Biomedical subjects

Matthias Schieker

Publications and source records attributed to Matthias Schieker.

7 recordsLinked to original sources

PubMed2007

New advances in fluorochrome sequential labelling of teeth using seven different fluorochromes and spectral image analysis.

Fluorochrome sequential labelling of mineralizing tissues is commonly used in different fields of clinical and basic research. Recently we improved polychrome fluorescent sequential labelling of bone by applying spectral image analysis to discriminate seven different fluorochromes. Although basic mineralization processes of bone and teeth follow comparable principles, the respective tissues differ in terms of matrix composition and mineral assembly. The aim of this study therefore was to investigate the feasibility of this new technique for polychrome sequential labelling of teeth and to demonstrate the advantages in the field of dentistry. Furthermore, the exact labelled area of each fluorochrome could be measured, even in regions of overlapping fluorochromes. The technique presented may provide a basis for further investigations of mineralization processes of different anatomical dental structures.

Animals↗

PubMed2005

Quantitative scanning acoustic microscopy compared to microradiography for assessment of new bone formation.

Recently, it has been shown that quantitative scanning acoustic microscopy (SAM) is a powerful tool to image the acoustic impedance of even inhomogeneous materials like bone. Therefore, the aim of our study was to compare SAM to conventional microradiography with respect to histomorphometrical assessment of undecalcified sections of newly formed bone. Forty specimens were harvested 12 weeks after implantation of either autogenous cancellous bone graft or 5.0 mg of Osteogenic Protein-1 (BMP-7) in a critical-sized defect model in sheep. Undecalcified transverse bone sections of 500 microm thickness were investigated with conventional microradiography and SAM. Linear regression analysis was carried out to compare the measurements of the area of new bone formation within the defect sites. Both methods allowed for good discrimination between newly formed bone and cortical bone at the edges of the former defect. Images obtained with SAM revealed a better resolution and sharpness compared to that of microradiographs since SAM imaging unlike microradiography does not depend on the thickness of bone sections. The results of quantitative histomorphometric analysis obtained by both methods showed no significant differences, and it was possible to predict 90% of the variability of each method (coefficient of determination r2 = 0.90; P < 0.0001). In conclusion, SAM offers comparable quantitative histomorphometric information with a better spatial resolution than conventional microradiography. Thus, SAM is a promising new micro-visualizing technique for basic bone research.

Animals↗

PubMed2005

Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self-renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms. METHODS: Here we describe a proof of concept approach of separate and combined gene transfer and gene silencing by nonviral DNA transfection of enhanced green fluorescent protein (EGFP) and EGFP-targeted small interfering RNAs (siRNAs) in hMSC. For optimization of nonviral DNA and siRNA transfer different liposomal-based transfection strategies were validated. RESULTS: The highest fraction of EGFP-expressing hMSC was obtained using Lipofectamine 2000 (50%) which also mediated the highest transfection rates of siRNAs into hMSC (>or=92%). Stably EGFP-expressing hMSC maintained their proliferation capacity paired with the ability to differentiate into different mesodermal lineages (bone, cartilage, and fat) without loss of transgene expression. Based on our nonviral nucleic acid delivery technique we showed efficient, functional, and long-term RNA interference (RNAi) in hMSC by gene specific knock-down of transiently and stably expressed EGFP (88-98%). CONCLUSIONS: This is the first demonstration of efficient nonviral transfer of both nucleic acids (DNA and siRNA) into hMSC, exhibiting the potential of targeted modification of hMSC. In particular, the combination of these techniques represents a powerful gene transfer/silencing strategy, thus facilitating detailed genetic approaches to study regulatory networks in stem cell differentiation processes.

Cell Culture Techniques↗

PubMed2005

Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing.

Nowadays, there is a significant need for synthetic bone replacement materials used in bone tissue engineering (BTE). Rapid prototyping and especially 3D printing is a suitable technique to create custom implants based on medical data sets. 3D printing allows to fabricate scaffolds based on Hydroxyapatite with complex internal structures and high resolution. To determine the in vitro behaviour of cells cultivated on the scaffolds, we designed a special test-part. MC3T3-E1 cells were seeded on the scaffolds and cultivated under static and dynamic setups. Histological evaluation was carried out to characterise the cell ingrowth. In summary, the dynamic cultivation method lead to a stronger population compared to the static cultivation method. The cells proliferated deep into the structure forming close contact to Hydroxyapatite granules.

3T3 Cells↗

PubMed2005

Polychrome labeling of bone with seven different fluorochromes: enhancing fluorochrome discrimination by spectral image analysis.

Bone formation and remodeling in vivo can be assessed by polychrome labeling using calcium-binding fluorescent dyes. The number of fluorochromes, however, limits this technique due to the fact that with increasing number, fluorescent spectra inevitably overlap, which makes discrimination more difficult. In order to enhance discrimination, we performed spectral image analysis. Non-critical size defects of the femur of male Wistar rats served as a model for bone formation. Eight different fluorochromes (calcein blue, xylenol orange, calcein, alizarine complexone, doxycycline, rolitetracycline, hematoporphyrin, and BAPTA) were administered sequentially subcutaneously every third day starting at day 4 after surgery. Following, bone specimen were embedded in methylmethacrylate and analyzed by spectral image acquisition using a Sagnac type interferometer (ASI, Israel). Seven of the eight applied fluorochromes could be resolved using spectral image examination. With BAPTA, we present a new fluorochrome suitable for bone labeling. Due to the superior sensitivity of the spectral image acquisition, the thickness of the bone sections could be reduced so that 5 mum thick sections could be analyzed. Spectral decomposition and subsequent linear unmixing allows depiction of each individual fluorochrome without interference of any other, enabling a reliable and superior morphometric analysis of labeled regions.

Animals↗

PubMed2004

The use of four-colour immunofluorescence techniques to identify mesenchymal stem cells.

In stem-cell research a major difficulty is caused by the lack of distinctive features that allow the identification of human mesenchymal stem cells (hMSC). Until now, there has been no specific marker and the most common way to identify hMSC is by their characteristic stem-cell properties: self-replication and differentiation potential. However, these findings can only be revealed retrospectively, and, once differentiated, hMSC lose their stem-cell character. The aim of this study was to establish four-colour immunofluorescence of several markers simultaneously in order to address the problem of how to identify hMSC on the single-cell level. The four markers collagen-I, collagen-IV, fibronectin and CD44 are known to be expressed by hMSC. Antibody binding was detected using secondary antibodies conjugated to FITC, Alexa546, TexasRed and AMCA. Because the distinction between Alexa546 and TexasRed was not possible on conventional digital images using standard filter sets, we performed spectral image acquisition. The image was subsequently decomposed into its pure spectral components, which permitted linear unmixing. Using this procedure we were able to demonstrate four-colour immunofluorescence on hMSC. With the possibility of using more sophisticated marker profiles and/or additional markers, four-colour immunofluorescence offers the opportunity of identifying hMSC on the single-cell level without performing differentiation assays.

Cell Separation↗

PubMedDate not supplied

Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts.

BACKGROUND: Osteosarcomas are malignant bone tumors consisting of cells with abnormal cellular functions. Although osteosarcoma-derived cells are commonly used for osteoblastic models, the molecular composition of the osteosarcoma extracellular matrix (ECM) is not well characterized. MATERIALS AND METHODS: We compared three osteosarcoma cell lines (MG-63, Saos-2 and U-2 OS) with normal human osteoblasts by immunocytochemistry. Cellular characteristics were assessed by morphometric analysis and proliferation kinetics. RESULTS: All investigated osteosarcoma cell lines exhibited very heterogeneous labelling profiles and each differed significantly from that of normal osteoblasts. Saos-2 cells revealed the most mature osteoblastic labelling profile while U-2 OS cells were negative for most of the investigated osteoblastic markers. CONCLUSION: We conclude that each osteosarcoma cell line exhibits a characteristic labelling profile and thus produces a differently composed extracellular matrix. This can be used in attempts to better characterize osteosarcoma, a as well as for their diagnosis.

Aged↗