Loss of ANK Causes Cementum Hyperplasia
As a first step toward understanding the developmental consequences of loss of ANK, the phenotypes of Ank KO mouse mandible and teeth were characterized during development. Days were selected to capture developmental time points of interest, i.e. before root formation (24 dpc), initiation of root/cementum formation (27 dpc), further developed root and tooth eruption (33 dpc), at completion of the root (45 dpc), and following more than 1 month in occlusion (79 dpc).
At the macroscopic level, no obvious defects were observed in mandible or tooth development for Ank KO mice, within limits of detection. Figure includes representative radiographs from WT and Ank KO hemimandibles at ages 27, 33, and 45 dpc. Molar tooth development and eruption in Ank KO appeared similar to WT for all ages.
Fig. 1 Mandible development and mineralization are unaffected in Ank KO mice. Representative radiographs of hemimandibles from 27, 33, and 45 dpc WT and Ank KO mice. Within detectable limits of procedures used here, no differences were noted in Ank KO mandible (more ...)
Detailed histological analyses of first mandibular molars and incisors were performed over the developmental course of 24–79 dpc. At the early age of 24 dpc, Ank
KO molar crowns and associated tissues exhibited no apparent differences from WT (online suppl. fig. 1A, B
With progression to root formation by 27 dpc, newly formed acellular cementum was present as a thin, basophilic layer covering the root dentin of WT molars (fig. ). In Ank KO mouse molars, regions of abnormally thick acellular cementum were visible under high magnification, beginning to embed adjacent cementoblasts (fig. , inset). Surrounding root tissues, dental follicle, and bone appeared similar in Ank KO mice and controls.
Fig. 2 Loss of ANK causes cementum hyperplasia. 27 dpc: root formation was underway by 27 dpc, with cervical cementum barely visible in WT at this stage (A) while Ank KO mouse molars (B) exhibited regions of abnormally thick cementum matrix (indicated by black (more ...)
With advanced root formation at 33 dpc, acellular cementum covered root dentin at a relatively uniform thickness in WT animals (fig. ). In the Ank KO mouse, the acellular cementum layer was several-fold increased (i.e. hypercementosis) compared to controls (fig. ). Unusually, cells were partially embedded in the cervical cementum of the Ank KO mouse. In the Ank KO, this tissue will be referred to as ‘cervical cementum’ as it diverged from the usual morphology of AEFC. Ank KO, dentin and bone appeared no different from WT at this age.
At 45 dpc, first-molar root development was complete, the tooth was erupted, and PDL had matured (fig. ). In the Ank KO mouse molar, cervical cementum thickness had increased (fig. ). The normally cell-free layer included numerous embedded cells (cementocytes) in the matrix. Ank KO apical cementum (cellular intrinsic fiber cementum) was not different from WT in regard to size or embedded cells (fig. vs. G). The PDL space in the Ank KO mouse remained nonmineralized. By 45 dpc, an apparent reduction in lingual alveolar bone height was noticed, as described further in the following section.
Importantly, at 79 dpc, with the first molar in functional occlusion for more than 1 month, the cervical cementum thickness had continued to expand in Ank
KO, yet the PDL space remained well maintained, comparable to age-matched WT controls (online suppl. fig. 1C, D
). Alveolar bone reduction in Ank
KO had advanced further by this age, a trend explored in more detail in the next section. This finding is in contrast to severe ectopic calcification found in joints of Ank
KO mice at this age [Gurley et al., 2006a
Examination of mandibular second and third molars revealed thick cervical cementum on these teeth. Cross-sections of the continually erupting mouse incisor exhibited cementum on the lingual ‘root analogue’ aspect, which was the acellular variety (online suppl. fig. 2A, C, E
). Like the cervical cementum of the molar, the Ank
KO incisor tooth exhibited a hypercementosis phenotype that was visible by 27 dpc and increasingly expanded versus WT over subsequent time points (online suppl. fig. 2B, D, F
). As with the molar, the dentin, pulp chamber and PDL of the incisor did not appear different from WT. Examination of thick cementum on both molars and incisor root analog indicates an intrinsic alteration in acellular cementum development.
The PDL Space in Ank KO Is Maintained by Alveolar Bone Remodeling
Histomorphometry was employed to detect and quantify any subtle changes in the dimensions of cementum and determine whether surrounding tissues were altered. Developmental age 45 dpc was chosen in order to measure molars at a mature root stage where teeth were erupted and PDL had become functional and organized. Measurements were made on histological sections cut in the buccal-lingual (frontal) direction, at distances of 100, 300 and 500 μm measured apically from the CEJ, the anatomical transition between crown and root. Tissues measured included alveolar bone, PDL, cervical cementum, dentin, predentin and pulp chamber.
Cervical cementum in Ank
KO mice was significantly thicker than in WT at all 3 points measured (p < 0.01 for all comparisons), on both buccal and lingual aspects of the tooth root (fig. ). Ank
KO cementum was from 8- to 12-fold thicker than in WT at the 3 points. Other tissues measured were not different in KO versus WT, with the notable exception that PDL was found to be slightly increased in Ank
KO versus WT (significant on lingual aspect at 300 μm and buccal aspect at 500 μm), a finding contrary to expectations in light of the widened cementum. Results from incisors paralleled those from molars, where cementum was significantly increased in Ank
KO versus WT, (approximately 10-fold, p < 0.005), while other tissues were not altered. In both molars and incisors of Ank
KO, measured dimensions of dentin, predentin, and pulp were similar to WT. Mean measurements and statistical analyses can be found in online supplementary table 2
Fig. 3 Histomorphometry confirms unique sensitivity of cementum to loss of ANK. A Measurements were made on 45 dpc molars (where tooth development was largely completed) at apical distances 100, 300, and 500 μm from the CEJ. Tissues measured included (more ...)
Histological examination indicated alteration of the alveolar bone around the first molars of Ank KO mice, therefore measurement of the distance from CEJ to ABC (the most coronal portion of the alveolar bone proper) was performed. Measurements confirmed a greater CEJ-ABC distance on both lingual and buccal sides (fig. ). The combination of bone alteration and maintenance of PDL width suggested PDL width was maintained by increased bone remodeling in Ank KO mice. TRAP staining confirmed approximately 3-fold increased numbers of TRAP-positive osteoclast-like cells on the alveolar bone proper (adjacent to PDL and tooth root) of both buccal and lingual alveolar bone in Ank KO versus WT (fig. vs. C). Further, identification of reversal lines in lingual alveolar bone (aided by immunohistochemical labeling of OPN) signified new bone apposition on the outer aspect of alveolar bone (fig. ), while WT bone tended to show apposition on both inner and outer aspects (fig. ). Altogether, maintenance of PDL and evidence for alveolar bone remodeling supports a role for PDL-bone interactions in order to accommodate increased cementum width in Ank KO molar teeth.
Though PDL was maintained around Ank KO molars, tissue masses consistent with cementicles (cementum-like masses forming in the PDL region) were observed in Ank KO mouse PDL, always localized to the most cervical portion of PDL on the buccal side, and separate from cementum and bone (data not shown). The late appearance and consistent localization of these cementicles suggest they result from some type of functional response rather than being a developmental defect.
Mineralization and Mechanical Properties of Dentoalveolar Tissues Are Unchanged in Ank KO Mice
Next, SEM in backscatter (BSE) mode was used to assess whether the quality of cementum formed was compromised in Ank KO versus WT. BSE mode indicates relative material density where a brighter image denotes denser material, reflecting composition and elemental content. BSE images from WT and Ank KO first molars in the cervical regions in both groups are shown in figure . Enamel, being the most mineralized, appeared brightest in both WT and KO. Dentin and cementum share similar composition and mineral content, and thus appeared at similar gray level contrast in SEM images. Within the limits of detection, no major difference was observed in any Ank KO mineralized tissues compared to WT, indicating that relative mineralization was likely not different.
Fig. 4 Mineralization and mechanical properties of dentoalveolar tissues are unchanged in Ank KO mice. SEM in BSE indicated no discernible difference in WT (A) versus Ank KO (B) mineralized tissues, suggesting percent mineralization and/or composition was not (more ...)
Mechanical properties of the mineralized dental tissues of WT and Ank KO were assessed by nanoindentation. No measureable differences were found in hardness (H) or reduced elastic modulus (Er) of cementum between WT (H = 0.6 ± 0.2 GPa; Er = 24 ± 3 GPa) and Ank KO cervical cementum (H = 0.7 ± 0.2; Er = 25 ± 3), as summarized in figure . Likewise, no significant difference was detected in WT versus KO comparisons of dentin and enamel.
Loss of ANK Alters Cementoblast Gene Expression and Cervical Cementum Extracellular Matrix Composition
IHC and ISH were used to characterize expression of selected cementum, bone and dentin markers (i.e. mineralized tissue extracellular matrix proteins and their respective genes) in Ank
KO and WT controls. Figure shows representative staining in WT and KO tissues, where 45 dpc was used to examine matrix produced over the course of cementogenesis, and the earlier age of 33 dpc was used to assay gene expression in situ during active cementum formation. Online supplementary figure 3
provides lower-magnification IHC images at 33, 45, and 79 dpc to illustrate the distribution of proteins in the periodontal region over a developmental to functional time course.
Fig. 5 Loss of ANK alters cementoblast gene expression and cervical cementum extracellular matrix composition. Histological sections from 45 dpc mice were used for IHC, while 33 dpc tissues were used for ISH. BSP: in WT (A), BSP protein staining was strong in (more ...)
BSP was localized to bone matrix and acellular and cellular cementum (fig. and online suppl. fig. 3
) [MacNeil et al., 1995
; McKee et al., 1996
]. In acellular cementum, BSP, a characteristic marker for this tissue, was present as a concentrated band defining the entire width of the cementum layer. The thick Ank
KO cervical cementum showed positive, but diffuse BSP staining, with some localization in the initial cementum, but not through the entire thickness (fig. , online suppl. fig. 3
). Results were consistent with two separate BSP antibodies, as well as following antigen retrieval using trypsin digestion (data not shown). Staining of BSP in bone and cellular cementum was similar between Ank
KO and controls. ISH revealed equivalent Bsp
mRNA expression in cementoblasts and osteoblasts in 33 dpc WT (fig. ) and Ank
KO (fig. ) tissues.
OPN protein, a known marker for acellular cementum, was observed in bone matrix, PDL, and in acellular and cellular cementum (fig. and online suppl. fig. 3
) [Bronckers et al., 1994
; MacNeil et al., 1995
; McKee et al., 1996
]. OPN localization in Ank
KO was notable for the strong, even immunostaining throughout the entire cervical cementum thickness, and high expression in cementoblasts lining the root (fig. ). Increased OPN protein in Ank
KO was also apparent by 33 dpc, and strong OPN localization in cervical cementum continued through 79 dpc (online suppl. fig. 3
). In WT, Opn
mRNA was localized to osteoblasts and some cementoblasts, especially those apically located on the molar where cementogenesis was ongoing (fig. ). In Ank
was intensely expressed by cementoblasts lining the entire root (fig. ), confirming increased and sustained Opn
versus WT molars. Opn
levels in other cells and tissue in Ank
KO, including osteoblasts, resembled WT.
DMP1 protein in WT was localized primarily to bone matrix around osteocytes (fig. ), with lower levels of staining apparent around some cementocytes in the apical cementum (online suppl. fig. 3
) [Butler et al., 2002
; Feng et al., 2003
; Baba et al., 2004
; Qin et al., 2007
; Ye et al., 2008
]. WT acellular cementum was not immunopositive for DMP1. In Ank
KO, DMP1 was heavily localized to the cervical cementum, especially perilacunar spaces around embedded cells, and in cells in the process of being embedded in cementum (fig. ). As with OPN, increased DMP1 was apparent by prefunctional age 33 dpc, and the trend continued through 79 dpc. DMP1 staining in Ank
KO cementum was even more intense than in bone, where DMP1 localization is usually greatest. Results were confirmed with 3 different DMP1 antibodies used, including N-terminal and C-terminal targeted antibodies. By ISH, Dmp1
mRNA was identified in WT odontoblasts and osteocytes (fig. ), while transcripts were clearly increased in cementoblasts along the molar root of the Ank
KO mouse (fig. ). In contrast, Dmp1
message levels were unchanged in odontoblasts, osteoblasts and osteocytes of Ank
KO versus WT.
All protein findings from Ank
KO mouse molars were consistent with staining in the incisor (online suppl. fig. 4
KO incisor cementum featured increased DMP1 protein and limited BSP compared to WT, while OPN immunostaining was robust in both WT and KO.
Several other extracellular matrix proteins were assayed by IHC and found to be no different in Ank KO versus WT (data not shown). These included OCN, dentin sialoprotein, MEPE, decorin, biglycan, and periostin.
ANK Is Expressed by Tooth Cells during Root Development
As a next step to identify the mechanism for unique sensitivity of the acellular cementum to loss of ANK, the pattern of gene and protein expression of this regulator was determined during mouse tooth development. To our knowledge, this was the first study of ANK expression in relation to tooth root development.
ANK protein and mRNA message were observed in several cells and tissues of the developing dentoalveolar complex at 33 dpc. ANK was localized to ameloblasts, cells of the pulp, odontoblasts, cells of the follicle and developing PDL, cementoblasts, osteoblasts, and some osteocytes (fig. ). This ubiquitous expression of ANK in the tooth agrees with previous reports demonstrating the presence of Ank
message in many organ systems [Ho et al., 2000
]. No special localization of ANK was noted in cementoblasts or cementum-associated PDL, though its widespread presence could make localized selective expression difficult to identify. The specificity of ANK staining was confirmed by using negative controls for IHC and ISH (fig. ), as well as multiple antibodies in WT and Ank
KO (online suppl. fig. 5
). To further confirm IHC and ISH results by an independent assay, we analyzed tooth-associated cell types for mRNA expression of Ank
, while also testing for Enpp1
(TNAP gene) expression. All cell lines robustly expressed Ank
as determined by RT-PCR (fig. ). While limited to gross tissue level of resolution, RT-PCR performed on RNA harvested from whole mouse molars established Ank
expression at the tissue level, in vivo, confirmed by positive reactions from mouse calvariae and femurs. Thus by multiple parallel techniques, Ank
were found to have wide-ranging expression in tooth cells and bone tissues.
Fig. 6 ANK is widely expressed during tooth root development. A, B In 33 dpc WT molars, ANK protein staining was observed in cells and tissues of the developing dentoalveolar complex, e.g. ameloblasts, odontoblasts, PDL, cementoblasts, osteoblasts and osteocytes. (more ...)
NPP1 Is Increased in Cementoblasts in Ank KO Mice
To determine whether loss of ANK altered expression of the parallel PPi regulator NPP1, IHC for NPP1 was performed. While low-level immunostaining of NPP1 was apparent in odontoblasts, osteoblasts and in some cells in the PDL, a discreet region of higher-intensity staining was apparent in cementoblasts lining the molar root in WT (fig. ). Compared to WT, NPP1 expression was remarkably increased in Ank KO cementoblasts lining the cervical root as well as those cells embedded in cementum (fig. ). In stark contrast to intense expression by Ank KO cementoblasts, NPP1 expression was similar in Ank KO osteoblasts, odontoblasts, or other cells, as compared to WT.
Fig. 7 NPP1 is more highly expressed by cementoblasts during tooth development and increased in Ank KO cementoblasts. A, B In 45 dpc WT molar, NPP1 staining was observed most strongly in cementoblasts, but was present at low levels in osteoblasts, odontoblasts (more ...)
Conditional Loss of ANK Confirms Local Function in Cementogenesis
is widely expressed in the body and in the dentoalveolar complex; therefore, in order to analyze whether loss of ANK is a systemic or local influence on the periodontium, conditional KO mice were prepared by crossing Gdf5-Cre
-expressing mice with mice harboring a floxed Ank
Gdf-5, a member of the transforming growth factor-β superfamily, is expressed in articular tissues, including the developing PDL [Morotome et al., 1998
; Sena et al., 2003
As described previously for joints [Gurley et al., 2006a
tissues exhibited variable phenotypic severity resulting from variation in extent of recombination. Mildly affected Gdf5-Cre
mice (n = 6) exhibited a slight but consistent increase in acellular cementum thickness versus controls, with occasional localized regions of hypercementosis (fig. ). Severely affected Gdf5-Cre
mice (n = 4) exhibited uniformly thick cementum indistinguishable from the global Ank
KO (fig. ). The ability for Gdf5
KO to recapitulate the cementum phenotype confirmed a local role for ANK in regulating cementum.
Fig. 8 Conditional loss of ANK confirms local function in cementogenesis. Compared to control molars (A), conditional KO of Ank in Gdf5-Cre-AnkfloxP resulted in mild phenotypes (B) marked by slight cementum (cc) thickening and local hypercementosis () (more ...)
DMP1 and NPP1 were assayed in Gdf5-Cre-AnkfloxP mice because of their markedly increased expression in cementoblasts in the global Ank KO. In mildly affected Gdf5-Cre-AnkfloxP molars, DMP1 staining in AEFC was primarily associated with regions of most increased thickness, while regions of thin cementum remained negative for DMP1 (fig. ). Regions of increased NPP1 were found in all mildly affected Gdf5-Cre-AnkfloxP molars, but ass for DMP1, the most intense NPP1 staining was consistently associated with increased AEFC apposition (fig. ). DMP1 and NPP1 staining in severely affected Gdf5-Cre-AnkfloxP molars mimicked that seen in global Ank KO (fig. ).
Ank and Enpp1 Are Expressed by Cementoblasts Coincident with in vitro Mineralization
Based on expression of ANK gene/protein and NPP1 protein in tooth cells at about the time of cementogenesis and the demonstration that ANK must function locally in cementoblast cells, in vitro experiments using OCCM.30 murine immortalized cementoblast cells were performed to determine whether these genes were expressed in temporal fashion during mineral formation. OCCM.30 cells were cultured in control media (DMEM with 2% FBS), media supplemented with 50 μg/ml AA, or mineralization media, which included 50 μg/ml AA and 10 mM BGP, for up to 10 days, with RNA isolated for gene expression (1, 3, 5–9 days) and von Kossa staining used in parallel samples to assay mineral nodule formation.
Cementoblasts incubated with mineralization media produced visible matrix by day 6, with beginnings of mineralized nodules by day 7, and increased staining at days 8–10 (fig. ). Cells receiving control media or AA alone did not produce mineral nodules. Expression of Akp2
peaked at day 3, during the early period of culture, consistent with the role of TNAP as an early bone/cementum marker involved in matrix modification prior to mineralization (fig. ) [Lian and Stein, 1992
]. Early, intense Akp2
expression required both AA and BGP (mineralizing conditions). Control or AA groups increased Akp2
slowly over the culture period, but did not produce mineral nodules. In contrast, Opn
expression was dramatically increased (more than 60-fold over day 1) at onset of mineralization on days 6–7, a trend previously reported in osteoblast cultures [Owen et al., 1990
; Lian and Stein, 1992
]. Notably, increased Opn
occurred only in the presence of AA and BGP, where mineral nodules were forming. Induction of Opn
in concert with mineral accumulation is consistent with OPN as a regulator of HAP mineralization.
Fig. 9 Ank and Enpp1 are expressed during the mineralization stage of cementoblasts, in vitro. Studies with OCCM.30 cementoblasts were performed to characterize the temporal developmental sequence of gene expression resulting in matrix mineralization. Von Kossa (more ...)
As observed for Opn expression, Ank and Enpp1 were induced at the onset of mineralization, both peaking at days 6–7, and only under mineralizing conditions. Ank levels reached 150% of day 1 levels, while Enpp1 peaked at 600% of day 1 levels. Results suggested Ank and Enpp1 were functionally coupled with mineralizing conditions, i.e. induction of these genes required formation of mineralized matrix. More specifically, the timing of gene expression and requirement for mineralizing media supported ANK and NPP1 as regulators of cementoblast extracellular matrix mineralization. In addition to a more dramatic increase for Enpp1 versus Ank during mineralization, relative levels of Enpp1 mRNA were much higher than Ank, more than 1,000-fold relative units (as normalized to Gapdh). Relatively high expression of Enpp1 mRNA in cementoblasts is in agreement with in vivo IHC observations of elevated NPP1 along the tooth root.