Rapid generation of single-insertion transgenics by Tol2 transposition in zebrafish.

BACKGROUND: The Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of the transgenic cassette in F1 fish. Each of these transgenes is susceptible to positional effects from the surrounding regulatory landscape, which can lead to altered expression and, consequently, activity. Scientists therefore must strike a balance between the need to maximize reproducibility by establishing single-insertion transgenic lines and the need to complete experiments within a reasonable timeframe. RESULTS: In this article, we introduce a simple competitive dilution strategy for rapid generation of single-insertion transgenics. By using cry:BFP reporter plasmid as a competitor, we achieved a nearly fourfold reduction in the number of the transgene of interest integrations while simultaneously increasing the proportion of single-insertion F1 generation transgenics to over 50%. We also observed variations in transgene of interest expression among independent single-insertion transgenics, highlighting that the commonly used ubiquitous ubb promoter is susceptible to position effects. CONCLUSIONS: Wide application of our competitive dilution strategy will save time, reduce animal usage, and improve reproducibility of zebrafish research.

Conditional mutagenesis strategies in zebrafish.

Gene disruption or knockout is an essential tool for elucidating gene function. Conditional knockout methodology was developed to further advance these studies by enabling gene disruption at a predefined time and/or in discrete cells. While the conditional knockout method is widely used in the mouse, technical limitations have stifled direct adoption of this methodology in other animal models including the zebrafish. Recent advances in genome editing have enabled engineering of distinct classes of conditional mutants in zebrafish. To further accelerate the development and application of conditional mutants, we will review diverse methods of conditional knockout engineering and discuss the advantages of different conditional alleles.

Dental pulp stem cell-derived extracellular matrix: autologous tool boosting bone regeneration.

BACKGROUND AIMS: To facilitate artificial bone construct integration into a patient's body, scaffolds are enriched with different biologically active molecules. Among various scaffold decoration techniques, coating surfaces with cell-derived extracellular matrix (ECM) is a rapidly growing field of research. In this study, for the first time, this technology was applied using primary dental pulp stem cells (DPSCs) and tested for use in artificial bone tissue construction. METHODS: Rat DPSCs were grown on three-dimensional-printed porous polylactic acid scaffolds for 7 days. After the predetermined time, samples were decellularized, and the remaining ECM detailed proteomic analysis was performed. Further, DPSC-secreated ECM impact to mesenchymal stromal cells (MSC) behaviour as well as its role in osteoregeneration induction were analysed. RESULTS: It was identified that DPSC-specific ECM protein network ornamenting surface-enhanced MSC attachment, migration and proliferation and even promoted spontaneous stem cell osteogenesis. This protein network also demonstrated angiogenic properties and did not stimulate MSCs to secrete molecules associated with scaffold rejection. With regard to bone defects, DPSC-derived ECM recruited endogenous stem cells, initiating the bone self-healing process. Thus, the DPSC-secreted ECM network was able to significantly enhance artificial bone construct integration and induce successful tissue regeneration. CONCLUSIONS: DPSC-derived ECM can be a perfect tool for decoration of various biomaterials in the context of bone tissue engineering.

DNA-DAPI Interaction-Based Method for Cell Proliferation Rate Evaluation in 3D Structures.

Effective cell number monitoring throughout the three-dimensional (3D) scaffold is a key factor in tissue engineering. There are many methods developed to evaluate cell number in 2D environments; however, they often encounter limitations in 3D. Therefore, there is a demand for reliable methods to measure cell proliferation in 3D surroundings. Here, we report a novel technique for the DNA content-based evaluation of cell proliferation using DNA-binding dye DAPI. We demonstrated the method's compatibility with four different cell cultures: cancer lines MCF-7 and MH-22a, embryonic fibroblast cell line Swiss 3T3, and primary mesenchymal stem cell culture isolated from rat's incisors. The DAPI based method was able to successfully evaluate cell proliferation in 2D, 2.5D, and 3D environments. Even though the proposed method does not discriminate between viable and dead cells, it might give a convenient snapshot of the cell number at a given time point. This should help to more reliably evaluate various processes proceeding in 2.5D and 3D cultures.

In vitro comparison of 3D printed polylactic acid/hydroxyapatite and polylactic acid/bioglass composite scaffolds: Insights into materials for bone regeneration.

3D printing of polylactic acid (PLA) and hydroxyapatite (HA) or bioglass (BG) bioceramics composites is the most promising technique for artificial bone construction. However, HA and BG have different chemical composition as well as different bone regeneration inducing mechanisms. Thus, it is important to compare differentiation processes induced by 3D printed PLA + HA and PLA + BG scaffolds in order to evaluate the strongest osteoconductive and osteoinductive properties possessing bioceramics. In this study, we analysed porous PLA + HA (10%) and PLA + BG (10%) composites' effect on rat's dental pulp stem cells fate in vitro. Obtained results indicated, that PLA + BG scaffolds lead to weaker cell adhesion and proliferation than PLA + HA. Nevertheless, osteoinductive and other biofriendly properties were more pronounced by PLA + BG composites. Overall, the results showed a strong advantage of bioceramic BG against HA, thus, 3D printed PLA + BG composite scaffolds could be a perspective component for patient-specific, cheaper and faster artificial bone tissue production.

The effect of larger than cell diameter polylactic acid surface patterns on osteogenic differentiation of rat dental pulp stem cells.

Topography of the scaffold is one of the most important factors defining the quality of artificial bone. However, the production of precise micro- and nano-structured scaffolds, which is known to enhance osteogenic differentiation, is expensive and time-consuming. Meanwhile, little is known about macro-patterns (larger than cell diameter) effect on cell fate, while this kind of structures would significantly facilitate the manufacturing of artificial skeleton. Therefore, this research is focused on polylactic acid scaffold's macro-pattern impact on rat's dental pulp stem cells (DPSCs) morphology, proliferation, and osteogenic differentiation. For this study, two types of scaffolds were 3D printed: wavy and porous. Wavy scaffolds consisted of 188 µm wide joined threads, meaning that cells might have been curved on the filament as well as compressed in the groove. Porous scaffolds were designed to avoid groove formation and consisted of 500 µm threads, arranged in the woodpile manner, forming 300 µm diameter pores. We found that both macro-surfaces influenced DPSC morphology compared to control. As a consequence, enhanced DPSC proliferation and increased osteogenic differentiation potential was registered in cells grown on these scaffolds. Finally, our results showed that the construction of an artificial bone did not necessarily require the precise structuring of the scaffold, because both types of macro-topographic PLA scaffolds were sufficient enough to induce spontaneous DPSC osteogenic differentiation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 174-186, 2019.