3D Printed Hierarchical Porous Poly(ε-caprolactone) Scaffolds from Pickering High Internal Phase Emulsion Templating.

In the realm of biomaterials, particularly bone tissue engineering, there has been a great increase in interest in scaffolds with hierarchical porosity and customizable multifunctionality. Recently, the three-dimensional (3D) printing of biopolymer-based inks (solutions or emulsions) has gained high popularity for fabricating tissue engineering scaffolds, which optimally satisfies the desired properties and performances. Herein, therefore, we explore the fabrication of 3D printed hierarchical porous scaffolds of poly(ε-caprolactone) (PCL) using the water-in-oil (w/o) Pickering PCL high internal phase emulsions (HIPEs) as the ink in 3D printer. The Pickering PCL HIPEs stabilized using hydrophobically modified nanoclay comprised of aqueous poly(vinyl alcohol) (PVA) as the dispersed phase. Rheological measurements suggested the shear thinning behavior of Pickering HIPEs having a dispersed droplet diameter of 3-25 µm. The pore morphology resembling the natural extracellular matrix and the mechanical properties of scaffolds were customized by tuning the emulsion composition and 3D printing parameters. In vitro biomineralization and drug release studies proved the scaffolds' potential in developing the apatite-rich bioactive interphase and controlled drug delivery, respectively. During in vitro osteoblast (MG63) growth experiments for up to 7 days, good adhesion and proliferation on PCL scaffolds confirmed their cytocompatibility, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis. This study suggests that the assembly of HIPE templates and 3D printing is a promising approach to creating hierarchical porous scaffolds potentially suitable for bone tissue engineering and can be stretched to other biopolymers as well.

Dual-functionalized Pickering HIPE templated poly(ɛ-caprolactone) scaffold for maxillofacial implants.

High internal phase emulsion (HIPE) templated poly (ɛ-caprolactone) (PCL) scaffolds have gained widespread attention for large-sized bone defects due to its tuneable 3D architecture and ease of fabricating crosslinked PCL (cPCL) scaffolds. However, extremely high stabilizer (surfactant or nanoparticle) concentration and negligence of microenvironment for regeneration sites like alveolar bones have restrained industrial acceptance of these scaffolds. Herein, we demonstrated the fabrication of nanocomposite cPCL scaffolds within Pickering HIPE templates stabilized using modified silica nanoparticles (mSiNP) concentrations as low as 0.1 to 1.0 wt%. Using an unconventional approach, the mSiNP Pickering stabilizer was added in dispersed phase, contradicting Bancroft's rule. The colloidal stability was attained due to faster drifting of mSiNP towards the interface when it was dispersed in silicone oil. Scaffolds with tuneable properties were fabricated by controlling the mSiNP concentration and ϕd. Further, cPCL scaffolds were functionalized using clove oil (CO) to improve their efficiency in eradicating S. aureus and E. coli by disrupting their cellular integrity. Additionally, formation of biofilm on the surface of the scaffolds was successfully inhibited by the incorporation of CO. CO-functionalized scaffolds demonstrated excellent cytocompatibility towards MG-63 cells allowing their successful adhesion and proliferation on the surface of the scaffolds.

Hypoxia-inducible miR-196a modulates glioblastoma cell proliferation and migration through complex regulation of NRAS.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in humans. Hypoxia has been correlated with the aggressive form of glial tumors, poor prognosis, recurrence and resistance to various therapies. MicroRNAs (miRNAs) have emerged as critical mediators of hypoxic responses and have shown great potential for cancer diagnostics and therapeutics. Here, we focus on the regulatory and functional characterization of miR-196a, a hypoxia-inducible miRNA, in GBM. METHODS: Hypoxia/HIF regulation of miR-196a was assessed by RT-qPCR, promoter-luciferase and ChIP assays in GBM cell lines. miR-196a levels were analyzed in The Cancer Genome Atlas (TCGA)-GBM, Chinese Glioma Genome Atlas (CGGA) and Indian GBM patient cohorts. miR-target interactions were studied using RNA/protein quantification and 3'UTR luciferase assays. The effect of miR-196a overexpression/inhibition was assessed on cellular viability, migration and apoptosis under hypoxia and normoxia. Microarray-based gene expression profiling studies were performrd to study the effect of miR-196a on the GBM cellular transcriptome under hypoxia. RESULTS: We identified miR-196a as a hypoxia-inducible and hypoxia-inducible factor (HIF)-regulated miRNA that plays an oncogenic role in GBM. miR-196a was found to be significantly up-regulated in TCGA-GBM, CGGA glioma as well as Indian GBM patient cohorts. miR-196a overexpression was found to induce cellular proliferation, migration, spheroid formation and colony formation and to inhibit apoptosis, while miR-196a inhibition using anti-miR-196a yielded opposite results, suggesting an oncogenic role of miR-196a in GBM. We further unveiled NRAS, AJAP1, TAOK1 and COL24A1 as direct targets of miR-196a. We also report a complex competitive regulation of oncogenic NRAS by miR-196a, miR-146a and let-7 in GBM. Analysis of microarray-based gene expression data obtained by miR-196a inhibition under hypoxia revealed a role of miR-196a in HIF, calcium adhesion, Wnt and cell adhesion pathways. Interestingly, miR-196a was found to positively regulate the expression of various genes involved in the induction or stabilization of HIFs and in maintenance of hypoxic conditions, thereby suggesting the existence of an indirect miR-196a/HIF positive feedback loop under hypoxia. CONCLUSIONS: Overall, our work identifies a novel association between hypoxia/HIF signalling and miR-196a in GBM and suggests its therapeutic significance.

Facile Fabrication of Composite Electrospun Nanofibrous Matrices of Poly(ε-caprolactone)-Silica Based Pickering Emulsion.

Functionalized matrices have been sought for their application in sensors, filtration, energy storage, catalysis, and tissue engineering. We report formation of an inorganic-organic composite matrix based on poly(ε-caprolactone) (PCL) functionalized with hydrophobically modified silica (m-silica) fabricated with reduced organic solvent usage. The matrix was obtained via electrospinning of a water-in-oil emulsion of PCL that was stabilized by judicious choice of m-silica as a Pickering agent resulting into an emulsifier free matrix. Inclusion of m-silica in PCL matrix resulted in enhancing tensile properties and cell proliferation efficiency. The electrospun composite matrix was free from any emulsifier or template polymer; thus any abrupt loss in mechanical properties was prevented when the matrix was subjected to aqueous conditions. The inorganic-organic biodegradable composite matrices thus produced using an emulsifier free emulsion find applications in tissue engineering and may further be evaluated for other areas including selective sorption and separation.

Electrospun composite matrices of poly(ε-caprolactone)-montmorillonite made using tenside free Pickering emulsions.

The production of composite electrospun matrices of poly(ε-caprolactone) (PCL) using an emulsifier-free emulsion, made with minimal organic solvent, as precursor is reported. Pickering emulsions of PCL were prepared using modified montmorillonite (MMT) clay as the stabilizer. Hydrophobic tallow group of the modified MMT clay resulted in analogous interaction of clay with oil and aqueous phase and its adsorption at the interface to provide stability to the resultant emulsion. Composite fibrous matrices of PCL and MMT were produced using electrospinning under controlled conditions. The fiber fineness was found to alter with PCL concentration and volume fraction of the aqueous and oil phases. A higher tensile strength and modulus was obtained with inclusion of MMT in PCL electrospun matrix in comparison to a matrix made using neat PCL. The presence of clay in the fibrous matrix did not change the cell proliferation efficiency in comparison to neat PCL matrix. Composite fibrous matrices of PCL/MMT bearing enhanced tensile properties may find applications in areas other than tissue engineering for example food packaging and filtration.

Analysis of EZH2: micro-RNA network in low and high grade astrocytic tumors.

Enhancer of Zeste homologue2 (EZH2) is an epigenetic regulator that functions as oncogene in astrocytic tumors, however, EZH2 regulation remains little studied. In this study, we measured EZH2 levels in low (Gr-II,DA) and high grade (Gr-IV,GBM) astrocytic tumors and found significant increased EZH2 transcript level with grade(median DA-8.5, GBM-28.9).However, a different trend was reflected in protein levels, with GBMs showing high EZH2 LI(median-26.5) compared to DA (median 0.3). This difference in correlation of EZH2 protein and RNA levels suggested post-transcriptional regulation of EZH2, likely mediated by miRNAs. We selected eleven miRNAs that strongly predicted to target EZH2 and measured their expression. Three miRNAs (miR-26a-5p,miR27a-3p and miR-498) showed significant correlation with EZH2 protein, suggesting them as regulators of EZH2, however miR-26a-5p levels decreased with grade. ChIP analyses revealed H3K27me3 modifications in miR-26a promoter suggesting feedback loop between EZH2 and miR26a. We further measured six downstream miRNA targets of EZH2 and found significant downregulation of four (miR-181a/b and 200b/c) in GBM. Interestingly, EZH2 associated miRNAs were predicted to target 25 genes in glioma-pathway, suggesting their role in tumor formation or progression. Collectively, our work suggests EZH2 and its miRNA interactors may serve as promising biomarkers for progression of astrocytic tumors and may offer novel therapeutic strategies.