Erratum: Addendum: Engineered extracellular vesicles from human periodontal-ligament stem cells increase VEGF/VEGFR2 expression during bone regeneration.

[This corrects the article DOI: 10.3389/fphys.2019.00512.].

Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples.

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.

Osteoblastic Differentiation on Graphene Oxide-Functionalized Titanium Surfaces: An In Vitro Study.

BACKGROUND: Titanium implant surfaces are continuously modified to improve biocompatibility and to promote osteointegration. Graphene oxide (GO) has been successfully used to ameliorate biomaterial performances, in terms of implant integration with host tissue. The aim of this study is to evaluate the Dental Pulp Stem Cells (DPSCs) viability, cytotoxic response, and osteogenic differentiation capability in the presence of GO-coated titanium surfaces. METHODS: Two titanium discs types, machined (control, Crtl) and sandblasted and acid-etched (test, Test) discs, were covalently functionalized with GO. The ability of the GO-functionalized substrates to allow the proliferation and differentiation of DPSCs, as well as their cytotoxic potential, were assessed. RESULTS: The functionalization procedures provide a homogeneous coating with GO of the titanium surface in both control and test substrates, with unchanged surface roughness with respect to the untreated surfaces. All samples show the deposition of extracellular matrix, more pronounced in the test and GO-functionalized test discs. GO-functionalized test samples evidenced a significant viability, with no cytotoxic response and a remarkable early stage proliferation of DPSCs cells, followed by their successful differentiation into osteoblasts. CONCLUSIONS: The described protocol of GO-functionalization provides a novel not cytotoxic biomaterial that is able to stimulate cell viability and that better and more quickly induces osteogenic differentiation with respect to simple titanium discs. Our findings pave the way to exploit this GO-functionalization protocol for the production of novel dental implant materials that display improved integration with the host tissue.

Synthesis of green fluorescent carbon dots from carbon nano-onions and graphene oxide.

In recent years, carbon dots (CDs) have triggered considerable interest due to their intriguing tunable photoluminescence properties. In this work, we report the synthesis of green-emitting CDs from two different carbon sources, namely carbon nano-onions and graphene oxide. We also investigate the effects of the two starting materials on the physico-chemical properties of the as-synthesised CDs. Our results show that both CDs exhibit remarkable emission properties and different fluorescence behaviour, which is attributed to the differences in size, surface defects, as well as the presence of different surface functional groups. Moreover, we propose an innovative, low-cost and time-saving method for the recovery of CDs from solution by acetone-mediated precipitation. We demonstrate that this methodology can rival the common dialysis-based purification approach; it shows excellent photostability, and the CD fluorescent properties are retained. Our work paves the way for the use of these particles for biomedical applications by exploiting their interesting fluorescent features as well as their oxygen-enriched surface for further functionalization strategies.

Optimization by response surface methodology of a dispersive magnetic solid phase extraction exploiting magnetic graphene nanocomposite coupled with UHPLC-PDA for simultaneous determination of new oral anticoagulants (NAOs) in human plasma.

In this paper a dispersive magnetic-solid phase extraction (MSPE) using a graphene nanocomposite (rG/Fe3O4) followed by ultra high performance liquid chromatography with photodiode array detection has been developed for the simultaneous analysis of new class of oral anticoagulants (NOAs) in human plasma. The performance of the nanocomposite graphene@Fe3O4 on the magnetic solid phase extraction of apixaban, rivaroxaban and dabigatran has been optimized using a Box-Behnken design of experiment. The amount of graphene nanocomposite, the sample pH and the adsorption time were the investigated parameters as a function of the extraction recovery. The analytical method was fully validated based on linearity, limit of detection (LOD), limit of detection (LOQ), inter- and intra-day precision and trueness, and extraction yield. Under optimal condition, excellent linearity (R2 > 0.9987) over the range (0.001-5.0 µg/mL), limit of detection (0.003 µg/mL), precision (0.81-8.97% RSD) and trueness (-5 to 9 % BIAS%) were observed for the target drugs. The average extraction recovery under optimal from plasma samples ranged between 96.6-98.6% for apixaban, rivaroxaban and dabigatran and the internal standard. The proposed method was developed, validated and successfully applied to the measurement of these NOAs in patients. The new approach offers an attractive alternative for the simultaneous analysis of the selected NOAs from plasma samples, providing several advantages including fewer sample preparation steps, ease of performance, and higher recoveries compared to traditional methodologies.

Engineered Extracellular Vesicles From Human Periodontal-Ligament Stem Cells Increase VEGF/VEGFR2 Expression During Bone Regeneration.

Bone regeneration represents still a challenge, in particular for calvarium defects. Recently, the development of biomaterials with the addiction of stem cells is giving promising results for the treatment of bone defects. In particular, it was demonstrated that scaffolds enriched with mesenchymal stem cells (MSCs) and/or their derivatives, such as conditioned medium (CM) and extracellular vesicles (EVs), may improve bone regeneration. Moreover, given the deep link between osteogenesis and angiogenesis, a successful approach must also take into consideration the development of vascularization. In this work we evaluated the bone regeneration capacity of a collagen membrane (3D-COL) enriched with human periodontal-ligament stem cells (hPDLSCs) and CM or EVs or EVs engineered with polyethylenimine (PEI-EVs) in rats subjected to a calvarial defect. We evaluated also their capacity to induce angiogenic factors. At first, in vitro results showed an increased expression of osteogenic markers in hPDLSCs cultured with the 3D-COL and PEI-EVs, associated also with the increased protein levels of Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). The increased expression of these proteins was confirmed also in vivo in rats implanted with the 3D-COL enriched with hPDLSCs and PEI-EVs. Moreover, histological examination evidenced in this group of rats the activation of bone regeneration and of the vascularization process. Also MicroCT imaging with morphometric analysis confirmed in rats transplanted with 3D-COL enriched with hPDLSCs and PEI-EVs an important regenerative process and a better integration level. All together, these results evidenced that the 3D-COL enriched with hPDLSCs and PEI-EVs may promote bone regeneration of calvaria defects, associated also with an increased vascularization.

Covalent Decoration of Cortical Membranes with Graphene Oxide as a Substrate for Dental Pulp Stem Cells.

(1) Background: The aim of this study was to optimize, through a cheap and facile protocol, the covalent functionalization of graphene oxide (GO)-decorated cortical membrane (Lamina®) in order to promote the adhesion, the growth and the osteogenic differentiation of DPSCs (Dental Pulp Stem Cells); (2) Methods: GO-coated Laminas were fully characterized by Scannsion Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) analyses. In vitro analyses of viability, membrane integrity and calcium phosphate deposition were performed; (3) Results: The GO-decorated Laminas demonstrated an increase in the roughness of Laminas, a reduction in toxicity and did not affect membrane integrity of DPSCs; and (4) Conclusions: The GO covalent functionalization of Laminas was effective and relatively easy to obtain. The homogeneous GO coating obtained favored the proliferation rate of DPSCs and the deposition of calcium phosphate.

Graphene Oxide Foils as an Osteoinductive Stem Cell Substrate.

The hydrophilic graphene derivative, graphene oxide (GO), is used to synthesize free-standing GO foils characterized by cross-linked GO sheets with enhanced mechanical properties and no tendency to release GO flakes in aqueous solution. These GO foils do not evidence cytotoxic effects toward dental pulp stem cells (DPSC). Rather, DPSC viability is significantly increased for cells grown on GO foil and SEM analyses evidence the synthesis of a consistent extracellular matrix by DPSCs with respect to cells grown on polystyrene. Gene expression of osteogenic markers and alkaline phosphatase (ALP) activity tests demonstrate DPSC differentiation toward the osteoblastic lineage. Indeed RUNX2, a key transcriptor factor associated with osteogenic differentiation, as well as SP7, responsible for triggering bone matrix mineralization, are significantly augmented after 7 and 14 days of culture on GO foil with respect to the control, respectively, underlying the capability of GO foil to promote a potential faster and better DPSC differentiation with respect to cells grown on polystyrene. This increase of rate differentiation is confirmed by SEM analyses of DPSCs evidencing a consistent extracellular matrix synthesis at the earliest time of culture (i.e., 3 and 14 days).