Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain.

Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration.

Fathers that are born small program alterations in the next-generation preimplantation rat embryos.

BACKGROUND: Low birth weight is associated with increased risk of adult cardiovascular and metabolic disease development, with recent studies highlighting transmission to subsequent generations via both maternal and paternal lines. However, the timing of parent-specific programming of disease risk to the next generation remains to be characterized. OBJECTIVE: The aim of this study was to examine how paternal low birth weight affects the cellular and molecular physiology of the next-generation [second-generation (F2)] blastocysts, before uterine implantation. METHODS: Uteroplacental insufficiency was surgically induced in Wistar Kyoto pregnant rats in late gestation, giving rise to first-generation restricted (born small) and sham-operated control (normal birth weight) male offspring, respectively. First-generation restricted and control male rats were naturally mated with normal females. RESULTS: Resultant F2 blastocysts derived from restricted males displayed reduced expression of growth regulatory genes of the mammalian target of rapamycin pathway compared with F2 control blastocysts (9-74%; P < 0.05). No differences were found in F2 restricted blastocyst structural characteristics, cell number, or carbohydrate utilization at the time of blastocyst retrieval or after 24 h of in vitro culture. However, histidine, methionine, pyruvate, serine, and tryosine consumption and aspartate and leucine production were greater in F2 restricted outgrowth than in controls (P < 0.05). CONCLUSIONS: The findings from this study clearly indicate that male rat offspring born small, arising from uteroplacental insufficiency, have physiologic alterations that manifest as modifications in gene expression levels and nutrient metabolism of F2 blastocysts, even in the absence of overt cellular growth differences. These data demonstrate that growth restriction and associated disease risk have the capacity to be transmitted to the next generation of offspring via the male germ line and is manifest as early as the blastocyst stage of development.

Low female birth weight and advanced maternal age programme alterations in next-generation blastocyst development.

Low birth weight is associated with an increased risk for adult disease development with recent studies highlighting transmission to subsequent generations. However, the mechanisms and timing of programming of disease transmission to the next generation remain unknown. The aim of this study was to examine the effects of low birth weight and advanced maternal age on second-generation preimplantation blastocysts. Uteroplacental insufficiency or sham surgery was performed in late-gestation WKY pregnant rats, giving rise to first-generation (F1) restricted (born small) and control offspring respectively. F1 control and restricted females, at 4 or 12 months of age, were naturally mated with normal males. Second-generation (F2) blastocysts from restricted females displayed reduced expression of genes related to growth compared with F2 control (P<0.05). Following 24 h culture, F2 restricted blastocysts had accelerated development, with increased total cell number, a result of increased trophectoderm cells compared with control (P<0.05). There were alterations in carbohydrate and serine utilisation in F2 restricted blastocysts and F2 restricted outgrowths from 4-month-old females respectively (P<0.05). F2 blastocysts from aged restricted females were developmentally delayed at retrieval, with reduced total cell number attributable to reduced trophectoderm number with changes in carbohydrate utilisation (P<0.05). Advanced maternal age resulted in alterations in a number of amino acids in media obtained from F2 blastocyst outgrowths (P<0.05). These findings demonstrate that growth restriction and advanced maternal age can alter F2 preimplantation embryo physiology and the subsequent offspring growth.

Soluble ligands and their receptors in human embryo development and implantation.

Extensive evidence suggests that soluble ligands and their receptors mediate human preimplantation embryo development and implantation. Progress in this complex area has been ongoing since the 1980s, with an ever-increasing list of candidates. This article specifically reviews evidence of soluble ligands and their receptors in the human preimplantation stage embryo and female reproductive tract. The focus will be on candidates produced by the human preimplantation embryo and those eliciting developmental responses in vitro, as well as endometrial factors related to implantation and receptivity. Pathways to clinical translation, including innovative diagnostics and other technologies, are also highlighted, drawing from this collective evidence toward facilitating joint improvements in embryo quality and endometrial receptivity. This strategy could not only benefit clinical outcomes in reproductive medicine but also provide broader insights into the peri-implantation period of human development to improve fetal and neonatal health.

Novel elastomeric fibrous networks produced from poly(xylitol sebacate)2:5 by core/shell electrospinning: fabrication and mechanical properties.

Fabrication of nonlinear elastic materials that resemble biological tissues remains a challenge in biomaterials research. Here, a new fabrication protocol to produce elastomeric fibrous scaffolds was established, using the core/shell electrospinning technique. A prepolymer of poly(xylitol sebacate) with a 2:5mol ratio of xylitol:sebacic acid (PXS2:5) was first formulated, then co-electrospun with polyvinyl alcohol (PVA - 95,000Mw). After cross-linking of core polymer PXS2:5, the PVA shells were rinsed off in water, leaving a porous elastomeric network of PXS2:5 fibres. Under aqueous conditions, the PXS2:5 fibrous scaffolds exhibited stable, nonlinear J-shaped stress-strain curves, with large average rupture elongation (76%) and Young׳s modulus (~1.0MPa), which were in the range of muscle tissue. Rupture elongation of PXS2:5 was also much higher when electrospun, compared to 2D solid sheets (45%). In direct contact with cell monolayers under physiological conditions, PXS2:5 scaffolds were as biocompatible as those made of poly-l-lactic acid (PLLA), with improvements over culture medium alone. In conclusion, the newly developed porous PXS2:5 scaffolds show tissue-like mechanical properties and excellent biocompatibility, making them very promising for bioengineering of soft tissues and organs.

Effects of calcium phosphate/chitosan composite on bone healing in rats: calcium phosphate induces osteon formation.

Vascularization of an artificial graft represents one of the most significant challenges facing the field of bone tissue engineering. Over the past decade, strategies to vascularize artificial scaffolds have been intensively evaluated using osteoinductive calcium phosphate (CaP) biomaterials in animal models. In this work, we observed that CaP-based biomaterials implanted into rat calvarial defects showed remarkably accelerated formation and mineralization of new woven bone in defects in the initial stages, at a rate of ∼60 µm/day (0.8 mg/day), which was considerably higher than normal bone growth rates (several µm/day, 0.1 mg/day) in implant-free controls of the same age. Surprisingly, we also observed histological evidence of primary osteon formation, indicated by blood vessels in early-region fibrous tissue, which was encapsulated by lamellar osteocyte structures. These were later fully replaced by compact bone, indicating complete regeneration of calvarial bone. Thus, the CaP biomaterial used here is not only osteoinductive, but vasculogenic, and it may have contributed to the bone regeneration, despite an absence of osteons in normal rat calvaria. Further investigation will involve how this strategy can regulate formation of vascularized cortical bone such as by control of degradation rate, and use of models of long, dense bones, to more closely approximate repair of human cortical bone.

Enzymatic and oxidative degradation of poly(polyol sebacate).

Poly(glycerol sebacate) (PGS) and poly(xylitol sebacate) (PXS) are biodegradable elastomers with tremendous potential in soft tissue engineering. This study was aimed at exploring the enzymatic degradation mechanisms of these polyesters, using biochemical conditions similar to those occurring in vivo. To this end, PGS and PXS (crosslinked at 130 for 2 or 7 (PGS)/12 days (PXS)) were incubated in vitro under physiological conditions in tissue culture media supplemented with either a biodegrading enzyme (esterase), an oxidant species (FeSO4/H2O2 with 0.11 molar ratio of Fe(2+/)H2O2), an oxidant generating enzyme (xanthine oxidase and xanthine) or combinations of these (FeSO4/H2O2 and esterase, or (v) xanthine oxidase/xanthine and esterase), based on their independent effects on polymer degradation. Testing was performed over 35 days of continuous incubation, during which mechanical properties, mass loss, biomaterial thickness and pH value of the culture medium were determined. Degradation kinetics of both PGS and PXS samples were primarily determined by the degree of crosslink density. Esterase and FeSO4/H2O2 accelerated the degradation of both polymers, by promoting hydrolysis and free-radical degradation, although this action was not affected by the presence of xanthine oxidase and xanthine. Degradation of PGS and PXS is primarily mediated by the action of esterase, with free-radical oxidation playing a secondary role, suggesting that both could synergistically affect the biodegradability of biomaterial implants, under more complex biological conditions.

Bone tissue engineering scaffolding: computer-aided scaffolding techniques.

Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).

Mechanically tissue-like elastomeric polymers and their potential as a vehicle to deliver functional cardiomyocytes.

One of the major challenges in the field of biomaterials engineering is the replication of the non-linear elasticity observed in soft tissues. In the present study, non-linearly elastic biomaterials were successfully fabricated from a chemically cross-linked elastomeric poly(glycerol sebacate) (PGS) and thermoplastic poly(L-lactic acid) (PLLA) using the core/shell electrospinning technique. The spun fibrous materials, containing a PGS core and PLLA shell, demonstrated J-shaped stress-strain curves, and having ultimate tensile strength, rupture elongation, and stiffness constants respectively comparable to muscle tissue properties. In vitro evaluations also showed that PGS/PLLA fibrous biomaterials possess excellent biocompatibility, capable of supporting human stem-cell-derived cardiomyocytes over several weeks in culture. Therefore, the core/shell electrospun elastomeric materials provide a new potential scaffold to support cells in the therapy of a wide range of soft tissues exposed to cyclic deformation, such as tendon, ligament, cardiac or smooth muscle and lung epithelium.

Method to impart electro- and biofunctionality to neural scaffolds using graphene-polyelectrolyte multilayers.

Electroactive scaffolds that are passively conductive and able to transmit applied electrical stimuli are of increasing importance for neural tissue engineering. Here, we report a process of rendering both 2D and 3D polymer scaffolds electrically conducting, while also enhancing neuron attachment. Graphene-heparin/poly-l-lysine polyelectrolytes were assembled via layer-by-layer (LbL) deposition onto 2D surfaces and 3D electrospun nanofibers. The employed LbL coating technique in this work enables the electro- and biofunctionalization of complex 3D scaffold structures. LbL assembly was characterized by a steady mass increase during the in situ deposition process in 2D, with regular step changes in hydrophobicity. Uniform coverage of the graphene/polyelectrolyte coatings was also achieved on nanofibers, with hydrodynamic flow and post-thermal annealing playing an important role in controlling sheet resistance of 2D surfaces and nanofibers. Cell culture experiments showed that both 2D and 3D graphene-PEMs supported neuron cell adhesion and neurite outgrowth, with no appreciable cell death. This electroactive scaffold modification may therefore assist in neuronal regeneration, for creating functional and biocompatible polymer scaffolds for electrical entrainment or biosensing applications.

Progress and challenges in biomaterials used for bone tissue engineering: bioactive glasses and elastomeric composites.

Driven by the increasing economic burden associated with bone injury and disease, biomaterial development for bone repair represents the most active research area in the field of tissue engineering. This article provides an update on recent advances in the development of bioactive biomaterials for bone regeneration. Special attention is paid to the recent developments of sintered Na-containing bioactive glasses, borate-based bioactive glasses, those doped with trace elements (such as Cu, Zn, and Sr), and novel elastomeric composites. Although bioactive glasses are not new to bone tissue engineering, their tunable mechanical properties, biodegradation rates, and ability to support bone and vascular tissue regeneration, as well as osteoblast differentiation from stem and progenitor cells, are superior to other bioceramics. Recent progresses on the development of borate bioactive glasses and trace element-doped bioactive glasses expand the repertoire of bioactive glasses. Although boride and other trace elements have beneficial effects on bone remodeling and/or associated angiogenesis, the risk of toxicity at high levels must be highly regarded in the design of new composition of bioactive biomaterials so that the release of these elements must be satisfactorily lower than their biologically safe levels. Elastomeric composites are superior to the more commonly used thermoplastic-matrix composites, owing to the well-defined elastic properties of elastomers which are ideal for the replacement of collagen, a key elastic protein within the bone tissue. Artificial bone matrix made from elastomeric composites can, therefore, offer both sound mechanical integrity and flexibility in the dynamic environment of injured bone.

In vitro enzymatic degradation of poly (glycerol sebacate)-based materials.

Enzymatic degradation is a major feature of polyester implants in vivo. An in vitro experimental protocol that can simulate and predict the in vivo enzymatic degradation kinetics of implants is of importance not only to our understanding of the scientific issue, but also to the well-being of animals. In this study, we explored the enzymatic degradation of PGS-based materials in vitro, in tissue culture medium or a buffer solution at the pH optima and under static or cyclic mechanical-loading conditions, in the presence of defined concentrations of an esterase. Surprisingly, it was found that the in vitro enzymatic degradation rates of the PGS-based materials were higher in the tissue culture medium than in the buffered solution at the optimum pH 8. The in vitro enzymatic degradation rate of PGS-based biomaterials crosslinked at 125°C for 2 days was approximately 0.6-0.9 mm/month in tissue culture medium, which falls within the range of in vivo degradation rates (0.2-1.5mm/month) of PGS crosslinked at similar conditions. Enzymatic degradation was also further enhanced in relation to mechanical deformation. Hence, in vitro enzymatic degradation of PGS materials conducted in tissue culture medium under appropriate enzymatic conditions can quantitatively capture the features of in vivo degradation of PGS-based materials and can be used to indicate effective strategies for tuning the degradation rates of this material system prior to animal model testing.

Fabrication and characterization of sol-gel derived 45S5 Bioglass®-ceramic scaffolds.

Although Bioglass® has existed for nearly half a century its ability to trigger bone formation and tuneable degradability is vastly superior to other bioceramics, such as SiO(2)-CaO bioactive glasses. The sol-gel process of producing glass foams is well established for SiO(2)-CaO compositions, but not yet established for 45S5 composites containing Na(2)O. In this work the sol-gel derived 45S5 Bioglass® has for the first time been foamed into highly porous three-dimensional scaffolds using a surfactant, combined with vigorous mechanical stirring and subsequent sintering at 1000°C for 2 h. It was found that the mechanical strength of the sintered sol-gel derived Bioglass® scaffolds was significantly improved, attributable to the small fraction of material on the pore walls. More importantly, the compressive strength of the three-dimensional scaffolds produced by this surfactant foaming method could be predicted using Gibson and Ashby's closed cell model of porous networks. A comparative experiment revealed that ion release from the sol-gel derived Bioglass® foams was faster than that of counterparts produced by the replication technique. In vitro evaluation using osteoblast-like cells demonstrated that the sol-gel derived 45S5 Bioglass foams supported the proliferation of viable cell populations on the surface of the scaffolds, although few cells were observed to migrate into the virtually closed pores within the foams. Further work should be focused on modifications of the reaction conditions or alternative foaming techniques to improve pore interconnection.

Optimizing interfacial features to regulate neural progenitor cells using polyelectrolyte multilayers and brain derived neurotrophic factor.

The development of biomaterials with controllable interfacial features which have the capability to instruct cellular behavior are required to produce functional scaffolds for the treatment of spinal cord injury (SCI). Here, poly-ɛ-caprolactone surfaces were biofunctionalized via layer-by-layer (LbL) deposition. The polyelectrolytes employed in this LbL technique were heparin and poly-L-lysine (PLL), the latter being chosen to improve cell adhesion and the subsequent cellular function of in vitrocultured neural progenitor cells. Material characterization results confirmed the deposition of well structured multilayers. Cell culture studies revealed significant differences in the cellular response to these adhesive/nonadhesive (PLL/heparin) polyelectrolyte multilayer (PEM)surfaces, with neurite outgrowth being significantly promoted on the PLL terminating layers. In addition, brain derived neurotrophic factor (BDNF) was adsorbed onto the LbL surfaces. This combined chemical and biological effect was then characterized in terms of neurite length along with the full length/truncated isoform 1 tyrosine kinase receptor (TrkB-FL/TrkB-T1) and growth associated protein-43 mRNA levels. Here, the authors report the differential effect of adsorbed and soluble BDNF of different concentrations. Adsorbed BDNF promoted neurite outgrowth and led to elevated, sustained TrkB mRNA levels. These findings highlight the potential of PEM biofunctionalized surfaces with integrated chemical and neurotrophin supportive cues to overcome SCI inhibitory environments and to promote regeneration.

The mechanical characteristics and in vitro biocompatibility of poly(glycerol sebacate)-bioglass elastomeric composites.

Biodegradable elastomeric materials have gained much recent attention in the field of soft tissue engineering. Poly(glycerol sebacate) (PGS) is one of a new family of elastomers which are promising candidates used for soft tissue engineering. However, PGS has a limited range of mechanical properties and has drawbacks, such as cytotoxicity caused by the acidic degradation products of very soft PGS and degradation kinetics that are too fast in vivo to provide sufficient mechanical support to the tissue. However, the development of PGS/based elastomeric composites containing alkaline bioactive fillers could be a method for addressing these drawbacks and thus may pave the way towards wide clinical applications. In this study, we synthesized a new PGS composite system consisting of a micron-sized Bioglass filler. In addition to much improved cytocompatibility, the PGS/Bioglass composites demonstrated three remarkable mechanical properties. First, contrary to previous reports, the addition of microsized Bioglass increases the elongation at break from 160 to 550%, while enhancing the Young's modulus of the composites by up to a factor of four. Second, the modulus of the PGS/Bioglass composites drops abruptly in a physiological environment (culture medium), and the level of drop can be tuned such that the addition of Bioglass does not harden the composite in vivo and thus the desired compliance required for soft tissue engineering are maintained. Third, after the abrupt drop in modulus, the composites exhibited mechanical stability over an extended period. This latter observation is an important feature of the new composites, because they can provide reliable mechanical support to damaged tissues during the lag phase of the healing process. These mechanical properties, together with improved biocompatibility, make this family of composites better candidates than plastic and related composite biomaterials for the applications of tissue engineering.

Sex-related physiology of the preimplantation embryo.

Male and female preimplantation mammalian embryos differ not only in their chromosomal complement, but in their proteome and subsequent metabolome. This phenomenon is due to a finite period during preimplantation development when both X chromosomes are active, between embryonic genome activation and X chromosome inactivation, around the blastocyst stage. Consequently, prior to implantation male and female embryos exhibit differences in their cellular phenotype. Manifestations of such differences include altered total activity of specific X-linked enzymes and the metabolic pathways they regulate. Subsequently, one would expect to be able to determine differences in the rate of consumption and utilization of specific nutrients between male and female embryos. Data to date on animal models support this, with sex-specific differences in glucose and amino acid utilization being reported for the mouse and cow blastocysts. Such differences in metabolic phenotype may logically be involved in the reported differences in growth rates between preimplantation embryos of different sex. As the fields of proteomics and metabolomics are being increasingly applied to human assisted conception it is prudent to consider how such technologies may be applied to identify sex differences in the human embryo. Such data would have implications far beyond current invasive technologies used to identify the sex of an embryo conceived in vitro for the diagnosis of X-linked diseases.

An elastomeric patch derived from poly(glycerol sebacate) for delivery of embryonic stem cells to the heart.

We hypothesize that a combinatorial approach of ventricle constraint and stem cell therapy would offer a greater benefit for the treatment of heart failure than either strategy alone. A heart patch would serve two therapeutic purposes: biomechanical support and cell delivery. In this study, we describe a hybrid heart patch engineered from a synthetic elastomer, poly(glycerol sebacate) (PGS), supplemented with cardiomyocytes differentiated from human embryonic stem cells (hESCs). In line with two therapeutically relevant considerations, i.e. biocompatibility and cell delivery efficiency, the PGS was (a) pre-conditioned in culture medium for 6 days, and (b) prepared without gelatin coatings to facilitate detachment and delivery of cardiomyocytes following patch implantation. Following pre-conditioning under physiological conditions, the PGS patch material without gelatin coating was found to satisfactorily support cardiomyocyte viability and attachment, with active cell beating for periods of longer than 3 months until interrupted. Dynamic culture studies revealed that cells detached more efficiently from the uncoated surface of PGS than from gelatin-coated PGS. No significant differences were detected between the beating rates of human embryonic stem cell-derived cardiomyocytes on tissue culture plate and the pre-conditioned and gelatin-uncoated PGS. PGS patches sutured over the left ventricle of rats in vivo remained intact over a 2 week period without any deleterious effects on ventricular function. We conclude that PGS is a suitable biomaterial for stem cell-based regeneration strategies to restore cardiomyocyte function, and the hybrid heart patch engineered under optimal conditions would be a promising support device for the cardiac repair.

Biomaterials for spinal cord regeneration: outgrowth of presumptive neuronal precursors on electrospun poly(epsilon)-caprolactone scaffolds microlayered with alternating polyelectrolytes.

The aim of this study was to assess the feasibility of electrospun poly(epsilon)-caprolactone (PCL) scaffolds treated with alternating paly-electrolytes as a controllable three-dimensional adhesive substrate for neuronal progenitors. Unmodified PCL surfaces were generally not supportive of mouse embryonic stem cell (mESC) colony adhesion. However, scaffolds surfaced using layer-by-layer (LbL) deposition of heparin/poly-L-lysine encouraged better local adhesion of mESC colonies, and networking of monolayers containing nestin-positive presumptive neurons, similar to laminin coated controls, as observed by immuno-fluorescence microscopy. Confocal microscopy further revealed depth-wise penetration of mESC nestin-positive cell populations, and orientation along grass topographical features in the LbL scaffolds. LbL deposition therefore appears to provide a satisfactory adhesive substrate for contact and mechanical guidance of neuronal outgrowth in three-dimensions.

Improved oxygen diffusion and mechanical aggregation of tumor colonies in a novel stirred mini-bioreactor.

The feasibility of a novel stirred bioreactor, the rotating aerial disk (RAD) design, was tested in this study. The novelty lies in its method of medium recirculation by convective airflow using a non-contact planar disc, a variation on a physically defined theoretical model. Computational predictions of improved oxygenation were confirmed by increases in measured dissolved oxygen, even at Reynolds numbers (100-200) where flow is mostly laminar. EL-4 mouse lymphoma cells grown for the first time as suspension cultures in the RAD bioreactor, were mechanically re-organization into dense, circular three-dimensional colonies (diameter 3-5 mm, thickness 5-800 microm), more rapidly than we have observed previously. Cell proliferation in the RAD vessels was similar to static cultures, although lactate production from glucose was significantly lower, suggesting a shift toward aerobic glycolysis. This possible reversal of the 'Warburg effect' was accompanied by a decrease in mitochondrial activity, perhaps reflecting a more quiescent cytoplasmic state. The RAD device may be useful as scalable, three-dimensional solid tumor model under more physiological conditions then static culture.

A bioreactor model of mouse tumor progression.

The present study represents an investigation of a novel stirred bioreactor for culture of a transformed cell line under defined hydrodynamic conditions in vitro. Cell colonies of the EL-4 mouse lymphoma cell line grown for the first time in a rotating disc bioreactor (RDB), were observed to undergo changes in phenotype in comparison to standard, static flask cultures. RDB cultures, with or without agitation, promoted the formation of adherent EL-4 cell plaques that merged to form contiguous tumor-like masses in longer-term cultures, unlike the unattached spheroid aggregates of flask cultures. Plaques grown under agitated conditions were further altered in morphology and distribution in direct response to fluid mechanical stimuli. Plaque colonies growth in RDBs with or without agitation also exhibited significant increases in production of interleukin-4 (IL-4) and lactate, suggesting an inducible "Warburg effect." Increases in cell biomass in RDB cultures were no different to flask cultures, though a trend toward a marginal increase was observed at specific rotational speeds. The RDB may therefore be a suitable alternative method to study mechanisms of tumor progression and invasiveness in vitro, under more complex physicochemical conditions that may approximate natural tissue environments.