The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/ poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml!1 Dexloaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.The authors thank the funds provided by Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes including project ProteoLight (PTDC/FIS/68517/2006). This work was also carried out with the support of the European Union funded STREP Project HIPPOCRATES (NMP3-CF-2003-505758) and European NOE EXPERTISSUES (NMP3-CT-2004-500283). The funding provided by Canon Foundation in Europe is gratefully acknowledged

Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation

Recently, our group has proposed a combinatorial strategy in tissue engineering principles employing carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellular release and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenic differentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if the Dex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation of osteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrow stromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml−1 Dexloaded CMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and 4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cells expanded in the presence and absence of 10−8 M Dex were used as controls. The effect of initial cell number seeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitative and quantitative new bone formation was evaluated in a non-destructive manner using micro-computed tomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used for the histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis of proteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explants were also quanti!ed, since these markers denote osteogenic differentiation. At 4 weeks post-implantation results have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles may be bene!cial as an intracellular nanocarrier, supplying Dex in a regimented manner and promoting superior ectopic de novo bone formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes (SFRH/BD/21786/2005) and by the Canon Foundation in Europe. We wish to thank P.B. Malafaya for the technical support during the micro-CT analyses, and to Materialise for providing the Mimics software. This work was also supported by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and European NoE EXPERTISSUES (NMP3-CT-2004-500283)

The Single-Particle Spectral Function of 16O^{16}{\rm O}

The influence of short-range correlations on the $p$-wave single-particle spectral function in $^{16}{\rm O}$ is studied as a function of energy. This influence, which is represented by the admixture of high-momentum components, is found to be small in the $p$-shell quasihole wave functions. It is therefore unlikely that studies of quasihole momentum distributions using the $(e,e'p)$ reaction will reveal a significant contribution of high momentum components. Instead, high-momentum components become increasingly more dominant at higher excitation energy. The above observations are consistent with the energy distribution of high-momentum components in nuclear matter.Comment: 5 pages, RevTeX, 3 figure

Integrin activation - the importance of a positive feedback

Integrins mediate cell adhesion and are essential receptors for the development and functioning of multicellular organisms. Integrin activation is known to require both ligand and talin binding and to correlate with cluster formation but the activation mechanism and precise roles of these processes are not yet resolved. Here mathematical modeling, with known experimental parameters, is used to show that the binding of a stabilizing factor, such as talin, is alone insufficient to enable ligand-dependent integrin activation for all observed conditions; an additional positive feedback is required.Comment: in press in Bulletin of Mathematical Biolog

The Role of Magnetic Resonance Imaging to Inform Clinical Decision-Making in Acute Spinal Cord Injury:A Systematic Review and Meta-Analysis

The clinical indications and added value of obtaining MRI in the acute phase of spinal cord injury (SCI) remain controversial. This review aims to critically evaluate evidence regarding the role of MRI to influence decision-making and outcomes in acute SCI. A systematic review and meta-analysis were performed according to PRISMA methodology to identify studies that address six key questions (KQs) regarding diagnostic accuracy, frequency of abnormal findings, frequency of altered decision-making, optimal timing, and differences in outcomes related to obtaining an MRI in acute SCI. A total of 32 studies were identified that addressed one or more KQs. MRI showed no adverse events in 156 patients (five studies) and frequently identified cord compression (70%, 12 studies), disc herniation (43%, 16 studies), ligamentous injury (39%, 13 studies), and epidural hematoma (10%, two studies), with good diagnostic accuracy (seven comparative studies) except for fracture detection. MRI findings often altered management, including timing of surgery (78%, three studies), decision to operate (36%, 15 studies), and surgical approach (29%, nine studies). MRI may also be useful to determine the need for instrumentation (100%, one study), which levels to decompress (100%, one study), and if reoperation is needed (34%, two studies). The available literature consistently concluded that MRI was useful prior to surgical treatment (13 studies) and after surgery to assess decompression (two studies), but utility before/after closed reduction of cervical dislocations was unclear (three studies). One study showed improved outcomes with an MRI-based protocol but had a high risk of bias. Heterogeneity was high for most findings (I(2) > 0.75). MRI is safe and frequently identifies findings alter clinical management in acute SCI, although direct evidence of its impact on outcomes is lacking. MRI should be performed before and after surgery, when feasible, to facilitate improved clinical decision-making. However, further research is needed to determine its optimal timing, effect on outcomes, cost-effectiveness, and utility before and after closed reduction

Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem.

Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause integrin activation

Momentum and Energy Distributions of Nucleons in Finite Nuclei due to Short-Range Correlations

The influence of short-range correlations on the momentum and energy distribution of nucleons in nuclei is evaluated assuming a realistic meson-exchange potential for the nucleon-nucleon interaction. Using the Green-function approach the calculations are performed directly for the finite nucleus $^{16}$O avoiding the local density approximation and its reference to studies of infinite nuclear matter. The nucleon-nucleon correlations induced by the short-range and tensor components of the interaction yield an enhancement of the momentum distribution at high momenta as compared to the Hartree-Fock description. These high-momentum components should be observed mainly in nucleon knockout reactions like $(e,e'p)$ leaving the final nucleus in a state of high excitation energy. Our analysis also demonstrates that non-negligible contributions to the momentum distribution should be found in partial waves which are unoccupied in the simple shell-model. The treatment of correlations beyond the Brueckner-Hartree-Fock approximation also yields an improvement for the calculated ground-state properties.Comment: 12 pages RevTeX, 7 figures postscript files appende

Production of cascade hypernuclei via the (K-,K+) reaction within a quark-meson coupling model

We study the production of bound cascade hypernuclei via the (K-,K+) reaction on 12C and 28Si targets within a covariant effective Lagrangian model, employing the cascade bound state spinors derived from the latest quark-meson coupling model as well as Dirac single particle wave functions. The K+-cascade production vertex is described by excitation, propagation and decay of Lambda and Sigma resonance states in the initial collision of a K- meson with a target proton in the incident channel. The parameters of the resonance vertices are fixed by describing the available data on total and differential cross sections for the cascade production in elementary (K-,K+) reaction. We find that both the elementary and hypernuclear production cross sections are dominated by the contributions from the Lambda(1520) intermediate resonant state. The 0 degree differential cross sections for the formation of simple s-state cascade particle-hole states peak at a beam momentum around 1.0 GeV/c, with a value in excess of 1 mub.Comment: 17 pages, 8 figures, version accepted for publication in Nucl. Phys.

Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.Peer ReviewedPostprint (published version

Momentum Distribution in Nuclear Matter and Finite Nuclei

A simple method is presented to evaluate the effects of short-range correlations on the momentum distribution of nucleons in nuclear matter within the framework of the Green's function approach. The method provides a very efficient representation of the single-particle Green's function for a correlated system. The reliability of this method is established by comparing its results to those obtained in more elaborate calculations. The sensitivity of the momentum distribution on the nucleon-nucleon interaction and the nuclear density is studied. The momentum distributions of nucleons in finite nuclei are derived from those in nuclear matter using a local-density approximation. These results are compared to those obtained directly for light nuclei like $^{16}O$.Comment: 17 pages REVTeX, 10 figures ps files adde