Growth factors regional patterned and photoimmobilized scaffold applied to bone tissue regeneration.

Bone diseases such as osteomalacia, osteoporosis, and osteomyelitis are major illnesses that threaten the health of human. This study aimed to provide an idea at the molecular level of material properties determined with UV specific surface approaches. The tert-butyl hydroperoxide (t-BHP) exposure aging model bone mesenchymal stem cells (BMSCs) were reverted by using a poly-hybrid scaffold (PS), which is a carbon nanotube (CNT) coated polycaprolactone (PCL) and polylactic acid (PLA) scaffold, combined with insulin-like growth factor-1 (IGF). Then, the region-specific PS photo-immobilized with different growth factors (GFs) was obtained by interference and diffraction of ultraviolet (UV) light. Additionally, the reverted BMSCs were regionally pattern differentiated into three kinds of cells on the GF immobilized PS (GFs/PS). In vivo, the GFs/PS accelerate bone healing in injured Sprague-Dawley (SD) rats. The data showed that GFs/PS effectively promoted the differentiation of reverted BMSCs in the designated area on 21st day. These results suggest region-specific interface immobilization of GFs concurrently differentiating reverted BMSCs into three different cells in the same scaffold. This method might be considered as a short-time, low cost, and simple operational approach to scaffold modification for tissue regeneration in the future.

FluidFM as a tool to study adhesion forces of bacteria - Optimization of parameters and comparison to conventional bacterial probe Scanning Force Spectroscopy.

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters (setpoint, z-speed, z-length, pause time, and relative underpressure) to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells (here the gram negative seawater bacterium Paracoccus seriniphilus and the gram positive bacterium Lactococcus lactis), we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell on a tipless cantilever. Even for the bacterium Lactococcus lactis, which is difficult to immobilze chemically (like seen in an earlier study), immobilization and the measurement of force-distance curves are possible by using the FluidFM technology.

Enhanced Degradation of Diesel Oil by Using Biofilms Formed by Indigenous Purple Photosynthetic Bacteria from Oil-Contaminated Coasts of Vietnam on Different Carriers.

Oil pollution in marine environment caused by oil spillage has been a main threat to the ecosystem including the ocean life and to the human being. In this research, three indigenous purple photosynthetic strains Rhodopseudomonas sp. DD4, DQ41, and FO2 were isolated from oil-contaminated coastal zones in Vietnam. The cells of these strains were immobilized on different carriers including cinder beads (CB), coconut fiber (CF), and polyurethane foam (PUF) for diesel oil removal from artificial seawater. The mixed biofilm formed by using CB, CF, and PUF as immobilization supports degraded 90, 91, and 95% of diesel oil (DO) with the initial concentration of 17.2 g/L, respectively, after 14 days of incubation. The adsorption of DO on different systems was accountable for the removal of 12-16% hydrocarbons for different carriers. To the best of our knowledge, this is the first report on diesel oil degradation by purple photosynthetic bacterial biofilms on different carriers. Moreover, using carriers attaching purple photosynthetic bacteria to remove diesel oil in large scale is considered as an essential method for the improvement of a cost-effective and efficient bioremediation manner. This study can be a promising approach to eliminate DO from oil-contaminated seawater.

Survival and Behavior of Encapsulated Probiotics (Lactobacillus plantarum) in Calcium-Alginate-Soy Protein Isolate-Based Hydrogel Beads in Different Processing Conditions (pH and Temperature) and in Pasteurized Mango Juice.

Hybrid alginate-soy protein isolate-based hydrogel beads were prepared and evaluated to enhance the survival of the encapsulated probiotics (Lactobacillus plantarum) during heat processing to incorporate in mango juice. The solutions of sodium alginate-soy protein isolate (SA-SPI) with probiotic cells were dropped into the gelation bath containing calcium chloride (3% w/v) solution to develop various types of hydrogel beads. The level of survival of probiotics in encapsulated beads under acidic conditions (pH 2, 3, and 6.5) and bile salt (0.5 and 1.0% w/v) was evaluated. The survival of the encapsulated probiotics to thermal processing was evaluated by treating the beads in saline solution (0.9% w/v) at 30, 50, 63, and 72°C. The encapsulated probiotic bacteria were found alive even after treatment at 72°C for 90 s. Most of the free cells did not survive at the temperature higher than 50°C and very low pH (pH 2 and 3). The survival of probiotic cells was found higher with the hybrid hydrogel beads containing alginate and soy protein isolate (1:8 w/w). Furthermore, mango juice fortified with encapsulated L. plantarum in hydrogel beads was subjected to thermal pasteurization at 72°C for 90 s.

Replica moulded poly(dimethylsiloxane) microwell arrays induce localized endothelial cell immobilization for coculture with pancreatic islets.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.

Design of capillary microfluidics for spinning cell-laden microfibers.

This protocol describes the design of capillary microfluidics for spinning bioactive (cell-laden) microfibers for three-dimensional (3D) cell culture and tissue-engineering applications. We describe the assembly of three types of microfluidic systems: (i) simple injection capillary microfluidics for the spinning of uniform microfibers; (ii) hierarchical injection capillary microfluidics for the spinning of core-shell or spindle-knot structured microfibers; and (iii) multi-barrel injection capillary microfluidics for the spinning of microfibers with multiple components. The diverse morphologies of these bioactive microfibers can be further assembled into higher-order structures that are similar to the hierarchical structures in tissues. Thus, by using different types of capillary microfluidic devices, diverse styles of microfibers with different bioactive encapsulation can be generated. These bioactive microfibers have potential applications in 3D cell culture, the mimicking of vascular structures, the creation of synthetic tissues, and so on. The whole protocol for device fabrication and microfiber spinning takes ~1 d.

Contribution of cellulose synthesis, formation of fibrils and their entanglement to the self-flocculation of Zymomonas mobilis.

Due to the unique Entner-Doudoroff pathway, Zymomonas mobilis has been acknowledged as a potential host to be engineered for biorefinery to produce biofuels and biobased chemicals. The self-flocculation of Z. mobilis can make the bacterial cells self-immobilized within bioreactors for high density to improve product productivities, and in the meantime enhance their tolerance to stresses, particularly product inhibition and the toxicity of byproducts released during the pretreatment of lignocellulosic biomass. In this work, we explored mechanism underlying such a phenotype with the self-flocculating strain ZM401 developed from the regular non-flocculating strain ZM4. Cellulase de-flocculation and the restoration of the self-flocculating phenotype for the de-flocculated bacterial cells subjected to culture confirmed the essential role of cellulose biosynthesis in the self-flocculation of ZM401. Furthermore, the deactivation of both Type I and Type IV restriction-modification systems was performed for ZM4 and ZM401 to improve their transformation efficiencies. Comparative genome analysis detected the deletion of a thymine from ZMO1082 in ZM401, leading to a frame-shift mutation for the putative gene to be integrated into the neighboring downstream gene ZMO1083 encoding the catalytic subunit A of cellulose synthase, and consequently created a new gene to encode a larger transmembrane protein BcsA_401 for more efficient synthesis of cellulose as well as the development of cellulose fibrils and their entanglement for the self-flocculation of the mutant. These speculations were confirmed by the morphological observation of the bacterial cells under scanning electron microscopy, the impact of the gene deletion on the self-flocculation of ZM401, and the restoration of the self-flocculating phenotype of ZM401 ΔbcsA by the gene complementation. The progress will lay a foundation not only for fundamental research in deciphering molecular mechanisms underlying the self-flocculation of Z. mobilis and stress tolerance associated with the morphological change but also for technological innovations in engineering non-flocculating Z. mobilis and other bacterial species with the self-flocculating phenotype.

Alginate-perlite encapsulated Pseudomonas putida A (ATCC 12633) cells: Preparation, characterization and potential use as plant inoculants.

Microbial immobilization can be used to prepare encapsulated inoculants. Here, we characterize and describe the preparation of Ca-alginate-perlite microbeads loaded with cells of plant growth-promoting Pseudomonas putida A (ATCC 12633), for their future application as agricultural inoculants. The microbeads were prepared by dropwise addition of a CaCl2-paraffin emulsion mixture to an emulsion containing alginate 2% (w/v), perlite 0.1-0.4% (w/v) and bacterial suspension in 0.9% NaCl (1010 CFU/mL). For all perlite concentrations used, microbead size was 90-120 µm, the trapped population was 108 CFU/g microbeads and the increase in mechanical stability was proportional to perlite concentration. Microbeads containing 0.4% (w/v) perlite were able to release bacteria into the medium after 30 days of incubation. When we evaluated how P. putida A (ATCC 12633) entrapped in Ca-alginate-perlite (0.4% (w/v)) microbeads colonized the Arabidopsis thaliana rhizosphere, an increase in colonization over time was detected (from an initial 2.1 × 104 to 9.2 × 105 CFU/g soil after 21 days). With this treatment, growth promotion of A. thaliana occurred with an increase in the amount of proteins, and in root and leaf biomass. It was concluded that the microbeads could be applied as possible inoculants, since they provide protection and a controlled release of microorganisms into the rhizosphere.

Single-Cell Microgels: Technology, Challenges, and Applications.

Single-cell-laden microgels effectively act as the engineered counterpart of the smallest living building block of life: a cell within its pericellular matrix. Recent breakthroughs have enabled the encapsulation of single cells in sub-100-µm microgels to provide physiologically relevant microniches with minimal mass transport limitations and favorable pharmacokinetic properties. Single-cell-laden microgels offer additional unprecedented advantages, including facile manipulation, culture, and analysis of individual cell within 3D microenvironments. Therefore, single-cell microgel technology is expected to be instrumental in many life science applications, including pharmacological screenings, regenerative medicine, and fundamental biological research. In this review, we discuss the latest trends, technical challenges, and breakthroughs, and present our vision of the future of single-cell microgel technology and its applications.

Hollow microcarriers for large-scale expansion of anchorage-dependent cells in a stirred bioreactor.

With recent advances in biotechnology, mammalian cells are used in biopharmaceutical industries to produce valuable protein therapeutics and investigated as effective therapeutic agents to permanently degenerative diseases in cell based therapy. In these exciting and actively expanding fields, a reliable, efficient, and affordable platform to culture mammalian cells on a large scale is one of the most vital necessities. To produce and maintain a very large population of anchorage-dependent cells, a microcarrier-based stirred tank bioreactor is commonly used. In this approach, the cells are exposed to harmful hydrodynamic shear stress in the bioreactor and the mass transfer rates of nutrients and gases in the bioreactor are often kept below an optimal level to prevent cellular damages from the shear stress. In this paper, a hollow microcarrier (HMC) is presented as a novel solution to protect cells from shear stress in stirred bioreactors, while ensuring sufficient and uniform mass transfer rate of gases and nutrients. HMC is a hollow microsphere and cells are cultured on its inner surface to be protected, while openings on the HMC provide sufficient exchange of media inside the HMC. As a proof of concept, we demonstrated the expansion of fibroblasts, NIH/3T3 and the expansion and cardiac differentiation of human induced pluripotent stem cells, along with detailed numerical analysis. We believe that the developed HMC can be a practical solution to enable large-scale expansion of shear-sensitive anchorage-dependent cells in an industrial scale with stirred bioreactors.

Microencapsulation extends mycelial viability of Streptomyces lividans 66 and increases enzyme production.

BACKGROUND: Filamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics. RESULTS: Here, we applied immobilization of Streptomyces lividans 66 using alginate as semi-solid matrix. This alginate-mediated micro-encapsulation increased the production of the extracellular enzyme tyrosinase more than three-fold. The increased production was accompanied by extended viability of the mycelium and a dramatic reduction in the release of intracellular proteins into the culture broth. CONCLUSIONS: Our data demonstrate the utility of micro-encapsulation as a powerful technique to achieve higher yields and lower downstream-processing costs of streptomycetes.

Bacterial electroactivity and viability depends on the carbon nanotube-coated sponge anode used in a microbial fuel cell.

The anode material is vital to improve the power generation of a microbial fuel cell (MFC). In this study, a carbon nanotube (CNT)-coated sponge with macro-porous structure, high surface area, and high conductivity was constructed as an anode to encapsulate Escherichia coli K12 (E. coli K12) cells. To achieve high power generation of the MFC, the optimal concentration of the CNT coating the sponge was found to be 30mgmL-1. At this concentration, a maximum power density of 787Wm-3 and a chemical oxygen demand (COD) removal of 80.9% were obtained with a long stable electricity generation process in batch mode. This indicates that the biofilm on the CNT (30mgmL-1)-coated sponge possessed excellent electroactivity and stability. Scanning electron microscope (SEM) images confirmed that the CNT-coated sponge provided a suitable microenvironment for E. coli K12 cells to maintain their attachment and colonization. Additionally, a CNT-dependent viability phenomenon of the E. coli K12 cells was discovered after electricity generation. This CNT-dependent viability of the E. coli K12 cells was stable and sustainable after storage at -20°C in a milk tube for one year.

Application of Hydrogels in Cartilage Tissue Engineering.

BACKGROUND: Cartilage has limited ability for self-repairing, prompting the search for cartilage substitutes that can repair cartilage defects. Hydrogels have attracted attention as cartilage substitutes, since their mechanical properties, swelling ability and lubricating behavior are similar to extracellular matrix of articular cartilage. Hydrogels can be of natural, synthetic or hybrid origin, and hydrogels can encapsulate stem cells and/or be loaded with growth factors to promote cell differentiation into a chondrogenic phenotype. OBJECTIVE AND RESULTS: This review summarizes basic research advances in using hydrogels to repair cartilage defects. The raw materials, stem cells and growth factors used to prepare hydrogels are discussed. CONCLUSION: Substantial success has been achieved in small animal models of cartilage repair and regeneration, but further research is needed to improve hydrogels' mechanical properties and their integration with surrounding tissues.

Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation.

BACKGROUND: Mesenchymal stem cells (MSCs) hold promising translational potential in cartilage regeneration. However, the efficacy of MSC-based tissue engineering is not satisfactory in the treatment of cartilage defect because of the inevitable cellular functional changes during ex vivo cell expansion. How to maintain the chondrogenic capacity of MSCs to improve their therapeutic outcomes remains an outstanding question. METHODS: Bone marrow-derived MSCs were firstly primed in chondrogenic induction medium which was then replaced with normal growth medium to attain the manipulated cells (M-MSCs). Methacrylated hyaluronic acid (MeHA) was synthesized as a scaffold to encapsulate the cells. The MSC- or M-MSC-laden constructs were treated with dynamic compressive loading (DL) in a bioreactor or with free loading (FL) for 14 days. Afterwards, the constructs were implanted in nude mice or rat models of osteochondral defects to test their efficiency in cartilage regeneration or repair. RESULTS: Data showed that the resulting M-MSCs exhibited superior chondrogenic differentiation potential and survivability compared with untreated MSCs. More importantly, we found that DL significantly promoted neocartilage formation in the MeHA hydrogel encapsulated with M-MSCs after 30 days of implantation in nude mice. Furthermore, the constructs laden with M-MSCs after DL for 14 days significantly enhanced cartilage healing in a rat model of osteochondral defect. CONCLUSIONS: Findings from this study highlight the importance of maintaining chondrogenic potential of MSCs by in-vitro chondrogenic preconditioning and a synergistic effect of mechanical stimulation in cartilage engineering, which may shed light on the stem cell-based tissue engineering for cartilage repair.

Microbial lipopeptide production and purification bioprocesses, current progress and future challenges.

Lipopeoptides are amphiphilic compounds combining interesting physicochemical properties and biological activities. Due to their high foaming capacity in aerated bioreactor, the development of scalable bioprocesses for their production is a major bottleneck. In addition, the genes involved in the biosynthesis of these lipopeptides are mainly regulated by the quorum sensing, a global regulatory mechanism depending on cell density and known to be activated in biofilms. Several approaches have thus been considered in literature taking into account two criteria, on one side, to favor, control or avoid foam formation and on the other side, to use planktonic or immobilized (biofilm) cells. These different bioprocesses are discussed in the present review along with the purification strategies proposed for extracting and concentrating these biosurfactants.

Repeated batch fermentation of immobilized E. coli expressing Vitreoscilla hemoglobin for long-term use.

This study describes an efficient and reusable process for ethanol production from medium containing whey powder, using alginate immobilized ethanologenic E. coli strains either expressing (TS3) or not expressing (FBR5) Vitreoscilla hemoglobin. Reuseabilities of the FBR5 and TS3 strains were investigated regarding their ethanol production capacities over the course of 15 successive 96-h batch fermentations. The ethanol production was fairly stable over the entire duration of the experiment, with strain TS3 maintaining a substantial advantage over strain FBR5. Storage of both strains in 2 different solutions for up to 60 d resulted in only a modest loss of ethanol production, with strain TS3 consistently outperforming strain FBR5 by a substantial amount. Strains stored for 15 or 30 d maintained their abilities to produce ethanol without dimunition over the course of 8 successive batch fermentations; again strain TS3 maintained a substantial advantage over strain FBR5 throughout the entire experiment. Thus, immobilization is a useful strategy to maintain the advantage in ethanol productivity afforded by expression of Vitreoscilla hemoglobin over long periods of time and large numbers of repeated batch fermentations, including, as in this case, using media with food processing wastes as the carbon source.

Further insights into the impact of mouse follicle stage on graft outcome in an artificial ovary environment.

STUDY QUESTION: Are mouse preantral follicles differently affected by isolation, encapsulation and/or grafting procedures according to stage? SUMMARY ANSWER: Isolated secondary follicles showed superior ability to survive and grow after transplantation, which was not related to a particular effect of the isolation and/or grafting procedure, but rather to their own ability to induce neoangiogenesis. WHAT IS KNOWN ALREADY: Isolated and encapsulated mouse preantral follicles can survive (6-27%) and grow (80-100%) in a fibrin matrix with a low concentration of fibrinogen and thrombin (F12.5/T1) after short-term transplantation. STUDY DESIGN, SIZE, DURATION: An in vivo experimental model using 20 donor Naval Medical Research Institute (NMRI) mice (6-25 weeks of age) and 14 recipient severe combined immunodeficient (SCID) mice (11-39 weeks of age) was applied. Each NMRI mouse underwent mechanical disruption of both ovaries and isolation of primordial-primary and secondary follicles with ovarian stromal cells, in order to encapsulate them in an F12.5/T1 matrix. Twelve out of 40 fibrin clots were immediately fixed as controls (D0) (10 for histology and 2 for scanning electron microscopy [SEM]) and the others (n = 28) were grafted to the inner part of the peritoneum for 2 (16 fibrin clots) or 7 (12 fibrin clots) days (D2 and D7). PARTICIPANTS/MATERIALS, SETTING, METHODS: This study involved the participation of the Gynecology Research Unit (Universitè Catholique de Louvain) and the Physiological Sciences Department (University of Brasília). Specific techniques were used to analyze the follicle recovery rate (hematoxylin-eosin staining), vascularization (CD34) and follicle ultrastructure (transmission electron microscopy [TEM] and SEM). MAIN RESULTS AND THE ROLE OF CHANCE: After follicle isolation and encapsulation, a statistically higher percentage of normal follicles was observed in the secondary group (62%) than in the primordial-primary group (47%). Follicle recovery rates were 34% and 62% for primordial-primary and secondary follicles on D2, respectively, and 12% and 42% on D7, confirming that secondary follicles survive better than primordial-primary follicles after grafting. Concerning vascularization, both follicle stages exhibited similar vascularization to that seen in control mouse ovary on D7, but a significantly higher number of vessels and greater vessel surface area were detected in the secondary follicle group. Despite structural differences in fiber density between fibrin clots and ovarian tissue observed by SEM and TEM, preantral follicles appeared to be well encapsulated in the matrix, also showing a normal ultrastructure after grafting. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results during the isolation procedure, we encapsulated a significantly higher number of round structures in the primordial-primary group than in the secondary group, which could partially explain the lower recovery rate of early-stage follicles in our previous study. However, it is not excluded that the physical and mechanical properties of the fibrin matrix may also play a role in follicle survival and growth, so further investigations are needed. WIDER IMPLICATIONS OF THE FINDINGS: This research represents one more key step in the creation of the artificial ovary. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) to C.A. Amorim as a research associate at FRS-FNRS and (grant 5/4/150/5 awarded to M.M. Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil) (grant #013/14 CAPES/WBI awarded to C.M. Lucci, with F. Paulini receiving a post-doctoral fellowship), and Wallonie-Bruxelles International, and donations from the Ferrero family. None of the authors have any competing interests to declare in relation to the topic.

Calcium pre-conditioning substitution enhances viability and glucose sensitivity of pancreatic beta-cells encapsulated using polyelectrolyte multilayer coating method.

Type I diabetics are dependent on daily insulin injections. A therapy capable of immunoisolating pancreatic beta-cells and providing normoglycaemia is an alternative since it would avoid the late complications associated with insulin use. Here, 3D-concave agarose micro-wells were used to culture robust pancreatic MIN-6 cell spheroids within 24 hours that were shown to exhibit cell-cell contact and uniform size (201 ± 2 µm). A polyelectrolyte multilayer (PEM) approach using alginate and poly-l-lysine was employed to coat cell spheroids. In comparison to conventional PEM, use of a novel Ca2+ pre-coating step enhanced beta-cells viability (89 ± 6%) and metabolic activity since it reduced the toxic effect of the cationic polymer. Pre-coating was achieved by treating MIN-6 spheroids with calcium chloride, which enabled the adhesion of anionic polymer to the cells surface. Pre-coated cells coated with four bilayers of polymers were successfully immunoisolated from FITC-mouse antibody and pro-inflammatory cytokines. Novel PEM coated cells were shown to secret significantly (P < 0.05) different amounts of insulin in response to changes in glucose concentration (2 vs. 20 mM). This work presents a 3D culture model and novel PEM coating procedure that enhances viability, maintains functionality and immunoisolates beta-cells, which is a promising step towards an alternative therapy to insulin.

Facile and effective synthesis of adsorbent - utilization of yeast cells immobilized in sodium alginate beads for the adsorption of phosphorus in aqueous solution.

We compared the adsorption efficiency of phosphates onto Ca-alginate immobilized yeast and freely suspended yeast under different conditions of pH and temperature. The results clearly demonstrated that the adsorption efficiency onto Ca-alginate immobilized yeast was better than that of freely suspended yeast, and reached a maximum at pH 9.17 and 35 °C. Scanning electron microscopy was used to characterize the morphology of Ca-alginate immobilized yeast. Fitting the adsorption equilibrium data to existing models showed that the Freundlich isotherm model described the process better than the Langmuir model, and the process of adsorption followed pseudo-first-order kinetics. During the initial period of experiment, external diffusion was a key rate-controlling step, and intraparticle diffusion also contributed to the mass transport. The thermodynamic properties (Gibbs free energy change of -15.143 kJ/mol, enthalpy change of 274.118 kJ/mol, and entropy change of 290 J/(mol K)) indicated that the adsorption process was endothermic.