Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration

To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.     

Comparative Study Assessing the Canal Cleanliness Using Automated Device and Conventional Syringe Needle for Root Canal Irrigation—An Ex-Vivo Study

The success of endodontic treatment relies on both apical and coronal sealing. To achieve a good three-dimensional seal, the removal of the smear layer becomes mandatory. This study aims to assess the difference in debris accumulation and smear layer formation while using automated root canal irrigation and conventional syringe needle irrigation. Single-rooted human mandibular premolar teeth (n = 30) which were indicated for orthodontic extractions were selected. An endodontic access cavity was prepared, and a glide path was created. Based on the irrigation protocol decided upon for the study, the teeth were randomly allocated into three study groups, namely Group 1, where the manual syringe needle irrigation method was adopted; Group 2, in which automated root canal irrigation was undertaken; and Group 3, in which teeth remained un-instrumented as it was considered the Control group. The teeth were decoronated at the cement-enamel junction (CEJ) and were subjected for scanning electron microscopy (SEM) examination. Debris and smear layers were viewed in 1000× magnification and scored. A statistically significant (p < 0.05) lower mean debris and smear layer score (p < 0.05) was observed in both study groups when compared with the control group. However, no significant difference (p > 0.05) in the debris and smear layer was observed between the manual syringe needle irrigation and automated irrigation, although automated irrigation devices can be a potential alternative. The present study concluded that the efficacy of smear layer removal remained the same with both automated irrigation and manual syringe irrigation.     

Control of steroidogenesis by the calcium messenger system in human adrenocortical cells

The involvement of the calcium messenger system in the control of steroidogenesis in the rat and bovine adrenal cortex has been studied extensively. However the role of these second messengers in the control of human adrenocortical function is not established. This was therefore studied by incubating collagenase-dispersed human adrenocortical cells with the calcium ionophore A23187 and the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). The effects of the calcium channel blocker verapamil on basal and stimulated steroidogenesis were also studied. Both TPA (1 pmol/l-10 mumol/l) and A23187 (1 nmol/l-10 mumol/l) caused a dose-dependent increase in cortisol, aldosterone and corticosterone production. Verapamil (10 mumol/l) inhibited the increase in aldosterone, corticosterone and cortisol produced in response to ACTH(1-24), potassium, and desacetyl-alpha MSH. Unlike previous results in the rat, these effects were not specific for aldosterone secretion. The results suggest that, as in other species, calcium mobilization and protein kinase C activation have a role in the control of steroidogenesis in the human adrenal cortex. However, in contrast to the rat, these mechanisms appear to be involved in the control of steroidogenesis in both the zona glomerulosa and inner zone cells.     

RNA sensing by conventional dendritic cells is central to the development of lupus nephritis

Glomerulonephritis is a common and debilitating feature of systemic lupus erythematosus (SLE). The precise immune mechanisms that drive the progression from benign autoimmunity to glomerulonephritis are largely unknown. Previous investigations have shown that a moderate increase of the innate Toll-like receptor 7 (TLR7) is sufficient for the development of nephritis. In these systems normalization of B-cell TLR7 expression or temporal depletion of plasmacytoid dendritic cells (pDCs) slow progression; however, the critical cell that is responsible for driving full immunopathology remains unidentified. In this investigation we have shown that conventional DC expression of TLR7 is essential for severe autoimmunity in the Sle1Tg7 model of SLE. We show that a novel expanding CD11b⁺ conventional DC subpopulation dominates the infiltrating renal inflammatory milieu, localizing to the glomeruli. Moreover, exposure of human myeloid DCs to IFN-α or Flu increases TLR7 expression, suggesting they may have a role in self-RNA recognition pathways in clinical disease. To our knowledge, this study is the first to highlight the importance of conventional DC-TLR7 expression for kidney pathogenesis in a murine model of SLE.The innate pathogen recognition receptor Toll-like receptor 7 (TLR7) has recently been implicated in the development of autoimmunity. This receptor, along with TLR8 and TLR9, was originally described by Beutler and colleagues (1), with ligand identification and functional characterization by Akira and colleagues just over a year later (2). TLR7 recognizes single-stranded RNA (ssRNA), inducing downstream activation of signaling molecules, including Jnk and NF-κB through a myeloid differentiation primary response gene 88 (MyD88)-dependent cascade (3). This process is central to host defense against invading viruses; however, TLR7 hyperactivity can also drive the initiation and progression of autoimmunity.Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the presence of antinuclear autoantibodies (ANA) (4). The ANAs form immune complexes with host-derived nuclear material, which accumulate as deposits within the tissues and organs. The ensuing inflammation can lead to organ failure, particularly glomerulonephritis (GN) (5). Prospective studies by Harley and colleagues (6) demonstrated that ANAs are detectable ∼6 years before clinical presentation of SLE, with only a small percentage of these individuals who have ANAs progressing to pathogenic autoimmunity. These data, together with murine studies, suggest that defects in multiple pathways contribute to the initiation and progression of systemic autoimmunity (7).Multiple investigations have shown that TLR7 and the MyD88 signaling pathway are critical for the initiation of autoimmunity and development of self-reactivity, because genetic ablation of either TLR7 or MyD88 prevents the development of both ANAs and subsequent immune-pathology (8–10). Furthermore, this signaling pathway is specifically required within B cells (11, 12). In TLR7-sufficient autoimmune prone systems, additional immune alterations, such as lpr (lymphoproliferation), sle3, or yaa (Y-linked autoimmune-accelerating locus) can lead to severe autoimmunity. The yaa murine susceptibility locus is a region comprising 16 genes, including TLR7, which translocated from the X chromosome to the Y (13, 14). The yaa-associated increase in TLR7 expression and function was determined to be critical for the development of severe disease, independent of the SLE-model (15–17). More recently, we demonstrated that a twofold increase in TLR7 alone on the Sle1 background is sufficient to drive disease in an almost identical manner to the addition of the yaa susceptibility locus (18). Normalization of B-cell TLR7 did not affect GN, suggesting that the increase in other cells drives severe pathology. Additional studies have suggested that although plasmacytoid dendritic cells (pDCs) contribute to pathology of a yaa-associated model, other cell types are critical for full disease pathogenesis (19). Determining these critical cell types will clarify the mechanisms of progression for therapeutic targeting.In this study we investigated the contribution of DCs to TLR7-mediated SLE-associated GN. Previous data has presented conflicting evidence on their role in lupus models. Although B-cell proliferation and antibody production can be enhanced by DC activation and genetic ablation diminishes disease progression (20, 21), DCs also play a protective role in regulatory T-cell development and their elimination can result in autoimmunity (22, 23). The overall response in each system may depend on the genetic background or incomplete ablation of DC subsets [discussed by Platt and Randolph (24)].We bred conditional SLE mice overexpressing TLR7 (Sle1Tg7) with a CD11c^Cre reporter strain (25) and demonstrated that the increase in TLR7 within CD11c⁺ cells was essential for all severe autoimmune traits, including splenomegaly, T- and B-cell activation, and GN. Purification of DCs and other leukocyte subsets confirmed normalization of TLR7 mRNA in CD11b⁺ conventional (c)DCs and pDCs. We identified and characterized a novel CD11b⁺ cDC infiltrating diseased kidneys, which was absent, together with all other pathological traits, upon restoration of TLR7. Taken together, our data suggest that the CD11b⁺ cDC, rather than the pDC, is playing the principle role in driving end organ disease. Finally, we determined that primary human BDCA1⁺ DCs or monocyte-derived DCs express extremely low levels of TLR7 mRNA, but this can be rapidly up-regulated on exposure to virus or IFN-α. This work has uncovered a key role for DCs in one of the most clinically important phases of SLE, the progression to lupus nephritis.     

Estradiol valerate and alcohol intake: dose-response assessments

Background

An injection of estradiol valerate (EV) provides estradiol for a prolonged period. Recent research indicates that a single 2.0 mg injection of EV modifies a female rat's appetite for alcoholic beverages. This research extends the initial research by assessing 8 doses of EV (from.001 to 2.0 mg/female rat), as well assessing the effects of 2.0 mg EV in females with ovariectomies.

Results

With the administration of EV, there was a dose-related loss of bodyweight reaching the maximum loss, when it occurred, at about 4 days after injections. Subsequently, rats returned to gaining weight regularly. Of the doses tested, only the 2.0 mg dose produced a consistent increase in intake of ethanol during the time previous research indicated that the rats would show enhanced intakes. There was, however, a dose-related trend for smaller doses to enhance intakes. Rats with ovariectomies showed a similar pattern of effects, to intact rats, with the 2 mg dose. After extensive histories of intake of alcohol, both placebo and EV-treated females had estradiol levels below the average measured in females without a history of alcohol-intake.

Conclusion

The data support the conclusion that pharmacological doses of estradiol can produce enduring changes that are manifest as an enhanced appetite for alcoholic beverages. The effect can occur among females without ovaries.     

Shilajit attenuates behavioral symptoms of chronic fatigue syndrome by modulating the hypothalamic-pituitary-adrenal axis and mitochondrial bioenergetics in rats

Ethnopharmacological relevance

Shilajit has been used as a rejuvenator for ages in Indian ancient traditional medicine and has been validated for a number of pharmacological activities.

Aim of the study

The effect of processed shilajit which was standardized to dibenzo-α-pyrones (DBPs;0.43% w/w), DBP-chromoproteins (DCPs; 20.45% w/w) and fulvic acids (56.75% w/w) was evaluated in a rat model of chronic fatigue syndrome (CFS). The mitochondrial bioenergetics and the activity of hypothalamus–pituitary–adrenal (HPA) axis were evaluated for the plausible mechanism of action of shilajit.

Materials and Methods

CFS was induced by forcing the rats to swim for 15 mins for 21 consecutive days. The rats were treated with shilajit (25, 50 and 100 mg/kg) for 21 days before exposure to stress procedure. The behavioral consequence of CFS was measured in terms of immobility and the climbing period. The post-CFS anxiety level was assessed by elevated plus maze (EPM) test. Plasma corticosterone and adrenal gland weight were estimated as indices of HPA axis activity. Analysis of mitochondrial complex chain enzymes (Complex I, II, IV and V) and mitochondrial membrane potential (MMP) in prefrontal cortex (PFC) were performed to evaluate the mitochondrial bioenergetics and integrity respectively.

Results

Shilajit reversed the CFS-induced increase in immobility period and decrease in climbing behavior as well as attenuated anxiety in the EPM test. Shilajit reversed CFS-induced decrease in plasma corticosterone level and loss of adrenal gland weight indicating modulation of HPA axis. Shilajit prevented CFS-induced mitochondrial dysfunction by stabilizing the complex enzyme activities and the loss of MMP. Shilajit reversed CFS-induced mitochondrial oxidative stress in terms of NO concentration and, LPO, SOD and catalase activities.

Conclusion

The results indicate that shilajit mitigates the effects of CFS in this model possibly through the modulation of HPA axis and preservation of mitochondrial function and integrity. The reversal of CFS-induced behavioral symptoms and mitochondrial bioenergetics by shilajit indicates mitochondria as a potential target for treatment of CFS.     

The same synaptic vesicles drive active and spontaneous release

Synaptic vesicles release neurotransmitter both actively (on stimulation) and spontaneously (at rest). It has been assumed that identical vesicles use both modes of release; however, recent evidence has challenged this view. Using several assays (FM dye imaging, pHluorin imaging and antibody-labeling of synaptotagmin) in neuromuscular preparations from Drosophila, frog and mouse, as well as rat cultured neurons, we found that the same vesicles participate in active and spontaneous release.     

Magnetic resonance imaging guided cardiovascular interventions in congenital heart diseases

The purpose of this article is to present some recent applications of diagnostic and interventional MRI in congenital heart disease. To date x-ray-based techniques have been the norm for most diagnostic and therapeutic applications. With the advent of ultrafast MRI and the development of MRI-compatible catheters and guide wires, the goal of achieving real-time guidance by MRI for interventions in congenital heart diseases has proven feasible. We briefly review the latest advances in cardiovascular MRI, and the development of MR-compatible devices for diagnostic and therapeutic applications such as ASD closure and pulmonary artery dilation.