Dosimetry of pulsed magnetic field towards attaining bacteriostatic effect on Enterococcus faecalis: Implications for endodontic therapy.

AIM: To examine in a laboratory setting the efficacy of moderate to high strength magnetic fields, as a potential bacteriostatic stimulus, against Enterococcus faecalis, one of the causative agents for infection during root canal treatments. METHODOLOGY: Four different strengths (1, 2, 3 and 4 T) of the pulsed magnetic field (PMF) were applied in thirty repetitions to bacterial suspension. A pickup coil setup was used to measure the electromotive force induced inside the bacterial suspensions. The optical density (OD) was monitored over time (for 16 h 40 min) during the post-treatment period to assess bacterial growth. Along with the change in OD values, live/dead assay, membrane depolarization study, atomic force microscopy (AFM), scanning electron microscopy (SEM) and reactive oxygen species (ROS) assay on selected samples were studied to evaluate the effect of PMFs. All results were analysed using one-way ANOVA followed by post hoc Tukey test and considered significant at p < .05. Regression analysis (at a confidence of 95%, α = 0.05) was performed on the bacterial growth and membrane depolarization studies to determine progressive changes of the outcomes. RESULTS: The peak value of the induced electromotive force was recorded as 0.25 V, for the 4 T magnetic field pulse with a pulse width of 16 ms. There was a significant arrest of bacterial cell growth after an exposure to PMFs of 1 T, 3 T and 4 T (ANOVA score: F (4, 495) =395.180 at p = .05). The image-based qualitative results of the live/dead assay using fluorescence microscopy techniques indicated that an exposure to higher PMFs (3 T/ 4 T) induced a bacteriostatic effect in a longer post-exposure timescale. Evidence of altered membrane potential within the 2 h of exposure to 4 T PMF was supported by the incidence of elevated ROS. For the ROS assay, a significant difference occurred for 4 T exposed samples (ANOVA score: calculated F (1, 3) =20.2749 at p = .05). SEM and AFM observations corroborated with the outcomes, by portraying significant membrane damage. CONCLUSION: In a laboratory setting, PMFs with higher magnitudes (3 T and 4 T) were capable of inducing bacteriostatic effects on E. faecalis.

UHMWPE-MWCNT-nHA based hybrid trilayer nanobiocomposite: Processing approach, physical properties, stem/bone cell functionality, and blood compatibility.

The development of polymeric nanocomposites for biomedical applications remains a major challenge in terms of tailored addition of nanoparticles to realize the simultaneous enhancement of fracture resistance and cell/blood compatibility. To address this, the present work has been planned to determine whether small addition of surface functionalized multiwalled-carbon-nanotube, MWCNT (<1.5 wt%) and egg-shell derived nanosized hydroxyapatite, nHA (<10 wt%) to ultrahigh-molecular-weight-polyethylene (UHMWPE) can significantly improve the physical properties as well as biocompatibility. The difference in mouse osteoblast and human mesenchymal stem cell (hMSc) proliferation has been validated using both the monolithic composite and a trilayered composite with two different UHMWPE nanocomposites on either face with pure polymer at the middle. The combination of rheology and micro-CT with fractography reveals the homogeneous dispersion of nanofillers, leading to mechanical property enhancement. The quantitative analysis of cell viability and cell spreading by immunocytochemistry method, using vinculin and vimentin expression, establish significant cytocompatibility with hMSc and osteoblast cells onto the trilayer hybrid nanobiocomposite substrates. The hemocompatibility of the investigated composites under the controlled flow of rabbit blood in a microfluidic device reveals the signature of reduced thrombogenesis with reduction of platelet activation on UHMWPE nanocomposite w.r.t. unreinforced UHMWPE. An attempt has been made to discuss the blood compatibility results in the backdrop of the bovine serum albumin adsorption kinetics. Summarizing, the present study establishes that the twin requirement of mechanical property and cyto/hemo-compatibility can be potentially realized in developing trilayer composites in UHMWPE-nHA-MWCNT system.

Acoustic Poration and Dynamic Healing of Mammalian Cell Membranes during Inkjet Printing.

We have investigated the effect of piezoelectric actuating voltage on cell behavior after drop on demand inkjet printing using mouse 3T3 cells as a model cell line. Cell viability after printing was assessed using a live/dead assay, Alamar Blue as an assay for cell proliferation, and propidium iodide (PI) and Texas Red labeled dextran molecular probes to assess cell membrane integrity. No significant difference was found for the cell death rate compared between an unprinted control population and after printing at 80, 90, and 100 V, respectively. However, cell proliferation was lower than that of the control population at all time points postprinting. Cell membrane integrity was quantified using PI and dextran probes of mean molecular weight of 3, 10, 40, and 70 kDa. Total membrane damage (assessed by PI) increased with increasing piezoelectric actuator driving voltage, and this was always greater than the unprinted control cells. The uptake of the labeled dextran only occurs after inkjet printing and was never observed with the control cells. The largest dextran molecular probe of 70 kDa was only taken up by cells after printing using the lower printing voltages of 80 and 90 V and was absent after printing at 100 V. At the two lower printing voltages, the membrane damage is recovered, and no dextran molecule penetrated the cells 2 h after printing. However, printing at 100 V leads to an increased uptake of 3 and 10 kDa dextran molecules, the retention of membrane porosity, and continued uptake of these 3 and 10 kDa dextran for greater than 2 h postprinting. We hypothesize that the change in membrane porosity with increasing actuation voltage can be explained by distinct nucleation and growth stages for pore formation in response to printing stress.

Neurogenesis-on-Chip: Electric field modulated transdifferentiation of human mesenchymal stem cell and mouse muscle precursor cell coculture.

A number of bioengineering strategies, using biophysical stimulation, are being explored to guide the human mesenchymal stem cells (hMScs) into different lineages. In this context, we have limited understanding on the transdifferentiation of matured cells to another functional-cell type, when grown with stem cells, in a constrained cellular microenvironment under biophysical stimulation. While addressing such aspects, the present work reports the influence of the electric field (EF) stimulation on the phenotypic and functionality modulation of the coculture of murine myoblasts (C2C12) with hMScs [hMSc:C2C12=1:10] in a custom designed polymethylmethacrylate (PMMA) based microfluidic device with in-built metal electrodes. The quantitative and qualitative analysis of the immunofluorescence study confirms that the cocultured cells in the conditioned medium with astrocytic feed, exhibit differentiation towards neural-committed cells under biophysical stimulation in the range of the endogenous physiological electric field strength (8 ±â€¯0.06  mV/mm). The control experiments using similar culture protocols revealed that while C2C12 monoculture exhibited myotube-like fused structures, the hMScs exhibited the neurosphere-like clusters with SOX2, nestin, ßIII-tubulin expression. The electrophysiological study indicates the significant role of intercellular calcium signalling among the differentiated cells towards transdifferentiation. Furthermore, the depolarization induced calcium influx strongly supports neural-like behaviour for the electric field stimulated cells in coculture. The intriguing results are explained in terms of the paracrine signalling among the transdifferentiated cells in the electric field stimulated cellular microenvironment. In summary, the present study establishes the potential for neurogenesis on-chip for the coculture of hMSc and C2C12 cells under tailored electric field stimulation, in vitro.

Recurrent Glioblastomas Exhibit Higher Expression of Biomarkers with Stem-like Properties.

BACKGROUND: Despite advances in the treatment of glioblastoma (GBM), the prognosis of patients continues to remain dismal. This unfavorable prognosis is mainly attributed to the tumor's propensity for progression and recurrence, which in turn is due to the highly aggressive nature of the persisting GBM cells that actively egress from the main tumor mass into the surrounding normal brain tissue. Such a recurrent tumor described to have a more malignant potential is highly invasive and resistant to current therapies, probably due to increased stemness and preferential selection of therapy-resistant clones of tumor cells. However, there is a paucity of literature on the expression of biomarkers in the recurrent GBM tumors that could have a role in conferring this aggressiveness. AIM: To identify the differences in the expression pattern of selected biomarkers in paired tissue samples of GBM. MATERIAL AND METHODS: A retrospective study on 30 paired samples of GBM (newly diagnosed/primary and recurrent) archived in the Department of Neuropathology, NIMHANS (2006-2009), was carried out. After obtaining clinical and demographic details, tumors were characterized histomorphologically and immunohistochemically on formalin-fixed paraffin-embedded tissues with reference to expression of biomarkers such as p53, epidermal growth factor receptor (EGFR), insulin-like growth factor binding protein 3 (IGFBP-3), sex determining region Y-box 2 (SOX2), and topoisomerase 2 A (Top2A). The results were statistically analyzed. RESULTS: It was observed that while p53 and IGFBP-3 expression remained unaltered in paired samples, a significant increase in the expression of EGFR (P < 0.01) was noted in the recurrent tumors. Among the other biomarkers, SOX2 expression was higher in the recurrent tumors when compared to the primary tumors (P < 0.01). Conversely, the expression of Top2A was reduced in recurrent tumors (P = 0.05). Mild elevation in the expression of IGFBP-3 was observed in recurrent tumors but was not statistically significant. CONCLUSION: A significant increase in the expression of SOX2 in recurrent tumors probably indicates the presence of undifferentiated cells with stem-like properties in these tumors. EGFR is known to mediate SOX2 expression thereby resulting in stemness of the glioma cancer cells, which could further explain its overexpression in recurrent GBMs. Furthermore, a decreased expression of TOP2A observed in the recurrent tumors could probably be due to reduction in chemosensitivity to temozolomide, which has been shown in earlier studies. We also noted that p53 expression remained unaltered in the recurrent tumors when compared to the primary, suggesting the absence of preferential clonal expansion of p53 mutant cells following exposure to radiochemotherapy. Our study reiterates the fact that GBM recurrences are associated with molecular alterations that probably contribute to radiochemoresistance, increased invasiveness, therapeutic efficacy, and stemness.

Controlled Shear Flow Directs Osteogenesis on UHMWPE-Based Hybrid Nanobiocomposites in a Custom-Designed PMMA Microfluidic Device.

The combinatorial influence of a biophysical cue (substrate stiffness) and biomechanical cue (shear flow) on the osteogenesis modulation of human mesenchymal stem cells (hMSCs) is studied for bone regenerative applications. In this work, we report stem cell differentiation on an ultra high molecular weight polyethylene (UHMWPE)-based hybrid nanobiocomposite [reinforced with a multiwalled carbon nanotube (MWCNT) and/or nanohydroxyapatite (nHA)] under a physiologically relevant shear flow (1 Pa) in a custom-built microfluidic device. Using a genotypic assessment with qRT-PCR and phenotypic assessment through analysis of cytoskeletal remodelling and marker proteins, the role of shear on the progression of osteogenesis modulation has been quantitatively established with statistically significant differences between nHA-reinforced and MWCNT-reinforced UHMWPE. Early-stage (alkaline phosphatase activity at day 8), middle-stage (matrix collagenation at day 14), and late-stage (matrix calcification at day 20) events were analyzed using mRNA expression changes of a limited cell volume after microfluidic culture experiments. The conventional Petri dish culture (static) exhibited an increased osteogenesis for nanoparticle-reinforced UHMWPE, irrespective of the type of nanoparticle. The shear-mediated culture experiments resulted in noticeable differences in the degree of osteogenesis with MWCNT being more effective than nHA reinforcement. The shear-mediated osteogenesis has been attributed to the skewed cellular morphology with a higher cell adhesion (vinculin expression) on UHMWPE and nHA than that of UHMWPE and MWCNT. The signatures of the cytoskeletal changes are reflected in terms of left-to-right (L-R) chirality as well as alignment and pattern of actin fibers. Moreover, stemness (vimentin expression) was found to be decreased because of differentiation. The electrophysiological analysis using patch clamp experiments also revealed a higher inward calcium current and intracellular calcium activity for the cells grown on the UHMWPE and nHA nanobiocomposite under shear. Overall, the present study conclusively establishes the synergistic role of substrate stiffness and shear on osteogenesis of hMSCs, in vitro.

On The Origin of Shear Stress Induced Myogenesis Using PMMA Based Lab-on-Chip.

One of the central themes in cell and tissue engineering is to develop an understanding as to how biophysical cues can influence cell functionality changes. The flow induced shear stress is regarded as one such biophysical cue to influence physiological changes in shear-sensitive tissues, in vivo. The origin of such phenomena is, however, poorly understood. While addressing such an issue, the present work demonstrates the intriguing synergistic effect of shear stress and spatial constraints in inducing aligned growth and differentiation of myoblast cells to myotubes. In a planned set of in vitro experiments, the regulation of laminar flow regime within a narrow window was obtained in a PMMA-based Lab-on-Chip (LOC) device, wherein the murine muscle cells (C2C12), chosen for their phenotypical differentiation stages, were cultured under graded shear conditions. The two factors of shear stress and spatial allowance were decoupled by another two sets of experiments. This aspect has been conclusively established using a PMMA device having a fixed width microchannel with varying shear and an identical amount of shear with different width of channels. On the basis of the extensive analysis of biochemical assays (WST-1, picogreen) together with gene expression using qRT-PCR and cell morphological changes (fluorescence/confocal microscopy), extensive differentiation of the myoblasts into myotubes is found to be dependent on both shear stress and spatial allocation with a maximum at an optimal shear of ca. 16 mPa. Quantitatively, the mRNA expression of myogenic biomarkers, i.e., myogenin, MyoD, and neogenin, exhibited 10- to 50-fold changes at ca. 16 mPa shear flow, compared to that under static conditions. Also, myotube aspect ratio and myotube density are modulated with shear stress and are in commensurate with gene expression changes. The flow cytometry analysis further confirmed that the cell cycle arrest at the G1/G0 phase triggers the onset of myogenesis. Taken together, the present study unambiguously establishes qualitative and quantitative biophysical basis for the origin of myogenesis toward the critical shear stress of murine myoblasts in a microfludic device, in vitro.

Modulation of Protein Adsorption and Cell Proliferation on Polyethylene Immobilized Graphene Oxide Reinforced HDPE Bionanocomposites.

The uniform dispersion of nanoparticles in a polymer matrix, together with an enhancement of interfacial adhesion is indispensable toward achieving better mechanical properties in the nanocomposites. In the context to biomedical applications, the type and amount of nanoparticles can potentially influence the biocompatibility. To address these issues, we prepared high-density polyethylene (HDPE) based composites reinforced with graphene oxide (GO) by melt mixing followed by compression molding. In an attempt to tailor the dispersion and to improve the interfacial adhesion, we immobilized polyethylene (PE) onto GO sheets by nucleophilic addition-elimination reaction. A good combination of yield strength (ca. 20 MPa), elastic modulus (ca. 600 MPa), and an outstanding elongation at failure (ca. 70%) were recorded with 3 wt % polyethylene grafted graphene oxide (PE-g-GO) reinforced HDPE composites. Considering the relevance of protein adsorption as a biophysical precursor to cell adhesion, the protein adsorption isotherms of bovine serum albumin (BSA) were determined to realize three times higher equilibrium constant (Keq) for PE-g-GO-reinforced HDPE composites as compared to GO-reinforced composites. To assess the cytocompatibility, we grew osteoblast cell line (MC3T3) and human mesenchymal stem cells (hMSCs) on HDPE/GO and HDPE/PE-g-GO composites, in vitro. The statistically significant increase in metabolically active cell over different time periods in culture for up to 6 days in MC3T3 and 7 days for hMSCs was observed, irrespective of the substrate composition. Such observation indicated that HDPE with GO or PE-g-GO addition (up to 3 wt %) can be used as cell growth substrate. The extensive proliferation of cells with oriented growth pattern also supported the fact that tailored GO addition can support cellular functionality in vitro. Taken together, the experimental results suggest that the PE-g-GO in HDPE can effectively be utilized to enhance both mechanical and cytocompatibility properties and can further be explored for potential biomedical applications.