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

Mutations in the Alpl gene in hypophosphatasia (HPP) reduce the function of tissue nonspecific alkaline phosphatase (TNAP), resulting in increased pyrophosphate (PPi) and a severe deficiency in acellular cementum. We hypothesized that exogenous phosphate (Pi) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP-diagnosed subjects, by correcting Pi/PPi ratio and modulating expression of genes involved with Pi/PPi metabolism. Ex vivo and in vitro analyses were employed to identify mechanisms involved in HPP-associated PDL/tooth root deficiencies. Constitutive expression of PPi-associated genes was contrasted in PDL versus pulp tissues obtained from healthy subjects. Primary PDL cell cultures from HPP subjects (monozygotic twin males) were established to assay alkaline phosphatase activity (ALP), in vitro mineralization, and gene expression. Exogenous Pi was provided to correct Pi/PPi ratio. PDL tissues obtained from healthy individuals featured higher basal expression of key PPi 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 HPP subjects enrolled in this study. Compared to controls, HPP-PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1mM Pi. Dysregulated expression of PPi regulatory genes Alpl, Ankh, and Enpp1 was also corrected by adding Pi, though other matrix markers evaluated in our study remained down-regulated. These findings underscore the importance of controlling Pi/PPi ratio toward development of a functional periodontal apparatus, and support Pi/PPi imbalance as the etiology of HPP-associated cementum defects.

低磷酸酯酶症（HPP）中ALPL基因的突变会降低组织非特异性碱性磷酸酶（TNAP）的功能，导致焦磷酸盐（PPi）增加以及无细胞牙骨质严重缺乏。我们假设外源性磷酸盐（Pi）可通过纠正Pi/PPi比值并调节参与Pi/PPi代谢的基因表达，来挽救从确诊为HPP的受试者获取的牙周膜（PDL）细胞的体外矿化能力。 采用体外和体内分析来确定与HPP相关的PDL/牙根缺陷所涉及的机制。对比了从健康受试者获取的PDL和牙髓组织中PPi相关基因的组成性表达。建立了来自HPP受试者（同卵双胞胎男性）的原代PDL细胞培养物，以测定碱性磷酸酶活性（ALP）、体外矿化和基因表达。提供外源性Pi以纠正Pi/PPi比值。 从健康个体获取的PDL组织与配对的牙髓组织相比，关键的PPi调节因子基因ALPL、进行性关节强直蛋白（Ankh）和外核苷酸焦磷酸酶/磷酸二酯酶1（Enpp1）的基础表达更高。在本研究纳入的双胞胎HPP受试者中发现了一种新的ALPL突变。与对照组相比，HPP - PDL细胞的ALP和矿化能力显著降低，添加1mM Pi可挽救这种情况。添加Pi也纠正了PPi调节基因ALPL、Ankh和Enpp1的失调表达，尽管我们研究中评估的其他基质标志物仍然下调。 这些发现强调了控制Pi/PPi比值对功能性牙周装置发育的重要性，并支持Pi/PPi失衡是HPP相关牙骨质缺陷的病因。