Search for: All records

Introduction: Directing mesenchymal stem cell (MSC) chondrogenesis by bioreactor cultivation provides fundamental insight towards engineering healthy, robust articular cartilage (AC). The mechanical environment is represented by compression, fluid shear stress, hydrostatic pressure, and tension which collectively contribute to the distinct spatial organization of AC. Mimicking this cell niche is necessary for dictating cell growth, fate, and role. Researchers have shown that different mechanical stimulus types improve MSC chondrogenic commitment demonstrated by increases in key chondrogenic gene and protein markers. However, challenges remain in manufacturing spatially, anisotropic AC consisting of defined regions such as native tissue. Our strategy towards furthering this effort involves exposing MSC-laden alginate scaffolds in a multi-chambered, perfusion bioreactor with controlled fluid shear stress magnitudes to better mimic the native AC microenvironment leading to defined regions throughout the scaffold marked by varied cellular phenotypes. Validations made from assessing biochemical content, mRNA expression, western blot analysis, and cell viability will provide meaningful insight towards regulating MSC chondrogenesis. Methods: MSCs grown up to passage 4 were expanded to confluency in a T-175 flask then released from the surface using trypsin. Cells were stored in -80 ℃ freezer until experimentation. Our bioreactor system was sterilized by UV radiation for 4 hours then perfused with 70% ethanol overnight. Cell-laden scaffolds were prepared by first dissolving 1.5% alginate into deionized water. The polymeric solution was sterilely filtered and stored until usage. Cryopreserved MSCs were thawed and suspended in α-MEM medium containing essential supplements. Cells were counted and resuspended in alginate at a density of 106 cells/mL. The mixture was transferred to our multi-chambered bioreactor where they were allowed to crosslink in CaCl2 solution for 45 min. Separate scaffolds (N = 3) were molded within an identical reactor system and removed to serve as a control to compare effects of fluid shear stress on MSC differentiation. All, structures were washed with PBS then supplied with DMEM/F-12 medium containing 10% FBS, 1% penicillin/streptomycin , 1% L-glutamine, 100 nM dexamethasone, 50 µg/mL L-ascorbic acid, and10 ng/mL TGF-β3. The flowrate for the bioreactor was adjusted to 20 mL/min which provided desired fluid shear ranges of 2-87 mPa to stimulate the cells . Cell cultures were grown for 7 days, and medium changed every 3 days. Sectioned samples were analyzed for biochemical content, mRNA expression, and western blot to understand the impact of fluid shear stress magnitudes on MSC differentiation. Results: Directed fluid shear stress across a cell-laden alginate scaffold contained within an individual chamber in our bioreactor indicates varied cellular behavior within the superficial and deep regions of the construct marked by spatially secreted biochemical content as well as mRNA expression. This observation is supported by superficial MSCs stimulated by high and medium mechanical stimulation which indicates a 1.3 and 1.2-fold increase in total collagen production, respectively, when directly compared to cells deep in the construct. A similar effect is supported by total GAG secretion where high and medium shear stress across the fluid hydrogel interface yielded 1.2 and 1.3-fold upregulation of protein secretion, respectively, when observed under similar conditions. Perfused MSCs show upregulation to 3 and 20-fold for Sox9 and aggrecan, respectively, compared to a static culture. Shear ranges distributed throughout our cell-laden alginate scaffold correlates to differential chondrogenic commitment shown by variance of Sox9 expression when assessed by location and depth. Additional information on COL10A1 expression demonstrates mechanical stimulation that reduces hypertrophic cell differentiation contrary to a static culture. Discussion: In this investigation we emphasize that cells respond differently to mechanical stimulation when located in either the superficial or deep region of an alginate scaffold. This observation is supported by enhanced matrix production of chondrogenic protein for cells near the perfused fluid and hydrogel interface compared to deeper areas when stimulated by high and medium fluid shear loading regimes. Most importantly, maintenance of a healthy fluid shear gradient in our TBR provides evidence of promoting MSC chondrogenesis by spatially upregulating anabolic cartilage-like markers in addition to diminishing the onset of cell hypertrophy. Our efforts in monitoring mRNA expression of our samples reveals enhancement of chondrogenic cell differentiation for a perfused sample marked by increases in Sox9 and aggrecan genes; whereas a static sample stimulated only by TGF-β3 leads to undesirable expression of COL10A1. Key takeaways from our study support the contributions from previous researchers in recreating the native AC mechanical environment to encourage MSC differentiation. The development of our TBR system for controlled delivery of fluid shear stresses to MSCs furthers efforts in spatially guiding MSC chondrogenesis which is critical for engineering zonally differentiated AC. 

Osteoarthritis, a chronic disease, remains an issue for adults that causes cartilage degradation within a joint . According to the Centers forDisease Control and Prevention (2023), over 32.5 million adults in the US are affected by osteoarthritis (OA). In this study we seek tounderstand the connection between tissue engineering and genetics to regenerate human articular cartilage (hAC). We purpose to validatea protocol for RNA isolation and characterize the transcriptome of hAC in a tri-layer fashion via bulk RNA sequencing (bulk-RNA-seq).Additionally, we aim to analyze the transcriptome of normal articular cartilage in comparison to the hAC chemical composition and physicalproperties. We are relating these properties to the tri-layers of hAC through histological staining with Safranin O—Fast green and imagingwith differential interference contrast (DIC) microscopy. We are relating these properties to superfic ial, middle, and deep zone with acryotome procedure, RNA extracted, and qualified by Bioanalyzer. Next, we generate bulk RNA sequencing of hAC layer-by-layer andcompare results to early passaging of Mesenchymal Stromal Cells (MSC) and tissues intended for Matrix-Induced Autologous ChondrocyteImplantation (MACI). We will use differential gene expression (DE) analysis by DESeq2 R package software for bulk-RNA-seq. The resultwill be interpreted in terms of differentiation from MSCs to gene expression patterns of tri-layer hAC. We will report on development andvalidation of protocols for isolating cells and their subsequent characterization with application in regenerating the tri-layered hACtranscriptome stimulatory bioreactors used in our laboratory and corresponding properties of the extracellular matrix (ECM) 

Ascomycota, the most speciose phylum of fungi, is a complex entity, comprising three diversesubphyla: Pezizomycotina, Saccharomycotina, and Taphrinomycotina. The largest and most diversesubphylum, Pezizomycotina, is a rich tapestry of 16 classes and 171 orders. Saccharomycotina, thesecond largest subphylum, is a diverse collection of seven classes and 12 orders, whileTaphrinomycotina, the smallest, is a unique assembly of six classes and six orders. Over the pastdecade, numerous taxonomic studies have focused on the generic, family, and class classifications ofAscomycota. These efforts, well-documented across various databases, are crucial for acomprehensive understanding of the classification. However, the study of taxonomy at the ordinallevel, a crucial tier in the taxonomic hierarchy, has been largely overlooked. In a global collaborationwith mycologists and lichenologists, this study presents the first comprehensive information on theorders within Pezizomycotina and Taphrinomycotina. The recent taxonomic classification ofSaccharomycotina has led to the exclusion of this subphylum from the present study, as an immediaterevision is not necessary. Each order is thoroughly discussed, highlighting its historical significance,current status, key identification characteristics, evolutionary relationships, ecological and economicroles, future recommendations, and updated family-level classification. Teaching diagrams for thelife cycles of several orders, viz. Asterinales, Helotiales, Hypocreales, Laboulbeniales, Meliolales,Mycosphaerellales, Ophiostomatales, Pezizales, Pleosporales, Phyllachorales, Rhytismatales,Sordariales, Venturiales, Xylariales (Pezizomycotina) and Pneumocystidales,Schizosaccharomycetales and Taphrinales (Taphrinomycotina) are provided. Each diagram is explained with a representative genus/genera of their sexual and asexual cycles of each order. WithinPezizomycotina, Dothideomycetes contains the highest number of orders, with 57, followed bySordariomycetes (52 orders), Lecanoromycetes (21 orders), Eurotiomycetes and Leotiomycetes (12orders each), Laboulbeniomycetes (3 orders), and Arthoniomycetes and Xylonomycetes (2 orderseach). Candelariomycetes, Coniocybomycetes, Geoglossomycetes, Lichinomycetes, Orbiliomycetes,Pezizomycetes, Sareomycetes, and Xylobotryomycetes each contain a single order, whileThelocarpales and Vezdaeales are treated as incertae sedis within Pezizomycotina. Notably, theclasses Candelariomycetes, Coniocybomycetes, Geoglossomycetes, Sareomycetes, andXylonomycetes, all recently grouped under Lichinomycetes, are treated as separate classes based onphylogenetic analysis and current literature. Within Lecanoromycetes, the synonymization ofSporastatiales with Rhizocarpales and Sarrameanales with Schaereriales is not supported in thephylogenetic analysis. These orders are retained separately, and the justifications are provided undereach section as well as in the discussion. Within Leotiomycetes, the order Medeolariales, which wasonce considered part of Helotiales, is treated as a distinct order based on phylogenetic evidence. Theclassification of Medeolariales may change as more data becomes available from different generegions. Lahmiales (Leotiomycetes) is not included in the phylogenetic analysis due to a lack ofmolecular data. Sareomycetes and Xylonomycetes are treated as separate classes. Spathulosporamixed with Lulworthiales and the inclusion of Spathulosporales within Lulworthiomycetidae issupported and extant molecular sampling is important to resolve the phylogenetic boundaries ofmembers of this subclass. The majority of the classes of Pezizomycotina and Taphrinomycotinaformed monophyletic clades in the phylogenetic analysis conducted based on SSU, LSU, 5.8S, TEFand RPB2 sequence data. However, Arthoniomycetes nested with the basal lineage ofDothideomycetes and formed a monophyletic clade also known as the superclass, Dothideomyceta.In Taphrinomycotina, a single order is accepted within each class. 

Abstract The superτ-charm facility (STCF) is an electron–positron collider proposed by the Chinese particle physics community. It is designed to operate in a center-of-mass energy range from 2 to 7 GeV with a peak luminosity of 0.5 × 10³⁵cm⁻²·s⁻¹or higher. The STCF will produce a data sample about a factor of 100 larger than that of the presentτ-charm factory — the BEPCII, providing a unique platform for exploring the asymmetry of matter-antimatter (charge-parity violation), in-depth studies of the internal structure of hadrons and the nature of non-perturbative strong interactions, as well as searching for exotic hadrons and physics beyond the Standard Model. The STCF project in China is under development with an extensive R&D program. This document presents the physics opportunities at the STCF, describes conceptual designs of the STCF detector system, and discusses future plans for detector R&D and physics case studies.