Magnetite nanoparticles, especially superparamagnetic iron oxide nanoparticles, are established contrast agents for magnetic resonance imaging. Magnetosomes, which are magnetite nanoparticles of biological origin, have been shown to have better contrast properties than current formulations possibly because of their larger size and high monodispersity. Here, we present an integrated study of magnetosomes and synthetic magnetite nanoparticles of varying size, hence, magnetic properties. We investigate not only the relaxation times as a measure for the contrast properties of these particles, but also their cytotoxicity and demonstrate the higher contrast of the larger particles. A theoretical model is presented that enables us to simulate the R-2/R-1 ratio of a contrast agent and confirm that larger particles offer higher contrast. The results from this study illustrate the possibility to obtain colloidal stability of large magnetic nanoparticles for magnetic resonance imaging applications and serve as an impetus for a more quantitative description of the contrast effect as a function of the size.

The main function of osteoclasts in vivo is the resorption of bone matrix, leaving behind typical resorption traces consisting of pits and trails. The mechanism of pit formation is well described, but less is known about trail formation. Pit-forming osteoclasts possess round actin rings. In this study we show that trail-forming osteoclasts have crescent-shaped actin rings and provide a model that describes the detailed mechanism. To generate a trail, the actin ring of the resorption organelle attaches with one side outside the existing trail margin. The other side of the ring attaches to the wall inside the trail, thus sealing that narrow part to be resorbed next (3–21 μm). This 3D configuration allows vertical resorption layer-by-layer from the surface to a depth in combination with horizontal cell movement. Thus, trails are not just traces of a horizontal translation of osteoclasts during resorption. Additionally, we compared osteoclastic resorption on bone and dentin since the latter is the most frequently used in vitro model and data are extrapolated to bone. Histomorphometric analyses revealed a material-dependent effect reflected by an 11-fold higher resorption area and a sevenfold higher number of pits per square centimeter on dentin compared to bone. An important material-independent aspect was reflected by comparable mean pit area (μm 2 ) and podosome patterns. Hence, dentin promotes the generation of resorbing osteoclasts, but once resorption has started, it proceeds independently of material properties. Thus, dentin is a suitable model substrate for data acquisition as long as osteoclast generation is not part of the analyses.

Sialic acid, which is located at the end of the carbohydrate moiety of cell surface glycoconjugates, is involved in many biologic responses, such as intercellular reactions and virus–cell fusion, especially in hematopoietic cells. Here we provide experimental evidence that the sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation. Lectin histochemical study demonstrated the existence of both alpha (2,3)-linked-sialic acid and alpha (2,6)-linked-sialic acid in mouse bone marrow-derived macrophages and in the RAW264.7 macrophage cell line, which are osteoclast precursors. Flow cytometric analysis of surface lectin staining revealed the kinetics of these sialic acids during osteoclastogenesis: alpha (2,3)-linked-sialic acid was abundantly expressed throughout osteoclastogenesis, whereas alpha (2,6)-linked-sialic acid levels declined at the terminal stage of osteoclast differentiation. To investigate the role of sialic acid in osteoclast differentiation, we performed an osteoclastogenesis assay with or without exogenous sialidase treatment. Desialylated cells formed TRAP-positive mononuclear cells, but did not become multinuclear cells despite the normal expression of osteoclast markers such as cathepsin K, integrin β3, and nuclear factor-ATc1. Flow cytometric analysis also demonstrated that exogenous sialidase effectively removed alpha (2,6)-linked-sialic acid, but only slightly changed the alpha (2,3)-linked-sialic acid content, suggesting that alpha (2,6)-linked-sialic acid might be involved in osteoclast differentiation. Findings from knockdown analysis using small interfering RNA oligonucleotides against alpha 2,6-sialyltransferase support this idea: alpha (2,6)-linked-sialic acid-deficient cells markedly inhibit the formation of multinuclear osteoclasts. Our findings suggest that alpha (2,6)-linked-sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation, possibly via its role in the cell–cell fusion process.

The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2−/−) mouse embryos.The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2−/− MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays.Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2−/− MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2−/− MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2.Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.

Glucorticoids (GCs) belong since their discovery to the standard therapy of rheumatoid arthritis (RA), a severe auto-inflammatory bone disease. One major side effect of GCs affects the bone itself leading to GC induced osteoporosis (GIO), the most secondary osteoporosis. GCs act via a receptor (GR) that can alter gene expression by binding as a dimer to GC responsible elements in the promoter region of target genes or by interacting with and thus interfering with other transcription factors as a monomer. We determined the contribution of molecular mechanisms and cell types critically involved in antiinflammatory effects of GCs in RA and on bone using conditional and function selective GR knockout mice. We showed that dimerization of the GR in IL-17 producing T cells is indispensable for the anti-inflammatory effect in the mouse model antigen-induced arthritis. Now we demonstrate that, surprisingly, in the Kbx/N induced arthritis model the GR dimer in mesenchymal cells is critical for anti-inflammatory effects of GCs. Thus, for immunosuppression of arthritis the GR is required in distinct cell types, which are of hematopoietic and mesenchymal origin. In a model of GIO we showed that unexpectedly interaction of the GR monomer with AP-1, but not NF-kB in osteoblasts is decisive for bone loss. Our findings define new criteria for SEGRM that act as an anti-inflammatory and protect the bone. Indeed we identified one lead-compound that still suppresses NF-kB dependent gene expression but does not affect osteoblast differentiation and activity. Furthermorewe identifiednovel GR target genes by functional genomics anddeveloped a screening platform for novel GR agonist-derivatives not affecting osteoblast function. Taken together, our approach gives new insights into GC action on arthritis and bone that can be translated into new concepts for anti-inflammatory therapies preventing GIO. doi:10.1016/j.bone.2012.08.022 O22 Osteoclasts on bone and dentin in vitro: Mechanism of degradation and comparison of resorption behaviour T. Wurger, P. Roschger, E. Zwettler, P. Fratzl, M.J. Rogers, K. Klaushofer, M. Rumpler Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKKand AUVATraumaCenter, 1stMed. Dept., HanuschHospital, Vienna, Austria Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Potsdam, Germany The Garvan Institute of Medical Research, Darlinghurst, New SouthWales,

The present paper was focused on the development of a new method of decellularized extracellular matrix (DECM) fabrication via a chemical treatment of a native bone tissue. Particular attention was paid to the influence of chemical treatment on the mechanical properties of native bones, sterility, and biological performance in vivo using the syngeneic heterotopic and orthotopic implantation models. The obtained data indicated that after a chemical decellularization treatment in 4% aqueous sodium chlorite, no noticeable signs of the erosion of compact cortical bone surface or destruction of trabeculae of spongy bone in spinal channel were observed. The histological studies showed that the chemical treatment resulted in the decellularization of both bone and cartilage tissues. The DECM samples demonstrated no signs of chemical and biological degradation in vivo. Thorough structural characterization revealed that after decellularization, the mineral frame retained its integrity with the organic phase; however clotting and destruction of organic molecules and fibers were observed. FTIR studies revealed several structural changes associated with the destruction of organic molecules, although all organic components typical of intact bone were preserved. The decellularization-induced structural changes in the collagen constituent resulted changed the deformation under compression mechanism: from the major fracture by crack propagation throughout the sample to the predominantly brittle fracture. Although the mechanical properties of radius bones subjected to decellularization were observed to degrade, the mechanical properties of ulna bones in compression and humerus bones in bending remained unchanged. The compressive strength of both the intact and decellularized ulna bones was 125–130 MPa and the flexural strength of humerus bones was 156 and 145 MPa for the intact and decellularized samples, respectively. These results open new avenues for the use of DECM samples as the replacement of wide bone tissue defects.

Thyroid hormones (T3,T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links. T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.

Mospd1 codes for a small protein with unknown physiological function, which is part of a family of genes, including Mospd2 and Mospd3, defined by the presence of the major sperm protein domain and two transmembrane domains. This work characterizes the Mospd1 gene, the intracellular location of the protein and its expression in different mouse tissues and mesenchymal cell lines during differentiation. The role of Mospd1 in mesenchymal cellular differentiation was studied by siRNA knockdown experiments in mouse osteoblastic MC3T3‐E1 cells. Transfection experiments of the targeted cDNA show MOSPD1 located in the endoplasmatic reticulum and in the Golgi apparatus. Removal of the last exon of the gene resulted in localization of the protein in the nucleus, which was attributed to a nuclear export sequence in the N‐terminal part. In mouse tissues the gene was generally strongly expressed while mesenchymal tissues showed the highest expression. In mesenchymal cell lines Mospd1 mRNA was higher expressed in cells with advanced differentiation status. In osteoblastic, myoblastic, and adipocytic cell lines Mospd1 was up‐regulated during differentiation. Genome‐wide gene expression analysis after knockdown of Mospd1 by siRNA in MC3T3‐E1 cells revealed a shift in the gene expression pattern from mesenchymal to epithelial genes featuring up‐regulation of the epithelial cadherin Cdh1 and down‐regulation of its inhibitors Snail1 and 2 and the mesenchymal cadherin Cdh11, suggesting a mesenchymal to epithelial transition. From these data we conclude that Mospd1 plays a pivotal role in the developmental regulation at the switch between mesenchymal and epithelial cells. J. Cell. Physiol. 226: 2505–2515, 2011. © 2010 Wiley‐Liss, Inc.

Kollagen Typ I, der Hauptbestandteil der extrazellularen Matrix des Knochens, weist eine super-molekulare Organisation auf. Die einzelnen Kollagenmolekule werden einer Reihe von intra- und extrazellularen Modifikationen unterzogen, die es ihnen ermoglicht extrazellular Kollagenfibrillen auszubilden. Ein wesentlicher Schritt dabei ist die Ausbildung von Kollagenquervernetzungen. Dieser Prozess ist gewebespezifisch und wird von vielen zellularen und matrix-abhangigen Signalen gesteuert. Hemmung der Lysyloxidase (Lox), ein Schlusselenzym der Kollagenvernetzung, fuhrt zu veranderten Quervernetzungen und gestorter Fibrillogenese. Ergebnisse aus in-vitro als auch in-vivo durchgefuhrten Experimenten bestatigten, dass solche Veranderungen zu Knochenmineralverlust, reduzierter Knochenfestigkeit und einer veranderten Mineralisation fuhren. Ein weiterer Faktor der eine wesentliche Rolle in der Organ und Gewebsentwicklung spielt ist die Regulierung der Expression von Genen durch epigenetische DNA-Methylierung. Bis zum jetzigen Zeitpunkt ist die Bedeutung von diesem Mechanismus in der Knochenentwicklung und Pathogenese nur wenig erforscht worden. In dieser Dissertation wurde daher auf die Rolle epigenetischer Genregulationen im Osteoblasten intensiv eingegangen. Nach Behandlung der pre-osteoblastaren MC3T3-E1 Maus Zelllinie mit den zwei Inhibitoren der Lox, beta aminopropionitrile (bAPN) und Homocysteine (hcys), haben wir den Effekt von diesen zwei Lathyrogenen auf die Zelllinie analysiert und verglichen. Die Expression von osteoblastaren Genen wurde mittels „real time polymerase chain reaction“ (qPCR) und „gene expression microarrays“ untersucht. Marker der osteoblastischen Aktivitat und Zellproliferation wurden durch Messung der Aktivitat der Alkalischen Phosphatase sowie Viabilitatstest bestimmt. Die Effekte der Substanzen auf die Kollagenquervernetzung wurden durch „Fourier-Transform-Infrarot-Spektrometrie“ (FTIR) gemessen. Die ersten Ergebnisse zeigten dass sowohl bAPN als auch hcys Lox nicht nur enzymatisch hemmen sondern auch dessen mRNA Expression vermindern. Unter Berucksichtigung der klinischen Bedeutung von hcys, haben wir den zellularen Signalweg fur die hcys-abhangige Verminderung der Lox Expression erforscht. Dabei nutzten wir Techniken wie „Enzyme-linked immunosorbent assay“ (ELISA), Immuno Blotting und Chromatin-Immunoprazipitation (ChIP). Auswertungen ergaben das Interleukin 6 (IL-6), der Transkriptionsfaktor „Friend leukemia integration 1“ (FLI1) und die DNA-Methyltransferse 1 (DNMT1) in der Repression von Lox durch hcys involviert sind. Untersuchungen an der Promotorregion des Lox-Gens ergaben eine DNA-methylierungsabhangige Regulation der Lox Expression durch hcys. Im letzten Teil der Arbeit wurde ein neuer Signal–Transduktionsweg aufgeklart, durch den die extrazellulare Matrix (ECM) die Proliferation und Differenzierung von Osteoblasten fordern kann. Beim Aussahen von MC3T3-E1 Zellen auf mit Kollagen Typ I beschichtete Platten haben wir eine erhohte Expression an osteoblastaren Genen und eine verminderte Expression des pro-apoptotischen Gens Fas beobachtet. Durch die Verwendung verschiedener Inhibitoren, qPCR und DNA-Methylierungsanalysen konnten wir zeigen, dass das extrazellulare Kollagen Typ I via FAK, MAPK und den Transkriptionsfaktor AP1 direkt die Expression des Gens Dnmt1 stimuliert was in weiterer Folge fur die Stilllegung des Gens Fas durch epigenetischer DNA-Methylierung verantwortlich ist.

Osteoblasts synthesize collagen matrix, which itself regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase (LOX), which is involved in the collagen cross-linking process. Lathyrogens, like ß-aminopropionitrile (ßAPN), inhibit the formation of a stable matrix. The aim of the present study was to investigate the influence of cross-linking on osteoblastic differentiation. MC3T3-E1 cells were seeded and treated with or without 400 μM ßAPN for 1 week. Thereafter, living cells were removed and, on this extracellular matrix, new MC3T3-E1 cells were seeded and cultured for 1 week without ßAPN. RNA was isolated, and expression of specific marker genes was determined by quantitative reverse transcription-polymerase chain reaction. Changes in specific cross-links after ßAPN treatment were measured with Fourier-transform infrared spectroscopy. The collagen matrix that formed showed a significant reduction of two major cross-links of bone collagen, deH-DHLNL and pyr, compared to control cultures. Gene expression studies showed an increase of collagen α1 (I) (COL1A1) to 150%. Expression of LOX and osteocalcin (OCN) mRNA was significantly downregulated to about 75%. When fresh MC3T3-E1 cells were seeded on this altered matrix without ßAPN, COL1A1 mRNA expression was upregulated (140%), OCN was downregulated (60%), and LOX mRNA expression remained unaffected. These results indicate that ßAPN treatment not only disrupts collagen cross-link formation but also affects osteoblastic activity and expression. In conclusion, the disrupted matrix produced in the presence of lathyrogen influences, even in its absence, the expression of osteoblastic genes.

The authors used rapid prototyping to produce three-dimensional hydroxylapatite scaffolds with controlled, fully interconnected porosity. The purpose of this study was to illuminate the effect of hormones on the osteogenic differentiation and to investigate how osteoblasts colonize the three-dimensional scaffold focusing on the formation of the cellular network. Preosteoblasts were seeded onto scaffolds, were optionally treated with the osteogenic hormones triiodo-L-thyronine (T3) and 1,25-dihydroxyvitamin-D3 (D3), and the expression of osteoblastic marker genes was investigated. Confocal laser scanning microscopy was used to investigate the three-dimensional growth behavior. Culturing cells on scaffolds strongly increased the expression of osteocalcin, osteoprotegerin, Runx2, and receptor activator of NFkB-ligand (RANKL). Treatment with T3 increased the expression of osteocalcin but did not change that of osteoprotegerin and Runx2. Treatment with D3 inhibited the expression of osteocalcin, Runx2, and osteoprotegerin. Both hormones had similar effects in the three-dimensional system as found in two-dimensional cultures although more accentuated, indicating that preosteoblasts behave more naturally on three-dimensional structures. The osteoblasts colonized the three-dimensional squared pores of scaffolds by forming a cellular network with a round central channel keeping it into the depth and depositing collagen fibrils. These results provide insight how osteoblasts colonize a three-dimensional system and underline the importance of this environment in osteoblastic differentiation studies.

Various orthopaedic methods for the fixation of fractured bone or after removal of bone tumours are currently in clinical use. Autografts, tissue obtained from another site in the same subject of the same species, are the gold standard for tissue repair and substitution. However, the use of autografts has some serious disadvantages, such as additional expense and trauma to the patient, possibility of donor site morbidity, and limited availability. In the case of allografts, in addition to limited supply and high costs, other complications such as viral transmission and immunogenicity are of serious concern. Therefore, there is a critical need to develop bone substitute materials approximating the properties of tissue, which should be replaced, but without the drawbacks of autografts or allografts. Degradable polymers that are already in clinical use usually consist of a copolymer of lactic and glycolic acid (Figure 1). These polyesters, which serve quite well for the fixation of fractured bone, cannot be used in the case of larger defects, e.g. after removal of a bone tumour, because of their hydrolytic degradation, which causes quite fast loss in mechanical strength. Moreover, the locally high concentration of free acids can result in tissue necrosis.