A Versatile Open-Source Printhead for Low-Cost 3D Microextrusion-Based Bioprinting.

Three-dimensional (3D) bioprinting promises to be essential in tissue engineering for solving the rising demand for organs and tissues. Some bioprinters are commercially available, but their impact on the field of Tissue engineering (TE) is still limited due to their cost or difficulty to tune. Herein, we present a low-cost easy-to-build printhead for microextrusion-based bioprinting (MEBB) that can be installed in many desktop 3D printers to transform them into 3D bioprinters. We can extrude bioinks with precise control of print temperature between 2-60 °C. We validated the versatility of the printhead, by assembling it in three low-cost open-source desktop 3D printers. Multiple units of the printhead can also be easily put together in a single printer carriage for building a multi-material 3D bioprinter. Print resolution was evaluated by creating representative calibration models at different temperatures using natural hydrogels such as gelatin and alginate, and synthetic ones like poloxamer. Using one of the three modified low-cost 3D printers, we successfully printed cell-laden lattice constructs with cell viabilities higher than 90% after 24-h post printing. Controlling temperature and pressure according to the rheological properties of the bioinks was essential in achieving optimal printability and great cell viability. The cost per unit of our device, which can be used with syringes of different volume, is less expensive than any other commercially available product. These data demonstrate an affordable open-source printhead with the potential to become a reliable alternative to commercial bioprinters for any laboratory.

Analysis of Spanish Radiometric Networks with the Novel Bias-Based Quality Control (BQC) Method.

Different types of measuring errors can increase the uncertainty of solar radiation measurements, but most common quality control (QC) methods do not detect frequent defects such as shading or calibration errors due to their low magnitude. We recently presented a new procedure, the Bias-based Quality Control (BQC), that detects low-magnitude defects by analyzing the stability of the deviations between several independent radiation databases and measurements. In this study, we extend the validation of the BQC by analyzing the quality of all publicly available Spanish radiometric networks measuring global horizontal irradiance (9 networks, 732 stations). Similarly to our previous validation, the BQC found many defects such as shading, soiling, or calibration issues not detected by classical QC methods. The results questioned the quality of SIAR, Euskalmet, MeteoGalica, and SOS Rioja, as all of them presented defects in more than 40% of their stations. Those studies based on these networks should be interpreted cautiously. In contrast, the number of defects was below a 5% in BSRN, AEMET, MeteoNavarra, Meteocat, and SIAR Rioja, though the presence of defects in networks such as AEMET highlights the importance of QC even when using a priori reliable stations.

Efficient Fabrication of Polycaprolactone Scaffolds for Printing Hybrid Tissue-Engineered Constructs.

Hybrid constructs represent substantial progress in tissue engineering (TE) towards producing implants of a clinically relevant size that recapitulate the structure and multicellular complexity of the native tissue. They are created by interlacing printed scaffolds, sacrificial materials, and cell-laden hydrogels. A suitable biomaterial is a polycaprolactone (PCL); however, due to the higher viscosity of this biopolymer, three-dimensional (3D) printing of PCL is slow, so reducing PCL print times remains a challenge. We investigated parameters, such as nozzle shape and size, carriage speed, and print temperature, to find a tradeoff that speeds up the creation of hybrid constructs of controlled porosity. We performed experiments with conical, cylindrical, and cylindrical shortened nozzles and numerical simulations to infer a more comprehensive understanding of PCL flow rate. We found that conical nozzles are advised as they exhibited the highest shear rate, which increased the flow rate. When working at a low carriage speed, conical nozzles of a small diameter tended to form-flatten filaments and became highly inefficient. However, raising the carriage speed revealed shortcomings because passing specific values created filaments with a heterogeneous diameter. Small nozzles produced scaffolds with thin strands but at long building times. Using large nozzles and a high carriage speed is recommended. Overall, we demonstrated that hybrid constructs with a clinically relevant size could be much more feasible to print when reaching a tradeoff between temperature, nozzle diameter, and speed.

Accurate Calibration in Multi-Material 3D Bioprinting for Tissue Engineering.

Most of the studies in three-dimensional (3D) bioprinting have been traditionally based on printing a single bioink. Addressing the complexity of organ and tissue engineering, however, will require combining multiple building and sacrificial biomaterials and several cells types in a single biofabrication session. This is a significant challenge, and, to tackle that, we must focus on the complex relationships between the printing parameters and the print resolution. In this paper, we study the influence of the main parameters driven multi-material 3D bioprinting and we present a method to calibrate these systems and control the print resolution accurately. Firstly, poloxamer hydrogels were extruded using a desktop 3D printer modified to incorporate four microextrusion-based bioprinting (MEBB) printheads. The printed hydrogels provided us the particular range of printing parameters (mainly printing pressure, deposition speed, and nozzle z-offset) to assure the correct calibration of the multi-material 3D bioprinter. Using the printheads, we demonstrated the excellent performance of the calibrated system extruding different fluorescent bioinks. Representative multi-material structures were printed in both poloxamer and cell-laden gelatin-alginate bioinks in a single session corroborating the capabilities of our system and the calibration method. Cell viability was not significantly affected by any of the changes proposed. We conclude that our proposal has enormous potential to help with advancing in the creation of complex 3D constructs and vascular networks for tissue engineering.

YAP regulates cell mechanics by controlling focal adhesion assembly.

Hippo effectors YAP/TAZ act as on-off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues.

Human stem cell decorated nanocellulose threads for biomedical applications.

Upon surgery, local inflammatory reactions and postoperative infections cause complications, morbidity, and mortality. Delivery of human adipose mesenchymal stem cells (hASC) into the wounds is an efficient and safe means to reduce inflammation and promote wound healing. However, administration of stem cells by injection often results in low cell retention, and the cells deposit in other organs, reducing the efficiency of the therapy. Thus, it is essential to improve cell delivery to the target area using carriers to which the cells have a high affinity. Moreover, the application of hASC in surgery has typically relied on animal-origin components, which may induce immune reactions or even transmit infections due to pathogens. To solve these issues, we first show that native cellulose nanofibers (nanofibrillated cellulose, NFC) extracted from plants allow preparation of glutaraldehyde cross-linked threads (NFC-X) with high mechanical strength even under the wet cell culture or surgery conditions, characteristically challenging for cellulosic materials. Secondly, using a xenogeneic free protocol for isolation and maintenance of hASC, we demonstrate that cells adhere, migrate and proliferate on the NFC-X, even without surface modifiers. Cross-linked threads were not found to induce toxicity on the cells and, importantly, hASC attached on NFC-X maintained their undifferentiated state and preserved their bioactivity. After intradermal suturing with the hASC decorated NFC-X threads in an ex vivo experiment, cells remained attached to the multifilament sutures without displaying morphological changes or reducing their metabolic activity. Finally, as NFC-X optionally allows facile surface tailoring if needed, we anticipate that stem-cell-decorated NFC-X opens a versatile generic platform as a surgical bionanomaterial for fighting postoperative inflammation and chronic wound healing problems.

Estimation of total soluble solids in grape berries using a hand-held NIR spectrometer under field conditions.

BACKGROUND: Recent studies have reported the potential of near infrared (NIR) spectral analysers for monitoring the ripeness of grape berries as an alternative to wet chemistry methods. This study covers various aspects regarding the calibration and implementation of predictive models of total soluble solids (TSS) in grape berries using laboratory and in-field collected NIR spectra. RESULTS: The performance of the calibration models obtained under laboratory conditions indicated that at least 700 berry samples are required to assure enough prediction accuracy. A statistically significant error reduction (ΔRMSECV = 0.1°Brix) with P < 0.001 was observed when measuring berries without epicuticular wax, which was negligible from a practical point of view. Under field conditions, the prediction errors (RMSEP = 1.68°Brix, and SEP = 1.67°Brix) were close to those obtained with the laboratory dataset (RMSEP = 1.42°Brix, SEP = 1.40°Brix). CONCLUSION: This work clarifies several methodological factors to develop a protocol for in-field assessing TSS in grape berries using an affordable, non-invasive, portable NIR spectral analyser. © 2015 Society of Chemical Industry.

Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix.

The use of animal products in the derivation and maintenance of human pluripotent stem cells (hPSCs) limits their possible applications in research and in clinics. Thus, one of the major goals in regenerative medicine is the establishment of animal-free conditions to support the culture and differentiation of human stem cells. Human fibroblasts produce an extracellular matrix (ECM) which can be extracted without the use of detergents, sterilized, and then used to coat tissue culture plates. We have shown that human embryonic stem cells (hESCs) grown on this matrix maintain their pluripotency in the presence of medium conditioned by fibroblast cells, and that these cells maintain expression of surface proteins (SSEA4, Tra1-60, Tra1-81), alkaline phosphatase activity, and specific intracellular markers (Nanog, Oct-4, Tert, FoxD3) in hESCs. This growth system reduces exposure of hPSCs to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally, facilitating their manipulation. Herein we present an improved version of our previous protocol for extracting ECM from human foreskin fibroblast using a different buffer. Our new hypotonic shock method is detergent-free, reduces costs, and preserves the integrity of the extracted ECM. This improved protocol has been validated for undifferentiated-state hPSC maintenance (more than 40 passages), stem cell differentiation, and for cell migration assays.

Heart valve tissue engineering: how far is the bedside from the bench?

Heart disease, including valve pathologies, is the leading cause of death worldwide. Despite the progress made thanks to improving transplantation techniques, a perfect valve substitute has not yet been developed: once a diseased valve is replaced with current technologies, the newly implanted valve still needs to be changed some time in the future. This situation is particularly dramatic in the case of children and young adults, because of the necessity of valve growth during the patient's life. Our review focuses on the current status of heart valve (HV) therapy and the challenges that must be solved in the development of new approaches based on tissue engineering. Scientists and physicians have proposed tissue-engineered heart valves (TEHVs) as the most promising solution for HV replacement, especially given that they can help to avoid thrombosis, structural deterioration and xenoinfections. Lastly, TEHVs might also serve as a model for studying human valve development and pathologies.

Different gDNA content in the subpopulations of prostate cancer extracellular vesicles: apoptotic bodies, microvesicles, and exosomes.

BACKGROUND: Extracellular vesicles (EVs) are cell-derived membrane vesicles. EVs contain several RNAs such as mRNA, microRNAs, and ncRNAs, but less is known of their genomic DNA (gDNA) content. It is also unknown whether the DNA cargo is randomly sorted or if it is systematically packed into specific EV subpopulations. The aim of this study was to analyze whether different prostate cancer (PCa) cell-derived EV subpopulations (apoptotic bodies, microvesicles, and exosomes) carry different gDNA fragments. METHODS: EV subpopulations were isolated from three PCa cell lines (LNCaP, PC-3, and RC92a/hTERT) and the plasma of PCa patients and healthy donors, and characterized by transmission electron microscopy, nanoparticle tracking analysis and total protein content. gDNA fragments of different genes were detected by real time quantitative PCR and confirmed by DNA sequencing. RESULTS: We report that the concentration of EVs was higher in the cancer patients than in the healthy controls. EV subpopulations differed from each other in terms of total protein and DNA content. Analysis of gDNA fragments of MLH1, PTEN, and TP53 genes from the PCa cell-derived EV subpopulations showed that different EVs carried different gDNA content, which could even harbor specific mutations. Altogether, these results suggest that both nucleic acids and proteins are selectively and cell-dependently packed into the EV subtypes. CONCLUSIONS: EVs derived from PCa cell lines and human plasma samples contain double-stranded gDNA fragments which could be used to detect specific mutations, making EVs potential biomarkers for cancer diagnostics and prognostics.

Assessment of flower number per inflorescence in grapevine by image analysis under field conditions.

BACKGROUND: Flowers, flowering and fruit set are key determinants of grapevine yield. Currently, practical methods to assess the flower number per inflorescence, necessary for fruit set estimation, are time and labour demanding. This work aims at developing a simple, cheap, fast, accurate and robust machine vision methodology to be applied to RGB images taken under field conditions, to estimate the number of flowers per inflorescence automatically. RESULTS: Ninety images of individual inflorescences of Vitis vinifera L. cultivars Tempranillo, Graciano and Carignan were acquired in the vineyard with a pocket RGB camera prior to flowering, and used to develop and test the 'flower counting' algorithm. Strong and significant relationships, with R(2) above 80% for the three cultivars were observed between actual and automated estimation of inflorescence flower numbers, with a precision exceeding 90% for all cultivars. CONCLUSION: The developed algorithm proved that the analysis of digital images captured by pocket cameras under uncontrolled outdoors conditions was able to automatically provide a useful estimation of the number of flowers per inflorescence of grapevines at early stages of flowering.

A xenogeneic-free protocol for isolation and expansion of human adipose stem cells for clinical uses.

Human adipose stem cells (HASCS) play a crucial role in the fields of regenerative medicine and tissue engineering for different reasons: the abundance of adipose tissue, their easy harvesting, the ability to multipotent differentiation and the fact that they do not trigger allogeneic blood response or secrete cytokines that act as immunosuppressants. The vast majority of protocols use animal origin reagents, with the underlying risk of transmitting infections by non-human pathogens. We have designed a protocol to isolate and maintain the properties of hASCs avoiding xenogeneic reagents. These changes not only preserve hASCs morphology, but also increase cell proliferation and maintain their stem cell marker profile. On the other hand, human serum albumin (HSA), Tryple® and human Serum (HS), do not affect hASCs multipotent differentiation ability. The amendments introduced do not trigger modifications in the transcriptional profile of hASCs, alterations in key biochemical pathways or malignization. Thus, we have proven that it is possible to isolate and maintain hASCs avoiding animal reagents and, at the same time, preserving crucial culture parameters during long term culture. Thereby we have revealed a novel and effective tool for the improvement of clinical, cell-based therapies.