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Hydrogel microparticles ranging from 0.1-100 µm, referred to as microgels, are attractive for biological applications afforded by their injectability and modularity, which allows facile delivery of mixed populations for tailored combinations of therapeutics. Significant efforts have been made to broaden methods for microgel production including via the materials and chemistries by which they are made. Via droplet-based-microfluidics we have established a method for producing click poly-(ethylene)-glycol (PEG)-based microgels with or without chemically crosslinked liposomes (lipo-microgels) through the Michael-type addition reaction between thiol and either vinyl-sulfone or maleimide groups. Unifom spherical microgels and lipo-microgels were generated with sizes of 74 ± 16 µm and 82 ± 25 µm, respectively, suggesting injectability that was further supported by rheological analyses. Super-resolution confocal microscopy was used to further verify the presence of liposomes within the lipo-microgels and determine their distribution. Atomic force microscopy (AFM) was conducted to compare the mechanical properties and network architecture of bulk hydrogels, microgels, and lipo-microgels. Further, encapsulation and release of model cargo (FITC-Dextran 5 kDa) and protein (equine myoglobin) showed sustained release for up to 3 weeks and retention of protein composition and secondary structure, indicating their ability to both protect and release cargos of interest.
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Hidrogeles , Microgeles , Animales , Caballos , Hidrogeles/química , Liposomas , Microfluídica , ReologíaRESUMEN
Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells.
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Materiales Biocompatibles , Biología Sintética , PolímerosRESUMEN
We perform coarse-grained (CG) molecular dynamics (MD) simulations to investigate the self-assembly of collagen-like peptide (CLP) triple helices into fibrillar structures and percolated networks as a function of solvent quality. The focus of this study is on CLP triple helices whose strands are different lengths (i.e., heterotrimers), leading to dangling 'sticky ends'. These 'sticky ends' are segments of the CLP strands that have unbonded hydrogen-bonding donor/acceptor sites that drive heterotrimeric CLP triple helices to physically associate with one another, leading to assembly into higher-order structures. We use a validated CG model for CLP in implicit solvent and capture varying solvent quality through changing strength of attraction between CG beads representing the amino acids in the CLP strands. Our CG MD simulations show that, at lower CLP concentrations, CLP heterotrimers assemble into fibrils and, at higher CLP concentrations, into percolated networks. At higher concentrations, decreasing solvent quality causes (i) the formation of heterogeneous network structures with a lower degree of branching at network junctions and (ii) increases in the diameter of network strands and pore sizes. We also observe a nonmonotonic effect of solvent quality on distances between network junctions due to the balance between heterotrimer end-end associations driven by hydrogen bonding and side-side associations driven by worsening solvent quality. Below the percolation threshold, we observe that decreasing solvent quality leads to the formation of fibrils composed of multiple aligned CLP triple helices, while the number of 'sticky ends' governs the spatial extent (radius of gyration) of the assembled fibrils.
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Colágeno , Péptidos , Solventes , Péptidos/química , Colágeno/química , Simulación de Dinámica Molecular , Estructura Secundaria de ProteínaRESUMEN
Collagen-like peptides (CLP) are multifunctional materials garnering a lot of recent interest from the biomaterials community due to their hierarchical assembly and tunable physicochemical properties. In this work, we present a computational study that links the design of CLP heterotrimers to the thermal stability of the triple helix and their self-assembly into fibrillar aggregates and percolated networks. Unlike homotrimeric helices, the CLP heterotrimeric triple helices in this study are made of CLP strands of different chain lengths that result in 'sticky' ends with available hydrogen bonding groups. These 'sticky' ends at one end or both ends of the CLP heterotrimer then facilitate inter-helix hydrogen bonding leading to self-assembly into fibrils (clusters) and percolated networks. We consider the cases of three sticky end lengths - two, four, and six repeat units - present entirely on one end or split between two ends of the CLP heterotrimer. We observe in CLP heterotrimer melting curves generated using coarse grained Langevin dynamics simulations at low CLP concentration that increasing sticky end length results in lower melting temperatures for both one and two sticky ended CLP designs. At higher CLP concentrations, we observe non-monotonic trends in cluster sizes with increasing sticky end length with one sticky end but not for two sticky ends with the same number of available hydrogen bonding groups as the one sticky end; this nonmonotonicity stems from the formation of turn structures stabilized by hydrogen bonds at the single, sticky end for sticky end lengths greater than four repeat units. With increasing CLP concentration, heterotrimers also form percolated networks with increasing sticky end length with a minimum sticky end length of four repeat units required to observe percolation. Overall, this work informs the design of thermoresponsive, peptide-based biomaterials with desired morphologies using strand length and dispersity as a handle for tuning thermal stability and formation of supramolecular structures.
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Colágeno , Simulación de Dinámica Molecular , Materiales Biocompatibles , Colágeno/química , Péptidos/química , TemperaturaRESUMEN
Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.
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Hidrogeles/uso terapéutico , Integrinas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Diferenciación Celular , HumanosRESUMEN
Assembling peptides allow the creation of structurally complex materials, where amino acid selection influences resulting properties. We present a synergistic approach of experiments and simulations for examining the influence of natural and non-natural amino acid substitutions via incorporation of charged residues and a reactive handle on the thermal stability and assembly of multifunctional collagen mimetic peptides (CMPs). Experimentally, we observed inclusion of charged residues significantly decreased the melting temperature of CMP triple helices with further destabilization upon inclusion of the reactive handle. Atomistic simulations of a single CMP triple helix in explicit water showed increased residue-level and helical structural fluctuations caused by the inclusion of the reactive handle; however, these atomistic simulations cannot be used to predict changes in CMP melting transition. Coarse-grained (CG) simulations of CMPs at experimentally relevant solution conditions, showed, qualitatively, the same trends as experiments in CMP melting transition temperature with CMP design. These simulations show that when charged residues are included electrostatic repulsions significantly destabilize the CMP triple helix and that an additional inclusion of a reactive handle does not significantly change the melting transition. Based on findings from both experiments and simulations, the sequence design was refined for increased CMP triple helix thermal stability, and the reactive handle was utilized for the incorporation of the assembled CMPs within covalently crosslinked hydrogels. Overall, a unique approach was established for predicting stability of CMP triple helices for various sequences prior to synthesis, providing molecular insights for sequence design towards the creation of bulk nanostructured soft biomaterials.
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Colágeno , Péptidos , Materiales Biocompatibles , Biomimética , HidrogelesRESUMEN
Photolabile moieties have been utilized in applications ranging from peptide synthesis and controlled protein activation to tunable and dynamic materials. The photochromic properties of nitrobenzyl (NB) based linkers are readily tuned to respond to cytocompatible light doses and are widely utilized in cell culture and other biological applications. While widely utilized, little is known about how the microenvironment, particularly confined aqueous environments (e.g., hydrogels), affects both the mode and rate of cleavage of NB moieties, leading to unpredictable limitations in control over system properties (e.g., rapid hydrolysis or slow photolysis). To address these challenges, we synthesized and characterized the photolysis and hydrolysis of NB moieties containing different labile bonds (i.e., ester, amide, carbonate, or carbamate) that served as labile crosslinks within step-growth hydrogels. We observed that NB ester bond exhibited significant rates of both photolysis and hydrolysis, whereas, importantly, the NB carbamate bond had superior light responsiveness and resistance to hydrolysis within the hydrogel microenvironment. Exploiting this synergy and orthogonality of photolytic and hydrolytic degradation, we designed concentric cylinder hydrogels loaded with different cargoes (e.g., model protein with different fluorophores) for either combinatorial or sequential release, respectively. Overall, this work provides new facile chemical approaches for tuning the degradability of NB linkers and an innovative strategy for the construction of multimodal degradable hydrogels, which can be utilized to guide the design of not only tunable materials platforms but also controlled synthetic protocols or surface modification strategies.
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Hidrogeles/química , Nitrobencenos/química , Albúmina Sérica Bovina/química , Animales , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/efectos de la radiación , Bovinos , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Colorantes Fluorescentes/química , Hidrogeles/efectos de la radiación , Hidrólisis/efectos de la radiación , Nitrobencenos/síntesis química , Nitrobencenos/efectos de la radiación , Fotólisis , Prueba de Estudio Conceptual , Rayos UltravioletaRESUMEN
Healthy function of the gut microenvironment is dependent on complex interactions between the bacteria of the microbiome, epithelial and immune (host) cells, and the surrounding tissue. Misregulation of these interactions is implicated in disease. A range of tools have been developed to study these interactions, from mechanistic studies to therapeutic evaluation. In this Digest, we highlight select tools at the cellular and molecular level for probing specific cell-microenvironment interactions. Approaches are overviewed for controlling and probing cell-cell interactions, from transwell and microfluidic devices to engineered bacterial peptidoglycan fragments, and cell-matrix interactions, from three-dimensional scaffolds to chemical handles for in situ modifications.
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Bacterias/química , Colorantes Fluorescentes/química , Interacciones Huésped-Parásitos , Intestinos/microbiología , Animales , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Microambiente Celular , Matriz Extracelular/metabolismo , Humanos , Inmunidad Innata , Intestinos/citología , Modelos BiológicosRESUMEN
The retro Michael-type addition and thiol exchange of thioether succinimide click linkages in response to thiol-containing environments offers a novel strategy for the design of glutathione-sensitive degradable hydrogels for controlled drug delivery. Here we characterize the kinetics and extent of the retro Michael-type addition and thiol exchange with changes in both the p Ka of the thiols and the identity of N-substituents of maleimides. A series of N-substituted thioether succinimides were prepared through typical Michael-type addition. Model studies (1H NMR, HPLC) of 4-mercaptophenylacetic acid (MPA, p Ka 6.6) conjugated to N-ethyl maleimide (NEM), N-phenyl maleimide (NPM), or N-aminoethyl maleimide (NAEM) and then incubated with glutathione showed half-lives of conversion from 3.1 to 18 h, with extents of conversion from approximately 12% to 90%. The variations in the rates of exchange and hydrolytic ring opening appear to be mediated by resonance effects, electron-withdrawing capacity of the N-substituted moiety, as well as the potential for intramolecular catalytic hydrogen bonding of amine substituents with water (particularly in the case of ring opening). Further model studies of 4-mercaptohydrocinnamic acid (MPP, p Ka 7.0) and N-acetyl-l-cysteine (NAC, p Ka 9.5) conjugated to selected N-substituted maleimides and then incubated with glutathione showed half-lives of conversion from 3.6 to 258 h, with extents of conversion from approximately 1% to 90%. A higher p Ka of the thiol decreased the rate of the exchange reaction and limited the impact of other electronic effects of N-substituents on the extents of conversion. Additional factors affecting the conversion kinetics were studied on NEM conjugates. The kinetics of the retro Michael-type addition and exchange reaction were not hindered by thiol traps of lower p Ka, but were retarded in conditions of lower pH. These studies shed light into details of thiol and maleimide design that could be used to tune the rates of degradation of drug and polymer conjugates for a variety of applications.
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Glutatión/química , Maleimidas/química , Compuestos de Sulfhidrilo/química , Cromatografía Líquida de Alta Presión , Semivida , Hidrólisis , Cinética , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
A method of cysteine alkylation using cyclopropenyl ketones is described. Due to the significant release of cyclopropene strain energy, reactions of thiols with cyclopropenyl ketones are both fast and irreversible and give rise to stable conjugate addition adducts. The resulting cyclopropenyl ketones have a low molecular weight and allow for simple attachment of amides via N-hydroxysuccinimide (NHS)-esters. While cyclopropenyl ketones do display slow background reactivity toward water, labeling by thiols is much more rapid. The reaction of a cyclopropenyl ketone with glutathione (GSH) proceeds with a rate of 595 M-1 s-1 in PBS at pH 7.4, which is considerably faster than α-halocarbonyl labeling reagents, and competitive with maleimide/thiol couplings. The method has been demonstrated in protein conjugation, and an arylthiolate conjugate was shown to be stable upon prolonged incubation in either GSH or human plasma. Finally, cyclopropenyl ketones were used to create PEG-based hydrogels that are stable to prolonged incubation in a reducing environment.
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Ciclopropanos/química , Cisteína/química , Cetonas/química , Alquilación , Glutatión/química , Humanos , Hidrogeles/síntesis química , Polietilenglicoles , Coloración y Etiquetado , Compuestos de Sulfhidrilo/química , Factores de TiempoRESUMEN
Hydrogel-based depots are of growing interest for release of biopharmaceuticals; however, a priori selection of hydrogel compositions that will retain proteins of interest and provide desired release profiles remains elusive. Toward addressing this, in this work, we have established a new tool for the facile assessment of protein release from hydrogels and applied it to evaluate the effectiveness of mesh size estimations on predicting protein retention or release. Poly(ethylene glycol) (PEG)-based hydrogel depots were formed by photoinitiated step growth polymerization of four-arm PEG functionalized with norbornene (PEG-norbornene, 4% w/w to 20% w/w, Mn â¼ 5 to 20 kDa) and different dithiol cross-linkers (PEG Mn â¼ 1.5 kDa or enzymatically degradable peptide), creating well-defined, robust materials with a range of mesh sizes estimated with Flory-Rehner or rubber elasticity theory (â¼5 to 15 nm). A cocktail of different model proteins was released from compositions of interest, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to facilely and quantitatively analyze temporal release profiles. Mesh size was predictive of retention of relatively large proteins and release of relatively small proteins. Proteins with diameters comparable to the mesh size, which is often the case for growth factors, were released by hindered diffusion and required experimental assessment of retention and release. With this knowledge, hydrogels were designed for the controlled release of a therapeutically relevant growth factor, PDGF-BB.
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Liberación de Fármacos , Hidrogeles/química , Proteínas Proto-Oncogénicas c-sis/química , Becaplermina , Reactivos de Enlaces Cruzados/química , Hidrogeles/síntesis química , Norbornanos/química , Polietilenglicoles/química , PorosidadRESUMEN
Stem cells reside in complex three-dimensional (3D) environments within the body that change with time, promoting various cellular functions and processes such as migration and differentiation. These complex changes in the surrounding environment dictate cell fate yet, until recently, have been challenging to mimic within cell culture systems. Hydrogel-based biomaterials are well suited to mimic aspects of these in vivo environments, owing to their high water content, soft tissue-like elasticity, and often-tunable biochemical content. Further, hydrogels can be engineered to achieve changes in matrix properties over time to better mimic dynamic native microenvironments for probing and directing stem cell function and fate. This review will focus on techniques to form hydrogel-based biomaterials and modify their properties in time during cell culture using select addition reactions, cleavage reactions, or non-covalent interactions. Recent applications of these techniques for the culture of stem cells in four dimensions (i.e., in three dimensions with changes over time) also will be discussed for studying essential stem cell processes.
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Despite advances in treatment and surveillance, melanoma continues to claim approximately 9,000 lives in the US annually (SEER 2013). The National Comprehensive Cancer Network currently recommends ipilumumab, vemurafenib, dabrafenib, and high-dose IL-2 as first line agents for Stage IV melanoma. Little data exists to guide management of cutaneous and subcutaneous metastases despite the fact that they are relatively common. Existing options include intralesional Bacillus Calmette-Guérin, isolated limb perfusion/infusion, interferon-α, topical imiquimod, cryotherapy, radiation therapy, interferon therapy, and intratumoral interleukin-2 injections. Newly emerging treatments include the anti-programmed cell death 1 receptor agents (nivolumab and pembrolizumab), anti-programmed death-ligand 1 agents, and oncolytic vaccines (talimogene laherparepevec). Available treatments for select sites include adoptive T cell therapies and dendritic cell vaccines. In addition to reviewing the above agents and their mechanisms of action, this review will also focus on combination therapy as these strategies have shown promising results in clinical trials for metastatic melanoma treatment.
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Inmunoterapia/métodos , Melanoma/secundario , Melanoma/terapia , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Productos Biológicos/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Ensayos Clínicos Fase I como Asunto , Terapia Combinada , Femenino , Humanos , Inyecciones Intralesiones , Interferones/uso terapéutico , Masculino , Melanoma/patología , Cirugía de Mohs/métodos , Terapia Molecular Dirigida/métodos , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto , Medición de Riesgo , Programa de VERF , Resultado del TratamientoRESUMEN
Degradable and cell-compatible hydrogels can be designed to mimic the physical and biochemical characteristics of native extracellular matrices and provide tunability of degradation rates and related properties under physiological conditions. Hence, such hydrogels are finding widespread application in many bioengineering fields, including controlled bioactive molecule delivery, cell encapsulation for controlled three-dimensional culture, and tissue engineering. Cellular processes, such as adhesion, proliferation, spreading, migration, and differentiation, can be controlled within degradable, cell-compatible hydrogels with temporal tuning of biochemical or biophysical cues, such as growth factor presentation or hydrogel stiffness. However, thoughtful selection of hydrogel base materials, formation chemistries, and degradable moieties is necessary to achieve the appropriate level of property control and desired cellular response. In this review, hydrogel design considerations and materials for hydrogel preparation, ranging from natural polymers to synthetic polymers, are overviewed. Recent advances in chemical and physical methods to crosslink hydrogels are highlighted, as well as recent developments in controlling hydrogel degradation rates and modes of degradation. Special attention is given to spatial or temporal presentation of various biochemical and biophysical cues to modulate cell response in static (i.e., non-degradable) or dynamic (i.e., degradable) microenvironments. This review provides insight into the design of new cell-compatible, degradable hydrogels to understand and modulate cellular processes for various biomedical applications.
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Hidrogeles/metabolismo , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Microambiente Celular , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Hidrogeles/química , Polímeros/química , Ingeniería de TejidosRESUMEN
Autoimmune diseases are caused by malfunctions of the immune system and generally impact women at twice the frequency of men. Many of the most serious autoimmune diseases are accompanied by a dysregulation of T-cell phenotype, both regarding the ratio of CD4+ to CD8+ T-cells and proinflammatory versus regulatory phenotypes. Biomaterials, in the form of particles and hydrogels, have shown promise in ameliorating this dysregulation both in vivo and ex vivo. In this review, we explore the role of T-cells in autoimmune diseases, particularly those with high incidence rates in women, and evaluate the promise and efficacy of innovative biomaterial-based approaches for targeting T-cells.
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Enfermedades Autoinmunes , Autoinmunidad , Masculino , Femenino , Humanos , Factores Sexuales , Linfocitos T CD8-positivos , FenotipoRESUMEN
Activation of fibroblasts is pivotal for wound healing; however, persistent activation leads to maladaptive processes and is a hallmark of fibrosis, where disease mechanisms are only partially understood. Human in vitro model systems complement in vivo animal models for both hypothesis testing and drug evaluation to improve the identification of therapeutics relevant to human disease. Despite advances, a challenge remains in understanding the dynamics of human fibroblast responses to complex microenvironment stimuli, motivating the need for more advanced tools to investigate fibrotic mechanisms. This work established approaches for assessing the temporal dynamics of these responses using genetically encoded fluorescent reporters of alpha smooth muscle actin expression, an indicator of fibroblast activation. Specifically, we created a toolset of human lung fibroblast reporter cell lines from different origins (male, female; healthy, idiopathic pulmonary fibrosis) and used three different versions of the reporter with the fluorescent protein modified to exhibit different temporal stabilities, providing temporal resolution of protein expression processes over a range of timescales. Using this toolset, we demonstrated that reporters provide insight into population shifts in response to both mechanical and biochemical cues that are not detectable by traditional end point assessments with differential responses based on cell origin. Furthermore, individual cells can also be tracked over time, with opportunities for comparison to complementary end point measurements. The establishment of this reporter toolset enables dynamic cell investigations that can be translated into more complex synthetic culture environments for elucidating disease mechanisms and evaluating therapeutics for lung fibrosis and other complex biological processes more broadly.
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The human body is complex and hierarchically structured, composed of cells residing within the extracellular matrix (ECM) of tissues that are assembled into organs, all working together to complete a given function. One goal of current biomaterials research is to capture some of this complexity outside of the body for understanding the underlying biology of development, repair, and disease and to devise new strategies for regenerative medicine or disease treatment. Polymeric materials have arisen as powerful tools to mimic the native ECM, giving experimenters a way to capture key aspects of the native cellular environment outside of the body. In particular, dynamic materials allow changes in the properties of these ECM mimics during an experiment, affording an additional degree of control for the experimenter. In this tutorial review, the basic cellular processes of cell migration, proliferation, and differentiation will be overviewed to motivate design considerations for polymeric ECM mimics, and examples will be given of how classes of dynamic materials are being used to study each cellular process.
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Chronic respiratory diseases and infections are among the largest contributors to death globally, many of which still have no cure, including chronic obstructive pulmonary disorder, idiopathic pulmonary fibrosis, and respiratory syncytial virus among others. Pulmonary therapeutics afford untapped potential for treating lung infection and disease through direct delivery to the site of action. However, the ability to innovate new therapeutic paradigms for respiratory diseases will rely on modeling the human lung microenvironment and including key cellular interactions that drive disease. One key feature of the lung microenvironment is the air-liquid interface (ALI). ALI interface modeling techniques, using cell-culture inserts, organoids, microfluidics, and precision lung slices (PCLS), are rapidly developing; however, one major component of these models is lacking-innate immune cell populations. Macrophages, neutrophils, and dendritic cells, among others, represent key lung cell populations, acting as the first responders during lung infection or injury. Innate immune cells respond to and modulate stromal cells and bridge the gap between the innate and adaptive immune system, controlling the bodies response to foreign pathogens and debris. In this article, we review the current state of ALI culture systems with a focus on innate immune cells and suggest ways to build on current models to add complexity and relevant immune cell populations.
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Enfermedad Pulmonar Obstructiva Crónica , Virus Sincitial Respiratorio Humano , Humanos , Pulmón , Macrófagos , Inmunidad , Inmunidad InnataRESUMEN
Enzymatically degradable peptides are commonly used as linkers within hydrogels for biological applications; however, controlling the degradation of these engineered peptides with different contexts and cell types can prove challenging. In this work, we systematically examined the substitution of d-amino acids (D-AAs) for different l-amino acids in a peptide sequence commonly utilized in enzymatically degradable hydrogels (VPMS↓MRGG) to create peptide linkers with a range of different degradation times, in solution and in hydrogels, and investigated the cytocompatibility of these materials. We found that increasing the number of D-AA substitutions increased the resistance to enzymatic degradation both for free peptide and peptide-linked hydrogels; yet, this trend also was accompanied by increased cytotoxicity in cell culture. This work demonstrates the utility of D-AA-modified peptide sequences to create tunable biomaterials platforms tempered by considerations of cytotoxicity, where careful selection and optimization of different peptide designs is needed for specific biological applications.
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Hidrogeles , Péptidos , Hidrogeles/farmacología , Sustitución de Aminoácidos , Péptidos/química , Materiales Biocompatibles , Microambiente CelularRESUMEN
Late recurrences of breast cancer are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after dormancy and occur most frequently with estrogen receptor-positive (ER+) breast cancer cells (BCCs) in bone marrow (BM). Interactions between the BM niche and BCCs are thought to play a pivotal role in recurrence, and relevant model systems are needed for mechanistic insights and improved treatments. We examined dormant DTCs in vivo and observed DTCs near bone lining cells and exhibiting autophagy. To study underlying cell-cell interactions, we established a well-defined, bioinspired dynamic indirect coculture model of ER+ BCCs with BM niche cells, human mesenchymal stem cells (hMSCs) and fetal osteoblasts (hFOBs). hMSCs promoted BCC growth, whereas hFOBs promoted dormancy and autophagy, regulated in part by tumor necrosis factor-α and monocyte chemoattractant protein 1 receptor signaling. This dormancy was reversible by dynamically changing the microenvironment or inhibiting autophagy, presenting further opportunities for mechanistic and targeting studies to prevent late recurrence.