Multiwalled carbon nanotubes enhance human bone marrow mesenchymal stem cells’ spreading but delay their proliferation in the direction of differentiation acceleration

Investigating the ability of films of pristine (purified, without any functionalization) multiwalled carbon nanotubes (MWCNTs) to influence human bone marrow mesenchymal stem cells’ (hBMSCs) proliferation, morphology, and differentiation into osteoblasts, we concluded to the following: A. MWCNTs delay the proliferation of hBMSCs but increase their differentiation. The enhancement of the differentiation markers could be a result of decreased proliferation and maturation of the extracellular matrix B. Cell spread on MWCNTs toward a polygonal shape with many thin filopodia to attach to the surfaces. Spreading may be critical in supporting osteogenic differentiation in pre-osteoblastic progenitors, being related with cytoskeletal tension. C. hBMSCs prefer MWCNTs than tissue plastic to attach and grow, being non-toxic to these cells. MWCNTs can be regarded as osteoinductive biomaterial topographies for bone regenerative engineering.Cellular interaction with substrate and neighboring cells plays a critical role in osteoblast survival, proliferation, differentiation as well as bone remodeling. Regulated biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration in the stem cell niche. Multiwalled carbon nanotubes (MWCNTs) represent a nanomaterial that has won enormous popularity in nanotechnology, exhibiting extraordinary physicochemical properties and supporting the growth of different kinds of cells.^1-3Simultaneous enhancement of osteoblast cells’ proliferation and differentiation,^4,5 decrease of proliferation rates along with decreased differentiation⁶ or increased differentiation accompanied with decreased proliferation⁷ have been reported. Contradictory results concerning osteoblast cell adhesion, and morphology have also been reported. Osteoblast cell lines on CNTs have been found to elongate but not widen or displayed a spindle-shaped morphology.^8,9 Spreading and surface area covered were reduced.^8-10 On the contrary, Tutak et al.⁷ reported robust spreading on medium roughness CNTs networks.This variable behavior on CNTs is probably due to the various cell types used in these works. It is reported that primary human marrow stromal cells and cell lines use substantially different mechanisms to regulate adhesion and spreading on the substrate.¹¹In a recent work of ours, published in Annals of Biomedical Engineering,¹² it was found that MWCNTs can create an osteogenic environment for human bone marrow mesenchymal stem cells (hBMSCs), even without addition of exogenous factors, representing a suitable reinforcement for bone tissue engineering scaffolds.In the following, we will highlight and discuss some aspects of this work''s results, in the context of literature findings, and provide additional material in order to elucidate issues on the influence of MWCNTs on hBMSCs’ proliferation, morphology, and differentiation into osteoblasts.     

Ester and carbamate ester derivatives of Biochanin A: synthesis and in vitro evaluation of estrogenic and antiproliferative activities

Biochanin A (BCA), a major isoflavone in red clover and many other legumes, has been reported to display estrogenic as well as cancer chemopreventive properties. Ingested BCA is known to display low bioavailability due to poor solubility, extensive metabolism and rapid clearance. Esters of bioactive isoflavones are known to increase metabolic stability and bioavailability following local rather than systemic administration. We synthesized BCA from phloroglucinol and p-methoxy-phenylacetic acid by a Friedel-Crafts reaction and cyclization. We also synthesized esters (1, 3) and carbamate esters (2, 4, 5) at position 7 of BCA using short aliphatic chains bearing a chlorine (1, 2) or a bromine atom (3, 4) or long aliphatic chains without such atoms (5). We tested the estrogenic and antiproliferative activities of 1-5 and BCA using human breast and endometrial adenocarcinoma cells. We found that 5 affects MCF-7 and Ishikawa cells in a manner providing for induction of gene expression to a level similar to 17β-estradiol and BCA but, unlike both of the latter, for suppression of cell proliferation as well. In addition, 5 appeared to display higher stability compared to 1-4 and BCA in both MCF-7 and Ishikawa cells. The inference is that 5 may represent a safer than BCA alternative to hormone replacement therapy.     

Role of Nox4 in murine models of kidney disease

Nox4 is a hydrogen peroxide-producing NADPH oxidase highly expressed in the kidney which has been linked to epithelial cell injury and diabetic-induced cellular dysfunction in cultured cells. The role of the enzyme for renal pathology in vivo, however, is unclear. To address this, three experimental animal models of renal injury (streptozotocin diabetes I, unilateral ureteral ligation (UUO), and 5/6 nephrectomy (5/6Nx)) were studied in either Nox4-inducible (Nox4(?/?)) or constitutive knockout (Nox4(-/-)) mice. Nox4 contributed more than 80% of diphenylene iodonium-sensitive H(2)O(2) formation of freshly isolated tubules determined by Amplex Red assay. In streptozotocin diabetes, acute deletion of Nox4 by tamoxifen-activated cre-recombinase increased albuminuria, whereas matrix deposition was similar between WT and Nox4(?/?) mice. Interestingly, renal Nox4 expression, mainly localized to tubular cells, decreased in the course of diabetes and this was not associated with a compensatory upregulation of Nox1 or Nox2. In the UUO model, renal expression of ICAM1, connective tissue growth factor, and fibronectin were higher in kidneys of Nox4(?/?) than control mice. Also in this model, levels of Nox4 decreased in the course of the disease. In the 5/6Nx model, which was performed in SV129 and SV129-Nox4(-/-) mice, no difference in the development of hypertension and albuminuria was found between the strains. Collectively, the first in vivo data of the kidney do not support the view that Nox4 is a main driver of renal disease. It rather appears that under specific conditions Nox4 may even slightly limit injury and disease progression.     

Analytical Performance of ELISA Assays in Urine: One More Bottleneck towards Biomarker Validation and Clinical Implementation

ELISA is the main approach for the sensitive quantification of protein biomarkers in body fluids and is currently employed in clinical laboratories for the measurement of clinical markers. As such, it also constitutes the main methodological approach for biomarker validation and further qualification. For the latter, specific assay performance requirements have to be met, as described in respective guidelines of regulatory agencies. Even though many clinical ELISA assays in serum are regularly used, ELISA clinical applications in urine are significantly less. The scope of our study was to evaluate ELISA assay analytical performance in urine for a series of potential biomarkers for bladder cancer, as a first step towards their large scale clinical validation. Seven biomarkers (Secreted protein acidic and rich in cysteine, Survivin, Slit homolog 2 protein, NRC-Interacting Factor 1, Histone 2B, Proteinase-3 and Profilin-1) previously described in the literature as having differential expression in bladder cancer were included in the study. A total of 11 commercially available ELISA tests for these markers were tested by standard curve analysis, assay reproducibility, linearity and spiking experiments. The results show disappointing performance with coefficients of variation>20% for the vast majority of the tests performed. Only 3 assays (for Secreted protein acidic and rich in cysteine, Survivin and Slit homolog 2 protein) passed the accuracy thresholds and were found suitable for further application in marker quantification. These results collectively reflect the difficulties in developing urine-based ELISA assays of sufficient analytical performance for clinical application, presumably attributed to the urine matrix itself and/or presence of markers in various isoforms.     

Diagnosis of metastatic tumors in cerebrospinal fluid samples using thin-layer cytology

OBJECTIVE: To evaluate the application of ThinPrep liquid-based cytology (LBC) and present our experience using LBC in the diagnosis of metastatic tumors in cerebrospinal fluid (CSF) samples. STUDY DESIGN: We examined 38 cytologic specimens of CSF, processed by ThinPrep technique. Of these, 18 presented with a previously diagnosed primary malignancy. Various immunocytochemical markers were performed. RESULTS: ThinPrep technology provided preservation of cytomorphologic features, high cellularity per slide and clear background. Analysis revealed 8 breast carcinomas, 5 lung carcinomas, 4 lymphomas, 3 adenocarcinomas of the gastrointestinal tract, 1 squamous cell carcinoma of uterine cervix and 1 urinary bladder carcinoma. Fifteen samples were negative for malignancy. CONCLUSION: CSF cytology is the only examination that verifies the presence of malignancy. Thin monolayer technology is suggested as an appropriate diagnostic method for metastatic tumors in CSF in everyday routine and seems to be a valuable tool for further management and planning of treatment.     

The influence of muscle type and dystrophin deficiency on murine expression profiles

The phenotypic differences among Duchenne muscular dystrophy patients, mdx mice, and mdx^5cv mice suggest that despite the common etiology of dystrophin deficiency, secondary mechanisms have a substantial influence on phenotypic severity. The differential response of various skeletal muscles to dystrophin deficiency supports this hypothesis. To explore these differences, gene expression profiles were generated from duplicate RNA targets extracted from six different skeletal muscles (diaphragm, soleus, gastrocnemius, quadriceps, tibialis anterior, and extensor digitorum longus) from wild-type, mdx, and mdx^5cv mice, resulting in 36 data sets for 18 muscle samples. The data sets were compared in three different ways: (1) among wild-type samples only, (2) among all 36 data sets, and (3) between strains for each muscle type. The molecular profiles of soleus and diaphragm separate significantly from the other four muscle types and from each other. Fiber-type proportions can explain some of these differences. These variations in wild-type gene expression profiles may also reflect biomechanical differences known to exist among skeletal muscles. Further exploration of the genes that most distinguish these muscles may help explain the origins of the biomechanical differences and the reasons why some muscles are more resistant than others to dystrophin deficiency. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Judith N. Haslett, Peter B. Kang These authors contributed equally to this work.     

Pollination ecology of Campanula species on Mt Olympos, Greece

Nine Campanula species occurring along the elevation gradient of Mt Olympos were studied regarding their pollination ecology. The main issues considered were 1) the relative importance of various insect taxa as Campanula pollinators, 2) the patterns of pollinators' size and activity as a function of altitude, 3) the effect of pollinator exclusion on floral longevity, and 4) the extent to which the morphological difference of C. versicolor from the other Campanula species on Mt Olympos is expressed in its pollinating fauna. The vast majority of Campanula pollinators were solitary bees. Andrenidae and Megachilidae bees (mainly Chelostoma campanulorum) dominated the pollinating fauna of most species, Melittidae and bumblebees were the commonest pollinators of high altitude species. Campanula versicolor differs from the other Campanula species in that its corolla is not bell-shaped but flat. Mainly Apis mellifera, syrphid flies, and carpenter bees, unlike all other Campanula species on Mt Olympos pollinated it. At the species level, rather large altitudinal differences of Campanula populations did not result into large diversification of their pollinating fauna. The insect visitation rate to flowers decreased with altitude. When pollinators were excluded, the floral longevity of the species examined increased three to five times. Neither flower phase (male of female) was consistently favoured in the absence of pollinators. The pollen loads of the different insect taxa (Apis mellifera included) were of variable purity. The majority of Megachilidae bees carried pollen loads of high purity. Pollen loads from insects visiting Campanula species at high altitudes did not differ significantly in their purity from those visiting lowland species. The distribution of Campanula pollinators' body size along the altitudinal gradient exhibited a U-shaped pattern. No relationship was found between insect-pollinator body size and corolla size of Campanula species.