Lateglacial and Holocene vegetation history in the Insubrian Southern Alps—New indications from a small-scale site

Fundamental uncertainties exist in the study region about the former lowland vegetation at local scales. All existing palaeoecological results are derived from sediments of medium- to large-sized sites (8-5000ha), which are thought to record mainly regional vegetation in their pollen content. Therefore the very small mire at Balladrum (0.05ha) was analysed for pollen, plant-macrofossils, and charcoal and the results compared with those of previous studies in the same region. Common regional signals were detected, but also new insights for the tree species Pinus cembra (L.), Abies alba (Mill.) and Castanea sativa (Mill.). Our palaeobotanical data reveal the local dominance of the timberline species P. cembra during the Lateglacial (16500-14250 cal b.p.) at low-altitudes. For A. alba an early presence in the area is suggested by pollen data, corroborating previous high-altitudinal studies indicating the presence of glacial refugia in the region. Occasional findings of C. sativa pollen throughout the Holocene may indicate the local but very rare presence of this species in the Insubrian Southern Alps, in contrast to the conventional opinion that C. sativa was introduced during the Roman Period. Altogether the results confirm the need of multiproxy palaeobotanical records from basins of variable size to assess the past composition of vegetation more accuratel

Noggin as a regulator of bone remodelling

Bone Morphogenetic Protein 2 (BMP2) is used in orthopaedic surgery to promote bone healing. The endogenous synthesis of BMP-2 antagonist family members, however, may limit the efficacy of exogenous BMP2. Noggin is one of these inhibitors that blocks the effects of BMP on the differentiation and activation of osteoblast (OB) in vitro and in vivo and inhibits OB-mediated osteoclast (OC) development. Furthermore, Noggin was found to modulate osteoclastogenesis through a direct effect on OC lineage cells. The present study aimed at elucidating the underlying mechanisms of these effects. Direct (conventional culture dishes) and indirect (transwell culture dishes) co-cultures of murine OB/OPC (Osteoclast Progenitor Cells) and cultures of OPC alone were supplemented with combinations of Noggin, BMP2, L51P (engineered, inactive variant of BMP2) and DMH1 (BMP receptor 1 inhibitor). In cultures of OPC, Noggin but not DMH1 caused an increase in the number of OC by a factor of 3 (p< 0.01). This effect could not be reversed by BMP2 and L51P, respectively. In contrast, in co-cultures of OB/OPC, exposure to Noggin attenuated OC development. In direct co-cultures, this inhibitory effect of Noggin was blocked by BMP2 and L51P. In both direct and indirect co-culture systems, exposure to Noggin induced the release of GM-CSF, a potent inhibitor of osteoclastogenesis, by a factor of 6 and 4, respectively (p< 0.01). Treatment of the cultures with αGM-CSF Ab, however, restored OC development in the indirect co-culture system only. The data suggests a previously unknown function of Noggin directly acting pro-differentiation on OC lineage cells independently of BMP signalling. In co-cultures, besides GM-CSF, cell-cell contact between OB and OPC is required for mediation of the maximal inhibitory effects of Noggin on OC development. The nature of potential interaction partners for Noggin, however, remains to be elucidated

Inositol Phosphatase SHIP1 – a Regulator of Osteoclast Lineage Cell Development and Activity

Introduction: Src-homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) is a negative regulator of the PI3K/Akt pathway that is expressed in hematopoietic cells. Osteoclast (OC) development depends on two essential pathways activated by receptor activator of NF-κB ligand (RANKL) and colony-stimulating factor-1 (CSF-1). Both pathways involve PI3K in their signalling and may therefore be regulated by SHIP1. SHIP1-deficient mice ((SHIPstyx/styx) are characterized by low bone density that has been suggested to be caused by an increased number of hyperactive OC. Purpose: This study aimed to investigate cellular mechanisms leading to low bone mass in SHIP1-deficient mice. Methods: MicroCT analysis of vertebrae and femora was performed to evaluate bone structure in vivo. To study OC development in vitro, progenitor cells (OPC) from SHIP1-deficient SHIPstyx/styx and control mice were cultured with RANKL and CSF-1. Osteoclastogenesis was assessed using an XTT cell viability assay and by determining TRAP activity. Furthermore, the capacity of OC to dissolve amorphous calcium phosphate (CaP) was determined. Results: In vivo, BV/TV of vertebrae and femora of SHIPstyx/styx mice was decreased compared to wt animals (40% and 35%, respectively, p<0.01). Trabecular number in vertebrae from SHIPstyx/styx mice was increased by 26%, while thickness was decreased by 30% (p<0.01). In femora from SHIPstyx/styx, trabecular thickness was reduced by 25% (p<0.05), whereas trabecular number remained unchanged. In vitro, SHIPstyx/styx OPC showed a 1.5-fold increased proliferation compared to controls (p<0.001), yet the number of OPC-derived OC was reduced by 40%. The capacity of SHIPstyx/styx OC to dissolve CaP was decreased by 60% compared to controls (p<0.001). Conclusions: Our data indicates a central role for SHIP1 in OC development and activity in vitro. The low bone mass phenotype in SHIPstyx/styx mice, however, may be caused by reduced bone formation or by the wasting disease and systemic inflammatory condition characteristic of SHIP1-deficient mice

The inositol phosphatase SHIP1 regulates skeletal development

Background/Introduction: Src-homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) is a lipid phosphatase expressed mainly in hematopoietic cells. SHIP1 regulates cell proliferation, differentiation, and survival via the PI3K/Akt signaling pathway. SHIP1-deficient (Styx) mice are osteoporotic, which is associated with an increased number of osteoclasts (OC). Purpose: This study aimed to investigate the underlying mechanisms through which SHIP1 controls osteoporosis. Methods: Osteoclast progenitor cells (OPC) were generated by incubating bone marrow cells with CSF-1. To develop OC, OPC from Styx, Styx het (heterozygous) and wt (wild type) mice were cultured with RANKL and CSF-1. Osteoclastogenesis was evaluated using an XTT cell viability assay, TRAP activity (OC marker) and qRT-PCR. Micro-computed tomography (Micro-CT) of vertebrae and femora were performed to evaluate the bone structure. Results: Deficiency in SHIP1 affected several aspects of bone. Compared to Styx het and wt controls, OPC-derived Styx OC presented several developmental defects, including a lower TRAP/XTT ratio and a 52% decrease in Calcr transcripts (encoding for the Calcitonin Receptor) (p<0.001). In vivo, there was a strong reduction of BV/TV in vertebrae and femora of Styx versus wt animals (39.6% and 35%, respectively, p<0.01). In particular, trabeculae in Styx vertebrae were increased by 8% (p<0.05) in numbers while decreased by 37% in thickness (p<0.001). In contrast, in Styx femora both the number and thickness of the trabeculae were decreased by 16% and 14%, respectively. These different phenotypes in Styx femora versus vertebrae indicate different paths to osteoporosis in bones with primary or secondary spongiosa. Conclusion(s): Taken together, our data indicate a central role for SHIP1-dependent PI3K/Akt signalling in bone remodeling. Further investigation will address the role of osteoblasts in the development of osteoporosis in SHIP1-deficient Styx mice

Gene expression by human monocytes from peripheral blood in response to exposure to metals

With increasing life expectancy and active lifestyles, the longevity of arthroplasties has become an important problem in orthopaedic surgery and will remain so until novel approaches to joint preservation have been developed. The sensitivity of the recipient to the metal alloys may be one of the factors limiting the lifespan of implants. In the present study, the response of human monocytes from peripheral blood to an exposure to metal ions was investigated, using the method of real-time polymerase chain reaction (PCR)-based low-density arrays. Upon stimulation with bivalent (Co2+ and Ni2+) and trivalent (Ti3+) cations and with the calcium antagonist LaCl3, the strength of the elicited monocytic response was in the order of Co2+ > or = Ni2+ > Ti3+ > or = LaCl3. The transcriptional regulation of the majority of genes affected by the exposure of monocytes to Co2+ and Ni2+ was similar. Some genes critically involved in the processes of inflammation and bone resorption, however, were found to be differentially regulated by these bivalent cations. The data demonstrate that monocytic gene expression is adapted in response to metal ions and that this response is, in part, specific for the individual metals. It is suggested that metal alloys used in arthroplasties may affect the extent of inflammation and bone resorption in the peri-implant tissues in dependence of their chemical composition

Rapid evaluation of bioactive Ti-based surfaces using an in vitro titration method

The prediction of implant behavior in vivo by the use of easy-to-perform in vitro methods is of great interest in biomaterials research. Simulated body fluids (SBFs) have been proposed and widely used to evaluate the bone-bonding ability of implant materials. In view of its limitations, we report here a rapid in vitro method based on calcium titration for the evaluation of in vivo bioactivity. Using four different titanium surfaces, this method identifies that alkaline treatment is the key process to confer bioactivity to titanium whereas no significant effect from heat treatment is observed. The presence of bioactive titanium surfaces in the solution during calcium titration induces an earlier nucleation of crystalline calcium phosphates and changes the crystallization pathway. The conclusions from this method are also supported by the standard SBF test (ISO 23317), in vitro cell culture tests using osteoblasts and in vivo animal experiments employing a pelvic sheep model

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation