Periodontal Defects in the A116T Knock-in Murine Model of Odontohypophosphatasia

Abstract

Mutations in ALPL result in hypophosphatasia (HPP), a disease causing defective skeletal mineralization. ALPL encodes tissue nonspecific alkaline phosphatase (ALP), an enzyme that promotes mineralization by reducing inorganic pyrophosphate, a mineralization inhibitor. In addition to skeletal defects, HPP causes dental defects, and a mild clinical form of HPP, odontohypophosphatasia, features only a dental phenotype. The Alpl knockout (Alpl(-/-)) mouse phenocopies severe infantile HPP, including profound skeletal and dental defects. However, the severity of disease in Alpl(-/-) mice prevents analysis at advanced ages, including studies to target rescue of dental tissues. We aimed to generate a knock-in mouse model of odontohypophosphatasia with a primarily dental phenotype, based on a mutation (c.346G>A) identified in a human kindred with autosomal dominant odontohypophosphatasia. Biochemical, skeletal, and dental analyses were performed on the resulting Alpl(+/A116T) mice to validate this model. Alpl(+/A116T) mice featured 50% reduction in plasma ALP activity compared with wild-type controls. No differences in litter size, survival, or body weight were observed in Alpl(+/A116T) versus wild-type mice. The postcranial skeleton of Alpl(+/A116T) mice was normal by radiography, with no differences in femur length, cortical/trabecular structure or mineral density, or mechanical properties. Parietal bone trabecular compartment was mildly altered. Alpl(+/A116T) mice featured alterations in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant differences in several bone properties measured by micro-computed tomography. Nonsignificant changes in acellular cementum did not appear to affect periodontal attachment or function, although circulating ALP activity was correlated significantly with incisor cementum thickness. The Alpl(+/A116T) mouse is the first model of odontohypophosphatasia, providing insights on dentoalveolar development and function under reduced ALP, bringing attention to direct effects of HPP on alveolar bone, and offering a new model for testing potential dental-targeted therapies in future studies.
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DOI: 10.1177/0022034515573273
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Research Reports: Biological
Introduction
Loss-of-function mutations in ALPL result in hypophosphata-
sia (HPP), an inborn error of metabolism that features defec-
tive mineralization of the skeleton and dentition (Whyte 2012).
ALPL encodes tissue nonspecific alkaline phosphatase (TNAP;
alternately, TNSALP), an enzyme that reduces local concentra-
tions of the mineralization inhibitor, inorganic pyrophosphate
(Millán 2013). Skeletal complications of HPP include rickets
and osteomalacia, with clinical severity ranging widely from
profound skeletal hypomineralization that is lethal at birth to
dental defects alone. Dental manifestations include cementum
deficiency, tooth loss, thin dentin, widened pulp chambers,
malformed roots, and enamel alterations (Foster, Nociti, et al.
2014; Foster, Ramnitz, et al. 2014). Dental hard tissues seem
exceptionally sensitive to HPP, as the clinical form odontohy-
pophosphatasia affects only the dentition (Reibel et al. 2009).
The Alpl knockout (Alpl-/-) mouse recapitulates the meta-
bolic and skeletal phenotype of severe infantile HPP (Narisawa
et al. 1997; Fedde et al. 1999). We demonstrated in Alpl-/- mice
573273JDRXXX10.1177/0022034515573273Journal of Dental ResearchA116T Knock-in Mouse Model
research-article2015
1National Institute of Arthritis and Musculoskeletal and Skin Diseases,
National Institutes of Health, Bethesda, MD, USA
2Sanford Children’s Health Research Center, Sanford-Burnham Medical
Research Institute, La Jolla, CA, USA
3Department of Orthodontics and Pediatric Dentistry, School of
Dentistry, University of Michigan, Ann Arbor, MI, USA
4Department of Bioengineering, University of California, San Diego, La
Jolla, CA, USA
5Center for Metabolic Bone Disease and Molecular Research, Shriners
Hospital for Children, St. Louis, MO, USA
6Division of Bone and Mineral Diseases, Washington University School
of Medicine, St. Louis, MO, USA
*Authors contributing equally to this article.
A supplemental appendix to this article is published electronically only at
http://jdr.sagepub.com/supplemental.
Corresponding Author:
J.L. Millán, Sanford Children’s Health Research Center, Sanford-Burnham
Medical Research Institute, La Jolla, CA, USA.
Email: millan@sanfordburnham.org
Periodontal Defects in the A116T
Knock-in Murine Model of
Odontohypophosphatasia
B.L. Foster1*, C.R. Sheen2*, N.E. Hatch3, J. Liu3, E. Cory4, S. Narisawa2,
T. Kiffer-Moreira2, R.L. Sah4, M.P. Whyte5,6, M.J. Somerman1,
and J.L. Millán2
Abstract
Mutations in ALPL result in hypophosphatasia (HPP), a disease causing defective skeletal mineralization. ALPL encodes tissue nonspecific
alkaline phosphatase (ALP), an enzyme that promotes mineralization by reducing inorganic pyrophosphate, a mineralization inhibitor. In
addition to skeletal defects, HPP causes dental defects, and a mild clinical form of HPP, odontohypophosphatasia, features only a dental
phenotype. The Alpl knockout (Alpl-/-) mouse phenocopies severe infantile HPP, including profound skeletal and dental defects. However,
the severity of disease in Alpl-/- mice prevents analysis at advanced ages, including studies to target rescue of dental tissues. We aimed
to generate a knock-in mouse model of odontohypophosphatasia with a primarily dental phenotype, based on a mutation (c.346G>A)
identified in a human kindred with autosomal dominant odontohypophosphatasia. Biochemical, skeletal, and dental analyses were
performed on the resulting Alpl+/A116T mice to validate this model. Alpl+/A116T mice featured 50% reduction in plasma ALP activity compared
with wild-type controls. No differences in litter size, survival, or body weight were observed in Alpl+/A116T versus wild-type mice. The
postcranial skeleton of Alpl+/A116T mice was normal by radiography, with no differences in femur length, cortical/trabecular structure or
mineral density, or mechanical properties. Parietal bone trabecular compartment was mildly altered. Alpl+/A116T mice featured alterations
in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant
differences in several bone properties measured by micro–computed tomography. Nonsignificant changes in acellular cementum did not
appear to affect periodontal attachment or function, although circulating ALP activity was correlated significantly with incisor cementum
thickness. The Alpl+/A116T mouse is the first model of odontohypophosphatasia, providing insights on dentoalveolar development and
function under reduced ALP, bringing attention to direct effects of HPP on alveolar bone, and offering a new model for testing potential
dental-targeted therapies in future studies.
Keywords: alkaline phosphatase, hypophosphatasia, bone, cementum, dentin, periodontium

2 Journal of Dental Research
defects in cementum, dentin, alveolar bone, and enamel
(McKee et al. 2011; Foster et al. 2012; Yadav et al. 2012;
Foster, Nagatomo, et al. 2013; Zweifler et al. 2014). However,
these mice die by 2 to 3 wk of age, preventing analysis of
milder forms of HPP at advanced ages, including studies to
rescue dental tissues.
We aimed to create a knock-in mouse model of odontohypo-
phosphatasia. The Alpl c.346G>A mutation, predicting an
A116T substitution, was selected for knock-in based on homol-
ogy with the predominantly dental phenotype of a large well-
characterized HPP kindred (Hu et al. 2000) and
dominant-negative effect in vitro of the causal mutation (Lia-
Baldini et al. 2001; Fauvert et al. 2009; Ishida et al. 2011). We
hypothesized that a mouse heterozygous for the A116T muta-
tion (Alpl+/A116T) would phenocopy odontohypophosphatasia
and serve as a model for further studies of HPP dental disease.
Materials and Methods
Knock-in Vector Design and Synthesis
Mouse experiments were approved by the Institutional Animal
Care and Use Committee of the Sanford Burnham Medical
Research Institute (La Jolla, CA, USA). Methods for vector
design and in vitro expression analysis are detailed in the
Appendix. The targeting construct to introduce the c.346G>A
base change into exon 5 of Alpl is shown in Figure 1A.
Sequencing confirmed germline transmission (Fig. 1B). The
AlplA116T mouse line was maintained by breeding wild-type
(WT) mice with heterozygote mice (Alpl+/A116T). Alpl+/A116T and
WT mice were analyzed at 27 d postnatal (dpn) and 4 and 14 mo.
Plasma Chemistry Analysis
Blood was collected by cardiac puncture, transferred into
lithium-heparinized tubes (Becton, Dickinson & Co., Franklin
Lakes, NJ, USA), and plasma was separated by centrifugation
at 3,000 × g for 10 min. Alkaline phosphatase (ALP) activity,
phosphorus, and calcium were measured using a VetScan
Comprehensive Diagnostic Profile rotor (Abaxis, Union City,
CA, USA).
Radiography
Radiographs of skeletons were obtained with an MX-20
Specimen Radiographic System (Faxitron X-ray Corp.,
Chicago, IL, USA) and inspected by 2 blinded independent
analysts familiar with HPP skeletal disease. Femoral length
was measured using MicroDicom software (Sofia, Bulgaria).
Hemimandibles were scanned in a cabinet X-ray (Faxitron
X-ray Corp.) at 30 kV for 40 s.
Micro–computed Tomography
Methods for micro–computed tomography (micro-CT) analy-
sis are detailed in the Appendix. Femora were scanned on a
Skyscan 1076 micro-CT scanner (Kontich, Belgium), and
regions of interest were determined using established guide-
lines (Bouxsein et al. 2010). Skulls were scanned on an eXplore
Locus SP micro-CT scanner (GE Healthcare, London, ON,
Canada), and regions of interest for the parietal and frontal
bones were determined as described previously (Liu et al.
2013) and measured using established algorithms (Meganck
et al. 2009; Umoh et al. 2009). For dentoalveolar analysis, dis-
sected mandibles were scanned on a Scanco Medical µCT 35
(Scanco Medical AG, Brüttisellen, Switzerland). DICM files
were reoriented using ImageJ software (1.48r), with coronal,
sagittal, and transverse planes of section chosen for
comparison.
Parameters analyzed by microCT included total cross-
sectional tissue area (Tt.Ar), cortical bone area (Ct.Ar),
cortical area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th),
tissue mineral density (TMD), tissue volume (TV), bone sur-
face (BS), bone volume (BV), trabecular thickness (Tb.Th),
trabecular spacing (Tb.Sp), trabecular number (Tb.N), struc-
ture model index (SMI), and bone mineral density (BMD),
bone mineral content (BMC), and tissue mineral content
(TMC) (Bouxsein et al. 2010).
Three-point Bone Bending
Three-point bone bending was performed to determine the
mechanical properties of the femur, using an Instron 3342
material testing machine (Instron, Norwood, MA, USA) fitted
with a 100-N load cell. The span was fixed at 10 mm, and the
cross-head was lowered at 1 mm/min. Load (N) and extension
(mm) were recorded every 0.2 s until fracture. Maximum stiff-
ness, work to fracture, and failure and fracture points were cal-
culated from load-extension curves as described previously
(Aspden 2003).
Histology
Mandibles used for histology were decalcified in AFS (10%
v/v glacial acetic acid, 4% v/v neutral buffered formalin, and
0.85% w/v sodium chloride in water) by stirring at 4 °C for 3
to 4 wk, then processed in paraffin to make serial 6-µm sec-
tions. To evaluate periodontal ligament collagen fiber organi-
zation, sections were stained by the picrosirius red method
with 0.2% phosphomolybdic acid hydrate, 0.4% Direct Red
80, and 1.3% 2,4,6-trinitrophenol (Polysciences, Inc.,
Warrington, PA, USA), as described previously (Foster 2012).
Staining for tartrate-resistant acid phosphatase to identify
osteoclasts was performed following the manufacturer’s
instructions (Wako Chemicals, Japan). Briefly, deparaffinized
sections were incubated at 37 °C for 60 min with staining solu-
tion containing sodium tartrate, followed by counterstaining
with nuclear stain, air-drying, and mounting.
Immunohistochemistry was performed on histologic sec-
tions using an avidin-biotinylated peroxidase-based kit
(Vectastain Elite, Vector Labs, Burlingame, CA) with a
3-amino-9-ethylcarbazole substrate (Vector Labs) to produce a
red product. Primary antibodies included rat monoclonal anti-
human ALPL (R&D Systems, Minneapolis, MN, USA;

A116T Knock-in Murine Model 3
Zweifler et al. 2014), rabbit polyclonal
anti-mouse bone sialoprotein (Dr. Renny
Franceschi, University of Michigan, Ann
Arbor, MI, USA; Foster, Soenjaya, et al.
2013), rabbit polyclonal LF-175 anti-
mouse osteopontin (Dr. Larry Fisher,
National Institute of Dental and
Craniofacial Research, Bethesda, MD,
USA; Foster 2012), goat polyclonal anti-
mouse receptor activator of nuclear factor
kappa-B ligand (RANKL; R&D Systems),
and rabbit polyclonal anti-human periostin
(POSTN; Abcam Inc., Cambridge, MA,
USA).
Samples used for undecalcified sec-
tioning were fixed in paraformaldehyde,
embedded in methylmethacrylate, and von
Kossa stained as described previously
(Foster, Soenjaya, et al. 2013).
Histomorphometry
Acellular cementum width was measured
on lingual and buccal surfaces of the first
mandibular molar mesial root 100 µm
from the cementoenamel junction and on
lingual surfaces of incisors. Mean values
for WT (n = 5 to 6) and Alpl+/A116T (n = 7 to
9) cementum thickness were compared by
the independent-samples t test. Serum
ALP levels were correlated to incisor and
molar acellular cementum thickness using
Pearson’s r coefficient. Cellular cementum
area was measured using ImageJ software.
Alveolar bone height was quantitated as
distance from cementoenamel junction to
the alveolar bone crest on the lingual and
buccal aspects. Statistical analyses were
performed using GraphPad Prism 6.01 (La
Jolla, CA, USA).
Results
Alpl A116T Mutation in Mice
Reduces Their Plasma ALP Activity
No significant differences in litter size,
survival to weaning, or survival to 120 dpn
were observed between WT and Alpl+/A116T
male mice (data not shown). Body weight
did not differ significantly between geno-
types (P > 0.05), averaging 31.6 ± 1.4 g for
WT and 30.4 ± 0.6 g for Alpl+/A116T male
mice at 120 dpn.
Plasma biochemistry analysis indicated
no differences between genotypes in cal-
cium and phosphorus concentrations
Figure 1. Postcranial bone phenotype is not altered by the Alpl A116T mutation in mice. (A)
Schematic representation of the targeting construct used to introduce the c.346G>A (A116T)
mutation into the Alpl locus. The construct consists of short and long arms (yellow) separated
by a neomycin gene under the control of the mouse phosphoglycerol kinase promoter
(red), flanked by loxP recognition sites (green lines), inserted into a BamHI restriction site
(blue lines) in intron 5 of Alpl. The nucleotide positions (nt) of the ends of the construct
on chromosome 4 are given. (B) Electropherogram of DNA extracted from knock-in mice
showing successful knock-in of the c.346G>A (highlighted) mutation into the Alpl locus. (C)
Plasma collected from wild-type (WT; n = 7) and Alpl+/A116T (n = 9) mice at 120 d postnatal
showed a 50% decrease in mean alkaline phosphatase (ALP) activity in Alpl+/A116T mice
(***P < 0.001 by 2-tailed Student’s t test). Values are reported on the scatter plot as individual
measurements (mean ± 95% confidence interval). (D–I) Radiography of WT and Alpl+/A116T
heterozygous male mice at 120 d postnatal showing whole animals (D, G), left forelimbs
(E, H), and left hind limbs (F, I). No overt skeletal pathology was detected in Alpl+/A116T mice at
this age. Scale bar indicates 10 mm for all panels (C–H).

4 Journal of Dental Research
(Appendix Table 2). However, ALP activity was significantly
reduced in Alpl+/A116T versus WT mice (Fig. 1C, Appendix
Table 2). Mean ALP was reduced by 50% in Alpl+/A116T mice
(25.6 ± 5.4 vs. 50 ± 10.2 U/L in WT), with ALP of individual
heterozygotes ranging from 34% to 68% of the WT mean. This
range in plasma ALP in Alpl+/A116T mice reflects a similarly
broad range reported in the human kindred (Hu et al. 2000).
Notably, ALP in Alpl+/A116T mice was higher than the activity
observed in vitro in cotransfected cells (with WT and mutant
alleles), which produced only 32.5% of WT activity (Appendix
Table 3).
Postcranial Skeletal Phenotype Is
Not Altered in Alpl+/A116T Mice
By radiography, no differences were observed between post-
cranial skeletons (i.e., the skeleton lying posterior to the skull)
of WT and Alpl+/A116T mice at 120 dpn, including no evidence
of malformations, fractures, rickets, calcific periarthritis,
chondrocalcinosis, or pseudogout
(Fig. 1D–I). Femur length did not
differ (P > 0.05) between groups
(17.1 ± 0.1 for both WT and Alpl+/A116T).
Similarly, radiographic analysis at
age 14 mo did not reveal any skel-
etal abnormalities (data not
shown). Micro-CT analysis of
femora indicated no differences in
cortical or trabecular bone struc-
ture or density (Appendix Table 4).
Three-point bending analysis indi-
cated no differences in failure or
fracture loads, work to fracture, or
stiffness between WT and Alpl+/
A116T femurs (Appendix Table 5).
Alterations in Cranial Bones
in Alpl+/A116T Mice
Frontal bones of Alpl+/A116T mice
were not different from WT mice
(Appendix Table 6). Parietal bone
mineral content, bone mineral den-
sity, tissue mineral content, and
BV/TV were not different from
controls. However, BS/BV and
trabecular number were increased,
while Tb.Th was decreased (P <
0.05 for all) in Alpl+/A116T com-
pared to WT mice.
Alpl+/A116T Mice Feature
Alveolar Bone Defects
Alpl-/- mice manifest mineraliza-
tion defects in alveolar bone, dentin, cementum, and enamel
(Millán et al. 2008; McKee et al. 2011; Foster et al. 2012;
Yadav et al. 2012; Foster, Nagatomo, et al. 2013). The
Appendix Figure is included for reference to dental defects in
Alpl-/- mice.
No gross differences in Alpl+/A116T versus WT mandibles
were indicated by radiography at 120 dpn (Fig. 2A, B).
Micro-CT revealed radiolucency in alveolar bone surrounding
the first molars of Alpl+/A116T mice, while molars and incisors
appeared unaltered (Fig. 2C vs. E). Alpl+/A116T mouse alveolar
bone featured regions consistent with resorptive lesions in lin-
gual and interproximal bone surrounding first and second
molars (Fig. 2D vs. F, G vs. I, H vs. J).
Quantitative micro-CT analysis was performed on 2 regions
of alveolar bone associated with first molars (Table). In the fur-
cation region, Alpl+/A116T mice featured significantly reduced
alveolar bone tissue mineral density (P < 0.01) indicating bone
hypomineralization and decreased Tb.Th (P < 0.05) and
increased BS/BV (P < 0.05) indicating alterations in trabecular
Figure 2. Alpl+/A116T mice feature alveolar bone (AB) defects. (A, B) Radiography reveals no overt
differences in wild-type control versus Alpl+/A116T mandibles, molars (M1 to M3), or incisors (INC) at
120 d postnatal. (C, E) Micro–computed tomography (micro-CT) cut sections in the coronal plane at
the first molar mesial root indicate radiolucency (white arrows in E) at the alveolar bone crest in
Alpl+/A116T versus wild-type controls. (D, F) Micro-CT coronal plane sections at the first molar root
furcation region reveal lesions consistent with osteoclastic resorption (white arrows in F) in the
alveolar bone of Alpl+/A116T mice. (G, I) Micro-CT sagittal plane sections show alterations in alveolar
bone in the furcation region (white asterisk in I) and resorptive type lesions in interproximal bone (IB)
between first and second molars of Alpl+/A116T mice (white arrow in I). (H, J) Micro-CT transverse plane
sections located 500 µm apical to the cementoenamel junction show extensive loss of interproximal
bone between first and second molar roots (white arrow in J).

A116T Knock-in Murine Model 5
bone (similar to patterns noted for parietal bone in Appendix
Table 6) compared with WT. In lingual alveolar bone, Alpl+/A116T
mice featured significantly reduced TV (P < 0.05), BV
(P < 0.01), and BV/TV (P < 0.05), reflecting reduced bone in
this region. Micro-CT analysis of dental tissues revealed reduced
BV/TV for Alpl+/A116T mouse molar enamel and root dentin but
no other differences (Appendix Table 7).
Delays in Alpl+/A116T mouse alveolar bone mineralization
were indicated by accumulation of osteoid (approximately 10
to 30 µm thick) on alveolar bone crests (Fig. 3A–D), consistent
with osteomalacia in the radiolucent regions observed by
micro-CT. Previously, we reported increased osteopontin
expression as a causative factor in mineralization defects in
Alpl-/- mice (Harmey et al. 2004; Harmey et al. 2006; McKee
et al. 2011; Foster, Nagatomo, et al. 2013). In Alpl+/A116T mice,
however, osteopontin deposition appeared unaltered in teeth
and surrounding bone (Fig. 3E–H). TNAP distribution by
immunohistochemistry also appeared normal in the periodon-
tium of Alpl+/A116T mice versus controls (Fig. 3I vs. K). Because
alterations in mineralization of tooth or bone, including HPP,
can lead to defective periodontal attachment, picrosirius red
staining was performed to evaluate collagen fiber organization.
PDL fibers were observed to be well organized, with no inser-
tion defects identified at the tooth or bone surfaces of
Alpl+/A116T mice (Fig. 3J vs. L).
Areas of apparent bone resorption were also determined on
histologic sections. Alpl+/A116T mice at 120 dpn featured local-
ized alveolar bone resorption associated with numerous
tartrate-resistant acid phosphatase–positive osteoclast-like
cells (Fig. 3M, O). RANKL was identified by immunohisto-
chemistry in some cells bordering areas of resorption, but the
levels were not remarkable in Alpl+/A116T versus WT tissues
(data not shown). Areas of extensive resorption featured a mix
of connective tissue with fibroblasts, blood vessels, and mar-
row space, indicated by periostin immunostaining (Fig. 3N, P).
Alveolar bone resorption was not observed in Alpl+/A116T mice
by micro-CT or histology at 27 dpn (data not shown).
In light of an apparent increase in resorption in Alpl+/A116T
alveolar bone, histomorphometry was performed to search for
changes in alveolar bone height. No differences were identified
in alveolar bone height in Alpl+/A116T compared to WT (data not
shown; P = 0.74 and 0.78 for lingual and buccal aspects,
respectively).
Acellular Cementum Thickness Is Correlated
with Serum ALP
HPP causes absent or reduced acellular cementum formation (as
in the Alpl-/- mouse; Appendix Fig.; Foster, Nociti, et al. 2014).
We confirmed presence of both acellular and cellular cementum
by histology (Fig. 4A–H) and immunohistochemistry (e.g., Fig.
3F, H). However, acellular cementum appeared thin in Alpl+/A116T
mouse molars and incisors, and histomorphometry suggested a
nonsignificant trend toward decreased thickness compared to
WT mice (P = 0.09 for incisor cementum; P = 0.07 and 0.14 for
molar lingual and buccal acellular cementum, respectively; Fig.
4I). Cellular cementum featured hypomineralized regions
(cementoid), confirmed by von Kossa staining at 27 dpn (Fig.
4G inset vs. 4E inset). Cellular cementum area was not altered in
Alpl+/A116T versus controls (P = 0.46 and 0.40 for buccal and lin-
gual sides, respectively; Fig. 4J). Plasma levels of circulating
residual ALP activity were correlated positively with incisor acel-
lular cementum thickness in WT and Alpl+/A116Tmice (P = 0.03),
with nonsignificant “trends” (P = 0.05 and 0.09) in molar acel-
lular cementum thickness (Fig. 4K, L).
Discussion
To define the long-term effects of relatively mild HPP on the
mineralized dentoalveolar tissues, we generated the first
knock-in mouse model of HPP. Our model featured a c.346G>A
point mutation in exon 5 of Alpl, resulting in an A116T amino
acid substitution. Alpl+/A116T mice featured a mean 50%
Table. Micro–computed Tomography Analysis of Alveolar Bone.
Furcation Lingual Aspect
Parameter WT Alpl+/A116T WT Alpl+/A116T
TV, mm30.31 ± 0.03 0.30 ± 0.02 0.13 ± 0.01 0.12 ± 0.01a
BV, mm30.20 ± 0.02 0.19 ± 0.01 0.11 ± 0.01 0.09 ± 0.02b
BV/TV, % 64.78 ± 5.56 62.57 ± 2.90 82.09 ± 3.50 73.65 ± 8.81a
TMD, g HA/cm31.07 ± 0.02 1.04 ± 0.01b0.98 ± 0.02 0.96 ± 0.02
Tb.N, 1/mm 6.68 ± 0.53 7.16 ± 0.92 N/A N/A
Tb.Th, mm 0.15 ± 0.02 0.13 ± 0.01aN/A N/A
Tb.Sp, mm 0.19 ± 0.02 0.17 ± 0.03 N/A N/A
BS/BV, mm2/mm319.55 ± 1.87 21.55 ± 1.07aN/A N/A
Alveolar bone associated with first mandibular molars was compared at 120 d postnatal in wild-type (WT; n = 7) and Alpl+/A116T mice (n = 9). Values are
reported as means ± SD.
BS, bone surface; BV, bone volume; Tb.N, trabecular number; Tb.Sp, trabecular spacing; Tb.Th, trabecular thickness; TMD, tissue mineral density; TV,
tissue volume.
aP < 0.05. Independent-samples t test.
bP < 0.01. Independent-samples t test.

6 Journal of Dental Research
Figure 3. Delayed mineralization and increased resorption in alveolar bone of Alpl+/A116T mice. (A–D) Histologic sections of mandibular dentoalveolar
tissues from 120 d postnatal mice feature overtly normal first molars (M1), while Alpl+/A116T mice feature accumulation of osteoid (area outlined
in dotted red line in D) at the alveolar bone (AB) crest. Yellow boxes in A and C represent regions shown in B and D, respectively. (E–H)
Immunohistochemistry (IHC) reveals no differences in osteopontin localization in tooth or bone of Alpl+/A116T mice versus wild-type controls. Yellow
boxes in E and G represent regions shown in F and H, respectively. No differences in Alpl+/A116T versus wild type are noted for (I, K) tissue nonspecific
alkaline phosphatase intensity or localization by IHC, or (J, L) collagen organization of the periodontal ligament (PDL), as shown by picrosirius
red staining under polarized light microscopy. (M, O) Regions of bone resorption in Alpl+/A116T mice are associated with numerous TRAP-positive
osteoclast-like (purple-red, multinucleated) cells on the bone surface. (N, P) IHC for periostin indicates that regions of extensive alveolar bone
resorption in Alpl+/A116T tissues feature a mix of connective tissue (white star) and blood vessels (black arrows). DE, dentin.

A116T Knock-in Murine Model 7
Figure 4. Acellular cementum thickness is correlated with serum alkaline phosphatase (ALP). (A–D) Histologic sections of mandibular first molar
teeth (M1) from 120 d postnatal Alpl+/A116T mice suggest thin acellular cementum (AC; arrows where notated) compared with controls. Yellow boxes in
A and C represent regions shown in B and D, respectively. (E, G) Compared with well-mineralized wild-type (WT) cellular cementum (CC), Alpl+/A116T
mice feature an accumulated cementoid layer (area outlined in dotted red line in G). Insets in E and G show von Kossa staining, where Alpl+/A116T mouse
molars feature a thick layer of unmineralized cementoid not observed in WT mice. (F, H) The acellular cementum of the Alpl+/A116T mandibular incisor
appears thin compared with WT. (I) Histomorphometry of acellular cementum width in first molars (bucal and lingual aspects) and incisors reveals no
statistically significant differences in Alpl+/A116T versus WT mice (P > 0.05; not significant [ns]). (J) Histomorphometry of cellular cementum area in first
molars (buccal and lingual aspects) reveals no differences in Alpl+/A116T versus WT mice. (K) Serum ALP is significantly positively correlated with incisor
acellular cementum width (P = 0.03), with nonsignificant positive relationships with (L) molar acellular cementum on lingual (P = 0.09) and buccal (P =
0.05) aspects. DE, dentin; PDL, periodontal ligament.

8 Journal of Dental Research
reduction in plasma ALP, with individual values ranging from
34% to 68% of WT. Postcranial skeletal elements were unaf-
fected in Alpl+/A116T mice, and parietal bones in the skull were
mildly affected in diploe (trabecular) measurements. The
A116T mutation caused hypomineralization of mouse alveolar
bone and cellular cementum, with a trend of reduced acellular
cementum. Acellular cementum of molars and incisors was
correlated with plasma ALP activity.
Dental Disease in HPP
The severity of human HPP is remarkably broad-ranging and
spans life-threatening forms (infantile and perinatal) to milder
forms (prenatal benign, childhood, adult, and odontohypo-
phosphatasia; Whyte 2012). Skeletal defects include rickets
during growth or osteomalacia in adult life, fractures, and bone
pain. Dental defects commonly manifest in HPP, affecting
cementum, dentin, enamel, and periodontal bone (Foster,
Nociti, et al. 2014; Foster, Ramnitz, et al. 2014). We hypothe-
sized that murine dental hard tissues are exceptionally sensi-
tive in HPP because odontohypophosphatasia affects only the
dentition, despite characteristic serum biochemical findings
(Reibel et al. 2009). At present, 280 ALPL mutations have been
identified for HPP (www.sesep.uvsq.fr), with often unclear
genotype-phenotype relationships, including that for odonto-
hypophosphatasia, where 21 ALPL mutations have been
recorded within 8 of the 12 exons. Mild HPP can result from
heterozygosity for missense mutations with a dominant-
negative effect (i.e., inhibiting the enzymatic activity of the
heterodimer), and these mutations sometimes localize to
domains of TNAP affecting dimerization or allosteric proper-
ties (Fauvert et al. 2009).
The Alpl-/- mouse, featuring loss of TNAP, has been a useful
model for studying the skeletal manifestations of the severe
infantile form of HPP (Narisawa et al. 1997; Fedde et al. 1999).
We have analyzed the dental pathologies in the Alpl-/- mouse,
demonstrating inhibition of acellular cementum formation,
delayed alveolar bone mineralization, disruption of odonto-
blast function and dentin mineralization, and enamel defects
(McKee et al. 2011; Foster et al. 2012; Yadav et al. 2012;
Foster, Nagatomo, et al. 2013; Zweifler et al. 2014). However,
the severity of disease and shortened life span of these mice
prevent long-term studies of the HPP skeleton and dentition,
including investigations to rescue dental tissues.
A116T Mouse Model for
Odontohypophosphatasia
To generate a mouse model of mildly decreased ALP and
odontohypophosphatasia, we selected the Alpl c.346G>A point
mutation, corresponding to an A116T substitution in a highly
conserved residue adjacent to the enzyme active site (Silvent
et al. 2014). Previous work by in vitro analysis reported a
dominant-negative effect of this mutation (Lia-Baldini et al.
2001; Fauvert et al. 2009; Ishida et al. 2011), and our in vitro
expression assays confirmed those findings. Plasma ALP of
Alpl+/A116T heterozygotes ranged from 34% to 68% of the WT,
with a mean reduction of 50%, consistent with the variation
observed in the original HPP kindred (Hu et al. 2000). Broad-
ranging ALP is likely related to instances of observed lack of
penetrance in human subjects with HPP, and the same applies
to mouse models. Still, the discovery of an observable dental
phenotype in Alpl+/A116T where ALP is mildly reduced has
prompted us to develop a full study on heterozygous Alpl+/-
mice, as it is possible that carriers of HPP mutations may not
be entirely asymptomatic.
Based on the presentation in this family, we predicted pri-
marily dental manifestations in Alpl+/A116T mice, including
defective formation of cementum, dentin, and enamel. Analysis
of these Alpl+/A116T mice revealed no detectable defects in the
skeleton except for alterations in cranial and alveolar bone and
teeth. These findings validate this Alpl mutation as primarily
affecting the murine craniofacial region. However, the
Alpl+/A116T mouse presented a milder-than-expected dental phe-
notype respective to the clinical presentation in the human kin-
dred. The Alpl+/A116T phenotype was primarily observed as
hypomineralization of alveolar bone and cellular cementum,
with minor changes in acellular cementum and no significant
dentin or enamel defects, or premature loss of teeth. The
importance of TNAP to cementogenesis was further supported
by a significant positive correlation of incisor acellular cemen-
tum to plasma ALP and nonsignificant trends for molars. We
previously identified mouse incisors as being more sensitive to
disturbance in Alpl/TNAP, possibly due to their rapid and con-
tinuous formation, and/or an altered Pi:PPi ratio (i.e., inorganic
phosphate to inorganic pyrophosphate; Foster, Nagatomo, et
al. 2013; Zweifler et al. 2014).
Two limitations of this model should be acknowledged—
namely, the mildly affected periodontal structure and function
and the lack of tooth loss. However, the model does provide a
viable mouse with dentoalveolar effects of HPP, and several
important insights were gained: (1) Teeth and periodontia
developed and functioned despite a moderate reduction in ALP;
(2) alterations in cementum and correlation of incisor cemen-
tum to residual plasma ALP activity supported cumulative data
indicating that this tissue is highly dependent on TNAP activity;
and (3) alveolar bone was identified as a target tissue in HPP,
where emphasis is more often placed on cementum and dentin
defects. Observed mineralization defects in alveolar bone in
Alpl+/A116T mice may be related to the continuous rapid turnover
rate of this tissue compared with other bones (Sodek and McKee
2000), prompting a higher requirement for local TNAP func-
tion. The cause for the apparent increase in resorption of alveo-
lar and interproximal bone in Alpl+/A116T mice is not clear,
although it may be related to altered tissue properties, constant
mechanical loads from chewing, and/or subtle changes in peri-
odontal attachment or function. The fact that resorptive lesions
were observed only with advanced age supports the hypothesis
that this is a functional response that gradually arises after teeth
enter occlusion.
Overall, the Alpl+/A116T mouse model provides valuable insights
into the effects of a relatively mild reduction of TNAP activity on

A116T Knock-in Murine Model 9
the dentoalveolar complex, highlighting changes in alveolar bone
and providing a new model for testing potential therapies.
Author Contributions
B.L. Foster, contributed to design, data acquisition, analysis, and
interpretation, drafted and critically revised the manuscript; C.R.
Sheen, contributed to conception, design, data acquisition, analysis,
and interpretation, drafted and critically revised the manuscript;
N.E. Hatch, E. Cory, R.L. Sah, contributed to data acquisition, anal-
ysis, and interpretation, critically revised the manuscript; J. Liu, T.
Kiffer-Moreira, contributed to data acquisition, analysis, and inter-
pretation, critically revised the manuscript; S. Narisawa, contributed
to conception, design, data acquisition, analysis, and interpretation,
critically revised the manuscript; M.P. Whyte, contributed to con-
ception, data acquisition, analysis, and interpretation, critically
revised the manuscript; M.J. Somerman, contributed to data inter-
pretation, critically revised the manuscript; J.L. Millán, contributed
to conception, design, data acquisition, analysis, and interpretation,
critically revised the manuscript. All authors gave final approval
and agree to be accountable for all aspects of the work.
Acknowledgments
This research was supported by a grant (DE 12889) to J.L.M. from
the National Institute for Dental and Craniofacial Research of the
National Institutes of Health (NIH; Bethesda, MD), a grant (AR
066110) to B.L.F. from the National Institute of Arthritis and
Musculoskeletal and Skin Diseases (NIAMS) / NIH, the Intramural
Research Program of NIAMS (M.J.S.), and support from Shriners
Hospitals for Children (M.P.W.). We thank Kenn Holmbeck
(National Institute for Dental and Craniofacial Research) and
Lyudmila Lukashova (Hospital for Special Surgery, New York,
NY, USA; NIH AR 046121) for assistance with micro–computed
tomography analysis, Nasrin Kalantari Pour (NIAMS) for assis-
tance with histology, and Helen Wimer (Smithsonian Institute,
Washington, DC) for assistance with plastic sectioning and stain-
ing. We thank Ling Wang of the Sanford-Burnham Medical
Research Institute Animal Facility for assistance with blastocyst
injection and Greg Martin and Sergey Kupriyanov of The Scripps
Research Institute Mouse Genetics Core for assistance with
embryonic stem cell electroporation. We also thank Carmen
Huesa for assistance with 3-point bending analysis. The authors
declare no potential conflicts of interest with respect to the author-
ship and/or publication of this article.
References
Aspden RM. 2003. Mechanical testing of bone ex vivo. Methods Mol Med.
80:369–379.
Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R.
2010. Guidelines for assessment of bone microstructure in rodents using
micro–computed tomography. J Bone Miner Res. 25:1468–1486.
Fauvert D, Brun-Heath I, Lia-Baldini AS, Bellazi L, Taillandier A, Serre JL,
de Mazancourt P, Mornet E. 2009. Mild forms of hypophosphatasia mostly
result from dominant negative effect of severe alleles or from compound
heterozygosity for severe and moderate alleles. BMC Med Genet. 10:51.
Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Weinstein RS,
Waymire K, Narisawa S, Millán JL, MacGregor GR, et al. 1999. Alkaline
phosphatase knock-out mice recapitulate the metabolic and skeletal defects
of infantile hypophosphatasia. J Bone Miner Res. 14:2015–2026.
Foster BL. 2012. Methods for studying tooth root cementum by light micros-
copy. Int J Oral Sci. 4:119–128.
Foster BL, Nagatomo KJ, Nociti FH Jr, Fong H, Dunn D, Tran AB, Wang W,
Narisawa S, Millán JL, Somerman MJ. 2012. Central role of pyrophosphate
in acellular cementum formation. PLoS One. 7(6):e38393.
Foster BL, Nagatomo KJ, Tso HW, Tran AB, Nociti FH Jr, Narisawa S, Yadav
MC, McKee MD, Millán JI, Somerman MJ. 2013. Tooth root dentin min-
eralization defects in a mouse model of hypophosphatasia. J Bone Miner
Res. 28:271–282.
Foster BL, Nociti FH Jr, Somerman MJ. 2014. The rachitic tooth. Endocr Rev.
35:1–34.
Foster BL, Ramnitz MS, Gafni RI, Burke AB, Boyce AM, Lee JS, Wright JT,
Akintoye SO, Somerman MJ, Collins MT. 2014. Rare Bone Diseases and
Their Dental, Oral, and Craniofacial Manifestations. J Dent Res. 93(7):7S-
19S.
Foster BL, Soenjaya Y, Nociti FH Jr, Holm E, Zerfas PM, Wimer HF,
Holdsworth DW, Aubin JE, Hunter GK, Goldberg HA, et al. 2013.
Deficiency in acellular cementum and periodontal attachment in bsp null
mice. J Dent Res. 92:166–172.
Harmey D, Hessle L, Narisawa S, Johnson K, Terkeltaub R, Millán J. 2004.
Concerted regulation of inorganic pyrophosphate and osteopontin by akp2,
enpp1, and ank: an integrated model of the pathogenesis of mineralization
disorders. Am J Pathol. 164:1199–1209.
Harmey D, Johnson KA, Zelken J, Camacho NP, Hoylaerts MF, Noda M,
Terkeltaub R, Millán JL. 2006. Elevated skeletal osteopontin levels con-
tribute to the hypophosphatasia phenotype in Akp2(-/-) mice. J Bone Miner
Res. 21:1377–1386.
Hu JC, Plaetke R, Mornet E, Zhang C, Sun X, Thomas HF, Simmer JP. 2000.
Characterization of a family with dominant hypophosphatasia. Eur J Oral
Sci. 108:189–194.
Ishida Y, Komaru K, Oda K. 2011. Molecular characterization of tissue-non-
specific alkaline phosphatase with an Ala to Thr substitution at position 116
associated with dominantly inherited hypophosphatasia. Biochim Biophys
Acta. 1812:326–332.
Lia-Baldini AS, Muller F, Taillandier A, Gibrat JF, Mouchard M, Robin B,
Simon-Bouy B, Serre JL, Aylsworth AS, Bieth E, et al. 2001. A molecular
approach to dominance in hypophosphatasia. Hum Genet. 109:99–108.
Liu J, Nam HK, Wang E, Hatch NE. 2013. Further analysis of the Crouzon
mouse: effects of the FGFR2(C342Y) mutation are cranial bone-dependent.
Calcif Tissue Int. 92:451–466.
McKee MD, Nakano Y, Masica DL, Gray JJ, Lemire I, Heft R, Whyte MP,
Crine P, Millán JL. 2011. Enzyme replacement therapy prevents dental
defects in a model of hypophosphatasia. J Dent Res. 90:470–476.
Meganck JA, Kozloff KM, Thornton MM, Broski SM, Goldstein SA. 2009.
Beam hardening artifacts in micro–computed tomography scanning can be
reduced by X-ray beam filtration and the resulting images can be used to
accurately measure BMD. Bone. 45:1104–1116.
Millán JL. 2013. The role of phosphatases in the initiation of skeletal mineral-
ization. Calcif Tissue Int. 93:299–306.
Millán JL, Narisawa S, Lemire I, Loisel TP, Boileau G, Leonard P, Gramatikova
S, Terkeltaub R, Camacho NP, McKee MD, et al. 2008. Enzyme replace-
ment therapy for murine hypophosphatasia. J Bone Miner Res. 23:777–787.
Narisawa S, Fröhlander N, Millán J. 1997. Inactivation of two mouse alkaline
phosphatase genes and establishment of a model of infantile hypophospha-
tasia. Dev Dyn. 208:432–446.
Reibel A, Manière MC, Clauss F, Droz D, Alembik Y, Mornet E, Bloch-Zupan
A. 2009. Orodental phenotype and genotype findings in all subtypes of
hypophosphatasia. Orphanet J Rare Dis. 4:6.
Silvent J, Gasse B, Mornet E, Sire JY. 2014. Molecular evolution of the tis-
sue-nonspecific alkaline phosphatase allows prediction and validation
of missense mutations responsible for hypophosphatasia. J Biol Chem.
289:24168–24179.
Sodek J, McKee MD. 2000. Molecular and cellular biology of alveolar bone.
Periodontol. 24:99–126.
Umoh JU, Sampaio AV, Welch I, Pitelka V, Goldberg HA, Underhill TM,
Holdsworth DW. 2009. In vivo micro-CT analysis of bone remodeling in a
rat calvarial defect model. Phys Med Biol. 54:2147–2161.
Whyte MP. 2012. Hypophosphatasia. In: Thakker RV, Whyte MP, Eisman
J, Igarashi T, editors. Genetics of bone biology and skeletal disease. San
Diego (CA): Elsevier. p. 337–360.
Yadav MC, de Oliveira RC, Foster BL, Fong H, Cory E, Narisawa S, Sah RL,
Somerman M, Whyte MP, Millán JL. 2012. Enzyme replacement prevents
enamel defects in hypophosphatasia mice. J Bone Miner Res. 27(8):1722–
1734.
Zweifler LE, Patel MK, Nociti FH, Wimer HF, Millan JI, Somerman MJ, Foster
BL. 2014. Counter-regulatory phosphatases TNAP and NPP1 temporally
regulate tooth root cementogenesis [published online December 12, 2014].
Int J Oral Sci. doi:10.1038/ijos.2014.62