Ameloblasts and Enamel Formation

• This review discusses recent discoveries related to enamel proteins (amelogenin, ameloblastin, and enamelin) and proteinases (MMP-20 and kallikrein-related peptidase-4).
• Focus is placed on their nomenclature, tissue expression, functions, proteinase activation, and substrate specificity.
• Null mice and human mutations are assessed regarding amelogenesis imperfecta.
• Pertinent controversies, such as the function of amelogenin in crystallite growth, are also addressed through thorough analysis of null mouse literature.
• A new mechanism by which enamel matrix proteins support and promote enamel development is proposed.

Introduction

• Tooth development is orchestrated, needing precise signaling to and from epithelial and mesenchymal cells.
• A high rate of incompletion reflects the complexity of the signaling pathways.
• This includes deficiency of third molars, second premolars, and lateral incisors, with agenesis of third molars occurring in roughly 20% of the world's population.
• Study of tooth development has revealed genes and tissues interact to form dental structures and the potential for developmental signaling pathways to go wrong.
• Teeth comprise cementum, dentin, and enamel:
  • Cementum is along the tooth root, anchoring the tooth with collagen fibers.
  • Dentin, a bone-like matrix, has dentinal tubules and is more elastic than enamel.
  • Enamel, covering the crown, is the hardest epithelial-derived calcified tissue in vertebrates.
• Enamel's hardness comes from its high mineral content.
• It contains very little protein (less than 1% organic material) compared to bone and dentin (20-30% organic material by weight).
• Teeth, due to enamel, are most resistant to deterioration, offering anthropological clues.
• Wear patterns, shapes, and carbon isotope compositions indicate past behaviors of our ancestors.

Overview of Enamel Development

• This review centers on enamel proteinases, their substrates, and discoveries that improve our understanding of enamel development.
• Enamel development (amelogenesis) has four stages: presecretory, secretory, transition, and maturation.
• The morphology and function of ameloblasts define the stages.
• Ameloblasts, responsible for enamel composition, are part of the enamel organ.
• The enamel organ comprises an outer epithelial layer, the stellate reticulum, the stratum intermedium, and the inner enamel epithelium (ameloblast layer).
• The basal end of the preameloblast connects via desmosomes to the stratum intermedium.
• The apical end attaches via hemidesmosomes to a basement membrane at the future DEJ site.
• The outer enamel epithelium covers the enamel organ and connects with ameloblasts (inner enamel epithelium) at the cervical loop.
• The stellate reticulum, between the outer dental epithelium and stratum intermedium, secretes glycosaminoglycans causing water to diffuse into the enamel organ.
• The stratum intermedium may shuttle ions to and from ameloblasts.
• Ameloblasts secrete enamel matrix proteins and proteinases, inducing mineral ribbons and arranging them into rod and interrod patterns.
• Deposition of predentin by odontoblasts at the future DEJ is an early event in the presecretory stage.
• Differentiating preameloblasts then form cytoplasmic projections, remove the basement membrane, and secrete proteins to initiate mineralization.
• Preameloblasts transform into secretory ameloblasts by elongating and forming Tomes' processes.
• Enamel matrix proteins secrete from one side of the Tomes' process (secretory face).
• The first enamel crystals grow between dentin crystals, mineralizing around dentin proteins.
• Protein rich secretory stage enamel is soft.
• The enamel layer thickens as ameloblasts secrete proteins.
• Mineral ribbons form normally to the secretory surface and eventually form into a rod (prism).
• Each ameloblast creates an enamel prism, collectively forming a 3D structure. Proteins forming interrod enamel exit near the "base" of the Tomes’ processes, whereas those involved in rod formation exit from the "tip" (secretory face), with protein cleavage products reabsorbed or accumulating between rod and interrod enamel. Crystalline development:
  • Rod crystallites grow parallel to one another in c-axis length.
  • Interrod crystallites have limited lengths.
• Ameloblasts move and secrete proteins into the enamel matrix:
  • Ameloblasts slide by one another during the secretory stage.
  • Movement leads to decussating enamel prism patterns in rodent incisors and entwined prism patterns in human molars.
  • Four different proteins are secreted into the enamel matrix.
• Three structural proteins and one proteinase make up the secretion:
  • Amelogenin (AMELX) comprises 80-90%.
  • Ameloblastin (AMBN) comprises roughly 5%.
  • Enamelin (ENAM) comprises 3-5%.
  • Matrix Metalloproteinase-20 (MMP20, enamelysin) is present in trace amounts.
• In the absence of ameloblastin or enamelin, enamel crystals do not form; in the absence of amelogenin, enamel crystals will form.
• The enamel layer achieves full thickness by the end of the secretory phase.
• At the end of the maturation stage, enamel achieves its final hardened form when proteins are removed. Transition and maturation stages:
  • The beginning of the transition stage depends on species and the specific developing tooth.
  • Before the enamel layer reaches its full thickness, ameloblasts stop moving relative to each other.
  • KLK4 helps actively remove the mass of secreted and hydrolyzed matrix proteins from the enamel layer.
• Enamel mineral composition is very similar to hydroxyapatite [Ca5OH(PO4)3], consisting of carbonate, sodium, and magnesium.
• The initial enamel ribbons have only a few apatitic unit cells in thickness (~10 nm) and a width of ~30 nm Structure of the enamel layer:
  • Thin organic matrix with no crystals exists between rod and interrod enamel.
  • Area called rod sheath/sheath space in humans.

Summary of enamel development

• Development has four defined stages:
  • Presecretory stage: ameloblasts secrete proteins at the forming DEJ after removing the basal lamina
  • Secretory stage: ameloblasts elongate, develop Tomes' processes, and secrete into the enamel matrix
  • Enamel crystallite ribbons form and lengthen.
  • The ameloblasts transition into shorter protein reabsorbing cells once the enamel reaches full thickness
• Ultimately the enamel will achieve its final hardened form by the end of the maturation stage.
• General features of amelogenesis are constant across species.

Enamel Proteinases

• Enamel had a high protein content during development but not when mature.
• Enamel matrix proteins get removed during the maturation stage.
• Protein percentage drops from 30% during the secretory stage to 2% during the early maturation stage.
• Profile shifts as forming enamel progresses from secretory to maturation stages. At least two classes of enzymes are present:
  • Metalloproteinase during the secretory stage.
  • Serine proteinase during the maturation stage.
• MMP20 is expressed during the secretory through early maturation stage
• KLK4 gets expressed from the transition through maturation stages.
• They are secreted into the enamel matrix:
  • MMP20 named “enamelysin”.
  • KLK4 originally named enamel matrix serine proteinase-1 (EMSP1) but renamed because the gene encoding KLK4 locates in the kallikrein gene cluster.

Matrix Metalloproteinase-20/Enamelysin

• MMP20 was cloned originally from a porcine enamel organ cDNA library.
• MMP20 tissue expression is highly restricted.
• MMP20 became the first proteinase definitively identified as expressed by the ameloblasts of the enamel organ.
• How MMP20 activates is an enigma.
• The MMP20 propeptide does not contain an RXXR furin consensus sequence.
• Membrane-type MMP1 (MT1-MMP, MMP14) has a transmembrane domain is a potential means of activation in vivo.
• A very important discovery: no strong evidence that MMP20 becomes activated within the enamel matrix to date.
• MMP20 Substrate Specificity was shown to cleave the most abundant enamel matrix protein, amelogenin.
• MMP20 expression may be restricted to tooth tissues due to its broad substrate specificity.
• MMP20 has been characterized for its ability to cleave amelogenin.
• Same group assessed ameloblastin, enamelin for MMP20 substrates.
• MMP20 likely responsible for generating the 32 kDa enamelin cleavage product in vivo.
• MMP20 plays an critical role in enamel development as demonstrating that all amelogenin cleavage products observed in vivo are expected MMP20 cleavage products. Human Mutations causes enamelin and dentin proteins.
• Also shown to cleave. -E-Cadherin. -Casein and /or gelatin. -Aggrecan and Cartilage Oligomeric Matrix Protein. -Type V collagen -Type XVIII collagen.
  • Fibronectin type IV collagen -Tenascin-C -And laminin-1/5 but not type 1,2 collagen.
• Reports confirm broad substrate specificity of matrix metalloproteinase because expression elsewhere caused tissue damage .

The Mmp20 Null Mouse

• Demonstrates catalytically Inactive and deletion Exon 5 highly conserved zinc binding site (HEXGHXXGXXH) active site members.
• MMP20 does not process amelogenin properly.
• Has altered structure proteins and enamel rod pattern.
• Has deteriorated enamel.
• Had hypoplastic enamel.
• Weight less percent in wild type controls and reduced overall and decreased Enamel hardness is all in enamel maturation.
• This leads to 2 decades of dramatic altered way we envision Amelogenesis .
• These findings may have altered our understanding of how to envision Amelogenesis as it has occurred in this span Tomes processes abnormal in mouse and normal ameloblastic processes. That does not occur in Mmp20 mice because primary reason that it shears off detention. The enamoel can only be characterized and only appears as two layers during histology The inner layer is is homogenous and not well mineralized.

However outer is large nodules and expressions

• normal thin mineralized layer of enamel begins forming
• during the secretory phase at dentino-enamel junction.
• MMP20 null that mineral layer is defective
• Ions precipated as secondary layer one top of the first
• Klk4 in mmp20 and Klk4 ablated micr covers but is uncertain but ball and dysplastic shapes could form and initiate normal functions with a very limited understanding that requires Conventional theories ,
• Theories also should support the note the nodule first to distrust smooth surface areas taken together to help learn MMP how about enamel formation but have much to work Human MMP20 mutations expressed from a gene of chromosome 11 q22 and It domain structures helps with Mmp20 Secretion as a enzyme propeptide function such that enzymes domain and latently hinge binds site terminal and Only post-translational modification is the connecting force.
• M M P doesn't shares other domain structures but has same as family members and Enamel defects are inherited and absence of generalized syndrome is an amelogenesis impefecta

Kallilrein-Related Peptidase-4

• KLK4 Nomenclature was original name to enamel Matrix Serine Proteinase named (EMSP 1), KLK-L1 tissue neutral term Serene Protease But not satisfaction and changed the official genes by cluster of proteins

• That requires Refinement is now refers to Peptidase Is on long term as it refers to new designation

• KLK 4 not publications.

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• In early stages that enzymes that activities.

• KLK4 activity substrate because klk4 can be stable and for its expressions but as the proteins akl4.

• If its high to stop the site will start kl4 activity

• Klk4 also be receptors and substrate expression during functions

• KlKL4 cannot initiate self and activity.

Human KLk4

• Mutations have expression domains in the catalytic state in areas where protein levels are at risk and not easily degraded
• Therefore no functional.

Other Matrix Proteinases

• Mutations were demonstrated has an all mutations in some expression
• However has some functions when it comes to expression Recent showed expressions.

Enamel Matrix Proteins

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Explore the function and key structural changes of Ameloblasts during enamel formation. Understand the role of enamel proteinases like MMP20 and the significance of high protein content in developing enamel. Examine the genetic inheritance patterns associated with Amelogenesis Imperfecta (AI).