Dentin, one of the four mineralized tissues of the craniofacial complex, forms sequentially from the deposition of an organic matrix to the nucleation of an inorganic phase within the matrix scaffold. Several promoters and inhibitors of mineralization support and regulate mineral nucleation. Clinical and experimental evidence suggest that dentin matrix protein 1 (DMP1) and phosphate-regulating neutral endopeptidase (PHEX) cooperate and are necessary for the formation of a cohesive dentin layer. The following study investigates the effect of PHEX loss-of-function on dentin matrix formation preceding mineralization. Using the Hyp mouse, an animal model for X-linked hypophosphatemia (XLH), we identified an irregular distribution of dentin extracellular matrix proteins. Likewise, dental pulp stem cells (DPSCs) from XLH patients exhibited altered proteolytic events with disrupted extracellular matrix deposition. Further differentiation assays demonstrated that XLH DPSCs exhibited impaired matrix mineralization. Overexpression of DMP1 in XLH DPSCs restored the irregular protein processing patterns to near-physiological levels. Our results support the hypothesis that hypophosphatemia resulting from PHEX loss-of-function affects the integrity of the organization of the dentin matrix and suggests that exogenous DMP1 can restore physiological processing of matrix proteins, in addition to its canonical role in mineralization.Keywords: dental pulp stem cells, X-linked hypophosphatemia, hypophosphatemia, dentin matrix protein 1, matrix biology Frontiers in Physiology | www.frontiersin.org February 2020 | Volume 11 | Article 82
Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG), including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1). Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5) expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration.
Summary/Abstract
This protocol is for the isolation of primary human dental pulp stem cells (DPSCs) from adult extracted molars, and for the generation of high titer lentivirus for in vitro infection of the DPSCs. Stable cell lines of dental pulp stem cells are generated, maintained in culture, and used for subsequent experiments.
Extracellular matrix of bone and dentin is highly complex and involves a dynamic process of deposition and removal. Cells are the main architect that build this designer matrix that is highly specialized to calcified tissues. Osteoblasts or odontoblasts secrete both collagen and noncollagenous proteins in a temporal and spatial manner. Type I collagen self-assembles and forms a fabric-like template onto which noncollagenous proteins and mineral bind in a well-regulated manner. Dentin matrix protein 1 (DMP1) is one such noncollagenous protein that contains several acidic groups that can bind calcium ions which in turn binds phosphate and initiates the calcification process. In this study, we demonstrate that DMP1 is localized at specific sites on the self-assembled collagen matrix of dentin. In vitro nucleation studies on demineralized and deproteinized dentin slice adsorbed with DMP1 show bundles of well-ordered needle-shaped nanohydroxyapatite deposited on the dentin matrix. The nucleated mineral structures had uniform length and width and their long axis was oriented parallel to the collagen fibril axis. Overall, the physiologically selfassembled collagen and DMP1 mediated ordered deposition of nanocrystalline HAP.
Matrix metalloproteinases (MMPs) have been implicated not only in the regulation of developmental processes but also in the release of biologically active molecules and in the modulation of repair during tertiary dentine formation. Although efforts to preserve dentine have focused on inhibiting the activity of these proteases, their function is much more complex and necessary for dentine repair than expected. The present review explores the role of MMPs as bioactive components of the dentine matrix involved in dentine formation, repair and regeneration. Special consideration is given to the mechanical properties of dentine, including those of reactionary and reparative dentine, and the known roles of MMPs in their formation. MMPs are critical components of the dentine matrix and should be considered as important candidates in dentine regeneration.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.