Biomedical subjects

Norbert Schütze

Publications and source records attributed to Norbert Schütze.

10 recordsLinked to original sources

PubMed2006

Gentamicin negatively influenced osteogenic function in vitro.

Local delivery of gentamicin is an accepted method of infection prophylaxis in the surgery of open fractures. However, the few reports of studies into the effect of locally applied gentamicin on osteoblasts used inadequate methods. In our study, we used the well-characterised C2C12 cell line with reproducible differentiation pathway into the osteoblast lineage. We investigated the viability, cell number, alkaline phosphatase activity, and the expression of osteogenic genes of C2C12 cells after exposure to gentamicin at concentrations of 12.5-800 microg/ml for 48 h. Exposure of C2C12 cells to gentamicin (12.5-800 mg/ml) for 48 h showed no significant changes in the cell number, but cell viability was decreased by one-third at the tested concentrations of 200-800 microg/ml. The alkaline phosphatase activity was significantly decreased by one-third to one-half at any tested concentration (12.5-800 microg/ml) of gentamicin. Any tested concentration of gentamicin up to 800 microg/ml for 48 h did not inhibit or decrease the osteogenic gene expression of osterix and alkaline phosphatase of the C2C12 cells. In conclusion, gentamicin at high concentrations as achieved by local application reduced cellular viability and alkaline phosphatase activity in vitro and therefore may be detrimental for bone healing and repair in vivo.

Alkaline Phosphatase↗

PubMed2006

Vitamin D signaling is modulated on multiple levels in health and disease.

Vitamin D signaling is dependent on the availability and turnover of the active Vitamin D receptor (VDR) ligand 1,25-dihydroxycholecalciferol and on the efficiency of VDR transactivation. Activating and inactivating secosteroid metabolizing p450 enzymes, e.g. 25-hydroxylases, 1alpha-hydroxylase and 24-hydroxylase, are responsible for ligand availability on the basis of substrate production in the skin and of nutritional intake of precursors. Net availability of active hormone depends on the delivery of substrate and the balance of activating and inactivating enzymes. 1Alpha-hydroxylase is the critical activating enzyme. It is expressed in the kidney for systemic supply and in target tissues for local secosteroid activation. It is upregulated in the kidney by low calcium intake and parathyroid hormone, downregulated by phosphatonins and proinflammatory signal transduction. Transactivation of VDR depends on the correct molecule structure, effective nuclear translocation and the presence of the unliganded heterodimer partner retinoid X-receptor (RXR) and other nuclear cofactors. Rapid Vitamin D-dependent membrane associated effects and consecutive second messenger activation exert an own pattern of gene regulation. A membrane receptor for these effects is hypothesized but not yet identified. Rickets is the long known clinical syndrome of impaired Vitamin D signaling due to Vitamin D3 deficiency. It can be caused by inherited defects of the cascade, nutritional deficits, lack of sunlight exposure, malabsorption and underlying diseases like chronic inflammation. It has been shown during the last decades that many modifiers of Vitamin D signaling are targets of disease in terms of inherited and acquired syndromes and that Vitamin D signaling is modulated at multiple levels and is more complex than mere mechanistic ligand/receptor/DNA interaction.

DNA↗

PubMed2006

CYR61 (CCN1) protein expression during fracture healing in an ovine tibial model and its relation to the mechanical fixation stability.

The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate to the mechanical fixation stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks), decreasing over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to a slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to a slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.

Animals↗

PubMed2005

Expression, purification, and functional testing of recombinant CYR61/CCN1.

The human cysteine-rich protein 61 (CYR61/CCN1) belongs to the CCN family of genes which plays an important role in cellular processes such as proliferation, migration, adhesion, and differentiation. These extracellular matrix signaling molecules consist of a modular structure and contain 38 conserved cysteine residues. Previously, we have shown that CYR61 is expressed in human osteoblasts and is regulated by bone-relevant growth factors. The protein also plays a role in angiogenesis. The open reading frame was cloned into a baculovirus expression vector and transfected into SF-21 insect cells. Recombinant protein was expressed as a fusion protein with the Fc-domain of human IgG and purified using affinity chromatography on protein G-Sepharose columns. The chorioallantoic membrane assay verified that blood vessel formation was stimulated by rCYR61. Additionally, human primary mesenchymal stem cells, osteoblasts, and endothelial cells responded to CYR61 treatment by a markedly stimulated proliferation. rCYR61-Fc represents a tool to elucidate its role in cells of the bone microenvironment.

Amino Acid Sequence↗

PubMed2004

Cyr61, a deregulated gene in endometriosis.

Gene expression profiling was performed to identify genes involved in the development of endometriosis. In the secretory phase of the menstrual cycle, several estrogen-regulated genes were up-regulated in endometria of women with endometriosis. The most consistent regulation with one of the highest factors was observed for the Cyr61 gene, which codes for a secreted, cysteine-rich, heparin-binding protein that promotes cell adhesion, migration, and neovascularization. Estrogen responsiveness of endometrial Cyr61 expression was suggested by the higher expression during the proliferative phase and the reduction observed in human endometrial fragments grafted into nude mice subsequently treated with an anti-estrogen. The expression level of Cyr61 was found to be further increased in ectopic endometriotic lesions, as compared to eutopic endometria. In these lesions, an imbalance in expression of the estrogen-converting enzymes 17beta-hydroxysteroid dehydrogenase type 1 and 2 was found, which might explain the elevated Cyr61 level. However, Cyr61 expression was not altered in endometriotic lesions of women treated with a GnRH agonist. These results suggest that Cyr61 may represent a gene characteristic for endometriosis and also play an important role in the development and persistence of endometriotic lesions.

17-Hydroxysteroid Dehydrogenases↗

PubMed2004

[Effects of clinically relevant alumina ceramic, zirconia ceramic and titanium particles of different sizes and concentrations on TNF-alpha release in a human macrophage cell line].

INTRODUCTION: Aseptic loosening is considered to be the main problem of modern endoprothesis. Tumor necrosis factor alpha (TNFalpha) seems to be the initiator protein of particle disease. The aim of our study was to investigate the TNFalpha response of macrophage like cells (MLC) after stimulation with periprosthetic particles, typically found during revision surgery. For this purpose alumina ceramic (Al2O3), zirconia ceramic (ZrO2) and titanium (Ti) particles of different sizes and concentrations were used. Important was to study the effects of different sizes due to TNFalpha secretion and the comparison of the biological effects of alumina ceramic and titanium. METHOD: To obtain an TNFalpha profile we used an established macrophage model (Rader et al.) with THP-1 cells (human monocytic cell line). Therefore 106 MLC were incubated with different particle concentrations and sizes for 6 h. The supernatant was then investigated for TNF using ELISA assay. RESULTS: Ti-particles provoked in both sizes (0.2 microm and 2.5 microm) the greatest TNFalpha response, 8 times and 17 times as high in comparison with control. But substantially more 0.2 microm sized Ti-particles were necessary to get the above mentioned results. Al2O3-particles were not as effective as Ti, but they released fourfold more TNFalpha compared to control. There was no difference in TNFalpha-secretion comparing Al2O3-particles of different sizes (0.6 microm and 2 microm), but a 1000 times greater concentration of the 0.6 microm sized particles were needed. Using Al2O3- and Ti-particles of the same size and concentration, Ti provoked a significant higher TNFalpha response. ZrO2 showed no effects on TNFalpha release. CONCLUSION: Because of our results we recommend ceramic articulating surfaces, which are superior to metal on metal matings ion term of biological reactions. Additionally bigger wear particles should be avoided. Revisionoperation should be done early to avoid huge amount of wear particles and to minimize local osteolysis.

Aluminum Oxide↗

PubMed2003

Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate.

One major problem of current cartilage repair techniques is that three-dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild-type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)-2 or -4 in alginate revealed that the formation of markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP-transfection. Cells were encapsulated in ultrahigh-viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously staining with haematoxylin-eosin or Alcian blue, immunohistochemical analysis, and quantitative RT-PCR confirmed the expression of chondrogenic markers (chondroitin-4- and -6-sulfate as well as type II collagen). Production of chondrogenic markers was particularly high in BMP-4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP-4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild-type cells and to some extent for BMP-2 transfected cells. The osteogenic markers, type I collagen, alkaline phosphatase, and Cbfa1 were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP-4 transfected cells as revealed quantitatively by real time RT-PCR. Thus, the in vitro results suggested that BMP-4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.

Alginates↗

PubMed2002

Expression and regulation of thioredoxin reductases and other selenoproteins in bone.

The expression of thioredoxin reductases and other selenoproteins in cells of the bone microenvironment may represent an important means of regulation of bone resorption and remodeling in health and disease. Selenoproteins and their substrates may influence intracellular and extracellular redox-dependent signaling, transcription factor activity, posttranslational modification of proteins, and general or compartmentalized scavenging from ROIs. However, the evaluation of their biological role in bone and their potential in terms of therapeutic approaches is just beginning.

Bone Diseases, Metabolic↗

PubMed2002

Testing of skeletal implant surfaces with human fetal osteoblasts.

The effect of standard orthopaedic implant materials on osteoblast proliferation and differentiation was investigated using a human osteoblast cell culture system. Human fetal osteoblasts 1.19 were cultured on stainless steel, cobalt-chrome-molybdenum, and commercially pure titanium for 12 days. Tissue culture polystyrene was used as a control. Cell proliferation was measured by electronic cell counting and by a colorimetric proliferation assay. To assess the degree of differentiation, levels of alkaline phosphatase activity, collagen Type I, and osteocalcin production were measured. Osteocalcin gene expression was measured by reverse transcriptase-polymerase chain reaction. Electronic cell counting and proliferation assays showed lower cell numbers and delayed proliferation on stainless steel and cobalt-chrome-molybdenum compared with titanium and polystyrene. Alkaline phosphatase and osteocalcin were measured higher on titanium than on stainless steel or cobalt-chrome-molybdenum. Differences in collagen Type I production were not found. Reverse transcriptase-polymerase chain reaction showed the highest osteocalcin gene expression on titanium. The human fetal osteoblast cell line 1.19 provides a rapidly proliferating and differentiating system for testing biomaterials in which differences in osteoblast proliferation and differentiation on orthopaedic implant materials could be revealed, suggesting that the chemistry of biomaterials has a dynamic effect on proliferation and differentiation of human osteoblasts.

Alkaline Phosphatase↗

PubMedDate not supplied

Characterization of mode II-wear particles and cytokine response in a human macrophage-like cell culture.

UNLABELLED: Informations about wear particles in metallosis (mode II wear) and their effects in vitro and in vivo are limited. The aim of this study was to characterize wear particles obtained intraoperatively and to analyse their effects on cytokine response in an established human macrophage-like cell culture model. METHOD: Wear particles were obtained intraoperatively from four patients with metallosis resulting from CrCoMo/PE/TiAIV-implants (mode II wear) (3 knee, 1 hip prosthesis). After purification, particles were characterized regarding to their composition and size (particle size analyser, electron microscopy, edx-analysis, histological slices). The effects of particles on the release of cytokines (PDGF, IL-1beta, IL-8, TNF alpha) were determined in an established human macrophage-like cell culture system by ELISA-assays. RESULTS: The metal wear particles consisted of TiAIV with a mean size of 0.1 +/- 0.15 microm, independent of the prosthesis location. CrCoMo particles could not be detected. In the cell culture model 1456 x 10(8) particles per 1 x 10(6) macrophages released maximum amounts of TNFalpha (8-fold) and IL-8 and IL-1beta (5-fold) while the survival rate of the cells was more than 90 percent. A particle-dependent increase of PDGF-levels could not be detected. CONCLUSION: As already shown for mode I wear particles (contact between primary bearing surfaces), also mode II wear particles cause release of bone resorbing cytokines in a macrophage-like cell culture model. Because their local and systemic effects in vivo are still not completely understood, we recommend a complete removal of wear particles in cases of metallosis to avoid possible immunological reactions of the body as well as periprosthetic osteolysis.

Aged↗