Decellularization of Wharton's Jelly Increases Its Bioactivity and Antibacterial Properties.

The field of regenerative medicine has recently seen an emerging trend toward decellularized extracellular matrix (ECM) as a biological scaffold for stem cell-delivery. Human umbilical cord represents a valuable opportunity from both technical and ethical point of view to obtain allogenic ECM. Herein, we established a protocol, allowing the full removal of cell membranes and nuclei moieties from Wharton's jelly (WJ) tissue. No alterations in the ECM components (i.e., collagen, GAG content, and growth factors), physical (i.e., porosity and swelling) and mechanical (i.e., linear tensile modulus) properties were noticed following WJ processing. Furthermore, no effect of the tissue processing on macromolecules and growth factors retention was observed, assuring thus a suitable bioactive matrix for cell maintenance upon recellularization. Based on the in vitro and in vivo biodegradability and stromal cell homing capabilities, decellularized WJ could provide an ideal substrate for stromal cells adhesion and colonization. Interestingly, the tissue processing increased the antibacterial and antiadhesive properties of WJ against Staphylococcus aureus and Staphylococcus epidermidis pathogens. Altogether, our results indicate that decellularized WJ matrix is able to limit Staphylococcus-related infections and to promote stromal cell homing, thus offering a versatile scaffold for tissue regenerative medicine.

Evaluation of mTHPC-loaded PLGA nanoparticles for in vitro photodynamic therapy on C6 glioma cell line.

Photodynamic therapy (PDT) is a very attractive strategy to complement or replace common cancer treatments such as radiotherapy, surgery, and chemotherapy. Some molecules have shown their efficiency as photosensitizers (PS), still many issues have to be solved such as the inherent cytotoxicity of the PS or its hydrophobic properties causing limitation in their solubility, leading to side effects. In this study, the encapsulation of an approved PS, the meso-tetra hydroxyphenylchlorine (mTHPC, Foscan®) within biocompatible and biodegradable poly(D, l-lactide-co-glycolide) acid (PLGA) NPs prepared by the nanoprecipitation method was studied. The mTHPC-loaded NPs (mTHPC ⊂ PLGA NPs) were analyzed by UV-vis spectroscopy to determine the efficiency of mTHPC encapsulation, and by dynamic light scattering (DLS) and atomic force microscopy (AFM) to determine mTHPC ⊂ PLGA NPs sizes, morphologies and surface charges. The longitudinal follow-up of mTHPC release from the NPs indicated that 50% of the encapsulated PS was retained within the NP matrix after a period of five days. Finally, the cytotoxicity and the phototoxicity of the mTHPC ⊂ PLGA NPs were determined in murine C6 glioma cell lines and compared to the ones of mTHPC alone. The studies showed a strong decrease of mTHPC cytotoxicity and an increase of mTHPC photo-cytotoxicity when mTHPC was encapsulated. In order to have a better insight of the underlying cellular mechanisms that governed cell death after mTHPC ⊂ PLGA NPs incubation and irradiation, annexin V staining tests were performed. The results indicated that apoptosis was the main cell death mechanism.

Bioinspired Nanofeatured Substrates: Suitable Environment for Bone Regeneration.

Bone mimicking coatings provide a complex microenvironment in which material, through its inherent properties (such as nanostructure and composition), affects the commitment of stem cells into bone lineage and the production of bone tissue regulating factors required for bone healing and regeneration. Herein, a bioactive mineral/biopolymer composite made of calcium phosphate/chitosan and hyaluronic acid (CaP-CHI-HA) was elaborated using a versatile simultaneous spray coating of interacting species. The resulting CaP-CHI-HA coating was mainly constituted of bioactive, carbonated and crystalline hydroxyapatite with 277 ± 98 nm of roughness, 1 µm of thickness, and 2.3 ± 1 GPa of stiffness. After five days of culture, CaP-CHI-HA suggested a synergistic effect of intrinsic biophysical features and biopolymers on stem cell mechanobiology and nuclear organization, leading to the expression of an early osteoblast-like phenotype and the production of bone tissue regulating factors such as osteoprotegerin and vascular endothelial growth factor. More interestingly, amalgamation with biopolymers conferred to the mineral a bacterial antiadhesive property. These significant data shed light on the potential regenerative application of CaP-CHI-HA bioinspired coating in providing a suitable environment for stem cell bone regeneration and an ideal strategy to prevent implant-associated infections.

In vitro antiproliferative effect and induction of apoptosis by Retama monosperma L. extract in human cervical cancer cells.

The antiproliferative effect of different extracts obtained from Retama monosperma L. was investigated on human SiHa and HeLa cervical cancer cell lines using a MTT colorimetric assay. The Retama monosperma L. dichloromethane fraction (Rm-DF) was the most active extract, exhibiting a significant cytotoxic activity on both cell lines in a dose-dependent manner, after 72 h of treatment. IC50 values obtained were 14.57 ± 4.15 µg/ml and 21.33 ± 7.88 µg/ml, for SiHa and HeLa cell lines respectively. The morphological features assessment of apoptosis in Rm-DF-treated cells showed a condensation of chromatin and apoptotic bodies, accompanied by a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species in both cell lines. The induction of apoptosis was further confirmed by Western blotting pro-caspase 3, Bcl2 and PARP; caspase 3 activity assay; and Annexin V labelling. Analysis of Rm-DF by CG/MS revealed the presence of five known quinolizidine alkaloids as well as, sparteine (10,97%), L-methyl cytisine (9.11%), 17-oxosparteine (3.49%), lupanine (0.93%) and anagyrine (39.63%). This study shows that Retama monosperma L. extract exhibits a potential anticancer activity against cervical cancer cell lines in vitro through the inhibition of proliferation and induction of apoptosis, which may involve a mitochondria-mediated signaling pathway.

Lumican inhibits B16F1 melanoma cell lung metastasis.

BACKGROUND: Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) involved in the control of melanoma growth and invasion. The aim of the present study was to analyse the role of lumican in the regulation of the development of lung metastasis. METHODS: B16F1 melanoma cells stably transfected with lumican expressing plasmid (Lum-B16F1) were injected to syngenic mice. The lung metastasis was compared to mice injected with mock-transfected B16F1 cells (Mock-B16F1). The expression of lumican, cyclin D1, apoptotic markers, vascular endothelium growth factor (VEGF) and Von Willebrand Factor (vWF) within lung metastasis nodules was investigated by immunohistochemistry. In parallel, cells cultured in presence of lumican were assayed for apoptosis and motility. RESULTS: We observed that the number and the size of lung metastasis nodules were significantly decreased in mice injected with Lum-B16F1 cells in comparison to Mock-B16F1 cells. This was associated with an increase of tumour cell apoptosis within metastasis nodules but the cell proliferation rate remained constant in the two mice groups. In contrast, the VEGF immunostaining and the number of blood vessels within the lung metastasis nodules were decreased in the lumican-expressing tumours. In vitro, a significant decrease of apoptotic markers in wild type B16F1 cells incubated with increasing amounts of lumican core protein was observed. In addition, pseudotubes formation on Matrigel(R) and the migratory capacity of endothelial cells was inhibited by lumican. Altogether, our results indicate that lumican decreases lung metastasis development not only by inducing tumour cell apoptosis but also by inhibiting angiogenesis.

Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells.

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope.

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2(n)x2(n) pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.