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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.

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 ± 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, βIII-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.

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.

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.