Biomedical subjects

Ulrich Nöth

Publications and source records attributed to Ulrich Nöth.

11 recordsLinked to original sources

PubMed2005

Activation of NF-kappaB signalling and TNFalpha-expression in THP-1 macrophages by TiAlV- and polyethylene-wear particles.

Wear particles are believed to induce periprosthetic inflammation which contributes to periprosthetic osteolysis. TNFalpha plays a pivotal role in the pathogenesis of this process. The molecular mechanisms leading to the development of periprosthetic inflammation with upregulated TNFalpha expression in monocytic cells in response to different wear particles have yet to be defined. In this study we evaluated the effects of polyethylene- and TiAlV-particles on activation of NF-kappaB signalling pathways and TNFalpha biosynthesis and release in monocytic cells with respect to periprosthetic osteoclastogenesis. THP-1 monocytic cells were differentiated to macrophage-like cells and exposed to LPS-detoxified polyethylene and prosthesis-derived TiAlV-particles. TNFalpha release was analyzed in culture supernatant by ELISA. NF-kappaB activation was examined by electrophoretic mobility shift assay (EMSA), and NF-kappaB target promoter activities including transactivation of the TNFalpha promoter were determined by luciferase reporter gene assays. Differentiated THP-1 macrophages were exposed to increasing numbers of particles for 0, 60, 180 and 360 min. Both, polyethylene- and TiAlV-particles induced a significant activation of both NF-kappaB and TNFalpha promoters at 180 min. A significant TNFalpha release was detected after 360 min exposure to polyethylene- and TiAlV-particles in a dose dependent manner. In comparison, LPS induced a much greater activation of NF-kappaB and TNFalpha promoters, and TNFalpha secretion into the supernatant was strongly induced. These results provide evidence that induction of the NF-kappaB signal transduction pathway in macrophages plays a major role in initiating and mediating the inflammatory response leading to periprosthetic osteolysis.

Alloys↗

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

Effects of polyethylene and TiAlV wear particles on expression of RANK, RANKL and OPG mRNA.

BACKGROUND: Wear debris has been associated with periprosthetic osteolysis and loosening of total joint arthroplasties. RANKL (receptor activator of NF-kappaB ligand), RANK (receptor activator of NF-kappaB) and OPG (osteoprotegerin) are three key molecules which regulate differentiation, survival, fusion, and activation of osteoclasts. MATERIAL AND METHODS: We evaluated the effect of TiAIV and polyethylene particles on expression of RANK, RANKL and OPG mRNA. We used a human monocytic leukemic cell line (THP-1) in this in vitro study. THP-1 monocytes were differentiated into macrophage-like cells and exposed to polyethylene particles and prosthesis-derived TiAIV particles. The supernantant was used for measurement of TNFalpha protein and total RNA was extracted from the cells. Expression of the genes coding for OPG, RANKL and RANK was analysed at the mRNA level using a semiquantitative RT-PCR method. RESULTS: Both polyethylene and TiAIV particles induced a significant release of TNFalpha after 6 h of exposure and a significant upregulation of RANK mRNA. OPG mRNA expression was transiently upregulated after exposure to polyethylene and TiAIV particles. These effects were dependent on particle dose. RANKL mRNA was not detectable in our THP-1 model. In contrast, LPS exhibited a different pattern of RANK/ RANKL/OPG mRNA expression. INTERPRETATION: Our findings provide evidence that both polyethylene and TiAIV particles induce upregulation of RANK expression in cells of the monocytic lineage, which may be important for periprosthetic osteoclastogenesis.

Alloys↗

PubMed2003

Activation of p38 and Smads mediates BMP-2 effects on human trabecular bone-derived osteoblasts.

The bone morphogenetic proteins (BMPs) are potent osteoinductive factors that accelerate osteoblast maturation, accompanied by increased cell-substrate adhesion. BMP-2 treatment of osteoblastic cells increases phosphorylation of the cytoplasmic BMP-2 signaling molecules, Smad1 and Smad5. We have previously reported that BMP-2 treatment increase cytoskeletal organization of human trabecular bone-derived osteoblast-like cells (osteoblasts), which is also accompanied by an activation of the focal adhesion kinase p125(FAK). We report here that activation of p125(FAK) occurs with the same kinetics as the phosphorylation of Smad1, suggesting that BMP-2 initiates cross-talk between Smad signaling and the adhesion-mediated signaling pathway. As an adjunct to these effects, we examined activation of mitogen-activated protein (MAP) kinase family members in response to focal adhesion contact formation. Although phosphorylated forms of all three kinases were apparent, only SAPK2alpha/p38 (p38) was activated in response to BMP-2 treatment. Inhibition of p38 kinase activity suppressed BMP-2 induced Smad1 phosphorylation, as well as its translocation to the nucleus, suggesting the integration of p38 activation with Smad1 signaling. Finally, inhibition of p38 in osteoblasts also led to the complete abrogation of BMP-2 induced osteocalcin gene expression and matrix mineralization. These findings suggest that BMP-2 must activate p38 in order to mediate osteogenic differentiation and maturation.

Bone Density↗

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

Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells.

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-beta1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated proteoglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, beta-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor gamma2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.

Adipose Tissue↗

PubMed2002

Prophylaxis of heterotopic ossification after total hip arthroplasty: a prospective randomized study comparing indomethacin and meloxicam.

We performed a randomized, prospective study on the prophylaxis of heterotopic ossification (HO) after total hip arthroplasty (THR), comparing indomethacin and the selective COX-2 inhibitor meloxicam. From the day after surgery, 272 patients were treated with 7.5 mg meloxicam, 15 mg meloxicam, or 2 x 50 mg indomethacin a day, for 14 days. After 6 months, radiographs of patients treated with 7.5 mg meloxicam showed that HO had occurred in one third. This treatment was therefore stopped after 26 patients have been assigned to this group. According to the intention-to-treat principle, patients given 15 mg meloxicam developed HO in 25% (20% Brooker grade I, 4% grade II and 1% grade III) and those given indomethacin in 10% (7% Brooker grade I, 1% grade II and 2% grade III), a statistically significant difference.

Aged↗

PubMed2002

In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human mesenchymal stem cells.

Cartilage constructs were fabricated by press-coating D,D-L,L-polylactic acid polymer blocks of 1 x 0.5 x 0.5 cm onto high-density cell pellets of 1.5 x 10(6) human mesenchymal stem cells (mhMSCs) isolated from the femoral head of patients undergoing total hip arthroplasty. Following attachment of the cell pellets to the polymer surfaces, chondrogenesis was induced by culturing the constructs for 3 weeks in a serum-free, chemically defined, chondrogenic differentiation medium supplemented with transforming growth factor beta-1 (TGF-beta1). Histochemical analysis showed that the press-coated pellets formed cell layers composed of morphologically distinct, chondrocyte-like cells, surrounded by a fibrous, sulfated proteoglycan-rich extracellular matrix. Immunohistochemical analysis detected collagen type II and cartilage proteoglycan link protein within the extracellular matrix. Expression of the cartilage-specific marker genes collagen types II, IX, X, and XI, and aggrecan was detected by RT-PCR. Scanning electron microscopy revealed organized and spatially distinct zones of cells within the cell-polymer constructs, with the superficial layer resembling compact hyaline cartilage. The fabrication method of press-coating biodegradable polymers with mhMSCs allows the in vitro production of cartilage constructs without harvesting chondrocytes from intact articular cartilage surfaces. These constructs may be applicable as prototypes for the reconstruction of articular cartilage defects in humans.

Adult↗

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↗

PubMed2002

Different osteochondral potential of clonal cell lines derived from adult human trabecular bone.

Cells derived from human trabecular bones have been shown to have multipotential differentiation ability along osteogenic, chondrogenic, and adipogenic lineages. In this study, we have derived two clonal sublines of human trabecular bone cells by means of stable transduction with human papilloma virus E6/E7 genes. Our results showed that these clonal sublines differ in their osteochondral potential, but are equally adipogenic, indicative of the heterogeneous nature of the parental cell population. The availability of these cell lines should be useful for the analysis of the mechanisms regulating the differentiation of adult mesenchymal progenitor cells.

Adult↗

PubMedDate not supplied

Human mesenchymal progenitor cell-based tissue engineering of a single-unit osteochondral construct.

A desirable strategy for articular cartilage repair is to surgically replace the damaged area with an in vitro-engineered osteochondral plug. We report here the development of a novel osteochondral construct using human trabecular bone-derived mesenchymal progenitor cells and a biodegradable poly-D,L-lactic acid scaffold. The cartilage layer was fabricated by press-coating a chondrifying high-density cell pellet onto the scaffold, which was then loaded with cells previously initiated to undergo osteogenesis. The composite was then cultured in a cocktail medium formulated to maintain both chondrogenesis and osteogenesis. Macroscopically, the construct consisted of a cartilage-like layer adherent to, and overlying, a dense bone-like component. RT-PCR, immunohistochemistry, and histology revealed hyaline-like cartilage and bone with an interface resembling the native osteochondral junction. All parameters, including mechanical properties, improved with increased culture time. The single-cell source nature of the construct, which minimizes handling while maximizing biocompatibility, suggests applicability for articular cartilage repair.

Absorbable Implants↗