Reduction of molecular gas diffusion through gaskets in leaf gas exchange cuvettes by leaf‐mediated pores

There is an ongoing debate on how to correct leaf gas exchange measurements for the unavoidable diffusion leakage that occurs when measurements are done in non‐ambient CO₂ concentrations. In this study, we present a theory on how the CO₂ diffusion gradient over the gasket is affected by leaf‐mediated pores (LMP) and how LMP reduce diffusive exchange across the gaskets. Recent discussions have so far neglected the processes in the quasi‐laminar boundary layer around the gasket. Counter intuitively, LMP reduce the leakage through gaskets, which can be explained by assuming that the boundary layer at the exterior of the cuvette is enriched with air from the inside of the cuvette. The effect can thus be reduced by reducing the boundary layer thickness. The theory clarifies conflicting results from earlier studies. We developed leaf adaptor frames that eliminate LMP during measurements on delicate plant material such as grass leaves with circular cross section, and the effectiveness is shown with respiration measurements on a harp of Deschampsia flexuosa leaves. We conclude that the best solution for measurements with portable photosynthesis systems is to avoid LMP rather than trying to correct for the effects.     

The Effects of 1α, 25-dihydroxyvitamin D3 and Transforming Growth Factor-β3 on Bone Development in an Ex Vivo Organotypic Culture System of Embryonic Chick Femora

Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D₃ (1α,25 (OH) ₂D₃) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)₂D₃ and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) ₂D₃ (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) ₂D₃ and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)₂D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.     

Effects of interferon alpha on human osteoprogenitor cell growth and differentiation in vitro.

The specific effects of interferon alpha (IFNalpha), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNalpha on osteogenic development by investigating cell differentiation, colony formation (colony forming unit-fibroblastic, CFU-F), cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow osteoprogenitor cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNalpha (5-1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 microM), which are agents known to affect osteogenic differentiation. IFNalpha produced a significant dose-dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNalpha (50-1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU-F showed dose- and time-dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase-positive CFU-F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP-26), was reduced in IFNalpha-treated cultures. Northern blot analysis showed a significant dose-dependent up-regulation of BMP-2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNalpha significantly inhibited BMP-2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNalpha inhibits human osteoprogenitor cell proliferation, CFU- F formation, HOP-26 expression, and alkaline phosphatase specific activity and modulates BMP-2 gene expression. These results suggest a role for IFNalpha in local bone turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation.     

Human bone marrow osteoprogenitors express estrogen receptor-alpha and bone morphogenetic proteins 2 and 4 mRNA during osteoblastic differentiation.

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.     

Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells

RNA interference (RNAi) is gene silencing induced by double-stranded RNA of 21-23 nucleotides in length, termed small interfering RNA, or siRNA. RNAi-based techniques have been widely applied to elucidate gene function, identify drug targets, and used in trials as a promising adjunct to silence disease-causing genes. However, emerging evidence suggests unexpected changes in expression of untargeted genes as a consequence of an off-target effect by RNAi in mammalian cells. To date, our understanding of such effects on stem cells is limited. We transfected human fetal femur-derived mesenchymal stem cells using commercially available nonspecific siRNA controls and examined adipocyte differentiation in the cells using morphology, histochemistry, and quantitative real-time PCR to examine the expression of key genes for adipogenic or osteogenic differentiation. We report here the induction of adipocyte differentiation in human mesenchymal stem cells using nonspecific siRNAs raising concerns as to the specificity of RNAi in stem cells and, critically, a need to understand and delineate the rules governing the specificity of RNAi.     

Effects of targeted overexpression of pleiotrophin on postnatal bone development

Pleiotrophin (PTN) is an extracellular matrix-associated growth/differentiation factor that, in post-natal life, is found mainly in bone and brain. Bone development was investigated in ptn-overexpressing mice between 1 and 30 weeks. In transgenics and controls, PTN (and its receptor syndecan-3) was synthesized by osteoblasts and was present in striated muscle. ptn over-expression enhanced intramembranous bone formation and had multiple effects on long-term bone growth. The pubertal growth spurt did not take place in transgenic mice, in which the growth trajectory was steady and continuous until 25 weeks. By 30 weeks, transgenic and control mice were of the same size, but the calcium content/mg bone was approximately 10% higher in the transgenics. PTN was also localized in growth plate and articular chondrocytes, but only in transgenic mice. In these, synthesis of type I collagen by articular chondrocytes was observed, as well as an encroachment of subchondral bone into the articular cartilage. The results suggest that PTN has multiple roles during in vivo bone formation and remodeling, probably acting as a co-factor or accessory protein that modulates the effects of primary signaling molecules.     

Vitamin A effects on UMR 106 osteosarcoma cells are not mediated by specific cytosolic receptors.

Retinol and retinoic acid at 20 microM altered cell morphology and inhibited cell proliferation of UMR 106 osteosarcoma cells in culture. No specific cytosolic binding proteins for retinol could be detected.     

Mathematical modelling of skeletal repair

Tissue engineering offers significant promise as a viable alternative to current clinical strategies for replacement of damaged tissue as a consequence of disease or trauma. Since mathematical modelling is a valuable tool in the analysis of complex systems, appropriate use of mathematical models has tremendous potential for advancing the understanding of the physical processes involved in such tissue reconstruction. In this review, the potential benefits, and limitations, of theoretical modelling in tissue engineering applications are examined with specific emphasis on tissue engineering of bone. A central tissue engineering approach is the in vivo implantation of a biomimetic scaffold seeded with an appropriate population of stem or progenitor cells. This review will therefore consider the theory behind a number of key factors affecting the success of such a strategy including: stem cell or progenitor population expansion and differentiation ex vivo; cell adhesion and migration, and the effective design of scaffolds; and delivery of nutrient to avascular structures. The focus will be on current work in this area, as well as on highlighting limitations and suggesting possible directions for future work to advance health-care for all.     

Identification of candidate regulators of multipotency in human skeletal progenitor cells

Stem cell differentiation is controlled intrinsically by dynamic networks of interacting lineage-specifying and multipotency genes. However, the relationship between internal genetic dynamics and extrinsic regulation of internal dynamics is complex and, in the case of skeletal progenitor cell differentiation, incompletely understood. In this study we elucidate a set of candidate markers of multipotency in human skeletal progenitor cells by systematic study of the relationships between gene expression and environmental stimulus. We used full genome cDNA microarrays to explore gene expression profiles in skeletal progenitor enriched populations derived from adult human bone marrow, minimally cultured in basal, osteogenic, chondrogenic, and adipogenic lineage-specifying culture conditions. We then used a variety of statistical clustering procedures to identify a small subset of genes which are related to these stromal lineages but are specific to none. For a selection of 11 key genes, conclusions of the microarray study were confirmed using quantitative real-time PCR.