Open AccessReview
Tumor Microenvironment on a Chip: The Progress and Future Perspective
Bioengineering 2017, 4(3), 64; doi:10.3390/bioengineering4030064 (registering DOI) -
Abstract
Tumors develop in intricate microenvironments required for their sustained growth, invasion, and metastasis. The tumor microenvironment plays a critical role in the malignant or drug resistant nature of tumors, becoming a promising therapeutic target. Microengineered physiological systems capable of mimicking tumor environments are
[...] Read more.
Tumors develop in intricate microenvironments required for their sustained growth, invasion, and metastasis. The tumor microenvironment plays a critical role in the malignant or drug resistant nature of tumors, becoming a promising therapeutic target. Microengineered physiological systems capable of mimicking tumor environments are one emerging platform that allows for quantitative and reproducible characterization of tumor responses with pathophysiological relevance. This review highlights the recent advancements of engineered tumor microenvironment systems that enable the unprecedented mechanistic examination of cancer progression and metastasis. We discuss the progress and future perspective of these microengineered biomimetic approaches for anticancer drug prescreening applications. Full article
Figures

Figure 1

Open AccessArticle
3D Printing and 3D Bioprinting in Pediatrics
Bioengineering 2017, 4(3), 63; doi:10.3390/bioengineering4030063 -
Abstract
Additive manufacturing, commonly referred to as 3D printing, is a technology that builds three-dimensional structures and components layer by layer. Bioprinting is the use of 3D printing technology to fabricate tissue constructs for regenerative medicine from cell-laden bio-inks. 3D printing and bioprinting have
[...] Read more.
Additive manufacturing, commonly referred to as 3D printing, is a technology that builds three-dimensional structures and components layer by layer. Bioprinting is the use of 3D printing technology to fabricate tissue constructs for regenerative medicine from cell-laden bio-inks. 3D printing and bioprinting have huge potential in revolutionizing the field of tissue engineering and regenerative medicine. This paper reviews the application of 3D printing and bioprinting in the field of pediatrics. Full article
Figures

Open AccessArticle
A Novel Multifunctional β-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove
Bioengineering 2017, 4(3), 62; doi:10.3390/bioengineering4030062 -
Abstract
The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from
[...] Read more.
The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a β-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a β-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-β-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-β-d-galactopyranoside, and 4-Nitrophenyl β-d-glucopyranoside, presenting β-N-acetylglucosaminidase, β-glucosidase, and β-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel β-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product. Full article
Figures

Figure 1

Open AccessArticle
Insights on Osmotic Tolerance Mechanisms in Escherichia coli Gained from an rpoC Mutation
Bioengineering 2017, 4(3), 61; doi:10.3390/bioengineering4030061 -
Abstract
An 84 bp in-frame duplication (K370_A396dup) within the rpoC subunit of RNA polymerase was found in two independent mutants selected during an adaptive laboratory evolution experiment under osmotic stress in Escherichia coli, suggesting that this mutation confers improved osmotic tolerance. To determine
[...] Read more.
An 84 bp in-frame duplication (K370_A396dup) within the rpoC subunit of RNA polymerase was found in two independent mutants selected during an adaptive laboratory evolution experiment under osmotic stress in Escherichia coli, suggesting that this mutation confers improved osmotic tolerance. To determine the role this mutation in rpoC plays in osmotic tolerance, we reconstructed the mutation in BW25113, and found it to confer improved tolerance to hyperosmotic stress. Metabolite analysis, exogenous supplementation assays, and cell membrane damage analysis suggest that the mechanism of improved osmotic tolerance by this rpoC mutation may be related to the higher production of acetic acid and amino acids such as proline, and increased membrane integrity in the presence of NaCl stress in exponential phase cells. Transcriptional analysis led to the findings that the overexpression of methionine related genes metK and mmuP improves osmotic tolerance in BW25113. Furthermore, deletion of a stress related gene bolA was found to confer enhanced osmotic tolerance in BW25113 and MG1655. These findings expand our current understanding of osmotic tolerance in E. coli, and have the potential to expand the utilization of high saline feedstocks and water sources in microbial fermentation. Full article
Figures

Figure 1

Open AccessArticle
Prospecting for Marine Bacteria for Polyhydroxyalkanoate Production on Low-Cost Substrates
Bioengineering 2017, 4(3), 60; doi:10.3390/bioengineering4030060 -
Abstract
Polyhydroxyalkanoates (PHAs) are a class of biopolymers with numerous applications, but the high cost of production has prevented their use. To reduce this cost, there is a prospect for strains with a high PHA production and the ability to grow in low-cost by-products.
[...] Read more.
Polyhydroxyalkanoates (PHAs) are a class of biopolymers with numerous applications, but the high cost of production has prevented their use. To reduce this cost, there is a prospect for strains with a high PHA production and the ability to grow in low-cost by-products. In this context, the objective of this work was to evaluate marine bacteria capable of producing PHA. Using Nile red, 30 organisms among 155 were identified as PHA producers in the medium containing starch, and 27, 33, 22 and 10 strains were found to be positive in media supplemented with carboxymethyl cellulose, glycerol, glucose and Tween 80, respectively. Among the organisms studied, two isolates, LAMA 677 and LAMA 685, showed strong potential to produce PHA with the use of glycerol as the carbon source, and were selected for further studies. In the experiment used to characterize the growth kinetics, LAMA 677 presented a higher maximum specific growth rate (µmax = 0.087 h−1) than LAMA 685 (µmax = 0.049 h−1). LAMA 677 also reached a D-3-hydroxybutyrate (P(3HB)) content of 78.63% (dry biomass), which was 3.5 times higher than that of LAMA 685. In the assay of the production of P(3HB) from low-cost substrates (seawater and biodiesel waste glycerol), LAMA 677 reached a polymer content of 31.7%, while LAMA 685 reached 53.6%. Therefore, it is possible to conclude that the selected marine strains have the potential to produce PHA, and seawater and waste glycerol may be alternative substrates for the production of this polymer. Full article
Figures

Open AccessArticle
Characterization of Chlorhexidine-Loaded Calcium-Hydroxide Microparticles as a Potential Dental Pulp-Capping Material
Bioengineering 2017, 4(3), 59; doi:10.3390/bioengineering4030059 -
Abstract
This study explores the delivery of novel calcium hydroxide [Ca(OH)2] microparticles loaded with chlorhexidine (CHX) for potential dental therapeutic and preventive applications. Herein, we introduce a new approach for drug-delivery to deep dentin-surfaces in the form of drug-loaded microparticles. Unloaded Ca(OH)
[...] Read more.
This study explores the delivery of novel calcium hydroxide [Ca(OH)2] microparticles loaded with chlorhexidine (CHX) for potential dental therapeutic and preventive applications. Herein, we introduce a new approach for drug-delivery to deep dentin-surfaces in the form of drug-loaded microparticles. Unloaded Ca(OH)2 [Ca(OH)2/Blank] and CHX-loaded/Ca(OH)2 microparticles were fabricated by aqueous chemical-precipitation technique. The synthesized-microparticles were characterized in vitro for determination of surface-morphology, crystalline-features and thermal-properties examined by energy-dispersive X-ray scanning and transmission electron-microscopy (EDX-SEM/TEM), Fourier-transform infrared-spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and differential scanning-calorimetry (DSC). Time-related pH changes, initial antibacterial/biofilm-abilities and cytotoxicity of CHX-loaded/Ca(OH)2 microparticles were evaluated. Microparticles were delivered to dentin-surfaces with subsequent SEM examination of treated dentin-substrates. The in vitro and ex vivo CHX-release profiles were characterized. Ca(OH)2/Blank were hexagonal-shaped with highest z-average diameter whereas CHX-inclusion evidenced micro-metric spheres with distinguishable surface “rounded deposits” and a negative-shift in diameter. CHX:Ca(OH)2/50 mg exhibited maximum encapsulation-efficiency with good antibacterial and cytocompatible properties. SEM examination revealed an intact layer of microparticles on exposed dentin-surfaces with retention of spherical shape and smooth texture. Microparticles loaded on dentin-surfaces showed prolonged release of CHX indicating substantial retention on dentin-substrates. This study validated the inherent-applicability of this novel drug-delivery approach to dentin-surfaces using micro-metric CHX-loaded/Ca(OH)2 microparticles. Full article
Figures

Open AccessArticle
The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation
Bioengineering 2017, 4(2), 58; doi:10.3390/bioengineering4020058 -
Abstract
Poly(hydroxyalkanoates) (PHAs) are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor
[...] Read more.
Poly(hydroxyalkanoates) (PHAs) are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization. Full article
Figures

Open AccessArticle
Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone
Bioengineering 2017, 4(2), 56; doi:10.3390/bioengineering4020056 -
Abstract
Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle
[...] Read more.
Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle tissues constructed on microdevices capable of measuring the contractile force and to verify if this model is suitable for drug screening using the contractile force as an index. Tissue engineered skeletal muscles containing striated myotubes were prepared on the microdevices for the study. The addition of 100 µM dexamethasone (Dex), which is used as a muscle atrophy inducer, for 24 h reduced the contractile force significantly. An increase in the expression of Atrogin-1 and MuRF-1 in the tissues treated with Dex was established. A decrease in the number of striated myotubes was also observed in the tissues treated with Dex. Treatment with 8 ng/mL Insulin-like Growth Factor (IGF-I) for 24 h significantly increased the contractile force of the Dex-induced atrophic tissues. The same treatment, though, had no impact on the force of the normal tissues. Thus, it is envisaged that the atrophic skeletal muscle tissues induced by Dex can be used for drug screening against atrophy. Full article
Figures

Open AccessReview
A Review of Microwave-Assisted Reactions for Biodiesel Production
Bioengineering 2017, 4(2), 57; doi:10.3390/bioengineering4020057 -
Abstract
The conversion of biomass into chemicals and biofuels is an active research area as trends move to replace fossil fuels with renewable resources due to society’s increased concern towards sustainability. In this context, microwave processing has emerged as a tool in organic synthesis
[...] Read more.
The conversion of biomass into chemicals and biofuels is an active research area as trends move to replace fossil fuels with renewable resources due to society’s increased concern towards sustainability. In this context, microwave processing has emerged as a tool in organic synthesis and plays an important role in developing a more sustainable world. Integration of processing methods with microwave irradiation has resulted in a great reduction in the time required for many processes, while the reaction efficiencies have been increased markedly. Microwave processing produces a higher yield with a cleaner profile in comparison to other methods. The microwave processing is reported to be a better heating method than the conventional methods due to its unique thermal and non-thermal effects. This paper provides an insight into the theoretical aspects of microwave irradiation practices and highlights the importance of microwave processing. The potential of the microwave technology to accomplish superior outcomes over the conventional methods in biodiesel production is presented. A green process for biodiesel production using a non-catalytic method is still new and very costly because of the supercritical condition requirement. Hence, non-catalytic biodiesel conversion under ambient pressure using microwave technology must be developed, as the energy utilization for microwave-based biodiesel synthesis is reported to be lower and cost-effective. Full article
Figures

Figure 1

Open AccessReview
Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production
Bioengineering 2017, 4(2), 55; doi:10.3390/bioengineering4020055 -
Abstract
Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses,
[...] Read more.
Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing. Full article
Figures

Figure 1

Open AccessArticle
Polyhydroxyalkanoate Production on Waste Water Treatment Plants: Process Scheme, Operating Conditions and Potential Analysis for German and European Municipal Waste Water Treatment Plants
Bioengineering 2017, 4(2), 54; doi:10.3390/bioengineering4020054 -
Abstract
This work describes the production of polyhydroxyalkanoates (PHA) as a side stream process on a municipal waste water treatment plant (WWTP) and a subsequent analysis of the production potential in Germany and the European Union (EU). Therefore, tests with different types of sludge
[...] Read more.
This work describes the production of polyhydroxyalkanoates (PHA) as a side stream process on a municipal waste water treatment plant (WWTP) and a subsequent analysis of the production potential in Germany and the European Union (EU). Therefore, tests with different types of sludge from a WWTP were investigated regarding their volatile fatty acids (VFA) production-potential. Afterwards, primary sludge was used as substrate to test a series of operating conditions (temperature, pH, retention time (RT) and withdrawal (WD)) in order to find suitable settings for a high and stable VFA production. In a second step, various tests regarding a high PHA production and stable PHA composition to determine the influence of substrate concentration, temperature, pH and cycle time of an installed feast/famine-regime were conducted. Experiments with a semi-continuous reactor operation showed that a short RT of 4 days and a small WD of 25% at pH = 6 and around 30 °C is preferable for a high VFA production rate (PR) of 1913 mgVFA/(L×d) and a stable VFA composition. A high PHA production up to 28.4% of cell dry weight (CDW) was reached at lower substrate concentration, 20 °C, neutral pH-value and a 24 h cycle time. A final step a potential analysis, based on the results and detailed data from German waste water treatment plants, showed that the theoretically possible production of biopolymers in Germany amounts to more than 19% of the 2016 worldwide biopolymer production. In addition, a profound estimation regarding the EU showed that in theory about 120% of the worldwide biopolymer production (in 2016) could be produced on European waste water treatment plants. Full article
Figures

Figure 1

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 -
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
[...] Read more.
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
Figures

Figure 1

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 -
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
[...] Read more.
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
Figures

Open AccessArticle
Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture
Bioengineering 2017, 4(2), 51; doi:10.3390/bioengineering4020051 -
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
[...] Read more.
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
Figures

Open AccessArticle
Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
Bioengineering 2017, 4(2), 52; doi:10.3390/bioengineering4020052 -
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)
[...] Read more.
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
Figures

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
[...] Read more.
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
Figures

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
[...] Read more.
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
Figures

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
[...] Read more.
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
Figures

Figure 1

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
[...] Read more.
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
Figures

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
[...] Read more.
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
Figures

Figure 1