Primary Enamel Research Articles

Comparison of the Effect of Bioglass, Chitosan, and SDF Compounds on Remineralization of Primary Caries Lesions in Primary Teeth: An in vitro Study.

Dental caries are among the most common oral and dental diseases affecting adults and children. To prevent caries, either the factors that cause caries should be reduced or the host resistance should be increased. Several compounds, such as bioglass, chitosan, and silver diamine fluoride (SDF), can enhance enamel remineralization. This study was conducted to investigate the effects of chitosan, bioglass, chitosan-bioglass, and SDF compounds on remineralizing primary enamel lesions. In this in vitro study, seventy-two primary canine teeth were collected. The teeth were exposed to a demineralization solution for 72 hours to create primary caries lesions. The primary Vickers microhardness test (VMT) was conducted to measure the initial values. The samples were randomly divided into six groups (n=12): Group 1: bioglass-chitosan solution; Group 2: chitosan; Group 3: bioglass solution; Group 4: SDF; Group 5: remineralization solution; Group 6: distilled water. The solutions of Groups 1, 2, and 3 were applied to the samples for 7 days, while the SDF solution was applied only once. The samples were immersed in an artificial saliva solution, which was refreshed daily. After the treatment, the final Vickers microhardness test (VMT) values were recorded. The data were analyzed statistically using a two-way ANOVA and Tukey's test (p< 0.05). The results indicated a statistically significant effect of remineralizing compounds on both pre-treatment and post-treatment microhardness (p< 0.0001). However, no significant difference in microhardness was observed between the groups studied (p= 0.225). All the compounds utilized in this study demonstrated a significant remineralizing effect on enamel lesions caused by primary caries in primary teeth. The chitosan-bioglass and bioglass groups exhibited the highest levels of remineralization, respectively. However, the comparison between the groups yielded insignificant results due to the dispersion of the samples. Therefore, further studies with larger sample sizes are recommended.

Surface and Mineral Changes of Primary Enamel after Laser Diode Irradiation and Application of Remineralization Agents: A Comparative In Vitro Study.

The purpose of this study is to evaluate the remineralization potential of primary teeth enamel after being exposed to different laser diode therapies. Ninety-six vestibular primary teeth enamel samples were divided into eight groups (n = 12) with varying treatments: control (G1), CPP-ACP-fluoride varnish (G2), diode lasers at 980 nm (G3), 808 nm (G4), 450 nm (G5), 980 nm + CPP-ACP-fluoride varnish (G6), 808 nm + CPP-ACP-fluoride varnish (G7), and 450 nm + CPP-ACP-fluoride varnish (G8). Each sample was assessed using a DIAGNOdent® (KaVo Dental, Biberach, Germany), at baseline, post-treatment, and post-pH cycle remineralization. SEM imaging was performed before and after treatment and following the pH cycle. The results indicated that the 980 nm and 808 nm diode lasers, both alone and in combination with CPP-ACP-fluoride varnish, either maintained or increased the calcium (Ca) weight percentage (Wt%) in the enamel. The 980 nm diode laser combined with CPP-ACP-fluoride varnish (G6) showed a significant increase in Ca Wt%, suggesting a strong remineralization effect. Similarly, the 808 nm diode laser alone (G4) also promoted a substantial increase in Ca Wt%. In contrast, the 450 nm diode laser, whether applied alone or in combination with CPP-ACP-fluoride varnish, resulted in a lower Ca Wt% and an increase in phosphorus (P) Wt%. Most groups, except for the CPP-ACP-fluoride varnish alone (G2), demonstrated an increase in P Wt%, indicating a complex interaction between laser therapy and enamel remineralization. The combined use of laser therapy with CPP-ACP-fluoride varnish significantly enhanced the remineralization of temporary teeth enamel. The 980 nm diode laser + CPP-ACP-fluoride varnish showed the most pronounced improvement in remineralization, while the 808 nm diode laser alone also effectively increased calcium solubility. These findings suggest that higher-wavelength diode lasers, particularly when combined with remineralizing agents, can effectively enhance the mineral content of primary teeth and promote enamel remineralization.

Effect of silver diamine fluoride combined with photodynamic therapy on primary enamel microhardness

ObjectivesThis study aimed to assess the effect of silver diamine fluoride (SDF) combined with photodynamic therapy (PDT) on primary enamel microhardness. Materials and MethodsThis in vitro, experimental study was conducted on 40 extracted primary molars with sound buccal/lingual surfaces. The baseline enamel microhardness of the teeth was initially measured by a Vickers hardness tester. Next, the teeth were randomly assigned to four groups (n = 10) for treatment with SDF, PDT, SDF plus PDT, and control (no intervention). After the intervention, the teeth underwent a 14-day pH-cycling, and enamel microhardness was measured again. The change in microhardness was calculated for each group, and comparisons were made by two-way ANOVA and t-test (alpha = 0.05). ResultsThe mean secondary microhardness of the three intervention groups was significantly higher than that of the control group (P<0.05). Despite slightly higher microhardness in SDF + PDT group, followed by the SDF group, the difference in secondary microhardness was not significant among the three intervention groups (P>0.05). ConclusionTreatment of primary enamel with SDF or PDT can increase its microhardness and enhance remineralization. However, combined use of SDF and PDT had no additional advantage over the use of SDF alone.

The effect of nano-hydroxyapatite and casein phosphopeptide-amorphous calcium phosphate with and without laser irradiation on the microhardness and surface morphology of demineralized primary enamel: An in vitro experimental study

The effect of nano-hydroxyapatite and casein phosphopeptide-amorphous calcium phosphate with and without laser irradiation on the microhardness and surface morphology of demineralized primary enamel: An in vitro experimental study

Efficacy of Self-assembling Peptide P11-4 in Remineralizing In Vitro Caries-like Lesions in Primary Enamel Samples in Combination with Calcium Phosphate-based Remineralization Agents.

The efficacy of self-assembling peptide P11-4 in combination with calcium-phosphate-based remineralization agents in remineralizing caries-like lesions in primary enamel was evaluated using a 21-day pH cycling experiment by Vickers microhardness [Vickers hardness number (VHN)] and scanning electron microscopy (SEM). A total of 120 primary enamel samples were made to undergo a demineralization cycle to produce caries-like lesions. They were divided into six groups, namely negative control (NC), positive control (P11-4), and four interventional groups in which each of the following calcium-phosphate-based agents were used in combination with P11-4-calcium sucrose phosphate (CSP), bioactive glass (BG), casein phosphopeptides, and casein phosphopeptides with fluoride. A 21-day pH cycling experiment was carried out with alternating demineralization and remineralization phases. The enamel samples were analyzed at baseline, post production of caries-like lesions, and post 21-day pH cycling using Vickers microhardness and SEM. Results were statistically analyzed using repeated measures of analysis of variance (ANOVA), keeping the level of significance at 0.05. Supplementing P11-4 with calcium-phosphate-based agents improved the surface hardness of the demineralized primary enamel samples, among which the fluoridated milk protein-based remineralization agent yielded a statistically significant improvement. P11-4 promoted the regeneration of incipient caries-like lesions. However, there is added benefit when this peptide is used in combination with a fluoridated calcium-phosphate-based agent. This study would help the clinician compose an effective regimen for the patient to follow at home posttreatment with P11-4, in-office treatment. Krishnamoorthi A, Shanbhog RS, Godhi BS, et al. Efficacy of Self-assembling Peptide P11-4 in Remineralizing In Vitro Caries-like Lesions in Primary Enamel Samples in Combination with Calcium Phosphate-based Remineralization Agents. Int J Clin Pediatr Dent 2024;17(5):552-557.

The Effect of Zamzam Water and ProShieldR Varnish on Microhardness of Eroded Primary Enamel Teeth Induced by Ironplus Syrup: An In Vitro Study

The Effect of Zamzam Water and ProShieldR Varnish on Microhardness of Eroded Primary Enamel Teeth Induced by Ironplus Syrup: An In Vitro Study

Comparison of the Remineralization Effect of Nano Herbal Toothpaste With Two Kinds of Toothpaste Containing Fluoride and Bioactive Glass on Primary Enamel Lesions: An In Vitro Study

Background: Considering the therapeutic properties of the compounds used in Albodent Iranian toothpaste and little evidence about the effect of NovaMin toothpaste on primary caries lesions, the purpose of this research was to compare the effect of toothpaste containing fluoride and toothpaste containing bioactive glass with Albodent toothpaste on enamel remineralization in a laboratory manner. Methods: Overall, 24 healthy human premolar teeth were selected and divided into buccal and lingual halves. The demineralization cycle of the samples was performed for 14 days. The samples were classified into four groups and were brushed twice a day for 28 days with tested toothpaste. A microhardness test was conducted, and changes in the enamel surface and morphology of one sample selected from each group were evaluated by scanning electron microscopy. The analysis of variance and Tukey’s tests were used for the statistical analysis of the data. Results: The average difference in microhardness in the studied groups was statistically significant (P&lt;0.05) when compared two-by-two with the control group. The most noticeable difference was found in the comparison of the Sensodyne group with the control group, while the lowest difference was observed in the comparison of the Albodent group with the control group. Conclusion: The presence of NovaMin in toothpaste along with fluoride can increase the efficiency of the remineralization rate of primary enamel lesions compared to fluoride toothpaste without bioactive glass. In addition, the effectiveness of herbal toothpastes, which do not contain essential artificial ingredients in the remineralization of primary enamel lesions was lower than that of non-herbal fluoride toothpastes.

Evaluation of the Effect of NovaMin and Er,Cr:YSGG Laser on Remineralization of Erosive Lesions of Primary Enamel Teeth: An In Vitro Study.

Introduction: The purpose of this in vitro study is to ascertain how NovaMin and Er,Cr:YSGG laser radiation affect the remineralization of primary tooth enamel lesions. Methods: 40 main teeth served as the study's sample size. These teeth were allocated into five groups at random. The first group served as a negative control (artificial saliva); the second group served as a positive control (APF gel 1.23%); the third group NovaMin, the fourth group Er,Cr:YSGG laser, the fifth group Er,Cr:YSGG laser and finally, the application of NovaMin. For the purpose of measuring microhardness, all samples were submitted to the dental materials laboratory three times. Results: The collected data were compared using the SPSS 28 program between the baseline measurement, after demineralization, and after remineralization. The data were analyzed using ANCOVA and Bonferroni tests. All groups demonstrated a considerable increase in microhardness as compared to the negative control group (P<0.05). With the exception of the third and fourth groups, there was no discernible difference in the rise in microhardness between the other groups. The third and fourth groups were compared, and the results revealed that NovaMin had a greater impact than laser therapy alone (P=0.023). Scanning electron microscopy (SEM) analysis backed up the findings. Conclusion: In comparison to using the Er,Cr:YSGG laser alone, using NovaMin alone can speed up the remineralization of erosion lesions on the surface of primary teeth.

Erosion behaviour of human, bovine and equine dental hard tissues

Dental hard tissues from different species are used in dental research, but little is known about their comparability. The aim of this study was to compare the erosive behaviour of dental hard tissues (enamel, dentin) obtained from human, bovine and equine teeth. In addition, the protective effect of the pellicle on each hard tissue under erosive conditions was determined. In situ pellicle formation was performed for 30 min on enamel and dentin samples from all species in four subjects. Calcium and phosphate release was assessed during 120 s of HCl incubation on both native and pellicle-covered enamel and dentin samples. SEM and TEM were used to examine surface changes in native enamel and dentin samples after acid incubation and the ultrastructure of the pellicle before and after erosive exposure. In general, bovine enamel and dentin showed the highest degree of erosion after acid exposure compared to human and equine samples. Erosion of human primary enamel tended to be higher than that of permanent teeth, whereas dentin showed the opposite behaviour. SEM showed that eroded equine dentin appeared more irregular than human or bovine dentin. TEM studies showed that primary enamel appeared to be most susceptible to erosion.

Preventive effect of children's toothpastes on erosive tooth wear of primary enamel and dentin

Preventive effect of children's toothpastes on erosive tooth wear of primary enamel and dentin

Erosive Effects of Pediatric Liquid Medicinal Syrups with Remineralising Agents on Primary Enamel: An In vitro Comparative Study

Background: The use of liquid medicinal syrups in childhood to the reasons that children suffer from illness quite often. The acidic component in the formulations may cause erosive to the dental tissues.&#x0D; Aims: The aim of study was to test the erosivity of common Paedtric medications and to assess the change of the erosive potential of the medications in the presence of commercial Remineralilising agents.&#x0D; Materials and Methods: A total of 27 extracted/exfoliated non carious Deciduous molars were used. 8 medical syrups were used.&#x0D; Statistical Analysis Used: ANOVA and post hoc analysis were applied. An Tukey alpha value of 0.05 was employed.&#x0D; Conclusion: The knowledge of the erosive potential of commonly used syrups is mandatory as erosion in children teeth.

Dynamic patterns of histone lactylation during early tooth development in mice.

Histone lactylation on its lysine (K) residues has been reported to have indispensable roles in lung fibrosis, embryogenesis, neural development, inflammation, and tumors. However, little is known about the lactylation activity towards histone lysine residue during tooth development. We investigated the dynamic patterns of lactate-derived histone lysine lactylation (Kla) using a pan-Kla antibody during murine tooth development, including lower first molar and lower incisor. The results showed that pan-Kla exhibited temporo-spatial patterns in both dental epithelium and mesenchyme cells during development. Notably, pan-Kla was identified in primary enamel knot (PEK), stratum intermedium (SI), stellate reticulum (SR), dental follicle cells, odontoblasts, ameloblasts, proliferating cells in dental mesenchyme, as well as osteoblasts around the tooth germ. More importantly, pan-Kla was also identified to be co-localized with neurofilament during tooth development, suggesting histone lysine lactylation may be involved in neural invasion during tooth development. These findings suggest that histone lysine lactylation may play important roles in regulating tooth development.

Immunohistochemical Localization of Endothelin- 1 and Endothelin A Receptor in Human Primary Tooth Enamel Organ.

Enamel organ (EO) is an ectodermal derived structure, which is involved in the different aspects of tooth development. Tooth development shares the same regulatory molecules and genes expressed in other developing organs. Endothelin- 1 (ET-1) and Endothelin A receptor (ETAR), (ET-1/ETAR) axis, are involved in differentiation of embryonic stem cells and organ development. The present study aimed to investigate the ET-1 and ETAR expression profiles during the development of human primary tooth EO with the relatively large sample size. In this experimental study, 33 human fetuses aged from 13 to 23 weeks (3 samples from each fetal age) were collected. The sampleswere divided into three age groups (<16 weeks, <19 weeks, ≥19 weeks) and cut for hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining. A two-way ANOVA test was conducted to examine the expression levels of ET-1 and ETAR in different layers of human primary tooth EO. The statistical significance was assumed at p ≤ 0.05. There were statistically significant differences between the expression levels of ET-1/ETAR axis in the four-layered human primary tooth EO in different fetal ages (13-23 weeks). Besides, there were significant differences between the expression levels of ET-1/ETAR axis in all layers of human primary enamel organ and types of teeth. Due to the profile of expression of ET-1/ETAR axis, it can be concluded that this axis contributes to the differentiation of all human primary EO layers and secretion of enamel. ET-1/ETAR axis is one of the signaling molecules, which may have crucial roles in tooth development.

The Effect of Bonding Strategy and Aging on Adhesion to Primary Enamel: An In-Vitro Study.

Resin composites are commonly used in pediatric dentistry, but there is limited evidence on adhesion to primary teeth, especially primary enamel. In this study, three bonding strategies were assessed - one-step self-etch (1-SE), two-step self-etch (2-SE), and three-step etch-and-rinse (3-ER) - by measuring the immediate and aged microshear bond strength (µSBS) to sound primary enamel. 120 extracted human primary molars with sound buccal surfaces were used for µSBS testing. Six adhesive protocols (two per bonding strategy) were selected and µSBS was measured either after 24 h or 10,000 thermal cycles (n = 10). Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to determine failure modes. Furthermore, 18 primary molars were etched using the different adhesive protocols (n = 3) for the measurement of surface roughness (Sa) using CLSM and morphological analysis using SEM. After 24 h, there was no significant difference in µSBS between 1-SE and 2-SE strategies (p = 0.96), but the 2-SE strategy yielded significantly higher µSBS after thermocycling (p < 0.001). The highest µSBS was obtained using the 3-ER strategy regardless of aging (p < 0.001). The 3-ER strategy clearly exposed enamel prisms and resulted in the highest Sa (p < 0.001). In contrast, if SE strategies were used, enamel prisms were barely recognizable, and Sa was not significantly different from baseline (p > 0.95). The 3-ER strategy is optimal for bonding to primary enamel. The etching effect of SE strategies is weaker, resulting in lower µSBS. Thermocycling revealed that the bonding durability of the 1-SE strategy is inferior to that of to multi-step strategies.

What is the most effective technique and composite for bonding aligner attachments to primary enamel?

Objectives: This in vitro study evaluated the shear bond strength (SBS) of aligner attachments bonded to primary teeth with different techniques and composites. Material and Methods: Eighty attachments were prepared using five composites: An aligner composite (GC aligner connect [GAC]), two flowable restorative composites (G-aenial universal injectable and Estelite universal super low flow), and two one-step orthodontic composites integrated with primer (GC ortho connect flow and GC ortho connect). Aligner and restorative composites were applied with self- or total-etch (SE or TE) modes of universal adhesive (G-Premio Bond). Self-adhering composites integrated with primer were applied after acid etching. Excessive composites were removed with tungsten carbide burs after the polymerization of resin attachments. All primary teeth were exposed to thermocycling. Bond strength was determined with a universal testing machine. Remnant resin attachment was scored with the resin attachment remnant index. Kruskal–Wallis, Mann–Whitney, and Chi-square tests were used for statistical analyses. Results: The self-etching technique demonstrated lower SBS values compared to total-etching and self-adhering groups regardless of composite type. The highest SBS was found with aligner composite (GAC) and the total-etching technique. Self-adhering composites showed sufficiently high SBS values comparable with GAC and TE bonding techniques. The amount of remnant attachment was significantly higher in the total-etching and self-adhering groups compared to SE bonding with GAC (P &lt; 0.05). Conclusion: Self-adhering orthodontic composites can be preferred to simplify the bonding of aligner attachments to primary enamel after acid etching.

Evaluation of Remineralization Potential of Natural Substances on Artificially Induced Carious Lesions in Primary Teeth: An In Vitro Study.

To evaluate the remineralizing potential of natural substances on artificially induced caries lesions in primary teeth. A total of 50 primary molar teeth were selected and subjected to a demineralization process. Then samples were randomly divided into five groups for the remineralization process. Group I-colophony, group II-5% sodium fluoride (NaF) + colophony, group III-grape seed extract (GSE) + colophony, group IV-5% NaF + colophony + 10% peptide, and group V- GSE + colophony + 10% peptide. All the groups were subjected to remineralization using a brushing stimulator for 3,000 cycles. Assessment was done using Vickers hardness testing machine for evaluating the enamel surface microhardness (SMH) and scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) for evaluating the surface morphology and mineral content, before and after demineralization and after remineralization, the obtained data was analyzed statistically using IBM Statistical Package for the Social Sciences (SPSS) software version 25. The enamel microhardness results of this study revealed that remineralization of enamel was highest in group V (212.83 ± 64.416) and least in group II (137.83 ± 26.324) p-value of 0.038. SEM-EDX analysis revealed high calcium (Ca) and fluoride (F) content in groups II and IV, which was significant (p-value of 0.001) from other groups. Surface morphology evaluated with SEM revealed spherical globular agglomerates and scaffolding deposits on the enamel surface in groups III and V resembling the remineralization process. Grape seed extract (GSE) with colophony and peptide is a superior natural alternative to NaF. Colophony also exhibited remineralizing potential in primary enamel. Natural remineralizing agents like GSE, colophony, and its combination serves as a potential alternative to overcome the toxic effect on long-term usage of F. These natural substances can be applicable in clinical conditions by incorporating toothpaste and varnish, which can be used as an alternative or adjuvant to the topical application of F. Pooja V Ravi, Rajakumar S, Kavitha Ramar. Evaluation of Remineralization Potential of Natural Substances on Artificially Induced Carious Lesions in Primary Teeth: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(2):244-250.

The role of O-GlcNAcylation mediated by OGT during tooth development.

To understand the mechanisms underlying tooth morphogenesis, we examined the developmental roles of important posttranslational modification, O-GlcNAcylation, which regulates protein stability and activity by the addition and removal of a single sugar (O-GlcNAc) to the serine or threonine residue of the intracellular proteins. Tissue and developmental stage-specific immunostaining results against O-GlcNAc and O-GlcNAc transferase (OGT) in developing tooth germs would suggest that O-GlcNAcylation is involved in tooth morphogenesis, particularly in the cap and secretory stage. To evaluate the developmental function of OGT-mediated O-GlcNAcylation, we employed an in vitro tooth germ culture method at E14.5, cap stage before secretory stage, for 1 and 2 days, with or without OSMI-1, a small molecule OGT inhibitor. To examine the mineralization levels and morphological changes, we performed renal capsule transplantation for one and three weeks after 2 days of in vitro culture at E14.5 with OSMI-1 treatment. After OGT inhibition, morphological and molecular alterations were examined using histology, immunohistochemistry, real-time quantitative polymerase chain reaction, in situ hybridization, scanning electron microscopy, and ground sectioning. Overall, inhibition of OGT resulted in altered cellular physiology, including proliferation, apoptosis, and epithelial rearrangements, with significant changes in the expression patterns of β-catenin, fibroblast growth factor 4 (fgf4), and sonic hedgehog (Shh). Moreover, renal capsule transplantation and immunolocalizations of Amelogenin and Nestin results revealed that OGT-inhibited tooth germs at cap stage exhibited with structural changes in cuspal morphogenesis, amelogenesis, and dentinogenesis of the mineralized tooth. Overall, we suggest that OGT-mediated O-GlcNAcylation regulates cell signaling and physiology in primary enamel knot during tooth development, thus playing an important role in mouse molar morphogenesis.

Nano-hydroxyapatite preparation for the remineralization of primary tooth enamel surface subjected to liquid medication: An observational study.

Acid-induced demineralization may be caused by the consumption of liquid medications routinely administered to children. Therefore, different remineralizing agents, such as fluorides and nano-hydroxyapatite, have been added to oral care products to remineralize erosive lesions.This study was conducted to compare the effectiveness of 1% nano-hydroxyapatite suspension and 2% sodium fluoride solution on the surface texture of primary teeth enamel previously exposed to liquid drugs. Thirty posterior primary teeth were extracted and grouped depending on the remineralizing agent used: (A) nano-hydroxyapatite and (B) sodium fluoride. Groups A and B were subjected to liquid medication in two subgroups: cephalexin (cephalexin monohydrate) and ParAzar (acetaminophen), followed by remineralization with 1% nano-hydroxyapatite suspension and 2% sodium fluoride solution. An atomic force microscope was used to analyze the surface texture of the primary tooth enamel by measuring the roughness and waviness parameters of the tested surfaces. The examination was performed at baseline, 7 days after exposure to drugs, and after exposure to remineralizing agents. A significant difference in enamel surface roughness between nano-hydroxyapatite-treated surfaces and sodium fluoride-treated surfaces was observed. A significant change was observed in the surface waviness of the primary enamel surface of sodium-fluoride-treated teeth that were subjected to cephalexin. Routine use of liquid medications could have a negative impact on primary enamel surface topography, because the primary tooth enamel is less mineralized than permanent tooth enamel. Our results show that compared with a 2% sodium fluoride solution, a 1% nano-hydroxyapatite suspension can be used for remineralizing and restoring defects of the enamel surface of primary teeth following exposure to liquid medication, thus reinforcing dental tissues with higher efficacy.

Effect of conditioning on microtensile bond strength of universal adhesives to primary enamel

Effect of conditioning on microtensile bond strength of universal adhesives to primary enamel

Eda controls the size of the enamel knot during incisor development.

Ectodysplasin (Eda) plays important roles in both shaping the developing tooth and establishing the number of teeth within the tooth row. Sonic hedgehog (Shh) has been shown to act downstream of Eda and is involved in the initiation of tooth development. Eda-/- mice possess hypoplastic and hypomineralized incisors and show changes in tooth number in the molar region. In the present study we used 3D reconstruction combined with expression analysis, cell lineage tracing experiments, and western blot analysis in order to investigate the formation of the incisor germs in Eda-/- mice. We show that a lack of functional Eda protein during early stages of incisor tooth germ development had minimal impact on development of the early expression of Shh in the incisor, a region proposed to mark formation of a rudimental incisor placode and act as an initiating signalling centre. In contrast, deficiency of Eda protein had a later impact on expression of Shh in the primary enamel knot of the functional tooth. Eda-/- mice had a smaller region where Shh was expressed, and a reduced contribution from Shh descendant cells. The reduction in the enamel knot led to the formation of an abnormal enamel organ creating a hypoplastic functional incisor. Eda therefore appears to influence the spatial formation of the successional signalling centres during odontogenesis.