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1.
Curr Microbiol ; 79(4): 99, 2022 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-35150334

RESUMO

Mycotoxins may be present in nuts, coffee, cereals, and grapes, among other products. Increasing concerns about human health and environmental protection have driven the application of biological control techniques that can inhibit fungal contaminants. In this study, the growth inhibition of the ochratoxigenic fungus Aspergillus carbonarius Ac 162 was evaluated using 5 lactic acid bacteria (LAB). The LAB studied were Lactobacillus plantarum MZ801739 (J), Lactobacillus plantarum MZ809351 (31) and Lactobacillus plantarum MZ809350 (34), isolated in the Ivory Coast, and Lactobacillus plantarum MN982928 (3) and Leuconostoc citreum MZ801735 (23), isolated in Mexico. J, 31, 34, 3 and 23 are the internal strain codes from our laboratory. LAB were cultivated in De Man, Rogosa and Sharpe (MRS) broth, and different polyols (glycerol, mannitol, sorbitol, and xylitol) were added to the culture broth to stimulate the production of antifungal compounds. The fungal inhibition studies were performed using the poisoned food technique. The highest inhibition of A. carbonarius growth was obtained by cultivating L. plantarum MZ809351 in the presence of xylitol and glycerol. Under these conditions, 1 L of the L. plantarum MZ809351 cultures were used to identify antifungal compounds. The compounds were concentrated by solid-phase extraction and then characterized by GC-MS. In addition to 9-octadecenoic acid, 3 diketopiperazines or cyclic dipeptides were identified, including cyclo (Leu-Leu), cyclo (Pro-Gly) and cyclo (Val-Phe), which were compounds related to microbial antifungal activities. Xylitol and glycerol induced the production of these antifungal compounds against A. carbonarius Ac 162. On the other hand, adding xylitol and glycerol to the MRS broth reduced the Ochratoxin A (OTA) content to 56.8 and 54.7%, respectively. This study shows the potential for using L. plantarum MZ809351 as a biocontrol agent to prevent the growth of A. carbonarius and reduce the production of OTA in foods.


Assuntos
Lactobacillus plantarum , Micotoxinas , Antifúngicos/farmacologia , Humanos , Polímeros
2.
J Oral Maxillofac Surg ; 79(1): 237-248, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32890473

RESUMO

PURPOSE: Oncological patients who undergo bilateral subtotal maxillectomies develop functional and esthetic sequelae that require immediate reconstruction. The purpose of this study is to evaluate the primary reconstruction of maxillary defects with fibula flap and dental implants assisted by virtual surgical planning (VSP) and to assess the postoperative outcomes compared with standard surgery. MATERIAL AND METHODS: A retrospective study was designed between January 2016 and April 2020 with 12 oncologic patients who underwent subtotal bilateral maxillectomy. Six consecutive patients were treated by standard surgical procedure (SS) at the beginning of the study. In 2018, the VSP was implemented, and 6 consecutive patients were treated using this technique. All patients were rehabilitated with Ticare implants and implant prostheses. Anatomic position of the bone, bone apposition, change of vertical distance, and horizontal shift, the operative and ischemia time, the esthetic results, and the functional rehabilitation were evaluated and compared. RESULTS: The position of the bone in anatomical position was 100% in the VSP group vs 66% in the SS group. The bone apposition was 100% in the VSP group vs 83.3%. The change of vertical distance and the horizontal shift were lower in the VSP group (P < .05). The ischemia time and operative time were shorter in the VSP group (P < .05). A good esthetic result was achieved in 83.3% in the VSP group vs 33.3% in the SS group; 81 dental implants and 1 zygomatic implant were placed. The success rate was 95% in the VSP group and 92.6% in the SS group. All patients were rehabilitated with implant prosthesis. CONCLUSIONS: VSP improves the accuracy of midface reconstruction (class IIC defect) with a better anatomical position of the bone, a higher rate of bone contact, and a lower change in vertical distance compared with standard surgery. It significantly improves the esthetic result, reduces ischemia time, and operation time.


Assuntos
Implantes Dentários , Retalhos de Tecido Biológico , Reconstrução Mandibular , Procedimentos de Cirurgia Plástica , Estética Dentária , Fíbula/cirurgia , Retalhos de Tecido Biológico/cirurgia , Humanos , Padrões de Referência , Estudos Retrospectivos
3.
J Craniofac Surg ; 30(4): 1149-1153, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31166261

RESUMO

OBJECTIVE: The aim of this study is to describe the importance of osteodistraction with transpalatal distractors for treating transversal maxillary hypoplasia in patients with cleft and lip palate. METHODS: The participants were 17 patients (9 females and 8 males) with cleft lip and palate. Among these, 10 presented unilateral cleft lip and palate, 4 bilateral cleft lip and palate, and 3 cleft palate only. RESULTS: All patients experienced a satisfactory palatal expansion and crossbite correction. The mean lengthening was 12.7 mm. The average increase of intercanine distance, intermolar distance, maxillary transverse dimension (MTD), facial transverse dimension (FTD) was 12.16, 8.45, 1.77, and 1.67 mm, respectively. The clinical follow-up was 29.7 months (range: 6-61 months). CONCLUSION: Palatal distraction is a safe and successful alternative for treating maxillary transversal alterations in patients with cleft lip and palate. This technique facilitates the establishment of an adequate transverse dimension of maxillary, and consequently a proper dental occlusion.


Assuntos
Fenda Labial/terapia , Fissura Palatina/terapia , Técnica de Expansão Palatina , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Má Oclusão/terapia , Maxila/anormalidades
4.
Nano Lett ; 18(1): 629-637, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29243484

RESUMO

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate-with molecular sensitivity-the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems.


Assuntos
Efrina-B1/metabolismo , Proteínas Imobilizadas/metabolismo , Nanoestruturas/química , Polimetil Metacrilato/química , Receptor EphB2/metabolismo , Efrina-B1/química , Células HEK293 , Humanos , Proteínas Imobilizadas/química , Ligantes , Nanoestruturas/ultraestrutura , Agregados Proteicos , Multimerização Proteica , Receptor EphB2/química
5.
Cleft Palate Craniofac J ; 56(4): 548-551, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30068230

RESUMO

OBJECTIVE: Describe the surgical repositioning of the premaxilla using an innovative minimally invasive endonasal approach and secondary bone graft at the same time. We want to emphasize the importance of virtual surgical planning in this technique. MATERIAL AND METHODS: A total of 6 patients with bilateral complete cleft lip and palate underwent a surgical repositioning of the premaxilla. Virtual surgical planning was performed in all cases. The ages varied between 8 and 12 years and all were male. Five patients were in the mixed dentition phase and 1 patient was in the definitive phase. Three of the patients had been prepared with presurgical nasoalveolar molding. The other 3 were not prepared for various reasons. All patients had primary repair of cleft lip and palate. INTERVENTIONS: An innovative minimally invasive endonasal approach is presented that has allowed a safe 3-D reposition of the premaxilla in patients with bilateral cleft palate. A simultaneous secondary alveoloplasty with the use of absorbable osteosynthesis is a good choice to achieve symmetry and stability. CONCLUSIONS: Virtual surgical planning is an exceptional instrument to make an appropriate presurgical selection of the patients in which combine the 2 procedures.


Assuntos
Fenda Labial , Fissura Palatina , Alveoloplastia , Criança , Humanos , Masculino , Maxila , Nariz
6.
Neuropharmacology ; 249: 109871, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38412889

RESUMO

Dopamine is unable to access the central nervous system through the bloodstream. Only its precursor can do so, and with an effectiveness below 100% of the dose administered, as it is metabolized before crossing the blood-brain barrier. In this study, we describe a new solid lipid nanocarrier system designed and developed for dopamine. The nanoparticles were prepared by the melt-emulsification method and then coated with chitosan. The nanocarriers developed had a droplet size of about 250 nm, a polydispersity index of 0.2, a positive surface charge (+30 mV), and a percentage encapsulation efficiency of 36.3 ± 5.4. Transmission and scanning electron microscopy verified uniformity of particle size with spherical morphology. Various types of tests were performed to confirm that the nanoparticles designed are suitable for carrying dopamine through the blood-brain barrier. In vitro tests demonstrated the ability of these nanocarriers to pass through endothelial cell monolayers without affecting their integrity. This study shows that the formulation of dopamine in chitosan-coated solid lipid nanoparticles is a potentially viable formulation strategy to achieve the bioavailability of the drug for the treatment of Parkinson's disease in the central nervous system.


Assuntos
Quitosana , Lipossomos , Nanopartículas , Portadores de Fármacos/metabolismo , Dopamina/metabolismo , Quitosana/metabolismo , Barreira Hematoencefálica/metabolismo
7.
Nanomedicine ; 9(5): 694-701, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23313904

RESUMO

Cells can respond to small changes in a varying concentration of exogenous signaling molecules. Here we propose the use of continuous surface chemical gradients for the in-depth study of dose-dependent effects on cells. A continuous surface gradient of bone morphogenetic protein-2 (BMP-2) is presented. The gradient covers a narrow range of surface densities (from 1.4 to 2.3 pmol/cm(2)) with a shallow slope (0.9 pmol/cm(3)). These characteristics represent a quasi-homogeneous surface concentration at the cell scale, which is crucial for cell screening studies. Cell fate evaluation at early stages of osteogenesis in C2C12 cells, indicates the potential of continuous gradients for in vitro screening applications. FROM THE CLINICAL EDITOR: The authors propose the use of surface-applied continuous chemical gradients for in-depth study of dose-dependent effects on cells. The method is demonstrated using BMP-2 proteins on C2C12 cells as a model system.


Assuntos
Proteína Morfogenética Óssea 2/química , Diferenciação Celular/genética , Osteogênese/efeitos dos fármacos , Polimetil Metacrilato/farmacologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Ouro/química , Humanos , Camundongos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Polimetil Metacrilato/química , Estreptavidina/química , Propriedades de Superfície
8.
ACS Appl Bio Mater ; 6(7): 2860-2874, 2023 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-37342003

RESUMO

The low endogenous regenerative capacity of the heart, added to the prevalence of cardiovascular diseases, triggered the advent of cardiac tissue engineering in the last decades. The myocardial niche plays a critical role in directing the function and fate of cardiomyocytes; therefore, engineering a biomimetic scaffold holds excellent promise. We produced an electroconductive cardiac patch of bacterial nanocellulose (BC) with polypyrrole nanoparticles (Ppy NPs) to mimic the natural myocardial microenvironment. BC offers a 3D interconnected fiber structure with high flexibility, which is ideal for hosting Ppy nanoparticles. BC-Ppy composites were produced by decorating the network of BC fibers (65 ± 12 nm) with conductive Ppy nanoparticles (83 ± 8 nm). Ppy NPs effectively augment the conductivity, surface roughness, and thickness of BC composites despite reducing scaffolds' transparency. BC-Ppy composites were flexible (up to 10 mM Ppy), maintained their intricate 3D extracellular matrix-like mesh structure in all Ppy concentrations tested, and displayed electrical conductivities in the range of native cardiac tissue. Furthermore, these materials exhibit tensile strength, surface roughness, and wettability values appropriate for their final use as cardiac patches. In vitro experiments with cardiac fibroblasts and H9c2 cells confirmed the exceptional biocompatibility of BC-Ppy composites. BC-Ppy scaffolds improved cell viability and attachment, promoting a desirable cardiomyoblast morphology. Biochemical analyses revealed that H9c2 cells showed different cardiomyocyte phenotypes and distinct levels of maturity depending on the amount of Ppy in the substrate used. Specifically, the employment of BC-Ppy composites drives partial H9c2 differentiation toward a cardiomyocyte-like phenotype. The scaffolds increase the expression of functional cardiac markers in H9c2 cells, indicative of a higher differentiation efficiency, which is not observed with plain BC. Our results highlight the remarkable potential use of BC-Ppy scaffolds as a cardiac patch in tissue regenerative therapies.


Assuntos
Miócitos Cardíacos , Polímeros , Polímeros/química , Pirróis/química , Diferenciação Celular
9.
Nat Commun ; 14(1): 1291, 2023 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-36894532

RESUMO

Antibiotic overuse has promoted the spread of antimicrobial resistance (AMR) with significant health and economic consequences. Genome sequencing reveals the widespread presence of antimicrobial resistance genes (ARGs) in diverse microbial environments. Hence, surveillance of resistance reservoirs, like the rarely explored oral microbiome, is necessary to combat AMR. Here, we characterise the development of the paediatric oral resistome and investigate its role in dental caries in 221 twin children (124 females and 97 males) sampled at three time points over the first decade of life. From 530 oral metagenomes, we identify 309 ARGs, which significantly cluster by age, with host genetic effects detected from infancy onwards. Our results suggest potential mobilisation of ARGs increases with age as the AMR associated mobile genetic element, Tn916 transposase was co-located with more species and ARGs in older children. We find a depletion of ARGs and species in dental caries compared to health. This trend reverses in restored teeth. Here we show the paediatric oral resistome is an inherent and dynamic component of the oral microbiome, with a potential role in transmission of AMR and dysbiosis.


Assuntos
Cárie Dentária , Microbiota , Masculino , Feminino , Humanos , Criança , Farmacorresistência Bacteriana/genética , Cárie Dentária/genética , Antibacterianos/farmacologia , Genes Bacterianos , Microbiota/genética
10.
Langmuir ; 28(38): 13688-97, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-22913232

RESUMO

In this work, we propose an easy method to produce highly tunable gradients of covalently bound proteins on topographically modified poly(methyl methacrylate). We used a microfluidic approach to obtain linear gradients with high slope (0.5 pmol·cm(-2)·mm(-1)), relevant at the single-cell level. These protein gradients were characterized using fluorescence microscopy and surface plasmon resonance. Both experimental results and theoretical modeling on the protein gradients generated have proved them to be highly reproducible, stable up to 7 days, and easily tunable. This method enables formation of versatile cell culture platforms combining both complex biochemical and physical cues in an attempt to approach in vitro cell culture methods to in vivo cellular microenvironments.


Assuntos
Técnicas Analíticas Microfluídicas , Polimetil Metacrilato/química , Proteínas/química , Animais , Adesão Celular , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Conformação Proteica , Ressonância de Plasmônio de Superfície
11.
Artigo em Inglês | MEDLINE | ID: mdl-35822641

RESUMO

We present a case of late mediastinitis following surgery for type A aortic dissection. After a thorough preoperative workup, the patient underwent a redo sternotomy, removal of all prosthetic material, and replacement of the aortic root with a homograft. The patient required venoarterial extracorporeal membrane oxygenation and delayed sternal closure for post-postoperative biventricular failure as well as prolonged antibiotic treatment. We present our institutional multidisciplinary approach for the management of such complex cases.


Assuntos
Dissecção Aórtica , Próteses Valvulares Cardíacas , Mediastinite , Dissecção Aórtica/cirurgia , Aorta/cirurgia , Humanos , Mediastinite/etiologia , Mediastinite/cirurgia , Polietilenotereftalatos
12.
ACS Appl Mater Interfaces ; 13(12): 13920-13933, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33739812

RESUMO

Tissue barriers play a crucial role in human physiology by establishing tissue compartmentalization and regulating organ homeostasis. At the interface between the extracellular matrix (ECM) and flowing fluids, epithelial and endothelial barriers are responsible for solute and gas exchange. In the past decade, microfluidic technologies and organ-on-chip devices became popular as in vitro models able to recapitulate these biological barriers. However, in conventional microfluidic devices, cell barriers are primarily grown on hard polymeric membranes within polydimethylsiloxane (PDMS) channels that do not mimic the cell-ECM interactions nor allow the incorporation of other cellular compartments such as stromal tissue or vascular structures. To develop models that accurately account for the different cellular and acellular compartments of tissue barriers, researchers have integrated hydrogels into microfluidic setups for tissue barrier-on-chips, either as cell substrates inside the chip, or as self-contained devices. These biomaterials provide the soft mechanical properties of tissue barriers and allow the embedding of stromal cells. Combining hydrogels with microfluidics technology provides unique opportunities to better recreate in vitro the tissue barrier models including the cellular components and the functionality of the in vivo tissues. Such platforms have the potential of greatly improving the predictive capacities of the in vitro systems in applications such as drug development, or disease modeling. Nevertheless, their development is not without challenges in their microfabrication. In this review, we will discuss the recent advances driving the fabrication of hydrogel microfluidic platforms and their applications in multiple tissue barrier models.


Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Técnicas Analíticas Microfluídicas/instrumentação , Engenharia Tecidual/instrumentação , Animais , Desenho de Equipamento , Humanos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Engenharia Tecidual/métodos
13.
Langmuir ; 26(17): 14154-61, 2010 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-20712344

RESUMO

This article describes a simple method for the construction of a universal surface chemical gradient platform based on the biotin-streptavidin model. In this approach, surface chemical gradients were prepared in poly(methyl methacrylate) (PMMA), a biocompatible polymer, by a controlled hydrolysis procedure. The physicochemical properties of the resulting modified surfaces were extensively characterized. Chemical analysis carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) showed the formation of a smooth, highly controllable carboxylic acid gradient of increasing concentration along the sample surface. Atomic force microscopy (AFM) and contact angle (CA) results indicate that, in contrast with most of the chemical gradient methods published in the literature, the chemical modification of the polymer surface barely affects its physical properties. The introduction of carboxylic acid functionality along the surface was then used for biomolecule anchoring. For this purpose, the surface was activated and derivatized first with biotin and finally with streptavidin (SAV) in a directed orientation fashion. The SAV gradient was qualitatively assessed by fluorescence microscopy analysis and quantified by surface plasmon resonance (SPR) in order to establish a quantitative relationship between SAV surface densities and the surface location. The usefulness of the fabrication method described for biological applications was tested by immobilizing biotinylated bradykinin onto the SAV gradient. This proof-of-concept application shows the effectiveness of the concentration range of the gradient because the effects of bradykinin on cell morphology were observed to increase gradually with increasing drug concentrations. The intrinsic characteristics of the fabricated gradient platform (absence of physicochemical modifications other than those due to the biomolecules included) allow us to attribute cell behavior unequivocally to the biomolecule surface density changes.


Assuntos
Biotina/química , Materiais Revestidos Biocompatíveis/química , Polimetil Metacrilato/química , Estreptavidina/química , Hidrólise , Propriedades de Superfície
14.
Nanomedicine ; 6(1): 44-51, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19501193

RESUMO

Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. However, novel applications in the biosensor field require materials to be compatible with cell growth and at the same time be suitable for technological processing. Technological polymers are key materials in the fabrication of disposable parts and other sensing elements. As such, it is essential to characterize the surface properties of technological polymers, especially after processing and sterilization. It is also important to understand how technological polymers affect cell behavior when in contact with polymer materials. Therefore, the aim of this research was to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly(methyl methacrylate), polystyrene, and poly(dimethylsiloxane). Glass was used as the control material. FROM THE CLINICAL EDITOR: Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. The aim of this research is to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly(methylmethacrylate) (PMMA), polystyrene (PS), and poly(dimethylsiloxane) (PDMS).


Assuntos
Proteínas Sanguíneas/metabolismo , Dimetilpolisiloxanos/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Polimetil Metacrilato/farmacologia , Poliestirenos/farmacologia , Adsorção/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Microscopia de Força Atômica , Propriedades de Superfície/efeitos dos fármacos , Termodinâmica
15.
Biofabrication ; 12(2): 025023, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32050182

RESUMO

The stiffness and topography of a cell's extracellular matrix (ECM) are physical cues that play a key role in regulating processes that determine cellular fate and function. While substrate stiffness can dictate cell differentiation lineage, migration, and self-organization, topographical features can change the cell's differentiation profile or migration ability. Although both physical cues are present and intrinsic to the native tissues in vivo, in vitro studies have been hampered by the lack of technological set-ups that would be compatible with cell culture and characterization. In vitro studies therefore either focused on screening stiffness effects in cells cultured on flat substrates or on determining topography effects in cells cultured onto hard materials. Here, we present a reliable, microfabrication method to obtain well defined topographical structures of micrometer size (5-10 µm) on soft polyacrylamide hydrogels with tunable mechanical stiffness (3-145 kPa) that closely mimic the in vivo situation. Topographically microstructured polyacrylamide hydrogels are polymerized by capillary force lithography using flexible materials as molds. The topographical microstructures are resistant to swelling, can be conformally functionalized by ECM proteins and sustain the growth of cell lines (fibroblasts and myoblasts) and primary cells (mouse intestinal epithelial cells). Our method can independently control stiffness and topography, which allows to individually assess the contribution of each physical cue to cell response or to explore potential synergistic effects. We anticipate that our fabrication method will be of great utility in tissue engineering and biophysics, especially for applications where the use of complex in vivo-like environments is of paramount importance.


Assuntos
Resinas Acrílicas/química , Técnicas de Cultura de Células/métodos , Hidrogéis/química , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Hidrogéis/farmacologia , Camundongos , Engenharia Tecidual
16.
Biofabrication ; 12(2): 025008, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31805546

RESUMO

Mounting evidence supports the importance of the intestinal epithelial barrier and its permeability both in physiological and pathological conditions. Conventional in vitro models to evaluate intestinal permeability rely on the formation of tightly packed epithelial monolayers grown on hard substrates. These two-dimensional models lack the cellular and mechanical components of the non-epithelial compartment of the intestinal barrier, the stroma, which are key contributors to the barrier permeability in vivo. Thus, advanced in vitro models approaching the in vivo tissue composition are fundamental to improve precision in drug absorption predictions, to provide a better understanding of the intestinal biology, and to faithfully represent related diseases. Here, we generate photo-crosslinked gelatine methacrylate (GelMA)-poly(ethylene glycol) diacrylate (PEGDA) hydrogel co-networks that provide the required mechanical and biochemical features to mimic both the epithelial and stromal compartments of the intestinal mucosa, i.e. they are soft, cell adhesive and cell-loading friendly, and suitable for long-term culturing. We show that fibroblasts can be embedded in the GelMA-PEGDA hydrogels while epithelial cells can grow on top to form a mature epithelial monolayer that exhibits barrier properties which closely mimic those of the intestinal barrier in vivo, as shown by the physiologically relevant transepithelial electrical resistance (TEER) and permeability values. The presence of fibroblasts in the artificial stroma compartment accelerates the formation of the epithelial monolayer and boosts the recovery of the epithelial integrity upon temporary barrier disruption, demonstrating that our system is capable of successfully reproducing the interaction between different cellular compartments. As such, our hydrogel co-networks offer a technologically simple yet sophisticated approach to produce functional three-dimensional (3D) in vitro models of epithelial barriers with epithelial and stromal cells arranged in a spatially relevant manner and near-physiological functionality.


Assuntos
Gelatina/química , Hidrogéis/química , Mucosa Intestinal/citologia , Metacrilatos/química , Polietilenoglicóis/química , Alicerces Teciduais/química , Animais , Fenômenos Biomecânicos , Células CACO-2 , Adesão Celular , Proliferação de Células , Células Epiteliais/citologia , Fibroblastos/citologia , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Impressão Tridimensional/instrumentação , Engenharia Tecidual/instrumentação
17.
Biofabrication ; 11(2): 025007, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30721885

RESUMO

Epithelial tissues contain three-dimensional (3D) complex microtopographies that are essential for proper performance. These microstructures provide cells with the physicochemical cues needed to guide their self-organization into functional tissue structures. However, most in vitro models do not implement these 3D architectural features. The main problem is the availability of simple fabrication techniques that can reproduce the complex geometries found in native tissues on the soft polymeric materials required as cell culture substrates. In this study reaction-diffusion mediated photolithography is used to fabricate 3D microstructures with complex geometries on poly(ethylene glycol)-based hydrogels in a single step and moldless approach. By controlling fabrication parameters such as the oxygen diffusion/depletion timescales, the distance to the light source and the exposure dose, the dimensions and geometry of the microstructures can be well-defined. In addition, copolymerization of poly(ethylene glycol) with acrylic acid improves control of the dynamic reaction-diffusion processes that govern the free-radical polymerization of highly-diluted polymeric solutions. Moreover, acrylic acid allows adjusting the density of cell adhesive ligands while preserving the mechanical properties of the hydrogels. The method proposed is a simple, single-step, and cost-effective strategy for producing models of intestinal epithelium that can be easily integrated into standard cell culture platforms.


Assuntos
Hidrogéis/química , Intestinos/fisiologia , Luz , Polimerização , Engenharia Tecidual/métodos , Acrilatos/química , Células CACO-2 , Colágeno/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Ligantes , Microtecnologia , Polietilenoglicóis/química , Fatores de Tempo , Alicerces Teciduais/química
18.
Sci Rep ; 9(1): 10140, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300688

RESUMO

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies.


Assuntos
Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Intestino Delgado/citologia , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Proliferação de Células , Colágeno , Meios de Cultivo Condicionados/farmacologia , Combinação de Medicamentos , Proteínas de Fluorescência Verde/genética , Laminina , Membranas Artificiais , Organoides , Proteoglicanas , Proteína Wnt3A/metabolismo
19.
Mater Sci Eng C Mater Biol Appl ; 97: 803-812, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30678971

RESUMO

In this article, we explored the selective antibiofouling capacity acquired by functional wrinkled hydrogel films via a fine tuning of their chemical structure through the gradual insertion of hydrophobic radical groups in their network. The hydrogel consists of three main components: hydroxyethyl methacrylate (HEMA, amphiphilic monomer), trifluoroethyl methacrylate (TFMA, hydrophobic monomer), and poly(ethylene glycol) diacrylate (PEGDA, hydrophilic crosslinking agent). Interestingly, the manipulation of the chemical composition affects both, surface morphology and physicochemical characteristics of the patterns, inducing transitions between different surface microstructures, i.e. from wrinkles to creases, to folds, and to crumples. Contact angle measurements show that the insertion of TFMA produces a slight decrease in surface wettability, remaining however highly hydrophilic. By using confocal Raman spectroscopy, important information about wrinkle formation mechanism could be obtained. The procedure presented in this article involves two consecutive thermal and photopolymerization steps, generating a "pseudo" two-layer system, which contracts at different extents when is exposed to external stimuli, leading to the formation of wrinkled surfaces. Finally, bacterial and cellular adhesion/proliferation studies were carried out, evidencing that the amount of TFMA included clearly reduce the bacterial adhesion while mammalian cells are able to still proliferate.


Assuntos
Materiais Biocompatíveis/química , Incrustação Biológica/prevenção & controle , Desenho de Fármacos , Metilgalactosídeos/química , Animais , Aderência Bacteriana/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Metacrilatos/química , Camundongos , Microscopia de Força Atômica , Polietilenoglicóis/química , Análise Espectral Raman , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Raios Ultravioleta , Molhabilidade
20.
Small ; 3(5): 871-9, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17394283

RESUMO

MG63 cells cultured on regular arrays of point microstructures (posts and holes) are shown to preferentially align at certain angles to the pattern of the structures, at 0 degrees, 30 degrees, and 45 degrees in particular. The effect is found to be more pronounced for post rather than hole structures (although no significant difference is found for the angles the cells make to the holes or posts) and is thought to be due to the fact that the cells use the posts as anchorage points to hold themselves to the surface. It is also shown that cells preferentially align with the structures depending on the dimensions of the structures and the distance between neighboring structures. This is important when designing structured surfaces for cell-surface interaction studies for materials to be used in, for example, drug delivery or tissue engineering.


Assuntos
Cristalização/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Osteoblastos/citologia , Osteoblastos/fisiologia , Polimetil Metacrilato/química , Engenharia Tecidual/métodos , Adesão Celular , Técnicas de Cultura de Células/métodos , Linhagem Celular , Polaridade Celular , Humanos , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Nanotecnologia/métodos , Tamanho da Partícula , Propriedades de Superfície
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