Application of electrical stimulation for functional tissue engineering in vitro and in vivo

The present invention provides new methods for the in vitro preparation of bioartificial tissue equivalents and their enhanced integration after implantation in vivo. These methods include submitting a tissue construct to a biomimetic electrical stimulation during cultivation in vitro to improve its structural and functional properties, and/or in vivo, after implantation of the construct, to enhance its integration with host tissue and increase cell survival and functionality. The inventive methods are particularly useful for the production of bioartificial equivalents and/or the repair and replacement of native tissues that contain electrically excitable cells and are subject to electrical stimulation in vivo, such as, for example, cardiac muscle tissue, striated skeletal muscle tissue, smooth muscle tissue, bone, vasculature, and nerve tissue

Biofabrication Enables Efficient Interrogation and Optimization of Sequential Culture of Endothelial Cells, Fibroblasts, and Cardiomyocytes for Formation of Vascular Cords in Cardiac Tissue Engineering

Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and, most lately, bioprinting. To be successful in this endeavor, it is crucial to provide in vitro micro-environmental clues for the cells resembling those in the organism. Therefore, scaffolds, populated with differentiated cells or stem cells, of increasing complexity and sophistication are being fabricated. However, no matter how sophisticated scaffolds are, they can cause problems stemming from their degradation, eliciting immunogenic reactions and other a priori unforeseen complications. It is also being realized that ultimately the best approach might be to rely on the self-assembly and self-organizing properties of cells and tissues and the innate regenerative capability of the organism itself, not just simply prepare tissue and organ structures in vitro followed by their implantation. Here we briefly review the different strategies for the fabrication of three-dimensional biological structures, in particular bioprinting. We detail a fully biological, scaffoldless, print-based engineering approach that uses self-assembling multicellular units as bio-ink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion

Vascular Endothelial Growth Factor Secretion by Nonmyocytes Modulates Connexin-43 Levels in Cardiac Organoids

We previously showed that the sequential, but not simultaneous, culture of endothelial cells (ECs), fibroblasts (FBs), and cardiomyocytes (CMs) resulted in elongated, beating cardiac organoids. We hypothesized that the expression of Cx43 and contractile function are mediated by vascular endothelial growth factor (VEGF) released by nonmyocytes during the preculture period. Cardiac organoids (200 μm diameter) were cultivated in microchannels to enable rapid screening. Three experimental groups were formed: (i) Simultaneous Preculture (ECs+FBs for 48 h, followed by CMs), (ii) Sequential Preculture (ECs for 24 h, FBs for 24 h, followed by CMs), and (iii) Simultaneous Triculture (ECs+FBs+CMs). Controls included CMs only, FBs only, and ECs only groups, and preculture with ECs only or FBs only. The highest VEGF levels were found in the Preculture groups [Simultaneous Preculture, 8.9 plus or minus 2.7 ng/(mL times h−1); Sequential Preculture, 16.6 plus or minus 3.4 ng/(mL times h−1)], as compared with Simultaneous Triculture where VEGF was not detectable, as shown by enzyme-linked immunosorbent assay. Analytical flow cytometry showed that VEGFR2 was expressed by ECs (86% plus or minus 2 VEGFR2+), FBs (44% plus or minus 1 VEGFR2+), and CMs (49% plus or minus 2 VEGFR2+), showing that all three cell types were capable of responding to changes in VEGF. Addition of anti-VEGF neutralizing IgG (0.4 μg/mL) to Simultaneous Preculture resulted in 3-fold decrease in Cx43 mRNA and 1.5-fold decrease in Cx43 protein, while Simultaneous Triculture supplemented with VEGF ligand (30 ng/mL) had a threefold increase in Cx43 mRNA and a twofold increase in Cx43 protein. Addition of a small molecule inhibitor of the VEGFR2 receptor (19.4 nM) to Sequential Preculture caused a 1.4-fold decrease in Cx43 mRNA and a 4.1-fold decrease in Cx43 protein. Cx43 was localized within CMs, and not within FBs or ECs. Enriched CM organoids and Sequential Preculture organoids grown in the presence of VEGFR2 inhibitor displayed low levels of Cx43 and poor functional properties. Taken together, these results suggest that endogenous VEGF-VEGFR2 signaling enhanced Cx43 expression and cardiac function in engineered cardiac organoids

Fitotoksični efekti soli natrijuma na klijavost i porast klice tritikalea

The limiting factor for increasing agricultural production for numerous crops is land salinity. The aim of the study was to determine stress effect of sodium salt (NaCl, NaHCO3, Na2SO3 i Na2CO3) on germination and growth triticale varieties – Vojvoda and Smaragd variety, in order to determine degree of tolerance of these varieties on the examined salts. Based on the toxic effect on percentage of germination, germination energy, root length and hypocotyl of the triticale Vojvoda variety and Smaragd variety, the most toxic effect has Na2CO3 and the weakest toxic effect has NaCl. The Smaragd variety is more susceptible to the presence of sodium salts in relation to theVojvoda variety.Limitirajući faktor za povećanje poljoprivredne proizvodnje za brojne biljne vrste je salinitet zemljišta, odnosno povećana koncentracija lako rastvorljivih soli u zemljištu, naročito natrijumovih. Cilj proučavanja bio je da se utvrdi efekat stresa soli natrijuma (NaCl, NaHCO3, Na2SO3 i Na2CO3) na klijanje i rast klijanaca tritikalea sorte Vojvoda i Smaragd, kako bi se odredio stepen tolerantnosti ovih sorti na ispitivane soli. Rezultati pokazuju obrnutu korelaciju između korišćenih koncentracija soli i procenta klijavosti i rasta korenka i hipokotila. Na osnovu toksičnog efekta na procenat klijavosti, energiju klijavosti, dužinu korenka i hipokotila semena triikalea sorte Vojvoda i sorte Smaragd najjači toksični efekat ima Na2CO3 a najslabiji NaCl. Sorta Smaragd osetljivija je na prisustvo soli natrijuma u odnosu na sortu Vojvoda

Bioakumulacija i translokacija metala u vrsti Alyssum markgrafii O. E. Schulz

The aim of this study was to determine the concentrations of 7 metals (Ca, Mg, Mn, Fe, Zn, Ni, Cr) in the soil and selected plant species Alyssum markgrafii O. E. Schulz in one serpentinite sites in the foothills of the mountain Goc. The concentrations of Ni and Cr in the investigated soil were above the maximum allowable concentration of substances in the soil, also above limit and remediation values for a given metals in the soil, according to regulation of Republic of Serbia. The great contents of Ni, Cr and Fe in plant tissues of species A. markgrafii were determined. Good translocation of Ca and Zn from under-ground to above-ground organs of species A. markgrafii has also been shown, and it can be considered suitable for the phytoextraction of investigated metals.Cilj ovog rada bio je da se odrede koncentracije nekih metala (Ca, Mg, Mn, Fe, Zn, Ni, Cr) u zemljištu i vrsti Alyssum markgrafii O. E. Schulz na lokalitetu u podnožju planine Goč. Koncentracije Ni i Cr u istraživanom zemljištu prelazile su propisane maksimalno dozvoljene koncentracije, granične vrednosti, kao i remedijacione vrednosti ovih metala u zemljištu, saglasno uredbi i pravilniku Republike Srbije. Listovi vrste A. markgrafii akumulirali su najviše Mg, Mn, Fe, Ni i Cr. Stablo ove vrste je sadržalo najviše Zn, a cvast najviše Ca. Utvrđeni su veliki sadržaji Ni, Cr i Fe u biljnim tkivima vrste A. markgrafii. Pokazana je i dobra translokacija Ca i Zn od podzemnih ka nadzemnim organima vrste A. markgrafii, kao i da se ona može smatrati pogodnom za fitoekstrakciju ispitivanih metala

Cardiac Tissue Engineering

We hypothesized that clinically sized (1-5 mm thick),compact cardiac constructs containing physiologically high density of viable cells (~108 cells/cm3) can be engineered in vitro by using biomimetic culture systems capable of providing oxygen transport and electrical stimulation, designed to mimic those in native heart. This hypothesis was tested by culturing rat heart cells on polymer scaffolds, either with perfusion of culture medium (physiologic interstitial velocity, supplementation of perfluorocarbons), or with electrical stimulation (continuous application of biphasic pulses, 2 ms, 5 V, 1 Hz). Tissue constructs cultured without perfusion or electrical stimulation served as controls. Medium perfusion and addition of perfluorocarbons resulted in compact, thick constructs containing physiologic density of viable, electromechanically coupled cells, in contrast to control constructs which had only a ~100 mm thick peripheral region with functionally connected cells. Electrical stimulation of cultured constructs resulted in markedly improved contractile properties, increased amounts of cardiac proteins, and remarkably well developed ultrastructure (similar to that of native heart) as compared to non-stimulated controls. We discuss here the state of the art of cardiac tissue engineering, in light of the biomimetic approach that reproduces in vitro some of the conditions present during normal tissue development

Reprocessing Clinically Used Rotary NiTi files - Cleanliness Validation Assays

Rotary NiTi instruments are routinely used in non-surgical endodontic treatment. Previous studies observed debris from manufacturing process adherent to new instruments, as well as presence of dentinal debris following clinical application and reprocessing. Therefore, rotary NiTi instruments that undergo multiple cycles of clinical use may represent a reprocessing challenge. This was a pilot study designed to quantify presence of dentinal debris on clinically used and reprocessed rotary NiTi instruments, using recently developed protein and hydroxyl-apatite quantification assays by ADA. Clinically used and reprocessed VortexBlue rotary instruments sizes 25/.04, 30/.04, 35/.04 and 40/.04 were selected. A reprocessing procedure included of ultrasonication in enzymatic solution followed by steam sterilization in autoclave. Comparable new instruments were subject to same reprocessing protocol as controls. Residual debris was visualized by observing under microscope (20x magnification) and phloxine B staining. Dentin residue was extracted and assessed via ARS assay for hydroxyl-apatite, and via OPA assay for protein, using recently developed ADA protocol (Gopal 2020, IADR). Data statistically analyzed through t-test (p<0.05). Residual dentin was visualized on the cutting edges and evident accumulation towards the shank and rubber stopper. Protein residue on used and reprocessed files could not be quantified. Amounts of HA around 450 ug/ml were extracted from used and reprocessed files. In addition, considerable amounts of debris were observed on rubber stoppers (approximately 500 ug/ml). The Alizarin Red Stain assay provides quantification of hydroxyl-apatite residues on used and reprocessed rotary NiTi instruments. In absence of more sophisticated clinical reprocessing protocol, it is advisable to discard them after single use