Defective enamel and bone development in sodium-dependent citrate transporter (NaCT) Slc13a5 deficient mice

Armando R Irizarry, Guirui Yan, Qingqiang Zeng, Jonathan Lucchesi, Matthew J Hamang, Yanfei L Ma, James Xiaojun Rong, Armando R Irizarry, Guirui Yan, Qingqiang Zeng, Jonathan Lucchesi, Matthew J Hamang, Yanfei L Ma, James Xiaojun Rong

Abstract

There has been growing recognition of the essential roles of citrate in biomechanical properties of mineralized tissues, including teeth and bone. However, the sources of citrate in these tissues have not been well defined, and the contribution of citrate to the regulation of odontogenesis and osteogenesis has not been examined. Here, tooth and bone phenotypes were examined in sodium-dependent citrate transporter (NaCT) Slc13a5 deficient C57BL/6 mice at 13 and 32 weeks of age. Slc13a5 deficiency led to defective tooth development, characterized by absence of mature enamel, formation of aberrant enamel matrix, and dysplasia and hyperplasia of the enamel organ epithelium that progressed with age. These abnormalities were associated with fragile teeth with a possible predisposition to tooth abscesses. The lack of mature enamel was consistent with amelogenesis imperfecta. Furthermore, Slc13a5 deficiency led to decreased bone mineral density and impaired bone formation in 13-week-old mice but not in older mice. The findings revealed the potentially important role of citrate and Slc13a5 in the development and function of teeth and bone.

Conflict of interest statement

Competing Interests: All authors are employees of Eli Lilly & Company. This commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Slc13a5 -/- mice were smaller…
Fig 1. Slc13a5-/- mice were smaller in size.
(A) Body weight and (B) femur length were less in 13-week-old Slc13a5-/- mice (n = 9/group) when compared to WT (Slc13a5+/+) or heterozygous (Slc13a5+/-) littermates. (C) Body weight of 32-week-old mice. ** P < 0.01; *** P < 0.001 compared with Slc13a5+/+ group.
Fig 2. Abnormal dental phenotypes in Slc13a5…
Fig 2. Abnormal dental phenotypes in Slc13a5-/- mice.
Representative pictures from WT (Slc13a5+/+) (A) and Slc13a5-/- (B-D) mice. Slc13a5-/- mice had white, opaque areas of discoloration on the incisors (B and C, solid arrows), fractured incisor (C, arrowhead), and/or abscess (D, solid arrow).
Fig 3. Slc13a5 -/- mice lacked mature…
Fig 3. Slc13a5-/- mice lacked mature enamel.
Representative images of ground thick plastic sections of the first molar (A-C) and incisor (D-F, at the level of the first molar) from the left mandible from 32-week-old Slc13a5+/+ (A, D), Slc13a5+/- (B, E) and Slc13a5-/- (C, F) mice. The first molars (C) and incisors (F) of Slc13a5-/- mice lacked mature mineralized enamel when compared to Slc13a5+/+ and Slc13a5+/- mice. Unmineralized to poorly mineralized aberrant matrix was present where mature enamel should have been located (F, arrows). Stevenel’s blue. Black arrows in A-B, D-E = mature enamel, black arrows in F = unmineralized (blue green material) or poorly mineralized aberrant enamel matrix. d = dentin; p = pulp.
Fig 4. The enamel organ epithelium of…
Fig 4. The enamel organ epithelium of the incisors of Slc13a5-/- mice was disorganized and dysplastic in all stages of amelogenesis.
Representative images of decalcified, paraffin-embedded sections of lower incisors (A-I) from 32- week-old Slc13a5+/+ (A, D, G), Slc13a5+/- (B, E, H) and Slc13a5-/- (C, F, I) mice. Images A to C were from the secretory stages of amelogenesis of the incisors near the apex of the tooth, D to F from the maturation stages of amelogenesis at the level of the first molar, and G to I from the post-maturation stages of amelogenesis near the site of eruption. Hematoxylin and Eosin. em = enamel matrix; es = enamel space.
Fig 5. μCT evaluation showed Slc13a5 -/-…
Fig 5. μCT evaluation showed Slc13a5-/- mice lacked mature, densely-mineralized enamel in the molars and incisors.
Representative μCT images of volumetric renderings (A-C) or slices (D-F) of first molars and incisors from 32-week-old WT (A, D), Slc13a5+/- (B, E) and Slc13a5-/- (C, F) mice. Tissue colored in red in panels A and B corresponds to mature enamel in molars or incisors. Tissue colored white in panels A to C corresponds to bone or dentin, both of which have generally similar radio-density. Enamel (e), dentin (d), and pulp (p) are indicated in molars (top) and incisors (bottom) in panels D through E. Size bar in panels A-C = 100 micrometers; size bar in panels D-E = 1 millimeter.
Fig 6. Slc13a5 -/- mice had decreased…
Fig 6. Slc13a5-/- mice had decreased enamel volume as measured with μCT.
(A) 32-week-old Slc13a5-/- mice lacked mature enamel as measured by μCT when compared to Slc13a5+/- and Slc13a5+/+. (B) Pulp volume was modestly increased in Slc13a5-/- mice. Mice were 32-week-old, n = 4~5/group. ** P < 0.01; **** P < 0.0001 compared with Slc13a5+/+ group. Volume was measured in mm3.
Fig 7. Measurements of bone mineral density,…
Fig 7. Measurements of bone mineral density, strength and formation.
At 13 weeks old, (A) Slc13a5-/- mice had similar BMD in distal femur (DF) and 5th lumbar vertebrae (LV5) to Slc13a5+/+ mice, but had decreased BMD in mid femur (MF), and a trend with decreased bone strength (B) (P = 0.096), and decreased calcein incorporation (C) compared with Slc13a5+/+ (n = 9 each group). At 32 weeks old, Slc13a5-/- mice had similar BMD in DF, LV5 and MF (D) and calcein incorporation (E) to Slc13a5+/+ (n = 5 each group). ** P < 0.01; *** P < 0.001 vs age-matched Slc13a5+/+ mice.
Fig 8. Serum concentrations of calcium, inorganic…
Fig 8. Serum concentrations of calcium, inorganic phosphorus, OCN, and CTX-I.
Serum concentrations of calcium (A), inorganic phosphorus (B), intact OCN (C), and CTX-I (D) were similar among 13-week-old Slc13a5+/+, Slc13a5+/- and Slc13a5-/- mice (n = 9/group).

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