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Defective Mineralization in X-Linked Hypophosphatemia Dental Pulp Cell Cultures.

ABSTRACT: X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro-computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.

SUBMITTER: Coyac BR

PROVIDER: S-EPMC6429567 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Defective Mineralization in X-Linked Hypophosphatemia Dental Pulp Cell Cultures.

Coyac B R BR Hoac B B Chafey P P Falgayrac G G Slimani L L Rowe P S PS Penel G G Linglart A A McKee M D MD Chaussain C C Bardet C C

Journal of dental research 20170907 2

X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activi ...[more]

PMID: 28880715

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Project description:Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide - a substrate for PHEX and a strong inhibitor of mineralization - derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.

Project description:BackgroundThe pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains.ObjectivesThe purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes.MethodThe literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review.ResultsMost published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent.DiscussionWhile this review highlights variability among experimental approaches, it does however identify a consensus experimental approach.ConclusionStandardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

Dataset Information

Defective Mineralization in X-Linked Hypophosphatemia Dental Pulp Cell Cultures.

Publications

Defective Mineralization in X-Linked Hypophosphatemia Dental Pulp Cell Cultures.

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