The spatial patterning of RGD and BMP-2 mimetic peptides at the subcellular scale modulates human mesenchymal stem cells osteogenesis.

Engineering artificial extracellular matrices, based on the biomimicry of the spatial distribution of proteins and growth factors within their native microenvironment, is of great importance for understanding mechanisms of bone tissue regeneration. Herein, photolithography is used to decorate glass surfaces with subcellular patterns of RGD and BMP-2 ligands; two mimetic peptides recognized to be involved in stem cells osteogenesis. The biological relevance of well-defined RGD and BMP-2 patterned surfaces is evaluated by investigating the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts, in the absence of induction media. The extent of hMSCs differentiation is revealed to be dependent on both the pattern shape and the ligand type. Indeed, the spatial patterning of BMP-2, but not RGD peptide, significantly enhances the extent of hMSCs differentiation, suggesting that geometric cues guide stem cells specification into specialized cells in a ligand type dependent manner. Such cell culture models provide an interesting tool to investigate how stem cells perceive and respond to their microenvironment and may contribute to the development of next-generation biomaterials capable of producing clinically relevant volume of bone tissue. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 959-970, 2018.

RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells.

UNLABELLED: Human bone marrow mesenchymal stem cells (hBMSCs) commitment and differentiation are dictated by bioactive molecules sequestered within their Extra Cellular Matrix (ECM). One common approach to mimic the physiological environment is to functionalize biomaterial surfaces with ECM-derived peptides able to recruit stem cells and trigger their linage-specific differentiation. The objective of this work was to investigate the effect of RGD and BMP-2 ligands crosstalk and density on the extent of hBMSCs osteogenic commitment, without recourse to differentiation medium. RGD peptide promotes cell adhesion via cell transmembrane integrin receptors, while BMP-2 peptide, corresponding to residues 73-92 of Bone Morphogenetic Protein-2, was shown to induce hBMSCs osteoblast differentiation. The immobilization of peptides on aminated glass was ascertained by X-ray Photoelectron Spectroscopy (XPS), the density of grafted peptides was quantified by fluorescence microscopy and the surface roughness was evaluated using Atomic Force Microscopy (AFM). The osteogenic commitment of hBMSCs cultured on RGD and/or BMP-2 surfaces was characterized by immunohistochemistry using STRO-1 as specific stem cells marker and Runx-2 as an earlier osteogenic marker. Biological results showed that the osteogenic commitment of hBMSCs was enhanced on bifunctionalized surfaces as compared to surfaces containing BMP-2, while on RGD surfaces cells mainly preserved their stemness character. These results demonstrated that RGD and BMP-2 mimetic peptides act synergistically to enhance hBMSCs osteogenesis without supplementing the media with osteogenic factors. These findings contribute to the development of biomimetic materials, allowing a deeper understanding of signaling pathways that govern the transition of stem cells towards the osteoblastic lineage. STATEMENT OF SIGNIFICANCE: For a long time, scientists thought that the differentiation of Mesenchymal Stem Cells (MSCs) into bone cells was dictated by growth factors. This manuscript shed light on other ligands that play a crucial role in regulating MSCs fate. In concrete terms, it was demonstrated that the osteoinductive effect of BMP-2 peptide is 2 folds improved in the presence of adhesive RGD peptide. Compared to previous works highlighting this synergistic cooperation between RGD and BMP-2 peptides, the main strength of this work lies to the use of primitive human cells (hMSCs) and well-defined biomimetic material surfaces (controlled surface roughness and peptide densities). This work provides valuable insights to develop custom-designed in vitro cell culture models, capable of targeting the desired cell response.

Neuroprotection mediated by glial cell line-derived neurotrophic factor: involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway.

The glial cell line-derived neurotrophic factor (GDNF) is first characterized for its trophic activity on dopaminergic neurons. Recent data suggested that GDNF could modulate the neuronal death induced by ischemia. The purpose of this study was to characterize the influence of GDNF on cultured cortical neurons subjected to two paradigms of injury (necrosis and apoptosis) that have been identified during cerebral ischemia and to determine the molecular mechanisms involved. First, we demonstrated that both neurons and astrocytes express the mRNA and the protein for GDNF and its receptor complex (GFRalpha-1 and c-Ret). Next, we showed that the application of recombinant human GDNF to cortical neurons and astrocytes induces the activation of the MAP kinase (MAPK) pathway, as visualized by an increase in the phosphorylated forms of extracellular signal-regulated kinases (ERKs). Thereafter, we demonstrated that GDNF fails to prevent apoptotic neuronal death but selectively attenuates slowly triggered NMDA-induced excitotoxic neuronal death via a direct effect on cortical neurons. To further characterize the neuroprotective mechanisms of GDNF against NMDA-mediated neuronal death, we showed that a pretreatment with GDNF reduces NMDA-induced calcium influx. This effect likely results from a reduction of NMDA receptor activity rather than an enhanced buffering or extrusion capacity for calcium. Finally, we also demonstrated that an ERKs activation pathway is necessary for GDNF-mediated reduction of the NMDA-induced calcium response. Together, these results describe a novel mechanism by which the activation of MAPK induced by GDNF modulates NMDA receptor activity, a mechanism that could be responsible for the neuroprotective effect of GDNF in acute brain injury.

Assessing exposure to cosmic radiation during long-haul flights.

The assessment of exposure to cosmic radiation on board aircraft is one of the concerns of organizations responsible for radiation protection. Cosmic-particle flux increases with altitude and latitude and depends on solar activity. To illustrate the effect of these parameters, exposure has been estimated on several airlines operating subsonic and supersonic aircraft on transatlantic, Siberian and transequatorial routes. Measurements have been made with a tissue-equivalent proportional counter using the microdosimetric technique. This type of system provides the absorbed dose, the ambient dose equivalent, the mean quality factor, and the dose distribution as a function of lineal energy. Data were collected at maximum solar activity in 1991-1992 and at minimum activity in 1996-1998. The lowest mean dose rate measured was 3 microSv h(-1) during a Paris-Buenos Aires flight in 1991. The highest rates were 6.6 microSv h(-1) during a Paris-Tokyo flight on a Siberian route and 9.7 microSv h(-1) on Concorde in 1996-1997. The mean quality factor is around 1.8. The corresponding annual effective dose, based on 700 h of flight for subsonic aircraft and 300 h for Concorde, can be estimated at between 2 mSv for the least-exposed routes and 5 mSv for the more-exposed routes.

Assessing exposure to cosmic radiation on board aircraft.

The assessment of exposure to cosmic radiation on board aircraft is one of the preoccupations of organizations responsible for radiation protection. The cosmic radiation particle flux increases with altitude and latitude and depends on the solar activity. The radiation exposure has been estimated on several airlines using transatlantic, Siberian and transequatorial routes on board subsonic and supersonic aircraft, to illustrate the effect of these parameters. Measurements have been obtained with a tissue equivalent proportional counter using the microdosimetric technique. Data have been collected at maximum solar activity in 1991-92 and at minimum in 1996-98. The lowest mean dose rate measured was 3 microSv/h during a Paris-Buenos Aires flight in 1991; the highest was 6.6 microSv/h during a Paris-Tokyo flight using a Siberian route and 9.7 microSv/h on Concorde in 1996-97. The mean quality factor is around 1.8. The corresponding annual effective dose, based on 700 hours of flight for subsonic aircraft and 300 hours for Concorde, can be estimated between 2 mSv for least-exposed routes and 5 mSv for more exposed routes.

Dosimetry during the first IBIS facility flight.

The dosimetry of cosmic rays was performed during the first experimental flight of the IBIS facility. Different thermoluminescent detectors (TLD) have been used to measure the contribution of the low linear energy transfer component (LET < 10 keV/micrometer) and plastic nuclear track detectors (PNTD) for the high linear energy tranfer (LET) component. Several parameters of tracks have been measured to determine the LET spectra of primary and secondary charged particles. The total absorbed dose rate (TLD+PNTD) during the flight was 0.23 mGy/day and the dose equivalent rate using the ICRP 60 was 0.52 mSv/day. The corresponding mean quality factor was 2.4. These results are in agreement with those obtained aboard the MIR station with a tissue equivalent proportional counter.