Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration.
Our findings identify the characteristic patterns about spatiotemporal morphogenesis of successional teeth in context of their predecessor and cascade initiation of additional molars in miniature pigs. Our study provides a basis toward better understanding the mechanisms underlying diphyodont replacement in human and also assists in tooth regeneration and tooth engineering in large animal.
MicroRNAs (miRNAs) play important roles in the regulation of rodent tooth development, but little is known about their role in tooth development in large mammals. We identified 637 unique miRNA sequences in a large-scale screen for miRNA expression profiles in the developing lower deciduous molars of miniature pigs (Sus scrofa) using Illumina Solexa deep sequencing. These candidate miRNAs and another 105 known Sus scrofa miRNAs were included in the custom-designed microarray and used to analyze the miRNA expression profile in the bud, cap, early bell, and late bell stages of tooth development. Microarray analysis revealed 166 transcripts that were differentially expressed in the four stages. Bioinformatic analysis identified 18 key miRNAs, including let-7f, miR-128, miR-200b, and miR-200c, that might play key roles in tooth development. Taken together, our results not only identified the specific microRNAome and expression profile in developing lower deciduous molars of the miniature pig, but they also provided useful information for investigating the molecular mechanism of tooth development in the miniature pig.
We developed a method of interfacing microfluidics with mass spectrometry (MS) using a robotic spotting system to automate the contact spotting process. We demonstrate that direct and automated spotting of analyte from multichannel microfluidic chips to a custom microstructured MALDI target plate was a simple, robust, and high-throughput method for interfacing parallel microchannels using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Using thermoplastic cyclic olefin copolymer (COC) polymer microfluidic chips containing eight parallel 100 lm 9 46 lm microchannels connected to a single input port, spotting volume repeatability and MALDI-MS signal uniformity are evaluated for a panel of sample peptides. The COC microfluidic chips were fabricated by hot embossing and solvent bonding techniques followed by chip dicing to create open ends for MS interfacing. Using the automatic robotic spotting approach, microfluidic chip-based reversed-phase liquid chromatography (RPLC) separations were interfaced with electrochemically etched nanofilament silicon (nSi) target substrate, demonstrating the potential of this approach toward chip-based microfluidic separation coupled with matrix-free laser desorption/ionization mass spectrometry.
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