1) Magnetic resonance microscopy was used to visualize and quantify the poly-L-lysine layer of alginate/poly-L-lysine/alginate microcapsules. The thickness of the poly-L-lysine layer was measured to be 40.6±6.2 μm regardless of alginate composition or coating time.
2) The T2 relaxation time and apparent diffusion coefficient of the alginate matrix were measured over a month-long culture to monitor changes in microstructure. Alginate beads with high guluronic content showed a decrease in T2 over time, while apparent diffusion coefficient was unaffected.
3) Changes in T2 were less pronounced for beads coated with
The document discusses an overview of livestock metabolomics. It defines metabolomics as the large-scale study of small molecules present in cells, biofluids, tissues and organs. Various techniques for metabolomic analysis are described including mass spectrometry, NMR spectroscopy, GC-MS and LC-MS. Applications of metabolomics in livestock include disease diagnosis, biomarker identification, monitoring drug and surgical impacts, and understanding gene-environment interactions. Specific examples include identifying metabolic biomarkers for mastitis resistance in dairy cows and detection of milk fever in cattle. The challenges and future prospects of metabolomics research are also outlined.
Genome-scale in silico atpE gene knockout in Escherichia coli could drive nov...Khadem2016
One of the applications of E. coli genome-scale model is in the biological discovery of underground metabolic functions of partially characterized genes and/or enzymes. Here we report for the first time, a failed prediction of atpE gene knockout of no growth in the most recent E. coli reconstruction iJ01366 model, and a positive experimental growth on glucose, enabling a model-driven biological discovery of the underground metabolic function of this gene in E. coli metabolism. These findings unfolded what could be described as either scope gaps in the reconstruction or true biological gaps (knowledge gaps) on the missing atpE gene function in E. coli metabolism. This study informs other studies that the gaps could be pursued into the E. coli metabolism, leading to a model-driven discovery in the future.
HYDROGELS FOR WOUND HEALING AND TISSUE ENGINEERING APPLICATIONSMunira Shahbuddin
This document summarizes research on hydrogels and their applications in tissue engineering, regenerative medicine, and wound healing. It describes several key findings:
1) Hydrogels from konjac glucomannan were shown to stimulate proliferation of fibroblasts and keratinocytes in a concentration-dependent manner and support their metabolic activity.
2) Crosslinking konjac glucomannan formed hydrogels that maintained viability of fibroblasts and keratinocytes. The hydrogels inhibited contraction and promoted re-epithelialization in a human tissue engineered skin model.
3) Analysis found the hydrogels modulated water content and interactions to influence cell-matrix interactions important for wound healing and tissue regeneration applications.
Ldb 145 Geni Mutanti_2014-11-19 Jamora - dalla ricerca al prodotto 5laboratoridalbasso
1) Fibulin-5 expression is elevated in the dermis of Snail transgenic skin, a model of tissue fibrosis. Deletion of Fibulin-5 decreases tissue stiffness and the amount of elastic fibers but not collagen fibers.
2) Depletion of Fibulin-5 decreases inflammation in the fibrotic skin as measured by reduced macrophage and T cell infiltration.
3) Fibulin-5 promotes the production of inflammatory chemokines from dermal fibroblasts and accentuates their activation both in vivo and in response to substrate stiffness in vitro.
This document summarizes a talk on organ development and the use of skin and hair as a model system. It discusses how organs form through morphometric modules guided by developmental signals. The skin and hair follicle are presented as an advantageous model due to the ability to culture stem cells, track defects in mice, and their dispensability. Key processes in hair follicle development highlighted include cadherin switching regulated by the Wnt and BMP signaling pathways and transcription factors like Lef1 and beta-catenin. Downregulation of E-cadherin, driven by these signals, is implicated in promoting polarity changes required for budding morphogenesis.
Ld b 145 geni mutanti_2014-11-18 jamora - ricerca scientifica 3laboratoridalbasso
This document discusses the role of the transcription factor Snail in wound healing and fibrosis. It finds that overexpression of Snail in the epidermis of mice induces dermal fibrosis through increased secretion of spondin-2/mindin by keratinocytes. Spondin-2 then activates dermal fibroblasts by stimulating the NF-kB pathway, leading to increased expression of genes associated with myofibroblast differentiation and extracellular matrix production. This establishes a paracrine signaling mechanism from epidermal Snail to induce dermal fibrosis through spondin-2.
This document discusses plant system biology, which analyzes plant systems as a whole by studying the interactions between their biological components. It describes various omics approaches like genomics, epigenomics, transcriptomics, proteomics, and metabolomics that are used to study different levels of biological organization. Integration of multi-omics data using bioinformatics tools and modeling approaches provides insights into how plants respond to stimuli by understanding the individual components and their interactions as a complex network.
MxiK is an essential component of the type III secretion system (T3SS) in Shigella bacteria. The researchers aim to purify and obtain soluble MxiK to allow for structural and protein interaction studies. Recombinant expression of MxiK in E. coli results in insoluble inclusion bodies. The researchers develop a purification method involving washing, denaturing, and nickel affinity chromatography to obtain highly purified denatured MxiK. However, extensive refolding attempts failed to obtain properly refolded soluble MxiK. In contrast, the closely related homolog PscK from Pseudomonas aeruginosa can be recombinantly expressed in a soluble form.
The document discusses an overview of livestock metabolomics. It defines metabolomics as the large-scale study of small molecules present in cells, biofluids, tissues and organs. Various techniques for metabolomic analysis are described including mass spectrometry, NMR spectroscopy, GC-MS and LC-MS. Applications of metabolomics in livestock include disease diagnosis, biomarker identification, monitoring drug and surgical impacts, and understanding gene-environment interactions. Specific examples include identifying metabolic biomarkers for mastitis resistance in dairy cows and detection of milk fever in cattle. The challenges and future prospects of metabolomics research are also outlined.
Genome-scale in silico atpE gene knockout in Escherichia coli could drive nov...Khadem2016
One of the applications of E. coli genome-scale model is in the biological discovery of underground metabolic functions of partially characterized genes and/or enzymes. Here we report for the first time, a failed prediction of atpE gene knockout of no growth in the most recent E. coli reconstruction iJ01366 model, and a positive experimental growth on glucose, enabling a model-driven biological discovery of the underground metabolic function of this gene in E. coli metabolism. These findings unfolded what could be described as either scope gaps in the reconstruction or true biological gaps (knowledge gaps) on the missing atpE gene function in E. coli metabolism. This study informs other studies that the gaps could be pursued into the E. coli metabolism, leading to a model-driven discovery in the future.
HYDROGELS FOR WOUND HEALING AND TISSUE ENGINEERING APPLICATIONSMunira Shahbuddin
This document summarizes research on hydrogels and their applications in tissue engineering, regenerative medicine, and wound healing. It describes several key findings:
1) Hydrogels from konjac glucomannan were shown to stimulate proliferation of fibroblasts and keratinocytes in a concentration-dependent manner and support their metabolic activity.
2) Crosslinking konjac glucomannan formed hydrogels that maintained viability of fibroblasts and keratinocytes. The hydrogels inhibited contraction and promoted re-epithelialization in a human tissue engineered skin model.
3) Analysis found the hydrogels modulated water content and interactions to influence cell-matrix interactions important for wound healing and tissue regeneration applications.
Ldb 145 Geni Mutanti_2014-11-19 Jamora - dalla ricerca al prodotto 5laboratoridalbasso
1) Fibulin-5 expression is elevated in the dermis of Snail transgenic skin, a model of tissue fibrosis. Deletion of Fibulin-5 decreases tissue stiffness and the amount of elastic fibers but not collagen fibers.
2) Depletion of Fibulin-5 decreases inflammation in the fibrotic skin as measured by reduced macrophage and T cell infiltration.
3) Fibulin-5 promotes the production of inflammatory chemokines from dermal fibroblasts and accentuates their activation both in vivo and in response to substrate stiffness in vitro.
This document summarizes a talk on organ development and the use of skin and hair as a model system. It discusses how organs form through morphometric modules guided by developmental signals. The skin and hair follicle are presented as an advantageous model due to the ability to culture stem cells, track defects in mice, and their dispensability. Key processes in hair follicle development highlighted include cadherin switching regulated by the Wnt and BMP signaling pathways and transcription factors like Lef1 and beta-catenin. Downregulation of E-cadherin, driven by these signals, is implicated in promoting polarity changes required for budding morphogenesis.
Ld b 145 geni mutanti_2014-11-18 jamora - ricerca scientifica 3laboratoridalbasso
This document discusses the role of the transcription factor Snail in wound healing and fibrosis. It finds that overexpression of Snail in the epidermis of mice induces dermal fibrosis through increased secretion of spondin-2/mindin by keratinocytes. Spondin-2 then activates dermal fibroblasts by stimulating the NF-kB pathway, leading to increased expression of genes associated with myofibroblast differentiation and extracellular matrix production. This establishes a paracrine signaling mechanism from epidermal Snail to induce dermal fibrosis through spondin-2.
This document discusses plant system biology, which analyzes plant systems as a whole by studying the interactions between their biological components. It describes various omics approaches like genomics, epigenomics, transcriptomics, proteomics, and metabolomics that are used to study different levels of biological organization. Integration of multi-omics data using bioinformatics tools and modeling approaches provides insights into how plants respond to stimuli by understanding the individual components and their interactions as a complex network.
MxiK is an essential component of the type III secretion system (T3SS) in Shigella bacteria. The researchers aim to purify and obtain soluble MxiK to allow for structural and protein interaction studies. Recombinant expression of MxiK in E. coli results in insoluble inclusion bodies. The researchers develop a purification method involving washing, denaturing, and nickel affinity chromatography to obtain highly purified denatured MxiK. However, extensive refolding attempts failed to obtain properly refolded soluble MxiK. In contrast, the closely related homolog PscK from Pseudomonas aeruginosa can be recombinantly expressed in a soluble form.
This document discusses using genomics, proteomics, and phenomics approaches to study plant physiology and improve traits like drought and heat tolerance. It provides examples of research using comparative genomics to identify drought tolerance genes in chickpeas. It also discusses using RNA interference to improve drought tolerance in rice by disrupting squalene synthase. Additionally, it examines improving the catalytic rate of Rubisco in rice through incorporating sorghum small subunits. The document concludes that integrating crop physiology with genomics, proteomics and phenomics can help develop climate-resilient crops.
Genomics and its application in crop improvementKhemlata20
meaning ,definition of genome ,genomics ,tools of genomics ,what is genome sequencing ,methods of genome sequencingand genome mapping ,advantage of genomics over traditional breeding program, examples of some crops whose genome has been sequenced, important points about genomics, work in the field of genomics ,applications of genomics .classification of genomics .different Omics in genomics like Proteomics ,Transcriptomics ,Metabolomics ,Need of genome sequencing
This study developed a new model using Drosophila melanogaster larvae to study chemotherapy-induced peripheral neuropathy (CIPN). The goals were to examine the effects of Taxol and Cisplatin chemotherapy drugs on mitochondrial dynamics and function, and determine if pretreatment with another drug could prevent the effects of chemotherapy. Behavioral assays showed that as drug concentration increased, motor and sensory function decreased and mortality rose. Imaging of larval neurons found that the chemotherapy drugs reduced mitochondrial movement, especially at higher Cisplatin doses. The results provide preliminary evidence that this model could help uncover mechanisms of CIPN and identify potential treatments.
The document summarizes a computational modeling approach for simulating synthetic microbial biofilms at a multiscale level. The approach combines 3D biophysical models of individual cells with models of genetic regulation and intercellular signaling. It was implemented in a software tool called CellModeller that uses parallel GPU computing to simulate over 30,000 cells in a typical biofilm colony within 30 minutes. Simulation results reproduced key features of experimentally observed E. coli biofilm colony morphologies. The modeling framework provides a way to predict the behavior of synthetic biofilms prior to experimental construction.
Optimization of Bacillus Subtilis Natto Immobilization Process on Alginate – ...inventionjournals
Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. In this study, Bacillus subtilis natto were immobilized in the alginate – chitosan complex for fermentation of nattokinase enzyme. Six factors affecting the efficiency of immobilization cells were screened by Plackett – Burman design including: concentration of alginate, concentration of chitosan, pH of chitosan, concentration of CaCl2, added cells density, shaking time after supplementing chitosan. Results of optimization have identified two factors affecting the efficiency of cell immobilization. They are concentration of alginate (2.5%) and added cells density (approximately 5.86 million colonies per milliliter). With these two factors optimized and others kept at the normal level, immobilization efficiency reached 90.73%. After Bacillus subtilis natto had been immobilized by optimization of parameters, we conducted application for fermenting nattokinase. For 24 hours of fermentation, nattokinase enzyme activity reached 71.80 ± 0.19 FU/ml. Immobilized Bacillus subtilis natto cells were reused 6 times and on the 6 th time of reuse, nattokinase enzyme activity only decreased 2.7% in compared with the 1st reuse.
This document summarizes Victoria Virador's presentation on engineering functional 3-D tissue models. It discusses using 3D tissue models to improve drug development and testing. Some key points include: (1) The drug development pipeline is slow, taking 9+ years and $1-2 billion; (2) 3D tissue models, like spheroids and organoids, can better model human physiology compared to traditional 2D cell cultures; (3) These models are being used for disease modeling, predictive toxicology screening, and angiogenesis research to aid drug development. The document provides several examples of 3D tissue models, including for skin, cancer, and validating model responses to toxic insults.
Mechanical stimulation plays an important role in ligament growth and maintenance, but the optimal stimulation parameters were previously unknown. This study used engineered ligament constructs from human donors to identify stretching parameters that maximize collagen production, as measured by ERK phosphorylation. A statistical model found that stretching at 0.9 Hz, 3% strain, 17%/s rate, and 5 minutes duration optimized ERK phosphorylation. This combination was validated in a second group of constructs. However, response varied between donors, and constructs from clots versus ligaments of one donor differed, highlighting the importance of cell source for engineered tissue phenotype. More research is needed to validate the model across diverse cell populations.
Historically, genetic toxicology has been comprised of bacterial and cell based in vitro assays such as the Ames assay (a bacterial mutagenicity assay), Micronucleus and Chromosomal Aberration assays (mammalian cytogenetic assays), and Mouse Lymphoma Assay (in vitro mammalian cell gene mutation assay). These were routinely used for safety evaluation and are still part of the standard core battery. The emergence of new technologies has facilitated the development of in vitro methods for safe and effective drug and chemical testing.
This BioReliance® toxicology services webinar will explore alternative models, including 3D skin models that comply with the EC Scientific Committee on Consumer Safety (SCCS) recommendations. It will also discuss how the 3Rs (Replace, Reduce, Refine) Principle advocates the exploration of such alternative methods while achieving required goals.
In this webinar, you will learn:
• About in vitro alternatives to animal toxicity testing in pharma, chemical, tobacco, and personal care products.
• How the 3Rs (Replace, Reduce, Refine) Principle advocates exploring alternative methods without compromising the required goals.
• Alternatives to comply with the 7th Amendment to the EC Cosmetics Directive.
This document describes a study that synthesized Fe3AlO6 nanoparticles using a soft chemical approach and characterized their structure and properties. It then assessed the cytotoxicity of the nanoparticles in human neural stem cells (hNSCs) through several assays. Specifically, Fe3AlO6 nanoparticles were synthesized via hydrolysis of a single-source molecular precursor under controlled conditions. X-ray diffraction and transmission electron microscopy confirmed the formation of monophasic nanocrystalline particles around 16.5 nm in size. Cytotoxicity assays including MTT, LDH and FDA were performed on hNSCs treated with varying concentrations of the nanoparticles. Scanning electron microscopy was also used to examine changes in cell morphology and nanoparticle distribution within
This document describes the development of a novel fluorescent protein-based sensor for detecting 2-oxoglutarate (2OG) levels in living cells. The sensor, termed mOGsor, was created by inserting the 2OG-binding domain GAF from the NifA protein into yellow fluorescent protein (YFP). mOGsor exhibits increased fluorescence intensity upon binding to 2OG in a concentration-dependent manner. Testing showed mOGsor has high specificity for 2OG and fast kinetics. Using mOGsor, the authors were able to monitor real-time changes in 2OG levels in E. coli cells under different nutrient conditions. mOGsor represents an improvement over previous FRET-based 2OG sensors by providing a
This study aimed to identify genes involved in tellurate reduction in the Enterobacter strain HRSW using transposon mutagenesis. The researchers obtained several mutants that lost the ability to reduce tellurate to elemental tellurium. They are currently locating the sites of transposon insertion and determining the identity of the genes required for tellurate reduction. Elucidation of the mechanism behind microbial tellurate reduction could improve the ability to predict how microorganisms interact with tellurium contaminants in the environment.
TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
Tilling and Ecotilling High throughput discovery of SNP variationFAO
Tilling and Ecotilling are reverse genetics techniques for discovering single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) in mutagenized plant populations. Tilling involves creating a mutagenized library, using locus-specific PCR and an endonuclease like CELI to detect mutations compared to a reference sequence. Ecotilling detects natural variation among individuals. Both techniques have been used in many species to discover novel genetic diversity and rare haplotypes. High throughput methods like DArT arrays can also discover polymorphisms across many individuals and loci simultaneously.
1) Transgenic fish models carrying bacteriophage λ and plasmid pUR288 vectors were developed to improve methods for assessing health risks from environmental mutagens and establish new animal models for studying in vivo mutagenesis.
2) The bacteriophage λ transgenic medaka model uses the cII and lacI genes as mutational targets, allowing detection of mutations through packaging of the phage vector and infection of E. coli. Spontaneous mutation frequencies in medaka were comparable to rodent models.
3) Exposure to chemical mutagens like ENU induced concentration-dependent, tissue-specific, and time-dependent increases in cII mutations, demonstrating the utility of the transgenic fish model for studying mut
The researchers fabricated poly(ethylene glycol) hydrogel matrices of varying stiffness by exposing a pre-polymer solution to UV light for different time periods (2.5, 4, and 6 minutes). They characterized the matrices and seeded breast cancer cells (MDA-MB-231) on them to investigate how the cells interact with and respond to matrix stiffness. They found that inhibiting a protein called ROCK reduced the cancer cells' actin organization, adhesion, and proliferation on stiffer matrices. This suggests that matrix stiffness influences aggressive cancer cell behavior through ROCK-mediated pathways.
1) The document discusses various methods for industrial biotechnology including strain engineering techniques like adaptive evolution, genome shuffling, and MAGE that aim to generate genetic diversity in strains.
2) It also covers the potential for algae-based biofuels but notes the current high production costs and need for further research and strain optimization over 10-15 years before being commercially viable.
3) The techniques discussed generate diversity in strains to increase metabolic capacity for industrial applications, though each method has strengths and weaknesses in terms of complexity, targeting abilities, and other factors.
Nuclear stability refers to a nucleus being stable and not spontaneously emitting radioactivity. A nucleus is stable when the forces binding the protons and neutrons together are balanced. Nuclear stability is greatest for nuclei with even numbers of both protons and neutrons, making them less radioactive. Nuclei with odd numbers of protons and/or neutrons are less stable and more radioactive. The document provides examples of predicting which nucleus in a pair would be radioactive based on whether they have odd or even numbers of protons and neutrons.
This document outlines a marketing plan to promote U-Glove, a product that protects hands from germs. The plan's goal is to educate women ages 25-45 about germs and make U-Glove their obvious choice for protection. Research shows women are more likely to use social media platforms like Facebook and Instagram, so the plan focuses on creating engaging content for these channels. The key strategy is to develop a friendly "Inner BFF" character that communicates care, relevance and humor to appeal to women's instinctual fears and influence their decisions at places like gas stations. Phase 1 of the plan focuses on building followers through posts with facts about health and germs across different social media platforms.
The document describes a proposed solution to limit vehicle speeds and reduce traffic accidents. A team of co-founders plans to develop a small electronic device using GPS, sensors, and cloud computing to control vehicle speeds based on the road zone. It will divide Egypt's map into zones and limit speeds accordingly to improve safety. The solution aims to reduce accident deaths by helping drivers keep appropriate speeds. It has the potential to save both lives and money for vehicle owners and insurance companies.
Surge soda is making a comeback targeted towards gamers. The document outlines strategies to promote Surge by partnering with gaming content creators on YouTube and Twitch, sponsoring gaming events, and creating augmented reality and social media games that feature Surge cans as characters. The goal is to position Surge as the energizing drink of choice for gamers and revive interest in the soda among millennials nostalgic for the 1990s brand identity.
This document discusses using genomics, proteomics, and phenomics approaches to study plant physiology and improve traits like drought and heat tolerance. It provides examples of research using comparative genomics to identify drought tolerance genes in chickpeas. It also discusses using RNA interference to improve drought tolerance in rice by disrupting squalene synthase. Additionally, it examines improving the catalytic rate of Rubisco in rice through incorporating sorghum small subunits. The document concludes that integrating crop physiology with genomics, proteomics and phenomics can help develop climate-resilient crops.
Genomics and its application in crop improvementKhemlata20
meaning ,definition of genome ,genomics ,tools of genomics ,what is genome sequencing ,methods of genome sequencingand genome mapping ,advantage of genomics over traditional breeding program, examples of some crops whose genome has been sequenced, important points about genomics, work in the field of genomics ,applications of genomics .classification of genomics .different Omics in genomics like Proteomics ,Transcriptomics ,Metabolomics ,Need of genome sequencing
This study developed a new model using Drosophila melanogaster larvae to study chemotherapy-induced peripheral neuropathy (CIPN). The goals were to examine the effects of Taxol and Cisplatin chemotherapy drugs on mitochondrial dynamics and function, and determine if pretreatment with another drug could prevent the effects of chemotherapy. Behavioral assays showed that as drug concentration increased, motor and sensory function decreased and mortality rose. Imaging of larval neurons found that the chemotherapy drugs reduced mitochondrial movement, especially at higher Cisplatin doses. The results provide preliminary evidence that this model could help uncover mechanisms of CIPN and identify potential treatments.
The document summarizes a computational modeling approach for simulating synthetic microbial biofilms at a multiscale level. The approach combines 3D biophysical models of individual cells with models of genetic regulation and intercellular signaling. It was implemented in a software tool called CellModeller that uses parallel GPU computing to simulate over 30,000 cells in a typical biofilm colony within 30 minutes. Simulation results reproduced key features of experimentally observed E. coli biofilm colony morphologies. The modeling framework provides a way to predict the behavior of synthetic biofilms prior to experimental construction.
Optimization of Bacillus Subtilis Natto Immobilization Process on Alginate – ...inventionjournals
Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. In this study, Bacillus subtilis natto were immobilized in the alginate – chitosan complex for fermentation of nattokinase enzyme. Six factors affecting the efficiency of immobilization cells were screened by Plackett – Burman design including: concentration of alginate, concentration of chitosan, pH of chitosan, concentration of CaCl2, added cells density, shaking time after supplementing chitosan. Results of optimization have identified two factors affecting the efficiency of cell immobilization. They are concentration of alginate (2.5%) and added cells density (approximately 5.86 million colonies per milliliter). With these two factors optimized and others kept at the normal level, immobilization efficiency reached 90.73%. After Bacillus subtilis natto had been immobilized by optimization of parameters, we conducted application for fermenting nattokinase. For 24 hours of fermentation, nattokinase enzyme activity reached 71.80 ± 0.19 FU/ml. Immobilized Bacillus subtilis natto cells were reused 6 times and on the 6 th time of reuse, nattokinase enzyme activity only decreased 2.7% in compared with the 1st reuse.
This document summarizes Victoria Virador's presentation on engineering functional 3-D tissue models. It discusses using 3D tissue models to improve drug development and testing. Some key points include: (1) The drug development pipeline is slow, taking 9+ years and $1-2 billion; (2) 3D tissue models, like spheroids and organoids, can better model human physiology compared to traditional 2D cell cultures; (3) These models are being used for disease modeling, predictive toxicology screening, and angiogenesis research to aid drug development. The document provides several examples of 3D tissue models, including for skin, cancer, and validating model responses to toxic insults.
Mechanical stimulation plays an important role in ligament growth and maintenance, but the optimal stimulation parameters were previously unknown. This study used engineered ligament constructs from human donors to identify stretching parameters that maximize collagen production, as measured by ERK phosphorylation. A statistical model found that stretching at 0.9 Hz, 3% strain, 17%/s rate, and 5 minutes duration optimized ERK phosphorylation. This combination was validated in a second group of constructs. However, response varied between donors, and constructs from clots versus ligaments of one donor differed, highlighting the importance of cell source for engineered tissue phenotype. More research is needed to validate the model across diverse cell populations.
Historically, genetic toxicology has been comprised of bacterial and cell based in vitro assays such as the Ames assay (a bacterial mutagenicity assay), Micronucleus and Chromosomal Aberration assays (mammalian cytogenetic assays), and Mouse Lymphoma Assay (in vitro mammalian cell gene mutation assay). These were routinely used for safety evaluation and are still part of the standard core battery. The emergence of new technologies has facilitated the development of in vitro methods for safe and effective drug and chemical testing.
This BioReliance® toxicology services webinar will explore alternative models, including 3D skin models that comply with the EC Scientific Committee on Consumer Safety (SCCS) recommendations. It will also discuss how the 3Rs (Replace, Reduce, Refine) Principle advocates the exploration of such alternative methods while achieving required goals.
In this webinar, you will learn:
• About in vitro alternatives to animal toxicity testing in pharma, chemical, tobacco, and personal care products.
• How the 3Rs (Replace, Reduce, Refine) Principle advocates exploring alternative methods without compromising the required goals.
• Alternatives to comply with the 7th Amendment to the EC Cosmetics Directive.
This document describes a study that synthesized Fe3AlO6 nanoparticles using a soft chemical approach and characterized their structure and properties. It then assessed the cytotoxicity of the nanoparticles in human neural stem cells (hNSCs) through several assays. Specifically, Fe3AlO6 nanoparticles were synthesized via hydrolysis of a single-source molecular precursor under controlled conditions. X-ray diffraction and transmission electron microscopy confirmed the formation of monophasic nanocrystalline particles around 16.5 nm in size. Cytotoxicity assays including MTT, LDH and FDA were performed on hNSCs treated with varying concentrations of the nanoparticles. Scanning electron microscopy was also used to examine changes in cell morphology and nanoparticle distribution within
This document describes the development of a novel fluorescent protein-based sensor for detecting 2-oxoglutarate (2OG) levels in living cells. The sensor, termed mOGsor, was created by inserting the 2OG-binding domain GAF from the NifA protein into yellow fluorescent protein (YFP). mOGsor exhibits increased fluorescence intensity upon binding to 2OG in a concentration-dependent manner. Testing showed mOGsor has high specificity for 2OG and fast kinetics. Using mOGsor, the authors were able to monitor real-time changes in 2OG levels in E. coli cells under different nutrient conditions. mOGsor represents an improvement over previous FRET-based 2OG sensors by providing a
This study aimed to identify genes involved in tellurate reduction in the Enterobacter strain HRSW using transposon mutagenesis. The researchers obtained several mutants that lost the ability to reduce tellurate to elemental tellurium. They are currently locating the sites of transposon insertion and determining the identity of the genes required for tellurate reduction. Elucidation of the mechanism behind microbial tellurate reduction could improve the ability to predict how microorganisms interact with tellurium contaminants in the environment.
TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
Tilling and Ecotilling High throughput discovery of SNP variationFAO
Tilling and Ecotilling are reverse genetics techniques for discovering single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) in mutagenized plant populations. Tilling involves creating a mutagenized library, using locus-specific PCR and an endonuclease like CELI to detect mutations compared to a reference sequence. Ecotilling detects natural variation among individuals. Both techniques have been used in many species to discover novel genetic diversity and rare haplotypes. High throughput methods like DArT arrays can also discover polymorphisms across many individuals and loci simultaneously.
1) Transgenic fish models carrying bacteriophage λ and plasmid pUR288 vectors were developed to improve methods for assessing health risks from environmental mutagens and establish new animal models for studying in vivo mutagenesis.
2) The bacteriophage λ transgenic medaka model uses the cII and lacI genes as mutational targets, allowing detection of mutations through packaging of the phage vector and infection of E. coli. Spontaneous mutation frequencies in medaka were comparable to rodent models.
3) Exposure to chemical mutagens like ENU induced concentration-dependent, tissue-specific, and time-dependent increases in cII mutations, demonstrating the utility of the transgenic fish model for studying mut
The researchers fabricated poly(ethylene glycol) hydrogel matrices of varying stiffness by exposing a pre-polymer solution to UV light for different time periods (2.5, 4, and 6 minutes). They characterized the matrices and seeded breast cancer cells (MDA-MB-231) on them to investigate how the cells interact with and respond to matrix stiffness. They found that inhibiting a protein called ROCK reduced the cancer cells' actin organization, adhesion, and proliferation on stiffer matrices. This suggests that matrix stiffness influences aggressive cancer cell behavior through ROCK-mediated pathways.
1) The document discusses various methods for industrial biotechnology including strain engineering techniques like adaptive evolution, genome shuffling, and MAGE that aim to generate genetic diversity in strains.
2) It also covers the potential for algae-based biofuels but notes the current high production costs and need for further research and strain optimization over 10-15 years before being commercially viable.
3) The techniques discussed generate diversity in strains to increase metabolic capacity for industrial applications, though each method has strengths and weaknesses in terms of complexity, targeting abilities, and other factors.
Nuclear stability refers to a nucleus being stable and not spontaneously emitting radioactivity. A nucleus is stable when the forces binding the protons and neutrons together are balanced. Nuclear stability is greatest for nuclei with even numbers of both protons and neutrons, making them less radioactive. Nuclei with odd numbers of protons and/or neutrons are less stable and more radioactive. The document provides examples of predicting which nucleus in a pair would be radioactive based on whether they have odd or even numbers of protons and neutrons.
This document outlines a marketing plan to promote U-Glove, a product that protects hands from germs. The plan's goal is to educate women ages 25-45 about germs and make U-Glove their obvious choice for protection. Research shows women are more likely to use social media platforms like Facebook and Instagram, so the plan focuses on creating engaging content for these channels. The key strategy is to develop a friendly "Inner BFF" character that communicates care, relevance and humor to appeal to women's instinctual fears and influence their decisions at places like gas stations. Phase 1 of the plan focuses on building followers through posts with facts about health and germs across different social media platforms.
The document describes a proposed solution to limit vehicle speeds and reduce traffic accidents. A team of co-founders plans to develop a small electronic device using GPS, sensors, and cloud computing to control vehicle speeds based on the road zone. It will divide Egypt's map into zones and limit speeds accordingly to improve safety. The solution aims to reduce accident deaths by helping drivers keep appropriate speeds. It has the potential to save both lives and money for vehicle owners and insurance companies.
Surge soda is making a comeback targeted towards gamers. The document outlines strategies to promote Surge by partnering with gaming content creators on YouTube and Twitch, sponsoring gaming events, and creating augmented reality and social media games that feature Surge cans as characters. The goal is to position Surge as the energizing drink of choice for gamers and revive interest in the soda among millennials nostalgic for the 1990s brand identity.
This document outlines a marketing strategy called "MixTape" for Corona Light beer. The strategy aims to associate Corona Light with cool, new music to appeal to "Self-Upgraders" ages 25-44 who seek new trends. It involves three phases: 1) Build credibility by featuring new bands on social media and playlists. 2) Encourage participation using hashtags. 3) Host exclusive live music sessions that are streamed online. The goal is to position Corona Light as a brand relevant to these consumers and increase loyalty.
Nuclear stability refers to a nucleus being stable and not spontaneously emitting radioactivity. A nucleus is stable when the forces binding the protons and neutrons together are balanced. Nuclei with an even number of both protons and neutrons are generally the most stable, while those with odd numbers of both protons and neutrons are the least stable and most radioactive. The nuclear stability can be predicted using the even-odd rule, where nuclides with even-even or odd-even combinations are more stable than odd-odd nuclides.
This study characterized the structural features of a glycoprotein (SJGP) isolated from the brown alga Saccharina japonica and evaluated its effects on probiotic properties of Lactobacillus plantarum. SJGP was found to contain various amino acids and monosaccharides. Pretreatment of L. plantarum with SJGP increased its adhesion, auto-aggregation, and growth in Caco-2 cells. SJGP also increased the auto-aggregation and hydrophobicity of L. plantarum. Adhesion genes in L. plantarum showed upregulated expression with SJGP. Therefore, SJGP improves the probiotic properties of L. plantarum and could potentially be used to develop functional foods.
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
Synergism effect between inoculum size and aggregate size on flavonoid produc...eSAT Journals
Abstract The present study aimed to investigate the effect of different culture conditions on biomass content and flavonoid production of the elite C. asiatica accession UPM03. When 0.1 g inoculum 25 mL-1 of cell was grown in Murashige & Skoog (MS) medium supplemented with 2 mg 2,4-D l-1 and 1 mg kinetin l-1, flavonoids were not significantly produced in cells or released into the culture medium. Production of flavonoid was correlated with the aggregation size and inoculum density. At aggregate size of 250-500 μm with 0.3 g inoculum density 25 mL-1, it gave the highest luteolin content with 35.45 μg g-1 DW. After investigating the effect of culture conditions, i.e. pH, inoculum density, light irradiation and plant growth regulator, we found that with the optimized condition (i.e. 250-500 μm aggregate size, 0.5 g wet cell 25 mL-1 supplemented with 3 mg L-1 2,4-D and 1 mg L-1 kinetin at pH 5.7 under 16h photoperiod) the luteolin content was 11-fold higher than the cell suspension cultured at an inoculum size of 0.3 g wet cell 25 mL-1 with 250-500 μm aggregate size. Index Terms: Centella asiatica, flavonoid, light irradiation, pH regime, plant growth regulators .
The document describes a high-throughput screen that identified small molecule compounds that can enhance the pharmacological effects of oligonucleotides. Several "hit" compounds were discovered that potentiated the actions of splice-switching oligonucleotides, antisense oligonucleotides, and siRNAs in cell culture. The hit compounds preferentially caused the release of fluorescent oligonucleotides from late endosomes. Studies in transgenic mice indicated the hit compounds could enhance the in vivo effects of a splice-switching oligonucleotide without significant toxicity. The findings suggest selected small molecules may help advance oligonucleotide-based therapeutics by modulating intracellular trafficking and endosomal release.
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
This document describes a method for fabricating porous poly(DL-lactic-co-glycolic acid) (PLGA) microspheres that fuse together at body temperature to form solid porous scaffolds, creating an injectable scaffold system. The microspheres were treated with ethanolic sodium hydroxide to increase surface porosity without disintegrating. When mixed with media and incubated at 37°C, the microspheres fused to form scaffolds with compressive strength of 0.9 MPa, porosity of 81.6%, and pore diameter of 54 μm, supporting NIH-3T3 cell attachment and proliferation in vitro. This study demonstrates a technique for producing an injectable and porous PLGA scaffold that solid
In the LaboratoryJChemEd.chem.wisc.edu • Vol. 76 No. 9 .docxbradburgess22840
In the Laboratory
JChemEd.chem.wisc.edu • Vol. 76 No. 9 September 1999 • Journal of Chemical Education 1283
Determination of Myoglobin Stability
by Visible Spectroscopy
Paul A. Sykes, Harn-Cherng Shiue, Jon R. Walker, and Robert C. Bateman Jr.*
Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS 39406-5043;
*[email protected]
Proteins are biopolymers that fold spontaneously into a
well-defined three-dimensional structure (1–5). The stability of
these intricate structures in solution is usually measured by
their resistance to denaturation in the presence of either heat
or chemical denaturants. These demonstrations of protein
stability are rarely encountered in the biochemistry teaching
laboratory despite publications in this Journal of denaturation
experiments involving chymotrypsin (6 ) and ribonuclease
(7 ). Both of those studies used as a denaturant the guani-
dinium ion, which increases the solubility of both polar and
nonpolar amino acid side chains and thereby reduces the hydro-
phobic effect on protein stability (8–10). The chymotrypsin
experiment is noteworthy in that both protein conformation
and enzyme activity are monitored simultaneously, directly
illustrating the structure–function relationship. However, the
protein conformation is monitored by intrinsic fluorescence,
a technique that may be difficult for a modestly equipped
teaching laboratory to utilize. The ribonuclease unfolding
experiment is monitored in the ultraviolet (287 nm) where the
total absorbance change upon denaturation is very small and
difficult to measure accurately. The simplest procedure would
be to utilize a visible protein that undergoes a significant
change in color intensity upon denaturation. This would allow
the denaturation to be followed with an inexpensive spectro-
photometer or possibly even a colorimeter. In our opinion,
the best candidate for such a protein is myoglobin.
Myoglobin (Mr 16,700) is a simple monomeric oxygen-
binding protein found within muscle cells. This protein’s
crystal structure was the first elucidated and its reversible
unfolding has been well studied (11, 12). Because of their
obvious historical and biological significance, myoglobin and
hemoglobin are discussed extensively in most biochemistry
texts. Myoglobin contains one polypeptide chain of 153
residues and a heme prosthetic group. The heme group is
buried in a hydrophobic pocket within the protein’s interior.
This interaction of the heme with a structural feature of the
protein results in the Soret band, a strong absorbance peak in
the visible spectrum at 409 nm for myoglobin. Upon dena-
turation of myoglobin, a decrease in absorbance at 409 nm
occurs owing to the exposure of heme to the polar aqueous
solvent (11–13). This denaturation is easily and reproducibly
measured in the teaching laboratory either by fluorescence
spectroscopy, as described in a recent review of protein folding
by Jones (13), or by visible spect.
Abstract book for the following conferences:
2013 2nd International Conference on Bioinformatics and Biomedical Science (ICBBS 2013)
2013 2nd International Conference on Environment, Energy and Biotechnology (ICEEB 2013)
2013 2nd International Conference on Chemical and Process Engineering (ICCPE 2013)
2013 2nd Journal Conference on Environmental Science and Development (JCESD 20132nd)
The conferences was held at Concorde Inn Kuala Lumpur International Airport, Malaysia on 09 June 2013
This thesis investigated using ellipsometry to analyze ligand binding to G-protein coupled receptors (GPCRs). GPCRs are important cell surface proteins linked to many diseases. Ellipsometry is an optical technique that can quantify ligand-receptor interactions by measuring changes at a surface when polarized light is reflected off. An A549 epithelial cell line expressing the CXCR4 GPCR and its ligand CXCL12 was used. Enzyme-linked immunosorbent assays were performed to determine optimal ligand and antibody concentrations. Baseline characterization of glass slides and binding experiments with ligand and antibody were conducted using ellipsometry. Psi-delta analysis of collected data showed trends in binding.
This study investigated whether S-methylcysteine (SMC), a metabolite of monohalomethanes, contributes to their neurotoxicity. The researchers found that:
1) High concentrations of SMC (10-2 M) reduced synaptic responses in hippocampal slices, an effect that was partially reversible.
2) In organotypic hippocampal cultures, 24 hour exposure to 5x10-5 M SMC compromised membrane integrity in the dentate gyrus, while lower concentrations increased population spike amplitudes and repetitive discharges without affecting membrane integrity.
3) In dissociated hippocampal neurons, SMC reduced GABA-induced currents, acting as a competitive GABAA receptor antagonist with
This document discusses materials and methods used in a study involving the chemical fipronil and zinc. Twenty male albino rats were divided into four groups of five rats each: a control group, a zinc group that received zinc supplementation, a fipronil group exposed to the insecticide fipronil, and a combination group exposed to both zinc and fipronil. Biochemical assays were conducted to assess oxidative stress markers like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione, lipid peroxidation, and total protein in the rats. Chemicals used including fipronil and zinc sulfate were obtained from reputable suppliers. Kits for the biochemical assays were purchased from a diagnostic
Tissue engineering uses scaffolds, cells, and signaling molecules to regenerate tissues and organs. Scaffolds provide a structure for cell attachment, growth, and tissue formation. Natural polymers like collagen and hyaluronic acid, and synthetic polymers like poly-lactic-co-glycolic acid are commonly used as scaffold materials. Scaffolds can be fabricated using various methods including freeze drying, electrospinning, 3D printing, and textile technologies to produce scaffolds with desirable properties like porosity and pore size for tissue growth. Scaffolds seeded with stem cells or tissue-specific cells aim to repair and regenerate tissues for applications in skin, bone, cartilage, and other organs.
Multiorgan Microdevices for ADME Evaluatio and Drug Design:-
Multi-organ micro-devices are microfluidic devices that gives the information of human metabolism by connecting the fluidic streams from several on-chip in vitro tissue cultures with each other in a physiologically relevant manner. Multi-organ micro-devices can predict tissue-tissue interactions that occur as a result of metabolite travel from one tissue to other tissues in vitro. These systems are capable of simulating human metabolism, including the conversion of a pro-drug to its effective metabolite as well as its subsequent active metabolite and toxic side effects. Since tissue-tissue interactions in the human body can play a significant role in determining the success of new pharmaceuticals, the development and use of multi-organ micro-devices present an opportunity to improve the drug development process. The devices have the potential to predict potential toxic side effects with higher accuracy before a drug enters the expensive and time consuming phase of clinical trials. Further, when operated with human biopsy samples, the devices could be a way for the development of individualized medicine. Since single organ devices are testing platforms for tissues that can later be combined with other tissues within multi-organ devices, we will also mention single organ devices where appropriate in the discussion those seems large area of interest in current research for individualized medicine and drug resistance study.
This document discusses several microfluidic separation methods for isolating circulating tumor cells (CTCs) from blood. It describes how microfluidics can accurately manipulate flow conditions to efficiently separate CTCs from blood cells based on differences in their biophysical properties such as size and deformability. Using these microfluidic approaches, viable CTCs can be retrieved from cancer patient blood samples with high isolation efficiency and purity. Identification of CTCs aids in cancer detection, disease monitoring, and insights into metastasis. The document also discusses using magnetic nanoparticles coupled with doxorubicin chemotherapy drug and an external magnetic field to more effectively deliver the drug to breast cancer cells and increase mortality rates.
This document describes the development of a novel growth factor delivery system using poly(lactic-co-glycolic acid) (PLGA) microparticles. The inclusion of a hydrophilic PLGA-PEG-PLGA triblock copolymer alters the release kinetics from the microparticles such that growth factor release can occur before polymer degradation. Three formulations are identified as promising candidates for delivering growth factors like BMP-2, with adjustable release profiles from 4 days to over 4 weeks. Mixing microparticles of different formulations provides another method to control release kinetics. This customized, localized delivery system has the potential to improve the efficacy and safety of recombinant growth factor therapies.
The interaction of QDs with RAW264.7 cells_ nanoparticle quantification, upta...Olga Gladkovskaya
This document summarizes a study that investigated the interaction of quantum dots (QDs) with RAW264.7 macrophage cells. Specifically, it quantified QD uptake kinetics over time using flow cytometry, studied the effect on cell function and viability, and examined immune responses. Small, green-emitting CdTe QDs with diameters of 2.1 nm were incubated with RAW264.7 cells. Uptake kinetics were quantified and optimal parameters like concentration and exposure time were identified. Cell viability, intracellular fluorescence, and inflammatory marker expression were analyzed at 12 and 24 hours. The QDs were quickly ingested and accumulated in cells, making them suitable for short-term assays, though longer term effects require further study.
Combined effects of PEGylation and particle size on uptake of PLGA particles ...Nanomedicine Journal (NMJ)
Abstract
Objective:
At the present study, relationship between phagocytosis of PLGA particles and combined effects of particle size and surface PEGylation was investigated.
Materials and Methods:
Microspheres and nanospheres (3500 nm and 700 nm) were prepared from three types of PLGA polymers (non-PEGylated and PEGylation percents of 9% and 15%). These particles were prepared by solvent evaporation method. All particles were labeled with FITC-Albumin. Interaction of particles with J744.A.1 mouse macrophage cells, was evaluated in the absence or presence of 7% of the serum by flowcytometry method.
Results:
The study revealed more phagocytosis of nanospheres. In the presence of the serum, PEGylated particles were phagocytosed less than non-PEGylated particles. For nanospheres, this difference was significant (P<0/05) and their uptake was affected by PEGylation degree. In the case of microsphere formulation, PEGylation did not affect the cell uptake. In the serum-free medium, the bigger particles had more cell uptake rate than smaller ones but the cell uptake rate was not influenced by PEGylation.
Conclusion:
The results indicated that in nanosized particles both size and PEgylation degree could affect the phagocytosis, but in micron sized particles just size, and not the PEGylation degree, could affect this.
This document summarizes research on modifying bacterial cellulose through the addition of hyaluronic acid and gelatin during fermentation to produce novel nanocomposites for use as biomaterials in stem cell therapy. Transmission infrared spectroscopy and scanning electron microscopy showed the influence of hyaluronic acid and gelatin on the bacterial cellulose structure. Cell viability studies with human dental pulp stem cells demonstrated higher cell adhesion to bacterial cellulose scaffolds containing hyaluronic acid or gelatin over time. Confocal microscopy confirmed cell adhesion and distribution within the scaffold fibers. This research presents a new approach for developing natural nanocomposites using bacterial cellulose as scaffolds for regenerative medicine applications.
Jonathan Lendrum, Dean's Distinguished Research Fellowship ApplicationJon Lendrum
The document summarizes an undergraduate research proposal that evaluates mouse sleep behavior and the function of the glymphatic system in response to manipulations of gut microbiota composition. The study involves orally administering antibiotics, probiotics, or a saline buffer to mice over two weeks. Sleep duration will be measured using pressure sensors. After treatment, a fluorescent tracer will be infused into mice to map the glymphatic system's waste clearance, allowing comparison between treatment groups. The researcher hypothesizes that antibiotics will impair waste clearance while probiotics will enhance it.
Jonathan Lendrum, Dean's Distinguished Research Fellowship Application
nihms-20590
1. Non-Invasive Evaluation of Alginate/Poly-L-lysine/Alginate
Microcapsules by Magnetic Resonance Microscopy
Ioannis Constantinidis1,2,*, Samuel C. Grant2,3,4,#,*, Susanne Celper5, Isabel Gauffin-
Holmberg5, Kristina Agering5, Jose A. Oca-Cossio1, Jonathan D. Bui4, Jeremy Flint3,4,
Christine Hamaty1, Nicholas E. Simpson1, and Stephen J. Blackband2,3,4
1 Division of Endocrinology, Department of Medicine, University of Florida, Gainesville, FL, 32610
2 National High Magnetic Field Laboratory, Tallahassee, FL, 32310
3 Department of Neuroscience, University of Florida, Gainesville, FL, 32610
4 McKnight Brain Institute, University of Florida, Gainesville, FL, 32610
5 Royal Institute of Technology and the Royal Karolinska Medical and Surgical Institute, Stockholm, Sweden.
Abstract
In this report, we present data to demonstrate the utility of 1H MR microscopy to noninvasively
examine alginate/poly-L-lysine/alginate (APA) microcapsules. Specifically, high-resolution images
were used to visualize and quantify the poly-L-lysine (PLL) layer, and monitor temporal changes in
the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/
PLL layer was quantified to be 40.6±6.2 μm regardless of the alginate composition used to generate
the beads or the time of alginate/PLL interaction (2, 6, or 20 minutes). However, there was a notable
difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate
and the alginate/PLL interaction time. The T2 relaxation time and the apparent diffusion coefficient
(ADC) of the alginate matrix were measured periodically throughout the month long culture period.
Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease
in T2 over the duration of the experiment, while ADC was unaffected. This decrease in T2 is attributed
to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a
regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease
in T2 was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial
alginate microstructure. Conversely, alginate beads generated with a high mannuronic content
alginate (with or without a PLL layer) did not display temporal changes in either T2 or ADC. This
observation suggests that the microstructure of high mannuronic content alginate beads is less
susceptible to culture conditions.
Keywords
MR imaging; alginate encapsulation; poly-L-lysine
Author to whom correspondence should be addressed: Ioannis Constantinidis Division of Endocrinology Department of Medicine PO
Box 100226 University of Florida Gainesville, FL, 32610-0226 consti@medicine.ufl.edu.
*Both authors have contributed equally to this study.
#Current address: Department of Chemical & Biomedical Engineering, The Florida State University & National High Magnetic Field
Laboratory, Tallahassee, FL 32310.
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NIH Public Access
Author Manuscript
Biomaterials. Author manuscript; available in PMC 2008 May 1.
Published in final edited form as:
Biomaterials. 2007 May ; 28(15): 2438–2445.
NIH-PAAuthorManuscriptNIH-PAAuthorManuscriptNIH-PAAuthorManuscript
2. INTRODUCTION
Evaluation of tissue engineered constructs by non-invasive imaging techniques can focus on
the cellular component of the constructs - providing information critical to their function - or
the biomaterials utilized in making the constructs - providing information critical to their
structural integrity. Both tasks are equally important and synergistic to understanding the
structure/function relationship of a construct under examination. Nuclear Magnetic Resonance
(NMR) is a versatile, non-invasive modality with a range of active nuclei and contrast
mechanisms that has the ability to provide both metabolic and structural information. Although
the use of NMR in medical and biological research is widespread, its application to tissue
engineering has been limited [1-3].
In the development of a bioartificial pancreas, most studies employing NMR techniques have
focused on 31P and 1H NMR spectroscopy to investigate the correlation between cellular
metabolism and insulin secretion under various environmental conditions [4-8]. One of the
conclusions derived by 1H NMR was that the resonance at 3.20 ppm, attributed to the
trimethylamine protons of choline and its phosphorylated mono- and diesters, was sensitive to
the overall metabolic activity of encapsulated cells maintained within a perfusion bioreactor
[8]. This resonance was subsequently utilized to quantify viable cell numbers within a disk-
shaped pancreatic construct both in vitro and in vivo [9,10]. Although these studies provide
invaluable information on the cellular function of the construct, they do not offer insight into
potential structural changes of the biomaterials utilized.
The ability to monitor the structural integrity of a tissue engineered construct either in vitro or
in vivo is critical in evaluating or predicting its structural demise. This assessment is particularly
important for alginate-based constructs that have been known to deteriorate with time. Alginate
is a naturally occurring biopolymer that has been used extensively as a vehicle to encapsulate
a variety of biological materials including enzymes and cells of both microbial and mammalian
origin. However, its long-term structural integrity is questioned given the soft gelatinous nature
of the material. In tissue engineering, and particularly in the development of a bioartificial
pancreas, alginates have been used to encapsulate islets [11-14] and transformed β-cells [4,
15-17] with considerable success. A layer of a polycation, such as poly-L-lysine (PLL),
followed by an additional layer of alginate is commonly used to coat the central alginate matrix,
providing mechanical stability to the matrix [18] and at least partial immunoprotection [19].
MR imaging at microscopic resolutions (<100 μm), commonly referred to as MR Microscopy,
has been used to monitor alginate beads [20-25] immediately following encapsulation or
shortly thereafter, but has not been used to track alginate beads during a long-term culture.
Because changes in the T2 relaxation time have been shown to correlate with changes in
material porosity [26], we postulated that MR microscopy can be used to examine alginate/
poly-L-lysine/alginate (APA) microcapsules integrity over time. In this report, 1H MR
microscopy was employed to (i) visualize and quantify the thickness of the PLL layer by taking
advantage of the contrast generated by the interaction between alginate and PLL and (ii)
monitor temporal changes in alginate gel microstructure by quantifying the MR properties of
the alginate matrix.
METHODS
Alginate Microcapsules
Two types of alginate were used in this study (FMC BioPolymer, Drammen, Norway): (i) a
high guluronic content alginate (MVG) composed of 73% guluronic acid (60% consecutive
guluronic residues) and 27% mannuronic acid (manufacturer's data); and (ii) a high mannuronic
content alginate (MVM) composed of 38% guluronic acid (20% consecutive guluronic
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3. residues) and 62% mannuronic acid (manufacturer's data). All alginate solutions were prepared
by dissolving powder alginate in physiological saline (0.85% NaCl) at a concentration of 2%
(w/v). The presence of non-crosslinking Na+ ions generates a more homogeneous gel bead
[27]. Alginate beads were generated with the aid of an electrostatic bead generator (Nisco,
Basel, Switzerland) by crosslinking alginate with 100 mM CaCl2. APA beads were produced
based on the protocol developed by Lim and Sun [13] and modified to suit our requirements
[16]. Briefly, alginate beads were washed with solutions of CaCl2, CHES, 0.05% PLL and
0.2% alginate (of the same type as that used for the core material) to create the final APA beads
at a diameter of 750±50 μm. The APA beads in this study were not treated with citrate to liquefy
the central alginate core.
To assess differences in the alginate/PLL interaction, two PLL polymers were tested; one of
low molecular weight (19,200 g/mol) and one of higher molecular weight (240,000 g/mol)
(Sigma, St. Louis, MO). Furthermore, alginate beads were exposed to a 0.05% PLL solution
for either 2, 6 or 20 minutes. Table 1 shows the various combinations of alginate composition,
PLL molecular weight and interaction times examined. From the time the beads were generated
to the end of the experiment, they were maintained in Dulbecco's Minimum Essential Medium
(DMEM; Sigma, St. Louis, MO).
Temporal effects due to a 30 days long in vitro culture on alginate gel microstructure were
assessed on both PLL coated and uncoated alginate beads composed of either MVG or MVM
alginate. For these longitudinal experiments, alginate beads were coated with the lower
molecular weight PLL using an exposure time of 6 minutes. The beads were maintained in a
fashion similar to the culture of encapsulated cells; the DMEM medium was changed with
fresh medium three times weekly. Because all experiments presented in this study were
performed with cell-free alginate beads, temporal changes reflect the effect of frequent media
switches, characteristic of in vitro cell culture, on the microstructure of the alginate gel without
the confounding effects of cell growth and/or metabolic activity, which may contribute to these
changes as well.
MR Microscopy
All MR images were acquired using a vertical 17.6-T 89-mm bore cryopumped magnet
equipped with a Bruker Avance console and Micro2.5 gradients (maximum strength of 1 T/
m). Three to six beads immersed in DMEM were loaded into a glass capillary (inner diameter,
1 mm) that was placed within a homebuilt solenoidal microcoil (6 mm length, 1.7 mm in
diameter). Coupled with the high magnetic field, these small RF solenoids greatly improve
the sensitivity of the NMR experiment and are susceptibility-matched to reduce magnetic field
perturbations [28].
The images presented here were acquired using a gradient recall echo (GRE) sequence with
averaging (NEX) = 4, echo time (TE) = 7.5, 15 and 25 ms, recovery time (TR) = 150 ms, matrix
(MTX) of 512×128×128, field of view (FOV) of 6.4×1.6×1.6 mm3. Thus, an isotropic spatial
resolution of 12.5 μm for each image was acquired in approximately 2.75 hrs. To assess the
contribution of T2* in the detection of the PLL layer, GRE images of high mannuronic content
APA beads were collected at variable TEs (4 - 40 msec) or variable bandwidths (15,000 -
100,000 Hz). These images were obtained by holding one of the two acquisition parameters
(i.e. TE and bandwidth) constant while varying the other. For these T2* assessments, APA
beads were composed of the lower molecular weight PLL and the time that the PLL solution
was allowed to interact with the alginate beads was 6 mins.
MR images were also acquired using a conventional spin-echo (SE) imaging sequence that
employed a bipolar read-refocusing gradient pair located after the second radio frequency pulse
to minimize the effect of unintended water diffusion resulting from the read gradient [29]. To
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4. quantify T2, separate images were acquired at echo times ranging from 15 to 120 ms. These
T2-weighted images were acquired in 25.5 min/image with NEX = 4, TR = 1.5 s, MTX =
512×256, field of view of 2.4×1.2 cm2 and slice thickness of 60 μm. Diffusion weighted images
were acquired at five diffusion weightings using the same spin echo sequence and by
incrementing the diffusion gradient strength (b values = 195, 507, 748, 977 and 1200 s/mm2).
All other imaging parameters remained constant between images. The nominal resolution of
both T2- and diffusion-weighted images was 47×47×60 μm3. Quantification of T2 relaxation
and the apparent diffusion coefficient (ADC) was performed based on a region of interest (ROI)
analysis. Circular ROIs encompassing the entire bead and the center of the bead were generated
and the average signal intensity over the entire ROI was measured. Based on these
measurements, the T2 relaxation time and the ADC were calculated by fitting signal intensity
as a function of TE or b value respectively using a non-linear Levenberg-Marquardt regression
analysis for a single decaying exponential function. The total acquisition time for quantitative
imaging experiments on a single bead preparation was 6.5 hrs.
Quantification of the PLL layer
The amount of PLL deposited on the alginate matrix was quantified by measuring the
concentration of the PLL solution before and after exposure to the alginate beads. Briefly,
samples of the PLL solution were collected before and after exposure with alginate beads,
lyophilized and dissolved in 100 μl of saline. Ten microliters of the resuspended solution were
placed in different wells within a 96-well-plate, and 100 μl of a fluorescamine (Sigma, St.
Louis, MO) solution (3 mg of fluorescamine per ml of DMSO) were added to each well. The
microwell plate was placed in a Synergy HT plate reader (Bio-Tek, Winooski, VT) and allowed
to incubate at 37 °C for 20 minutes. Fluorescence was measured using an excitation filter
centered at 360 nm with a bandwidth of 40 nm and an emission filter centered at 460 nm with
a bandwidth of 10 nm. The values measured from our samples were compared against a
calibration curve of known PLL concentrations. The amount of PLL deposited per ml of beads
was calculated by subtracting the measurements before and after exposure. Thus, when small
quantities of PLL are deposited on the beads, the error in this measurement is large. Each
measurement was performed twice and a t-test analysis was used to determine the statistical
significance of the results.
RESULTS
Visualization and Quantification of the PLL layer
Figure 1 illustrates SE images (TE = 25 and 65 ms) of four MVM beads. The bottom two beads
are coated with a PLL layer (lower molecular weight PLL with 6 minutes exposure time) while
the top two are not coated with PLL. The non-coated beads show a faint ring around the bead
attributed to a small susceptibility (T2*) effect induced by the gel/media interface. The PLL
coated beads show a dark circle at the periphery of the alginate matrix that clearly demarcates
the beads from the surrounding medium. Compared to the signal intensity of the (a) inner bead
and (b) external media, the PLL layer in Figure 1 shows a contrast enhancement of (a) 27±5%
and (b) 39±2%, respectively, for a TE = 25 ms and an enhancement of (a) 54±0.8% and (b) 70
±0.07% for a TE = 65 ms. For reference, the periphery of the uncoated beads in Figure 1 displays
a contrast enhancement of only 3.5±0.8 to 4.8±8.8 % compared to the interior of the bead.
These averages and standard deviations were determined based on n=7 measurements.
Additionally, the thickness of the observed layer did not increase by increasing either TE or
decreasing bandwidth and was the same for both SE and GRE images (data not shown). These
observations suggest that the contrast generated by the PLL layer is largely due to intrinsic
T2 relaxation with a small contribution by local field inhomogeneities. Conversely, the contrast
generated by the small air bubble located on the top PLL coated bead, is dominated by T2>*
as demonstrated by the increase in its size with increasing TE. However, it is possible that
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5. T2* contribution may be underestimated given the limited number of pixels across the PLL
layer.
Figure 2 shows GRE images illustrating differences in the alginate/PLL contrast as a function
of the alginate composition and the exposure time between alginate beads and the PLL solution.
Specifically, for alginate beads coated with the lower molecular weight PLL, the contrast due
to the PLL layer increased as the interaction time between alginate and PLL increased from 2
to 6 to 20 minutes. This effect was observed for both alginate compositions examined, but it
was more pronounced for beads generated with MVM alginate. However, when the high
molecular weight PLL polymer was used, there were no distinguishable differences in contrast
as a function of either interaction times or alginate composition. To quantify these alterations
in contrast, the signal-to-noise ratio (SNR) of the PLL layer was calculated for each exposure
time and echo time. For the images of Figure 2, the SNRs of the low molecular weight PLL
layer MVM beads were 23±0.6, 20±0.8 and 17±0.1 for exposure times of 2, 6 and 20 minutes,
respectively. These values correspond to a contrast enhancement relative to the minimal
exposure time of 12% and 26% with the longer PLL exposures. This enhancement also is
reflected in the calculated T2* coefficients for the low molecular weight PLL layer of the MVM
beads: 21.2, 20.2 and 16.6 ms for exposures of 2, 6 and 20 minutes, respectively. By
comparison, the high molecular weight PLL layer of the MVM beads of Figure 2 displayed
only an average contrast enhancement relative to the minimum exposure of 2.3±1.0% and
T2* coefficient of 18.1 ms. Meanwhile, the PLL enhancement seen in the MVG beads relative
to the minimum exposure improved by 3.8±0.1% and 8.6±0.02% with increasing exposure
times while the T2* coefficient was calculated as 24.0, 23.3 and 22.7 ms for PLL exposures of
2, 6 and 30 minutes, respectively. Clearly, though not evident with the high molecular weight
PLL, contrast enhancements could be identified and quantified for both alginates for the lower
molecular weight PLL as a function of the exposure time.
In support of these images, the amount of PLL deposited on the alginate beads was quantified
for the various experimental conditions examined in this study. Figure 3 shows a bar graph
depicting the amount of PLL deposited per milliliter of APA beads. These data show that when
the lower molecular weight PLL was used, there was a statistically significant (p<0.05) increase
in the amount of PLL deposited on the alginate beads with increasing time of exposure.
Furthermore, the quantities of PLL deposited on MVM based beads exposed for either 6 or 20
minutes to PLL were statistically higher (p<0.05) than the corresponding MVG based beads.
Conversely, when the higher molecular weight PLL was used, the amount of PLL deposited
on the alginate beads could not be reliably quantified regardless of exposure time or alginate
composition.
Figure 4 shows a profile of the alginate/PLL thickness measured on four MVM APA beads
(lower molecular weight PLL with 6 minutes exposure time) imaged with a GRE sequence and
TE = 25 ms. Each point corresponds to a different slice through the bead, and thus, the values
on the x-axis correspond to the relative location of the PLL measurement proceeding from one
edge of a bead to the diametrically opposed side. The PLL thickness in each slice was measured
by using a spatially defined histogram of MR signal intensity to count individual pixels in the
MR microimage. Pixels were counted in the PLL thickness measurement if their signal intensity
decreased by more than 20% from the signal intensity of the center of the bead. In Figure 4,
the center point of the graph corresponds to the equator of the bead and represents the most
accurate measurement of the PLL thickness at 40.6 ± 6.2 μm. The increased apparent thickness
at each edge of the profile is attributed to the curvature of the bead and the slice thickness of
the images. Despite the detected changes in contrast intensity and PLL deposited on the beads,
the thickness of the PLL layer measured by MR was constant for all combinations of PLL
molecular weight, exposure time and alginate composition examined. In all cases, the thickness
at the equator of the bead ranged between 35-50 μm. Furthermore, the thickness of the PLL
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6. layer did not change during the 30 days of culture, suggesting that the PLL layer is stable during
this period.
Using 3D volume rendering, the PLL layer was segmented to produce 3D images of the entire
bead to assess the uniformity of the PLL coat, as seen in Figure 5. The left panel shows a GRE
image (a single slice from a 3D dataset acquired with TE = 25 ms) of a single APA bead (MVM
alginate, lower molecular weight PLL, 6 min exposure). The panel on the right shows the 3D
segmentation of the PLL shell generated from the GRE data set. The darker hue represents the
PLL shell while the lighter hue represents the surrounding media. Segmentation was performed
using AMIRA 3.1 (Mercury Computer Systems, Inc., Chelmsford MA). To simplify
segmentation, a Gaussian filter was applied directly to the processed image dataset to enhance
contrast between the internal bead and PLL layer and reduce the number of false positive points
included in the rendered PLL layer. Segmentation was based on an automated selection of
pixels determined by MR signal intensity thresholds (PLL layer fell between 15 and 30% of
the peak signal intensity of the dataset), with nominal manual correction. The resulting image
shows that there are no visible defects in the coating of the alginate beads with PLL, which
underscores the continuity of this layer even at short exposure times.
Longitudinal changes in alginate NMR relaxation parameters due to long term culture
Figure 6A illustrates the temporal changes in T2 relaxation monitored over a month for MVG
based alginate beads, while Figure 6B shows the temporal changes for MVM based alginate
beads. Our data show that MVG beads exhibit a decrease in T2 relaxation time reaching a
minimum value within 15 days. This decrease is markedly higher in non-PLL coated beads.
In contrast, MVM beads did not exhibit an appreciable change in T2 relaxation for either PLL
coated or non-coated beads. Diffusion-weighted images did not yield statistically significant
temporal changes in ADC for any of the culture combinations examined (data not shown). This
observation is in agreement with previous studies reporting a lack of change in ADC as a
function of alginate composition [21,22].
DISCUSSION
Visualization of the PLL layer in APA beads is not new; it was recently demonstrated with the
use of fluorescent-labeled PLL and the aid of a confocal laser scanning microscope [30].
However, the MR techniques utilized in this study do not require the synthesis of fluorescent-
labeled PLL. Commonly purchased PLL and standard SE and GRE MR sequences were utilized
throughout this study. Our data show that T2- and T2*-weighted MR images are sensitive
measures of the presence and thickness of the PLL layer. MR-based measurements of PLL
thickness ranged from 35-50 μm for all combinations of PLL exposure times, PLL molecular
weights and alginate compositions examined. This thickness is in agreement with data
presented by Strand, et al. (30-50 μm for exposure times up to 30 minutes), and it is attributed
to the infiltration of PLL into the alginate matrix [30]. Such PLL infiltration is also reported
in the studies by de Vos, et al. and Tam, et al. [31,32]. This agreement between MR and non-
MR methods further indicates that susceptibility effects do not significantly contribute to the
thickness of the PLL layer in the MR images acquired under these conditions, which
underscores the robust nature of the technique.
The contrast observed in either GRE or SE images is sensitive to changes in the interaction
between lower molecular weight PLL and alginate beads regardless of their composition.
Specifically, MVM or MVG beads displayed higher contrast (darker bead periphery) when
exposed for 20 minutes than when exposed for 2 minutes. Similarly, the contrast of the PLL
layer is higher (i.e. darker) in MVM beads than in corresponding MVG beads at a given
exposure time. These observations were confirmed by the quantification of the PLL deposited
on the beads. Thus, for the lower molecular weight PLL utilized in this study, the MR contrast
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7. of the PLL layer and the quantity of PLL deposited onto alginate beads correlated to the
guluronic content of the beads and the exposure intervals.
Previous studies describing the interaction of similarly low molecular weight PLL and alginate
are contradictory. The studies by Thu, et al. demonstrated that the binding affinity of PLL to
alginate was lower for high guluronic content alginates, suggesting that more PLL can be
deposited on alginates with a high mannuronic content [33,34]. Conversely, the study by van
Hoogmoed, et al. demonstrated that alginate capsules made with a high guluronic content
alginate contained 20% more PLL than similar capsules made with an intermediate guluronic
content alginate [35]. The cause of this discrepancy is not fully understood. However, a new
study by Tam, et al. suggests that the conformation of the alginate/PLL complex depends on
the strength of the interaction between the two molecules [32]. Specifically, when the
interaction between alginate and PLL is weak (e.g., for high guluronic content alginates), the
alginate/PLL complex has a random coil conformation. Alternatively, when the interaction
between the two molecules is strong (e.g., for high mannuronic content alginates), the alginate/
PLL complex has a specific helical conformation, namely an α–helix of interwoven with
alginate [32]. Because the helical conformation is tighter than the random coil conformation,
it will lead to more spin-spin interactions and shorten the T2 relaxation time (i.e., darker contrast
for the PLL/alginate interface). Therefore, it is possible that the difference in the contrast of
the PLL layer in APA beads is attributed to the conformational differences of PLL/alginate
complex and not solely to the quantity of PLL deposited on the beads.
In a similar vein, these data also demonstrate that the correlation between MR contrast and
PLL exposure time on alginate does not hold for the higher molecular weight PLL utilized in
the study. This is best illustrated by comparing preparations of MVG based beads. The contrast
of the PLL layer on all MVG preparations is similar, regardless of the molecular weight of the
PLL used to coat the beads. This observation suggests that similar quantities of PLL are
deposited on these MVG beads. However, quantification of PLL showed statistically higher
quantities of PLL were deposited on the beads when the lower molecular weight PLL was
utilized. This is attributed to the restricted diffusion of the higher molecular weight PLL as it
penetrates and interacts with alginate and it is in agreement with the study by Thu, et al. [33].
Therefore, MR Microscopy provides an excellent method to assess non-invasively and non-
destructively the interaction and dynamics of a polycation such as PLL with anionic hydrogels
such as alginate.
A key concern with the use of alginate in tissue engineered constructs is the long-term stability
of the alginate gel. Given that alginate gel is formed by the ionic interaction between negatively
charged alginate molecules and positively charged calcium ions (or other similarly charged
cations), it is reasonable to hypothesize that calcium ions may diffuse out of the alginate matrix
during a prolonged culture. This process may cause the alginate/calcium bond to weaken,
resulting in a weaker gel that may dissolve or break. A recent study by Simpson et al. [36]
demonstrated that alginates with a higher guluronic content are more susceptible to changes
in Ca+2 ion concentration. Thus, it is reasonable to assume that the “egg-box” configuration
[37] of the gelled MVG alginate will weaken with time in culture, due to the diffusion of
Ca+2 ions, causing a collapse of the gel structure and a decrease in gel porosity. From the NMR
perspective, this decrease should manifest as a decrease in T2 values, which is indicated by the
data presented in Figure 6. The fact that the observed decrease in T2 is more pronounced in
non-PLL-coated MVG beads than in PLL coated beads supports a previous study suggesting
that the PLL layer hinders the transport of Ca+2 ions across the PLL layer [22]. Thus, the MVG
gel microstructure is better maintained in PLL-coated beads than in non-coated beads.
Metabolic studies with βTC3 cells encapsulated in MVG APA beads show that encapsulated
cells begin to grow after 20 days in culture, whereas βTC3 cells encapsulated in MVM grow
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8. continuously from the onset of the culture [38]. T2 data presented in Figure 6, show that MVG
beads undergoes a gel microstructure re-organization during the first 2 weeks of culture. It is
particularly interesting to note that the minimum T2 values measured from MVG APA beads
are similar to those measured from MVM beads throughout the study. These observations
suggest that MVG gels re-organize during in vitro culture, reaching a configuration that is more
conducive to cell growth. One way to prevent the re-organization of the MVG gel is to
periodically wash the alginate beads in CaCl2 as was recently demonstrated [36]. Unlike MVG
alginate, MVM alginate does not experience the same change in porosity due to the weaker
Ca+2/alginate interaction. Consequently, the MVM microstructure does not undergo a
significant re-organization over the culture period, which is consistent with the static T2 value
measured in this study.
Finally, T2 values for MVG and MVM alginate beads measured 24 hours after encapsulation
and maintained in DMEM media were compared to measurements reported previously with
similar beads maintained in CaCl2 for a day [22]. While T2 values of MVG beads were similar
in both media conditions, T2 values for MVM beads maintained in DMEM were higher than
those reported for beads maintained in CaCl2. This observation suggests that MVG alginate
beads maintained their initial organization after 24 hours of DMEM incubation while MVM
alginate beads underwent a rapid alteration during this period. This difference may be attributed
to the difference swelling properties between MVG and MVM alginates. High guluronic
content alginates maintain their rigid structure initially due to their strong Ca+2/alginate
interaction, while high mannuronic content alginates swell more readily resulting in an increase
in T2 once the presence of external CaCl2 is removed. Notably, the swelling of the MVM
alginate takes place rapidly during this first 24-hour timeframe, after which the structure of the
MVM bead remains very constant during the remainder of the culture time as reflected by the
T2 measurements over the 30 day period.
CONCLUSION
In summary, we have demonstrated that MR microscopy can non-invasively and non-
destructively visualize and quantify the PLL layer and monitor temporal changes in the
structure of alginate beads during a prolonged culture. The data show that the thickness of the
PLL layer at the periphery of alginate beads is between 35-50 μm regardless of the alginate
composition or the time of exposure to the PLL solution (up to 20 minutes). The data also show
that high guluronic content alginate beads undergo reorganization of their gel structure during
an in vitro culture, while high mannuronic content alginate beads are not susceptible to such
changes under similar culture conditions. It should be emphasized that the imaging techniques
utilized in this study are generic and can be applied to study other hydrogels and/or other
polycation layers.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the financial support of the NIH through grants R01 DK56890, R01 DK47858,
and P41 RR16105. All MR data were obtained at the Advanced Magnetic Resonance Imaging and Spectroscopy
(AMRIS) facility in the McKnight Brain Institute of the University of Florida. Pilot funds for imaging time were
provided by AMRIS and the National High Magnetic Field Laboratory.
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11. FIGURE 1.
Spin echo MR microimages of MVM alginate beads with and without PLL coating (6 minute
exposure to low molecular weight PLL) at two different echo times. Note that the dark circle
around the alginate beads coated with PLL does not significantly change in size with longer
TE – unlike the air bubble (white arrow)
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12. FIGURE 2.
GRE MR microimages of MVM APA beads showing the intensity of the PLL layer as a
function of alginate composition and interaction time. The isotropic resolution of these images
is 12.5 μm.
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13. FIGURE 3.
A bar graph depicting the amount of PLL deposited per milliliter of alginate beads. Solid black,
grey and white bars represent measurements based on 2, 6 and 20 minutes of exposure
respectively between the various alginate and PLL combinations. Each bar is the average of
duplicate measurements within the same experiment. This experiment was repeated three times
with consistent results. An (*) indicates statistically significant differences (P<0.05) between
the 6 or 20 minute exposures and to the corresponding alginate exposed for 2 minute to PLL.
A (#) indicates statistically significant differences (P<0.05) between MVM and MVG alginates
at the corresponding time exposures to PLL.
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14. FIGURE 4.
A profile of PLL layer thickness measurements performed on MVM beads coated with the
lower molecular weight PLL following a 6 minute exposure. The beads were imaged with a
GRE sequence and a TE = 25 ms. The x-axis represents arbitrary slice numbers with effective
thickness of 50 μm. The error bars represent the standard deviation of the mean based on four
measurements.
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15. FIGURE 5.
The MR image on the left shows a single slice from a 3D GRE dataset with a TE = 25 ms of
a single MVM alginate bead coated with the lower molecular weight PLL following a 6 minute
exposure. The dark contrast generated by the PLL layer has been segmented in the 3D rendering
shown on the right. The darker hue of the 3D rendering represents the PLL shell while the
lighter hue represents the surrounding media. Segmentation was performed based on pixel
intensity values. Although a 3D Gaussian filter was applied to the image for the purpose of
segmentation, the acquisition voxel size in the 3D dataset was 12.5×12.5×12.5 μm3.
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16. FIGURE 6.
A: Temporal changes in the T2 relaxation time of MVG alginate beads. Solid diamonds
represent data acquired from MVG APA beads and open diamonds represent data acquired
from MVG beads that were not coated with PLL. B: Temporal changes in the T2 relaxation
time of MVM alginate beads. Solid diamonds represent data acquired from MVM APA beads
and open diamonds represent data acquired from MVM beads that were not coated with PLL.
The error bars represent the standard deviation of the T2 relaxation measurement.
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17. NIH-PAAuthorManuscriptNIH-PAAuthorManuscriptNIH-PAAuthorManuscript
Constantinidis et al. Page 17
TABLE 1
Combinations of alginate composition, PLL molecular weight and time of interaction between alginate and PLL
examined
Alginate Type MVM MVG
Molecular Weight of PLL
(Daltons)
Low
(19,200)
High
(240,000)
Low
(19,200)
High
(240,000)
Exposure Time
(minutes)
2 6 20 2 6 20 2 6 20 2 6 20
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