RESUMEN
OBJECTIVE: Dental implants provide support for dental crowns and bridges by serving as abutments for the replacement of missing teeth. To prevent bacterial accumulation and growth at the site of implantation, solutions such as systemic antibiotics and localized delivery of bactericidal agents are often employed. The objective of this study was to demonstrate a novel method of controlled localized delivery of antibacterial agents to an implant site using a biodegradable custom fabricated ring. METHODS: The study involved incorporating a model antibacterial agent (metronidazole) into custom designed poly-ε-caprolactone/alginate (PCL/alginate) composite rings to produce the intended controlled release profile. The rings can be designed to fit around the body of any root form dental implants of various diameters, shapes and sizes. RESULTS: In vitro release studies indicate that pure (100%) alginate rings exhibited an expected burst release of metronidazole in the first few hours, whereas Alginate/PCL composite rings produced a medium burst release followed by a sustained release for a period greater than 4 weeks. By varying the PCL/alginate weight ratios, we have shown that we can control the amount of antibacterial agents released to provide the minimal inhibitory concentration (MIC) needed for adequate protection. The fabricated composite rings have achieved a 50% antibacterial agent release profile over the first 48 h and the remaining amount slowly released over the remainder of the study period. The PCL/alginate agent release characteristic fits the Ritger-Peppas model indicating a diffusion-based mechanism during the 30-day study period. SIGNIFICANCE: The developed system demonstrates a controllable drug release profile and the potential for the ring to inhibit bacterial biofilm growth for the prevention of diseases such as peri-implantitis resulting from bacterial infection at the implant site.
Asunto(s)
Implantes Absorbibles , Alginatos/química , Antiinfecciosos/administración & dosificación , Implantes Dentales , Sistemas de Liberación de Medicamentos , Metronidazol/administración & dosificación , Poliésteres/química , Antiinfecciosos/química , Materiales Biocompatibles/química , Preparaciones de Acción Retardada , Difusión , Sistemas de Liberación de Medicamentos/instrumentación , Módulo de Elasticidad , Elasticidad , Diseño de Equipo , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Hidrogeles/química , Metronidazol/química , Pruebas de Sensibilidad Microbiana , Estrés Mecánico , Factores de TiempoRESUMEN
Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds.
Asunto(s)
Alginatos/química , Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Hidrogeles/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Pruebas de Toxicidad/métodosRESUMEN
In this study, we have evaluated the use of ultra-sterile alginate hydrogels encapsulated with HepG2 liver cells for applications in high throughput drug screening. We have studied the cellular viability and metabolic capacity of the encapsulated cells in two different alginate structures SLM100 (G:M::40:60) and SLG100 (G:M::60:40). We have also developed protocols to characterize the encapsulated cells within the alginate structure using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Further we have studied the Phase-I/II metabolic characteristics of the encapsulated cells in monolayer and 3D culture. Our results indicate that cells encapsulated within SLM100 and SLG100 class of alginates have shown high cellular viability with >80% even after 14 days in culture. As expected, the proliferation rates of the encapsulated cells are held steady and do not proliferate within the gels. Production of liver-specific enzymes such as CYP1A1 and CYP3A4 after 14 days in culture indicates the viability and functionality of the encapsulated HepG2 cells. Phase-II Glutathione activity of the encapsulated cells were also maintained in 3D culture conditions. The encapsulated cells within the 3D gels were also capable of metabolizing the pro-drug EFC (7-ethoxy-4-trifluoromethyl coumarin) to HFC (7-hydroxy-4-trifluoromethyl) in a linear fashion over a period of time. These results have provided us with baseline results to benchmark future improvements in material and design configurations for optimal pharmacokinetic response of in vitro tissue model systems.
Asunto(s)
Alginatos , Hidrogeles , Pruebas de Toxicidad/métodos , Alginatos/ultraestructura , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Cumarinas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Glutatión/metabolismo , Células Hep G2 , Profármacos/metabolismoRESUMEN
Vibrio parahaemolyticus is a common food-borne pathogen that is normally associated with seafood. In 1996, a pandemic O3:K6 strain abruptly appeared and caused the first pandemic of this pathogen to spread throughout many Asian countries, America, Europe, and Africa. The role of temperate bacteriophages in the evolution of this pathogen is of great interest. In this work, a new temperate phage, VP882, from a pandemic O3:K6 strain of V. parahaemolyticus was purified and characterized after mitomycin C induction. VP882 was a Myoviridae bacteriophage with a polyhedral head and a long rigid tail with a sheath-like structure. It infected and lysed high proportions of V. parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae strains. The genome of phage VP882 was sequenced and was 38,197 bp long, and 71 putative open reading frames were identified, of which 27 were putative functional phage or bacterial genes. VP882 had a linear plasmid-like genome with a putative protelomerase gene and cohesive ends. The genome does not integrate into the host chromosome but was maintained as a plasmid in the lysogen. Analysis of the reaction sites of the protelomerases in different plasmid-like phages revealed that VP882 and PhiHAP-1 were highly similar, while N15, PhiKO2, and PY54 made up another closely related group. The presence of DNA adenine methylase and quorum-sensing transcriptional regulators in VP882 may play a specific role in this phage or regulate physiological or virulence-associated traits of the hosts. These genes may also be remnants from the bacterial chromosome following transduction.
Asunto(s)
Plásmidos , Profagos/genética , Profagos/aislamiento & purificación , Vibrio parahaemolyticus/virología , Secuencia de Aminoácidos , ADN Viral/química , ADN Viral/genética , Orden Génico , Genes Virales , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Myoviridae/clasificación , Myoviridae/genética , Myoviridae/aislamiento & purificación , Myoviridae/ultraestructura , Sistemas de Lectura Abierta , Profagos/clasificación , Profagos/ultraestructura , Alineación de Secuencia , Análisis de Secuencia de ADN , Vibrio cholerae/virología , Vibrio parahaemolyticus/genética , Vibrio vulnificus/virología , Virión/ultraestructuraRESUMEN
Large thick tissue constructs have reported limited success primarily due to the inability of cells to survive deep within the scaffold. Without access to adequate nutrients, cells placed deep within the tissue construct will die out, leading to non-uniform tissue regeneration. Currently, there is a necessity to design nutrient conduit networks within the tissue construct to enable cells to survive in the matrix. However, the design of complex networks within a tissue construct is challenging. In this paper, we present the Lindenmayer system, an elegant fractal-based language algorithm framework, to generate conduit networks in two- and three-dimensional architecture with several degrees of complexity. The conduit network maintains a parent-child relationship between each branch of the network. Several L-system parameters have been studied-branching angle, branch length, ratio of parent to child branch diameter, etc-to simulate several architectures under a given L-system notation. We have also presented a layered manufacturing-based UV-photopolymerization process using the Texas Instruments DLP system to fabricate the branched structures. This preliminary work showcases the applicability of L-system-based construct designs to drive scaffold fabrication systems.
Asunto(s)
Diseño Asistido por Computadora , Modelos Cardiovasculares , Ingeniería de Tejidos/métodos , Andamios del Tejido , Algoritmos , Animales , Biotecnología , Humanos , Riñón/irrigación sanguínea , Procesos Fotoquímicos , Porosidad , Ratas , Rayos UltravioletaRESUMEN
Vibrio parahaemolyticus, an important seafood-associated enteropathogen, usually encounters different adverse conditions in its native or food-processing environment, and the stresses resulting from these conditions may affect the survival of this pathogen and thus change its risk with regard to food hygiene. In this study, we investigated the thermotolerance of V. parahaemolyticus under sublethal heat shock and characterized this response by examining the changes in protein profiles and toxin production. Logarithmically grown cells heat shocked at 42 degrees C for 30 min were more resistant to thermal inactivation at 47 degrees C than were unshocked cells. After the 25 degrees C culture was heat shocked, 24 species of proteins were induced, while 13 species were inhibited, as indicated by polyacrylamide gel electrophoresis. DnaJ-, GroEL-, and GroES-like proteins with molecular sizes of 47, 62, and 12 kDa, respectively, were detected by immunoblotting with antibodies raised against the Escherichia coli proteins. During 1 to 8 h of heat shock, GroEL-like protein was produced in substantial amounts and was present in the periplasmic and extracellular fractions, while DnaJ- and GroES-like proteins were present mainly in the total cellular fraction. DnaK-like protein was not detected; nevertheless, the presence of the dnaK-like genetic element was revealed by Southern blotting. Production of thermostable direct hemolysin, the major virulence factor in V. parahaemolyticus, was enhanced in the cells heat shocked at 42 degrees C but not in those heat shocked at 37 degrees C.