Biological activity of rhBMP-2 released from PLGA microspheres

Human recombinant bone morphogenetic protein-2 (rhBMP-2) has been proven effective in stimulating the regeneration of bone in both skeletal and extraskeletal locations. Through encapsulation within, and release from, biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) microspheres, a proven vehicle for sustained delivery of various proteins, the local concentrations of rhBMP-2 could be maintained at optimal levels to stimulate bone regeneration and remodeling at the site of healing in diverse clinical settings. Thus the purpose of this work was to investigate the encapsulation of rhBMP-2 in PLGA microspheres and its biologic activity upon release. Using in vitro tests in simulated body fluids, the effect of rhBMP-2 released from PLGA microspheres upon osteoblast cell cultures was found to be statistically similar to the effect produced by positive controls consisting of nonencapsulated aqueous rhBMP-2 in simulated body fluids. This clarifies an important step in skeletal tissue engineering strategies aimed at the use of encapsulated rhBMP-2 to stimulate bone regeneration and remodeling.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Disentangling Woodland Caribou Movements in Response to Clearcuts and Roads across Temporal Scales

Although prey species typically respond to the most limiting factors at coarse spatiotemporal scales while addressing biological requirements at finer scales, such behaviour may become challenging for species inhabiting human altered landscapes. We investigated how woodland caribou, a threatened species inhabiting North-American boreal forests, modified their fine-scale movements when confronted with forest management features (i.e. clearcuts and roads). We used GPS telemetry data collected between 2004 and 2010 on 49 female caribou in a managed area in Québec, Canada. Movements were studied using a use – availability design contrasting observed steps (i.e. line connecting two consecutive locations) with random steps (i.e. proxy of immediate habitat availability). Although caribou mostly avoided disturbances, individuals nonetheless modulated their fine-scale response to disturbances on a daily and annual basis, potentially compromising between risk avoidance in periods of higher vulnerability (i.e. calving, early and late winter) during the day and foraging activities in periods of higher energy requirements (i.e. spring, summer and rut) during dusk/dawn and at night. The local context in which females moved was shown to influence their decision to cross clearcut edges and roads. Indeed, although females typically avoided crossing clearcut edges and roads at low densities, crossing rates were found to rapidly increase in greater disturbance densities. In some instance, however, females were less likely to cross edges and roads as densities increased. Females may then be trapped and forced to use disturbed habitats, known to be associated with higher predation risk. We believe that further increases in anthropogenic disturbances could exacerbate such behavioural responses and ultimately lead to population level consequences.     

Engineering a polymeric gene delivery vector based on poly(ethylenimine) and hyaluronic acid

In this work, the effects of primary amines, ligand targeting, and overall charge on the effectiveness of branched poly(ethylenimine)-hyaluronic acid conjugate (bPEI-HA) zwitterionic gene delivery vectors are investigated. To elucidate the relative importance of each of these parameters, we explored the zeta potential, cytotoxicity, and transfection efficiency for a variety of formulations of bPEI-HA. It was found that the length of the hyaluronic acid (HA) oligosaccharide had the most significant effect on cytotoxicity and transfection efficiency with human mesenchymal stem cells. Test groups of bPEI incorporating HA with a length of 10 saccharides had significantly higher transfection efficiency (14.6 ± 2.0%) and lower cytotoxicity than other formulations tested, with the cytotoxicity of the group containing the greatest mass of 10 saccharide showing similar results as the positive controls at the highest polymer concentration (100 μg/mL). Additionally, molar incorporation of HA, as opposed to the saccharide length and HA mass incorporation, had the greatest effect on zeta potential but a minor effect on both cytotoxicity and transfection efficiency. This work demonstrates the relative importance of each of these tunable design criteria when creating a zwitterionic polymeric gene delivery vector and provides useful specific information regarding the design of bPEI-HA gene delivery vectors.     

Differentiation of naive CD4<Superscript>+</Superscript> T cells towards T helper 2 cells is not impaired in rheumatoid arthritis patients

An impaired differentiation of naive CD4+ T cells towards Th2 cells may contribute to the chronic tissue-destructive T-cell activity in rheumatoid arthritis (RA). The differentiation of naive CD4+ T cells into memory Th2 cells by IL-7 in comparison with that by IL-4 was studied in RA patients and in healthy controls. Naive CD4+ T cells from peripheral blood were differentiated by CD3/CD28 costimulation in the absence of or in the presence of IL-7 and/or IL-4. The production of IFN-gamma and IL-4 was measured by ELISA and by single-cell FACS analysis to indicate Th1 and Th2 cell activity. CD3/CD28 costimulation and IL-7 were early inducers of IL-4 production, but primarily stimulated IFN-gamma production. In contrast, in short-term cultures exogenously added IL-4 did not prime for IL-4 production but suppressed IL-7-induced IFN-gamma production. Upon long-term stimulation of naive CD4+ T cells, IFN-gamma production was differentially regulated by IL-7 and IL-4, but IL-4 production was increased by both IL-7 and IL-4. IL-7 and IL-4 additively induced polarization towards a Th2 phenotype. This susceptibility of naive CD4+ T cells to become Th2 cells upon culture with IL-7 and IL-4 was increased in RA patients compared with that in healthy controls. These findings demonstrate that, in RA patients, differentiation of naive CD4+ T cells towards a Th2 phenotype by CD3/CD28 costimulation, IL-7 and IL-4 is not impaired. The perpetuation of arthritogenic T-cell activity in RA therefore seems not to be the result of intrinsic defects of naive CD4+ T cells to develop towards suppressive memory Th2 cells.     

The growth and phenotypic expression of human osteoblasts

The care of patients with a skeletal deficiency currently involves the use of bone graft or a non-biologic material such as a metal or polymer. There are alternate possibilities in development which involve the growth of bone cells (osteoblasts) on degradable polymer scaffolds. These tissue engineering strategies require production of the polymeric scaffold, cellular harvest followed by either ex vivo or in vivo growth of the cells on the scaffold, and exploration of the interaction between the cell and scaffold. Research into these strategies utilizes cells from a variety of species, but clinical applications will likely require human osteoblasts. This study explores the process whereby human osteoblasts are harvested under sterile conditions during joint replacement surgery from normally discarded cancellous bone, transported from the operating room to the lab, and grown in culture. This process is feasible, and the cells express their phenotype via the production of alkaline phosphatase and collagen in culture.     

Procathepsin and acid phosphatase are stored in Musca domestica yolk spheres

Yolk spheres present in mature invertebrate oocytes are composed of yolk proteins and proteolytic enzymes. In the fly Musca domestica, yolk proteins are degraded during embryogenesis by a cathepsin-like proteinase that is stored as a zymogen. An acid phosphatase is also active in the yolk spheres during Musca embryogenesis. In this paper we show that procathepsin and acid phosphatase are initially stored by a different pathway from the one followed by yolk protein precursors. Both enzymes are taken up by the oocytes and transitorily stored into small vesicles (lysosomes) surrounding the early yolk spheres. Fusion of both structures, the early yolk spheres and lysosomes, creates the mature yolk spheres.     

In vitro cytotoxicity of injectable and biodegradable poly(propylene fumarate)-based networks: unreacted macromers,cross-linked networks,and degradation products

This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric network based on poly(propylene fumarate) (PPF) and the cross-linking agent PPF-diacrylate (PPF-DA). Using a methyl tetrazolium (MTT) assay, the effect of the concentrations of PPF and PPF-DA on the cytotoxicity of its unreacted macromers, cross-linked networks, and degradation products was examined. The influence of network structure properties on cell viability and attachment to the cross-linked material was also investigated. The unreacted macromers exhibited a time- and dose-dependent cytotoxic response that increased with more PPF-DA in the mixture. Conversely, the cross-linked networks formed with more PPF-DA did not demonstrate an adverse response because increases in conversion and cross-linking density prevented the extraction of toxic products. Fibroblast attachment was observed on the PPF/PPF-DA networks with the highest double bond conversions. The degradation products, obtained from the complete breakdown of the networks in basic conditions, displayed a dose-dependent cytotoxic response. These results show that there are concerns regarding the biocompatibility of injectable, biodegradable PPF/PPF-DA networks but also sheds light onto potential mechanisms to reduce the cytotoxic effects.     

7th SOSORT consensus paper: conservative treatment of idiopathic & Scheuermann's kyphosis

Abstract

Thoracic hyperkyphosis is a frequent problem and can impact greatly on patient's quality of life during adolescence. This condition can be idiopathic or secondary to Scheuermann disease, a disease disturbing vertebral growth. To date, there is no sound scientific data available on the management of this condition. Some studies discuss the effects of bracing, however no guidelines, protocols or indication's of treatment for this condition were found. The aim of this paper was to develop and verify the consensus on managing thoracic hyperkyphosis patients treated with braces and/or physiotherapy.

Methods

The Delphi process was utilised in four steps gradually modified according to the results of a set of recommendations: we involved the SOSORT Board twice, then all SOSORT members twice, with a Pre-Meeting Questionnaire (PMQ), and during a Consensus Session at the SOSORT Lyon Meeting with a Meeting Questionnaire (MQ).

Results

There was an unanimous agreement on the general efficacy of bracing and physiotherapy for this condition. Most experts suggested the use of 4-5 point bracing systems, however there was some controversy with regards to physiotherapeutic aims and modalities.

Conclusion

The SOSORT panel of experts suggest the use of rigid braces and physiotherapy to correct thoracic hyperkyphosis during adolescence. The evaluation of specific braces and physiotherapy techniques has been recommended.