Protective effect and mechanism of lycopene on endothelial progenitor cells (EPCs) from type 2 diabetes mellitus rats.

Endothelial progenitor cells (EPCs), widely existing in bone marrow and peripheral blood, are involved in the repair of injured vascular endothelium and angiogenesis which are important to diabetic mellitus (DM) patients with vascular complications. The number and the function of EPCs are related to the advanced glycation end products (AGEs) generated in DM patients. Lycopene (Lyc) is an identified natural antioxidant that protects EPCs under the microenvironment of AGEs from damage. However, the underlying mechanism remains unclear. To investigate the effect of Lyc on EPCs, we isolated EPCs from DM rat bone marrow and determined cell proliferation, cell cycle,apoptosis and autophagy of EPCs. The present study showed that 10µg/mL Lyc improved cell proliferation and had low cytotoxicity in the presence of AGEs. In addition, Lyc rescued S phase of the cell cycle arrest, reduced apoptosis rate and decreased autophagic reaction including ROS and mitochondrial membrane potential (MMP) of EPCs. Moreover, Lyc combined use of autophagy inhibitors, 3-MA, had better protective effects. Taken together, our data suggests that Lyc promotes EPCs survival and protect EPCs from apoptosis and oxidative autophagy induced by AGEs, further remaining the number and function of EPCs. This study provides new insights into Lyc protective mechanism of AGEs-induced oxidative autophagy in EPCs from DM patients and offers a new therapy for DM vascular complications.

Effects of Astragalus Combined with Angelica on Bone Marrow Hematopoiesis Suppression Induced by Cyclophosphamide in Mice.

Danggui Buxue Tang (DBT), a combination of Astragalus and Angelica at a 5 : 1 ratio, mainly promotes hematopoiesis. However, in the clinic, the combination ratio of Astragalus and Angelica to treat low hematopoietic function is not an absolute 5 : 1 ratio, suggesting that the herbs may promote hematopoiesis better after being combined at a certain range of ratios. The objective of this study is to investigate the effect of different ratio combinations of Astragalus and Angelica on bone marrow hematopoiesis suppression induced by cyclophosphamide (CTX) and to probe the interaction and mechanism of Astragalus combined with Angelica in promoting hematopoiesis. Following establishment of the model, mice were administered with Astragalus (6.00 g·kg-1), Angelica (3.00 g·kg-1), and combinations of Astragalus and Angelica at different ratios, including 10 : 1 (Astragalus 9.81 g·kg-1+Angelica 0.98 g·kg-1), 5 : 1 (Astragalus 9.00 g·kg-1+Angelica 1.80 g·kg-1), 2 : 1 (Astragalus 7.71 g·kg-1+Angelica 3.08 g·kg-1), 1 : 1 (Astragalus 5.40 g·kg-1+Angelica 5.40 g·kg-1), 1 : 2.5 (Astragalus 3.08 g·kg-1+Angelica 7.71 g·kg-1), 1 : 5 (Astragalus 1.80 g·kg-1+Angelica 9.00 g·kg-1), and 1 : 10 (Astragalus 0.98 g·kg-1+Angelica 9.81 g·kg-1). Our results suggested that Astragalus mixed with Angelica synergistically promoted hematopoiesis best when the combination ratio of Astragalus and Angelica was 1 : 1, 1 : 2.5 or 1 : 5; moreover, the effect of Angelica was greater than that of Astragalus. The potential mechanisms of the combinations of Astragalus and Angelica that promote hematopoiesis include the dissolution of the effective components, promoting the synthesis and secretion of hematopoietic growth factor (HGF) and the proliferation of hematopoietic progenitor cells (HPCs).

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages.

BACKGROUND: The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. RESULTS: Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. CONCLUSION: Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent.

Genotoxicity testing of peptides: folate deprivation as a marker of exaggerated pharmacology.

The incidence of micronucleated-cells is considered to be a marker of a genotoxic event and can be caused by direct- or indirect-DNA reactive mechanisms. In particular, small increases in the incidence of micronuclei, which are not associated with toxicity in the target tissue or any structurally altering properties of the compound, trigger the suspicion that an indirect mechanism could be at play. In a bone marrow micronucleus test of a synthetic peptide (a dual agonist of the GLP-1 and GIP receptors) that had been integrated into a regulatory 13-week repeat-dose toxicity study in the rat, small increases in the incidence of micronuclei had been observed, together with pronounced reductions in food intake and body weight gain. Because it is well established that folate plays a crucial role in maintaining genomic integrity and pronounced reductions in food intake and body weight gain were observed, folate levels were determined from plasma samples initially collected for toxicokinetic analytics. A dose-dependent decrease in plasma folate levels was evident after 4 weeks of treatment at the mid and high dose levels, persisted until the end of the treatment duration of 13-weeks and returned to baseline levels during the recovery period of 4 weeks. Based on these properties, and the fact that the compound tested (peptide) per se is not expected to reach the nucleus and cause DNA damage, the rationale is supported that the elevated incidence of micronucleated polychromatic erythrocytes is directly linked to the exaggerated pharmacology of the compound resulting in a decreased folate level.

Biochemical, radiologic, ultrastructural, and genetic evaluation of iron overload in acute leukemia and iron-chelation therapy.

Iron overload in hereditary hemochromatosis and hematologic malignancy has unfavorable effects on morbidity. Herein, 53 children (age 108.4±58.3 mo, 25 girls and 28 boys) with acute myeloblastic and lymphoblastic leukemia, who received 4 different chemotherapy protocols, were evaluated for iron overload throughout chemotherapy. Iron overload arose: (1) before chemotherapy, which was dependent on neither chemotherapy nor packed red blood cell transfusions and (2) after chemotherapy, which was dependent on the duration and nature of chemotherapy and partially on transfusion of packed red blood cells. Iron overload was documented in 75% of patients with a ferritin level >1000 ng/mL, by liver and heart magnetic resonance imaging, and they were administered iron-chelation therapy with success. Three of 10 radiologically iron-overloaded patients were heterozygous for H63D mutation. Aminolevulinic acid and porphobilinogen levels were normal. Light microscopic examination of the bone marrow revealed increased iron granules in erythroblasts, platelets, and megakaryocytes, iron-laden macrophages, free iron in the matrix, dyshematopoiesis, and apoptotic cells. Electron microscopic examination revealed iron-laden secondary lysosomes and autolysosomes in normoblasts and iron-laden primary granules in promyelocytes, irrelevant to the ferritin level, implying autophagia due to chemotherapy as a source of the excess iron. We think that iron overload, which is an important complication of acute leukemia, should be evaluated separately from "transfusion overload," and the management principles specific to leukemia should be implemented.

Role of recombinant human erythropoietin in mitomycin C-induced genotoxicity: analysis of DNA fragmentation, chromosome aberrations and micronuclei in rat bone-marrow cells.

Mitomycin C (MMC) is one of the most effective chemotherapeutic agents. However, during clinical use several side effects may occur. Recombinant human erythropoietin (rhEPO), a glycoprotein that regulates haematopoiesis, has been shown to exert an important cyto-protective effect in many tissues. The aim of this study was to explore whether rhEPO protects against MMC-induced genotoxicity in rat bone-marrow cells. Adult male Wistar rats were divided into six groups of 18 animals each: a control group, a 'rhEPO alone' group, an 'MMC alone' group and three 'rhEPO+MMC' groups (pre-, co- and post-treatment conditions). Our results show that MMC induced a noticeable genotoxic effect in rat bone-marrow cells. rhEPO reduced the effects of MMC significantly in every type of experiment conducted, such as the frequency of micronuclei, the percentage of chromosome aberrations and the level of DNA damage measured with the comet assay. The protective effect of rhEPO was more efficient when it was given 24h prior to MMC treatment.

Maturation of murine bone marrow dendritic cells induced by acidic Ginseng polysaccharides.

In this study, we report that a acidic polysaccharide (AGP) isolated from a Chinese medicinal herb, named Ginseng (Panax giseng C.A. Meyer), induces maturation of bone marrow dendritic cells (BMDCs) via concrete changes both inside and outside BMDCs. The impacts of AGP on BMDCs were assessed with use of conventional scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) for morphology, flow cytometry (FCM) for key surface molecules, cytochemistry assay, FITC-dextran, bio-assay for phagocytosis and enzyme linked immunosorbent assay (ELISA) for production of cytokines. Our results elucidated that PPS promoted maturation of BMDCs via changes as reflected by the down-regulation of acid phosphatase (ACP) activity inside the BMDCs, which occurs when phagocytosis of BMDCs to antigen decreased, while antigen presentation increased upon maturation, higher expression of key surface molecules of MHC II, CD80, CD86, CD83, and CD40, and releasing higher level of cytokines IL-12 and low level of TNF-α. Our study suggest that AGP play marked immunostimulating role on the maturation of murine BMDCs through precise regulation of phagocytosis and enzyme activities inside the BMDCs.

Effects of micropitted/nanotubular titania topographies on bone mesenchymal stem cell osteogenic differentiation.

Micro/nanotopographical modification of biomaterials constitutes a promising approach to direct stem cell osteogenic differentiation to promote osseointegration. In this work, titania nanotubes (NTs) 25 and 80 nm in size with the acid-etched Ti topography (AcidTi) and hierarchical hybrid micropitted/nanotubular topographies (Micro/5VNT and Micro/20VNT) are produced to mimic the structure of the natural bone extracellular matrix (ECM). The effects on bone mesenchymal stem cell (MSC) osteogenic differentiation are studied systematically by various microscopic and biological characterization techniques. Cell adhesion is assayed by nucleus fluorescence staining and cell proliferation is studied by CCK-8 assay and flow cytometry. Osteogenic differentiation is assayed by alkaline phosphatase (ALP) expression, collagen secretion, matrix mineralization, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis on the osteogenesis related gene expression. All the topographies are observed to induce MSC osteogenic differentiation in the absence of osteogenic supplements. The nanotube surfaces significantly promote cell attachment and spread, collagen secretion and ECM mineralization, as well as osteogenesis-related gene expression. Among them, Micro/20VNT shows the best ability to simultaneously promote MSC proliferation and osteogenic differentiation. Our results unambiguously demonstrate their excellent ability to support MSC proliferation and induce MSC osteogenic differentiation, especially those with the micropitted topography.

Evaluation of mutagenic and antimutagenic activities of neem (Azadirachta indica) seed oil in the in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test.

AIM OF THE STUDY: The possible mutagenic and antimutagenic activity of neem oil (NO) and its DMSO extract (NDE) were, examined in the Ames Salmonella/microsome mutagenicity test and the mouse bone marrow micronucleus assay. MATERIALS AND METHODS: Eight different strains of Salmonella typhimurium were, used to study the genotoxicity of neem oil both in the presence and absence of Aroclor-1254 induced rat liver homogenate (S9). Two-dose treatment protocol was, employed to study the cytogenetic activity in micronucleus assay. Similarly, the antimutagenic activity of neem oil and NDE was studied against mitomycin (MMC) and 7,12-dimethylbenz[a]anthracene (DMBA) in the above two test systems. RESULTS: Neem oil was non-mutagenic in all the eight tester strains of Salmonella typhimurium both in the presence and absence of S9 mix. In the present study, there was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in neem oil treated groups over the negative control (DMSO) group of animals, indicating the non-clastogenic activity of neem oil in the micronucleus test. Neem oil showed good antimutagenic activity against DMBA induced mutagenicity compared to its DMSO extract. However, neem oil showed comparatively less antimutagenicity against MMC in the Ames assay. In vivo anticlastogenic assays shows that neem oil exhibited better activity against DMBA induced clastogenicity. CONCLUSION: These results indicate non-mutagenic activity of neem oil and significant antimutagenic activity of neem oil suggesting its pharmacological importance for the prevention of cancer.

Genotoxicity assessment of S-equol in bacterial mutation, chromosomal aberration, and rodent bone marrow micronucleus tests.

Intervention studies in man have shown that dietary soy isoflavones may provide certain health benefits. One of the possible reasons for this benefit is that the daidzein contained in soy is converted to S-equol. As part of a drug development program for S-equol, three genotoxicity studies were conducted. The studies comprised bacterial mutation, chromosomal aberration, and in vivo bone marrow micronucleus tests conducted according to Good Laboratory Practices (GLP). No evidence of genotoxic activity was observed in the in vitro tests at concentrations up to those associated with cell toxicity. In addition, no evidence of cytotoxicity or genotoxicity was seen in the rat bone marrow micronucleus test in animals dosed at levels up to the standard limit of 2000 mg/kg. It is concluded that S-equol is not active in the standard battery of genotoxicity assays recommended by the International Conference on Harmonisation (ICH) for registration of new pharmaceuticals. The current results support the further development of S-equol.

Ovine bone marrow mesenchymal stem cells: isolation and characterization of the cells and their osteogenic differentiation potential on embroidered and surface-modified polycaprolactone-co-lactide scaffolds.

The current study was undertaken with the goal being isolation, cultivation, and characterization of ovine mesenchymal stem cells (oMSC). Furthermore, the objective was to determine whether biological active polycaprolactone-co-lactide (trade name PCL) scaffolds support the growth and differentiation of oMSC in vitro. The oMSC were isolated from the iliac crest of six merino sheep. Three factors were used to demonstrate the MSC properties of the isolated cells in detail. (1) Their ability to proliferate in culture with a spindle-shaped morphology, (2) presence of specific surface marker proteins, and (3) their capacity to differentiate into the three classical mesenchymal pathways, osteoblastic, adipogenic, and chondrogenic lineages. Furthermore, embroidered PCL scaffolds were coated with collagen I (coll I) and chondroitin sulfate (CS). The porous structure of the scaffolds and the coating with coll I/CS allowed the oMSC to adhere, proliferate, and to migrate into the scaffolds. The coll I/CS coating on the PCL scaffolds induced osteogenic differentiation of hMSC, without differentiation supplements, indicating that the scaffold also has an osteoinductive character. In conclusion, the isolated cells from the ovine bone marrow have similar morphologic, immunophenotypic, and functional characteristics as their human counterparts. These cells were also found to differentiate into multiple mesenchymal cell types. This study demonstrates that embroidered PCL scaffolds can act as a temporary matrix for cell migration, proliferation, and differentiation of oMSC. The data presented will provide a reliable model system to assess the translation of MSC-based therapy into a variety of valuable ovine experimental models under autologous settings.

Vitalized guided bone regeneration membrane from marrow stromal cells.

PURPOSE: Various materials have been used to make guided bone regeneration membranes. The purpose of this study was to create a novel osteogenic membrane without synthetic material. The osteogenic potential of the membrane was evaluated by both in vitro and in vivo testing. MATERIALS AND METHODS: The membrane was obtained by continuous culture of marrow stromal cells. The structure of the membrane was characterized by staining with hematoxylin-eosin, von Kossa, and carboxyfluorescein diacetate; immunohistochemical staining against collagen type I; electron microscopy; and energy-dispersive spectrometry. The osteogenic potential and bone augmentation effect of the membrane were investigated by implantation of the membrane and a membrane/natural coral composite into nude mice, respectively. RESULTS: The membrane was composed of living cells and a dense matrix of collagen type I. Mineral deposition was apparent through electronic microscopic observation and von Kossa staining. Energy-dispersive spectrometry indicated that the calcium:phosphorus ratio of mineral was 1.71 in the membrane. The membrane had formed a thin layer of bone 2 months after implantation subcutaneously. In the bone augmentation specimens, new bone was observed histologically on the surface and in the pores of natural coral in all specimens of membrane-coral composite. CONCLUSIONS: This study developed a novel strategy to produce a vital guided bone regeneration membrane without synthetic material. Membrane derived from marrow stromal cells was osteogenic and had an optimizing bone augmentation effect.

A minimal common osteochondrocytic differentiation medium for the osteogenic and chondrogenic differentiation of bone marrow stromal cells in the construction of osteochondral graft.

To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approach, controllable differentiation of bone marrow stromal cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. This study proposes to test a minimum common osteochondrocytic differentiation medium (MCDM) formulated by including common soluble supplements (dexamethasone and ascorbic acid) used to induce chondrogenic and osteogenic differentiation. The MCDM coupled with supplemented growth factors was tested for its ability to differentiate BMSCs into osteogenic and chondrogenic lineages in both two-dimensional and three-dimensional culture systems. When transforming growth factor beta3 was added to MCDM, BMSCs differentiated to chondrocyte-like cells, evidenced by the expression of glycosaminoglycans and type II collagen, whereas osteogenic differentiation was induced by supplementing osteogenic protein-1, resulting in detectable expression of osteopontin and osteocalcin. These chondrogenic and osteogenic differentiation markers were significantly enhanced in the three-dimensional cultures compared to the two-dimensional monolayer cultures. The results achieved in this study lay a foundation for future development of osteochondral graft, which could be engineered from bilayered scaffold with spatially loaded growth factors to control BMSC differentiation.

Inhibition of cyclophoshamide-induced mutagenicity by microsized powder of selenium-enriched green tea in mice.

Previously, the antioxidant activity of Se-enriched green tea extracts has been studied in vitro. In the present study, an in vivo micronuclei test was employed to assess the antimutagenic effect of microsized Se-enriched green tea powder (MSTP) in mice bone marrow. Pretreatments of MSTP, micrometer-sized regular tea powder (MRTP), selenite, and MRTP + selenite were given by gavage for 29 consecutive days prior to cyclophoshamide (CP) treatment. Certain key antioxidant enzymes were also investigated to elucidate the mechanism of antimutagenic effect. Results indicated that MSTP and MRTP or selenite alone did not significantly induce micronuclei at either concentration, confirming its nonmutagenicity. In the CP-treated groups, significant suppressions in the micronuclei were recorded following pretreatment with MSTP, MRTP, and selenite administration. The antimutagenic effect of MSTP was evidently observed by significant reduction in the frequencies of micronuclei in bone marrow cells when compared to a positive control group. The administration of MSTP, selenite, and MRTP + selenite also increased the levels of selenium concentration, glutathione peroxidase (GPx), and superoxide dismutase (SOD) enzymes in both blood and liver. However, no pronounced differences in activities of GPx and SOD were found among MSTP, selenite, and MRTP + selenite. The present findings demonstrate that the antimutagenic potential of MSTP could not be solely related to the enhancment of antioxidant enzymes of GPx and SOD.

Studies on the genotoxic effect of beryllium chloride and the possible protective role of selenium/vitamins A, C and E.

The genotoxic potential of beryllium chloride (BeCl2) was evaluated in vivo in mice using different endpoints. Chromosomal aberrations in bone marrow cells and in spermatocytes as well as sperm abnormalities were determined in the tested mice. The protective role of an orally administered drug consisting of selenium and vitamins A, C and E (selenium-ACE) was also studied. For analysis of chromosomal aberrations, both single and repeated oral treatments for a period of 3 weeks were performed. The doses used were 93.75, 187.50, 375, and 750 mg BeCl2/kg bw, which corresponds to 1/16, 1/8, 1/4, and 1/2 of the experimental LD50. BeCl2 induced a statistically significant increase in the percentage of chromosomal aberrations in both somatic and germ cells, with a dose- and time-response. The percentage of induced chromosomal aberrations was significantly reduced in all BeCl2-treated groups after oral administration of selenium-ACE. Beryllium chloride also induced a significant increase in the percentage of abnormal sperm. This percentage reached values of 9.62 +/- 0.32 and 5.56 +/- 0.31 in mice treated with the highest test dose of BeCl2 and with BeCl2+selenium-ACE, respectively, compared with 1.96 +/- 0.14 for the control. In conclusion, the results demonstrate the genotoxic effect of beryllium chloride and confirm the protective role of selenium-ACE against the genotoxicity of beryllium chloride.

Behaviour of human osteoblastic cells cultured on plasma-sprayed titanium implants in the presence of nicotine.

OBJECTIVES: The aim of this work was to analyse the behaviour of human bone marrow osteoblastic cells cultured on the surface of routinely used plasma-sprayed titanium implants in the presence of plasmatic and salivary nicotine levels reported in smokers. MATERIAL AND METHODS: Human bone marrow cells (first subculture) were seeded on titanium implants and cultured for 35 days in alpha-minimal essential medium supplemented with 10% foetal bovine serum, 50 microg/ml ascorbic acid, 10 mM beta-glycerophosphate and 10 nM dexamethasone. Seeded implants were exposed to nicotine, 10-1 mg/ml, from days 1 to 35, and characterized for cell morphology, viability/proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. RESULTS: Low levels of nicotine, 10 and 50 ng/ml, representative of the plasma concentrations reported in smokers, did not cause significant effects in the cell behaviour, although a small induction in cell growth and functional activity appeared to occur. Higher nicotine levels, 0.01-1 mg/ml, within those attained in saliva through tobacco use, caused evident dose-dependent effects in osteoblastic cell behaviour, i.e., a stimulatory effect in cell growth, ALP activity and matrix mineralization, at concentrations up to 0.2 mg/ml, and a deleterious effect at higher levels. CONCLUSIONS: Considering the high tissue diffusion potential of nicotine, the results suggest the possibility of a direct modulation of the osteoblast activity as a contributing factor to the overall effect of nicotine in the bone microenvironment around dental implants.

Preclinical safety evaluation of IFNalpha2a-NGR.

IFNalpha2a-NGR is an antitumor agent of bacterial origin. The report presents the preclinical toxicity studies with IFNalpha2a-NGR in mice, rats and monkeys. The single-dose toxicity study showed no effect on general signs, body weight, food consumption, ophthalmology, hematology and clinical chemistry and necropsy analysis. In repeated-dose toxicity studies, increase in HB was noted both in rat and monkey, showed that IFNalpha2a-NGR may not cause the suppression of hematopoiesis. Decrease in AST, A/G, GLU, T-Bil in rat and AST, TP, GLO in monkey were noted, accompanied by increase in TP and GLB in rat and BUN in monkey. All the clinical chemistry changes were mild, reversible and considered to be incidental, since no related abnormal parameters or results were found. Increase in spleen and thymus organ-to-body weight ratios and decrease in menses were mild, reversible and likely related to pharmacology activity of IFNalpha2a. Ames, chromosomal aberration and bone marrow micronulecus test were conducted and the results were negative. The degree of irritation caused by various concentration of IFNalpha2a-NGR was determined to be the same as that induced by normal saline. In conclusion, preclinical safety studies that IFNalpha2a-NGR was well tolerated at pharmacologically active doses in mice, rats and monkeys.

Efficacy of a plant extract (Chelidonium majus L.) in combating induced hepatocarcinogenesis in mice.

Ethanolic whole plant extract of Chelidonium majus, extensively used in traditional systems of medicine against various liver ailments, has been tested for its possible anti-tumor, hepato-protective and anti-genotoxic effects in p-dimethylaminoazobenzene (p-DAB) induced hepatocarcinogenesis in mice through multiple assays: cytogenetical, biochemical, histological and electron microscopical. Different sets of mice, 5 (for 7, 15 and 30 days' treatment) or 10 (for 60, 90 and 120 days) each, were chronically fed a diet suitably mixed with p-DAB and phenobarbital to develop liver tumors. One sub-group of carcinogen fed mice was also fed C. majus extract; 0.1 ml daily (drug-treated) while the other equal amount of dilute ethyl alcohol ("vehicle" of plant extract) (positive control). A separate group of mice was maintained with normal diet without any carcinogen treatment (negative control). Data of several cytogenetical endpoints and biochemical assay of some toxicity marker enzymes at all fixation intervals and histology of liver sections through ordinary, scanning and transmission electron microscopy at 60 and 120 days and that of spleen and kidney at 90 days were critically analyzed in the treated lots vis-a-vis controls. The results suggest anti-tumor, anti-genotoxic and hepato-protective effects of the plant extract, showing potentials for use in cancer therapy.

Activation of mononuclear bone marrow cells treated in vitro with a complex homeopathic medication.

Canova is a Brazilian homeopathic medication with immunomodulatory properties, recommended for patients where the immune system is depressed. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix and progenitor cells that differentiate into mature blood cells. We now report the effect of in vitro administration of the medication on the mononuclear differentiation of the bone marrow cell. Swiss mice femurs were dissected cleaned and the cells of the marrow were flushed. The cells were plated, treated or not, incubated for different times and processed for light, transmission and scanning electron, and confocal microscopy analysis. Bone marrow cells showed an enhanced proliferation in vitro in response to Canova medication and Canova plus M-CSF and an increase was also observed in the numbers of the cell niches and ring-shaped nuclei cells. Confocal and transmission and scanning electron microscopy showed the stages of monocyte maturation, with resting and activated cells. With Canova treatment there was a marked increase in cell size, which is mainly attributable to the augmented cytoplasm, an increase in the number of mitochondria, expansion of the RER and an enlarged Golgi. The response to Canova treatment indicates that it influences mononuclear differentiation and activation of bone marrow progenitor and stromal cells.

Dose-dependent osteogenic effect of octacalcium phosphate on mouse bone marrow stromal cells.

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.