Klaus-Wolfgang Wenzel

Collagen type I‐coating of Ti6Al4V promotes adhesion of osteoblasts

The initial contact of osteoblasts with implant surfaces is an important event for osseointegration of implants. Osseointegration of Ti6Al4V may be improved by precoating of its surface with collagen type I. In this study, the adhesion of rat calvarial osteoblasts to uncoated and collagen type I-coated titanium alloy was investigated over a period of 24 h. Collagen type I-coating accelerates initial adhesion of osteoblasts in the presence of fetal calf serum. One hour after plating, no differences in the percentage of adherent cells between the surfaces investigated were found. Adhesion of osteoblasts to uncoated surfaces was reduced by the GRGDSP peptide by about 70%, whereas adhesion to collagen type I-coated surfaces remained unaffected by treatment of the cells with the peptide. Cell adhesion to coated materials was reduced by about 80% by anti-integrin beta1 antibody. The integrin beta1 antibody did not influence the adhesion to uncoated titanium alloy. The results suggest that osteoblasts adhere to collagen type I-coated materials via integrin beta1 but not by interacting with RGD peptides, whereas adhesion to uncoated titanium alloy is mediated by RGD sequences but not via integrin beta1. Fibronectin does not seem to be involved in the adhesion of osteoblasts to either coated or uncoated titanium alloy.

Transforming growth factor-β1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells

© 1997 Federation of European Biochemical Societies.

Loss of caveolin expression in type I pneumocytes as an indicator of subcellular alterations during lung fibrogenesis

Abstract Caveolin is a major structural protein of caveolae, also known as plasmalemmal vesicles, which are particularly abundant in type I pneumocytes and capillary endothelial cells of lung parenchyma. Here we demonstrate that caveolin expression in the alveolar epithelium of rats and mini pigs is strikingly downregulated after irradiation-induced lung injury. Indirect immunoperoxidase staining with polyclonal anti-caveolin antibodies, confirmed by double fluorescence studies with type I cell-specific monoclonal anti-cytokeratin antibodies or lectins, revealed a dramatic loss of caveolin immunoreactivity in type I pneumocytes. In contrast, caveolin expression increased in endothelial cells. Immunoblotting of lung homogenates from normal and irradiated rats using specific anti-caveolin antibodies confirmed the presence of caveolin in normal tissue and its marked decrease of expression in fibrotic tissue. The loss of caveolin as an important structural protein of caveolae in alveolar epithelial cells may be an early indicator of serious type I cell injury during fibrogenesis. The increase of caveolin immunoreactivity in endothelia of blood vessels may indicate that different types of caveolae and/or different regulatory mechanisms of caveolin expression exist.

Transforming growth factor β1 immobilized adsorptively on Ti6Al4V and collagen type I coated Ti6Al4V maintains its biological activity

Abstract Titanium and titanium alloys are often used for orthopedic and dental implants. Osseointegration of Ti6Al4V may be improved not only by precoating of the surface with extracellular matrix proteins like collagen type I but also by additional immobilization of growth factors. In the present study, transforming growth factor β1 (TGF-β1) which is known as an inducer of collagen synthesis was immobilized adsorptively on uncoated and collagen type I coated Ti6Al4V surfaces. TGF-β1 was found immobilized slightly faster to collagen type I coated than to uncoated Ti6Al4V and released slower from the collagen coated material. Immobilized TGF-β1 is biologically active for at least 3 weeks storage at 4°C. Sterilization by ethylene oxide inactivates immobilized TGF-β1. In osteoblasts cultured on implants with adsorptively immobilized TGF-β1, mRNA level and specific catalytic activity of alkaline phosphatase as well as accumulation of calcium and phosphate were found reduced, whereas procollagen α1(I) mRNA level and the rate of collagen synthesis were increased.

Upregulation of gap junction protein connexin43 in alveolar epithelial cells of rats with radiation-induced pulmonary fibrosis

The degree of immunoreactive connexin43 (Cx43) in rat lung was evaluated during the development of radiation-induced pulmonary fibrosis in rat by a double immunofluorescence technique using polyclonal antisera of Cx43 and monoclonal antibodies to cytokeratins on cryostat sections. In normal rat lungs, Cx43 was detected in pneumocytes type II and I, in large blood vessel endothelia, in peribronchial smooth muscle cells, and in some peribronchial and perivascular interstitial cells. As early as 1 week after irradiation, enhanced immunoreactivity for Cx43 in the epithelial cells was detected. In severely injured lungs (about 3 months after irradiation), Cx43 was found also in the cytoplasm of type II pneumocytes. These findings were confirmed by western blot data. Western blot analysis also revealed increased phosphorylation of Cx43. It remains to be investigated whether the increased content of Cx43 in irradiated rat lung may be due to an enhanced number of gap junction between type I and II alveolar epithelial cells.

Immunohistochemical evidence for loss of ICAM-1 by alveolar epithelial cells in pulmonary fibrosis

ICAM-1 is an intercellular adhesion molecule of the immunoglobulin supergene family involved in adherence of leukocytes to the endothelium and in leukocytic accumulation in pulmonary injury. In the current study, the antigen retrieval technique was used to detect ICAM-1 immunohistochemically in paraffin sections of lungs from human, mouse and rat as well as in bleomycin- or radiation-induced fibrotic lungs from rat and human. In normal lung tissue, the expression of ICAM-1 on alveolar type I epithelial cells is stronger than on alveolar macrophages and on endothelial cells. Preembedding immuno-electron microscopy of normal rat, mouse and human lung samples revealed sclective ICAM-1 expression on the surface of type I alveolar epithelial cells and, to a lesser extent, on the pulmonary capillary endothelium and on alveolar macrophages. In fibrotic specimens, both focal lack and strengthening of immunostaining on the surface of type I cells was found. Alveolar macrophages were found focally lacking ICAM-1 immunoreactivity. In some cases, rat type II pneumocytes exhibited positive immunoreactions for ICAM-1. Immunoelectron microscopy with preembedded rat lungs (bleomycin-exposed cases) confirmed the altered ICAM-1 distribution at the alveolar epithelial surface. In the alveolar fluid of fibrotic rat lungs, in contrast to that from untreated controls, soluble ICAM-1 was detected by western blot analysis.

Cellular distribution of c-Jun and c-Fos in rat lung before and after bleomycin induced injury

Abstract C-Jun and c-Fos transcription factors have been associated with enhanced cellular proliferation. We studied their cellular distribution in normal and fibrotic rat lung. Pulmonary fibrosis was induced by intratracheal administration of bleomycin. In normal rat lung, c-Jun and c-Fos are present in alveolar macrophages and type II pneumocytes, in the bronchiolar epithelium and in smooth muscle cells of bronchioli and blood vessels. Subcellular fractionation of proteins revealed a predominant presence of both c-Jun and c-Fos in the heavy membrane fraction containing mitochondria and secretory granules. This was confirmed by immunoelectron microscopy, which also revealed a different localization of c-Jun and c-Fos in different cell types. Whereas in type II pneumocytes and in macrophages cytoplasmic c-Jun and c-Fos is associated with mitochondria, in Clara cells of the bronchial epithelium only secretory granules contain c-Jun and c-Fos. In addition, c-Jun is strongly present in the nuclear fraction. In the fibrotic rat lung c-Jun and c-Fos are located in the same cell types as in control lungs. In addition, fibroblasts contain c-Jun and c-Fos in areas of proliferation whereas in areas of complete fibrosis there is only a very weak expression of c-Jun and c-Fos.

Proteinases and proteinase inhibitors during the development of pulmonary fibrosis in rat

Changes in the activities of several proteinases and their inhibitors were investigated during the development of bleomycin-induced pulmonary fibrosis in rat. Studies on the proteinase-anti-proteinase-ratio may contribute to the understanding of the mechanism of the development of pulmonary fibrosis and may help to develop therapeutic strategies to prevent tissue damage by proteolytic attack. In the acute inflammatory period the activity of metalloelastase in lung tissue increased by about 10-fold. The time course of changes in the activity of 72 kD gelatinase indicates that this gelatinase accounts at least partially for the elastolytic activity. Elastase inhibitory activity in lung tissue showed maxima at days 1 and 5 and high levels in the fibrotic phase. The increase of the elastase inhibitory activity at the beginning of the fibrotic period corresponds with elevated activity of alpha 2-macroglobulin. Alveolar fluid and alveolar macrophages did not contain elastase activity but contained high elastase inhibitory activity. During the period of chronic inflammation, the activities of the cathepsins L, B, H and S in lung tissue and in isolated alveolar macrophages were found to be strongly increased.

Localization of surfactant protein A (SP-A) in alveolar macrophage subpopulations of normal and fibrotic rat lung.

The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N-acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of .DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1+/SP-A+ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages.