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


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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. © International & American Associations for Dental Research 2015.
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DOI: 10.1177/0022034515573273
Research Reports: Biological
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
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
Corresponding Author:
J.L. Millán, Sanford Children’s Health Research Center, Sanford-Burnham
Medical Research Institute, La Jolla, CA, USA.
Periodontal Defects in the A116T
Knock-in Murine Model of
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
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,
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
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).
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,
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).
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).
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,
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).
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 (, 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
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.
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.
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... Alpl −/− mice phenocopy severe infantile HPP with <1% of circulating ALP activity compared to control mice, reflecting the range of severe dental defects observed in individuals with HPP, and serving as an important model to study disease mechanisms (20,21,31,33). Compared to wild-type (WT) controls, Alpl −/− mice show severe enamel and dentin hypoplasia and reduced alveolar bone volume and mineral density (Figures 1A-E) (30,(35)(36)(37)(38)(39). ...
... In summary, while effects of HPP are highly variable, they can potentially impact all dentoalveolar mineralized tissues, in part because TNAP is highly expressed by all mineralizing cells during dental development and tissue mineralization. A mouse model that harbored a knock-in Alpl A116T substitution mutation associated with odonto-HPP (42) showed about 50% decreased circulating ALP levels and mild effects on acellular cementum and alveolar bone, possibly the two most sensitive tissues (33). ...
Full-text available
Mineralization of the skeleton occurs by several physicochemical and biochemical processes and mechanisms that facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Two key phosphatases, phosphatase, orphan 1 (PHOSPHO1) and tissue-non-specific alkaline phosphatase (TNAP), play complementary roles in the mineralization process. The actions of PHOSPHO1 on phosphocholine and phosphoethanolamine in matrix vesicles (MVs) produce inorganic phosphate (P i ) for the initiation of HA mineral formation within MVs. TNAP hydrolyzes adenosine triphosphate (ATP) and the mineralization inhibitor, inorganic pyrophosphate (PP i ), to generate P i that is incorporated into MVs. Genetic mutations in the ALPL gene-encoding TNAP lead to hypophosphatasia (HPP), characterized by low circulating TNAP levels (ALP), rickets in children and/or osteomalacia in adults, and a spectrum of dentoalveolar defects, the most prevalent being lack of acellular cementum leading to premature tooth loss. Given that the skeletal manifestations of genetic ablation of the Phospho1 gene in mice resemble many of the manifestations of HPP, we propose that Phospho1 gene mutations may underlie some cases of “pseudo-HPP” where ALP may be normal to subnormal, but ALPL mutation(s) have not been identified. The goal of this perspective article is to compare and contrast the loss-of-function effects of TNAP and PHOSPHO1 on the dentoalveolar complex to predict the likely dental phenotype in humans that may result from PHOSPHO1 mutations. Potential cases of pseudo-HPP associated with PHOSPHO1 mutations may resist diagnosis, and the dental manifestations could be a key criterion for consideration.
... 3,20 It has been suggested that pathogenic variants in ALPL can cause several alterations in alveolar bone properties including defective or delay in bone mineralization and resorptive lesions thereby increasing the risk for periodontal disease and early loss of teeth due to poor attachment of tooth roots to bone via periodontal ligament. 22 Additional dental findings such as tooth enamel hypoplasia or hypomineralization, dental caries, enlarged pulp chambers, and short roots have also been described in permanent dentition among adults with HPP. 20 Enamel hypoplasia may lead to occlusal problems (e.g., tooth wear and cracked tooth), increase susceptibility to dental caries, and early pulpal involvement. ...
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Hypophosphatasia (HPP) is a genetic condition with broad clinical manifestations caused by alkaline phosphatase (ALP) deficiency. Adults with HPP exhibit a wide spectrum of signs and symptoms. Dental manifestations including premature tooth loss are common. Much of the published literature reporting dental manifestations consists of case reports and series of symptomatic patients, likely biased towards more severe dental manifestations. The objective of this study was to systematically explore the dental manifestations among adults with HPP by conducting a comprehensive dental evaluation. To minimize bias, the study explored dental manifestations in an unselected cohort of adults with HPP. Participants were identified searching electronic health record (EHR) data from a rural health system to discover adults with persistent ALP deficiency. Heterozygotes with pathogenic (P), likely pathogenic (LP), or uncertain variants (VUS) in ALPL and at least one elevated ALP substrate were defined as adults with HPP and underwent genetic, dental, oral radiographic, and biomarker evaluation. Twenty‐seven participants completed the study. Premature tooth loss was present in 63% (17/27); 19% (5/27) were missing eight or more teeth. Statistically significant associations were found between premature permanent tooth loss and HPP biomarkers ALP (p = 0.049) and bone‐specific ALP (p = 0.006). Serum ALP (ρ = −0.43, p = 0.037) and bone‐specific ALP (ρ = −0.57, p = 0.004) were negatively correlated with number of teeth lost prematurely. As noted with tooth loss, periodontal breakdown was associated with bone‐specific ALP. An inverse association between periodontal breakdown and bone‐specific ALP was observed (p = 0.014). These findings suggest a role for ALP in maintenance of dentition.
... Methods for measuring trabecular microstructure parameters are as previously described [25]. Brief, femurs were cleaned of soft tissue and fixed in 4% paraformaldehyde. ...
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Background Rhizoma drynariae, a traditional Chinese herb, is commonly used in treatment of bone healing in osteoporotic fractures. However, whether the Rhizoma drynariae total flavonoids (RDTF) can promote the absorption of calcium and enhance the bone formation is unclear. The aim of the present study was to investigate the preventive effects of RDTF combined with calcium carbonate (CaCO3) on estrogen deficiency-induced bone loss. Methods Three-month-old Sprague–Dawley rats were ovariectomized (OVX) and then treated with CaCO3, RDTF, and their admixtures for ten weeks, respectively. The bone trabecular microstructure, bone histopathological examination, and serum biomarkers of bone formation and resorption were determined in the rat femur tissue. The contents of osteoprotegerin (OPG), receptor activator of the NF-κB (RANK), and its ligand (RANKL) in marrow were analyzed by ELISA, and the protein expressions of Wnt3a, β-catenin, and phosphorylated β-catenin (p-β-catenin) were analyzed by Western blot. Statistical analysis was conducted by using one-way analysis of variance (ANOVA) followed by LSD post hoc analysis or independent samples t test using the scientific statistic software SPSS version 20.0 Results RDTF combined with CaCO3 could promote osteosis and ameliorate bone loss to improve the repair of cracked bone trabeculae of OVX rats. Furthermore, RDTF combined with CaCO3 also could prevent OVX-induced decrease in collagen fibers in the femoral tissue of ovariectomized rats and promote the regeneration of new bone or cartilage tissue, while CaCO3 supplementation promoted the increase in bone mineral content. Nevertheless, there was no difference in the expression of Wnt3a, β-catenin and p-β-catenin between osteopenic rats and RDTF treated rats, but RDTF combined with CaCO3 could activate the Wnt3a/β-catenin pathway. Conclusions RDTF combined with CaCO3 could ameliorate estrogen deficiency-induced bone loss via the regulation of Wnt3a/β-catenin pathway.
... In 2015, a murine model for odontohypophosphatasia (odonto-HPP) was first generated by Foster et al. [55]. Odonto-HPP is a mild form of HPP characterized only by oral manifestations, including premature exfoliation of deciduous teeth. ...
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Alkaline phosphatase (ALP) is a ubiquitous membrane-bound glycoprotein capable of providing inorganic phosphate by catalyzing the hydrolysis of organic phosphate esters, or removing inorganic pyrophosphate that inhibits calcification. In humans, four forms of ALP cDNA have been cloned, among which tissue-nonspecific ALP (TNSALP) (TNSALP) is widely distributed in the liver, bone, and kidney, making it an important marker in clinical and basic research. Interestingly, TNSALP is highly expressed in juvenile cells, such as pluripotent stem cells (i.e., embryonic stem cells and induced pluripotent stem cells (iPSCs)) and somatic stem cells (i.e., neuronal stem cells and bone marrow mesenchymal stem cells). Hypophosphatasia is a genetic disorder causing defects in bone and tooth development as well as neurogenesis. Mutations in the gene coding for TNSALP are thought to be responsible for the abnormalities, suggesting the essential role of TNSALP in these events. Moreover, a reverse-genetics-based study using mice revealed that TNSALP is important in bone and tooth development as well as neurogenesis. However, little is known about the role of TNSALP in the maintenance and differentiation of juvenile cells. Recently, it was reported that cells enriched with TNSALP are more easily reprogrammed into iPSCs than those with less TNSALP. Furthermore, in bone marrow stem cells, ALP could function as a “signal regulator” deciding the fate of these cells. In this review, we summarize the properties of ALP and the background of ALP gene analysis and its manipulation, with a special focus on the potential role of TNSALP in the generation (and possibly maintenance) of juvenile cells.
... Individuals provided blood or saliva samples and were tested using next-generation sequencing (NGS) with a multi-gene panel consisting of 13 genes that were selected based on associations with hypophosphatemia as reported in the medical literature: ALPL, (12)(13)(14)(15)(16)(17)(18) CLCN5, (19)(20)(21)(22) CYP2R1, (23)(24)(25)(26) CYP27B1, (27)(28)(29)(30) DMP1, (31)(32)(33)(34)(35)(36)(37)(38) ENPP1, (39)(40)(41)(42) FAH, (43)(44)(45) FAM20C, (46)(47)(48)(49)(50)(51)(52) FGF23, (10,(53)(54)(55)(56)(57)(58)(59) FGFR1, (60)(61)(62)(63) PHEX, (64)(65)(66)(67)(68) SLC34A3, (69)(70)(71) and VDR. (72,73) Each gene in the NGS panel was targeted with oligonucleotide baits (Agilent Technologies, Santa Clara, CA, USA; Roche, Pleasanton, CA, USA; IDT, Coralville, IA, USA) that were designed to capture exons, the 10 bases flanking exonic sequences, and certain noncoding regions of interest (PHEX c.*231A>G). ...
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X‐linked hypophosphatemia (XLH), a dominant disorder caused by pathogenic variants in the PHEX gene, affects both sexes of all ages and results in elevated serum FGF23 and below‐normal serum phosphate. In XLH, rickets, osteomalacia, short stature and lower limb deformity may be present with muscle pain and/or weakness/fatigue, bone pain, joint pain/stiffness, hearing difficulty, enthesopathy, osteoarthritis, and dental abscesses. Invitae and Ultragenyx collaborated to provide a no‐charge sponsored testing program using a 13‐gene next‐generation sequencing panel to confirm clinical XLH or aid diagnosis of suspected XLH/other genetic hypophosphatemia. Individuals aged ≥6 months with clinical XLH or suspected genetic hypophosphatemia were eligible. Of 831 unrelated individuals tested between February 2019 and June 2020 in this cross‐sectional study, 519 (62.5%) individuals had a pathogenic or likely pathogenic variant in PHEX (PHEX‐positive). Among the 312 PHEX‐negative individuals, 38 received molecular diagnoses in other genes, including ALPL, CYP27B1, ENPP1 and FGF23; the remaining 274 did not have a molecular diagnosis. Among 319 patients with a provider‐reported clinical diagnosis of XLH, 88.7% (n=283) had a reportable PHEX variant; 81.5% (n=260) were PHEX‐positive. The most common variant among PHEX‐positive individuals was an allele with both the gain of exons 13‐15 and c.*231A>G (3’UTR variant) (n=66/519). Importantly, over 80% of copy number variants would have been missed by traditional microarray analysis. A positive molecular diagnosis in 41 probands (4.9%; 29 PHEX positive, 12 non‐PHEX positive) resulted in at least one family member receiving family testing. Additional clinical or family member information resulted in VUS reclassification to P/LP in 48 individuals, highlighting the importance of segregation and clinical data. In one of the largest XLH genetic studies to date, 65 novel PHEX variants were identified and a high XLH diagnostic yield demonstrated broad insight into the genetic basis of XLH. This article is protected by copyright. All rights reserved.
Background and objective: Although cementum plays an essential role in tooth attachment and adaptation to occlusal force, the regulatory mechanisms of cementogenesis remain largely unknown. We have previously reported that Axin2-expressing (Axin2+ ) mesenchymal cells in periodontal ligament (PDL) are the main cell source for cementum growth, and constitutive activation of Wnt/β-catenin signaling in Axin2+ cells results in hypercementosis. Therefore, the aim of the present study was to further evaluate the effects of β-catenin deletion in Axin2+ cells on cementogenesis. Materials and methods: We generated triple transgenic mice to conditionally delete β-catenin in Axin2-lineage cells by crossing Axin2CreERT2/+ ; R26RtdTomato/+ mice with β-cateninflox/flox mice. Multiple approaches, including X-ray analysis, micro-CT, histological stainings, and immunostaining assays, were used to analyze cementum phenotypes and molecular mechanisms. Results: Our data revealed that loss of β-catenin in Axin2+ cells led to a cementum hypoplasia phenotype characterized by a sharp reduction in the formation of both acellular and cellular cementum. Mechanistically, we found that conditional removal of β-catenin in Axin2+ cells severely impaired the secretion of cementum matrix proteins, for example, bone sialoprotein (BSP), dentin matrix protein 1 (DMP1) and osteopontin (OPN), and markedly inhibited the differentiation of Axin2+ mesenchymal cells into osterix+ cementoblasts. Conclusions: Our findings confirm the vital role of Axin2+ mesenchymal PDL cells in cementum growth and demonstrate that Wnt/β-catenin signaling shows a positive correlation with cementogenic differentiation of Axin2+ cells.
Hypophosphatasia (HPP) is the inherited error‐of‐metabolism caused by mutations in ALPL, reducing the function of tissue‐nonspecific alkaline phosphatase (TNAP/TNALP/TNSALP). HPP is characterized by defective skeletal and dental mineralization and is categorized into several clinical subtypes based on age of onset and severity of manifestations, though premature tooth loss from acellular cementum defects is common across most HPP subtypes. Genotype‐phenotype associations and mechanisms underlying musculoskeletal, dental, and other defects remain poorly characterized. Murine models that have provided significant insights into HPP pathophysiology also carry limitations including monophyodont dentition, lack of osteonal remodeling of cortical bone, and differing patterns of skeletal growth. To address this, we generated the first gene edited large animal model of HPP in sheep via CRISPR/Cas9‐mediated knock‐in of a missense mutation (c.1077C>G; p.I359M) associated with skeletal and dental manifestations in humans. We hypothesized that this HPP sheep model would recapitulate the human dentoalveolar manifestations of HPP. Compared to wild‐type (WT), compound heterozygous (cHet) sheep with one null allele and the other with the targeted mutant allele exhibited the most severe alveolar bone, acellular cementum, and dentin hypomineralization defects. Sheep homozygous for the mutant allele (Hom) showed alveolar bone and hypomineralization effects and trends in dentin and cementum, while sheep heterozygous (Het) for the mutation did not exhibit significant effects. Important insights gained include existence of early alveolar bone defects that may contribute to tooth loss in HPP, observation of severe mantle dentin hypomineralization in an HPP animal model, association of cementum hypoplasia with genotype, and correlation of dentoalveolar defects with ALP levels. The sheep model of HPP faithfully recapitulated dentoalveolar defects reported in individuals with HPP, providing a new translational model for studies into etiopathology and novel therapies of this disorder, as well as proof‐of‐principle that genetically engineered large sheep models can replicate human dentoalveolar disorders.
The periodontium supports and attaches teeth via mineralized and nonmineralized tissues. It consists of two, unique mineralized tissues, cementum and alveolar bone. In between these tissues, lies an unmineralized, fibrous periodontal ligament (PDL), which distributes occlusal forces, nourishes and invests teeth, and harbors progenitor cells for dentoalveolar repair. Many unanswered questions remain regarding periodontal biology. This review will focus on recent research providing insights into one enduring mystery: the precise regulation of the hard‐soft tissue borders in the periodontium which define the interfaces of the cementum–PDL–alveolar bone structure. We will focus on advances in understanding the molecular mechanisms that maintain the unmineralized PDL “between a rock and a hard place” by regulating the mineralization of cementum and alveolar bone.
Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), the latter of which has “mouse-like” arms but extremely long metatarsals of the feet. Intersecting gene-expression differences in metatarsals and forearms of the two species revealed that about 10% of orthologous genes are associated with the disproportionately rapid elongation of neonatal jerboa feet. These include genes and enriched pathways not previously associated with endochondral elongation as well as those that might diversify skeletal proportion in addition to their known requirements for bone growth throughout the skeleton. We also identified transcription regulators that might act as “nodes” for sweeping differences in genome expression between species. Among these, Shox2, which is necessary for proximal limb elongation, has gained expression in jerboa metatarsals where it has not been detected in other vertebrates. We show that Shox2 is sufficient to increase mouse distal limb length, and a nearby putative cis-regulatory region is preferentially accessible in jerboa metatarsals. In addition to mechanisms that might directly promote growth, we found evidence that jerboa foot elongation may occur in part by de-repressing latent growth potential. The genes and pathways that we identified here provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.
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Human-treated dentin matrix (hTDM) is a biomaterial scaffold, which can induce implant cells to differentiate into odontoblasts and then form neo-dentin. However, hTDM with long storage or prepared by high-speed handpiece would not to form neo-dentin. In this research, we developed two fresh hTDM with different grinding speeds, which were low-speed hTDM (LTDM) with maximum speed of 500 rpm and high-speed hTDM (HTDM) with a speed of 3,80,000 rpm. Here, we aim to understand whether there were induced regeneration capacity differences between LTDM and HTDM. Scanning electron microscope showed that DFCs grew well on both materials, but the morphology of DFCs and the extracellular matrix was different. Especially, the secreted extracellular matrixes on the inner surface of LTDM were regular morphology and ordered arrangement around the dentin tubules. The transcription-quantitative polymerase chain reaction (qRT-PCR), western blot and immunofluorescence assay showed that the dentin markers DSPP and DMP-1 were about 2× greater in DFCs induced by LTDM than by HTDM, and osteogenic marker BSP was about 2× greater in DFCs induced by HTDM than by LTDM. Histological examinations of the harvested grafts observed the formation of neo-tissue were different, and there were neo-dentin formed on the inner surface of LTDM and neo-cementum formed on the outer surface of HTDM. In summary, it found that the induction abilities of LTDM and HTDM are different, and the dentin matrix is directional. This study lays a necessary foundation for searching the key factors of dentin regeneration in future.
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Cementum is critical for anchoring the insertion of periodontal ligament fibers to the tooth root. Several aspects of cementogenesis remain unclear, including differences between acellular cementum and cellular cementum, and between cementum and bone. Biomineralization is regulated by the ratio of inorganic phosphate (Pi) to mineral inhibitor pyrophosphate (PPi), where local Pi and PPi concentrations are controlled by phosphatases including tissue-nonspecific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). The focus of this study was to define the roles of these phosphatases in cementogenesis. TNAP was associated with earliest cementoblasts near forming acellular and cellular cementum. With loss of TNAP in the Alpl null mouse, acellular cementum was inhibited, while cellular cementum production increased, albeit as hypomineralized cementoid. In contrast, NPP1 was detected in cementoblasts after acellular cementum formation, and at low levels around cellular cementum. Loss of NPP1 in the Enpp1 null mouse increased acellular cementum, with little effect on cellular cementum. Developmental patterns were recapitulated in a mouse model for acellular cementum regeneration, with early TNAP expression and later NPP1 expression. In vitro, cementoblasts expressed Alpl gene/protein early, whereas Enpp1 gene/protein expression was significantly induced only under mineralization conditions. These patterns were confirmed in human teeth, including widespread TNAP, and NPP1 restricted to cementoblasts lining acellular cementum. These studies suggest that early TNAP expression creates a low PPi environment promoting acellular cementum initiation, while later NPP1 expression increases PPi, restricting acellular cementum apposition. Alterations in PPi have little effect on cellular cementum formation, though matrix mineralization is affected.
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ALPL encodes the tissue non-specific alkaline phosphatase, TNSALP, which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions out of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, ...). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time-consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction.
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Hereditary diseases affecting the skeleton are heterogeneous in etiology and severity. Though many of these conditions are individually rare, the total number of people affected is great. These disorders often include dental-oral-craniofacial (DOC) manifestations, but the combination of the rarity and lack of in-depth reporting often limit our understanding and ability to diagnose and treat affected individuals. In this review, we focus on dental, oral, and craniofacial manifestations of rare bone diseases. Discussed are defects in 4 key physiologic processes in bone/tooth formation that serve as models for the understanding of other diseases in the skeleton and DOC complex: progenitor cell differentiation (fibrous dysplasia), extracellular matrix production (osteogenesis imperfecta), mineralization (familial tumoral calcinosis/hyperostosis hyperphosphatemia syndrome, hypophosphatemic rickets, and hypophosphatasia), and bone resorption (Gorham-Stout disease). For each condition, we highlight causative mutations (when known), etiopathology in the skeleton and DOC complex, and treatments. By understanding how these 4 foci are subverted to cause disease, we aim to improve the identification of genetic, molecular, and/or biologic causes, diagnoses, and treatment of these and other rare bone conditions that may share underlying mechanisms of disease.
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Crouzon syndrome is a debilitating congenital disorder involving abnormal craniofacial skeletal development caused by mutations in fibroblast growth factor receptor-2 (FGFR2). Phenotypic expression in humans exhibits an autosomal dominant pattern that commonly involves premature fusion of the coronal suture (craniosynostosis) and severe midface hypoplasia. To further investigate the biologic mechanisms by which the Crouzon syndrome-associated FGFR2(C342Y) mutation leads to abnormal craniofacial skeletal development, we created congenic BALB/c FGFR2(C342Y/+) mice. Here, we show that BALB/c FGFR2(C342Y/+) mice have a consistent craniofacial phenotype including partial fusion of the coronal and lambdoid sutures, intersphenoidal synchondrosis, and multiple facial bones, with minimal fusion of other craniofacial sutures. This phenotype is similar to the classic and less severe form of Crouzon syndrome that involves significant midface hypoplasia with limited craniosynostosis. Linear and morphometric analyses demonstrate that FGFR2(C342Y/+) mice on the BALB/c genetic background differ significantly in form and shape from their wild-type littermates and that in this genetic background the FGFR2(C342Y) mutation preferentially affects some craniofacial bones and sutures over others. Analysis of cranial bone cells indicates that the FGFR2(C342Y) mutation promotes aberrant osteoblast differentiation and increased apoptosis that is more severe in frontal than parietal bone cells. Additionally, FGFR2(C342Y/+) frontal, but not parietal, bones exhibit significantly diminished bone volume and density compared to wild-type mice. These results confirm that FGFR2-associated craniosynostosis occurs in association with diminished cranial bone tissue and may provide a potential biologic explanation for the clinical finding of phenotype consistency that exists between many Crouzon syndrome patients.
This book identifies and analyzes the genetic basis of bone disorders in humans and demonstrates the utility of mouse models in furthering the knowledge of mechanisms and evaluations of treatments. The book is aimed at all students of bone biology and genetics, and with this in mind, it includes general introductory chapters on genetics and bone biology and more specific disease-orientated chapters, which comprehensively summarize the clinical, genetic, molecular genetic, animal model, functional and molecular pathology, diagnostic, counselling and treatment aspects of each disorder. Saves academic, medical, and pharma researchers time in quickly accessing the very latest details on a broad range of genetic bone issues, as opposed to searching through thousands of journal articles. Provides a common language for bone biologists and geneticists to discuss the development of bone cells and genetics and their interactions in the development of disease Researchers in all areas bone biology and genetics will gain insight into how clinical observations and practices can feed back into the research cycle and will, therefore, be able to develop more targeted genomic and proteomic assays For those clinical researchers who are also MDs, correct diagnosis (and therefore correct treatment) of bone diseases depends on a strong understanding of the molecular basis for the disease.
Hypophosphatasia is the rare, heritable form of rickets or osteomalacia that features low serum alkaline phosphatase (ALP) activity (hypophosphatasemia). Hypophosphatasemia in HPP is explained by a generalized reduction of activity of the "tissue-non-specific" isoenzyme of ALP (TNSALP) caused by loss-of-function mutation(s) within the TNSALP gene. The tissue-specific ALP isoenzyme genes are intact. In HPP, extracellular accumulation of the TNSALP substrate inorganic pyrophosphate (PPi), an inhibitor of mineralization, explains the skeletal disease. Characterization of this inborn error-of-metabolism verified a role for ALP in bone formation. The discoveries of elevated endogenous levels of three phospho compounds in HPP clarified the metabolic basis for this inborn-error-of-metabolism and the physiological role of TNSALP. HPP occurs worldwide. It is especially prevalent for inbred Mennonite families in Manitoba, Canada, where approximately 1 in 2500 newborns manifests severe disease, and approximately 1 in 25 individuals is a carrier. The prognosis for the various forms of HPP typically reflects the severity of the skeletal disease that corresponds with the age at presentation.
Teeth are mineralized organs comprised of three unique hard tissues, enamel, dentin, and cementum, and supported by the surrounding alveolar bone. While odontogenesis differs from osteogenesis in several respects, tooth mineralization is susceptible to similar developmental failures as bone. Here we discuss conditions fitting under the umbrella of "rickets," which traditionally referred to skeletal disease associated with vitamin D deficiency, but has been more recently expanded to include newly identified factors involved in endocrine regulation of vitamin D, phosphate, and calcium, including phosphate regulating endopeptidase homolog, X-linked (PHEX), fibroblast growth factor 23 (FGF23), and dentin matrix protein 1 (DMP1). Systemic mineral metabolism intersects with local regulation of mineralization, and factors including tissue nonspecific alkaline phosphatase (TNAP) are necessary for proper mineralization, where rickets can result from loss of activity of TNAP. Individuals suffering from rickets often bear the additional burden of a defective dentition, and transgenic mouse models have aided in understanding the nature and mechanisms involved in tooth defects, which may or may not parallel rachitic bone defects. This report reviews dental effects of the range of rachitic disorders, including discussion of etiologies of hereditary forms of rickets, a survey of resulting bone and tooth mineralization disorders, and a discussion of mechanisms, known and hypothesized, involved in the observed dental pathologies. Descriptions of human pathology are augmented by analysis of transgenic mouse models, and new interpretations are brought to bear on questions of how teeth are affected under conditions of rickets. In short, the "rachitic tooth" will be revealed.
Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene cause hypophosphatasia, a heritable form of rickets and osteomalacia, caused by an arrest in the propagation of hydroxyapatite (HA) crystals onto the collagenous extracellular matrix due to accumulation of extracellular inorganic pyrophosphate (PPi), a physiological TNAP substrate and a potent calcification inhibitor. However, TNAP knockout (Alpl –/– ) mice are born with a mineralized skeleton and have HA crystals in their chondrocyte- and osteoblast-derived matrix vesicles (MVs). We have shown that PHOSPHO1, a soluble phosphatase with specificity for two molecules present in MVs, phosphoethanolamine and phosphocholine, is responsible for initiating HA crystal formation inside MVs and that PHOSPHO1 and TNAP have nonredundant functional roles during endochondral ossification. Double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality, despite normal systemic phosphate and calcium levels. This strongly suggests that the Pi needed for initiation of MV-mediated mineralization is produced locally in the perivesicular space. As both TNAP and nucleoside pyrophosphohydrolase-1 (NPP1) behave as potent ATPases and pyrophosphatases in the MV compartment, our current model of the mechanisms of skeletal mineralization implicate intravesicular PHOSPHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.
Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null ((-/-)) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp(-/-) mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp(-/-) mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp(-/-) mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function.