ERS Mediated by GRP-78/PERK/CHOP Signaling Is Involved in Fluoride-Induced Ameloblast Apoptosis.

Fluoride can be widely ingested from the environment, and its excessive intake could result in adverse effects. Dental fluorosis is an early sign of fluoride toxicity which can cause esthetic and functional problems. Though apoptosis in ameloblasts is one of the potential mechanisms, the specific signal cascade is in-conclusive. High-throughput sequencing and molecular biological techniques were used in this study to explore the underlying pathogenesis of dental fluorosis, for its prevention and treatment. A fluorosis cell model was established. Viability and apoptosis rate of mouse ameloblast-derived cell line (LS8 cells) was measured using cell counting kit-8 (CCK-8) assay and flow cytometry analysis. Cells were harvested with or without 2-mM sodium fluoride (NaF) stimulation for high-throughput sequencing. Based on the sequencing data, subcellular structures, endoplasmic reticulum stress (ERS), and apoptosis related biomarkers were verified using transmission electron microscopy, quantitative real-time polymerase chain reaction, and Western blotting techniques. Expression of ERS markers, apoptosis related proteins, and enamel formation enzymes were detected using Western blotting after addition of 4-phenylbutyrate (4-PBA). NaF-inhibited LS8 cells displayed time- and dose- dependent viability. Additionally, apoptosis and morphological changes were observed. RNA-sequencing data showed that protein processing in endoplasmic reticulum was obviously affected. ERS and apoptosis were induced by excessive NaF. Downregulation of kallikrein-related peptidase 4 (KLK4) was also observed. Inhibition of ERS by 4-PBA rescued the apoptotic and functional protein changes in cells. Excessive fluoride induces apoptosis by activating ERS, which is mediated by GRP-78/PERK/CHOP signaling. Key proteinase is present in maturation-stage enamel; KLK4 was also affected by fluoride, but rescued by 4-PBA. This study presents a possibility for therapeutic strategies for dental fluorosis, while further exploration is required.

In vitro study in the endothelial cell compatibility and endothelialization of genipin-crosslinked biological tissues for tissue-engineered vascular scaffolds.

To overcome the cytotoxicity of the chemical reagents used to fix bioprostheses, genipin, a naturally occurring crosslinking agent, was used to fix biological tissues in present study. We prepared the biological vascular scaffolds through cell extraction and fixing the porcine thoracic arteries with 1% (by w/v) genipin solution for 3 days, and then examined their mechanical properties and microstructures; glutaraldehyde- and epoxy-fixed counterparts were used as controls. HUVECs were seeded on the type I collagen-coated surface of different modified acellular vascular tissues (fixed with different crosslinking agents), and the growths of HUVECs on the specimens were demonstrated by means of MTT test, the secretion of PGI2 and vWF by HUVECs on the various specimens was also measured. Finally, HUVECs were seeded on the luminal surface of acellular biological vascular scaffolds (<6 mm internal diameter) which were, respectively, treated in the same manner described above, and then cultured for 9 days. On the ninth day, the HUVECs on the luminal surface of these vascular scaffolds were examined morphologically and by immunohistochemistry. Genipin-fixation can markedly diminish antigenicity of the vascular tissues through partially getting rid of cell or reducing the level of free amino groups in the vascular tissues. Genipin-fixed acellular vascular tissues mimicked the natural vessels due to the maintenance of the integrity of total structure and the large preservation of the microstructures of collagen fibers and elastic fibers; therefore, it appeared suitable to fabricate vascular scaffolds in mechanical properties. Compared to controls, the genipin-fixed acellular vascular tissues were characterized by low cytotoxicity and good cytocompatibility. The HUVECs can not only proliferate well on the genipin-fixed acellular vascular tissues, but also preserve the activities and function of endothelial cells, and easily make it endothelialized in vitro. The results showed that the genipin-fixed acellular porcine vascular scaffolds should be promising materials for fabricating vascular grafts or the scaffolds of tissue-engineered blood vessels.

[Minimal invasive microscopic tooth preparation based on endodontic, periodontal and functional health].

Tooth preparation is the primary and core operation technique for dental esthetic restoration treatment, due to its effect of providing restoration space, bonding interfaces and marginal lines for dental rehabilitation after tooth tissue reduction. The concept of microscopic minimal invasive dentistry put forward the issue of conducting high-quality tooth preparation, conserve tooth-structure, protect vital pulp and periodontal tissue simultaneously. This study reviewed the concepts, physiology background, design and minimal invasive microscopic tooth preparation, and in the meantime, individualized strategies and the two core elements of tooth preparation (quantity and shape) are listed.