RESUMO
The evolving US opioid crisis is complex and requires myriad different interventions. These include reducing opioid overprescribing and curtailing the supply of illicit opioids, overdose rescue interventions, and treatment and recovery support services for those with opioid use disorders. To date, more distal primary prevention strategies that have an evidence base are underutilized. Yet, the impact of early environments on later substance use disorder risk is increasingly well understood, including knowledge of the mechanistic linkages between brain development and subsequent risk behaviors. Applying this developmental framework to prevention shows promise, and some middle-school interventions have demonstrated significant reductions in prescription opioid misuse. Reducing these risks of initial misuse of opioids may be the "ounce of prevention" that makes a substantial difference in a society now reeling from the worst drug crisis our country has seen. The challenge is to continue to develop and test promising distal interventions and to support implementation fidelity through frameworks that ensure their cultural appropriateness and sustainability. In addition, research is needed to develop new prevention strategies for adults, including patients with pain at risk for transitioning from prescription to illicit opioids.
Assuntos
Causas de Morte/tendências , Intervenção Educacional Precoce/organização & administração , Promoção da Saúde/organização & administração , Transtornos Relacionados ao Uso de Opioides/epidemiologia , Transtornos Relacionados ao Uso de Opioides/mortalidade , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Adolescente , Adulto , Previsões , Humanos , Estados Unidos/epidemiologia , Adulto JovemRESUMO
PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.
Assuntos
Adesão Celular , Células Imobilizadas/fisiologia , Técnicas de Cocultura/métodos , Dimetilpolisiloxanos/metabolismo , Células Endoteliais/fisiologia , Células Secretoras de Glucagon/fisiologia , Células Secretoras de Insulina/fisiologia , Humanos , Ilhotas Pancreáticas , Impressão Tridimensional , Propriedades de SuperfícieRESUMO
Ex vivo-expanded buccal mucosal epithelial (BME) cell transplantation has been used to reconstruct the ocular surface. Methods for enrichment and maintenance of BME progenitor cells in ex vivo cultures may improve the outcome of BME cell transplantation. However, the parameter of cell seeding density in this context has largely been neglected. This study investigates how varying cell seeding density influences BME cell proliferation and differentiation on tissue culture polystyrene (TCPS). The highest cell proliferation activity was seen when cells were seeded at 5×104 cells/cm2. Both below and above this density, the cell proliferation rate decreased sharply. Differential immunofluorescence analysis of surface markers associated with the BME progenitor cell population (p63, CK19, and ABCG2), the differentiated cell marker CK10 and connexin 50 (Cx50) revealed that the initial cell seeding density also significantly affected the progenitor cell marker expression profile. Hence, this study demonstrates that seeding density has a profound effect on the proliferation and differentiation of BME stem cells in vitro, and this is relevant to downstream cell therapy applications.
Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Mucosa Bucal/citologia , Células-Tronco/citologia , Animais , Contagem de Células , Proliferação de Células , Células Cultivadas , Feminino , Poliestirenos/química , Ratos Wistar , Alicerces Teciduais/químicaRESUMO
One of the most significant hurdles to the affordable, accessible delivery of cell therapy is the cost and difficulty of expanding cells to clinically relevant numbers. Immunotherapy to prevent autoimmune disease, tolerate organ transplants or target cancer critically relies on the expansion of specialized T cell populations. We have designed 3D-printed cell culture lattices with highly organized micron-scale architectures, functionalized via plasma polymerization to bind monoclonal antibodies that trigger cell proliferation. This 3D technology platform facilitate the expansion of therapeutic human T cell subsets, including regulatory, effector, and cytotoxic T cells while maintaining the correct phenotype. Lentiviral gene delivery to T cells is enhanced in the presence of the lattices. Incorporation of the lattice format into existing cell culture vessels such as the G-Rex system is feasible. This cell expansion platform is user-friendly and expedites cell recovery and scale-up, making it ideal for translating T cell therapies from bench to bedside.
Assuntos
Técnicas de Cultura de Células/instrumentação , Impressão Tridimensional/instrumentação , Subpopulações de Linfócitos T/citologia , Alicerces Teciduais/química , Anticorpos Imobilizados/farmacologia , Bioimpressão/instrumentação , Bioimpressão/métodos , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Desenho de Equipamento , Humanos , Imunoterapia Adotiva , Subpopulações de Linfócitos T/efeitos dos fármacosRESUMO
The propensity of glycosaminoglycans to mediate cell-cell and cell-matrix interactions opens the door to capture cells, including circulating blood cells, onto biomaterial substrates. Chondroitin sulfate (CS)-B is of particular interest, since it interacts with the receptor (EGF)-like module-containing mucin-like hormone receptor-like 2 precursor (EMR2) displayed on the surface of leukocytes and endothelial progenitor cells. Herein, CS-B and its isomer CS-A were covalently immobilized onto heptylamine plasma polymer films via three different binding chemistries to develop platform technology for the capture of EMR2 expressing cells onto solid carriers. Surface characterization verified the successful immobilization of both glycosaminoglycans. The EMR2 expressing human myeloid cell line U937 preferentially bound onto CS-B-modified substrates, and U937 cells preincubated with CS-B in solution exhibited reduced affinity for the substrate. The direct capture of hematopoietic and blood-circulating endothelial cell types via a glycosaminoglycan-binding surface receptor opens an unexplored route for the development of biomaterials targeted at these cell types.
Assuntos
Separação Celular/métodos , Materiais Revestidos Biocompatíveis/química , Dermatan Sulfato/química , Receptores Acoplados a Proteínas G/metabolismo , Aminas/química , Adesão Celular , Sulfatos de Condroitina/química , Materiais Revestidos Biocompatíveis/metabolismo , Dermatan Sulfato/metabolismo , Expressão Gênica , Humanos , Gases em Plasma , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Propriedades de Superfície , Células U937RESUMO
A label-free porous silicon (pSi) based, optical biosensor, using both an antibody and aptamer bioreceptor motif has been developed for the detection of insulin. Two parallel biosensors were designed and optimised independently, based on each bioreceptor. Both bioreceptors were covalently attached to a thermally hydrosilylated pSi surface though amide coupling, with unreacted surface area rendered stable and low fouling by incorporation of PEG moieties. The insulin detection ability of each biosensor was determined using interferometric reflectance spectroscopy, using a range of different media both with and without serum. Sensing performance was compared in terms of response value, response time and limit of detection (LOD) for each platform. In order to demonstrate the capability of the best performing biosensor to detect insulin from real samples, an in vitro investigation with the aptamer-modified surface was performed. This biosensor was exposed to buffer conditioned by glucose-stimulated human islets, with the result showing a positive response and a high degree of selectivity towards insulin capture. The obtained results correlated well with the ELISA used in the clinic for assaying glucose-stimulated insulin release from donor islets. We anticipate that this type of sensor can be applied as a rapid point-of-use biosensor to assess the quality of donor islets in terms of their insulin production efficiency, prior to transplantation.
Assuntos
Anticorpos Imobilizados/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Insulina/análise , Ilhotas Pancreáticas/química , Sequência de Bases , Células Cultivadas , Glucose/metabolismo , Humanos , Insulina/metabolismo , Interferometria/instrumentação , Ilhotas Pancreáticas/metabolismo , Limite de Detecção , Dispositivos Ópticos , Porosidade , Silício/química , Análise Espectral/instrumentaçãoRESUMO
Current organ transplantation protocols require the rapid transport of freshly isolated donor tissue to the recipient patient at the site where the procedure is to be conducted. During transport, the tissue graft can quickly deteriorate as a result of oxygen starvation. In this study, we report the fabrication of oxygen-releasing coatings for improved tissue preservation. The coatings were prepared via the encapsulation of calcium peroxide or urea peroxide microparticles between layers of octadiene plasma polymer films. By varying the thickness of the plasma polymer coating and type of peroxide, formulations were obtained that generate oxygen upon contact with aqueous solutions, while at the same time limiting the amount of toxic reactive oxygen species produced. The optimized coatings were tested under hypoxic conditions using the MIN6 ß-cell line, which resulted in a 3-fold increase in the viability of cultured cells. These thin oxygen-releasing coatings can be deposited on a wide range of surfaces, creating a platform for oxygen delivery with the potential to extend the viability of transported tissues and increase the time frame available for graft transport.
RESUMO
Ordered arrays of silicon nano- to microscale pillars are used to enable biomolecular trafficking into primary human cells, consistently demonstrating high transfection efficiency can be achieved with broader and taller pillars than reported to date. Cell morphology on the pillar arrays is often strikingly elongated. Investigation of the cellular interaction with the pillar reveals that cells are suspended on pillar tips and do not interact with the substrate between the pillars. Although cells remain suspended on pillar tips, acute local deformation of the cell membrane was noted, allowing pillar tips to penetrate the cell interior, while retaining cell viability.
Assuntos
Silício/química , Movimento Celular , Sobrevivência Celular , Humanos , Microtecnologia , TransfecçãoRESUMO
Porous silicon (pSi) substrates are a promising platform for cell expansion, since pore size and chemistry can be tuned to control cell behavior. In addition, a variety of bioactives can be loaded into the pores and subsequently released to act on cells adherent to the substrate. Here, we construct a cell microarray on a plasma polymer coated pSi substrate that enables the simultaneous culture of human endothelial cells on printed immobilized protein factors, while a second soluble growth factor is released from the same substrate. This allows three elements of candidate pSi scaffold materials-topography, surface functionalization, and controlled factor release-to be assessed simultaneously in high throughput. We show that protein conjugation within printed microarray spots is more uniform on the pSi substrate than on flat glass or silicon surfaces. Active growth factors are released from the pSi surface over a period of several days. Using an endothelial progenitor cell line, we investigate changes in cell behavior in response to the microenvironment. This platform facilitates the design of advanced functional biomaterials, including scaffolds, and carriers for regenerative medicine and cell therapy.
Assuntos
Materiais Biocompatíveis/química , Sistemas de Liberação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Polímeros/química , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/química , Humanos , Polímeros/farmacologia , Porosidade , Silício/química , Análise Serial de TecidosRESUMO
Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response.
RESUMO
Underage drinking and its associated problems have profound negative consequences for underage drinkers themselves, their families, their communities, and society as a whole, and contribute to a wide range of costly health and social problems. There is increased risk of negative consequences with heavy episodic or binge drinking. Alcohol is a factor related to approximately 4,300 deaths among underage youths in the U.S. every year. Since the mid-1980s, the nation has launched aggressive underage drinking prevention efforts at the federal, state, and local levels, and national epidemiologic data suggest that these efforts are having positive effects. For example, since 1982, alcohol-related traffic deaths among youth aged 16-20 years have declined by 79%. Evidence-based or promising strategies for reducing underage drinking include those that limit the physical, social, and economic availability of alcohol to youth, make it illegal for drivers aged <21 years to drive after drinking, and provide mechanisms for early identification of problem drinkers. Strategies may be implemented through a comprehensive prevention approach including policies and their enforcement, public awareness and education, action by community coalitions, and early brief alcohol intervention and referral programs. This paper focuses on underage drinking laws and their enforcement because these constitute perhaps the most fundamental component of efforts to limit youth access to and use of alcohol.
Assuntos
Acidentes de Trânsito/tendências , Serviços Preventivos de Saúde/economia , Consumo de Álcool por Menores/legislação & jurisprudência , Consumo de Álcool por Menores/prevenção & controle , Consumo de Álcool por Menores/tendências , Acidentes de Trânsito/prevenção & controle , Adolescente , Fatores Etários , Governo Federal , Feminino , Humanos , Aplicação da Lei , Masculino , Estados Unidos , Adulto JovemRESUMO
Cell microarrays are a novel platform for the high throughput discovery of new biomaterials. By re-creating a multitude of cell microenvironments on a single slide, this approach can identify the optimal surface composition to drive a desired cell response. To systematically study the effects of molecular microenvironments on stem cell fate, we designed a cell microarray based on parallel exposure of mesenchymal stem cells (MSCs) to surface-immobilised collagen I (Coll I) and bone morphogenetic protein 2 (BMP 2). This was achieved by means of a reactive coating on a slide surface, enabling the covalent anchoring of Coll I and BMP 2 as microscale spots printed by a robotic contact printer. The surface between the printed protein spots was passivated using poly (ethylene glycol) bisamine 10,000Da (A-PEG). MSCs were then captured and cultured on array spots composed of binary mixtures of Coll I and BMP 2, followed by automated image acquisition and quantitative, multi-parameter analysis of cellular responses. Surface compositions that gave the highest osteogenic differentiation were determined using Runx2 expression and calcium deposition. Quantitative single cell analysis revealed subtle concentration-dependent effects of surface-immobilised proteins on the extent of osteogenic differentiation obscured using conventional analysis. In particular, the synergistic interaction of Coll I and BMP 2 in supporting osteogenic differentiation was confirmed. Our studies demonstrate the value of cell microarray platforms to decipher the combinatorial interactions at play in stem cell niche microenvironments.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Células-Tronco Mesenquimais/citologia , Análise em Microsséries/métodos , Osteogênese/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Fosfatos de Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Imunofluorescência , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Análise de Componente Principal , Análise Serial de Proteínas , Ratos Wistar , Espectrometria de Massa de Íon SecundárioRESUMO
Control over particle self-assembly is a prerequisite for the colloidal templating of lithographical etching masks to define nanostructures. This work integrates and combines for the first time bottom-up and top-down approaches, namely, particle self-assembly at liquid-liquid interfaces and metal-assisted chemical etching, to generate vertically aligned silicon nanowire (VA-SiNW) arrays and, alternatively, arrays of nanoscale pores in a silicon wafer. Of particular importance, and in contrast to current techniques, including conventional colloidal lithography, this approach provides excellent control over the nanowire or pore etching site locations and decouples nanowire or pore diameter and spacing. The spacing between pores or nanowires is tuned by adjusting the specific area of the particles at the liquid-liquid interface before deposition. Hence, the process enables fast and low-cost fabrication of ordered nanostructures in silicon and can be easily scaled up. We demonstrate that the fabricated VA-SiNW arrays can be used as in vitro transfection platforms for transfecting human primary cells.
Assuntos
Nanoestruturas/química , Nanotecnologia/métodos , Nanofios/química , Transfecção/métodos , Humanos , Metais/química , Nanoporos , Cultura Primária de Células , Silício/químicaRESUMO
Zero-valent, or elemental, silicon nanostructures exhibit a number of properties that render them attractive for applications in nanomedicine. These materials hold significant promise for improving existing diagnostic and therapeutic techniques. This review summarizes some of the essential aspects of the fabrication techniques used to generate these fascinating nanostructures, comparing their material properties and suitability for biomedical applications. We examine the literature in regards to toxicity, biocompatibility and biodistribution of silicon nanoparticles, nanowires and nanotubes, with an emphasis on surface modification and its influence on cell adhesion and endocytosis. In the final part of this review, our attention is focused on current applications of the fabricated silicon nanostructures in nanomedicine, specifically examining drug and gene delivery, bioimaging and biosensing.
Assuntos
Nanoestruturas , Silício/química , Animais , HumanosRESUMO
Nanomaterials promise to improve disease diagnosis and treatment by enhancing the delivery of drugs, genes, biomolecules and imaging agents to specific subcellular targets. In order to optimize nanomaterial design for this purpose, a comprehensive understanding of how these materials are taken up and transported within the cell is required. In this review, we discuss the endocytic pathways employed by different types of nanoparticles with emphasis on the influence of nanoparticle surface modification. The use of pharmacological inhibition to probe internalization and intracellular trafficking pathways of nanoparticles is critically evaluated. Finally, approaches to target-specific delivery of therapeutics via nanoparticles into the cytoplasm and nucleus are addressed.
Assuntos
Sistemas de Liberação de Medicamentos , Endocitose , Nanomedicina , Nanopartículas , Preparações Farmacêuticas/metabolismo , Tecnologia Farmacêutica/métodos , Transporte Ativo do Núcleo Celular , Animais , Transporte Biológico , Química Farmacêutica , Portadores de Fármacos , Sistemas de Liberação de Medicamentos/métodos , Humanos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/químicaRESUMO
In this study, the ability of porous silicon nanoparticles (PSi NPs) to entrap and deliver nitric oxide (NO) as an effective antibacterial agent is tested against different Gram-positive and Gram-negative bacteria. NO was entrapped inside PSi NPs functionalized by means of the thermal hydrocarbonization (THC) process. Subsequent reduction of nitrite in the presence of d-glucose led to the production of large NO payloads without reducing the biocompatibility of the PSi NPs with mammalian cells. The resulting PSi NPs demonstrated sustained release of NO and showed remarkable antibacterial efficiency and anti-biofilm-forming properties. These results will set the stage to develop antimicrobial nanoparticle formulations for applications in chronic wound treatment.
RESUMO
Advanced approaches to direct the differentiation of embryonic stem cells are highly sought after. The surface-bound chemical gradient format is a powerful screening approach that can be deployed to study changes in stem cell behavior as a function of subtle changes in surface chemistry. Here, we investigate the spontaneous differentiation of cells derived from differentiating mouse embryoid body (mEB) cells into endoderm, mesoderm, and ectoderm following culture on surface-bound gradients of chemical functional groups in the absence of differentiation-biasing bioactive factors. Gradients were created using a diffusion-controlled plasma polymerization technique. The generated coating ranged from hydrophobic 1,7-octadiene (OD) plasma polymer at one end of the gradient to a more hydrophilic allylamine (AA) plasma polymer on the opposite end. The gradient surface was divided into seven equal regions of progressively increasing AA plasma polymer content and mEB cell response within these regions was compared. Cells adhered preferentially to the central regions of the gradient; however, cell proliferation increased toward AA-plasma-polymer-rich end of the gradient. Variation in the expression of germ layer markers was noted across the gradient surface. High AA:OD plasma polymer ratios triggered cell differentiation toward both mesoderm and ectoderm. Expression of tissue-specific markers, in particular, KRT18, AFP, and TNNT2, was strikingly responsive to subtle changes in surface chemistry, exhibiting vastly different expression levels between adjacent regions. Our results suggest that the surface-bound gradient platform is well suited to screening surface chemistries for use in the field of stem cell technologies and regenerative medicine.
Assuntos
Alilamina/farmacologia , Diferenciação Celular , Corpos Embrioides/citologia , Alcenos/farmacologia , Animais , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Imunofluorescência , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camundongos , Espectroscopia Fotoeletrônica , Propriedades de SuperfícieRESUMO
In the field of stem cell technology, future advancements rely on the effective isolation, scale-up and maintenance of specific stem cell populations and robust procedures for their directed differentiation. The stem cell microenvironment - or niche - encompasses signal inputs from stem cells, supporting cells and from the extracellular matrix. In this context, the contribution of physicochemical surface variables is being increasingly recognised. This paradigm can be exploited to exert control over cellular behaviour. However, the number of parameters at play, and their complex interactions, presents a formidable challenge in delineating how the decisions of cell fate are orchestrated within the niche. Additionally, in the case of mesenchymal stem cells (MSC), more than one type of stem cell niche has been identified. By employing high throughput screening (HTS) strategies, common and specific attributes of each MSC niche can be probed. Here, we explore biological, chemical and physical parameters that are known to influence MSC self-renewal and differentiation. We then review techniques and strategies that allow the HTS of surface properties for conditions that direct stem cell fate, using MSC as a case study. Finally, challenges in recapturing the niche, particularly its three dimensional nature, in surface-based HTS formats are discussed.
Assuntos
Células-Tronco Mesenquimais/citologia , Nicho de Células-Tronco/fisiologia , HumanosRESUMO
Isatin, 1H-indole-2,3-dione, is a heterocyclic compound of significant importance in medicinal chemistry. It is a synthetically versatile molecule, a precursor for a large number of pharmacologically active compounds. Isatin and its derivatives have aroused great attention in recent years due to their wide variety of biological activities, relevant to application as insecticides and fungicides and in a broad range of drug therapies, including anticancer drugs, antibiotics and antidepressants. The purpose of this review is to provide an overview of the pharmacological activities of isatin and its synthetic and natural derivatives. Molecular modifications to tailor the properties of isatin and its derivatives are also discussed.
Assuntos
Desenho de Fármacos , Isatina/farmacologia , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antidepressivos/química , Antidepressivos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Humanos , Isatina/análogos & derivados , Isatina/química , Praguicidas/química , Praguicidas/farmacologia , Relação Estrutura-AtividadeRESUMO
The cell microarray format can recreate a multitude of cell microenvironments on a single chip using only minimal amounts of reagent. In this study, we describe surface modifications to passivate cell microarrays, aiming to adapt the platform to the study of stem cell behavior over long-term culture periods. Functionalization of glass slides with (3-glycidyloxypropyl) trimethoxysilane enabled covalent anchoring of extracellular matrix proteins on microscale spots printed by a robotic contact printer. Subsequently, the surface was passivated by bovine serum albumin (BSA) or poly(ethylene glycol)bisamine (A-PEG) with molecular weights of 3000, 6000, and 10 000 Da. Cloud-point conditions for A-PEG grafting were attained that were compatible with protein deposition. Passivation strategies were assessed by culturing mesenchymal stem cells on the microarray platform. While both BSA and A-PEG passivation initially blocked cell adhesion between the printed spots, only A-PEG grafting was able to maintain cell pattern integrity over the entire culture period of 3 weeks.