Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture.

Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as "mini tissues" to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D(3) induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D(3) has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D(3) and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.

Adhesion of fibroblasts on micro- and nanostructured surfaces prepared by chemical vapor deposition and pulsed laser treatment.

The development of micro- and nanostructured surfaces which improve the cell-substrate interaction is of great interest in today's implant applications. In this regard, Al/Al2O3 bi-phasic nanowires were synthesized by chemical vapor deposition of the molecular precursor (tBuOAlH2)2. Heat treatment of such bi-phasic nanowires with short laser pulses leads to micro- and nanostructured Al2O3 surfaces. Such surfaces were characterized by scanning electron microscopy (SEM), electron dispersive spectroscopy and x-ray photoelectron spectroscopy. Following the detailed material characterization, the prepared surfaces were tested for their cell compatibility using normal human dermal fibroblasts. While the cells cultivated on Al/Al2O3 bi-phasic nanowires showed an unusual morphology, cells cultivated on nanowires treated with one and two laser pulses exhibited morphologies similar to those observed on the control substrate. The highest cell density was observed on surfaces treated with one laser pulse. The interaction of the cells with the nano- and microstructures was investigated by SEM analysis in detail. Laser treatment of Al/Al2O3 bi-phasic nanowires is a fast and easy method to fabricate nano- and microstructured Al2O3-surfaces for studying cell-surface interactions. It is our goal to develop a biocompatible Al2O3-surface which could be used as a coating material for medical implants exhibiting a cell selective response because of its specific physical landscape and especially because it promotes the adhesion of osteoblasts while minimizing the adhesion of fibroblasts.

Nanoscale analysis of biofunctional molecules and components.

In basic medical research, as well as in concrete clinical applications, the nanoscale analysis of biofunctional units is of strongly increasing relevance. Dedicated investigations on molecules, molecular composites or functional units can efficiently be performed ex or in situ by scanning probe methods and, namely, by scanning force microscopy. In the following investigations, the folding behavior of DNA is presented as an example of basic research in medicine. In contrast, the investigation of bioadsorption phenomena in systems on which apheresis therapies are based provides a real contribution to applied medicine.

Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes.

A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.

Polyploidization and centrosome hyperamplification in inflammatory bronchi.

OBJECTIVE AND DESIGN: Inflammatory and tumorous bronchi were screened in order to obtain new tumor relevant cytogenetic parameters. MATERIAL OR SUBJECTS: Bronchial cells of 32 patients were cultivated by standard cell culture procedures. METHODS: Tetraploidy and aneuploidy was determined by enumeration of chromosome 7 and 8 versus the number of centrosomes. The resulting data were correlated with histopathological data. RESULTS: Tetra- and aneuploidy of epithelial cells were detectable in 76% of tumor cell cultures, 75% of high grade inflammatory tissues and 40% of non- and low grade-inflammatory tissues. Additionally, we observed centrosome hyper-amplification and multipolar mitoses not only in the tumor but also in the early stages of inflammation. CONCLUSION: Inflammatory bronchi already show tumor-specific features and may consequently represent the preliminary genetic stage of cancer development in bronchi.

The scanning near-field optical microscope as a tool for proteomics.

The identification of the entire genetic code of human DNA is more or less completed. With this knowledge, research in identifying the real information lying in the genes, will begin. This information is contained in the proteins, which are the main biological actors in the cell. For this reason proteins will be targeted in biological investigations in the future. The structure, affinity and reactivity of each identified protein has to be determined, which is a primary goal in the field of proteomics. This will require new and better strategies to identify protein-protein interaction. Our approach, based on the detection and visualization of single proteins by scanning near-field optical microscopy (SNOM), has allowed us to visualize various fixed and fluorochrome-labelled proteins at the nanometer scale. Subsequently SNOM may then be developed to efficiently detect the specific behavior of a certain protein in response to other biomolecules.

Polyploidization: a Janus-faced mechanism.

Normal human somatic cells are diploid. But sometimes certain tissues of the human body contain elevated numbers of tetraploid cells. This tetraploid cell population seems to represent the first step of an ongoing process of polyploidization. All tissues containing tetraploid cells have in common the fact that they are subjected to stress, which is caused by a variety of circumstances like inflammation, elevated metabolism, ageing, repair processes or selection pressure. Tetraploid cells are supposed to play a beneficial role in these stress situations, because they are known to be more resistant in general and because they are characterized by an elevated biosynthetic activity. In contrast to their beneficial character, they have a big potential concerning the malignant development of a tissue: they play a crucial role in early morphological stages of the pathway hyperplasia-metaplasia-dysplasia-carcinoma. This report links several intracellular mechanisms with each other, which potentially determine the real fate of tetraploid cells.

Centrosome multiplication accompanies a transient clustering of polyploid cells during tissue repair.

Cells from different human wounds were analyzed concerning their degree of ploidy. The experiments showed an increased tetraploidization rate in well-healing wounds especially during inflammation and proliferation. Recent data described a polyploidization in different tissues, which is accompanied and maybe caused by the multiplication of the centrosome. We show here for the first time that cells from nonmalignant tissue, namely human wound cells, are characterized by an extensive centrosome multiplication. In an effort to identify a certain mechanism, by which the centrosome may act as a modulator of the cells' ploidy, we focused our interest on p53, whose interaction with the centrosome was recently described. Applying a wound model onto p53-wildtype (wt) and p53-knockout (ko) mice, we could show that polyploidization was not reversible in p53-ko mice during wound healing. The lack of p53, the centrosome multiplication, and the polyploidization therefore may contribute to the physiological process of tissue repair in physiologically "normal" tissue.

Tetraploidization is a physiological enhancer of wound healing.

To investigate the role of chromosomal alterations in the process of wound healing on the cellular level, we analyzed biopsies from well-healing (10) and chronic defect (8) wounds. Classical chromosome preparation and fluorescence in situ hybridization were performed with cultured cells and smear preparations. Results from both techniques showed an unusual high rate of tetraploid cells (4 n) in granulation tissue of well-healing wounds (6.5-60%), whereas we found only a low amount of tetraploid cells (from 0 to 5.5%) in chronic wounds. In fibroblast control cultures, there was a percentage of 2-5.5%. In chromosome preparations, we noticed an increased number of nonclonal structural and numerical chromosome aberrations in both well-healing and chronic wounds. Our data show clearly that especially tetraploidization is a typical phenomenon in the well-healing wound, where it apparently supports the healing process.

Differential expression of heat shock protein 70 in well healing and chronic human wound tissue.

Heat shock protein 70 (hsp 70) is an important member of the heat shock protein family, which is induced by different forms of stress. We attempted to find out if hsp 70 is also involved in wound healing, which likewise resembles a stress situation for cells too. Therefore we collected tissue samples from well healing and chronic human wound tissue. We used Northern- and Western-blot analysis to study the expression of hsp 70. At the protein level we found a strong correlation between well healing wounds and high expression of hsp 70, whereas chronic wounds showed no or weak expression. Interestingly hsp 70 mRNA did not show this significant correlation, displaying a variant expression pattern in the same kind of wound tissue, possibly due to unknown posttranscriptional regulating step, which has to be investigated in further studies. To localize hsp 70 mRNA and protein was used insitu hybridization and immunohistochemistry. Both displayed an overexpression in endothelial cells of capillary vessels.