Matrix Metalloproteinase-20 immunolocalization in rat first molar tooth development after treatment with amoxicillin

Amoxicillin is one of the most commonly prescribed antibiotics in children. It is, therefore, administered as the first choice of antibiotics for respiratory, gastrointestinal, neuronal, and skin infections. This study aimed to determine whether amoxicillin use affects the formation of dentin and enamel during the secretion and early phases of mineralization. Sixteen pregnant adult Wistar rats were equally divided into two groups. The first group that did not receive the drug was prescribed a saline solution (control group), and the other group received 250 mg/kg/day amoxicillin (study group). The treatments were administered daily by oral gavage from the 13 th gestation day to the end of gestation. After birth, the newborn also received the same treatment as their mothers from the first day of birth until 7 or 12 days after birth. The newborns will be sacrificed at 7 and 12 days postnatally. The jaws will be dissected, and the maxilla, where the samples are fixed in 10% formaldehyde solution, and the upper first molars will be analyzed. Immunostaining with MMP-20 on day 7 and 12 were performed. A significant difference in the positive expression of ameloblast for MMP20 antibody between groups was observed at 7 days (P = 0.014) and no significant difference at 12 days (P = 0.347). A significant difference for positive expression of odontoblast for MMP20 antibody between groups in both durations was observed at 7 days (P = 0.178) and not a significant difference at 12 days (P = 0.143) and powerful expression in enamel matrix during early mineralization stage in the control group and strong expression in the study group. The current findings indicate that amoxicillin affects the expression of MMP20 during the secretory stage by decreasing the expression of MMP20 in ameloblast, and the expression of MMP20 decrease in cells during the early mineralization stage in both groups at different rate.


Introduction
The mammalian dentition comprises three types of teeth: incisors, canines, and molars, all of which come from separate parts of the oral epithelium 1 .The dental lamina is the earliest sign of tooth formation in humans. 2 .During the bud, cap, and bell phases, epithelial morphogenesis determines the size and form of the tooth crown.Ameloblasts and odontoblasts, which differentiate at the intersection between the epithelium and the mesenchyme, secrete the tooth-specific hard tissue enamel and dentin 3 .Dentinogenesis happens both prenatally and postnatally and may be seen to a lesser level throughout one's life when secondary and tertiary dentin is created. 4.Dentin is a crystalline substance that is somewhat tougher than bone but less hard than enamel 5 .The Odontoblasts are specialized cells that generate dentin and have distinct morphological features, such as the extension of cytoplasmic processes into dentinal tubules 6 .Enamel formation is a two-step process.When formed initially, enamel only mineralizes to around 30% of its original state.After the whole thickness of the enamel layer has been established, losing the organic matrix and water and adding minerals amplifies to achieve higher than 96% mineral content.Amelogenesis comprises three functional stages: presecretory, secretory, and maturity stages 7 .In the secretory stage, ameloblasts secrete four separate proteins into the enamel matrix while this happens.Amelogenin (AMELX), ameloblastin (AMBN), and enamelin (ENAM) are structural proteins, while matrix metalloproteinase-20 is a proteinase (MMP20, enamelysin).During the maturation stage of tooth formation, after the opposition stage, when the enamel matrix is only partly mineralized at around 30%, it completes its mineralization process to a whole level of 96 percent 5 .Amoxicillin is a bactericidal antibiotic with a wide range of activity against grampositive and gram-negative bacteria.Amoxicillin is one of the most regularly prescribed antibiotics in primary care 8.According to the former FDA categorization system, amoxicillin is a pregnancy category B medicine, indicating no studies establishing a definite danger 9 .According to previous studies, amoxicillin disrupted the early phases of amelogenesis, resulting in a loss of enamel matrix and morphological changes in ameloblasts.Amoxicillin may delay the development of ameloblasts into secretory cells 10,11.MMP20 (matrix metallopeptidase 20) and KLK4 (kallikrein-related peptidase 4 ) are two principal proteinases released by ameloblasts expressed at different phases during enamel development 12 .Secretory stage ameloblasts primarily express MMP20, and its expression drops dramatically as the ameloblasts mature 13,14 .The current investigation aimed to ascertain the effects of giving Wistar rats a 250 mg/kg dose of amoxicillin before and after birth on the enamel and dentin structures.

Materials and Methods
All experimental procedures were carried out according to the ethical principles of animal experimentation of the College of Dentistry, University of Baghdad.Sixteen female rats (Rattus norvegicus albinus), aged 2-3 months and weighing approximately 175-225 gm, were used in this study.The rat was kept under controlled temperature (23± °C) and humidity (55 ± 5%), with a 12-h /12-h light / dark cycle.Food ad libitum and water were available.The first preparation of amoxicillin dose according to the animal weight, followed by animal preparation, which was done in the animal house of the College of Science/ University of Kufa, and then immunohistochemical preparation was done in a technical laboratory in Najaf.Sixteen pregnant rats were randomly assigned into two groups.The control group (8 pregnant rats) received normal saline twice weekly.The experimental group (8 pregnant rats) received 250 mg/ kg amoxicillin (2mg/ kg ) daily from 13 days to 21 days of pregnancy because amoxicillin can pass through the placenta and the umbilical cord during this time.It also starts to build the first maxillary molar in rats, which only have a permanent dental system 15.After birth, the 48 pups from each 16 mother, from postnatal day 1 until postnatal day 7 or day 12, received the same treatment as their respective mother.Choosing the time of postnatal periods in this study related to the scientific base, as at 7 days old, rats represent the time in which hard tissue, dentin and enamel deposition occurred 16 .12-day-old rats show the end of the secretory stage in the cervical area and the enamel mineralization stage in the cusp (the final period of secretion and beginning of enamel maturation) 10 .
After scarification of 7-and 12-day-old neonatal rats in each group, the head of the rats was separated from the body, and all the soft tissue of the head was removed.Then, the head was cut into two parts through the sagittal section, the maxilla only that contained the upper first molar, and then specimens were placed in 10% formalin for 24 hours.After thorough decalcification, tissue samples were processed in the standard paraffin blocks technique.All analyzed samples' paraffin-embedded blocks were cut into serial sections of 4 μm thickness and put on glass slides for H&E staining.Other 4 μm thick slices were placed on positively charged slides for immunohistochemical localization of MMP20.The IHC assay procedure was performed according to the manufacturer's instructions, Rabbit polyclonal antibody (MMP-20) from Abcam company USA (ab198815).The cellular localization of this MMP20 antibody is secreted in extracellular space > extracellular matrix.Furthermore, the Mouse/Rabbit PolyDetector Plus HRP/DAB detection IHC kit from BIOSCIENCE, USA, was used for MMP-20 detection.The scoring was done under a light microscope.Subjective methods were performed; the percentage of the positive cells for the MMP20 antibody was scored and estimated visually by calculating the number of positive ameloblast cells and odontoblast cells, dividing them by the number of total cells and multiplying them by 100.This procedure was repeated for the six sections of the same block, and then the mean value of the 4 fields of each section and mean score were calculated using magnifying power lens X 40.To evaluate MMP-20 immunoexpression in each field, Cusp tips and the medial and cervical portions of the upper first molar were considered 10 .The scoring was graded as follows 17 : : "0" score = 0%, "1" score =1-25% 26-50% , "3" score = 51-75% , "4" score =75-100% score ="2"

Statistical analysis:
Expression MMP-20 in ameloblast and odontoblast in both groups was scored using the Wilcoxon sum rank test.All analyses were performed using Statistical Package for Social Science (SPSS version 21, Chicago In press, Illinois, USA).The level of significance was P ≤ 0.05.

Results
The immunohistochemical finding of the upper first molar tooth germ in a 7-day control group shows a powerful positive expression of MMP-20 in ameloblast and odontoblast.Also, the result showed mild expression in presenting and negative expression in the enamel matrix and dentin Figure 1, as in the study group, The immunohistochemical localization of MMP20 was strong in the distal ends of the ameloblasts, odontoblasts and stratum intermedium (SI).While weak expression in dentin and negative expression in enamel Figure 2. Immunohistochemical view of the upper first molar tooth germ at 12 days in the control group shows a powerful expression of MMP20 in the enamel matrix And strong expression in ameloblasts, odontoblast and stratum intermedium, while negative expression in dentin, mild expressions in predentin.The Immunohistochemical findings of tooth germ in a 12-day study group showed robust expression of MMP20 in the enamel matrix and negative expression in dentin.The immuno-reactive products were primarily concentrated in the ameloblasts' distal side of the cytoplasm, and positive expression of MMP20 was seen in dental pulp and odontoblasts Figure 3 A and B.

Figure 3. A. View of the upper first molar in a 12day control rat shows positive expression of MMP20 in enamel matrix (EM), ameloblasts (AM), odontoblasts (OD), and stratum intermedium (SI) while negative expression in dentin(D), B. View of upper first molar in 12day study rat show positive expression of MMP20 in enamel matrix (EM) and negative expression in dentin (D) DAB stain with counter stain hematoxylin×40.
Table 1.shows group comparison differences by Wilcoxon sum rank test for Ameloblasts scores.The score results showed that positive expression of ameloblast for the MMP20 antibody was more in control than in the study group in both periods.A significant difference in the positive expression of ameloblast for MMP20 antibody between groups was observed at 7 days (P = 0.014) and no significant difference at 12 days (P = 0.347).While Table (2) shows the duration comparison difference in both groups, the score results showed a highly significant duration difference in ameloblast expression for MMP20 antibody in both the control and study groups, where the p-value in the control group is (P = 0.000201) and in the study group is (P = 0.006).

Days Groups
Control Study  Shows that ameloblast expression for MMP20 antibody in the control group was more than that in the study in both 7 and 12 days duration with a higher score value of positive expression of ameloblast for MMP20 seen in the control group at 7 days duration and lower score value in study group at the same period.Table 3 shows group comparison differences by Wilcoxon sum rank test for odontoblast cells.The score showed that the positive expression of odontoblast for the MMP20 antibody was more in control than in the study group in both periods.Nonsignificant differences for positive expression of odontoblast for MMP20 antibody between groups in both durations were observed at 7 days (P = 0.178) and 12 days (P = 0.143).At the same time, Table (4) shows duration comparison differences between groups for odontoblast expression for MMP20 antibody.The score results showed no significant difference in odontoblast expression for MMP20 antibody in both the control and study groups, where the p-value in the control group is (P = 0.478) and in the study group is (P = 0.178).

Days Groups
Control Study  show that odontoblast expression for MMP20 antibody in the control group was more than that in the study in both 7-and 12-day duration with a higher score value of positive expression of odontoblast for MMP20 seen in the control group at 12 days duration and lower score value in study group at the same period.

Discussion
Recent years have brought increased attention to the role of enamel-forming enzymes in tooth development and how their expression changes can result in disorders such as amelogenesis imperfecta 18,19 .Evidence suggests that MMP-20 and other enzymes, which function during the secretion and early phases of mineralization and break down enamel proteins to promote the formation of hydroxyapatite crystals, are essential to the enamel mineralization process.KLK-4 works to thicken crystals during the late mineralization/maturation stage 20,10 .The primary proteins secreted during the secretory phase of the protein matrix are amelogenins, which are crucial for the formation and development of hydroxyapatite crystals 10 .This matches 21, which exhibits more intense amelogenin in the secretory stage and weak amelogenin expression in the control group's enamel matrix in the early maturation stage.Enamelysin (MMP-20) is the foremost enamel matrix-processing enzyme.Its expression initiates prior to the onset of dentin mineralization and continues throughout the secretory stage of amelogenesis 22.In the present study, it has been found that there was a positive expression of MMP20 in Ameloblast cells and odontoblast but at different rates between groups.The immunoreactivity for MMP20 was more intense in the control group than amoxicillin -the treated group over 7 days period, Especially in ameloblast cytoplasm, This finding is consistent with 23 , Who showed that the antibiotic amoxicillin reduces the development of the enzyme matrix metallo-peptidase-20 (MMP20), which is essential for the extraction and destruction of enamel proteins.The immunoreactivity for MMP20 was more intense in 7 days (secretory stage) in both groups than in 12 days because MMP-20 is a protease that is secreted with enamel proteins during the early (secretory) stage of amelogenesis when the crystallites are growing predominantly in length 24 .I also agree with 25,26 , who mentioned that secretory stage ameloblasts primarily express MMP20, and its expression drops dramatically as the ameloblasts mature.Furthermore, I agree with 27 , who mentioned that As enamel thickens during the secretory stage of development, matrix metalloproteinase-20 (MMP-20, enamelysin) is first expressed, and kallikrein-4 (KLK-4, EMSP1) is later expressed during the maturation stage as the enamel hardens.MMP20 expression was positive in ameloblast and odontoblast in both groups.This corresponds to 28 .who mentioned that ameloblasts and odontoblasts both produce MMP20.During the secretory stage, ameloblasts synthesize MMP20, and their cleavage products are absorbed and destroyed by the same cells 29,17 .At the same time, the role of MMP20 in dentinogenesis is unknown 12 .The expression of MMP20 was strong in the enamel matrix in the early mineralization stage because the roles of MMP-20 and KLK4 in the development of dental enamel are to promote the orderly substitution of mineral for organic matrix, resulting in an enamel layer that is tougher, less porous, and free of residual enamel proteins 24 .Also, the role of MMP20 in the enzymatic activity of the breakdown of the freshly deposited matrix is required to replace enamel matrix proteins with hydroxyapatite crystals 29,17 .During this stage, when the first molars of 12-day-old rats were examined for MMP-20 immunoexpression in ameloblast and odontoblast, there were no significant differences among the treated and control groups, and that coincided with 10 .MMP20 shows positive expression in ameloblast and odontoblast.However, to a lesser degree than 7, the old group agrees with 20 , who mentioned that MMP-20 plays a role in enamel mineralization by degrading enamel proteins during the secretory and early mineralization stages, allowing hydroxyapatite crystals to form.During the late stages of mineralization/maturation, Kallikrein4 (KLK-4) acts on crystal thickening.

Conclusion
The current findings indicate that amoxicillin affects the expression of MMP20 during the secretory stage by decreasing the expression of MMP20 in ameloblast, and the expression of MMP20 decrease in cells during the early mineralization stage in both groups at different rate.

Figure 4 .
Figure 4. Shows that ameloblast expression for MMP20 antibody in the control group was more than that in the study in both 7 and 12 days duration with a higher score value of positive expression of ameloblast for MMP20 seen in the control group at 7 days duration and lower score value in study group at the same period.

Figure 4 .
Figure 4. Groups comparison difference for expression of ameloblast for MMP20 in both healing periods.

Figure 5 .
Figure 5.show that odontoblast expression for MMP20 antibody in the control group was more than that in the study in both 7-and 12-day duration with a higher score value of positive expression of odontoblast for MMP20 seen in the control group at 12 days duration and lower score value in study group at the same period.

Figure 5 .
Figure 5. Groups comparison difference for expression of odontoblast for MMP20 in both healing periods.