**2.1 Amelogenesis and developmental defects of enamel**

Tooth development is strictly genetically controlled but sensitive to environmental disturbances (Suckling et al., 1988) since teeth have been formed they do not undergo remodeling. (Brook, 2009) During dental development, a single layer of inner enamel epithelial cells undergoes a remarkable change in cell shape in preparation for the secretion of enamel extracellular matrix. These cells develop into tall ameloblasts with cellular extensions called Tomes' processes, which function during enamel matrix secretion. Following generation of the enamel layer, the ameloblasts shorten and reorganize during the transition stage; they then enter maturation, where they change histologically from ruffle-ended to smooth-ended at the location where Tomes' processes have retracted. These cells reduce the enamel protein content and increase the mineral content so that the enamel layer can develop into the hardest tissue in the body. Finally, the cells shorten further and adhere to the enamel surface until just before eruption of the tooth into the oral cavity (Smith , 1979). In other words, enamel formation occurs in three stages:


The mineralization of the enamel matrix is described as a two-step process. Firstly, the ameloblasts secrete an organic matrix that is immediately mineralized to about 30% by weight. Secondly, when the full thickness of enamel has been secreted by an ameloblast, a progressive increases in mineral content begin. Smooth-ended ameloblasts remove of water and proteins from the enamel matrix, whereas ruffle-ended ameloblasts participate in the active transport of calcium and phosphate into the matrix. The principal proteins acted in the enamel matrix are:


According to BROOK, 2009 (Brook, 2009) in the secretory stage the enamel protein matrix deposited by the ameloblasts is predominantly formed of amelogenin (85%). At the midsecretory stage for appositional crystal growth and structural maintenance amelogenin is essential. However, while enamelin contributes less than 5% of the matrix it plays a major role in controlling the initiation of hydroxyapatite formation in early amelogenesis, being necessary for creating and maintaining enamel crystallite elongation at the mineralization front immediately adjacent to ameloblasts. The further enamel protein ameloblastin is a celladhesion molecule that maintains the differentiation stage of secreting ameloblasts and

Molar Incisor Hypomineralization:

(Jalevik et al., 2005)

increase in C content. (Fearne et al., 2004)

Morphological, Aetiological, Epidemiological and Clinical Considerations 429

no evidence of defective structure. At a more occlusal level, the defect is confined to the inner enamel while the outer enamel does not appear to be affected. As move occlusally, the hypomineralisation becomes more evident, eventually spreading to span the entire thickness of the enamel. The defects usually did not involve the cusp tips; but if a marginal

Microstructural analysis of sound and hypomineralised enamel showed two marked changes in microstructure in the MIH affected enamel region; less dense prism structure with loosely packed apatite crystals and wider sheath regions. (Xie et al., 2008) These changes appear to occur during enamel maturation and may be responsible for the marked reduction in hardness and elastic modulus of the affected enamel. (Fagrell et al., 2010) In addition, the enamel in the transitional region adjacent to the demarcated defects in MIH has also notable alterations in their prism sheaths. Despite the translucent, normal appearance, the transitional region between the affected and unaffected regions in MIH teeth had weakened prism sheaths which compromised its overall mechanical properties. (Chan et al., 2010) The reason for this is unclear but may be also related to the lack of organization of the enamel crystals due poorly demarcated prism boundaries in the affected regions (Mahoney et al., 2004) and the packing of the crystals seemed to be less tight and less well organized in the porous parts. The borders of the enamel rods were indistinct and the interrods zones hardly visible, or the rods were very thin with wide interrod zones.

Semi-quantitative analysis by energy dispersive X-ray spectrometry in extracted MIH affected teeth showed that the mineral composition of this type of enamel is low (Javelik & Norén, 2001), on average the mineral density is about 19 % lower than sound enamel (Baroni & Marchionni, 2011, Farah et al., 2010a, Jalevik & Noren, 2000, Schulze et al., 2004), there is a decrease in Ca:P ratio in the enamel (Rodd et al., 2007a, Jalevik, 2001) related to an

Also, MIH enamel has substantially higher protein content than normal enamel, but a nearnormal level of residual amelogenins. This characteristic distinguishes MIH from hypomaturation defects that contain high residual amelogenins such as *Amelogenesis Imperfecta* or Fluorosis (Mangum et al., 2010a, Wright et al., 1996, Wright et al., 1997) and in turn typifies MIH as a hypocalcification defect as mentioned above. Pathogenically, it points to a pre-eruptive disturbance of mineralization involving albumin probably due to an overabundance of albumin that interferes with the mineralisation process. It justifies the porosities exhibited in the subsurface (Jalevik & Noren, 2000) because albumin degradation may be a prerequisite for maximal crystal growth in the maturation stage of enamel. (Farah et al., 2010b, Farah et al., 2010c, Mangum et al., 2010b) The presence of excessive albumin seemed to be promote KLK4 inactivity resulting in enamel with elevated protein content and reduced mineral content. In cases of MIH with post-eruptive breakdown, on the exposed surface there is a subsequent protein adsorption on the exposed hydroxyapatite matrix. An indicator of the severity of MIH affected teeth is the actual organic content of its enamel (Farah et al., 2010a) Brown enamel, the most severe MIH lesion, has the highest protein content (15–21-fold greater), whilst the protein content of white/opaque and yellow enamel are both markedly higher (8-fold greater) than sound enamel. (Farah et al., 2010a) For sound enamel, when subjected to mechanical forces the controlling deformation mechanism was distributed shearing within nanometer thick protein layer between its

ridge was involved, its maximum height was affected. (Farah et al., 2010a)

controls their secretion. The subsequent breakdown and removal of matrix proteins by means of proteolytic processing is essential for further development and mineralisation. Enamelysin (Mmp20), a matrix metalloproteinase, and the enamel serine protease kallikrein 4 (Klk4) are two major molecules involved in this process. (Wright et al., 2009, Bartlett et al., 2011) Mmp20 is expressed in secretory stage ameloblasts and also has effects on them maturation stage as well as on the mineralisation of mantle dentine. Klk4, present in both ameloblasts and odontoblasts, is expressed at the enamel transition and maturation phase. KLK4 which is secreted into the enamel by ameloblasts during the transition and maturation stages of amelogenesis. Klk4 degrades the organic matrix remaining from the secretion stage. This facilitates the continued deposition of minerals into enamel required for full mineralisation of hard enamel. Amelogenin is cleaved by Mmp20 and later degraded during maturation by Klk4. Within the ameloblasts Dlx3 and Dlx6 are expressed throughout the presecretory, secretory and maturation stages. During secretion Dlx2 is switched off and Dlx1 expression is upregulated. The Dlx homeobox genes may influence enamel formation by the regulation of amelogenin expression. Normal enamel thickness may be achieved by Runx2 suppressing enamel protein expression at the end of the secretory stage to give normal enamel thickness. In the maturation phase Runx2 induces Klk4 and upregulates basal membrane protein expression to induce ameloblast attachment to the enamel matrix. (Brook, 2009, Wrightet al., 2009, Bartlett et al., 2011)

In general, systemic factors that disturb the ameloblasts during the secretory stage cause restrictions of crystal elongation and result in pathologically thin, or hypoplastic enamel. On the other hand, disturbances during the transitional and/or maturation stage of amelogenesis result in pathologically soft (hypomaturated, hypomineralised) enamel of normal thicknesses. (Suga, 1989) According to REID AND DEAN, 2006 (Reid & Dean, 2006), enamel formation as a whole takes approximately one thousand days. Two thirds of this time is devoted to the maturation stage of amelogenesis. Considering this, the most critical period for enamel defects of first permanent molars and incisors is the first year of life coinciding with their early maturation (Alaluusua, 2010). In this period ameloblasts are highly sensitive to environmental disturbances. (Suckling, 1989) Hypomineralisation may also develop later because enamel maturation in the first permanent molars takes several years (later maturation stage). (Alaluusua, 2010)

### **2.2 Amelogenesis and MIH**

As mentioned previously, there are no hypoplastic defects in MIH affected teeth because there is not any discernable reduction in enamel thickness teeth. (Farah et al., 2010a, Fearne et al., 2004) It suggests that any reduction in enamel thickness seen clinically is indicative of post-eruption disintegration of enamel. Furthermore, this clarify that whatever insult affects the developing tooth it happens after the enamel secretion is completed and affects the maturation phase of the mineralization process in a localized area of enamel. (Farah et al., 2010a)

#### **2.3 Characteristics of MIH affected teeth**

MIH is a qualitative defective enamel classified as hypomineralised type that follows the natural incremental lines of enamel formation, from cuspal to cement-enamel junction. (Farah et al., 2010a, Fearne et al., 1994) In the most cervical section, the enamel is sound with

controls their secretion. The subsequent breakdown and removal of matrix proteins by means of proteolytic processing is essential for further development and mineralisation. Enamelysin (Mmp20), a matrix metalloproteinase, and the enamel serine protease kallikrein 4 (Klk4) are two major molecules involved in this process. (Wright et al., 2009, Bartlett et al., 2011) Mmp20 is expressed in secretory stage ameloblasts and also has effects on them maturation stage as well as on the mineralisation of mantle dentine. Klk4, present in both ameloblasts and odontoblasts, is expressed at the enamel transition and maturation phase. KLK4 which is secreted into the enamel by ameloblasts during the transition and maturation stages of amelogenesis. Klk4 degrades the organic matrix remaining from the secretion stage. This facilitates the continued deposition of minerals into enamel required for full mineralisation of hard enamel. Amelogenin is cleaved by Mmp20 and later degraded during maturation by Klk4. Within the ameloblasts Dlx3 and Dlx6 are expressed throughout the presecretory, secretory and maturation stages. During secretion Dlx2 is switched off and Dlx1 expression is upregulated. The Dlx homeobox genes may influence enamel formation by the regulation of amelogenin expression. Normal enamel thickness may be achieved by Runx2 suppressing enamel protein expression at the end of the secretory stage to give normal enamel thickness. In the maturation phase Runx2 induces Klk4 and upregulates basal membrane protein expression to induce ameloblast attachment to the enamel matrix.

In general, systemic factors that disturb the ameloblasts during the secretory stage cause restrictions of crystal elongation and result in pathologically thin, or hypoplastic enamel. On the other hand, disturbances during the transitional and/or maturation stage of amelogenesis result in pathologically soft (hypomaturated, hypomineralised) enamel of normal thicknesses. (Suga, 1989) According to REID AND DEAN, 2006 (Reid & Dean, 2006), enamel formation as a whole takes approximately one thousand days. Two thirds of this time is devoted to the maturation stage of amelogenesis. Considering this, the most critical period for enamel defects of first permanent molars and incisors is the first year of life coinciding with their early maturation (Alaluusua, 2010). In this period ameloblasts are highly sensitive to environmental disturbances. (Suckling, 1989) Hypomineralisation may also develop later because enamel maturation in the first permanent molars takes several

As mentioned previously, there are no hypoplastic defects in MIH affected teeth because there is not any discernable reduction in enamel thickness teeth. (Farah et al., 2010a, Fearne et al., 2004) It suggests that any reduction in enamel thickness seen clinically is indicative of post-eruption disintegration of enamel. Furthermore, this clarify that whatever insult affects the developing tooth it happens after the enamel secretion is completed and affects the maturation phase of the mineralization process in a localized area of enamel. (Farah et al.,

MIH is a qualitative defective enamel classified as hypomineralised type that follows the natural incremental lines of enamel formation, from cuspal to cement-enamel junction. (Farah et al., 2010a, Fearne et al., 1994) In the most cervical section, the enamel is sound with

(Brook, 2009, Wrightet al., 2009, Bartlett et al., 2011)

years (later maturation stage). (Alaluusua, 2010)

**2.3 Characteristics of MIH affected teeth** 

**2.2 Amelogenesis and MIH** 

2010a)

no evidence of defective structure. At a more occlusal level, the defect is confined to the inner enamel while the outer enamel does not appear to be affected. As move occlusally, the hypomineralisation becomes more evident, eventually spreading to span the entire thickness of the enamel. The defects usually did not involve the cusp tips; but if a marginal ridge was involved, its maximum height was affected. (Farah et al., 2010a)

Microstructural analysis of sound and hypomineralised enamel showed two marked changes in microstructure in the MIH affected enamel region; less dense prism structure with loosely packed apatite crystals and wider sheath regions. (Xie et al., 2008) These changes appear to occur during enamel maturation and may be responsible for the marked reduction in hardness and elastic modulus of the affected enamel. (Fagrell et al., 2010) In addition, the enamel in the transitional region adjacent to the demarcated defects in MIH has also notable alterations in their prism sheaths. Despite the translucent, normal appearance, the transitional region between the affected and unaffected regions in MIH teeth had weakened prism sheaths which compromised its overall mechanical properties. (Chan et al., 2010) The reason for this is unclear but may be also related to the lack of organization of the enamel crystals due poorly demarcated prism boundaries in the affected regions (Mahoney et al., 2004) and the packing of the crystals seemed to be less tight and less well organized in the porous parts. The borders of the enamel rods were indistinct and the interrods zones hardly visible, or the rods were very thin with wide interrod zones. (Jalevik et al., 2005)

Semi-quantitative analysis by energy dispersive X-ray spectrometry in extracted MIH affected teeth showed that the mineral composition of this type of enamel is low (Javelik & Norén, 2001), on average the mineral density is about 19 % lower than sound enamel (Baroni & Marchionni, 2011, Farah et al., 2010a, Jalevik & Noren, 2000, Schulze et al., 2004), there is a decrease in Ca:P ratio in the enamel (Rodd et al., 2007a, Jalevik, 2001) related to an increase in C content. (Fearne et al., 2004)

Also, MIH enamel has substantially higher protein content than normal enamel, but a nearnormal level of residual amelogenins. This characteristic distinguishes MIH from hypomaturation defects that contain high residual amelogenins such as *Amelogenesis Imperfecta* or Fluorosis (Mangum et al., 2010a, Wright et al., 1996, Wright et al., 1997) and in turn typifies MIH as a hypocalcification defect as mentioned above. Pathogenically, it points to a pre-eruptive disturbance of mineralization involving albumin probably due to an overabundance of albumin that interferes with the mineralisation process. It justifies the porosities exhibited in the subsurface (Jalevik & Noren, 2000) because albumin degradation may be a prerequisite for maximal crystal growth in the maturation stage of enamel. (Farah et al., 2010b, Farah et al., 2010c, Mangum et al., 2010b) The presence of excessive albumin seemed to be promote KLK4 inactivity resulting in enamel with elevated protein content and reduced mineral content. In cases of MIH with post-eruptive breakdown, on the exposed surface there is a subsequent protein adsorption on the exposed hydroxyapatite matrix. An indicator of the severity of MIH affected teeth is the actual organic content of its enamel (Farah et al., 2010a) Brown enamel, the most severe MIH lesion, has the highest protein content (15–21-fold greater), whilst the protein content of white/opaque and yellow enamel are both markedly higher (8-fold greater) than sound enamel. (Farah et al., 2010a) For sound enamel, when subjected to mechanical forces the controlling deformation mechanism was distributed shearing within nanometer thick protein layer between its

Molar Incisor Hypomineralization:

Fig. 3. Multifactorial aetiology of MIH

Morphological, Aetiological, Epidemiological and Clinical Considerations 431

period, socioeconomic factors and nutrition during first 3 years of child life were entered into databank. Besides, in this study, randomly, there were two-age and sex-matched children to each MIH child. After a regression logistic analyses, the results showed a positive association between severe demarcated opacities in permanent first molars with breastfeeding for more than 6 months, late introduction of gruel and late introduction of infant formula. Moreover, a combination of these variables increased the risk to develop severe demarcated opacities by more five times. According these results, the authors concluded that nutritional conditions during first 6 months of life may influence the risk to

develop severe demarcated opacities in first permanent molars. (Fagrell et al., 2011)

constituent mineral crystals; whereas for hypomineralised enamel micro cracking and subsequent crack growth were more evident in its less densely packed microstructure. (Xie et al., 2009) Thereafter, the ability of dental enamel to absorb energy and sustain deformation without catastrophic failure is attributed to its viscoelastic protein layers. Thus, the change in the protein content in teeth with MIH induces the enamel fracture when subjected to the masticatory efforts.

In relation to the dentin of MIH affected teeth it was observed that the Ca/P ratios for dentin below hypomineralized enamel were in principle identical to those of normal enamel; but when the Ca/C ratio was analyzed, dentin below hypomineralized enamel had the lowest values and the level of C was highest for dentin below hypomineralized enamel. In addition, O and P levels in dentin below normal enamel were higher compared with values in dentin below hypomineralized and N values for dentin below hypomineralized enamel are the highest. (Heijs et al., 2007)

This enhanced knowledge concerning the microstructural changes in hypomineralised enamel improves the understanding of some of the problems associated with the clinical management of these teeth. In particular, the frequent occurrence of enamel fractures and inadequate retention of adhesive materials both of which are recognized as significant clinical challenges preventing successful restoration of these compromised teeth. It is known that organic matter such as proteins have poor acid solubility. The presence of increased amounts of organic matter in the hypomineralised enamel, specifically within both prism structure and sheath regions may inhibit the creation of an adequate etch profile which in turn compromises the adhesion between resin based restorative materials and the defective enamel. (William et al., 2006b) Improved clinical outcomes are likely to depend, at least in part, on the successful treatment of these proteins prior to any enamel etching or adhesive strategies. (Baroni & Marchionni, 2011, Xie et al., 2008)
