Open AccessArticle
Production of Polyhydroxyalkanoates Using Hydrolyzates of Spruce Sawdust: Comparison of Hydrolyzates Detoxification by Application of Overliming, Active Carbon, and Lignite
Bioengineering 2017, 4(2), 53; doi:10.3390/bioengineering4020053 (registering DOI) -
Abstract
Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the
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Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the price of the carbon source used in the fermentation. Burkholderia cepacia and Burkholderia sacchari are generally considered promising candidates for PHA production from lignocellulosic hydrolyzates. The wood waste biomass has been subjected to hydrolysis. The resulting hydrolyzate contained a sufficient amount of fermentable sugars. Growth experiments indicated a strong inhibition by the wood hydrolyzate. Over-liming and activated carbon as an adsorbent of inhibitors were employed for detoxification. All methods of detoxification had a positive influence on the growth of biomass and PHB production. Furthermore, lignite was identified as a promising alternative sorbent which can be used for detoxification of lignocellulose hydrolyzates. Detoxification using lignite instead of activated carbon had lower inhibitor removal efficiency, but greater positive impact on growth of the bacterial culture and overall PHA productivity. Moreover, lignite is a significantly less expensive adsorbent in comparison with activated charcoal and; moreover, used lignite can be simply utilized as a fuel to, at least partially, cover heat and energetic demands of fermentation, which should improve the economic feasibility of the process. Full article
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Open AccessArticle
Utilization of Sugarcane Bagasse by Halogeometricum borinquense Strain E3 for Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
Bioengineering 2017, 4(2), 50; doi:10.3390/bioengineering4020050 (registering DOI) -
Abstract
Sugarcane bagasse (SCB), one of the major lignocellulosic agro-industrial waste products, was used as a substrate for biosynthesis of polyhydroxyalkanoates (PHA) by halophilic archaea. Among the various wild-type halophilic archaeal strains screened, Halogeometricum borinquense strain E3 showed better growth and PHA accumulation as
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Sugarcane bagasse (SCB), one of the major lignocellulosic agro-industrial waste products, was used as a substrate for biosynthesis of polyhydroxyalkanoates (PHA) by halophilic archaea. Among the various wild-type halophilic archaeal strains screened, Halogeometricum borinquense strain E3 showed better growth and PHA accumulation as compared to Haloferaxvolcanii strain BBK2, Haloarcula japonica strain BS2, and Halococcus salifodinae strain BK6. Growth kinetics and bioprocess parameters revealed the maximum PHA accumulated by strain E3 to be 50.4 ± 0.1 and 45.7 ± 0.19 (%) with specific productivity (qp) of 3.0 and 2.7 (mg/g/h) using NaCl synthetic medium supplemented with 25% and 50% SCB hydrolysate, respectively. PHAs synthesized by strain E3 were recovered in chloroform using a Soxhlet apparatus. Characterization of the polymer using crotonic acid assay, X-ray diffraction (XRD), differential scanning calorimeter (DSC), Fourier transform infrared (FT-IR), and proton nuclear magnetic resonance (1H-NMR) spectroscopy analysis revealed the polymer obtained from SCB hydrolysate to be a co-polymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] comprising of 13.29 mol % 3HV units. Full article
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Open AccessArticle
Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture
Bioengineering 2017, 4(2), 51; doi:10.3390/bioengineering4020051 (registering DOI) -
Abstract
The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control
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The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control over the mechanical environment. Therefore, we developed a miniaturized, parallelizable perfusion bioreactor system with two different bioreactor chambers. Pressure sensors were also implemented to determine the permeability of biomaterials which allows us to approximate the shear stress conditions. To characterize the flow velocity and shear stress profile of a porous scaffold in both bioreactor chambers, a computational fluid dynamics analysis was performed. Furthermore, the mixing behavior was characterized by acquisition of the residence time distributions. Finally, the effects of the different flow and shear stress profiles of the bioreactor chambers on osteogenic differentiation of human mesenchymal stem cells were evaluated in a proof of concept study. In conclusion, the data from computational fluid dynamics and shear stress calculations were found to be predictable for relative comparison of the bioreactor geometries, but not for final determination of the optimal flow rate. However, we suggest that the system is beneficial for parallel dynamic cultivation of multiple samples for 3D cell culture processes. Full article
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Open AccessArticle
Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
Bioengineering 2017, 4(2), 52; doi:10.3390/bioengineering4020052 (registering DOI) -
Abstract
The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC)
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The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)—a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity. Full article
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Open AccessArticle
Additive Manufacturing of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/poly(ε-caprolactone) Blend Scaffolds for Tissue Engineering
Bioengineering 2017, 4(2), 49; doi:10.3390/bioengineering4020049 -
Abstract
Additive manufacturing of scaffolds made of a polyhydroxyalkanoate blended with another biocompatible polymer represents a cost-effective strategy for combining the advantages of the two blend components in order to develop tailored tissue engineering approaches. The aim of this study was the development of
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Additive manufacturing of scaffolds made of a polyhydroxyalkanoate blended with another biocompatible polymer represents a cost-effective strategy for combining the advantages of the two blend components in order to develop tailored tissue engineering approaches. The aim of this study was the development of novel poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/ poly(ε-caprolactone) (PHBHHx/PCL) blend scaffolds for tissue engineering by means of computer-aided wet-spinning, a hybrid additive manufacturing technique suitable for processing polyhydroxyalkanoates dissolved in organic solvents. The experimental conditions for processing tetrahydrofuran solutions containing the two polymers at different concentrations (PHBHHx/PCL weight ratio of 3:1, 2:1 or 1:1) were optimized in order to manufacture scaffolds with predefined geometry and internal porous architecture. PHBHHx/PCL scaffolds with a 3D interconnected network of macropores and a local microporosity of the polymeric matrix, as a consequence of the phase inversion process governing material solidification, were successfully fabricated. As shown by scanning electron microscopy, thermogravimetric, differential scanning calorimetric and uniaxial compressive analyses, blend composition significantly influenced the scaffold morphological, thermal and mechanical properties. In vitro biological characterization showed that the developed scaffolds were able to sustain the adhesion and proliferation of MC3T3-E1 murine preosteoblast cells. The additive manufacturing approach developed in this study, based on a polymeric solution processing method avoiding possible material degradation related to thermal treatments, could represent a powerful tool for the development of customized PHBHHx-based blend scaffolds for tissue engineering. Full article
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Open AccessArticle
Assessing and Resolving Model Misspecifications in Metabolic Flux Analysis
Bioengineering 2017, 4(2), 48; doi:10.3390/bioengineering4020048 -
Abstract
Metabolic flux analysis (MFA) is an indispensable tool in metabolic engineering. The simplest variant of MFA relies on an overdetermined stoichiometric model of the cell’s metabolism under the pseudo-steady state assumption to evaluate the intracellular flux distribution. Despite its long history, the issue
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Metabolic flux analysis (MFA) is an indispensable tool in metabolic engineering. The simplest variant of MFA relies on an overdetermined stoichiometric model of the cell’s metabolism under the pseudo-steady state assumption to evaluate the intracellular flux distribution. Despite its long history, the issue of model error in overdetermined MFA, particularly misspecifications of the stoichiometric matrix, has not received much attention. We evaluated the performance of statistical tests from linear least square regressions, namely Ramsey’s Regression Equation Specification Error Test (RESET), the F-test, and the Lagrange multiplier test, in detecting model misspecifications in the overdetermined MFA, particularly missing reactions. We further proposed an iterative procedure using the F-test to correct such an issue. Using Chinese hamster ovary and random metabolic networks, we demonstrated that: (1) a statistically significant regression does not guarantee high accuracy of the flux estimates; (2) the removal of a reaction with a low flux magnitude can cause disproportionately large biases in the flux estimates; (3) the F-test could efficiently detect missing reactions; and (4) the proposed iterative procedure could robustly resolve the omission of reactions. Our work demonstrated that statistical analysis and tests could be used to systematically assess, detect, and resolve model misspecifications in the overdetermined MFA. Full article
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Open AccessArticle
Hypoxic Three-Dimensional Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Reactor
Bioengineering 2017, 4(2), 47; doi:10.3390/bioengineering4020047 -
Abstract
Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced
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Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced differentiation capacity are observed after in vitro expansion and reduce therapeutic success in clinical studies. Remarkably, cultivation of MSCs in three-dimensional aggregates preserve stem cell properties. Hence, the large scale formation and cultivation of MSC aggregates is highly desirable. Besides other effects, MSCs cultivated under hypoxic conditions are known to display increased proliferation and genetic stability. Therefore, in this study we demonstrate cultivation of adipose derived human MSC aggregates in a stirred tank reactor under hypoxic conditions. Although aggregates were exposed to comparatively high average shear stress of 0.2 Pa as estimated by computational fluid dynamics, MSCs displayed a viability of 78–86% and maintained their surface marker profile and differentiation potential after cultivation. We postulate that cultivation of 3D MSC aggregates in stirred tank reactors is valuable for large-scale production of MSCs or their secreted compounds after further optimization of cultivation parameters. Full article
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Open AccessReview
Microtechnology-Based Multi-Organ Models
Bioengineering 2017, 4(2), 46; doi:10.3390/bioengineering4020046 -
Abstract
Drugs affect the human body through absorption, distribution, metabolism, and elimination (ADME) processes. Due to their importance, the ADME processes need to be studied to determine the efficacy and side effects of drugs. Various in vitro model systems have been developed and used
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Drugs affect the human body through absorption, distribution, metabolism, and elimination (ADME) processes. Due to their importance, the ADME processes need to be studied to determine the efficacy and side effects of drugs. Various in vitro model systems have been developed and used to realize the ADME processes. However, conventional model systems have failed to simulate the ADME processes because they are different from in vivo, which has resulted in a high attrition rate of drugs and a decrease in the productivity of new drug development. Recently, a microtechnology-based in vitro system called “organ-on-a-chip” has been gaining attention, with more realistic cell behavior and physiological reactions, capable of better simulating the in vivo environment. Furthermore, multi-organ-on-a-chip models that can provide information on the interaction between the organs have been developed. The ultimate goal is the development of a “body-on-a-chip”, which can act as a whole body model. In this review, we introduce and summarize the current progress in the development of multi-organ models as a foundation for the development of body-on-a-chip. Full article
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Open AccessArticle
OpenDrop: An Integrated Do-It-Yourself Platform for Personal Use of Biochips
Bioengineering 2017, 4(2), 45; doi:10.3390/bioengineering4020045 -
Abstract
Biochips, or digital labs-on-chip, are developed with the purpose of being used by laboratory technicians or biologists in laboratories or clinics. In this article, we expand this vision with the goal of enabling everyone, regardless of their expertise, to use biochips for their
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Biochips, or digital labs-on-chip, are developed with the purpose of being used by laboratory technicians or biologists in laboratories or clinics. In this article, we expand this vision with the goal of enabling everyone, regardless of their expertise, to use biochips for their own personal purposes. We developed OpenDrop, an integrated electromicrofluidic platform that allows users to develop and program their own bio-applications. We address the main challenges that users may encounter: accessibility, bio-protocol design and interaction with microfluidics. OpenDrop consists of a do-it-yourself biochip, an automated software tool with visual interface and a detailed technique for at-home operations of microfluidics. We report on two years of use of OpenDrop, released as an open-source platform. Our platform attracted a highly diverse user base with participants originating from maker communities, academia and industry. Our findings show that 47% of attempts to replicate OpenDrop were successful, the main challenge remaining the assembly of the device. In terms of usability, the users managed to operate their platforms at home and are working on designing their own bio-applications. Our work provides a step towards a future in which everyone will be able to create microfluidic devices for their personal applications, thereby democratizing parts of health care. Full article
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Open AccessArticle
Comparative N-Glycosylation Analysis of the Fc Portions of a Chimeric Human Coagulation Factor VIII and Immunoglobulin G1
Bioengineering 2017, 4(2), 44; doi:10.3390/bioengineering4020044 -
Abstract
Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion
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Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor’s quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor’s properties, most notably its binding to Fcγ receptors. Full article
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Open AccessArticle
In Vitro Growth of Human Keratinocytes and Oral Cancer Cells into Microtissues: An Aerosol-Based Microencapsulation Technique
Bioengineering 2017, 4(2), 43; doi:10.3390/bioengineering4020043 -
Abstract
Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT) cell line and an oral squamous cell carcinoma (OSCC) cell line
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Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT) cell line and an oral squamous cell carcinoma (OSCC) cell line (ORL-48) based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 μL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 μm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM), fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies. Full article
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Open AccessArticle
Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy
Bioengineering 2017, 4(2), 42; doi:10.3390/bioengineering4020042 -
Abstract
IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG
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IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated. Full article
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Open AccessArticle
Uncertainty Quantification in Irreversible Electroporation Simulations
Bioengineering 2017, 4(2), 41; doi:10.3390/bioengineering4020041 -
Abstract
One recent area of cancer research is irreversible electroporation (IRE). Irreversible electroporation is a minimally invasive procedure where needle electrodes are inserted into the body to ablate tumor cells with electricity. The aim of this paper is to investigate how uncertainty in tissue
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One recent area of cancer research is irreversible electroporation (IRE). Irreversible electroporation is a minimally invasive procedure where needle electrodes are inserted into the body to ablate tumor cells with electricity. The aim of this paper is to investigate how uncertainty in tissue and tumor conductivity propagate into final ablation predictions used for treatment planning. Two dimensional simulations were performed for a circular tumor surrounded by healthy tissue, and electroporated from two monopolar electrodes. The conductivity values were treated as random variables whose distributions were taken from published literature on the average and standard deviation of liver tissue and liver tumors. Three different Monte Carlo setups were simulated each at three different voltages. Average and standard deviation data was reported for a multitude of electrical field properties experienced by the tumor. Plots showing the variability in the electrical field distribution throughout the tumor are also presented. Full article
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Open AccessArticle
Nonstandard Finite Difference Method Applied to a Linear Pharmacokinetics Model
Bioengineering 2017, 4(2), 40; doi:10.3390/bioengineering4020040 -
Abstract
We extend the nonstandard finite difference method of solution to the study of pharmacokinetic–pharmacodynamic models. Pharmacokinetic (PK) models are commonly used to predict drug concentrations that drive controlled intravenous (I.V.) transfers (or infusion and oral transfers) while pharmacokinetic and pharmacodynamic (PD) interaction models
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We extend the nonstandard finite difference method of solution to the study of pharmacokinetic–pharmacodynamic models. Pharmacokinetic (PK) models are commonly used to predict drug concentrations that drive controlled intravenous (I.V.) transfers (or infusion and oral transfers) while pharmacokinetic and pharmacodynamic (PD) interaction models are used to provide predictions of drug concentrations affecting the response of these clinical drugs. We structure a nonstandard finite difference (NSFD) scheme for the relevant system of equations which models this pharamcokinetic process. We compare the results obtained to standard methods. The scheme is dynamically consistent and reliable in replicating complex dynamic properties of the relevant continuous models for varying step sizes. This study provides assistance in understanding the long-term behavior of the drug in the system, and validation of the efficiency of the nonstandard finite difference scheme as the method of choice. Full article
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Open AccessReview
The Role of Microfluidics for Organ on Chip Simulations
Bioengineering 2017, 4(2), 39; doi:10.3390/bioengineering4020039 -
Abstract
A multichannel three-dimensional chip of a microfluidic cell culture which enables the simulation of organs is called an “organ on a chip” (OC). With the integration of many other technologies, OCs have been mimicking organs, substituting animal models, and diminishing the time and
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A multichannel three-dimensional chip of a microfluidic cell culture which enables the simulation of organs is called an “organ on a chip” (OC). With the integration of many other technologies, OCs have been mimicking organs, substituting animal models, and diminishing the time and cost of experiments which is better than the preceding conventional in vitro models, which make them imperative tools for finding functional properties, pathological states, and developmental studies of organs. In this review, recent progress regarding microfluidic devices and their applications in cell cultures is discussed to explain the advantages and limitations of these systems. Microfluidics is not a solution but only an approach to create a controlled environment, however, other supporting technologies are needed, depending upon what is intended to be achieved. Microfluidic platforms can be integrated with additional technologies to enhance the organ on chip simulations. Besides, new directions and areas are mentioned for interested researchers in this field, and future challenges regarding the simulation of OCs are also discussed, which will make microfluidics more accurate and beneficial for biological applications. Full article
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Open AccessArticle
Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans
Bioengineering 2017, 4(2), 38; doi:10.3390/bioengineering4020038 -
Abstract
Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one
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Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy. Full article
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Open AccessArticle
Bio-Inspired Multi-Functional Drug Transport Design Concept and Simulations
Bioengineering 2017, 4(2), 37; doi:10.3390/bioengineering4020037 -
Abstract
In this study, we developed a microdevice concept for drug/fluidic transport taking an inspiration from supramolecular motor found in biological cells. Specifically, idealized multi-functional design geometry (nozzle/diffuser/nozzle) was developed for (i) fluidic/particle transport; (ii) particle separation; and (iii) droplet generation. Several design simulations
[...] Read more.
In this study, we developed a microdevice concept for drug/fluidic transport taking an inspiration from supramolecular motor found in biological cells. Specifically, idealized multi-functional design geometry (nozzle/diffuser/nozzle) was developed for (i) fluidic/particle transport; (ii) particle separation; and (iii) droplet generation. Several design simulations were conducted to demonstrate the working principles of the multi-functional device. The design simulations illustrate that the proposed design concept is feasible for multi-functionality. However, further experimentation and optimization studies are needed to fully evaluate the multifunctional device concept for multiple applications. Full article
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Open AccessArticle
An Approach to In Vitro Manufacturing of Hypertrophic Cartilage Matrix for Bone Repair
Bioengineering 2017, 4(2), 35; doi:10.3390/bioengineering4020035 -
Abstract
Devitalized hypertrophic cartilage matrix (DCM) is an attractive concept for an off-the-shelf bone graft substitute. Upon implantation, DCM can trigger the natural endochondral ossification process, but only when the hypertrophic cartilage matrix has been reconstituted correctly. In vivo hypertrophic differentiation has been reported
[...] Read more.
Devitalized hypertrophic cartilage matrix (DCM) is an attractive concept for an off-the-shelf bone graft substitute. Upon implantation, DCM can trigger the natural endochondral ossification process, but only when the hypertrophic cartilage matrix has been reconstituted correctly. In vivo hypertrophic differentiation has been reported for multiple cell types but up-scaling and in vivo devitalization remain a big challenge. To this end, we developed a micro tissue-engineered cartilage (MiTEC) model using the chondrogenic cell line ATDC5. Micro-aggregates of ATDC5 cells (approximately 1000 cells per aggregate) were cultured on a 3% agarose mold consisting of 1585 microwells, each measuring 400 µm in diameter. Chondrogenic differentiation was strongly enhanced using media supplemented with combinations of growth factors e.g., insulin, transforming growth factor beta and dexamethasone. Next, mineralization was induced by supplying the culture medium with beta-glycerophosphate, and finally we boosted the secretion of proangiogenic growth factors using the hypoxia mimetic phenanthroline in the final stage of in vivo culture. Then, ATDC5 aggregates were devitalized by freeze/thawing or sodium dodecyl sulfate treatment before co-culturing with human mesenchymal stromal cells (hMSCs). We observed a strong effect on chondrogenic differentiation of hMSCs. Using this MiTEC model, we were able to not only upscale the production of cartilage to a clinically relevant amount but were also able to vary the cartilage matrix composition in different ways, making MiTEC an ideal model to develop DCM as a bone graft substitute. Full article
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Open AccessArticle
Fed-Batch Synthesis of Poly(3-Hydroxybutyrate) and Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate) from Sucrose and 4-Hydroxybutyrate Precursors by Burkholderia sacchari Strain DSM 17165
Bioengineering 2017, 4(2), 36; doi:10.3390/bioengineering4020036 -
Abstract
Based on direct sucrose conversion, the bacterium Burkholderia sacchari is an excellent producer of the microbial homopolyester poly(3-hydroxybutyrate) (PHB). Restrictions of the strain’s wild type in metabolizing structurally related 3-hydroxyvalerate (3HV) precursors towards 3HV-containing polyhydroxyalkanoate (PHA) copolyester calls for alternatives. We demonstrate the
[...] Read more.
Based on direct sucrose conversion, the bacterium Burkholderia sacchari is an excellent producer of the microbial homopolyester poly(3-hydroxybutyrate) (PHB). Restrictions of the strain’s wild type in metabolizing structurally related 3-hydroxyvalerate (3HV) precursors towards 3HV-containing polyhydroxyalkanoate (PHA) copolyester calls for alternatives. We demonstrate the highly productive biosynthesis of PHA copolyesters consisting of 3-hydroxybuytrate (3HB) and 4-hydroxybutyrate (4HB) monomers. Controlled bioreactor cultivations were carried out using saccharose from the Brazilian sugarcane industry as the main carbon source, with and without co-feeding with the 4HB-related precursor γ-butyrolactone (GBL). Without GBL co-feeding, the homopolyester PHB was produced at a volumetric productivity of 1.29 g/(L•h), a mass fraction of 0.52 g PHB per g biomass, and a final PHB concentration of 36.5 g/L; the maximum specific growth rate µmax amounted to 0.15 1/h. Adding GBL, we obtained 3HB and 4HB monomers in the polyester at a volumetric productivity of 1.87 g/(L•h), a mass fraction of 0.72 g PHA per g biomass, a final PHA concentration of 53.7 g/L, and a µmax of 0.18 1/h. Thermoanalysis revealed improved material properties of the second polyester in terms of reduced melting temperature Tm (161 °C vs. 178 °C) and decreased degree of crystallinity Xc (24% vs. 71%), indicating its enhanced suitability for polymer processing. Full article
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Open AccessArticle
Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC
Bioengineering 2017, 4(2), 34; doi:10.3390/bioengineering4020034 -
Abstract
There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a
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There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which the organism exhibits a distinct tropism for. Stable clonal populations of cells expressing a construct encoding for a truncated version of MUC5AC containing N- and C-termini interspersed with two native tandem repeat sequences (N + 2TR + C) were generated. Binding of H. pylori to protein immunoprecipitated from cell lysates and supernatants was assessed. High molecular weight mucin could be detected in both cell lysates and supernatants of transfected cells. Recombinant protein formed high molecular weight oligomers, was both N and O glycosylated, underwent cleavage similar to native MUC5AC and was secreted from the cell. H. pylori bound better to secreted mucin than intracellular mucin suggesting that modifications on extracellular MUC5AC promoted binding. Lectin analysis demonstrated that secreted mucin was differentially glycosylated compared to intracellular mucin. H. pylori also bound to a recombinant C-terminus MUC5AC protein, but binding to this protein did not inhibit binding to the N + 2TR + C protein. This study demonstrates the feasibility of using recombinant mucins containing tandem repeat sequences to assess microbial mucin interactions. Full article
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