Search

Graphene paper (GP), a macroscopic self-supporting material, has exceptional flexibility and preserves the excellent physical and chemical properties of graphene nanomaterials. But its applications in regenerative medicine remain to be further explored. Here, we biologically functionalized GP with hydroxyapatite (HA) nanorods by the use of GP-binding peptides as an affinity linker. This strategy solved two daunting challenges for regenerative medicine applications of GP: the lack of good hydrophilicity for supporting cell growth and the difficulty in forming composites by binding with nanobiomaterials. Briefly, we first screened a high-affinity GP-binding peptide (TWWNPRLVYFDY) by the phage display technique. Then we chemically conjugated the GP-binding peptide to the synthetic HA nanorods. The GP-binding peptide on the resultant HA nanorods enabled them to be bound and assembled onto the GP substrate with high affinity, forming a GP-peptide-HA composite with significantly improved hydrophilicity of GP. The composite promoted the attachment and proliferation of mesenchymal stem cells (MSCs), demonstrating its outstanding biocompatibility. Due to the unique compositions of the composite, it was also found to induce osteogenic differentiation of MSCs in vitro in the absence of other inducers in the medium, by verifying the expression of the osteogenic markers including collagen-1, bone morphogenetic proteins 2, runx-related transcription factor 2, osteocalcin, and alkaline phosphatase. Our work suggests that the GP-binding peptide can be used to link inorganic nanoparticles onto GP to facilitate the biomedical applications of GP.

[EN] Low cost and user-friendly paper microfluidic devices, combined with DNA-based biosensors with binding capacities for specific molecules, have been proposed for the developing of novel platforms that ease and speed-up the process of cell secretion monitoring. In this work, we present the first cellulose microfluidic paper-based analytical device for the single-step detection of cell secreted Vascular Endothelial Growth Factor through a self-reporting Structure Switching Signaling Aptamer. A three-part Structure Switching Signaling Aptamer was designed with an aptameric sequence specific for VEGF, which provides a quantifiable fluorescent signal through the displacement of a quencher upon VEGF recognition. The VEGF biosensor was integrated in cellulose paper, enabling the homogenous distribution of the sensor in the paper substrate and the detection of as low as 0.34ng of VEGF in 30min through fluorescence intensity analysis. As a proof-of-concept, the biosensor was incorporated in a microfluidic paper-based analytical device format containing a VEGF detection zone and a control zone, which was applied for the detection of cell secreted VEGF in the supernatant of mesenchymal stem cells culture plates, demonstrating its potential use in cell biology research. Authors acknowledge funding support from Basque Government, under Grupos Consolidados with Grant No. IT1271-19, from “Ministerio de Ciencia y Educación de España” under grant PID2020-120313GB-I00 /AIE /10.13039/501100011033, from the University of the Basque Country and the Spanish Government under the program “Margarita Salas” funded by “Unión Europea-Next Generation EU” . LBD and FBL personally acknowledge funds from the DNASURF (H2020-MSCA-RISE-778001) project. The authors thank for technical and human support provided by PhD Maite Alvarez from DNA Bank Service (SGIker) of the University of the Basque Country (UPV/EHU) and European funding (ERDF and ESF). Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN). Authors thank Dr. Alberto Gorrochategui from “Clínica Dermatológica Ercilla (Bilbao)” for providing the hair follicles from where hHF-MSCs were extracted. Finally, authors also wish to thank the technical assistance from the Nanopharmagene research group at the University of the Basque Country (UPV/EHU). FB-L and LBD acknowledge the “Red de Microfluídica Española” RED2018-102829-T.

Significance: We introduce an application of machine learning trained on optical phase features of epithelial and mesenchymal cells to grade cancer cells’ morphologies, relevant to evaluation of cancer phenotype in screening assays and clinical biopsies. Aim: Our objective was to determine quantitative epithelial and mesenchymal qualities of breast cancer cells through an unbiased, generalizable, and linear score covering the range of observed morphologies. Approach: Digital holographic microscopy was used to generate phase height maps of noncancerous epithelial (Gie-No3B11) and fibroblast (human gingival) cell lines, as well as MDA-MB-231 and MCF-7 breast cancer cell lines. Several machine learning algorithms were evaluated as binary classifiers of the noncancerous cells that graded the cancer cells by transfer learning. Results: Epithelial and mesenchymal cells were classified with 96% to 100% accuracy. Breast cancer cells had scores in between the noncancer scores, indicating both epithelial and mesenchymal morphological qualities. The MCF-7 cells skewed toward epithelial scores, while MDA-MB-231 cells skewed toward mesenchymal scores. Linear support vector machines (SVMs) produced the most distinct score distributions for each cell line. Conclusions: The proposed epithelial–mesenchymal score, derived from linear SVM learning, is a sensitive and quantitative approach for detecting epithelial and mesenchymal characteristics of unknown cells based on well-characterized cell lines. We establish a framework for rapid and accurate morphological evaluation of single cells and subtle phenotypic shifts in imaged cell populations.

The aim of this study was to prepare cellulose nanowhiskers (CNWs) from wastepaper powder (WPP), as an environmentally friendly approach for obtaining the source material, which is a highly available and low-cost precursor for cellulose nanomaterial processing. Acid hydrolysis and calcification treatments were employed for extraction of CNWs and preparation of novel phospho-calcified cellulose nanowhiskers (PCCNWs). CNWs and PCCNWs were analyzed through optical microscopy (OM), scanning electron microscopy (SEM), Fourier-transformed infrared spectra (FTIR) and X-ray diffraction analysis (XRD). Cell behaviors in the presence of CNWs and PCCNWs were studied by MTT assay and live-dead staining. Finally, the effect of these particles on osteogenic differentiation of stem cells was evaluated based on alkaline phosphatase activity (ALP), calcium mineralization as well as von Kossa and alizarin red staining. Based on the results, PCCNWs had a positive effect on osteogenic differentiation of human mesenchymal stem cells (hMSCs) and can be used for developing new approaches for bone tissue engineering.

Significance: Currently, various scaffolds with immobilized cells are widely used in tissue engineering and regenerative medicine. However, the physiological activity and cell viability in such constructs might be impaired due to a lack of oxygen and nutrients. Photobiomodulation (PBM) is a promising method of preconditioning cells to increase their metabolic activity and to activate proliferation or differentiation. Aim: Investigation of the potential of PBM for stimulation of cell activities in hydrogels. Approach: Mesenchymal stromal cells (MSCs) isolated from human gingival mucosa were encapsulated in modified fibrin hydrogels with different thicknesses and concentrations. Constructs with cells were subjected to a single-time exposure to red (630 nm) and near-infrared (IR) (840 nm) low-intensity irradiation. After 3 days of cultivation, the viability and physiological activity of the cells were analyzed using confocal microscopy and a set of classical tests for cytotoxicity. Results: The cell viability in fibrin hydrogels depended both on the thickness of the hydrogels and the concentration of gel-forming proteins. The PBM was able to improve cell viability in hydrogels. The most pronounced effect was achieved with near-IR irradiation at the 840-nm wavelength. Conclusions: PBM using near-IR light can be applied for stimulation of MSCs metabolism and proliferation in hydrogel-based constructs with thicknesses up to 3 mm.

Clinical imaging techniques for the anterior segment of the eye provide excellent anatomical information, but molecular imaging techniques are lacking. Molecular photoacoustic imaging is one option to address this need, but implementation requires use of contrast agents to distinguish molecular targets from background photoacoustic signals. Contrast agents are typically selected based on a priori knowledge of photoacoustic properties of tissues. However, photoacoustic properties of anterior ocular tissues have not been studied yet. Herein, anterior segment anatomy and corresponding photoacoustic signals were analyzed in brown and blue porcine eyes ex vivo. Measured photoacoustic spectra were compared to known optical absorption spectra of endogenous chromophores. In general, experimentally measured photoacoustic spectra matched expectations based on absorption spectra of endogenous chromophores reported in the literature, and similar photoacoustic spectra were observed in blue and brown porcine eyes. However, unique light–tissue interactions at the iris modified photoacoustic signals from melanin. Finally, we demonstrated how the measured PA spectra established herein can be used for one application of molecular PA imaging, detecting photoacoustically labeled stem cells in the anterior segment for glaucoma treatment.