Concentration of MMP20 and KLK4 during enamel development in MIH
Zilberman U, Shama A, Amara R and Hassan J
Published on: 2023-07-10
Abstract
Objectives:
To determine the concentration of matrix of metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4) during the maturation stage of amelogenesis on MIH/SPMH development. During the secretory stage of amelogenesis the ameloblasts start secreting large amounts of enamel matrix proteins. The enamel proteins are removed during the maturation stage by two proteases: MMP20 and KLK4.
Design:
During 2018, concentration of MMP20 and KLK4 were determined from blood samples of 500 children aged 0-5 years old. Four years latter a sample of 89 children (aged 6-7 years) were clinically examined for MIH/SPMH signs on their permanent or deciduous molars by trained residents, according to EAPD criteria.
Results:
25 children showed signs of MIH/SPMH out of 89. The concentration of MMP20 and KLK4 were statistically analized in the children with MIH/SPMH in comparison with children without. In children with clinical signs of MIH/SPMH the concentrations of of KLK4 was significantly lower (4.99 in comparison with 7.84 in control group; P value=0.02). The concentration of MMP20 was higher in the MIH/SPMH group but without statistical significance (P value=0.24).
Conclusions:
KLK4 is expressed during the transitional and maturation stages and is responsible for protein degradation. In the absence of KLK4 substantial retention of enamel proteins occurs in the enamel. It may be postulated that the retention of enamel proteins in MIH/SPMH teeth was caused by reduced concentration of KLK4 during transitional and maturation stages of amelogenesis.
Keywords
Molar incisor hypomineralization (MIH), Matrix metalloproteinase 20 (MMP20), Kallikrein 4 (KLK4), Enamel, HypomineralizationIntroduction
Molar Incisor Hypomineralization (MIH), defined in 2001 as hypomineralization of systemic origin of one to four first permanent molars frequently associated with affected permanent incisors, is described as demarcated qualitative defects of enamel [1]. It may affect one to four permanent molars with varying degrees of severity [2]. Hypomineralization of second primary molars (SPMH) is a predictor for MIH in permanent dentition. The prevalence of MIH, based on a meta-analysis that included 70 studies estimated the global average prevalence of 14.2% [3], and may be the most common of all dental developmental defects [4]. A systematic review found a prevalence of of 13.5% for MIH and 3.6% for SPMH, and affected incisors were observed in 36.6% of the MIH cases [5]. America has the highest prevalence (15.3%) and Asia had the lowest prevalence (10.7%). In Israel the prevalence of MIH was 17.1% in the Jewish children and 18% in the Arab children and prevalence of SPMH was 2.7% [6]. More intriguing was the finding that in the youngest group (6-10 years) the MIH prevalence was 2-3% higher than in the older group (10-16 years). A similar result of increased prevalence of MIH after 6 years was reported from Brazil [7]. The aetiology of MIH/SPMH remains unclear, although both environmental and genetic factors are suspected to play roles [8,9]. Perinatal factors, such as prematurity and caesarean delivery, were associated with an increase risk of developing MIH. Postnatal factors, like measels, urinary tract infections, otitis media, gastric disorders, bronchitis, kidney diseases, pneumonia and asthma, were also associated with MIH. An increasing number of studies highlight the genetic and epigenetic influences in the development of MIH [10]. Through a family-based association study, it was suggested that variations in genes related to amelogenesis were associated with the susceptibility to develop MIH [11]. In first molars affected by MIH, the mineral content was significantly lower. The reduction of calcium was by 35% and of phosphate by 60%. The oxygen and carbon content was increased by more than 30%. In primary molars affected by hypomineralization, the reduction in minerals was by 10% of phosphate and 40% of calcium on demarcated opacities and by 64% phosphate and 72% calcium at the border of breakdown of enamel [12].
Mineralization during amelogenesis is not completed in enamel with MIH lesions. Enamel development (amelogenesis) has four stages, defined by the morphology and function of the ameloblasts: pre-secretory, secretory, transition and maturation. During the pre-secretory stage the ameloblasts develop an extensive protein synthetic apparatus and prepare to secrete the organic matrix of enamel. Secretory stage enamel is protein rich and has a soft consistency. The ameloblasts starts secreting large amounts of enamel matrix proteins and elaborate and organize the entire enamel thickness. In association with newly secreted proteins, long thin mineral ribbons form. The ameloblasts secrete several proteins into the enamel matrix: amelogenin (AMELX) (80 - 90% of the organic matter), ameloblastin (AMBN) (only 5%) and enamelin (ENAM) (3 - 5%). During the maturation stage the previously secreted matrix proteins are removed and the crystallites expand in volume [13,14,15]. All three matrix proteins are crucial for healthy enamel formation and their orchestrated removal is critical to allow for expansion of the mineral phase and completion of enamel mineralization [16]. During enamel formation almost all of the protein supporting mineral formation is removed by two proteases: matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4) [17]. During the secretory stage MMP20 is secreted and its role is to cleave the most abundant enamel matrix protein, amelogenin [18,19,20]. KLK4 is secreted during the transition and maturation stages of amelogenesis, immediately preceding the point where the quantity of enamel proteins in the matrix drops. Its role is to cleave the residual enamel matrix, facilitating its removal from the enamel layer and allowing the crystals to grow in width and thickness until adjacent crystals contact. This activity is essential for hardening of the enamel layer [21].
The affectd permanent or primary molars shows hypomineralization at the occlusal two thirds of the crowns, while the affectd incisors showed hypomineralization at the incisal third of the crown. The mineralization of the second primary molar begins at 18th week in utero and of first permanent molars at birth and the crown is completed at 2.5-3 years. The mineralization of the permanent incisors begins at 3-4 months and the crown is completed at 4-5 years [22]. So, it may be postulated that the hypomineralization occurs during the last trimester of pregnancy till 5-6 months for primary molars and from birth till age of two years for first permanent molars and incisors.
The aim of this research was to correlate between the concentration of MMP20 and KLK4 to appearance of MIH/SPMH in a healthy group of children without significant childhood diseases.
Material And Methods
The main problem faced is that the primary molars errupts by the age of 20-30 months and first permanent molars errupts at the age of 6-7 years while the hypomineralization takes place during pregnancy till first two years of life. Due to that, the research was performed in two phases. Both phases of the research were approved by the Helsinki committee of the French Medical Center, Nazareth, Israel (no. 0049-17-EMC).
The first phase included examination of enzymatic concentration of MMP20 and KLK4 in serum of 500 children 0-5 years old during 2018, and the mathodology and results were published in 2020 [23]. No clinical examination of the oral cavity of these children was performed.
The second phase was performed four years latter, during 2022. 89 children, aged 1-3 years old during the first phase were clinically examined at the age of 5-7. The clinical examinations were performed by trained residents in pediatric dentistry, according to EAPD criteria for the judgement of MIH [24]. The concentration of MMP20 and KLK4 in children with/without MIH were statistically anlyzed using an independent sample statistics of Student's t test of the SPSS program.
Results
In 25 children MIH/SPMH was clinically detected, in 14 out of 48 boys (29%) and in 11 out of 41 girls (27%). In the boys group, 9 showed hypomineralization of permanent molars and 5 showed hypomineralization of second primary molars. In the girls group, 5 showed MIH and 6 showed SPMH. No significant differences were found between boys and girls. The concentration of MMP20 and KLK4 at the age of 1-3 years were correlated to the clinically appearance of MIH/SPMH. Table 1 shows the statistical analysis. The concentration of MMP20 was higher in children with MIH/SPMH but without any statistical significance (P=0.24). The concentration of KLK4 was significantly lower in children with MIH/SPMH (P=0.02).
Discussion
This two phase’s research showed that the concentration of KLK4 during the first years of life was significantly lower in children with clinical MIH/SPMH. MMP20 is secreted early during the secretory stage, and cleaves enamel matrix proteins, amelogenin and ameloblastin, at a limited number of sites [19,25]. Uncleaved enamel proteins are prominent in the secretory stage of MMP20 null mice [26]. MMP20 null mice show severe enamel malformation, and mutations in human MMP20 cause a nonsyndromic form of amelogenesis imperfecta [27,28,29].
The beginning of KLK4 expresion occurs during the maturation stage of amelogenesis [30], and serves a more degradative function. KLK4 can be activated by MMP20, but not only. It is active also in MMP20 ablated mice. [26]. Its cleavage pattern complements that of MMP20, so that the matrix proteins are degradated further into small polypeptides that diffuse to the enamel surface where they are reabsorbed by the ameloblasts. KLK4 null mouse enamel is normal in thickness, but the enamel is hypomineraized. Two different human KLK4 mutations are known to cause autosomal recessive hypomaturation Amelogenesis Imperfecta. The enamel was normal in thickness, but showed only a slight increase in opacity over the underlying dentin, due to decreased mineral content. This soft enamel fractured from the occlusal surfaces of molars [31]. The mineral level reaches approximately 80% of normal enamel, and showed increase surface roughness and a susceptibility to attrition and enamel fracture in mice [20,32]. The mineral level in human affected molars showed higher reduction of calcium and phosphate, and in many cases breakdown of enamel occurred after eruption of affected molars [12], than reported in KLK4 null mices. Both humans and mice have shown us that KLK4 is essential for enamel to achieve its final hardened form.
The results of this research are the first indication that the reduced mineralization in MIH/SPMH affected teeth may be caused by a temporary reduction of KLK4 concentration during the second half of pregnancy till 2 years of age. Why it happens and how the activity of KLK4 returns to normal or maybe some other proteases will take its function, are the subjects of more researches.
Table 1: The concentration of MMP20 and KLK4 in affected and healthy children.
|
MMP20 |
KLK4 |
||
|
MIH/SPMH-yes |
MIH/SPMH-no |
MIH/SPMH-yes |
MIH/SPMH-no |
N. |
25 |
64 |
25 |
64 |
Mean |
52.22 |
33.06 |
4.99 |
7.84 |
SD |
55.79 |
25.19 |
2.36 |
5.84 |
test |
t-test |
|
t-test |
|
statistic |
|
1.24 |
|
-2.43 |
DF |
|
15.17 |
|
46.96 |
P value |
|
0.24 |
|
0.02 |
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