composite allows for macroscale and microscale patterning without significant swelling, enabling the fabrication of structures using photolithography and laser cutting techniques. PFG can be patterned with electrically conductive materials, making it suitable for cell guidance and stimulation. The versatility, mechanical robustness, bioactivity, and electrochemical properties of PFG are shown for skeletal muscle tissue engineering using C2C12 cells as a model. Overall, such composite biomaterials with tunable properties have broad potential in flexible bioelectronics, wound healing, regenerative medicine, and food systems.

Protease-Sensitive, VEGF-Mimetic Peptide, and IKVAV Laminin-Derived Peptide Sequences within Elastin-Like Recombinamer Scaffolds Provide Spatiotemporally Synchronized Guidance of Angiogenesis and Neurogenesis.

Spatiotemporal control of vascularization and innervation is a desired hallmark in advanced tissue regeneration. For this purpose, we design a 3D model scaffold, based on elastin-like recombinamer (ELR) hydrogels. This contains two interior and well-defined areas, small cylinders, with differentiated bioactivities with respect to the bulk. Both are constructed on a protease sensitive ELR with a fast-proteolyzed domain, but one bears a VEGF-mimetic peptide (QK) and the other a laminin-derived pentapeptide (IKVAV), to promote angiogenesis and neurogenesis, respectively. The outer bulk is based on a slow proteolytic sequence and RGD cell adhesion domains. In vitro studies show the effect of QK and IKVAV peptides on the promotion of endothelial cell and axon spreading, respectively. The subcutaneous implantation of the final 3D scaffold demonstrates the ability to spatiotemporally control angiogenesis and neurogenesis in vivo. Specifically, the inner small cylinder containing the QK peptide promotes fast endothelialization, whereas the one with IKVAV peptide promotes fast neurogenesis. Both, vascularization and innervation take place in advance of the bulk scaffold infiltration. This scaffold shows that it is possible to induce vascularization and innervation in predetermined areas of the scaffold well ahead to the bulk infiltration. That significantly increases the efficiency of the regenerative activity.

The Relationship between Mutations in Gene-Specific Domains of Salivary Fibronectin (cFn) and Dynamin-2 (Dynm-2) and the Development of Porphyromonas gingivalis-Initiated Periodontitis

Periodontitis is a chronic inflammatory disease characterized by the destruction of the supporting structures of the teeth. Its high prevalence and negative effects on quality of life make it one of the current problems in dentistry. Porphyromonas gingivalis (P. gingivalis) is the predominant periodontal pathogen that expresses a number of virulence factors involved in the pathogenesis of periodontitis. P. gingivalis fimbriae are a critical factor in the interaction between the organism and the host tissue. They promote both bacterial adhesion and invasion into the target sites. Fimbriae are capable of binding to human saliva components, extracellular matrix proteins, and commensal bacteria, as well as firmly binding to the cellular integrin α5β1. After attachment to α5β1-integrin, P. gingivalis is captured by cellular pseudopodia, which makes invagination through an actin-mediated pathway possible. It has been proven that the invagination event also requires the participation of the host cell dynamin, actin fibers, microtubules and lipid rafts. Work has emerged investigating mutations in the proline-rich terminal domain (PRD) and their impact on disease development. Salivary antimicrobial peptides are early protective factors against microbial attack. Of great interest is fibronectin (FN) as the main competitor of P. gingivalis fimbriae. The FN can interact with cells in three different regions: the central cell-binding domain (CCBD), the COOH terminal heparin-binding domain (Hep2), and the type III connecting segment (IIICS), including the CS1 region (Yamada, 1991). CCBD is the major cell-adhesion domain of FN and contains an Arg–Gly–Asp (RGD) motif that is recognized by members of the cell adhesion receptor integrin family, including a5b1, which is the primary FN receptor in many cell types. The work focuses on identifying the relationship between the development of periodontitis and the presence of mutations in the adhesion domains of salivary proteins such as cellular fibronectin (cFN) and dynamin-2 (DYNM2).

Role of ADGRG1/GPR56 in Tumor Progression.

Cellular communication plays a critical role in diverse aspects of tumorigenesis including tumor cell growth/death, adhesion/detachment, migration/invasion, angiogenesis, and metastasis. G protein-coupled receptors (GPCRs) which constitute the largest group of cell surface receptors are known to play fundamental roles in all these processes. When considering the importance of GPCRs in tumorigenesis, the adhesion GPCRs (aGPCRs) are unique due to their hybrid structural organization of a long extracellular cell-adhesive domain and a seven-transmembrane signaling domain. Indeed, aGPCRs have been increasingly shown to be associated with tumor development by participating in tumor cell interaction and signaling. ADGRG1/GPR56, a representative tumor-associated aGPCR, is recognized as a potential biomarker/prognostic factor of specific cancer types with both tumor-suppressive and tumor-promoting functions. We summarize herein the latest findings of the role of ADGRG1/GPR56 in tumor progression.

Synthetic 3D PEG-Anisogel Tailored with Fibronectin Fragments Induce Aligned Nerve Extension.

An enzymatically cross-linked polyethylene glycol (PEG)-based hydrogel was engineered to promote and align nerve cells in a three-dimensional manner. To render the injectable, otherwise bioinert, PEG-based material supportive for cell growth, its mechanical and biochemical properties were optimized. A recombinant fibronectin fragment (FNIII9*-10/12-14) was coupled to the PEG backbone during gelation to provide cell adhesive and growth factor binding domains in close vicinity. Compared to full-length fibronectin, FNIII9*-10/12-14 supports nerve growth at similar concentrations. In a 3D environment, only the ultrasoft 1 w/v% PEG hydrogels with a storage modulus of ∼10 Pa promoted neuronal growth. This gel was used to establish the first fully synthetic, injectable Anisogel by the addition of magnetically aligned microelements, such as rod-shaped microgels or short fibers. The Anisogel led to linear neurite extension and represents a large step in the direction of clinical translation with the opportunity to treat acute spinal cord injuries.

Cell-Laden Electroconductive Hydrogel Simulating Nerve Matrix To Deliver Electrical Cues and Promote Neurogenesis.

Natural nerve tissue is composed of nerve bundles with multiple aligned assembles, and matrix electroconductivity is beneficial to the transmission of intercellular electrical signals, or effectively deliver external electrical cues to cells. Herein, aiming at the biomimetic design of the extracellular matrix for neurons, we first synthesized electroconductive polypyrrole (PPy) nanoparticles with modified hydrophilicity to improve their uniformity in collagen hydrogel. Next, cell-laden collagen-PPy hybrid hydrogel microfibers with highly oriented microstructures were fabricated via a microfluidic chip. The hydrogel microfibers formed a biomimetic three-dimensional microenvironment for neurons, resulting from the native cell adhesion domains, oriented fibrous structures, and conductivity. The oriented fibrous microstructures enhanced neuron-like cells aligning with fibers' axon; the matrix conductivity improved cell extension and upregulated neural-related gene expression; moreover, external electrical stimulation further promoted the neuronal functional expression. This mechanism was attributed to the electroconductive matrix and its delivered electrical stimulation to cells synergistically upregulated the expression of an L-type voltage-gated calcium channel, resulting in an increase in the intracellular calcium level, which in turn promoted neurogenesis. This approach has potential in constructing the biomimetic microenvironment for neurogenesis.

The three dimensional cues-integrated-biomaterial potentiates differentiation of human mesenchymal stem cells

Alginate (Alg) hydrogels, the most popular natural biomaterials, mimic the extracellular matrix (ECM) microenvironment and offer potential biomedical applications. Despite their excellent properties such as biocompatibility, hydrophilicity and ionic crosslinking, the absence of an intrinsic cell adhesion domain lessens their cell-carrier applications in tissue engineering. Herein, we suggest a three-dimensional (3D) cell culture system by integrating Alg hydrogel and fibroblast-derived matrix (FDM). FDM including cell-adhesion motifs, signaling, and physico-mechanical cues is prepared by the decellularization process by avoiding unfavorable chemical reactions. This cues-integrated-biomaterials (CiB) 3D platform shows increased cell viability, proliferation, chondrogenic and osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) in situ. The results show that the Alg/FDM hydrogel (CiB) matrix provides an excellent microenvironment for cell adhesion and can control the differentiation of hMSCs into specific lineages. Thus, these results suggest the potential applications of the Alg/FDM hydrogel matrix as a viable 3D culture system for tissue regeneration.

Spatial control and cell adhesion selectivity on model gold surfaces grafted with elastin-like recombinamers

A simple system for cell selectivity and spatially controlled cell adhesion has been developed using model gold surfaces grafted with a combination of two ELRs containing into their backbone cell-adhesion domains such as RGD and REDV. Grafting onto gold was achieved via redox reaction through thiol groups present in amino terminal cysteine tails of the ELRs. The correlation among contact angle, SEM micrographs, AFM, XPS and QCM-D have been carried out.After in-depth adhesion studies, a mixture of 75% ELR-REDV and 25% ELR-RGD was found to exhibit high selectivity for endothelial cells, promoting strong adhesion thereof.Consequently, certain areas of gold surfaces (strips) were cleaned by laser ablation and functionalized with the mixture 75% ELR-REDV – 25% ELR-RGD leading to a spatial segregation of the co-culture made of HUVEC and HFF1 cells. This platform therefore exhibits selective spatial control over cell adhesion associated with the bioactive epitopes (RGD and REDV) contained in the ELR sequence, since each functionalized surface (including strips) have similar topographic and hydrophobic characteristics and mechanical properties are in the same order of magnitude.

All-in-one trifunctional strategy: A cell adhesive, bacteriostatic and bactericidal coating for titanium implants.

Strategies to inhibit initial bacterial adhesion are extremely important to prevent infection on biomaterial surfaces. However, the simultaneous attraction of desired eukaryotic cells remains a challenge for successful biomaterial-host tissue integration. Here we describe a method for the development of a trifunctional coating that repels contaminating bacteria, kills those that adhere, and promotes osteoblast adhesion. To this end, titanium surfaces were functionalized by electrodeposition of an antifouling polyethylene glycol (PEG) layer and subsequent binding of a peptidic platform with cell-adhesive and bactericidal properties. The physicochemical characterization of the samples via SEM, contact angle, FTIR and XPS analysis verified the successful binding of the PEG layer and the biomolecules, without altering the morphology and topography of the samples. PEG coatings inhibited protein adsorption and osteoblast-like (SaOS-2) attachment; however, the presence of cell adhesive domains rescued osteoblast adhesion, yielding higher values of cell attachment and spreading compared to controls (p < 0.05). Finally, the antibacterial potential of the coating was measured by live/dead assays and SEM using S. sanguinis as a model of early colonizer in oral biofilms. The presence of PEG layers significantly reduced bacterial attachment on the surfaces (p < 0.05). This antibacterial potential was further increased by the bactericidal peptide, yielding values of bacterial adhesion below 0.2% (p < 0.05). The balance between the risk of infection and the optimal osteointegration of a biomaterial is often described as "the race for the surface", in which contaminating bacteria and host tissue cells compete to colonize the implant. In the present work, we have developed a multifunctional coating for a titanium surface that promotes the attachment and spreading of osteoblasts, while very efficiently inhibits bacterial colonization, thus holding promise for application in bone replacing applications.

Random and oriented electrospun fibers based on a multicomponent, in situ clickable elastin-like recombinamer system for dermal tissue engineering

Herein we present a system to obtain fibers from clickable elastin-like recombinamers (ELRs) that crosslink in situ during the electrospinning process itself, with no need for any further treatment to stabilize them. These ELR-click fibers are completely stable under in vitro conditions. A wrinkled fiber morphology is obtained. In addition to a random fiber orientation, oriented fibers with a high degree of alignment and coherence can also be obtained by using a rotational electrode. The production of multicomponent fibers means that different functionalities, such as cell-adhesion domains (RGD peptides), can be incorporated into them. In a subsequent study, two main cell lines present in the dermis and epidermis, namely keratinocytes and fibroblasts, were cultured on top of the ELR-click fibers. Adhesion, proliferation, fluorescence, immunostaining and histology studies showed the cytocompatibility of these scaffolds, thus suggesting their possible use for wound dressings in skin tissue engineering applications. Statement of significanceFor the first time stable electrospun bioactive fibers are obtained by the in situ mixing of two “clickable” ELR components previously described by Gonzalez et al (Acta Biomaterialia 2014). This work describes an efficient system to prepare fibrous scaffolds based on peptidic polymers by electrospinning without the need of crosslinking agents that could be harmful for cells or living tissues. These bioactive fibers support cell growth due to the inclusion of RGD motifs (Staubli et al. Biomaterials 2017). Finally, the in vitro biocompatibility of the two main cell types found in the outer layers of skin, fibroblasts and keratinocytes, indicates that this system is of great interest to prepare elastic artificial skin substitutes for wound healing applications.

Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin

Human P-cadherin is a cell adhesion protein of the family of classical cadherins, the overexpression of which is correlated with poor prognosis in various types of cancer. Antibodies inhibiting cell-cell adhesion mediated by P-cadherin show clear therapeutic effect, although the mechanistic basis explaining their effectiveness is still unclear. Based on structural, physicochemical, and functional analyses, we have elucidated the molecular mechanism of disruption of cell adhesion by antibodies targeting human P-cadherin. Herein we have studied three different antibodies, TSP5, TSP7, and TSP11, each recognizing a different epitope on the surface of the cell-adhesive domain (EC1). Although all these three antibodies recognized human P-cadherin with high affinity, only TSP7 disrupted cell adhesion. Notably, we demonstrated that TSP7 abolishes cell adhesion by disabling the so-called X-dimer (a kinetic adhesive intermediate), in addition to disrupting the strand-swap dimer (the final thermodynamic state). The inhibition of the X-dimer was crucial for the overall inhibitory effect, raising the therapeutic value of a kinetic intermediary not only for preventing, but also for reversing, cell adhesion mediated by a member of the classical cadherin family. These findings should help to design more innovative and effective therapeutic solutions targeting human P-cadherin.

Elastin-like-recombinamers multilayered nanofibrous scaffolds for cardiovascular applications

Coronary angioplasty is the most widely used technique for removing atherosclerotic plaques in blood vessels. The regeneration of the damaged intima layer after this treatment is still one of the major challenges in the field of cardiovascular tissue engineering. Different polymers have been used in scaffold manufacturing in order to improve tissue regeneration. Elastin-mimetic polymers are a new class of molecules that have been synthesized and used to obtain small diameter fibers with specific morphological characteristics. Elastin-like polymers produced by recombinant techniques and called elastin-like recombinamers (ELRs) are particularly promising due to their high degree of functionalization. Generally speaking, ELRs can show more complex molecular designs and a tighter control of their sequence than other chemically synthetized polymers Rodriguez Cabello et al (2009 Polymer 50 5159–69, 2011 Nanomedicine 6 111–22). For the fabrication of small diameter fibers, different ELRs were dissolved in 2,2,2-fluoroethanol (TFE). Dynamic light scattering was used to identify the transition temperature and get a deep characterization of the transition behavior of the recombinamers. In this work, we describe the use of electrospinning technique for the manufacturing of an elastic fibrous scaffold; the obtained fibers were characterized and their cytocompatibility was tested in vitro. A thorough study of the influence of voltage, flow rate and distance was carried out in order to determine the appropriate parameters to obtain fibrous mats without beads and defects. Moreover, using a rotating mandrel, we fabricated a tubular scaffold in which ELRs containing different cell adhesion sequences (mainly REDV and RGD) were collected. The stability of the scaffold was improved by using genipin as a crosslinking agent. Genipin-ELRs crosslinked scaffolds show a good stability and fiber morphology. Human umbilical vein endothelial cells were used to assess the in vitro bioactivity of the cell adhesion domains within the backbone of the ELRs.

Distortion of the normal function of synaptic cell adhesion molecules by genetic variants as a risk for autism spectrum disorders.

Synaptic cell adhesion molecules (SCAMs) are a functional category of cell adhesion molecules that connect pre- and postsynapses by the protein-protein interaction via their extracellular cell adhesion domains. Countless numbers of common genetic variants and rare mutations in SCAMs have been identified in the patients with autism spectrum disorders (ASDs). Among these, NRXN and NLGN family proteins cooperatively function at synaptic terminals both of which genes are strongly implicated as risk genes for ASDs. Knock-in mice carrying a single rare point mutation of NLGN3 (NLGN3 R451C) discovered in the patients with ASDs display a deficit in social interaction and an enhancement of spatial learning and memory ability reminiscent of the clinical phenotype of ASDs. NLGN4 knockout (KO) and NRXN2α KO mice also show a deficit in sociability as well as some specific neuropsychiatric behaviors. In this review, we selected NRXNs/NLGNs, CNTNAP2/CNTNAP4, CNTN4, ITGB3, and KIRREL3 as strong ASD risk genes based on SFARI score and summarize the protein structures, functions at synapses, representative discoveries in human genetic studies, and phenotypes of the mutant model mice in light of the pathophysiology of ASDs.

Copolymer-Mediated Cell Aggregation Promotes a Proangiogenic Stem Cell Phenotype In Vitro and In Vivo.

Material-induced cell aggregation drives a proangiogenic expression profile. Copolymer substrates containing cell-repellent and cell-adhesive domains force the aggregation of human mesenchymal stem cells, which results in enhanced tubulogenesis in vitro and stabilization of vasculature in vivo. These findings can be used to design instructive biomaterial scaffolds for clinical use.

Engineering thick cell sheets by electrochemical desorption of oligopeptides on membrane substrates.

We developed a gold-coated membrane substrate modified with an oligopeptide layer that can be used to grow and subsequently detach a thick cell sheet through an electrochemical reaction. The oligopeptide CCRRGDWLC was designed to contain a cell adhesive domain (RGD) in the center and cysteine residues at both terminals. Cysteine contains a thiol group that forms a gold–thiolate bond on a gold surface. Cells attached to gold-coated membrane substrates via the oligopeptide layer were readily and noninvasively detached by applying a negative electrical potential to cleave the gold–thiolate bond. Because of the effective oxygen supply, fibroblasts vigorously grew on the membrane substrate and the thickness of the cell sheets was ∼60 μm at 14 days of culture, which was 2.9-fold greater than that of cells grown on a conventional culture dish. The cell sheets were detached after 7 min of electrical potential application. Using this approach, five layers of cell sheets were stacked sequentially with thicknesses reaching >200 μm. This approach was also beneficial for rapidly and readily transplanting cell sheets. Grafted cell sheets secreted collagen and remained at the transplanted site for at least 2 months after transplantation. This simple electrochemical cell sheet engineering technology is a promising tool for tissue engineering and regenerative medicine applications.

Physicochemical properties of microfabricated hydrogel scaffolds modulate tumor dormancy and chemotherapy resistance in a hepatic all-human microphysiologic model of metastasis

Event Abstract Back to Event Physicochemical properties of microfabricated hydrogel scaffolds modulate tumor dormancy and chemotherapy resistance in a hepatic all-human microphysiologic model of metastasis Alex J. Wang1, Marianna Sofman1, Jorge Valdez1, Sarah E. Wheeler2, Amanda M. Clark2, Venkateswaran C. Pillai3, Raman Venkataramanan2, 3, Carissa L. Young1, Douglas A. Lauffenburger1, Donna B. Stoltz2, Alan Wells2 and Linda G. Griffith1 1 MIT, Department of Biological Engineering, United States 2 University of Pittsburgh, Department of Pathology, United States 3 University of Pittsburgh, Department of Pharmaceutical Sciences, United States Introduction: Micrometastases are clinically chemoresistant, particularly during presumed dormancy, which prevents clearance of these cells that often emerge later to kill patients. Liver is a common site of metastasis for many cancers, including the particularly fatal “triple negative” breast cancer (TNBC). We have previously developed a microscale reactor for long-term highly functional culture of 3D liver-like tissue for use in drug metabolism and toxicity studies [1],[2]. In previous work, we used a thin polystyrene scaffold that fostered formation of 3D tissue structures when seeded with primary human liver cells comprising a mixture of hepatocytes and non-parenchymal cells (NPCs). Adaptation of this reactor system to study TNBC micrometastases in the liver microenvironment requires attention to environmental factors, such as scaffold biophysical properties, that may influence the phenotype of the liver cells. Polystyrene scaffolds are stiff and induce a mild inflammatory phenotype in the liver cells, and this inflammatory response may stimulate growth of the micrometastases. To eliminate this deleterious effect, we developed an entirely new approach based on microfabrication of functionalized PEG hydrogels. We hypothesized that providing a more physiological environment for liver cell seeding and subsequent introduction of cancer cells would promote breast cancer cell dormancy and provide increased protection from chemotherapy as seen in patients, enabling the study of this elusive but critical stage in cancer progression. Materials and Methods: Hydrogel scaffolds with defined channel geometries were formed by micromolding of polyethylene glycol macromers (700 MW linear PEG-diacrylate or 20K MW 8-arm PEG-norbornene) with thiol crosslinkers using UV-initiated polymerization of macromer solutions. Hydrogels were functionalized with fibronectin-derived cell adhesive domains [3]. Physiochemical properties, including swelling, stiffness, and peptide composition, were systematically varied [4],[5]. Hydrogel crosslinking strategies included the use of peptides susceptible to proteolytic degradation and/or controlled dissolution of gel upon addition of a bacterial transpeptidase that releases the trapped cells for further characterization. 3D liver co-cultures employing primary human liver cells were initiated in microperfused bioreactors and seeded with fluorescently-labelled MDA-MB-231 or MCF-7 breast cancer cells (500-1000 cells/well) as described previously [1]. Functional behavior of liver cells, inflammatory cytokines, and growth of tumor cells were monitored over 1-3 weeks of culture in either homeostatic or simulated inflammatory (addition of LPS) conditions. Results: Hydrogel scaffolds seeded with human hepatocytes and NPCs were comparable in efficiency and overall markers of hepatocellular health as assessed using injury markers, functional markers, and tissue morphology when compared to traditional polystyrene scaffolds. Interestingly, the LPS-induced inflammatory response was enhanced in hydrogel compared to standard scaffolds, presumably due to reduced basal activation. Cancer cell numbers and markers for proliferation in hydrogel were decreased on day 15 in hydrogel compared to rigid scaffolds, indicating a dormant phenotype. Treatment of cultures with chemotherapy showed a decline in cancer cell numbers in rigid scaffolds, which contained more proliferating cells. Conversely, cancer cell numbers in hydrogel scaffolds were unaffected, which implies increased dormancy. Discussion: The PEG hydrogel microenvironment more closely reflects physiologically relevant properties and appears to better maintain NPC survival and health, as well as increasing hepatic tissue density within each channel. Cancer cell proliferation is attenuated in hydrogels, possibly due to decreased cellular stress and inflammatory response by NPCs. The use of hydrogels in this system allows for the first model of metastasis escape from chemotherapy in an all-human system. Ongoing work is elucidating the signaling pathways involved in this response, as well as continuing to design more robust and modular hydrogel scaffolds for the liver bioreactor. We thank Linda Stockdale for assistance with micromolding of hydrogels; We thank Kasper Renggli for insightful discussions

Assessment of collagen crosslinking and denaturation for the design of regenerative scaffolds.

Crosslinking and denaturation were two variables that deeply affected the performance of collagen-based scaffolds designed for tissue regeneration. If crosslinking enhances the mechanical properties and the enzymatic resistance of collagen, while masking or reducing the available cell binding sites, denaturation has very opposite effects, as it impairs the mechanical and the enzymatic stability of collagen, but increases the number of exposed cell adhesive domains. The quantification of both crosslinking and denaturation was thus fundamental to the design of collagen-based scaffolds for selected applications. The aim of this work was to investigate the extents of crosslinking and denaturation of collagen-based films upon dehydrothermal (DHT) treatment, that is, one of the most commonly employed methods for zero-length crosslinking that shows the unique ability to induce partial denaturation. Swelling measurements, differential scanning calorimetry, Fourier transform infrared spectroscopy, colorimetric assays for the quantification of primary amines, and mechanical tests were performed to analyze the effect of the DHT temperature on crosslinking and denaturation. In particular, chemically effective and elastically effective crosslink densities were evaluated. Both crosslinking and denaturation were found to increase with the DHT temperature, although according to different trends. The results also showed that DHT treatments performed at temperatures up to 120°C maintained the extent of denaturation under 25%. Coupling a mild DHT treatment with further crosslinking may thus be very useful not only to modulate the crosslink density, but also to induce a limited amount of denaturation, which shows potential to partially compensate the loss of cell binding sites caused by crosslinking.

The height of cell-adhesive nanoposts generated by block copolymer/surfactant complex systems influences the preosteoblast phenotype

In tissue engineering, the nanoscale topography of the substrate is important, because transplanted cells can recognize and respond to topographical patterns, allowing control of gene expression and tissue formation. In this study, we hypothesized that the height of cell-adhesive nanoposts could regulate cell phenotype. Nano-patterned surfaces were generated via self-assembly of polystyrene-b-poly(ethylene oxide)/dodecylbenzenesulfonic acid (PS-b-PEO/DBSA) complex systems. The height of PS nanoposts, which are considered to be cell-adhesion domains, was varied from 11 to 43nm, while nanopost size and the center-to-center distance between nanoposts were kept constant. Adhesion, growth, and differentiation of mouse preosteoblasts (MC3T3-E1) cultured on the nano-patterned surfaces were significantly influenced by nanopost height. This approach therefore holds great promise for the design of biomedical devices, as well as tissue engineering scaffolds.

Potency of Fish Collagen as a Scaffold for Regenerative Medicine

Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine.