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Biomedical subjects

Thomas Scheper

Publications and source records attributed to Thomas Scheper.

13 recordsLinked to original sources
PubMed2007

Fast and efficient screening system for new biomaterials in tissue engineering: a model for peripheral nerve regeneration.

The use of three-dimensional biodegradable matrices is one major issue in tissue engineering. Numerous materials, fabrication techniques, and modifications have been used and tested in different areas of tissue engineering recently. But nevertheless, technology is far from being optimized and optimal constructs with bioidentical and mechanical properties have not been described in the literature so far. Hence, there is great demand of new suitable biomaterials for tissue engineering applications. In this study, a fast and efficient screening system for initial testing of biomaterials for cell culture application was developed. The set up for the screening system and the decision criteria applied for the determination of suitability of new materials are presented. Hep-G2 and PC-12 cells were seeded onto different matrices and cultured over a period of 2 weeks. The viability of the cells was monitored via the MTT assay. Cell spreading was investigated by DAPI-staining of cell nuclei. Furthermore, the adhesion of the cells on the different matrices was examined by counting the number of attached cells. With these general assays a classification of materials is possible with regard to their suitability. Optimal cell models must be chosen for the defined applications and at least two cell lines are necessary for a differentiating interpretation.

Animals↗
PubMed2007

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.

3T3 Cells↗
PubMed2006

Isolation of bovine lactoferrin, lactoperoxidase and enzymatically prepared lactoferricin from proteolytic digestion of bovine lactoferrin using adsorptive membrane chromatography.

A new downstream procedure for the isolation of bovine lactoferrin (bLf), lactoperoxidase and bovine lactoferricin (LfcinB) from sweet cheese whey was developed at the laboratory scale, based on membrane adsorber technology. The procedure was upscaled later on to an industrially relevant scale for the purificationof sweet whey concentrate with a recovery yield for lactoferrin of more than 90%. Based on these results the industrial process for 1 x 10(8) kg whey per year was projected. These high-value proteins were downstreamed by using cation-exchange membrane systems (Sartobind S, Sartorius, Göttingen, Germany). These strongly acidic membranes trap proteins in its anionic form. The dynamic loading capacity for both proteins as well as the optimal elution profiles with sodium chloride gradients were derived from laboratory experiments using membrane modules with 15-75 cm2 membrane material. Further investigations were performed with 1 m2 modules in a continuous process mode. The enzymatic preparation of LfcinB from bLf was performed by pepsin hydrolysis and the isolation of LfcinB was directly carried out from the enzymatic digest mixture. The identification of the proteins was performed with matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS). LfcinB and bLf were both tested afterwards in biological assays in order to show not only the efficiency of the downstreaming process in regard to product quantity but also to product quality (biological activity).

Adsorption↗
PubMed2006

State variables monitoring by in situ multi-wavelength fluorescence spectroscopy in heterologous protein production by Pichia pastoris.

State variables throughout non-induced and induced cultivations of Pichia pastoris for the heterologous Rhizopus oryzae lipase (ROL) production were monitored with a multi-wavelength on-line fluorescence sensor. Based on this work, the use of in situ multi-wavelength fluorometry combined with chemometrics models (PLS-1 models) provided a quantitative prediction of biomass and substrates (glycerol and methanol) during non-induced and induced ROL production. The mean prediction errors for both variables were about 7% and 10%, respectively. ROL is also quite satisfactory estimated in the exponential growth phase with prediction errors similar to biomass and substrate variables. However, in the stationary phase, where proteolytic degradation of ROL is observed, the prediction error could get a value about 20%. This fact is due to the lower reproducibility of protein production from batch to batch.

Biomass↗
PubMed2006

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.

Animals↗
PubMed2005

Application of 2D-fluorescence spectroscopy for on-line monitoring of pseudoenantiomeric transformations in supercritical carbon dioxide systems.

2D-Fluorescence spectroscopy has been shown to be effective for the on-line monitoring of spectroscopic detectable substrates L-phenylalanine-7-amido-4-methylcoumarine (L-PheAMC) and D-phenylalanine-7-amido-4-trifluoromethylcoumarine (D-PheAFC) in supercritical carbon dioxide. Earlier investigations with the coumarine substrates in watery and organic phases showed their potential for on-line enantiomeric evaluations of enzymatic reactions in different reaction media. The solubility of the different substrates and their fluorescence maximums were investigated in SCCO2. The sole hydrolyzations of L-PheAMC and D-PheAFC with alpha-chymotrypsin and the esterase from porcine liver were tracked on-line in the supercritical medium; however, different solubility characteristics of the methyl- and trifluoromethyl-substituted coumarins influence the simultaneous detection of the L- and D-substrate within the applied high-pressure reactor system.

Carbon Dioxide↗
PubMed2005

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.

Adsorption↗
PubMed2004

Flow cytometry: interesting tool for studying binding behavior of DNA on inorganic layered double hydroxide (LDH).

BACKGROUND: A new method was established to characterize the binding kinetics of DNA toward layered double hydroxides (LDHs). The setup consisted of a newly developed sampling tube that allows the injection of analyte during the flow cytometric measurement. METHODS: Layered double hydroxides consist of cationic metal hydroxide layers and exchangeable interlayer anions. This negatively charged structure permits biomolecules such as DNA to adsorb, and a so-called DNA-LDH hybrid is formed. The hydroxide layers can be removed in acidic media and the DNA will be released. CERATOFIX (a registered trademark of Sud-Chemie AG NA that belongs to the family of LDHs, produced by Sud-Chemie AG). The chemical structure can be summarized as [Mg(2)Al(OH)(6)](CO(3))(0.5). The binding capacity and kinetic characteristics of different types of CERATOFIX NA for a model DNA was determined by flow cytometry. RESULTS: The static binding capacities of the different LDHs were determined after 1- and 16-h incubation with DNA solution, showing different binding patterns between the LDH materials. The binding kinetics were revealed by flow cytometric measurements in short-term and long-term kinetic experiments, showing that the majority of DNA adsorbs within the first 60 s. CONCLUSIONS: DNA removal from cell culture supernatants is one of the major concerns in downstream processing. Due to the anion exchange capabilities of LDHs it seemed a very interesting approach to use these materials for binding of DNA for elimination purposes.

Adsorption↗
PubMed2003

Chemometric modelling with two-dimensional fluorescence data for Claviceps purpurea bioprocess characterization.

Modern bioprocess control requires fast data acquisition and in-time evaluation of bioprocess variables. On-line fluorescence spectroscopy and the application of chemometric methods accomplish these goals. In order to demonstrate how time-consuming off-line analysis methods can be replaced for bioprocess monitoring, fluorescence measurements were performed during different cultivations of the fungus Claviceps purpurea. To predict process variables like biomass, protein, and alkaloid concentrations, chemometric models were developed on the basis of the acquired fluorescence spectra. The results of these investigations are presented and the applicability of this approach for bioprocess monitoring is discussed.

Alkaloids↗
PubMed2003

The total synthesis of (-)-callystatin A.

Callystatin A is a prominent member of a class of natural products which display promising growth inhibition of cancer cells in their biological profile. The challenging structure and the interesting biological activity of (-)-callystatin A fueled our interest in the synthesis of this marine natural product. We achieved the total synthesis using a highly convergent approach joining four subunits together with a Wittig olefination, a selective Heck reaction and an aldol reaction as the pivotal steps. The aldol reaction as one of the final transformations during the synthesis opens fast access to a variety of structural analogues and circumvents tedious protecting group manipulations. Here we report an improved synthesis utilizing a modified vinyl iodide which shortens the synthesis by two steps. Additionally, first biological results will be reported.

Animals↗
PubMed2002

Controlled enzymatic removal of damaging casein layers on medieval wall paintings.

A new, gentle enzymatic method was developed for a controlled removal of casein layers from medieval wall paintings. These casein layers were applied over the last 60 years on wall paintings in order to decrease substantial damage due to a peeling off of the frescoes from the roughcast surface due to environmental effects. However, due to the aging of the casein layers (at 40-50 years), a more drastic peeling occurred and the danger of total destruction of the wall paintings is severe. Thus, screening was performed to find the most suitable enzyme for casein digestion. Alcalase 2.5 DX L was the most appropriate enzyme for an effective proteolysis reaction. The enzyme was immobilized on functionalized cellulose membrane. A membrane pad system with immobilized enzymes was developed which could be pressed on the casein layers on the wall painting. A controlled removal of the casein layers by proteolytic digestion was observed and it was possible to continuously wash off the hydrolyzed casein fragments from the wall painting surface by an aqueous carbonate buffer flowing through the membrane pad. The removal and the digestion was monitored by reverse HPLC. Additionally, an on-line monitoring system was set up in order to continuously follow the casein layer removal and the digestion procedure directly on the wall painting. This technique is based on noninvasive 2D-fluorescence monitoring. Optical fiber systems were used to continuously monitor the fluorescence intensity of casein-bound tryptophan. The off-line data were verified with the on-line 2D-fluorescence data. Based on the scientific result an appropriate technique for the controlled enzymatic removal of damaging casein layers on the surface of medieval wall paintings using immobilized enzyme is now available. It is now applied to remove such casein layers from medieval wall paintings in the Allerheiligen-Kapelle Cloister, Wienhausen, Germany, and the St. Alexander Kirche, Wildeshausen, Germany.

Caseins↗
PubMedDate not supplied

Innovative modular membrane adsorber system for high-throughput downstream screening for protein purification.

To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8-strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8-strip anion exchange and the cation exchange membrane. The isolation of His(6)-tagged proteins, Bgl-His and GFP-His from fermentation broth and lysate, respectively, was performed using an 8-strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl-His) was studied in 8-strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu(2+) ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale-up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.

Adsorption↗