Full Length ArticleTissue-specific mineralization defects in the periodontium of the Hyp mouse model of X-linked hypophosphatemia
Introduction
Disorders of mineral metabolism result in both skeletal and extraskeletal consequences. Skeletal disorders have to be considered not only with regard to the bone itself, but also in the context of disturbed mineral ion metabolism occurring at extraskeletal sites, where alterations can include any one, or a combination, of the following: abnormalities in i) calcium, phosphorus, parathyroid hormone (PTH), and vitamin D metabolism, ii) bone turnover, mineralization, volume, linear growth, and strength, and iii) vascular or soft tissue calcification [1]. In bones and teeth, these abnormalities lead to deficient mineralization known as rickets, osteomalacia and odonto/dentinomalacia.
Although rickets appeared in epidemic form during the industrial revolution from deficiency of vitamin D, in the 1930s, a familial inheritance pattern was proposed for patients displaying vitamin D-resistant rickets [2]. In the past four decades, different mechanisms involved in the initiation and maintenance of the disturbances in bone and mineral metabolism have been uncovered, and X-linked hypophosphatemia (XLH) has been identified as the most prevalent heritable rickets/osteomalacia (~ 1:20,000) [3], [4]. This genetic disorder of calcium and phosphate metabolism is caused by inactivating PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) gene mutations, with XLH being characterized by renal phosphate wasting and defective mineralization of the skeleton and dentition [5], [6], [7]. Loss-of-function mutations in the PHEX gene in XLH cause reduced (or absent) enzymatic activity of PHEX, a zinc metallo-endopeptidase [8], [9], [10] highly expressed by bone (osteoblasts and osteocytes) and tooth (odontoblasts) cells [11]. Skeletal complications in XLH patients include rickets and osteo/odontomalacia, with clinical manifestations including delayed walking, leg bowing (genu varum) or knock knees (genu valgum), growth failure and dental issues such as the occurrence of “spontaneous” tooth abscesses in the absence of caries or trauma [7], [12], [13]. Ultrastructural studies have revealed that the appendicular bone in XLH patients presents with hypomineralized perilacunar matrix around osteocytes often referred to as “halos” (or periosteocytic lesions) [14], and the XLH dentin appears poorly mineralized with large, unmineralized interglobular spaces surrounding unmerged mineralization foci [12]. The mineralization-inhibiting protein osteopontin (OPN) has been proposed as a key player in the pathobiology of these mineralization defects as it accumulates in XLH bone in periosteocytic lesions [15], [16], and in interglobular dentin [17], [18].
Recently, accumulating evidence suggests a higher susceptibility of XLH patients to periodontitis [6], [19], [20], with the recent report of reduced acellular cementum thickness and a higher frequency of intrabony defects displayed in patients with XLH [20]. Consistent with these observations, cementum hypoplasia has been described in the murine model of XLH [21], [22]. Dental cementum is a mineralized tissue covering the tooth root that functions in ligamentous tooth attachment to mandibular and maxillary bone, and post-eruptive adjustment of tooth position in the jaws. During formation of cellular cementum near the tips of tooth roots, some cementoblasts become embedded in the cementoid matrix and become cementocytes. Although cementum presents specific features [23], [24], [25], in some ways it is considered as a bone-like tissue with terminally differentiated cells (cementocytes) embedded in a mineralized extracellular matrix, similar to osteocytes in bone [26]. In the context of genetic disorders disturbing calcium and phosphate metabolism, it still remains to be determined whether cementum formation and function are affected, as previously reported in XLH for bone and dentin.
In the present study, we analyzed how PHEX deficiency affects matrix mineralization of cementum and alveolar bone in the mandible and maxilla under normal and experimental conditions. The periodontal phenotype associated with XLH was examined in human samples, and characterized in greater depth using observations from experiments on the Hyp mouse – the XLH mouse model harboring a Phex mutation that phenocopies the biochemical and clinical features of XLH. Two experimental manipulations (alveolar bone healing and induction of cementum apposition) were performed in Hyp mice to explore the pathobiology of XLH oral bone and cementum.
Section snippets
Human teeth
Human permanent teeth from XLH patients and from gender- and age-matched control individuals extracted prior to orthodontic treatments were collected in the dental departments of Paris Nord Val de Seine Hospitals, AP-HP, France. Informed consent was obtained from patients in agreement with French law (agreement n°DC-2009-927, Cellule Bioéthique DGRI/A5). Teeth were fixed for 7 days at 4 °C in 4% paraformaldehyde solution at pH 7.2–7.4, followed by microwave-assisted decalcification in 4.13% EDTA
Periodontal phenotype of XLH patients and Hyp mice
PHEX was found to be expressed in the periodontium, not only by odontoblasts, osteoblasts and osteocytes, but also by the cellular cementum-forming cells – the cementoblasts and cementocytes (Supplemental Fig. 1).
Observations of tooth roots revealed that XLH teeth had a thin and poorly mineralized acellular cementum (panel A in Fig. 1), consistent with what has been reported previously [20]. In Hyp mice, the acellular cementum also was thin (panel B in Fig. 1a, b). Sirius red staining of molar
Discussion
Here, we show that both cementum and bone formation and healing are altered in XLH/Hyp periodontium. We demonstrate that loss of PHEX activity dramatically alters the periodontal phenotype in humans and in the Hyp mouse (the murine model of XLH), supporting recent clinical studies showing that adult patients with XLH have a higher susceptibility to periodontitis [19], [20]. Interestingly, although cementum and bone mineralization appears similarly altered, each tissue presents a tissue-specific
Conflict of interest statement
BRC, GF, BB, LS, JS, GP, MB, TS, MDM, CC and CB have declared that no conflict of interest exists.
AL: Research grant and/or consulting from Ultragenyx Pharmaceutical, Novato, CA.
Fundings sources
This work was supported by a grant from "La Fondation pour la Recherche Médicale" for Life Imaging Facility of Paris Descartes University (Plateforme d'Imageries du Vivant - PIV) (FRM DGE20111123012). BRC was supported by "La Fondation pour la Recherche Médicale" (PhD scholarship FDM20140731354) (France).
Authors' contribution
BRC, CC and CB designed the study. BRC and GF performed the Raman spectroscopy experiments. BRC and CB performed the surgeries. BRC, LS and JS performed the in vivo radiographic follow-up of all samples. BRC and BB performed other experiments of the study. TS provided the mouse model. BRC, MBD, AL and CC followed the patients and collected the bone and tooth specimens. BRC, MDM, CC and CB contributed to the analysis of the data and the drafting of the manuscript. All authors reviewed and
Acknowledgements
The authors thank Jean-Marc Masse and Alain Schmitt (Institut Cochin, Paris, France) and Annie Llorens (EA2496, Montrouge, France) for their help with the microscopy methods. MDM is a member of the FRQ-S Network for Oral and Bone Health Research, and the McGill Centre for Bone and Periodontal Research.
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2020, Journal of Structural BiologyCitation Excerpt :Very few reports on XLH have analyzed cementum. Acellular cementum was shown to be reduced in thickness in a study on the permanent dentition in XLH (Biosse Duplan et al., 2017) and cellular cementum mineralization was defective (Coyac et al., 2017). Reduced acellular cementum thickness also appears to accompany enamel and dentin defects in primary teeth from individuals with XLH (Fig. 2B).
Alterations of bone material properties in adult patients with X-linked hypophosphatemia (XLH)
2020, Journal of Structural BiologyCitation Excerpt :Our study suggests clearly that some abnormalities in the bone material properties and in bone matrix mineralization in XLH cannot solely be explained by disturbances in phosphate and vitamin D metabolism. It is well-recognized that hydroxyapatite crystals of our skeleton do not grow in isolation but are controlled by a multitude of biologically active agents and there is robust evidence that OPN accumulates in bone (and teeth) matrix of XLH patients (Addison and McKee, 2010; Barros et al., 2013; Boukpessi et al., 2017; Coyac et al., 2017). But it is unclear why the lack of PHEX results in increased secretion of bioactive FGF23 hormone by osteocytes.
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