Correction of hypophosphatasia-associated mineralization deficiencies in vitro by phosphate/pyrophosphate modulation in periodontal ligament cells

Abstract

Background: Mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene in hypophosphatasia (HPP) reduce the function of tissue non-specific alkaline phosphatase (ALP), resulting in increased pyrophosphate (PP(i)) and a severe deficiency in acellular cementum. We hypothesize that exogenous phosphate (P(i)) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP-diagnosed patients, by correcting the P(i)/PP(i) ratio and modulating expression of genes involved with P(i)/PP(i) metabolism.

Methods: Ex vivo and in vitro analyses were used to identify mechanisms involved in HPP-associated PDL/tooth root deficiencies. Constitutive expression of PP(i)-associated genes was contrasted in PDL versus pulp tissues obtained from healthy individuals. Primary PDL cell cultures from patients with HPP (monozygotic twin males) were established to assay ALP activity, in vitro mineralization, and gene expression. Exogenous P(i) was provided to correct the P(i)/PP(i) ratio.

Results: PDL tissues obtained from healthy individuals featured higher basal expression of key PP(i) regulators, genes ALPL, progressive ankylosis protein (ANKH), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), versus paired pulp tissues. A novel ALPL mutation was identified in the twin patients with HPP enrolled in this study. Compared to controls, HPP-PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1 mM P(i). Dysregulated expression of PP(i) regulatory genes ALPL, ANKH, and ENPP1 was also corrected by adding P(i), although other matrix markers evaluated in our study remained downregulated.

Conclusion: These findings underscore the importance of controlling the P(i)/PP(i) ratio toward development of a functional periodontal apparatus and support P(i)/PP(i) imbalance as the etiology of HPP-associated cementum defects.

Figure 1. Hypophosphatasia (HPP) patients

(A) At 14 years old, patient A experienced spontaneous exfoliation of a permanent mandibular central incisor (tooth 31 by FDI notation) during brushing. (B) Corresponding panoramic radiograph, showing effects of odonto-HPP on the permanent dentition, including delayed eruption of several permanent teeth (e.g. mandibular premolars 34, 35, 44, and 45), enlarged pulp chambers, and thin dentin. (C) SEM imaging of the apical root region of tooth 31 demonstrates lack of cementum and exposed dentin surface with absence of attached collagenous PDL fibers (+), as well as evidence of root dentin resorption (*). Arrow indicates the occlusal direction. (D) Representative DNA sequencing electropherogram showing sequencing analysis of exon 5 of the Alpl gene in control versus patients A and B. The control subject is homozygous for C in the second position of codon 135 (454 nt), while the HPP patients are heterozygous, presenting both a normal (C) and altered allele (T) in the 454-nt position (R135C). Arrow indicates the position of nucleotide substitution in codon 135 GCG (Arginine) > GTG (Cysteine). (E) Confirmation of Alpl missense mutation was performed by enzymatic digestion, demonstrating the Hha I restriction site was abolished. MW: molecular weight marker.

Figure 2. Expression of pyrophosphate and mineralization related genes in PDL and pulp tissues

Gene expression was assayed in non-pooled PDL and pulp tissues from 9 normal subjects (17–22 years old, 2 males and 7 females). Mean and SD for mRNA levels of Alpl, Ankh, Enpp1, Ocn, Dmp1 and Col1. PDL expressed significantly higher mRNA levels for PP_i regulators Alpl, Ankh, and Enpp1, and for mineralization associated genes Ocn and Col1. In contrast, Dmp1 was expressed at higher levels in pulp. Both left and right axes represent relative gene expression, with the left axis representing genes Alpl to Dmp1, and the right axis representing more highly expressed Col1. (*): statistically different by the Student t-test (α=0.05).

Figure 3. Rescue of HPP mineralization deficiency by addition of inorganic phosphate

(A) Control and HPP-PDL cells grown in 2% FBS were counted by MTS method. Values corresponding to relative cell numbers are shown as mean ± SD (absorbance at 490nm). Proliferation and cell viability were equivalent between HPP and control cells, except at day 2. (*): Intragroup significant differences versus day 0 by Kruskal-Wallis one-way ANOVA followed by the Student-Newman-Keuls method (α=0.05), and (&) intergroup significant differences within the same period of time by the Student t-test. (B) Relative alkaline phosphatase activity (ALP) was significantly lower in HPP-PDL cells versus controls. (*): statistically different by the Student t-test (α=0.05). (C and D) Alizarin red assay for in vitro mineralization. Control and HPP-PDL cells grown in 2% FBS, and compared to controls, HPP-PDL cells displayed a severely limited mineralization capacity with βGP as a phosphate source. The HPP mineralization deficiency was rescued when 1mM P_i was used as the phosphate source. Different capital letters indicate intergroup statistical differences (control versus HPP) for α = 0.05, using the Kruskal-Wallis test followed by Student-Newman-Keuls method.

Figure 4. Addition of phosphate corrects pyrophosphate regulatory genes in HPP-PDL cells

Gene expression for PP_i regulating factors (Alpl, Ankh, Enpp1) and genes associated with regulation of mineralization (Bsp, Opn, Dmp1) in control versus HPP-PDL cells, with or without 1mM P_i treatment. While addition of P_i rescued expression of PP_i associated genes in HPP cells, other mineral markers were not corrected. Different capital letters indicate intergroup statistical differences (control vs. HPP, +/− Pi treatment)within the same time point (day 1, 5, 15, or 20)for α = 0.05, using the Kruskal-Wallis test followed by Student-Newman-Keuls method.