Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

A genetic model for the secretory stage of dental enamel formation.

The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane.

Effect of over-the-counter brimonidine tartrate 0.025% ophthalmic solution on pupil size in healthy adults.

PURPOSE: To evaluate the effect of brimonidine tartrate 0.025% ophthalmic solution on pupil size under scotopic conditions in healthy adults METHODS: Pupil size was measured in 56 eyes of 28 volunteer participants using a pupillometer under scotopic conditions. Age, gender, and iris color were recorded. Subjects using any ophthalmic medications other than artificial tears were excluded. The pupil size was subsequently measured again under scotopic conditions 60 min after instillation of brimonidine tartrate 0.025% ophthalmic solution. RESULTS: Statistically significant miosis was seen after instillation of brimonidine tartrate 0.025% (p = 0.04). Average pupil size prior to brimonidine 0.025% instillation was 7.28 ± 1.05 mm, and average pupil size after instillation of brimonidine 0.025% was 6.36 ± 1.68 mm, a reduction of - 23.7% in pupil area. Subjects with light irides demonstrated a greater miotic effect than subjects with dark irides (1.55 mm vs. 0.67 mm, p < 0.0001), with a pupil area reduction of - 37.6% and - 17.4%, respectively. The amount of miosis was independent of initial pupil size. CONCLUSIONS: Brimonidine tartrate 0.025% causes significant miosis in scotopic settings, although the effect is not as great in darker colored eyes. Further studies are needed to determine the latency and duration of the effect and whether the amount of miosis is clinically significant.

Mutations in RELT cause autosomal recessive amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt-/- mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt-/- enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.

Sirt1 overexpression suppresses fluoride-induced p53 acetylation to alleviate fluoride toxicity in ameloblasts responsible for enamel formation.

Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo. Here we assessed SIRT1 function on fluoride-induced Ac-p53 formation using CRISPR/Cas9-mediated Sirt1 knockout (LS8Sirt/KO) cells or CRISPR/dCas9/SAM-mediated Sirt1 overexpressing (LS8Sirt1/over) cells. NaF (5 mM) induced Ac-p53 formation and increased cell cycle arrest via Cdkn1a/p21 expression in Wild-type (WT) cells. However, fluoride-induced Ac-p53 was suppressed by the SIRT1 activator resveratrol (50 µM). Without fluoride, Ac-p53 persisted in LS8Sirt/KO cells, whereas it decreased in LS8Sirt1/over. Fluoride-induced Ac-p53 formation was also suppressed in LS8Sirt1/over cells. Compared to WT cells, fluoride-induced Cdkn1a/p21 expression was elevated in LS8Sirt/KO and these cells were more susceptible to fluoride-induced growth inhibition. In contrast, LS8Sirt1/over cells were significantly more resistant. In addition, fluoride-induced cytochrome-c release and caspase-3 activation were suppressed in LS8Sirt1/over cells. Fluoride induced expression of the DNA double strand break marker γH2AX in WT cells and this was augmented in LS8Sirt1/KO cells, but was attenuated in LS8Sirt1/over cells. Our results suggest that SIRT1 deacetylates Ac-p53 to mitigate fluoride-induced cell growth inhibition, mitochondrial damage, DNA damage and apoptosis. This is the first report implicating Ac-p53 in fluoride toxicity.

The economics of femtosecond laser-assisted cataract surgery.

PURPOSE OF REVIEW: Femtosecond lasers for use during cataract surgery carry significant purchase and use costs. The aim of this article is to help surgeons and surgery centers anticipate financial issues related to implementing femtosecond laser-assisted cataract surgery (FLACS). Such scenarios hopefully can help to inform decision making around the purchase and use of these lasers. RECENT FINDINGS: FLACS has several potential advantages over traditional phacoemulsification. However, although studies have demonstrated noninferiority of FLACS, there continues to be few data to support significantly improved visual outcomes. The literature does show a significantly higher cost for FLACS. As this cost can be passed on to patients under Medicare rules, there is the potential for increased physician revenue, which can be a motivator for adoption of this new technology. The magnitude of this increase is heavily influenced by the financial details of the implementation, like the cost of the laser, the volume of surgery performed, and the incremental increase in revenue. SUMMARY: A financial analysis should be performed prior to purchasing a femtosecond laser. This analysis can help predict if FLACS is going to be a financial windfall or a money loser.

Beta-catenin is essential for ameloblast movement during enamel development.

Beta-catenin is a multifunctional protein that plays key roles in cadherin-based cell adherens junctions and in the Wnt signaling pathway. The canonical Wnt/ß-catenin pathway can regulate transcription factors that control cell movement/invasion. We investigated whether ß-catenin regulates ameloblast movement through canonical Wnt signaling. The morphological and physical properties of enamel were assessed in enamel from control and ß-catenin conditional knockout (cKO) mice. Ameloblast-lineage cells (ALC) were used to investigate the potential roles of ß-catenin in cell migration and in E-cadherin expression. Compared with controls, incisors from ß-catenin cKO mice were short, blunt, and where enamel was present, it was soft and malformed. Scanning electron microscopy revealed a dysplastic rod pattern within the enamel of incisors from ß-catenin cKO mice, and Vickers microhardness measurements confirmed that mice with ß-catenin ablated from their enamel organ had enamel that was significantly softer than normal. Amelogenesis was disrupted in the absence of ß-catenin and the ameloblasts did not differentiate properly. We further demonstrated that migration of ALCs was inhibited in vitro and that E-cadherin expression was significantly up-regulated when ALCs were treated with the ß-catenin inhibitor, ICG-001. Beta-catenin ablation causes enamel malformation in mice and this phenotype may occur, in part, by a lack of ameloblast differentiation and/or movement necessary to form the decussating enamel rod structure.

23-mm iodine-125 plaque for uveal melanoma: benefit of vitrectomy and silicone oil on visual acuity.

PURPOSE: To review outcomes in mostly large uveal melanoma treated with a 23-mm-diameter iodine-125 plaque, the largest size available at our center, and the influence of vitrectomy and silicone oil 1000 centistokes for radiation attenuation. METHODS: A one-to-one matched case-control comparison was performed. Case patients were treated with a 23-mm-diameter iodine-125 plaque and vitrectomy with silicone oil 1000-cSt placement. Control cases, treated with 23-mm plaque alone, were matched to cases with respect to tumor size and distance from tumor apex to optic nerve and fovea. Postoperative complications, visual acuity and metastasis were reviewed. RESULTS: Twenty case patients with uveal melanoma treated with a 23-mm plaque were identified. The final logMAR vision was 0.83 in case patients and 2.06 in control patients (P = 0.0064); the change from pre-treatment to last follow-up logMAR vision was 0.70 in cases and 1.62 in controls (P = 0.019). Of good vision outcomes, 65 % of cases and 25 % of controls achieved vision ≥20/200 (P = 0.025). Of poor vision outcomes, 35 % of cases and 80 % of controls achieved vision <20/200 (P = 0.0053), and 5 % of cases and 35 % of controls achieved "light perception" or "no light perception" vision (P = 0.044). Thirty-nine of the 40 eyes (98 %) achieved local tumor control. Metastasis occurred in 15 % of cases and 45 % of controls (P = 0.082). CONCLUSIONS: Iodine-125 brachytherapy for mostly large uveal melanoma is effective in achieving local tumor control. Furthermore, combining brachytherapy with vitrectomy and silicone oil 1000-cSt for radiation attenuation significantly improves vision over the use of plaque alone.

Sirtuin1 and autophagy protect cells from fluoride-induced cell stress.

Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum (ER) stress and oxidative stress. Previously, we reported that fluoride induces ER-stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augment SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50, 100 and 125ppm) in drinking water for 6weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis.

Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats.

Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F(-)) exposure generates reactive oxygen species (ROS) that can cause endoplasmic reticulum (ER)-stress. We therefore screened oxidative stress arrays to identify genes regulated by F(-) exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F(-)-treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F(-) (∼1.5 & 2.0 fold for the 50 or 100 ppm F(-) treatment groups, respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F(-) treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride-treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E-enriched diets within a given F(-) treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F(-) and activation of this gene may adversely affect ameloblast function.

Mobility gene expression differences among wild-type, Mmp20 null and Mmp20 over-expresser mice plus visualization of 3D mouse ameloblast directional movement.

Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel development. Matrix metalloproteinase-20 (MMP20) is a tooth specific proteinase essential for proper enamel formation. We previously reported that MMP20 cleaves cadherins and may regulate ameloblast movement. Here, we used an Amelx promoter driven tdTomato reporter to label mouse ameloblasts. With these transgenic mice, we assessed ameloblast mobility group dynamics and gene expression. Three-dimensional imaging of mouse ameloblasts were observed in hemi-mandibles by using a tissue clearing technique. The three-dimensional ameloblast layer in Tg(Amelx-Mmp20) mice that overexpress MMP20 was uneven and the ameloblasts migrated away from this layer. Mouse ameloblast movement toward incisal tips was monitored by ex vivo time-lapse imaging. Gene expression related to cell migration and adhesion was analyzed in ameloblasts from wild-type mice, Mmp20-/- mice with no functional MMP20 and from Tg(Amelx-Mmp20) overexpressing mice. Gene expression was altered in Mmp20-/- and Tg(Amelx-Mmp20) mice compared to wild type. Among the genes assessed, those encoding laminins and a gap junction protein were upregulated in Mmp20-/- mice. New techniques and findings described in this study may lead to an improved understanding of ameloblast movement during enamel formation.

Mesenchymal cell TRPM7 expression is required for bone formation via the regulation of chondrogenesis.

Transient receptor potential melastatin-subfamily member 7 (TRPM7) is a bifunctional protein containing a kinase fused to an ion channel permeated with cations, including Ca2+ and Mg2+. Trpm7-null mice show embryonic lethality. Paired related homeobox 1 (Prx1) is expressed in undifferentiated mesenchymal cells such as the progenitor cells of both chondrocytes and osteoblasts involved in limb skeleton formation. Prx1-Cre-dependent Trpm7 mesenchymal-deleted mice were generated to examine the role of TRPM7 in bone development. We found that Prx1-Cre;Trpm7fl/fl mice had shortened bones and impaired trabecular bone formation. Trabecular bone parameters, such as the bone volume (BV/TV), and trabecular number (Tb.N), were decreased in Prx1-Cre;Trpm7fl/fl mice. The cortical bone parameters of cortical bone area (Ct.Ar) and cortical bone thickness (Ct.Th) were also down-regulated in these mice. The bone formation rate in Prx1-Cre;Trpm7fl/fl mice was unchanged, but the hypertrophic area and cell size of the zone were smaller, and the expression of Col2a1, Col10a1 and Mmp13 was downregulated compared with control mice. These findings suggest impaired chondrogenesis in Prx1-Cre;Trpm7fl/fl mice compared to control mice. The receptor activator of nuclear factor-kappa B ligand (RANKL) expression was increased, and RANKL-positive cells and osteoclasts were markedly accumulated in the boundary region between the growth plate and trabecular bone. In contrast, TRPM7 KR mice, which are kinase-dead mutants in which the TRPM7 ion channel function has not been altered, showed no marked differences in trabecular or cortical bone parameters compared to wild-type mice. These findings suggest that TRPM7 is critical as a cation channel rather than as a kinase in bone development via the regulation of chondrogenesis.

Effect of kallikrein 4 loss on enamel mineralization: comparison with mice lacking matrix metalloproteinase 20.

Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase. Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4), a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo.

Tetracycline antibiotics, including doxycycli\e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.

ADAM10: Possible functions in enamel development.

ADAM10 is A Disintegrin And Metalloproteinase (ADAM) family member that is membrane bound with its catalytic domain present on the cell surface. It is a sheddase that cleaves anchored cell surface proteins to shed them from the cell surface. ADAM10 can cleave at least a hundred different proteins and is expressed in most tissues of the body. ADAM10 is best characterized for its role in Notch signaling. Interestingly, ADAM10 is transported to specific sites on the cell surface by six different tetraspanins. Although the mechanism is not clear, tetraspanins can regulate ADAM10 substrate specificity, which likely contributes to the diversity of ADAM10 substrates. In developing mouse teeth, ADAM10 is expressed in the stem cell niche and subsequently in pre-ameloblasts and then secretory stage ameloblasts. However, once ameloblasts begin transitioning into the maturation stage, ADAM10 expression abruptly ceases. This is exactly when ameloblasts stop their movement that extends enamel crystallites and when the enamel layer reaches its full thickness. ADAM10 may play an important role in enamel development. ADAM10 can cleave cadherins and other cell-cell junctions at specific sites where the tetraspanins have transported it and this may promote cell movement. ADAM10 can also cleave the transmembrane proteins COL17A1 and RELT. When either COL17A1 or RELT are mutated, malformed enamel may occur in humans and mice. So, ADAM10 may also regulate these proteins that are necessary for proper enamel development. This mini review will highlight ADAM10 function, how that function is regulated by tetraspanins, and how ADAM10 may promote enamel formation.

MMP20 cleaves E-cadherin and influences ameloblast development.

Dental enamel development occurs in stages as observed by the changing morphology of the ameloblasts that are responsible for enamel formation. During the secretory stage of development, proteins including MMP20 are secreted into the enamel matrix. MMP20 is required for proper enamel formation as mutation of the Mmp20 gene causes autosomal recessive amelogenesis imperfecta. Here, we examined in detail the morphology of the Mmp20-null ameloblast cell layer. Intriguingly, we found that the Mmp20-null mouse secretory stage ameloblasts retract their Tomes' processes as if preparing to enter the maturation stage but later reextend their Tomes' processes as if resuming the secretory stage. We also demonstrated that MMP20 cleaves epithelial cadherin, i.e. E-cadherin. Cadherins are transmembrane proteins with extracellular domains that provide adhesive contacts between neighboring cells. Their intracellular domains bind to the cell cytoskeleton through catenins, including ß-catenin. When specific MMPs cleave the cadherin extracellular domain, ß-catenin is released and may locate to the cell nucleus as a transcription factor. Therefore, MMP20 may influence ameloblast developmental progression through hydrolysis of cadherin extracellular domains with associated release of transcription factor(s).

Why does enamel in Klk4-null mice break above the dentino-enamel junction?

BACKGROUND: The enamel layerof kallikrein 4 (Klk4)-null mice has a normal thickness and a decussating pattern of enamel rods, but it contains residual enamel proteins, is less highly mineralized, and fractures in its deepest part just above the dentino-enamel junction (DEJ). The plane of fracture is puzzling because the deepest enamel is deposited earliest and, through the action of the secretory stage enamel protease (Mmp20), is the most mature part of the enamel layer at the time of the onset of Klk4 expression. OBJECTIVES: To characterize the planes of fracture in Mmp20- and Klk4-null mice and to localize Klk4 expression in developing teeth. METHODS: Klk4- and Mmp20-null mice were sacrificed at 7 weeks and their mandibular incisors were characterized by scanning electron microscopy. Klk4(+/)(lac)(Z) mice were mated with Klk4(+/)(lac)(Z) mice. Offspring were genotyped by polymerase chain reaction. Klk4(+/)(+), Klk4(+/)(lac)(Z), and Klk4(lac)(Z/)(lac)(Z) (null) littermates on postnatal days 5, 8, 11, and 14 were processed for ß-galactosidase histochemistry. RESULTS: The enamel layer fractures at the DEJ in Mmp20-null mice, and fractures occur in enamel above the DEJ in Klk4-null mice. Klk4 is not expressed by secretory-stage ameloblasts, murine odontoblasts beneath the secretory stage, or maturation-stage ameloblasts. Klk4 is specifically expressed by transition and maturation-stage ameloblasts. CONCLUSIONS: The breakage of enamel near the DEJ in Klk4-null mice is not due to a failure of odontoblasts to express Klk4, but it relates to a progressive hypomineralization of enamel with depth.

Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino-enamel junction, and a decussating enamel rod pattern.

Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.

Kallikrein-related peptidase 4, matrix metalloproteinase 20, and the maturation of murine and porcine enamel.

The crowns of matrix metalloproteinase 20 (Mmp20) null mice fracture at the dentino-enamel junction (DEJ), whereas the crowns of kallikrein-related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9-wk-old wild-type, Klk4 null, and Mmp20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild-type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in Mmp20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the Mmp20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid-maturation wild-type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in Mmp20 null mice because of hypomineralization at the DEJ.