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Clinically, increased breast tumor stiffness is associated with metastasis and poorer outcomes. Yet, in vitro studies of tumor cells in 3D scaffolds have found decreased invasion in stiffer environments. To resolve this apparent contradiction, MDA-MB-231 breast tumor spheroids were embedded in ‘low’ (2 kPa) and ‘high’ (12 kPa) stiffness 3D hydrogels comprised of methacrylated gelatin/collagen I, a material that allows for physiologically-relevant changes in stiffness while matrix density is held constant. Cells in high stiffness materials exhibited delayed invasion, but more abundant actin-enriched protrusions, compared to those in low stiffness. We find that cells in high stiffness had increased expression of Mena, an invadopodia protein associated with metastasis in breast cancer, as a result of EGFR and PLCγ1 activation. As invadopodia promote invasion through matrix remodeling, we examined matrix organization and determined that spheroids in high stiffness displayed a large fibronectin halo. Interestingly, this halo did not result from increased fibronectin production, but rather from Mena/α5 integrin dependent organization. In high stiffness environments, FN1 knockout inhibited invasion while addition of exogenous cellular fibronectin lessened the invasion delay. Analysis of fibronectin isoforms demonstrated that EDA-fibronectin promoted invasion and that clinical invasive breast cancer specimens displayed elevated EDA-fibronectin. Combined, our data support a mechanism by which breast cancer cells respond to stiffness and render the environment conducive to invasion. More broadly, these findings provide important insight on the roles of matrix stiffness, composition, and organization in promoting tumor invasion.

Recent evidence supports the fimbriae of the fallopian tube as a potential origin site for high-grade serous ovarian cancer (HGSOC). The progression of many solid tumors is accompanied by changes in the microenvironment, including alterations of the extracellular matrix (ECM). The ECM of fallopian tube and HGSOC has not been well characterized. Therefore, we sought to determine the ECM composition of the benign fallopian tube and how it changes with the onset of serous intraepithelial carcinomas (STICs), precursor of HGSOC. The ECM composition of benign human fallopian tube was first defined from a meta-analysis of published proteomic datasets and identified 190 ECM proteins. We then conducted de novo proteomics using ECM enrichment and identified 88 proteins, 7 of which were not identified in prior studies. We further investigated the levels and localization of seven of these ECM proteins (type I, III, and IV collagens, fibronectin, laminin, versican, perlecan) and hyaluronic acid using multi-spectral immunohistochemical staining of fimbriae from patients with benign conditions or STICs. Quantification revealed an increase in stromal fibronectin and a decrease in epithelial versican in STICs. Our results provide an in-depth picture of the ECM in the benign fallopian tube and identified ECM changes that accompany STIC formation.

Angiogenesis is a hallmark of fibrocalcific aortic valve disease (CAVD). An imbalance of pro- and anti-angiogenic factors is thought to play a role in driving this disease process, and valvular interstitial cells (VICs) may act as a significant source of these factors. CAVD is also known to exhibit sexual dimorphism in its presentation, and previous work suggested that VICs may exhibit cellular-scale sex differences in the context of angiogenesis. The current study sought to investigate the production of angiogenesis-related factors by male and female VICs possessing quiescent (qVIC) or activated (aVIC) phenotypes. Production of several pro-angiogenic growth factors was elevated in porcine aVICs relative to qVICs, with sex differences found in both the total amounts secreted and their distribution across media vs. lysate. Porcine valvular endothelial cells (VECs) were also sex-separated in culture and found to behave similarly with respect to metabolic activity, viability, and tubulogenesis, but male VECs exhibited higher proliferation rates than female VECs. VECs responded to sex-matched media conditioned by VICs with increased tubulogenesis, but decreased proliferation, particularly upon treatment with aVIC-derived media. It is likely that this attenuation of proliferation resulted from a combination of decreased basic fibroblast growth factor and increased thrombospondin-2 (TSP2) secreted by aVICs. Overall, this study indicates that VICs regulate angiogenic VEC behavior via an array of paracrine molecules, whose secretion and sequestration are affected by both VIC phenotype and sex. Moreover, strong sex differences in TSP2 secretion by VICs may have implications for understanding sexual dimorphism in valve fibrosis, as TSP2 is also a powerful regulator of fibrosis.

Summary Clinical evidence supports a role for the extracellular matrix (ECM) in cancer risk and prognosis across multiple tumor types, and numerous studies have demonstrated that individual ECM components impact key hallmarks of tumor progression (e.g., proliferation, migration, angiogenesis). However, the ECM is a complex network of fibrillar proteins, glycoproteins, and proteoglycans that undergoes dramatic changes in composition and organization during tumor development. In this review, we will highlight how engineering approaches can be used to examine the impact of changes in tissue architecture, ECM composition (i.e., identity and levels of individual ECM components), and cellular- and tissue-level mechanics on tumor progression. In addition, we will discuss recently developed methods to model the ECM that have not yet been applied to the study of cancer., Graphical Abstract, Highlights • The extracellular matrix (ECM) is dramatically altered during cancer progression • Engineering methods can model the resulting changes in tissue architecture in vitro • Natural and synthetic biomaterials mimic changes in ECM composition and mechanics • Fiber-based and dynamic materials not yet used to study tumor ECM are highlighted, Tissue Engineering; Cancer

Collagen is the most abundant protein in mammals, accounting for approximately one-third of the total protein in the human body. Thus, it is a logical choice for the creation of biomimetic environments, and there is a long history of using collagen matrices for various tissue engineering applications. However, from a biomaterial perspective, the use of collagen-only scaffolds is associated with many challenges. Namely, the mechanical properties of collagen matrices can be difficult to tune across a wide range of values, and collagen itself is not highly amenable to direct chemical modification without affecting its architecture or bioactivity. Thus, many approaches have been pursued to design scaffold environments that display critical features of collagen but enable improved tunability of physical and biological characteristics. This paper provides a brief overview of approaches that have been employed to create such engineered collagen matrices. Specifically, these approaches include blending of collagen with other natural or synthetic polymers, chemical modifications of denatured collagen, de novo creation of collagen-mimetic chains, and reductionist methods to incorporate collagen moieties into other materials. These advancements in the creation of tunable, engineered collagen matrices will continue to enable the interrogation of novel and increasingly complex biological questions.

Re‐epithelialization is a critical step in wound healing and results from the collective migration of keratinocytes. Previous work demonstrated that immobilized, but not soluble, epidermal growth factor (EGF) resulted in leader cell‐specific activation of phospholipase C gamma 1 (PLCγ1) in HaCaT keratinocytes, and that this PLCγ1 activation was necessary to drive persistent cell migration. To determine the mechanism responsible for wound edge‐localized PLCγ1 activation, we examined differences in cell area, cell–cell interactions, and EGF receptor (EGFR) localization between wound edge and bulk cells treated with vehicle, soluble EGF, or immobilized EGF. Our results support a multistep mechanism where EGFR translocation from the lateral membrane to the basolateral/basal membrane allows clustering in response to immobilized EGF. This analysis of factors regulating PLCγ1 activation is a crucial step toward developing therapies or wound dressings capable of modulating this signal and, consequently, cell migration.

Microarchitectural features of collagen-rich extracellular matrices provide the mechanical foundation for tissue function and exhibit topographical cues that influence cellular behavior including proliferation, migration and protein expression. Preservation of tissue microarchitecture is required for accurate evaluation of tissue characteristics and pathology. It is unclear whether common tissue preservation methods possess equal ability to preserve microarchitecture. We investigated collagen microarchitecture in samples that had been flash frozen, fixed in formalin or preserved in RNAlater®, and which contained both collagen-rich and collagen-sparse regions. Fibrillar collagen organization was characterized using picrosirius red staining and second harmonic generation (SHG) microscopy. Maintenance of collagen fiber characteristics compared to the gold standard of flash freezing depended on both the method of preservation and the local collagen content of the tissue. Both formalin fixation and RNAlater® preserved collagen fiber characteristics similar to flash freezing in collagen-rich areas of the tissue, but not in collagen-sparse regions. Analysis using picrosirius red staining indicated preservation-dependent changes in overall tissue architecture and suprafibrillar organization. Together with considerations of cost, ease of use, storage conditions and ability to use the preserved tissue for RNA or protein analysis, our quantitative characterization of the effects of preservation method on collagen microarchitecture may help investigators select the most appropriate preservation approach for their needs.

An insufficient understanding of calcific aortic valve disease (CAVD) pathogenesis remains a major obstacle in developing treatment strategies for this disease. The aim of the present study was to create engineered environments that mimic the earliest known features of CAVD and apply this in vitro platform to decipher relationships relevant to early valve lesion pathobiology. Glycosaminoglycan (GAG) enrichment is a dominant hallmark of early CAVD, but culture of valvular interstitial cells (VICs) in biomaterial environments containing pathological amounts of hyaluronic acid (HA) or chondroitin sulfate (CS) did not directly increase indicators of disease progression such as VIC activation or inflammatory cytokine production. However, HA-enriched matrices increased production of vascular endothelial growth factor (VEGF), while matrices displaying pathological levels of CS were effective at retaining lipoproteins, whose deposition is also found in early CAVD. Retained oxidized low-density lipoprotein (oxLDL), in turn, stimulated myofibroblastic VIC differentiation and secretion of numerous inflammatory cytokines. OxLDL also increased VIC deposition of GAGs, thereby creating a positive feedback loop to further enrich GAG content and promote disease progression. Using this disease-inspired in vitro platform, we were able to model a complex, multistep pathological sequence, with our findings suggesting distinct roles for individual GAGs in outcomes related to valve lesion progression, as well as key differences in cell-lipoprotein interactions compared with atherosclerosis. We propose a pathogenesis cascade that may be relevant to understanding early CAVD and envision the extension of such models to investigate other tissue pathologies or test pharmacological treatments.

Host-defense peptides (HDPs) are produced by eukaryotes to defend against bacterial infection, and diverse synthetic polymers have recently been explored as mimics of these natural peptides. HDPs are rich in both hydrophobic and cationic amino acid residues, and most HDP-mimetic polymers have therefore contained binary combinations of hydrophobic and cationic subunits. However, HDP-mimetic polymers rarely duplicate the hydrophobic surface and cationic charge density found among HDPs ( Hu , K. ; et al. Macromolecules 2013 , 46 , 1908 ); the charge and hydrophobicity are generally higher among the polymers. Statistical analysis of HDP sequences ( Wang , G. ; et al. Nucleic Acids Res. 2009 , 37 , D933 ) has revealed that serine (polar but uncharged) is a very common HDP constituent and that glycine is more prevalent among HDPs than among proteins in general. These observations prompted us to prepare and evaluate ternary nylon-3 copolymers that contain a modestly polar but uncharged subunit, either serine-like or glycine-like, along with a hydrophobic subunit and a cationic subunit. Starting from binary hydrophobic-cationic copolymers that were previously shown to be highly active against bacteria but also highly hemolytic, we found that replacing a small proportion of the hydrophobic subunit with either of the polar, uncharged subunits can diminish the hemolytic activity with minimal impact on the antibacterial activity. These results indicate that the incorporation of polar, uncharged subunits may be generally useful for optimizing the biological activity profiles of antimicrobial polymers. In the context of HDP evolution, our findings suggest that there is a selective advantage to retaining polar, uncharged residues in natural antimicrobial peptides.

Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp(3) carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications.

The high failure rate of small diameter vascular grafts continues to drive the development of new materials and modification strategies that address this clinical problem, with biomolecule incorporation typically achieved via surface-based modification of various biomaterials. In this work, we examined whether the method of biomolecule incorporation (i.e., bulk versus surface modification) into a polyurethane (PU) polymer impacted biomaterial performance in the context of vascular applications. Specifically, hyaluronic acid (HA) was incorporated into a poly(ether urethane) via bulk copolymerization or covalent surface tethering, and the resulting PU-HA materials characterized with respect to both physical and biological properties. Modification of PU with HA by either surface or bulk methods yielded materials that, when tested under static conditions, possessed no significant differences in their ability to resist protein adsorption, platelet adhesion, and bacterial adhesion, while supporting endothelial cell culture. However, only bulk-modified PU-HA materials were able to fully retain these characteristics following material exposure to flow, demonstrating a superior ability to retain the incorporated HA and minimize enzymatic degradation, protein adsorption, platelet adhesion, and bacterial adhesion. Thus, despite bulk methods rarely being implemented in the context of biomolecule attachment, these results demonstrate improved performance of PU-HA upon bulk, rather than surface, incorporation of HA. Although explored only in the context of PU-HA, the findings revealed by these experiments have broader implications for the design and evaluation of vascular graft modification strategies.

Nylon-3 co-polymers containing both hydrophobic and cationic subunits can mimic the activity profile of host-defense peptides, if subunit identity and proportion are carefully selected. These sequence- and stereo-random co-polymers inhibit bacterial growth at relatively low concentrations, apparently via disruption of bacterial membranes, but they are relatively non-disruptive toward eukaryotic cell membranes (low hemolytic activity). In all previous examples, the hydrophobic subunits have contained cycloalkyl groups that incorporate the backbone Cα-Cβ bond. Here we have explored the effects of using analogous acyclic hydrophobic subunits. The results indicate that the replacing cyclic with acyclic hydrophobic subunits has a modest influence on biological properties. This influence appears to arise from differences in subunit flexibility.

Host-defense peptides inhibit bacterial growth but show little toxicity toward mammalian cells. A variety of synthetic polymers have been reported to mimic this antibacterial selectivity; however, achieving comparable selectivity for fungi is more difficult because these pathogens are eukaryotes. Here we report nylon-3 polymers based on a novel subunit that display potent antifungal activity (MIC = 3.1 μg/mL for Candida albicans) and favorable selectivity (IC10 > 400 μg/mL for 3T3 fibroblast toxicity; HC10 > 400 μg/mL for hemolysis).

Mechanical mismatch, along with inadequate hemocompatibility and endothelialization, contribute to the high failure rate of many synthetic vascular grafts. However, due to the dueling nature of these requirements (i.e., inhibiting platelet adhesion frequently means inhibiting endothelial cell (EC) adhesion), the creation of materials that simultaneously satisfy the mechanical and biological design criteria needed for small diameter vascular grafts has been an elusive goal. In this work, we demonstrate the ability of polyurethane (PU) containing hyaluronic acid (HA) in its backbone structure to reduce protein adsorption, platelet and bacterial adhesion, and fibroblast and macrophage proliferation while allowing the retention of both ECs and vascular-appropriate mechanics. Irrespective of HA molecular weight (MW), PU-HA materials selectively supported the growth of ECs relative to fibroblasts, reduced platelet adhesion, and performed comparably to negative controls with respect to bactericidal activity. The extent of EC growth on the PU-HA materials did differ with HA MW, with a lower HA MW yielding improved EC growth in both two-dimensional (2-D) films and 3-D electrospun fibrous scaffolds. These findings illustrate that HA incorporated into the backbone of a synthetic polymer structure can retain bioactivity, with subtle differences in HA MW significantly impacting the physical and biological properties of the biomaterial; in particular, PU modified with low-MW HA appears promising for vascular graft applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 2870–2882, 2013.

The ability to design biomaterials that interact with biological environments in a predictable manner necessitates an improved understanding of how surface chemistry influences events such as protein adsorption and cell adhesion. In this work, we examined mechanisms governing the interactions between 3T3 fibroblasts and nylon-3 polymers, which have a protein-like polyamide backbone and are highly amenable to tuning of chemical and physical properties. Protein adsorption and cell adhesion to a library of nylon-3 polymers were characterized and analyzed by partial least squares regression. This analysis revealed that specific chemical features of the nylon-3 polymers correlated with the extent of protein adsorption, which, in turn, correlated with cell adhesion in a serum-containing environment. In contrast, in a serum-free environment, cell adhesion could be predicted solely from chemical properties. Enzymatic treatments of 3T3 cells prior to plating indicated that proteins bound to the cell surface mediated cell-nylon-3 polymer interactions under serum-free conditions, with additional analysis suggesting that cell-associated fibronectin played a dominant role in adhesion in the absence of serum. The mechanistic insight gained from these studies can be used to inform the design of new polymer structures in addition to providing a basis for continued development of nylon-3 copolymers for tissue engineering applications.

Growth factors play a critical role in regulating processes involved in cellular differentiation and tissue regeneration, and are therefore considered essential elements in many tissue engineering strategies. The covalent immobilization of growth factors to biomaterial matrices addresses many of the challenges associated with delivering freely-diffusible growth factors and has thus emerged as a promising method of achieving localized and sustained growth factor delivery. This Feature Article discusses methods that have been used to immobilize growth factors to substrates, followed by an overview of several tissue repair and regeneration applications in which immobilized growth factors have been used.

Disruption of the extracellular matrix (ECM) is frequently found in calcific aortic valve disease (CAVD), yet the role of ECM components in valvular interstitial cell (VIC) function and dysfunction remains poorly understood. This study examines the contributions of exogenous and endogenous hyaluronic acid (HA), in both two-dimensional (2D) and 3D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2D setting in which the cells were exposed to different molecular weights of exogenous HA presented in either an immobilized or soluble form. Delivery of HA suppressed nodule formation in a molecular weight-dependent manner, while blocking VIC recognition of HA via an antibody to CD44 abolished these nodule-suppressive effects and stimulated other hallmarks of valvular dysfunction. These 2D results were then validated in a more physiologically-relevant setting, using an approach that allowed the characterization of VIC phenotype in response to HA alterations in the native 3D environment. In this approach, leaflet organ cultures were analyzed following treatment with anti-CD44 or with hyaluronidase to specifically remove HA. Disruption of VIC-HA interactions upregulated markers of VIC disease and induced leaflet mineralization. Similarly, HA-deficient leaflets exhibited numerous hallmarks of CAVD, including increased VIC proliferation, apoptosis, increased expression of disease-related markers, and mineralization. These findings suggest that VIC-HA interactions are crucial in maintaining a healthy VIC phenotype. Identification of ECM components that can regulate VIC phenotype and function has significant implications for understanding of native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering.

The range of application of polyurethanes has been limited by their poor hemocompatibility and inability to resist non-specific binding of biomolecules and cells. In this work, a non-adhesive PU-based material was synthesized via the copolymerization of PU with dermatan sulfate. Incorporation of DS into the PU backbone dramatically increased material hydrophilicity and decreased protein adsorption. The in vitro adhesion of several cell types, including platelets, also significantly decreased with increasing DS content. Both the physical and biological properties of the DS contributed to the anti-adhesive properties of the PU/DS copolymer, and this anti-adhesive nature of PU/DS renders this new biomaterial attractive for blood-contacting or non-fouling applications.

Despite the rapidly growing body of work on stem cell-based adipose tissue engineering, there remains much to be learned about the role of the scaffold and culture environments in directing the adipogenic differentiation of cells. The present study examined how various culture environments and differentiation stimuli (traditional differentiation medium [DM] and coculture with mature adipocytes) impacted the adipogenic differentiation of human preadipocytes, with studies progressing from two-dimensions (2D) to three-dimensions (3D) in vitro. Assays for adipogenic markers (leptin, adiponectin, and glycerol) and Oil Red O staining were used to assess differentiation. After 16 days of 2D culture, adipogenesis was substantially greater when preadipocytes were cocultured with adipocytes rather than treated with DM. In a 3D in vitro environment, the production of adipogenic markers was significantly elevated relative to 2D conditions, and the coculture condition continued to stimulate greater adipogenesis. Alterations in 3D scaffold physical properties had only a minimal effect on the function of mature adipocytes, but significantly impacted the ability of preadipocytes to undergo adipogenic differentiation in vitro. These alterations in scaffold environment and in medium conditions, particularly the application of adipocyte/preadipocyte coculture methods in lieu of traditional DM, may provide further means for optimizing adipogenic outcomes in vitro and in vivo.

Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (

Valvular interstitial cells (VICs) possess many properties that make them attractive for use in the construction of a tissue-engineered valve; however, we have found that the surfaces to which VICs will adhere and spread are limited. For example, VICs adhere and spread on collagen and laminin-coated surfaces, but display altered morphology and do not proliferate. Interestingly, fibronectin (FN) was one adhesion protein that facilitated VIC adhesion and proliferation. Yet VICs did not spread on surfaces modified with RGD, a ubiquitous cell-adhesive peptide, nor with other FN-specific peptide sequences such as EILDV and PHSRN. Hyaluronic acid (HA) is a highly elastic polysaccharide that is involved in natural valve morphogenesis and possesses binding interactions with FN. Hyaluronic acid was modified to form photopolymerizable hydrogels, and VICs were found to spread and proliferate on HA-based gels, forming a confluent monolayer on the gels within 4 days. Modified HA retained its ability to specifically bind FN, allowing for the formation of gels containing both HA and FN. Valvular interstital cells cultured on HA surfaces displayed significantly increased production of extracellular matrix proteins, indicating that HA-based scaffolds may provide useful biological cues to stimulate heart valve tissue formation.

Candida albicans is the most common fungal pathogen in humans, and most diseases produced by C. albicans are associated with biofilms. We previously developed nylon-3 polymers with potent activity against planktonic C. albicans and excellent C. albicans versus mammalian cell selectivity. Here we show that these nylon-3 polymers have strong and selective activity against drug-resistant C. albicans in biofilms, as manifested by inhibition of biofilm formation and by killing of C. albicans in mature biofilms. The best nylon-3 polymer (poly-βNM) is superior to the antifungal drug fluconazole for all three strains examined. This polymer is slightly less effective than amphotericin B (AmpB) for two strains, but the polymer is superior against an AmpB-resistant strain.

Healing of chronic wounds such as diabetic foot ulcers is a significant clinical problem. Methods of accelerating healing in these difficult lower extremity sites include use of growth factor-loaded gels, hyperbaric oxygen, grafts, and artificial skin replacements. Nitric oxide (NO) has been proposed as a possible active agent for enhancing wound healing. This study examines the in vitro and in vivo responses to a novel hydrogel that produces therapeutic levels of NO. A hydrogel wound dressing was fabricated using ultraviolet light-initiated polymerization from poly(vinyl alcohol) with a NO donor covalently coupled to the polymer backbone. NO release from the NO-modified hydrogel was shown to occur over a time period of up to 48 hours, and there was no associated decrease in fibroblast growth or viability in vitro associated with NO hydrogels. Fibroblasts in culture with NO hydrogels had an increased production of extracellular matrix compared with cells cultured without the NO hydrogels. Preliminary animal studies in a diabetic mouse, impaired wound healing model were conducted comparing low (0.5 mM) and high (5 mM) doses of NO. Time to complete closure was similar in control wounds and NO-treated wounds; however, at 8 days control wounds were significantly smaller than NO-treated wounds. By days 10 to 13 this delay was no longer apparent. Granulation tissue thickness within the wounds at days 8 and 15 and scar tissue thickness after wound closure were increased in animals exposed to higher dose NO hydrogels. The results of this study suggest that exogenous NO released from a hydrogel wound dressing has potential to modulate wound healing.

Fungal infections are a major challenge to human health that is heightened by pathogen resistance to current therapeutic agents. Previously, we were inspired by host-defense peptides to develop nylon-3 polymers (poly-β-peptides) that are toxic toward the fungal pathogen Candida albicans but exert little effect on mammalian cells. Based on subsequent analysis of structure–activity relationships among antifungal nylon-3 polymers, we have now identified readily prepared cationic homopolymers active against strains of C. albicans that are resistant to the antifungal drugs fluconazole and amphotericin B. These nylon-3 polymers are nonhemolytic. In addition, we have identified cationic–hydrophobic copolymers that are highly active against a second fungal pathogen, Cryptococcus neoformans, and moderately active against a third pathogen, Aspergillus fumigatus.

Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells, but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

Statin drugs are prescribed primarily for their ability to lower cholesterol, but may also exert beneficial side effects unrelated to cholesterol metabolism. Previous work has described a “statin paradox,” where statin treatment decreased osteoblastic markers in valve myofibroblasts while increasing those same markers in preosteoblasts. However, valvular interstitial cells (VICs) themselves are a multipotent cell type, capable of differentiating into activated, myofibroblastic VICs (aVICs) and osteoblastic VICs (obVICs), motivating the question of whether the statin paradox can exist within an individual valve containing these phenotypically distinct VIC subpopulations. In the current study, a heterogeneous initial population of porcine VICs was differentiated into aVICs or obVICs and treated with simvastatin. Gene expression analysis was conducted daily over an 8-day time course to capture temporally dynamic changes in cell phenotype induced by statin treatment. These studies demonstrated that the two VIC populations, aVICs and obVICs, exhibited differential responses to statin treatment. Specifically, simvastatin increased the expression of osteoblastic markers in obVICs, but not in aVICs, while also suppressing the myofibroblastic phenotype in both aVICs and obVICs. These results indicate that the statin paradox can exist within the heterogeneous VIC population of an individual diseased valve and that statin efficacy in the context of calcific aortic valve disease (CAVD) may be dependent upon the cellular composition of the valve. These findings may have implications for clinical usage of statins, shedding light on how statin efficacy in CAVD may be dependent upon the disease stage or why some individuals exhibit better responsiveness to statin therapy.

Nylon-3 polymers have a polyamide backbone reminiscent of that found in proteins (β- vs α-amino acid residues, respectively), which makes these materials interesting for biological applications. Because of the versatility of the ring-opening polymerization process and the variety of β-lactam starting materials available, the structure of nylon-3 copolymers is highly amenable to alteration. A previous study showed that relatively subtle changes in the structure or ratio of hydrophobic and cationic subunits that comprise these polymers can result in significant changes in the ability of nylon-3-bearing surfaces to support cell adhesion and spreading. In the present study, we have exploited the highly tailorable nature of these polymers to synthesize new versions possessing a wide range of chain lengths, with the intent of optimizing these materials for use as cell-supportive substrates. We find that longer nylon-3 chains lead to better fibroblast attachment on modified surfaces and that at the optimal chain lengths less hydrophobic subunits are superior. The best polymers we identified are comparable to an RGD-containing peptide in supporting fibroblast attachment. The results described here will help to focus future efforts aimed at refining nylon-3 copolymer substrates for specific tissue engineering applications.

Polymers in the nylon-3 family contain subunits derived from beta-amino acids, which are linked to one another via amide bonds. Thus, the nylon-3 backbone is homologous to the alpha-amino acid-based backbone of proteins. This molecular-level homology suggests that nylon-3 materials might be intrinsically protein-mimetic. The experiments described here explore this prospect in the context of cell adhesion, with tissue engineering as a long-range goal. We have evaluated a small library of sequence-random nylon-3 copolymers for the ability to render surfaces attractive to NIH 3T3 fibroblast adhesion and spreading. Library screening was accomplished in a high-throughput, parallel mode via attachment of the copolymers in a two-dimensional array to a modified glass surface. Significant variations in fibroblast adhesion and spreading were observed as a function of nylon-3 subunit identity and proportion. Several of the nylon-3 copolymers supported cell adhesion and morphology that was comparable, or even superior, to that achieved on positive control substrates such as tissue culture polystyrene and collagen-coated glass. Moreover, studies conducted under serum-free conditions demonstrated that specific nylon-3 derivatives supported cell adhesion independently of serum protein adsorption. Although cell adhesion was diminished in the absence of serum, particular copolymers demonstrated an ability to support substantially greater cell adhesion than any of the other conditions, including the positive controls. The nylon-3 copolymers that were most effective at promoting adhesion to a modified glass surface proved also to be effective at promoting adhesion when attached to a PEG-based hydrogel, demonstrating the potential for these copolymers to be used in tissue engineering applications.

Acceleration of wound closure results not only in decreased patient suffering and cost of wound treatment, but may also minimize scarring and lead to formation of a more stable closed wound. Cell migration is a critical element in wound healing, and it is believed that the ability to control the migration direction of cells will lead to accelerated closure of wounds. Thus, we have synthesized surfaces that are covalently modified with gradients of epidermal growth factor (EGF), a key molecule in the native wound-healing process, in order to create a platform that promotes directed cell migration. Standard photo-patterning techniques used herein enabled precise control over the spatial location of tethered EGF and the fabrication and quantitative characterization of gradient patterns of different types and slopes. Under serum-free conditions, human epidermal keratinocytes on immobilized EGF gradients preferentially migrated in the direction of higher EGF concentrations, and exhibited unidirectional migration speed and distance that was over five-fold greater than that observed on control surfaces. Treatment of migrating cells with an inhibitor of the EGF receptor resulted in immediate cessation of migration, thus verifying that the observed migration trends were directly attributable to keratinocyte interactions with immobilized EGF.

Despite decades of research into haemocompatible biomaterials, there remain surprisingly few materials that can be used in blood-contacting applications. We have synthesized copolymers of polyurethane (PU) with hyaluronic acid (HA) with the goal of creating materials that incorporate an inherently non-thrombogenic, biological component into the bulk polymer structure. HA was incorporated into the polymer backbone as a chain extender during PU synthesis, and the physical and biological properties of the resulting copolymer were directly controlled by the HA content. Increases in HA content led to a linear increase in hydrophilicity (R(2)=0.993) and corresponding increase in surface energy compared to PU controls. Elastic modulus also increased with HA content (p

A highly sensitive (pM), efficient (t20 min) detection assay was developed by designing surfaces with grafted antibodies. Through this approach, a short half-life antigen, glucagon, was rapidly detected in a biologically complex plasma/blood environment. Tailoring of graft composition eliminates the need for time-consuming blocking steps, significantly reducing antigen-antibody incubation times, while maintaining antibody specificity and activity toward target antigen. Grafted antibodies were bound through solvated, mobile polymer chains, thereby circumventing problems associated with antibody accessibility, analyte diffusion, and steric limitations. The efficiency of this assay is provided through grafting synthesized, acrylated antibodies in the presence of PEG monoacrylate. This procedure eliminates the need for blocking steps, due to a decrease in nonspecific protein interactions. These polymerizable antibodies were tethered with a range of densities while retaining biological activity. Moreover, biological activity of acrylated antibodies was compared to that of unmodified antibodies and remained comparable. The acrylated antibodies were grafted from substrate surfaces using controlled radical photopolymerization, maintaining the advantages of classical antibody immobilization techniques while providing improved detection. Through integrating this antibody conjugation chemistry and immunoassay approach with photolithographic techniques, construction of spatial patterns on a microfluidic device was demonstrated for efficient, parallel screening of multiple antigens.

Acrylate monomers were photografted from polymer substrates to create cell responsive chemically and biologically active surfaces that manipulate cell response. Three monomers, polyethylene glycol monoacrylate (MW 375 g/mol) (PEG375A), a monomeric extra-cellular matrix protein, and a cell-cleavable fluorescent monomer, were spatially photopatterned from a base substrate. The base substrate consisted of a dithiocarbamate (DTC) functionalized urethane diacrylate/tri(ethylene glycol)diacrylate copolymer and was shown to non-specifically support NIH 3T3 fibroblast cell adhesion. The DTC-containing polymer was further modified by grafting PEG375A to demonstrate selective blocking of cell-material interactions. Next, acrylated collagen type I was patterned onto polymer substrates to further promote specific cell interactions (i.e. by presenting cell-adhesive moieties). Hydrophilic PEG375A grafted patterns were shown to prevent 3T3 fibroblast adhesion to polymer in spatially grafted regions, while biologically active acrylated collagen type I promoted cell-surface interactions. Collagen type I was grafted at varying densities (0–7.5 pmol/grafted square), and the extent of cell adhesion and spreading were evaluated for each of these graft densities using fluorescence microscopy. Finally, methacrylated carboxyfluorescein diacetate (CFDA) was synthesized and photografted onto a cell-adhesive substrate as a cell sensing mechanism. The acetate groups found in the structure of CFDA cleave in the presence of cells. This cell-responsive substrate results in fluorescence indicative of acetate-group cleavage associated with cell interactions that occurs in patterned regions on polymer surfaces. Collectively, the results herein show the utility and application of a spatially and temporally controlled photografting process for designing cell responsive polymer surfaces.

Publisher Summary The nature of cell adhesion to substrate materials has a tremendous effect on cell function and tissue development. Signaling cascades initiated by cell adhesion have the ability to regulate a variety of events, including embryogenesis, tissue differentiation, and cell migration. Signaling via receptor–ligand interactions provides the cell with vital information about its extracellular environment. Coordinated cellular responses to these signals enable cells to adapt their function to their specific environment. Understanding how cells interact with a substrate is crucial in the development of functional biomaterials. The field of tissue engineering requires the creation of materials that are capable of directing tissue development, and one approach in achieving this control is through the manipulation of biological interactions between cell and material. An example of how cell–surface interactions influence cell function is the in vitro culture of mammary epithelial cells on tissue culture polystyrene (TCPS) vs laminin-coated surfaces. In vivo, epithelial cells are found as a monolayer on a basal lamina. When cultured on untreated TCPS in vitro, these cells lose their normal cuboidal morphology and ability to secrete milk proteins. However, upon addition of laminin to the surface, normal function and morphology are retained

Valvular interstitial cells (VICs) were isolated from porcine aortic heart valves and cultured in vitro on a variety of natural and synthetic surfaces to identify suitable scaffolds for tissue engineering a heart valve. VICs possess many properties that make them attractive for use in the construction of a tissue engineered valve; however, the surfaces to which VICs will adhere and spread are very limited. For example, VICs adhere and spread on collagen and laminin-coated surfaces, but display greatly altered morphology and do not, proliferate. Interestingly, fibronectin was one adhesion protein that facilitated VIC adhesion and proliferation. Yet, VICs did not spread on surfaces modified with RGD, a ubiquitous cell-adhesive peptide, nor to EILDV, a fibronectin-specific peptide sequence. Hyaluronic acid (HA), a highly elastic polysaccharide, was modified to form photopolymerizable hydrogels. VICs were found to spread and proliferate on these gels, forming a confluent monolayer on the gels within four days. Because HA alone experiences rapid enzymatic degradation, it was also combined with photopolymerizable poly(ethylene glycol) (PEG) to form gels with better mechanical properties and extended degradation times. These HA-PEG hydrogels possess desirable macroscopic properties while simultaneously providing a suitable cellular environment for VICs to form a tissue engineered heart valve.

Pharmacologically active materials offer the advantage of providing sitespecific pharmacotherapy for prolonged periods of time. The use of biomaterials as drug delivery vehicles is a continuing area of research, due to the desire not only to provide localized drug therapy but to improve the body’s acceptance of the implanted materials or initiate desired cell responses at the implantation site. However, it is also possible to covalently modify materials such that the “drug” remains attached to the biomaterial, providing a truly localized therapy, and is pharmacologically active for indefinite periods of time. In this manner, not only do the materials retain their biological activity, but the activity of the incorporated biological molecule or drug is often better preserved when bound to a substrate than when free in solution. Many pharmacological agents have extremely short half-lives in vivo. Attachment to materials often helps to stabilize the active substance while allowing it to interact with its environment and perform its intended functions. A wide range of biologically active molecules can be covalently attached to synthetic biomaterials in a manner such that the substance retains its bioactivity, and is sometimes more effective than its soluble counterpart.