[Diagnosis of periprosthetic hip infections]. / Diagnostik der infizierten Hüftendoprothese.

The diagnosis of periprosthetic infection requires a clear definition itself and structured procedure concerning anamnesis, clinical examination, laboratory findings, puncture and imaging diagnostics. The clinical presentation may vary considerable due to the time of their occurrence as early, delayed, or late infection. Recognition of risk factors and knowledge of differential diagnoses facilitate and confirm the diagnosis. The synovial fluid is assessed with regard to leukocyte count, protein content, and glucose. Intraoperative tissue specimen sampling has to be performed correctly; the histopathological and microbiological studies must be assessed using specific criteria. The examination and classification of periprosthetic membranes make discrimination of the causal pathological mechanism possible, especially distinction between septic and aseptic loosening. In this manner statements with regard to etiology and prosthesis durability are possible. Different causative microorganisms appear postoperatively at specific times. Pathogens that grow as biofilms are of great significance, as they may compound diagnosis and therapy. Early infections are often caused by virulent microorganisms (S. aureus) with acute onset. Delayed (low grade) infections are usually caused by less virulent microorganisms, such as S. epidermidis or coagulase-negative staphylococci. Many diagnostic imaging methods have been used in the assessment of periprosthetic infection: plain radiographs, arthrography, ultrasonography, computed tomography, and magnetic resonance imaging. Nuclear medicine with bone scintigraphy or positron-emission tomography enhance diagnostic capabilities. Cultures of samples obtained by sonication of prostheses are more sensitive than conventional periprosthetic tissue culture. Multiplex PCR of sonication fluid is a promising test for diagnosis of periprosthetic joint infection. The promising diagnostic accuracy for interleukin-6 and procalcitonin has yet not been affirmed.

Cyr61 is a target for heparin in reducing MV3 melanoma cell adhesion and migration via the integrin VLA-4.

The integrin VLA-4 is important for the metastatic dissemination of melanoma cells. We could recently show that heparin can block VLA-4 binding, which contributes, next to blocking P- and L-selectin, to the understanding of antimetastatic activities of heparin. The matricellular ligand Cyr61, secreted by numerous tumours, is responsible for increased tumourigenicity and metastasis. This has been attributed to Cyr61 binding to, and thus activating integrins. However, a VLA-4/Cyr61 axis has not yet been reported. Since Cyr61 possesses heparin binding capabilities, Cyr61 can be supposed as potential target for heparin to indirectly interfere with integrin functions. The present in vitro studies address (i) the existence of a Cyr61/VLA-4 axis and (ii) the functional relevance of heparin interference via Cyr61. The C-terminal module III of Cyr61 could be exposed as nanomolar affine binding site for VLA-4. A shRNA-based knockdown of Cyr61 in MV3 human melanoma cells reduced VLA-4-mediated cell binding to VCAM-1, migration on fibronectin, and integrin signalling functions significantly. Using a biosensor approach we provide insight into heparin interference with this process. The low-molecular-weight heparin tinzaparin, but not the pentasaccharide fondaparinux, binds module IV of Cyr61 with micromolar affinity. But tinzaparin cannot interfere with Cyr61 accumulation onto syndecan-4, indicating different Cyr61 binding sites for heparin and other GAGs. Nonetheless, tinzaparin affects the VLA-4 binding and signalling functions selectively via Cyr61 already at very low concentration most likely by blocking the cellular secreted free Cyr61. This study emphasises Cyr61 as promising, and hitherto not considered target for heparin to selectively influence integrin functions.

Inhibition of endotoxin-induced perinatal asthma protection by pollutants in an experimental mouse model.

BACKGROUND: One of the most promising strategies to face the increasing asthma prevalence and to prevent disease development might be an early contact with microbial compounds. However, little is known about an interaction between an early-life contact to microbial compounds leading to asthma protection in the offspring and a co-exposure to allergy-promoting pollutants. METHODS: Pregnant BALB/c mice were repeatedly exposed to aerosolized endotoxin (lipopolysaccharide, LPS). The offspring was further exposed to aerosolized LPS before allergen sensitization with ovalbumin (OVA). Some of the mice were co-exposed to mycotoxins or diesel exhaust particles (DEP) during pregnancy. The 6-week-old offspring was immunized with OVA and analyzed in a murine asthma model. RESULTS: While the offspring of naïve mothers developed an asthma-like phenotype, the offspring of mice perinatally exposed to LPS was significantly protected. Co-exposure of mice to mycotoxins or DEP during pregnancy inhibited the LPS-induced protection leading to the development of eosinophilic airway inflammation, airway hyperactivity, and increased antigen-specific IgE levels in the offspring. Furthermore, the asthma-preventive effect of perinatal LPS exposure was IFN-gamma dependent. Additionally, the IFN-gamma promoter of CD4+ T cells in the LPS-exposed offspring revealed a significant protection against loss of histone 4 acetylation, which was abolished after prenatal co-exposure to pollutants. Prenatal treatment of mice with the antioxidant N-acetylcysteine reversed the pollutant-induced increased asthma risk in the offspring. CONCLUSION: Our results show that exposure to pollutants during pregnancy may cause the development of allergic asthma in the offspring by inhibiting the endotoxin-induced perinatal asthma protection.

Connective tissue growth factor does not affect transforming growth factor-beta 1-induced Smad3 phosphorylation and T lymphocyte proliferation inhibition.

Transforming growth factor-beta 1 (TGF-beta(1)) is a key regulator of immune tolerance. TGF-beta(1) controls T lymphocyte activation and is involved in the immunosuppressive function and generation of regulatory T lymphocytes. Connective tissue growth factor (CTGF) has an essential role in the formation of connective tissue and blood vessels. CTGF expression is induced by TGF-beta(1) in several cell types and CTGF mediates several of the downstream actions of TGF-beta(1). Since little is known about the potential synergy between CTGF and TGF-beta(1) in T lymphocyte biology, the purpose of the present study was to determine whether CTGF can modulate TGF-beta(1)-mediated effects on human CD4+ T lymphocytes. Human recombinant CTGF was expressed in HEK293 cells. rCTGF was biologically active demonstrated by induction of proliferation in the endothelial cell line EA hy 926. rCTGF alone did not potentiate or diminish anti-CD3-induced CD4+ T lymphocyte proliferation and did not activate the Smad signaling pathway in CD4+ T lymphocytes. Furthermore, rCTGF did not attenuate TGF-beta(1)-mediated inhibition of CD4+ T lymphocyte proliferation and TGF-beta(1)-induced Smad signaling in CD4+ T lymphocytes. These results indicate that rCTGF had no detectable effects of its own on human CD4+ T lymphocytes and did not potentiate the effects of low amounts of TGF-beta(1) on human CD4+ T lymphocytes. Overall, these data support the hypothesis that CTGF does not act on CD4+ T lymphocytes.

Coexpression of osteogenic and adipogenic differentiation markers in selected subpopulations of primary human mesenchymal progenitor cells.

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age-related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP-activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator-activated receptor-gamma2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor-beta1 (TGF-beta1) and promoted by bone morphogenetic protein 2 (BMP-2). Osteogenic culture conditions including BMP-2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development.

Expression of the angiomatrix and angiogenic proteins CYR61, CTGF, and VEGF in osteonecrosis of the femoral head.

Angiogenesis and bone repair are closely linked processes. VEGF, CYR61, and CTGF have been identified as signaling factors that control angiogenesis and could be important in fracture healing. The purpose of this study was to investigate the expression of these signaling factors in osteonecrosis of the femoral head. Twenty-one bone cylinders were retrieved from hips of patients with osteonecrosis of the femoral head at different ARCO stages. Immunohistochemistry for CD34, CYR61, CTGF, and VEGF expression was done on each bone cylinder representing the different regions of osteonecrosis (necrosis, fibrosis, transition zone, and edematous area). VEGF, CYR61, and CTGF were expressed in samples with osteonecrosis. Particularly VEGF and CYR61 were highly expressed in the edematous area. CYR61 was also highly expressed in the transition zone. CTGF was expressed mainly in the area of marrow fibrosis and edema. CYR61, CTGF, and VEGF are expressed to different degrees in the different repair zones of osteonecrosis. Particularly, the high expression of VEGF and CYR61 in the edematous area may represent a consequence of hypoxia and indicate a role of these proteins in the repair processes ongoing in osteonecrosis.

Anterior cruciate ligament constructs fabricated from human mesenchymal stem cells in a collagen type I hydrogel.

BACKGROUND: Disruptions of the anterior cruciate ligament (ACL) of the knee joint are common and are currently treated using ligament or tendon grafts. In this study, we tested the hypothesis that it is possible to fabricate an ACL construct in vitro using mesenchymal stem cells (MSC) in combination with an optimized collagen type I hydrogel, which is in clinical use for autologous chondrocyte transplantation (ACT). METHODS: ACL constructs were molded using a collagen type I hydrogel containing 5 x 10(5) MSC/mL and non-demineralized bone cylinders at each end of the constructs. The constructs were kept in a horizontal position for 10 days to allow the cells and the gel to remodel and attach to the bone cylinders. Thereafter, cyclic stretching with 1 Hz was performed for 14 days (continuously for 8 h/day) in a specially designed bioreactor. RESULTS: Histochemical analysis for H and E, Masson-Goldner and Azan and immunohistochemical analysis for collagen types I and III, fibronectin and elastin showed elongated fibroblast-like cells embedded in a wavy orientated collagenous tissue, together with a ligament-like extracellular matrix in the cyclic stretched constructs. No orientation of collagen fibers and cells, and no formation of a ligament-like matrix, could be seen in the non-stretched control group cultured in a horizontal position without tension. RT-PCR analysis revealed an increased gene expression of collagen types I and III, fibronectin and elastin in the stretched constructs compared with the non-stretched controls. DISCUSSION: In conclusion, ACL-like constructs from a collagen type I hydrogel, optimized for the reconstruction of ligaments, and MSC have been fabricated. As shown by other investigators, who analyzed the influence of cyclic stretching on the differentiation of MSC, our results indicate a ligament-specific increased protein and gene expression and the formation of a ligament-like extracellular matrix. The fabricated constructs are still too weak for animal experiments or clinical application and current investigations are focusing on the development of a construct with an internal augmentation using biodegradable fibers.

siRNA technology.

RNA interference (RNAi) represents a mechanism invented by nature to protect the genome. In the past few years the field has emerged at a surprisingly high pace. The underlying molecular mechanism of gene silencing provides us with short interfering RNAs (siRNAs) which allows to target any gene with high specificity and efficiency. siRNAs can now be obtained in various ways allowing for numerous in vitro and in vivo applications. Successful knock-downs of disease-related genes indicate that siRNAs open the door for novel therapeutic procedures.

Detection of differentially expressed genes in particle disease using array-filter analysis.

UNLABELLED: The precise cellular mechanism of osteolysis in particle disease is still unknown. The aim of the study was to screen for new gene products in macrophages during particle contact. METHOD: In an established macrophage model THP1-cells (human monocytic cells) were differentiated under the influence of vitamin D3 and GM-CSF into macrophage-like cells (MLC). MLCs were incubated each with different concentrations of polyethylene particles, Lipopolysaccharids (LPS) and controls. Isolated RNA was transcribed into complementary radioactive 32P labeled cDNA. This probe was hybridised on an human cDNA expression array and analysed by autoradiography. To obtain a more reliable method quantifying mRNA, the reverse transcriptase polymerase chain reaction (RT-PCR) was used. RESULTS: The arrays showed an upregulation of the following genes by particles: TNF-Rezeptor 2, IL-1 Receptor Antagonist, Bone Morphogenic Protein 4 and HM 145. This was proven three times using RT-PCR and statistically significant in comparison to the controls. LPS induced the same upregulation except for HM145 whereas particles caused downregulation of this mRNA expression. CONCLUSION: Our results prove that the model of differentiated THP-1 cells treated with PE particles is a suitable system to analyse differential gene expression patterns, since the induction of the major positive control genes TNF alpha and IL1 beta were detected by this approach. BMP 4 is known as signal protein which mediates ectopic bone formation and can also be interpreted as a contra regulatory gene. HM 145 belongs to the leukocyte chemotactic peptide receptor family. HM 145 seems to be one of the first genes that is enhanced along the septical pathway but less expressed by contact with particles. Analysis of HM 145 expression might help to diagnose septic versus aseptic loosening of prosthesis.

Formation of cartilage matrix proteins by BMP-transfected murine mesenchymal stem cells encapsulated in a novel class of alginates.

Proliferation and differentiation of wild-type, BMP-2 and BMP-4 transfected cells of C3H10T1/2, a mouse mesenchymal stem cell line that can differentiate into chondrocytes, were studied under monolayer (2D-) and encapsulation (3D-) conditions. Cells were encapsulated in a novel class of alginate. The alginate was of clinical grade (CG) because of complete removal of mitogenic and cytotoxic contaminants by chemical means. Compared to commercial alginates used so far for encapsulation it was characterized by ultra-high viscosity (UHV; viscosity of a 0.1% w/v solution of about 20 cP). In contrast to monolayer cultures, proliferation of cells was prevented when the cells were encapsulated in UHV/CG alginate at the same suspension density. As revealed by immunohistochemistry and quantitative RT-PCR, transfected and wild-type monolayer cells showed synthesis of type I collagen after transfer into differentiation medium, while culture in an alginate scaffold resulted in an upregulation of type II collagen and other hyaline cartilage proteins. BMP-4 transfected cells produced considerably more type II collagen than BMP-2 transfected and wild-type cells. BMP-4 transfected cells were also characterized by type I collagen production up to Day 10 and exhibited transient alkaline phosphatase activity levels that were much higher than the peak values observed for the other two cell lines. The coincidence of the ALP peak values with downregulation of type I collagen in BMP-4 transfected cells suggested that C3H10T1/2 cells differentiate into chondrocytes via a chondroprogenitor-like cell.

[Standardized testing of skeletal implant surfaces with an osteoblast cell culture system. IV. Specific gene expression during differentiation]. / Standardisierte Testung von Skelett-Implantatoberflächen mit einem Osteoblasten-Zellkultursystem. IV. Spezifische Genexpression während der Differenzierung.

Successful osseointegration of an implant depends on the properties of the material of which it is made. A standardized cell culture system for the assessment of the biological effect of material surfaces has already been described. In the present study, this system has been extended to include the quantitative analysis of the material-dependent osteoblast gene expression. Human foetal osteoblasts (hFOB 1.19) were cultured for 3 weeks on titanium surfaces of varying roughness, and on surfaces of chromium-cobalt-molybdenum alloy (CrCoMo). Using a real time RT-PCR technique, expressions of alkaline phosphatase, collagen 1 and osteocalcin were determined as parameters of osteoblast differentiation. In comparison with CrCoMo, differentiation was accelerated on titanium. While the smooth titanium surface leads to earlier cell growth, the rough surface induces more prolonged and stronger cell proliferation. Our results confirm at the molecular level the excellent clinical biocompatibility of titanium surfaces. The real-time RT-PCR provides a new method for the quantitative assessment of material-dependent osteoblastic differentiation.

IL-12p40-dependent agonistic effects on the development of protective innate and adaptive immunity against Salmonella enteritidis.

To study a potential IL-12p40-dependent but IL-12p75-independent agonistic activity regulating the immune response against Salmonella Enteritidis, the course of infection in IL-12p35-deficient mice (IL-12p35(-/-), capable of producing IL-12p40) was compared with that of IL-12p40(-/-) mice. Mice lacking IL-12p40 revealed a higher mortality rate and higher bacterial organ burden than mice capable of producing IL-12p40. This phenotype was found in both genetically susceptible (BALB/c, Ity(s)) and resistant mice (129Sv/Ev, Ity(r)) indicating Ity-independent mechanisms. The more effective control of bacteria in the IL-12p35(-/-) mice was associated with elevated serum IFN-gamma and TNF-alpha levels. In contrast, IL-12p40(-/-) mice showed reduced IFN-gamma production, which was associated with significantly elevated serum IgE levels. Early during infection (days 3 and 4 postinfection), as well as late (day 20 postinfection), the number of infected phagocytes was strongly increased in the absence of IL-12p40 indicating impaired bactericidal activity when IL-12p40 was missing. Liver histopathology revealed a decreased number of mononuclear granulomas in IL-12p40(-/-) mice. Depletion of CD4(+) or CD8(+) T lymphocytes in vivo suggested that both T cell subpopulations contribute to the IL-12p40-dependent protective functions. Analysis of IL-12p40 vs IL-23p19 mRNA expression revealed an up-regulation of only IL-12p40 mRNA during Salmonella infection. Together these data indicate that IL-12p40 can induce protective mechanisms during both the innate and the adaptive type 1 immune response in Salmonella infection. This novel activity of IL-12p40 complements the well described dominant and essential role of IL-12p75 in protective immunity to Salmonella infection.

5' flanking sequence of the human immediate early responsive gene ccn1 (cyr61) and mapping of polymorphic CA repeat sequence motifs in the human ccn1 (cyr61) locus.

AIMS: The human ccn1 (hccn; hcyr61) gene has been identified previously at the mRNA and protein level as a 1,25-dihydroxyvitamin D(3) and growth factor regulated gene in human osteoblasts. This study aimed to analyse genomic clones containing the human ccn1 (cyr61) gene and to provide the 5' flanking region. METHODS: Genomic clones were isolated by screening a lambda library and by array filter hybridisations of a genomic library. Sequencing was performed using the dye terminator method. Promoter activity was measured after transient transfection using a beta galactosidase assay. CA repeat motifs were studied by a combined PCR/fragment analysis protocol. RESULTS: The human 5' flanking region of 870 nucleotides contains several stretches with high homology to the mouse promoter as well as CA repeat motifs. This first report on the human 5' flanking sequence of the hccn1 (hcyr61) gene provides important insights into regulation pathways for the expression of this 1,25-dihydroxyvitamin D(3) and growth factor responsive early gene. A genomic clone containing the hccn1 (hcyr61) gene region also yielded a CA sequence located 3' of the ccn1 (cyr61) gene. This CA repeat and one of the CA repeat motifs in the promoter were studied in detail and found to be polymorphic. CONCLUSIONS: The 5' flanking sequence of the hccn1 (hcyr61) gene provides insights into the mechanisms of regulation of this immediate early gene product. The CA repeat polymorphisms within the gene region will be useful in the genetic study of disorders affecting bone metabolism.

[Cytotoxicity study of high gold content Degutan surfaces of various degrees of roughness with fibroblasts (BALB 3T3) and osteoblasts (hFOB 1.19)]. / Zytotoxizitätsuntersuchung von hochgoldhaltigen Degutan-Oberflächen unterschiedlicher Rauhigkeit mit Fibroblasten (BALB 3T3) und Osteoblasten (hFOB 1.19).

The cytotoxicity of Degutan surfaces with different degrees of roughness, and the effect of surface structures on osteoblast proliferation and differentiation, was investigated with standardised cell culture systems. Fibroblast cell lines (BALB/3T3) and osteoblast cell lines (hFOB 1.19) were used. The number and variability of the cells were determined for assessment of proliferation and alkaline phosphatase activity, collagen I and osteocalcin production were used as parameters for differentiation. In the early phase, the largest numbers of cells and greatest proliferation were measured on polished Degutan surfaces. In the late phase, however, larger numbers of cells and a greater degree of proliferation were to be seen on sandblasted and sandblasted/heat-treated Degutan surfaces. No differences were found for collagen I, osteocalcin production or alkaline phosphatase activity. Neither the osteoblasts nor the fibroblasts revealed a toxic effect of Degutan. The results for osteoblast differentiation correlate with recent studies on identical structured titanium surfaces. In view of the immeasurable amount of ion release, Degutan may be considered an ideal model for an inert material surface.

The immediate early gene product hCYR61 localizes to the secretory pathway in human osteoblasts.

The human cysteine-rich protein 61 (hCYR61) belongs to the growing CCN (CYR61/CTGF/NOV) family of immediate early genes, which modulate cell growth and differentiation. hCYR61 is regulated by 1alpha, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and growth factors in fetal human osteoblasts (hFOB cells). The murine homologue CYR61 was characterized as an extracellular matrix-associated protein that modulates basic fibroblast growth factor signaling, angiogenesis, and binds to integrin alpha(v)beta(3). Here we report the intracellular localization of the human CYR61 gene product by overexpressing fusion proteins with green fluorescent protein (GFP) in primary osteoblasts and the hFOB cell line. Full-length hCYR61-GFP localizes to the Golgi apparatus and cytoplasmatic granules, indicating targeting to the secretory pathway. Secretion of hCYR61 from osteoblasts is verified by Western blot detection from cellular supernatants. A truncated hCYR61-GFP fusion protein containing only the 34 N-terminal amino acids of hCYR61 also localizes to the Golgi apparatus mainly in the perinuclear region, which suggests that the N-terminus of hCYR61 is sufficient to target the protein to the secretory pathway. In summary, our results present the first evidence that human CYR61 localizes to the secretory pathway in primary osteoblasts and hFOB cells, and that it is secreted from these cells. The N-terminal 34 amino acids of hCYR61 provide a sufficient Golgi targeting sequence. Together with the immediate early regulation of hCYR61 mRNA by 1,25-(OH)(2)D(3), this suggests that hCYR61 might function as an extracellular signaling molecule in human bone.

[Standardized testing of bone implant surfaces with an osteoblast cell culture cyste. III. PVD hard coatings and Ti6Al4V]. / Standardisierte Testung von Skelett-Implantatoberflächen mit einem Osteoblasten-Zellkultursystem. III. PVD-Hartstoffbeschichtungen und Ti6Al4V.

The effect of titanium-based PVD coatings and a titanium alloy on the proliferation and differentiation of osteoblasts was investigated using a standardised cell culture system. Human fetal osteoblasts (hFOB 1.19) were cultured on titanium-niobium-nitride ([Ti,Nb]N), titanium-niobium-oxy-nitride coatings ([Ti,Nb]ON) and titanium-aluminium-vanadium alloy (Ti6Al4V) for 17 days. Cell culture polystyrene (PS) was used as reference. For the assessment of proliferation, the numbers and viability of the cells were determined, while alkaline phosphatase activity, collagen I and osteocalcin synthesis served as differentiation parameters. On the basis of the cell culture experiments, a cytotoxic effect of the materials can be excluded. In comparison with the other test surfaces, [Ti,Nb]N showed greater cell proliferation. The [Ti,Nb]N coating was associated with the highest level of osteocalcin production, while all other differentiation parameters were identical on all three surfaces. The test system described reveals the influence of PVD coatings on the osteoblast differentiation cycle. The higher oxygen content of the [Ti,Nb]ON surface does not appear to have any positive impact on cell proliferation. The excellent biocompatibility of the PVD coatings is confirmed by in vivo findings. The possible use of these materials in the fields of osteosynthesis and articular surfaces is still under discussion.