Mitochondrial function contributes to oxysterol-induced osteogenic differentiation in mouse embryonic stem cells

AbstractOxysterols, oxidized derivatives of cholesterol, are biologically active molecules. Specific oxysterols have potent osteogenic properties that act on osteoprogenitor cells. However, the molecular mechanisms underlying these osteoinductive effects on embryonic stem cells (ESCs) are unknown. This study investigated the effect of an oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on osteogenic differentiation of ESCs and the alterations to mitochondrial activity during differentiation. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, matrix mineralization, mRNA expression of osteogenic factors, runt-related transcription factor 2, osterix, and osteocalcin, and protein levels of collagen type IA (COLIA) and osteopontin (OPN). Treatment of cells with SS increased osteoinductive activity compared to the control group. Intracellular reactive oxygen species production, intracellular ATP content, mitochondrial membrane potential, mitochondrial mass, mitochondrial DNA copy number, and mRNA expression of peroxisome proliferator-activated receptor-γ coactivators 1α and β, transcription factors involved in mitochondrial biogenesis, were significantly increased during osteogenesis, indicating upregulation of mitochondrial activity. Oxysterol combinations also increased protein levels of mitochondrial respiratory complexes I–V. We also found that SS treatment increased hedgehog signaling target genes, Smo and Gli1 expression. Inhibition of Hh signaling by cyclopamine suppressed mitochondrial biogenesis and ESC osteogenesis. Subsequently, oxysterol-induced Wnt/β-catenin pathways were inhibited by repression of Hh signaling and mitochondrial biogenesis. Transfection of β-catenin specific siRNA decreased the protein levels of COLIA and OPN, as well as ALP activity. Collectively, these data suggest that lipid-based oxysterols enhance differentiation of ESCs toward the osteogenic lineage by regulating mitochondrial activity, canonical Hh/Gli, and Wnt/β-catenin signaling

Skin-Integrated wearable systems and implantable biosensors: a comprehensive review

Biosensors devices have attracted the attention of many researchers across the world. They have the capability to solve a large number of analytical problems and challenges. They are future ubiquitous devices for disease diagnosis, monitoring, treatment and health management. This review presents an overview of the biosensors field, highlighting the current research and development of bio-integrated and implanted biosensors. These devices are micro- and nano-fabricated, according to numerous techniques that are adapted in order to offer a suitable mechanical match of the biosensor to the surrounding tissue, and therefore decrease the body’s biological response. For this, most of the skin-integrated and implanted biosensors use a polymer layer as a versatile and flexible structural support, combined with a functional/active material, to generate, transmit and process the obtained signal. A few challenging issues of implantable biosensor devices, as well as strategies to overcome them, are also discussed in this review, including biological response, power supply, and data communication.This research was funded by FCT- FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA, grant numbers: PTDC/EMD-EMD/31590/2017 and PTDC/BTM-ORG/28168/2017

3D bioprinting of gellan gum-based hydrogels tethered with laminin-derived peptides for improved cellular behavior

The treatment of skeletal muscle defects is still a topic of noteworthy concern since surgical intervention is not capable of recovering muscle function. Herein, we propose myoblasts laden in laminin-inspired biofunctionalized gellan gum hydrogels as promising tissue-engineered skeletal muscle surrogates. Gellan gum-based hydrogels were developed by combining native gellan gum (GG) and GG tethered with laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T) or RKRLQVQLSIRTC (Q)), using different polymer content (0.75%â 1.875%). Hydrogels were characterized in terms of compressive modulus, molecules trafficking, and C2C12 adhesion. Hydrogels with higher polymeric content (1.125%â 1.875%) showed higher stiffness whereas hydrogels with lower polymer content (0.75%â 1.125%) showed higher fluorescein isothiocyanate-dextran molecules diffusion. Cell spreading was achieved regardless of the laminin-derived peptide but preferred in hydrogels with higher polymer content (1.125%â 1.875%). Taken together, hydrogels with 1.125% of polymer content were selected for printability analysis. GG-based inks showed a non-newtonian, shear-thinning, and thixotropic behavior suitable for printing. Accordingly, all inks were printable, but inks tethered with T and Q peptides presented some signs of clogging. Cell viability was affected after printing but increased after 7â days of culture. After 7â days, cells were spreading but not showing significant signs of cellâ cell communications. Therefore, cell density was increased, thus, myocytes loaded in V-tethered GG-based inks showed higher cellâ cell communication, spreading morphology, and alignment 7, 14â days post-printing. Overall, myoblasts laden in laminin-inspired biofunctionalized GG-based hydrogels are a promising skeletal muscle surrogate with the potential to be used as in vitro model or explored for further in vivo applications.CEEC Individual, Grant/Award Number: 2020.01541.CEECIND/CP1600/CT0024; Fundacao para a Ciencia e a Tecnologia, Grant/Award Number: PD/BD/128090/201

Improved blastocyst development of single cow OPU-derived presumptive zygotes by group culture with agarose-embedded helper embryos

<p>Abstract</p> <p>Background</p> <p>The <it>in vitro </it>culture of presumed zygotes derived from single cow ovum pick-up (OPU) is important for the production of quality blastocysts maintaining pedigree. The aim of the present study was to evaluate the agar chip-embedded helper embryo coculture system for single cow OPU-derived zygotes by assessing embryo quality.</p> <p>Methods</p> <p>Cumulus oocyte complexes (COCs) were collected from Hanwoo cows with high genetic merit twice a week using the ultra-sound guided OPU technique and from slaughterhouse ovaries. The Hanwoo cow COCs and slaughterhouse ovaries were matured <it>in vitro</it>, fertilized <it>in vitro </it>with thawed Hanwoo sperm and cultured for 24 h. The presumed zygotes were subsequently placed in three different culture systems: (1) control OPU (controlOPU) with single cow OPU-derived presumed zygotes (2~8); (2) agar chip-embedded slaughterhouse helper embryo coculture (agarOPU) with ten presumed zygotes including all presumed zygotes from a cow (2~8) and the rest from agar chip-embedded slaughterhouse presumed zygotes (8~2); and (3) slaughterhouse <it>in vitro </it>embryo production (sIVP) with ten slaughterhouse ovary-derived presumed zygotes, each in 50 μL droplets. Day 8 blastocysts were assayed for apoptosis and gene expression using real time PCR.</p> <p>Results</p> <p>The coculture system promoted higher blastocyst development in OPU zygotes compared to control OPU zygotes cultured alone (35.2 vs. 13.9%; P < 0.01). Genes predicted to be involved in implantation failure and/or embryo resorption were down-regulated (P < 0.05) in control OPU zygotes (<it>CD9</it>, 0.4-fold; <it>AKRAB</it>1, 0.3-fold) and in cocultured zygotes (<it>CD9</it>, 0.3-fold; <it>AKRAB</it>1, 0.3-fold) compared to sIVP blastocysts (1.0-fold). Moreover, genes involved in implantation and/or normal calf delivery were up-regulated (P < 0.05 to P < 0.01) in control OPU zygotes (<it>PGSH</it>2, 5.0-fold; <it>TXN</it>, 4.3-fold; <it>PLAU</it>, 1.7-fold) and cocultured zygotes (<it>PGSH</it>2, 14.5-fold; <it>TXN</it>, 3.2-fold; <it>PLAU</it>, 6.8-fold) compared to sIVP (1.0-fold) blastocysts. However, the expression of <it>PLAC8, TGF-β1, ODC1</it>, <it>ATP5A1 </it>and <it>CASP3 </it>did not differ between the three culture groups.</p> <p>Conclusions</p> <p>Results show that the agar chip-embedded helper embryo coculture system enhances developmental competence and embryo quality in cultures of limited numbers of high pedigree single cow OPU presumed zygotes.</p

3D biosensors in advanced medical diagnostics of high mortality diseases

Cardiovascular diseases, cancer, and diabetes are high mortality diseases, which account for almost two thirds of all deaths worldwide. Their early detection and continuous evaluation is fundamental for an improved patient prognosis and reduced socioeconomic impact. Current biosensor technologies are typically based on the analysis of whole blood samples from patients for the detection of disease-specific biomarkers. However, these technologies display serious shortcomings, such as reduced sensitivity and dynamic range, limited in vivo applicability, and lack of continuous monitoring. There is the urgent need for new diagnostic and treatment follow-up tools, which allow for the early detection of the pathology as well as for the continuous monitoring of the physiological response to specific therapies. During the last years, a new generation of biosensor technologies with improved performance has emerged in the biomedical sector. The combination of advanced biomaterial methods, biochemical tools, and micro/nanotechnology approaches has resulted in the development of innovative three-dimensional (3D) biosensor platforms for advanced medical diagnosis. In this review, we report the most recent advances in the field of 3D biosensors for clinical applications, focusing on the diagnosis and monitoring of cardiovascular diseases, cancer, and diabetes. We discuss about their clinical performance compared to standard biosensor technologies, their implantable capability, and their integration into microfluidic devices to develop clinically-relevant models. Overall, we anticipate that 3D biosensors will drive us toward a new paradigm in medical diagnosis, resulting in real-time in vivo biosensors capable to significantly improve patient prognosis.V.M.C., S.C.K, and D.C. acknowledge thefinancial support from theEuropean Union Framework Programme for Research and InnovationHorizon 2020 on Forefront Research in 3D Disease Cancer Models asinvitroScreening Technologies (FoReCaST) under Grant agreement no.668983. V.M.C also thanks the Portuguese Foundation for Science andTechnology (FCT) for his distinction attributed under the FCTInvestigator program (IF/01214/2014). D.C. and S.C.K also acknowl-edge the support from the FCT under the scope of the project ModellingCancer Metastasis into the Human Microcirculation System using aMulti-organ-on-a-Chip Approach (2MATCH) (PTDC/BTM-ORG/28070/2017) funded by the Programa Operacional Regional do Norte sup-ported by Fundo Europeu de Desenvolvimento Regional (FEDER). A.I.B.acknowledges thefinancial support of project FROnTHERA (NORTE-01-0145-FEDER-000023

Embryoid body size-mediated differential endodermal and mesodermal differentiation using polyethylene glycol (PEG) microwell array

Embryoid bodies have a number of similarities with cells in gastrulation, which provides useful biological information about embryonic stem cell differentiation. Extensive research has been done to study the control of embryoid body-mediated embryonic stem cell differentiation in various research fields. Recently, microengineering technology has been used to control the size of embryoid bodies and to direct lineage specific differentiation of embryonic stem cells. However, the underlying biology of developmental events in the embryoid bodies of different sizes has not been well elucidated. In this study, embryoid bodies with different sizes were generated within microfabricated PEG microwell arrays, and a series of gene and molecular expressions related to early developmental events was investigated to further elucidate the size-mediated differentiation. The gene and molecular expression profile suggested preferential visceral endoderm formation in 450 μm embryoid bodies and preferential lateral plate mesoderm formation in 150 μm embryoid bodies. These aggregates resulted in higher cardiac differentiation in 450 μm embryoid bodies and higher endothelial differentiation in 150 μm embryoid bodies, respectively. Our findings may provide further insight for understanding embryoid body size-mediated developmental progress.National Science Foundation (U.S.) (CAREER Award DMR0847287)United States. Office of Naval Research (Naval Research Young National Investigator Award)National Institutes of Health (U.S.) (HL092836, EB02597, AR057837

Micropatterned silk-fibroin/eumelanin composite films for bioelectronic applications

There has been growing interest in the use of natural bionanomaterials and nanostructured systems for diverse biomedical applications. Such materials can confer unique functional properties as well as address concerns pertaining to sustainability in production. In this work, we propose the biofabrication of micropatterned silk fibroin/eumelanin composite thin films to be used in electroactive and bioactive applications in bioelectronics and biomedical engineering. Eumelanin is the most common form of melanin, naturally derived from the ink of cuttlefish, having antioxidant and electroactive properties. Another natural biomaterial, the protein silk fibroin, is modified with photoreactive chemical groups, which allows the formation of electroactive eumelanin thin films with different microstructures. The silk fibroin/eumelanin composites are fabricated to obtain thin films as well as electroactive microstructures using UV curing. Here, we report for the first time the preparation, characterization, and physical, electrochemical, and biological properties of these natural silk fibroin/eumelanin composite films. Higher concentrations of eumelanin incorporated into the films exhibit a higher charge storage capacity and good electroactivity even after 100 redox cycles. In addition, the microscale structure and the cellular activity of the fibroin/eumelanin films are assessed for understanding of the biological properties of the composite. The developed micropatterned fibroin/eumelanin films can be applied as natural electroactive substrates for bioapplications (e.g., bioelectronics, sensing, and theranostics) because of their biocompatible properties.The authors acknowledge the FRONTHERA project (Frontiers of technology for theranostics of cancer, metabolic and neurodegenerative diseases) n degrees NORTE-01-0145-FEDER0000232, the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement n degrees 668983. FoReCaST (Forefront Research in 3D Disease Cancer Models as in vitro Screening Technologies), and FCT grants POCI-01-0145-FEDER-031590, PD/BD/150546/2019 and PTDC/BTM-ORG/28168/2017. VKY acknowledges support from the National Science Foundation (CBET1704435)

In vitro and in vivo assessments of an optimal polyblend composition of polycaprolactone/gelatin nanofibrous scaffolds for Achilles tendon tissue engineering

In this study, we manufactured various ratios of polycaprolactone (PCL)/gelatin (GE) highly aligned electrospun nanofibrous scaffolds (ENs) to investigate the effects of polymer ratio on tenogenic differentiation activity. For biological assessments, the cell proliferation rate was optimal in the PCL/GE (9:1) group. Interestingly, however, the tenogenic differentiation rate was best for the PCL/GE (7:3) group. From our outcomes, we established that a poly-blending mix of PCL/GE (7:3) is a promising ratio for tenogenic differentiation. Thus, our findings may provide for an effective mesh to promote tenogenic differentiation of ENs in future tendon tissue engineering applications.This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & funded by the Korean government (MSIP&MOHW) (No. 2017M3A9E4048170)