Development and optimization of the production procedure of biphasic scaffolds for osteochondral tissue engineering

Scaffolds used for osteochondral tissue engineering should comprise two distinct regions: a bottom region with characteristics corresponding to bone tissue, such as a porous structure with mineral components (predominantly hydroxyapatite), and a top region with characteristics of articular cartilage, which is gelatinous with high water content. In this work, we have investigated possibilities to formulate and optimize a procedure for obtaining such biphasic scaffolds based on gellan gum (GG). A porous base layer of the scaffold was obtained by lyophilization of the 2 % GG hydrogel with dispersed bioactive glass nanoparticles, as hydroxyapatite precursors. Next, different procedures were investigated to produce the upper GG hydrogel such as partial immersion of the porous layer in the GG solution and pouring the GG solution over the porous layer at different moisture conditions and temperatures. A simple mathematical model was derived and subsequently experimentally validated to find optimal temperatures of the porous layer, GG solution and the surrounding environment to provide adequate gelation rate to form the GG hydrogel on top of the porous layer with a thin interfacial zone

Optimization of in vitro conditions for 3D culture of rat glioma cells

Cancer is the second leading cause of death globally, making the search for its cure one of the most important challenges of the 21st century. With ethical questions regarding animal testing and inconsistency of results of cancer drug testing in standard two-dimensional (2D) monolayer cell cultures with the results in vivo, there is a pressing need for better in vitro models of human cancers that will provide more relevant systems for cancer drug screening. Three-dimensional (3D) in vitro systems based on natural polymers with immobilized cancer cells that mimic cancerous tissue and bioreactors that provide relevant chemical and physical signals could close the gap between 2D in vitro and in vivo cancer models. The aim of this study was to optimize culture conditions for the rat glioma cell line C6 immobilized in alginate microfibers in perfusion bioreactors in terms of cell density and perfusion rate. In this study we investigated following sets of parameters: perfusion rate of 0.12, 0.25 and 0.30 ml min-1 coupled with the cell density of 4∙106 cells ml-1 , and perfusion rate of 0.30 ml min-1 coupled with the cell density of 8∙106 cells ml-1 . Microfiber cultures under static conditions in Petri dishes served as controls. The results have shown that the perfusion rate of 0.30 ml min-1 in combination with the cell density of 8∙106 cells ml-1 yields higher cell viability and proliferation compared to the control static culture. These results indicate the importance of culture medium perfusion in the bioreactor for improved mass transfer of nutrients and oxygen to alginate microfibers so that the investigated system shows potentials for use as a model system in cancer research

Optimization of Bioreactor Cultures of Glioblastoma Cells Immobilized in Alginate Microfibers

Glioblastoma is the most common and aggressive malignant brain tumor in adults. Existing treatment choices that include surgery, radiation and chemotherapy are not successful in long-term survival, while development of new anticancer drugs is being held back by the lack of adequate model systems for anticancer drug testing. Namely, in traditionally used two-dimensional (2D) monolayer cancer cell cultures the native cell morphology, polarity and interactions between both cells and cells and extracellular components are either changed or absent, while studies on animals often produce misleading results due to interspecies differences. Hence, there is a pressing need for new glioblastoma model systems that provide more in vivo-like environment for investigation and development of new anticancer drugs. The aim of this work was to develop a biomimetic 3D environment for cultivation of glioblastoma cells based on alginate microfibers as cell carriers and perfusion bioreactors. Previous studies have shown that static cultures of cervical cancer cells SiHa immobilized in alginate microfibers may be diffusion limited while perfusion, which enhanced mass transport, has induced negative effects on human embryonic teratocarcinoma cells NTERA-2 in superficial zones of alginate microbeads by hydrodynamic shear stresses. Thus, in the present study, the specific focus was on optimization of cell concentration within microfibers and regimes of cultivation to achieve beneficial effects of fluid flow in perfusion bioreactors. A series of experiments were conducted in which the concentration of rat glioma cells C6 was varied between 2 and 8 × 10 6 cell cm -3 at several flowrates and regimens of static and perfusion culture periods. Mixed results were obtained implying that efficient mass transport has a higher effect in microfiber cultures at lower cell concentrations (i.e. ~2 × 10 6 cell cm -3 ). In specific, medium flow at the superficial velocity of 100 µm s -1 induced considerable cell proliferation as compared to control static cultures, which maintained the initial cell numbers. Mathematical modelling indicated that the convective transport of substances with low diffusion coefficients (~10 -19 m 2 s -1 ) may have induced the observed positive effects. Still, exact relations of cultivation conditions and cell responses in terms of viability, proliferation and metabolic activity should be further investigated

Optimization of 3d cancer cell culture conditions by application of chemical engineering principles

Cancer cell immobilization in polymer hydrogels serving as extracellular matrices and cultivation in perfusion bioreactors that provide appropriate chemical signals, efficient mass transfer and hydrodynamic shear stresses is a promising strategy for development of physiologically relevant tumor models. In this work, perfusion cultures of 2 cancer cell types (C6 rat glioma and embryonal carcinoma NT2/D1 cells) immobilized in alginate microgels were established, while static cultures served as controls. Continuous perfusion had different effects on the cultured cells inducing enhanced proliferation of the glioma cells immobilized in microfibers (8x10^6 cell/ml), while reducing the viability of the NT2/D1 cells immobilized in microbeads (1x10^6 cell/ml). In order to elucidate the observed effects, chemical engineering principles were applied to assess mass transfer and hydrodynamic conditions. The second Fick’s law was solved analytically while the diffusionadvection-reaction equation was solved numerically to model mass transport in the static and bioreactor cultures, respectively. Moreover, Reynolds numbers, pressure drops and shear stresses in bioreactor cultures were calculated for assessment of flow regime and hydrodynamic conditions. The modeling results have indicated that oxygen transport is diffusion-controlled through the alginate hydrogel, while medium perfusion improves mass transfer of larger compounds having smaller diffusion coefficients (∼10^(-13) m^2/s), which possibly stimulated glioma cell proliferation. On the other hand, the obtained shear stress (~50 mPa) in the perfused packed bed of microbeads was above physiological levels, which provided the explanation of the poor NT2/D1 cell survival. This study stresses the importance of multidisciplinary approach in addressing such multifactorial diseases as cancer

Chemical engineering methods in analyses of 3D cancer cell cultures: Hydrodinamic and mass transport considerations

A multidisciplinary approach based on experiments and mathematical modeling was used in biomimetic system development for three-dimensional (3D) cultures of cancer cells. Specifically, two cancer cell lines, human embryonic teratocarcinoma NT2/D1 and rat glioma C6, were immobilized in alginate microbeads and microfibers, respectively, and cultured under static and flow conditions in perfusion bioreactors. At the same time, chemical engineering methods were applied to explain the obtained results. The superficial medium velocity of 80 μm s-1 induced lower viability of NT2/D1 cells in superficial microbead zones, implying adverse effects of fluid shear stresses estimated as ∼67 mPa. On the contrary, similar velocity (100 μm s-1) enhanced the proliferation of C6 glioma cells within microfibers compared to static controls. An additional study of silver release from nanocomposite Ag/honey/alginate microfibers under perfusion indicated that the medium partially flows through the hydrogel (interstitial velocity of ∼10 nm s-1). Thus, a diffusion-advection-reaction model described the mass transport to immobilized cells within microfibers. Substances with diffusion coefficients of ∼10-9-10-11 m2 s-1 are sufficiently supplied by diffusion only, while those with significantly lower diffusivities (∼10-19 m2 s-1) require additional convective transport. The present study demonstrates the selection and contribution of chemical engineering methods in tumor model system development

An integrative approach in developing scaffolds based on gellan gum and bioactive glass aimed for osteochondral tissue engineering

Bilayer scaffolds based on gellan gum (GG) and nanoparticulate bioactive-glass (BAG) were developed by an integrative approach based on engineering principles and characterization in biomimetic bioreactors. The osteo-inductive GG-BAG layer containing 2 % w/w GG and 2 % w/w BAG (composition: 70 n/n % SiO2, 30 n/n % CaO) was produced by gelation followed by freeze-drying to obtain open porosity in axial and radial directions. The chondral layer was obtained by dispensing a warm 2 % w/w GG solution at 60˚C over the frozen macroporous GG-BAG layer at -25˚C. The temperatures were optimized by applying a one-dimensional unsteady-state heat transfer model so to obtain a thin integration zone, 0.5 – 1 mm thick. The scaffolds were evaluated regarding bioactivity in a biomimetic bioreactor with specially designed chambers to provide supply of two media relevant for chondral and bone tissues. In the present experiment, simulated body fluid (SBF) was supplied countercurrently continuously during 14 days of the experiment (1.1 ml min-1 flowrate), while dynamic compression (5 % deformation, 0.68 Hz frequency, 337.5 µm s-1 loading rate, 1 h / day) was applied on the chondral layer, from day 7 to day 14. SEM analyses have confirmed the retained integrity of the scaffolds, as well as formation of hydroxyapatite (HAp) uniformly throughout the osteo-layer of the scaffolds.Significantly higher bioactivity under biomimetic conditions compared to static controls resulted in slightly but significantly increased compression modulus. These results indicated a high potential of the applied integrative strategy for the development of biomimetic bilayer scaffolds

The Effect of Preparation Method on Ni/Ce/Al Catalyst for High Temperature Water-Gas Shift Reaction

High temperature water gas shift (HT-WGS) is an important catalytic process connected with reforming process in hydrogen production. Ni/CeO2-Al2O3 (or Al2O3) catalysts were studied in this work on the effect of catalyst preparation method toward the physicochemical properties and the HT-WGS activity. Ni/CeO2-Al2O3 were prepared by sol-gel and impregnation methods whereas Ni/Al2O3 was prepared by impregnation method. The catalyst samples were characterized by XRD, H2-TPR and H2-TPD techniques. The catalytic activities of HT-WGS catalysts was demonstrated at 550°C, GHSV of 2x105 mLh-1gcat-1and steam-to-CO ratio of 3. Nickel was detected as a nickel aluminate phase in the calcined catalyst. Ni strongly interacted with support in the calcined catalyst prepared by sol-gel method. The strong metal-support interaction can be resisted by preparing catalyst via impregnation and CeO2 can promote the H2O dissociation in HT-WGS mechanism. The highest metal dispersion, largest metal surface area and greatest HT-WGS activity were consequently achieved by Ni/CeO2-Al2O3 prepared from impregnation method

Development of 3D microenvironment for engineering of glioblastoma brain tumor

The aim of this work was to develop a 3D microenvironment for glioblastoma brain tumor engineering based on alginate hydrogels as a matrix for cell immobilization followed by cultivation in a biomimetic perfusion bioreactor. Alginate microfibers with immobilized cells were obtained by a simple extrusion technique. We have examined the influence of the needle diameter (22G - 28G), cell density in alginate solution (1 x 106 - 8 x 106 cells/ml) and different cancer cell lines (rat C6 and human U251 and U87) on cell immobilization efficiency and viability. The best alginate microfibers (500 µm in diameter) with all immobilized cells were obtained by applying a 25G needle with a minimal cell density of 4 x 106 cells/ml. The obtained microfibers with immobilized cells (C6 and U87) were cultivated in a perfusion bioreactor at the continuous medium flowrate in the range 0.05-0.30 ml/min over short- and long-term cultivation periods. The results have shown that the flowrate of 0.30 ml/min, corresponding to the superficial velocity of 100 µm/s, in combination with the C6 cell density of 8 x 106 cells/ml in short-term studies yielded higher cell viabilities and proliferation as compared to the control static culture. In addition, U87 cells immobilized in alginate microfibers at the density of 4 x 106 cells/ml after long-term cultivation at the medium flowrate of 0.05 ml/min (superficial velocity of 15 µm/s) stayed viable. The overall results have shown potentials of the applied approach for tumor engineering provided optimization of cultivation conditions for each cell type

Development of a physiologically relevant 3D in vitro model for osteosarcoma cell cultivation comprising alginate composite scaffolds and a perfusion bioreactor system

Osteosarcoma is the most common type of bone cancer, which affects both children and adults. Treatment of osteosarcoma exhibits slow progress due to inadequacy of both in vivo animal models and 2D in vitro models regularly used for antitumor drug testing. Our approach is to create a physiologically relevant 3D in vitro model for osteosarcoma cell cultivation, which has the potential to overcome inherent weaknesses of 2D in vitro and animal models. In order to imitate native osteosarcoma microenvironment, macroporous alginate scaffolds with incorporated hydroxyapatite/β-tricalcium phosphate (HAp/β-TCP) powder were produced with two compositions: 1 wt% alginate, 1 wt% powder and 2 wt.% alginate, 2 wt% powder. Bioactivity and stability of the scaffolds were investigated under biomimetic conditions of continuous flow of the culture medium in perfusion bioreactor at the superficial medium velocity of 400 µm/s, which was reported in literature to be beneficial for osteogenesis. Scaffolds with the higher alginate concentration was shown to be more stable in the culture medium, since the scaffolds with the lower alginate concentration disintegrated after 5-7 days under flow conditions. Biocompatibility of the obtained scaffolds was investigated in short-term cultivation studies of murine osteosarcoma cells K7M2-wt seeded onto the scaffolds. The scaffolds were cultivated in perfusion bioreactors at the superficial flow velocity of 15 µm/s, while static cultures served as a control. After cultivation, osteosarcoma cells remained adhered to the scaffold surface, expressed metabolic activity and retained their initial proliferation ability while the flow was shown to positively affect the cultivated cells