Separation of patatins and protease inhibitors from potato fruit juice with clay minerals as cation exchangers.

Potato fruit juice as a by-product of the starch industry contains proteins with interesting functionalities such as protease inhibitors or patatin with its high nutritional value. Due to their functional properties, these proteins are principally of industrial interest. A drawback for the application of these potato proteins is the separation and isolation under maintenance of the biological activity. So far, there are no methods in literature, which are satisfying concerning the costs or the separation performance. In this study, we show a chromatographic approach using natural clay minerals as cation exchangers to separate two protein fractions in potato fruit juice. Additionally, the content of glycoalkaloids naturally occurring in potatoes is significantly reduced in a single step together with the separation of the patatins and the protease inhibitors.

Adsorption and separation of proteins by a synthetic hydrotalcite.

In this study, the potential use of a synthetic Mg/Al hydrotalcite (layered double hydroxide) as a novel chromatography material for protein purification was investigated. The hydrotalcite is present in its carbonate form and is characterized by an Al/Mg-ratio of 1.85. Zetapotential measurements confirm a positive surface potential up to pH 10 suggesting applicability as anion exchanger. The binding of model proteins covering a broad range of isoelectric points and molecular weights was performed at different pH-values under batch conditions to evaluate the binding behaviour of the hydrotalcite. Furthermore, static binding capacities were exemplarily determined for hemoglobin and human serum albumin. Additionally, the adsorption and elution of hemoglobin was studied under dynamic conditions. The binding behaviour of the hydrotalcite was compared to commercially available anion exchangers and was found to be a function of pH, depending on the model protein. Variant adsorption behaviour is explained by further interactions like hydrogen bonds and by an unequal charge distribution over the protein surfaces. The hydrotalcite reveals high adsorption capacities under static (260 mg/g) as well as under dynamic conditions (88 mg/g at 34 cm/h; 61 mg/g at 340 cm/h). With appropriate buffers like 500 mM carbonate (pH 10) the adsorbed proteins can be nearly completely desorbed making regeneration possible. Due to the binding and elution properties it is concluded, that the hydrotalcite can serve anion exchange material for chromatographic protein separations.

A rotating bed system bioreactor enables cultivation of primary osteoblasts on well-characterized Sponceram regarding structural and flow properties.

The development of bone tissue engineering depends on the availability of suitable biomaterials, a well-defined and controlled bioreactor system, and on the use of adequate cells. The biomaterial must fulfill chemical, biological, and mechanical requirements. Besides biocompatibility, the structural and flow characteristics of the biomaterial are of utmost importance for a successful dynamic cultivation of osteoblasts, since fluid percolation within the microstructure must be assured to supply to cells nutrients and waste removal. Therefore, the biomaterial must consist of a three-dimensional structure, exhibit high porosity and present an interconnected porous network. Sponceram, a ZrO(2) based porous ceramic, is characterized in the presented work with regard to its microstructural design. Intrinsic permeability is obtained through a standard Darcy's experiment, while Young's modulus is derived from a two plates stress-strain test in the linear range. Furthermore, the material is applied for the dynamic cultivation of primary osteoblasts in a newly developed rotating bed bioreactor.

Design and characterization of a rotating bed system bioreactor for tissue engineering applications.

The main challenge in the development of bioreactors for tissue engineering is the delivery of a sufficient nutrient and oxygen supply for cell growth in a 3D environment. Thus, a new rotating bed system bioreactor for tissue engineering applications was developed. The system consists of a culture vessel as well as an integrated rotating bed of special porous ceramic discs and a process control unit connected with the reactor to ensure optimal culturing conditions. The aim of the project was the design and construction of a fully equipped rotating bed reactor, and in particular, the characterization and optimization of the system with regard to technical parameters such as mixing time and pH-control to guarantee optimal conditions for cell growth and differentiation. Furthermore, the applicability of the developed system was demonstrated by cultivation of osteoblast precursor cells. The porous structure of the ceramic discs and the external medium circulation loop provide an optimal environment for tissue generation in long-term cultivations. Mass transfer limitations were minimized by the slow rotation, which also provides the cells with sufficient nutrients and oxygen through alternate contact to air and medium. An osteoblast precursor cell line was successfully cultivated in this bioreactor for 28 days.

Cultivation of MC3T3-E1 cells on a newly developed material (Sponceram) using a rotating bed system bioreactor.

The influence of a 3D macroporous scaffold (Sponceram) on the differentiation process into bone cells was investigated under static conditions in cell culture dishes. Furthermore, cultivations were performed using a new bioreactor system in the presence or absence of bone morphogenetic protein 2 (BMP-2). Preosteoblastic MC3T3-E1 cells were first cultured on Sponceram scaffolds in 96-well dishes using standard medium, differentiation medium and BMP-2 medium. Cell proliferation showed a similar course for all conditions used. Alkaline phosphatase (AP) activity resulted in a maximum at day 5 in the presence of BMP-2. Two bioreactor cultivations were performed in a BIOSTAT Bplus RBS (rotating bed system) 500 on Sponceram carrier discs. One cultivation was performed using standard medium. The second one was used with the same medium with BMP-2 substituted. Significant calcification of the extracellular matrix in the presence of BMP-2 occurred but even in the absence of BMP-2 mineralization was observed. mRNA expression of collagen I, osteocalcin and bone sialoprotein was detected after both reactor cultivations. This study demonstrates that macroporous Sponceram is suitable for the cultivation and differentiation of MC3T3-E1 cells into the osteoblastic phenotype. The results of the bioreactor cultivation revealed that the scaffold promoted the differentiation process even in the absence of BMP-2.

Application of collagen matrices for cartilage tissue engineering.

Articular cartilage shows little capacity for self-repair once it has been damaged. The aim of this study was to investigate different collagen matrices regarding their applicability for cartilage tissue engineering. The matrices consist of collagen I and small amounts of elastine, were crosslinked with carbodiimide or glucose. Primary chondrocytes were seeded onto these different collagen matrices and cultured with or without differentiation medium. The viability of the cells was monitored via MTT test. The arrangement of the cells onto the scaffold was investigated by histological staining. Furthermore, extracellular matrix synthesis was studied by immunohistological staining, especially the expression of the typical chondrogenic marker collagen II. Moreover gene expression for collagen type II was analysed by RT-PCR. The chondrocytes showed high viability on all matrices used. The results for the histological staining revealed a three-dimensional arrangement of the chondrocytes in the collagen matrices. Moreover, the matrices also supported chondrogenic differentiation. On the matrix MATRIDERM 2 mm the synthesis of collagen II was stimulated without adding any differentiation supplements to the cell culture medium, as observed by immunohistological staining and by gene expression analysis of collagen II.

Fast and efficient protein purification using membrane adsorber systems.

The purification of proteins from complex cell culture samples is an essential step in proteomic research. Traditional chromatographic methods often require several steps resulting in time consuming and costly procedures. In contrast, protein purification via membrane adsorbers offers the advantage of fast and gentle but still effective isolation. In this work, we present a new method for purification of proteins from crude cell extracts via membrane adsorber based devices. This isolation procedure utilises the membranes favourable pore structure allowing high flow rates without causing high back pressure. Therefore, shear stress to fragile structures is avoided. In addition, mass transfer takes place through convection rather than diffusion, thus allowing very rapid separation processes. Based on this membrane adsorber technology the separation of two model proteins, human serum albumin (HSA) and immungluboline G (IgG) is shown. The isolation of human growth hormone (hGH) from chinese hamster ovary (CHO) cell culture supernatant was performed using a cation exchange membrane. The isolation of the enzyme penicillin acylase from the crude Escherichia coli supernatant was achieved using an anion exchange spin column within one step at a considerable purity. In summary, the membrane adsorber devices have proven to be suitable tools for the purification of proteins from different complex cell culture samples.

Development of multifunctional polymer-mineral composite materials for bone tissue engineering.

The main goal of this article is the development of a novel approach to construct multifunctional composite scaffolds for bone tissue engineering. For this purpose, different kinds of mineral macroporous supports, water-soluble aldehyde-containing copolymers of N-vinylpyrrolidone, as well as different nonspecific and biospecific ligands governing cell adhesion and growth have been used. The composite materials were tested initially for cytotoxicity in cell culture experiments using a model cell line.