Bojana Obradovic

Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering.

Cell seeding of three‐dimensional polymer scaffolds is the first step of the cultivation of engineered tissues in bioreactors. Seeding requirements of large scaffolds to make implants for potential clinical use include: (a) high yield, to maximize the utilization of donor cells, (b) high kinetic rate, to minimize the time in suspension for anchorage‐dependent and shear‐sensitive cells, and (c) high and spatially uniform distribution of attached cells, for rapid and uniform tissue regeneration. Highly porous, fibrous polyglycolic acid scaffolds, 5–10 mm in diameter and 2–5 mm thick, were seeded with bovine articular chondrocytes in well‐mixed spinner flasks. Essentially, all cells attached throughout the scaffold volume within 1 day. Mixing promoted the formation of 20–32‐μm diameter cell aggregates that enhanced the kinetics of cell attachment without compromising the uniformity of cell distribution. The kinetics and possible mechanisms of cell seeding were related to the formation of cell aggregates by a simple mathematical model that can be used to optimize seeding conditions for cartilage tissue engineering.

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage.

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.

Method for Quantitative Analysis of Glycosaminoglycan Distribution in Cultured Natural and Engineered Cartilage

AbstractCartilage tissue engineering can provide a valuable tool for controlled studies of tissue development. As an example, analysis of the spatial distribution of glycosaminoglycans (GAG) in sections of cartilaginous tissues engineered under different culture conditions could be used to correlate the effects of environmental factors with the structure of the regenerated tissue. In this paper we describe a computer-based technique for quantitative analysis of safranin-O stained histological sections, using low magnification light microscopy images. We identified a parameter to quantify the intensity of red color in the sections, which in turn was proportional to the biochemically determined wet weight fraction of GAG in corresponding tissue samples, and to describe the spatial distribution of GAG as a function of depth from the section edge. A broken line regression model was then used to determine the thickness of an external region, with lower GAG fractions, and the spatial rate of change in GAG content. The method was applied to the quantitatation of GAG distribution in samples of natural and engineered cartilage, cultured for 6 weeks in three different vessels: static flasks, mixed flasks, and rotating bioreactors.

Electrostatic generation of alginate microbeads loaded with brewing yeast

Abstract The substantial concern with the possible use of immobilized yeast cells for beer production is reduction of internal mass transfer resistance during continuous fermentation. One way to minimise this problem is to use small-diameter beads. The effects of bead diameters in the range 0.3–2.0 mm on yeast cell immobilization and growth over a short-term cultivation were investigated. Bead diameters in the range 0.5–0.6 mm were optimal and provided rapid cell growth and the highest final cell concentration (2.33×10 9 cells/ml of beads). Electrostatic droplet generation was investigated as a technique for production of alginate microbeads. The effects of applied potential, internal needle diameter and electrode position on bead diameter were assessed. The results have shown that this method can be used for controlled production of small-size microbeads loaded with yeast. Depending on applied conditions it was possible to produce the beads in the range 250 μm–2.0 mm in diameter.

Alginate-immobilized lipase by electrostatic extrusion for the purpose of palm oil hydrolysis in lecithin/isooctane system

Lipase from Candida rugosa was immobilized in alginate beads for possible application in non-aqueous or microaqueous reaction systems. An electrostatic droplet generation technique was used for production of small diameter (<1 mm) lipase-alginate beads. This technique provided negligible loss of the lipase (immobilization efficiencies were 98.2–99.2%). Under optimal immobilization conditions (applied potential 4.9 kV, needle gauge 21, 2% sodium alginate solution) the lipase-alginate beads, 0.65 mm in diameter, retained enzyme activity equivalent to 75% that of free lipase. The activity of the immobilized lipase was verified in the reaction of palm oil hydrolysis in a lecithin/isooctane system. The reaction rate with alginate-immobilized lipase was lower than with the free enzyme but the final conversions were approximately the same (∼74%). Immobilized lipase could be used for up to three reaction cycles with little loss of activity.

Novel kinetic model of the removal of divalent heavy metal ions from aqueous solutions by natural clinoptilolite

Removal of heavy metal ions from aqueous solutions using zeolites is widely described by pseudo-second order kinetics although this model may not be valid under all conditions. In this work, we have extended approaches used for derivation of this model in order to develop a novel kinetic model that is related to the ion exchange mechanism underlying sorption of metal ions in zeolites. The novel model assumed two reversible steps, i.e. release of sodium ions from the zeolite lattice followed by bonding of the metal ion. The model was applied to experimental results of Cu(II) sorption by natural clinoptilolite-rich zeolitic tuff at different initial concentrations and temperatures and then validated by predictions of ion exchange kinetics of other divalent heavy metal ions (i.e. Mn(II), Zn(II) and Pb(II)). Model predictions were in excellent agreements with experimental data for all investigated systems. In regard to the proposed mechanism, modeling results implied that the sodium ion release rate was constant for all investigated metals while the overall rate was mainly determined by the rate of heavy metal ion bonding to the lattice. In addition, prediction capabilities of the novel model were demonstrated requiring one experimentally determined parameter, only.

A comprehensive approach to in vitro functional evaluation of Ag/alginate nanocomposite hydrogels.

In this work, we present a comprehensive approach to evaluation of alginate microbeads with included silver nanoparticles (AgNPs) at the concentration range of 0.3-5mM for potential biomedical use by combining cytotoxicity, antibacterial activity, and silver release studies. The microbeads were investigated regarding drying and rehydration showing retention of ∼ 80-85% of the initial nanoparticles as determined by UV-vis and SEM analyses. Both wet and dry microbeads were shown to release AgNPs and/or ions inducing similar growth delays of Staphylococcus aureus and Escherichia coli at the total released silver concentrations of ∼ 10 μg/ml. On the other hand, these concentrations were highly toxic for bovine chondrocytes in conventional monolayer cultures while nontoxic when cultured in alginate microbeads under biomimetic conditions in 3D perfusion bioreactors. The applied approach outlined directions for further optimization studies demonstrating Ag/alginate microbeads as potentially attractive components of soft tissue implants as well as antimicrobial wound dressings.

A validated model of GAG deposition, cell distribution, and growth of tissue engineered cartilage cultured in a rotating bioreactor.

In this work a new phenomenological model of growth of cartilage tissue cultured in a rotating bioreactor is developed. It represents an advancement of a previously derived model of deposition of glycosaminoglycan (GAG) in engineered cartilage by (i) introduction of physiological mechanisms of proteoglycan accumulation in the extracellular matrix (ECM) as well as by correlating (ii) local cell densities and (iii) tissue growth to the ECM composition. In particular, previously established predictions and correlations of local oxygen concentrations and GAG synthesis rates are extended to distinguish cell secreted proteoglycan monomers free to diffuse in cell surroundings and outside from the engineered construct, from large aggrecan molecules, which are constrained within the ECM and practically immovable. The model includes kinetics of aggregation, that is, transformation of mobile GAG species into immobile aggregates as well as maintenance of the normal ECM composition after the physiological GAG concentration is reached by incorporation of a product inhibition term. The model also includes mechanisms of the temporal evolution of cell density distributions and tissue growth under in vitro conditions. After a short initial proliferation phase the total cell number in the construct remains constant, but the local cell distribution is leveled out by GAG accumulation and repulsion due to negative molecular charges. Furthermore, strong repulsive forces result in expansion of the local tissue elements observed macroscopically as tissue growth (i.e., construct enlargement). The model is validated by comparison with experimental data of (i) GAG distribution and leakage, (ii) spatial‐temporal distributions of cells, and (iii) tissue growth reported in previous works. Validation of the model predictive capability—against a selection of measured data that were not used to construct the model—suggests that the model successfully describes the interplay of several simultaneous processes carried out during in vitro cartilage tissue regeneration and indicates that this approach could also be attractive for application in other tissue engineering systems. Biotechnol. Bioeng. 2010. 105: 842–853.

Principles of tissue culture and bioreactor design

In this chapter, we review the principles of cell culture and bioreactor design in the context of tissue engineering. In the first part, we describe the principles of cell and tissue culture, with emphasis on the initiation of culture, transformation, preservation, and validation of the cells and differentiation methods that utilize simple and well-established petri dish protocols. In the second part, we describe the design and operation of tissue engineering bioreactors, with focus on mass transport considerations associated with environmental control, and the provision of biophysical signals associated with the modulation of cell differentiation and functional tissue assembly. We provide eight examples of representative bioreactor designs that illustrate how some of the differentiation factors are provided in a physiologically relevant fashion.

Flow regimes and liquid mixing in a draft tube gas-liquid-solid fluidized bed

Abstract Flow behavior of gas bubbles, liquid and particles, and mixing in the liquid phase were studied in draft and annulus of a draft tube gas-liquid-solid fluidized bed. The experimental unit was a 80mm ID × 1.5 m column with an 50mm ID internal draft tube. A 2-D unit of the same size and geometry was used to assess the local concentrations and velocities of liquid, particles and air bubbles. Fluid and particle dynamics in the draft and annular regions were analyzed using the drift-flux model extended to the 3- phase flow, and in terms of the effective buoyancy of solid particles. Liquid mixing was studied based on the tracer-response technique, using the model of axial dispersion.