The Beneficial Role of Filipendula ulmaria Extract in Prevention of Prodepressant Effect and Cognitive Impairment Induced by Nanoparticles of Calcium Phosphates in Rats.

Mineral components of dental composites are used in many medical and dental applications, including preventive, restorative, and regenerative dentistry. To evaluate the behavioural alterations induced by nanosized particles of novel dental composites, by means of depressive level and cognitive functions, experimental groups of rats were chronically administered with nanosized hydroxyapatite (HA), tricalcium phosphate (TCP), and amorphous calcium phosphate (ACP) with or without simultaneous application of Filipendula ulmaria L. (FU) methanolic extract. The significant prodepressant action was observed in groups solely treated with HA and ACP. Besides, prolonged treatment with ACP also resulted in a significant decline in cognitive functions estimated in the novel object recognition test. The adverse impact of calcium phosphates on estimated behavioural functions was accompanied by increased oxidative damage and apoptotic markers in the prefrontal cortex, as well as diminished specific neurotrophin (BDNF) and gabaergic expression. The results of our investigation showed that simultaneous antioxidant supplementation with FU extract prevented calcium phosphate-induced behavioural disturbances, as well as prooxidative and apoptotic actions, with the simultaneous restoration of BDNF and GABA-A receptors in the prefrontal cortex. These findings suggest that FU may be useful in the prevention of prodepressant impact and cognitive decline as early as the manifestation of calcium phosphate-induced neurotoxicity.

IL (Interleukin)-33 Suppresses Abdominal Aortic Aneurysm by Enhancing Regulatory T-Cell Expansion and Activity.

Objective- Inflammation occurs during the progression of abdominal aortic aneurysm (AAA). IL (interleukin)-33 is a pleiotropic cytokine with multiple immunomodulatory effects, yet its role in AAA remains unknown. Approach and Results- Immunoblot, immunohistochemistry, and immunofluorescent staining revealed increased IL-33 expression in adventitia fibroblasts from mouse AAA lesions. Daily intraperitoneal administration of recombinant IL-33 or transgenic IL-33 expression ameliorated periaorta CaPO4 injury- and aortic elastase exposure-induced AAA in mice, as demonstrated by blunted aortic expansion, reduced aortic wall elastica fragmentation, enhanced AAA lesion collagen deposition, attenuated T-cell and macrophage infiltration, reduced inflammatory cytokine production, skewed M2 macrophage polarization, and reduced lesion MMP (matrix metalloproteinase) expression and cell apoptosis. Flow cytometry analysis, immunostaining, and immunoblot analysis showed that exogenous IL-33 increased CD4+Foxp3+ regulatory T cells in spleens, blood, and aortas in periaorta CaPO4-treated mice. Yet, ST2 deficiency muted these IL-33 activities. Regulatory T cells from IL-33-treated mice also showed significantly stronger activities in suppressing smooth muscle cell inflammatory cytokine and chemokine expression, macrophage MMP expression, and in increasing M2 macrophage polarization than those from vehicle-treated mice. In contrast, IL-33 failed to prevent AAA and lost its beneficial activities in CaPO4-treated mice after selective depletion of regulatory T cells. Conclusions- Together, this study established a role of IL-33 in protecting mice from AAA formation by enhancing ST2-dependent aortic and systemic regulatory T-cell expansion and their immunosuppressive activities.

Investigation of the Genotoxicity of Aluminum Oxide, ß-Tricalcium Phosphate, and Zinc Oxide Nanoparticles In Vitro.

The aim of this study was to investigate the genotoxicity of aluminum oxide (Al2O3), ß-tricalcium phosphate (ß-TCP) (Ca3(PO4)2), and zinc oxide (ZnO) nanoparticles (NPs) that were 4.175, 9.058, and 19.8 nm sized, respectively, on human peripheral blood lymphocytes using micronucleus (MN) and chromosome aberration (CA) techniques. Aluminum oxide and ß-TCP NPs did not show genotoxic effects on human peripheral blood cultures in vitro, even at the highest concentrations; therefore, these materials may be suitable for use as biocompatible materials. It was observed that, even at a very low dose (≥12.5 ppm), ZnO NPs had led to genotoxicity. In addition, at high concentrations (500 ppm and above), ZnO NPs caused mortality of lymphocytes. For these reasons, it was concluded that ZnO NPs are not appropriate for using as a biocompatible biomaterial.

A Short Overview on the Biomedical Applications of Silica, Alumina and Calcium Phosphate-based Nanostructured Materials.

This article reviews the use of silica, alumina and calcium phosphate-based nanostructured materials with biomedical applications. A short introduction on the use of the materials in Science, Nanotechnology and Health is included followed by a revision of each of the selected materials. A description of the principal synthetic methods used in the preparation of the materials in nanostructured form is included. The most widely used applications in biomedicine are reviewed including, for example drug-delivery, bone regeneration, imaging, sensoring amongst others. Finally, a short description of the toxicity and cytotoxicity associated with each of the materials of this revision is presented. This short literature revision serves to demonstrate the very promising future ahead of nanosystems based on silica, alumina and calcium phosphate for biological and biomedical applications.

Basic calcium phosphate crystals and osteoarthritis pathogenesis: novel pathways and potential targets.

PURPOSE OF REVIEW: Basic calcium phosphate (BCP) crystals have long been associated with the pathogenesis of osteoarthritis. As our knowledge concerning BCP crystals in osteoarthritis expands, so does the potential to develop targeted therapies. The present review discusses recent advances in this field and attempts to summarize our current understanding regarding the role of BCP crystals in osteoarthritis pathogenesis. RECENT FINDINGS: BCP crystals injected into the knees of mice induce osteoarthritis-like changes, further evidence of their pathogenic properties. Interleukin-6 has emerged as a key cytokine involved in BCP crystal-induced inflammation that could represent a potential therapeutic target. The role of BCP crystal-induced osteoclastogenesis has also recently been explored and may also hold the key to future targeted therapies. Although tools to detect BCP crystals remain limited, dual energy computerized tomography scanning has emerged as a useful noninvasive means of quantifying intra-articular calcium crystal deposition. SUMMARY: BCP crystals can activate a number of inflammatory pathways which in turn may lead to cartilage degradation and osteoarthritis. Understanding of these pathways may ultimately yield targeted therapies for osteoarthritis, for which none currently exists.

Development of calcium phosphate/sulfate biphasic cement for vital pulp therapy.

OBJECTIVES: Bioactive calcium phosphate cement (CPC) has been used widely to repair bone defects because of its excellent biocompatibility and bioactivity. However, the poor handling properties, low initial mechanical strength, and long setting time of CPC limit its application in vital pulp therapy (VPT). The aim of this study was to synthesize biphasic calcium phosphate/sulfate cements and evaluate the feasibility of applying these cements in VPT. METHODS: The physical, chemical, and mechanical properties of CPC were improved by mixing the cement with various amounts of α-calcium sulfate hemihydrate (CSH). The hydration products and crystalline phases of the materials were characterized using scanning electron microscopy and X-ray diffraction analysis. In addition, the physical properties, such as the setting time, compressive strength, viscosity, and pH were determined. Water-soluble tetrazolium salt-1 and lactase dehydrogenase were used to evaluate cell viability and cytotoxicity. RESULTS: The developed CPC (CPC/CSH cement), which contains 50wt% CSH cement, exhibited no obvious temperature increase or pH change during setting when it was used as a paste. The initial setting time of the CPC/CSH biphasic cement was substantially shorter than that of CPC, and the initial mechanical strength was 23.7±5.6MPa. The CPC/CSH cement exhibited higher viscosity than CPC and, thus, featured acceptable handling properties. X-ray diffraction analysis revealed that the relative peak intensity for hydroxyapatite increased, and the intensity for calcium sulfate dehydrate decreased as the amount of CPC was increased. The cell viability and cytotoxicity test results indicated that the CPC/CSH cement did not harm dental pulp cells. SIGNIFICANCE: The developed CPC/CSH biphasic cement exhibits substantial potential for application in VPT.

Zn- and Mg- containing tricalcium phosphates-based adjuvants for cancer immunotherapy.

Zn-, and Mg-containing tricalcium phosphates (TCPs) loaded with a hydrothermal extract of a human tubercle bacillus (HTB) were prepared by immersing Zn-TCP and Mg-TCP in HTB-containing supersaturated calcium phosphate solutions. The in vitro and in vivo immunogenic activities of the HTB-loaded Zn-, and Mg-TCPs (Zn-Ap-HTB and Mg-Ap-HTB, respectively) were evaluated as potential immunopotentiating adjuvants for cancer immunotherapy. The Zn-Ap-HTB and Mg-Ap-HTB adjuvants showed no obvious cytotoxicity and more effectively stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion by macrophage-like cells than unprocessed HTB or HTB-loaded TCP (T-Ap-HTB) in vitro. Zn-Ap-HTB and Mg-Ap-HTB mixed with liquid-nitrogen-treated tumor tissue markedly inhibited the in vivo development of rechallenged Lewis lung carcinoma (LLC) cells compared with T-Ap-HTB and the unprocessed HTB mixed liquid-nitrogen-treated tumor tissue. Zn-Ap-HTB and Mg-Ap-HTB contributed to eliciting potent systemic antitumor immunity in vivo.

Influence of calcium phosphate crystal assemblies on the proliferation and osteogenic gene expression of rat bone marrow stromal cells.

Calcium phosphates (CaPs) have been investigated as substrates to promote bone formation both in vitro and in vivo. The aim of this study was to examine the proliferation and differentiation of rat bone marrow stromal cells (BMSCs) cultured on three-dimensional (3D) octacalcium phosphate (OCP) crystal assemblies. The cytotoxicity of OCP crystal assemblies was evaluated by measuring the lactate dehydrogenase (LDH) release from BMSCs during 10h of incubation with OCP crystal assemblies. The proliferation of BMSCs on OCP crystal assemblies in medium with or without osteogenic supplements was also investigated using the MTT assay with tissue culture treated plastic (TP) as the control. The tissues formed by BMSCs cultured on OCP crystal assemblies for 24 days were examined following staining with haematoxylin and eosin (H&E), alkaline phosphatase (ALP) and Van Gieson's techniques. The influence of OCP crystal assemblies on mRNA expression of alpha chain of collagen type I (Coll-Ia), ALP and osteocalcin (OC), osteonectin (ON), osteopontin (OP), lumican, Cbfa1, EST317 and EST350 by the BMSCs were also investigated using semi-quantitative RT-PCR. Although OCP crystals were relatively cytotoxic compared with TP, proliferation of BMSCs occurred when seeded onto OCP crystal assemblies. BMSCs cultured on OCP demonstrated similar proliferation rates as found on the control and no significant difference (P<0.05) in the number of cells cultured in medium supplemented with or without osteogenic additives on TP and OCP. The deposition of collagen and ALP were detected in tissue synthesised by BMSCs cultured on OCP crystals assemblies. OCP crystal assemblies down-regulated basal bone ECM proteins, including Coll-Ia, ON and lumican, in the first week of culture, whilst up-regulation of the same genes was observed after 24 days of culture. The observed down-regulation of Cbfa1 on OCP substrates was consistent with the negative effect of OCP crystal assemblies on the genes encoding bone ECM proteins. The up-regulation of OC mRNA expression by OCP crystal assemblies could be related to the requirement for synthesis of more OC proteins to control the concentration of calcium ions in culture medium.

Calcium phosphate nanoparticle adjuvant.

Vaccination to protect against human infectious diseases may be enhanced by using adjuvants that can selectively stimulate immunoregulatory responses. In a murine model, a novel nanoparticulate adjuvant composed of calcium phosphate (CAP) was compared with the commonly used aluminum (alum) adjuvants for its ability to induce immunity to herpes simplex virus type 2 (HSV-2) and Epstein-Barr virus (EBV) infections. Results indicated that CAP was more potent as an adjuvant than alum, elicited little or no inflammation at the site of administration, induced high titers of immunoglobulin G2a (IgG2a) antibody and neutralizing antibody, and facilitated a high percentage of protection against HSV-2 infection. Additional benefits of CAP include (i) an insignificant IgE response, which is an important advantage over injection of alum compounds, and (ii) the fact that CAP is a natural constituent of the human body. Thus, CAP is very well tolerated and absorbed. These studies were performed with animal models. By virtue of the potency of this CAP adjuvant and the relative absence of side effects, we believe that this new CAP formulation has great potential for use as an adjuvant in humans.

Local tissue irritating effects and adjuvant activities of calcium phosphate and aluminium hydroxide with different physical properties.

Effects of calcium phosphate and aluminium hydroxide adjuvants with different physical properties were examined in guinea pigs for local histopathological reactions, electron-microscopical changes of macrophages and adjuvanticity on total IgG antibody response to subcutaneously administered ovalbumin (OVA) and tetanus toxoid (TT). Calcium phosphate gel (Ca-gel) induced active inflammatory reactions consisting of neutrophils (pseudoeosinophils) and foamy macrophages associated with many multinuclear giant cells for at least 4 weeks. Aluminium hydroxide gel (Al-gel) also elicited granulomatous inflammatory reactions consisting mainly of macrophages with foamy cytoplasm, small lymphocytes and giant cells at the injection sites for up to 8 weeks or longer. Severity of local tissue irritation due to calcium phosphate gel (Ca-gel) was similar to that due to Al-gel except for the duration of the inflammatory reactions. Calcium phosphate suspension (Ca-sus)-induced local reactions completely ceased by the 4th week, while aluminium hydroxide suspension (Al-sus)-induced reactions were seen up to the 8th week. Electron-microscopical observations showed that both Al-gel and Al-sus caused damage of macrophages. The adjuvant activity of Al-gel for OVA or TT was significantly stronger than that of any other adjuvant material, whereas those of Ca-gel and Ca-sus were not seen at a dose of 3 mg calcium phosphate per millilitre. Al-sus-TT at a dose of 3 mg aluminium hydroxide per millilitre induced very low levels of antibody. These results suggest that calcium phosphate adjuvant may not be an useful alternative to Al adjuvant.

Relationship between hemolytic activity and adsorption capacity of aluminum hydroxide and calcium phosphate as immunological adjuvants for biologicals.

Aluminum hydroxide (Al) and calcium phosphate (Ca) gels have been used as vaccine adjuvants for many years. We investigated mechanism of the hemolytic activities of both adjuvant materials. The hemolytic activity of each gel depended on the gel dose. The adsorption capacities and the hemolytic activities of both adjuvants decreased as the concentration of phosphate increased in a gel-washing solution. A positive correlation between the hemolytic activity and the adsorption capacity was found in Al-gel. A disruptive effect of Ca-gel on membrane of erythrocytes was shown by electron microscopy. Ca-gel required more than 10 times as much pre-adsorbed ovalbumin as did Al-gel to inhibit the hemolysis. These results suggest that the hemolytic activity of both adjuvant materials depended mainly on the adsorption ability, and it may be useful to control the adsorption ability of adjuvants to reduce their hemolytic activity.

Studies on the toxicities of aluminium hydroxide and calcium phosphate as immunological adjuvants for vaccines.

Aluminium hydroxide (Al) and calcium phosphate (Ca) have been used for many years as immunological adjuvants for biologicals. We investigated the toxic effects of both adjuvants with different physical properties. Al-gel elicited vascular permeability-increasing and toxic effects to macrophages (M phi), while its haemolytic effect was weak. Ca-gel elicited a significantly stronger haemolytic effect, but no other toxic effect. Incubation of M phi or polymorphonuclear leucocytes with Al-suspension resulted in the largest release of lactate dehydrogenase. Ca-suspension caused haemolysis of about 50% of that caused by Ca-gel.

Toxicity of calcium sodium metaphosphate fiber. II. Chronic inhalation and oncogenicity study.

Male and female Fischer 344 rats (80/sex/group) were exposed to CSM fiber 6 hr/day, 5 days/week at target-exposure levels of 0, 1, 5, or 25 mg/m3 for 24 months, corresponding to 0, 27, 80, and 513 fibers/cc, respectively. Number and size of the airborne fibers were determined during the course of the study. At 3 and 12 months, 10 rats/sex/group were euthanized and at 18 and 24 months 5 rats/sex/group were euthanized. In addition, 5 rats/sex/group were removed from exposure at 18 months and maintained for a 6-month recovery period. All animals surviving at the completion of the exposure period were maintained in a clean environment for up to 5 additional months. Clinical laboratory examinations were performed on 10 animals/sex/group at 3, 12, and 24 months. The number of fibers in the lung were also determined at 3, 12, 18, and 24 months. Body weight and survival did not appear to be affected by treatment. There were no biologically significant effects on clinical parameters. There was a dose-related increase in lung weight during the exposure period which was generally reversible during the recovery periods. There also was a dose-related increase in the number of fibers/milligram of lung, but no increase in lung fiber burden after the first 3 months. The number of fibers in the lungs of animals exposed to CSM fiber for 18 months and allowed 6-month recovery period showed a decrease especially at the high dose. No increase in tumors (benign or malignant) was observed in this study. Microscopic changes considered reflective of an irritant response were observed in the nasal turbinates notably at the 5 and 25 mg/m3 levels. Histological changes were also observed in the lungs at the 5 and 25 mg/m3 levels. The incidence and/or severity of histopathological changes in the 1 mg/m3 group was considered to be essentially comparable to controls.