Enamel - structure and development

Enamel is a wonderful component of tooth, hardest material but at the same time is lifeless. I have added on biomimetics and enamel regeneration

1. ENAMEL PRESENTER: LEKSHMY JAYAN I MDS, ORAL AND MAXILLOFACIAL PATHOLOGY

2. INTRODUCTION Ectodermally derived structure produced by ameloblasts Hardest substance in the body Wear resistant outer layer of the dental crown Forms insulating barrier – protects the tooth [ Rodrigo.S.Lacruz et al: Dental enamel formation and for oral health and disease, May 2017]

3. [Michel Goldberg el al : Dentin: structure, composition and mineralisation, January 2011]

4. composition ( robinson et al, 1971)

5. Inorganic components – various minerals Organic components – forms enamel matrix ( non collagenous proteins and enzymes) Primary function of organic material- direct growth of enamel crystals . Enamel proteins Non- amelogenins in enamel formation- Ameloblastin, Enamelin, Tuftelin AMELOGENINS (90%) NON- AMELOGENINS (10%)

6. Inorganic component – mainly hydroxyapatite crystals , carbonates and trace elements. Enamel hydroxyapatite crystals- largest of all calcified tissues Susceptible to dissolution of acid- basis of dental caries. Water is present as a part of hydroxyapatite crystal, boundaries of rods. [ Jayasudha et al: Enamel regeneration- Current progress and challenges, September 2014]

7. INORGANIC ORGANIC (ENAMEL PROTEINS) OXYGEN (43.4%) AMELOGENIN (90%) CALCIUM (36.6%) NON AMELOGENIN (10%) PHOSPHORUS (7.7%) AMELOBLASTIN SODIUM (0.67%) ENAMELIN CARBON (0.64%) TUFTELIN MAGNESIUM (0.35%) AMELOTIN FLUORIDE, STRONTIUM, LEAD ODAM, DSPP

8. Hydroxyapatite crystals Augustin Alexis Damur , 1856 Naturally occurring mineral form of calcium apatite Chemical formula- Ca5 (PO4 )3 (OH) Ca10 (PO4 )6 (OH)2 Hydroxyl end member of complex apatite group. Hydroxyl group replaced by F,Cl,CO3 – Fluroapatite or Chlorapatite

9. Carbonated calcium deficient hydroxyapatite- tooth and bone Also seen in calcification within pineal gland - Corpora arenacea (Brain sand) CO3 substitution for OH or PO4 – susceptible to acidic dissolution- progression of caries F substitution – resistant to dissolution- caries prevention and erosion reduction [ Rodrigo.S.Lacruz et al: Dental enamel formation and implication for oral health and disease, May 2017]

10. HYDROXYAPATITE CRYSTALS IN ENAMEL Closely packed, long, ribbon like carbonate apatite crystals Width =60-70 nm, Thickness =25-30 nm Length span the entire thickness of enamel layer. Maturing enamel- hexagonal crystal Matured enamel- irregular

11. Hydroxyapatite crystal in bone

12. physical characteristics Protective covering Hardest calcified tissue

13. Resistant covering Brittle Modulus of elasticity- 1338.2 + 307 MPa Hardness – 274.8 + 18.1 kg/mm Specific gravity- 2.8 [ K.J.Chun et al: Comparison of mechanical properties and role between enamel and dentin in the human teeth, 2014]

14. Semipermeable membrane demonstrated by radioactive tracers and certain dyes (C- labelled urea, Iodine). The organic matrix and water in enamel is in a network of micropores- dynamic connection between enamel and systemic, pulpal or dentinal tubule fluids - Micropores or cracks allow the penetration of fluids. -Permeability decreases and hardness increases with age. [Jansen et al: Permeability of normal enamel, 1951] Colour of enamel – yellowish white to greyish white determined by translucency [ K.J.Chun et al: Comparison of mechanical properties and role between enamel and dentin in the human teeth, 2014]

15. Structure of enamel Rods or prisms Hunter Schreger bands Incremental lines Gnarled enamel Surface structures Enamel tufts and lamellae Enamel cuticle DEJ Enamel cuticle

16. Enamel rods Fundamental organisational units of enamel Cylindrical in LS Number- 5 million to 12 million Length is greater than thickness Increased area of enamel in surface than at DEJ

17. Diameter- 4 μ m Clear crystalline appearance Arcade outline - hexagonal , round, oval, fish-scale near DEJ Keyhole shape outline near enamel surface

18. ULTRASTRUCTURE: Key hole pattern or paddle shaped pattern Width =5 μ m Thickness= 9 μ m In LS, sections pass through heads or bodies of one row of rods and tails of an adjacent row, producing an appearance of rods separated by interrod substance. Bizarre pattern- packed tightly together.

19. Head (1) occlusal and incisal surface One rod is formed by 4 ameloblast Tail (3) cervical region EM- apatite crystals, parallel to long axis of the rods, heads deviate about 65° Length- 0.05-1 μ m Thickness-30nm Width- 90nm

20. CROSS STRIATIONS: Each enamel rod is built up of segments, separated by dark lines- striated appearance Demarcate rod segments More pronounced in hypocalcified enamel Formed by diurnal rhythm in enamel matrix formation Length of each segment-4 μ m

21. DIRECTION OF RODS: CLINICAL CORRELATION: Unsupported Enamel Rods: -Un supported enamel rods are brittle and susceptible to fracture-break to produce leakage at the margins -lodging of food/bacteria in these spaces- secondary caries.

22. Other patterns that complicate enamel structures: -Irregular bending in transverse plane of tooth, cervical- straight -Intertwine in inner 2/3 rd - dissimilar local orientation - Wavy course in clockwise and anticlockwise direction in cuspal and incisal edge - Developmental pits and fissures- converge in outward course CLINICAL CORRELATION: Wavy course of enamel rods: The wavy course, oblique direction and interlocking of the enamel rods render prevention from enamel fracture

23. Rod sheath Thin peripheral layer Darker than rod Relatively acid resistant Less calcified than rod Often incomplete

24. Interprismatic substance Cement enamel rods together More calcified than rod sheath but less than rod Minimum in human teeth

25. Hunter-Schreger bands Optical phenomenon seen in LS Found in inner 2/3 rd of enamel More or less regular change in direction of rods- functional adaptation Parazones -dark bands Diazones -light bands Angle between parazone and diazone-40°

26. Enamel crystals aggregate in each zone, deviated in opposite direction and tilted to 50° with respect to central axis. Controversies in the formation of Hunter Schreger bands: 1. Change in the direction between adjacent group of rods 2. Variation in calcification of enamel 3. Composed of alternate zones of different permeability and different content of organic material

27. incremental lines INCREMENTAL LINES NEONATAL LINE OF RETZIUS

28. INCREMENTAL LINES OF RETZIUS Brownish bands in GS of enamel calcified teeth and in forming enamel Illustrate incremental pattern of enamel LS- surround tip of dentin Cervical part- run obliquely from DEJ to surface, deviate occlusally Transverse section- concentric circle Represents 6-11 days of rhythmic deposition of enamel

29. Other proposed causes: 1. Periodic bending of enamel rods 2. Variations in basic organic structure 3. Physiologic calcification rhythm MEAN DAILY FORMATION OF ENAMEL = 4 μ m CLINICAL CORRELATION : Accentuated incremental lines can also be pathological, caused by metabolic and systemic disturbances such as exanthematous fever that affects enamel formation .

30. neonatal line or ring Boundary marked between enamel formed before and after birth Accentuated striae of Retzius ETIOLOGY - Sudden change in environment and nutrition - Antenatal enamel is better calcified than postnatal enamel

31. Used to identify enamel formed before and after birth Seen in all deciduous teeth and in permanent first molars Frequently seen in first molars of girls than boys Location also varies in pre and post term birth

32. gnarled enamel Wavy pattern in the enamel at the cuspal region Optical appearance of enamel cut in oblique plane Bundle of rods intertwine more regularly Makes enamel more stronger Not hypomineralised !

33. Surface structures Prismless enamel Perikymata Enamel pits and caps Cracks or enamel lamellae Enamel tufts Rod ends

34. Prismless enamel Relatively structureless layer of enamel , approximately 30 μ m thick Seen in 70% of permanent and all deciduous teeth Most commonly in cervical areas of enamel surface Surface, prismless , hydroxyapatite parallel to each other and perpendicular to Striae of Retzius Hypermineralised

35. Perikymata (imbrication lines) Transverse, wave like grooves, external manifestation of Striae of Retzius Lie parallel to each other and CEJ 30 per mm in CEJ 10 per mm near occlusal or incisal edge of a surface COURSE- regular, irregular in cervical region

36. Enamel rod ends

37. Enamel pits and caps Enamel pits- 1-1.5 μ m in diameter, depressed ends of ameloblast Caps- small elevation of about 10-15 μ m, enamel deposition on non- mineralizable debris Enamel brochs - large enamel elevations

38. enamel lamellae Thin, leaf like structures that extend from the enamel surface toward the DEJ May extend or penetrate dentin Consist more of organic and less of inorganic Confused with cracks Develop in areas of tension- when rods cross, donot calcify Disturbance severe- crack develop – filled by surrounding cells or organic substance from oral cavity

39. TYPES OF ENAMEL LAMELLAE

40. Cells from enamel organ fill a crack in enamel - in depth degeneration - close to surface, remain vital for sometime – HORNIFIED CUTICLE From connective tissue- cementum formation CLINICAL CORRELATION- SITE OF WEAKNESS !! – pathways for cariogenic bacteria

41. enamel tufts Resembles tufts of grass in GS Arise at DEJ and reach into the enamel to about 1/5 th to 1/3 rd of its thickness Narrow, ribbon-like structure , inner end arises at the dentin Tufts in different planes are projected into one plane- TUFT OF GRASS Extent in direction of long axis of the crown

42. Hypocalcified enamel rods and interprismatic substance SEM- tubular structure with cross striation TEM- plate like structure in centre of tufts originating from superficial layer of dentin, crossing DEJ and entering tufts

43. Enamel cuticle PRIMARY ENAMEL CUTICLE- Nasmyth’s membrane SECONDARY ENAMEL CUTICLE- Afibrillar cementum PELLICLE- Precipitate of salivary protein

44. enamel cuticle (nasmyth’s membrane/ primary enamel cuticle) Covers entire crown of newly erupted tooth Removed by mastication Basal lamina secreted by ameloblasts when enamel formation is complete Protects enamel surface from resorption by adjacent vascular tissue prior to eruption of teeth

45. Secondary enamel cuticle Cover cervical area of the enamel Thickness=upto 10 μ m Continuous with cementum Probably of mesodermal origin or may be elaborated by attachment epithelium Secreted after enamel organ is retracted from cervical region during tooth development

46. pellicle Reform within hours after mechanical cleaning May be colonised by microorganisms to form a bacterial plaque Plaque may be calcified forming calculus CLINICAL CORRELATION- If not removed, the pellicle can get colonized by microbes to form plaque which subsequently lead to caries.

47. Dentinoenamel junction Surface of dentin at DEJ –pitted, depression fit rounded projection of enamel, holds the enamel firmly on the dentin. Scalloped, convexity towards dentin Crystals of enamel and dentin mix with each other Series of ridges- more pronounced in occlusal area- greater masticatory stress Hypermineralised zone about 30 μ m thick at DEJ- prominent before mineralisation is complete

48. enamel spindles Odontoblastic process that cross the DEJ into enamel, thickened at their end before mineralisation Hypomineralised Direction corresponds to direction of ameloblast(90° to dentin) Enamel rods and spindles are divergent GS- dark in transmitted light Width=5nm Length=70nm Diameter=2 μ m Seen in cusp tip

49. age changes Attrition Generalised loss of enamel rods Flattening of perikymata Decreased permeability

50. amelogenesis

51. amelogenesis Enamel first forms, 30% mineralised Organic matrix breaks down and removed – crystals grow wider and thicker- 96% mineralisation Ameloblast secrete matrix protein Ameloblast has unique lifecycle- phenotypic changes Enamel formation – differentiation of IEE, OEE – cuspal tips – all cells are differentiated into ameloblast Dentin and enamel formation cuts off blood supply to enamel organ Reversal of nutritional source

52. LIFECYCLE OF AMELOBLAST

53. MORPHOGENIC STAGE Ameloblast- short, columnar, large oval nuclei ,almost fill the cell body Terminal bars appear during differentiation Migration of mitochondria to basal region of the cell IEE separated by dental papilla basal lamina Adjacent pulp layer- cell free, narrow, light zone

54. organising stage IEE interacts with adjacent connective tissue which differentiates into ODONTOBLASTS Reversal of polarity Cell free zone between IEE and dental papilla disappear Preameloblasts secrete protein similar to enamel matrix- phagocytosed by odontoblast- EPITHELIAL MESENCHYMAL INTERACTION Terminal phase- DENTIN FORMATION , reversal of nutritional source

55. formative stage After dentin formation Presence of dentin is necessary for formation of enamel ( Reciprocal induction ) Formation – enamel matrix retain same length and arrangement Intiation – secretion of enamel matrix- change in organisation and number of cytoplasmic organelles and inclusion Earliest change- development of blunt cell processes ameloblast surfaces

56. maturative stage Maturation/ full mineralisation after most of the thickness of enamel matrix formed in occlusal/ incisal area, cervical area – progressing Ameloblast-slightly reduced in length and closely attached to enamel matrix microvilli at distal extension, cytoplasmic vacuoles containing enamel matrix like material (absorptive function) Cells of stratum intermedium- assume spindle shape Smooth and Ruffle ended ameloblasts

57. protective stage Enamel fully developed and calcified Ameloblast- indistinguishable from OEE and stratum intermedium OEE + AMELOBLAST + STRATUM INTERMEDIUM = REE REE protect mature enamel, separates it from connective tissue till eruption Retraction of enamel organ from cervical edge CLINICAL CORRELATION- Contact occurs – afibrillar / coronal cementum formation on enamel or resorption

58. desmolyic stage REE – separates oral epithelium and CT Elaborate desmolytic enzymes- destroy CT fibres Premature degeneration of REE- prevent eruption of tooth

59. 1. FORMATION ENAMEL MATRIX Secretory activity after dentin deposition Lose projection penetrating basal lamina (separation between predentin and ameloblast) Islands of enamel matrix deposited along predentin Continuous layer of enamel formed along dentin

60. ENAMEL MATRIX PROTEINS: AMELOGENIN(1983) - Major component Extracellular degradation by MMP – tyrosine and leucine rich amelogenins Regulate cell growth Genes coding present in both X and Y chromosome Most of amelogenins secreted is removed during maturation Maintain space between enamel crystals Remains between and around the crystals CLINICAL CORRELATION- AMELOGENESIS IMPERFECTA- Mutations in human Amelogenin gene located on the X chromosome (AIH1) -hypoplastic or hypomineralized.

61. KALLIKREIN-4 - Secreted in late enamel formation by ameloblast Remove Amelogenin scaffold prior to mineralisation. CLINICAL CORRELATION- Over expression- Hypocalcified enamel AMELOBLASTIN(1996 ) - Nucleation and growth of crystals Cell-matrix attachment Maintenance of ameloblast in differentiated state CLINICAL CORRELATION- Lack of expression- Termination of amelogenesis Failure to produce any enamel.

62. ENAMELIN(1997)- Original enamel protein CLINICAL CORRELATION- Lack of expression- No true enamel formed Thin, highly irregular mineralised crust covered the dentin AMELOTIN – New protein – secreted by mature ameloblast Late stage of enamel formation CLINICAL CORRELATION- Over expression- Extremely soft enamel hypomineralisation of inner enamel and structural defects in outer enamel. TUFTELIN - Localised to DEJ Involved in cell signalling

63. Odontogenic Ameloblast associated gene(ODAM) (2006)- Secreted in late secretory, transition stage, maturation stage CLINICAL CORRELATION- Lack of expression- No enamel defect but altered junctional epithelial attachment Predispose tooth to periodontal infection DENTIN SIALOPHOSPHOPROTEIN - Localised to DEJ Transient in ameloblast and in odontoblast till dentin formation is complete Proteolytic cleavage into Dentin Sialoprotein and dentin Phosphoprotein Regarded as transition zone between dentin and enamel

64. CLINICAL CORRELATION- Over expression DSP- Increase enamel hardness DPP- Soften and weakened bulk enamel NBCe1- Electrogenic sodium bicarbonate cotransporter Also seen in renal proximal tubule, pancreas, eye, heart, brain, developing tooth. Ameloblast- maintain pH buffering during mineralisation CLINICAL CORRELATION- Lack of expression- Enamel too soft to even measure Dentin is significantly softened

65. Development of tomes’ process Projection of ameloblast into enamel matrix Partly delineated by incomplete septa Junctional complex encircle ameloblast at proximal and distal ends – form webs controls substances that pass between ameloblast and enamel Distal terminal bars – separate Tomes’ process from cell proper Secretion from areas close to junctional complex and adjacent ameloblast – INTERROD ENAMEL Distal portion of Tomes’ process lengthens and narrower

66. AMELOBLAST COVERING MATURING ENAMEL Shorter than ameloblast over incompletely formed enamel TRANSITION STAGE- changes occurring in ameloblast after secretory stage and prior to onset of maturation process

67. During Maturative stage, ameloblast cycle between RUFFLED and SMOOTH border – MODULATION - every 5 to 7 hours/ day Period of maturation more than secretion

68. Ruffle-ended Ameloblasts Smooth-ended Ameloblasts Leaky proximal and tight distal junctions Tight proximal and leaky distal junctions involved in exchange of molecules Numerous lysosomes exhibit considerable endocytosis Little endocytic activity Presence of organelles which promote pumping of calcium ions into maturing enamel No calcium pumping activity CLINICAL CORRELATION- Enamel hypoplasia Chronological Enamel Hypoplasia

69. Membrane bound proteins present in ameloblast – CD63, annexins A2, lysosomal associated glycoprotein 1 – removal of organic matrix 90% of protein lost during maturation, remaining protein envelopes around individual crystals

70. mineralisation and maturation of enamel matrix

71. IMMEDIATE PARTIAL MINERALISATION OF MATRIX Occurs in matrix segments and interprismatic substance as they are laid down No matrix vesicles, no unmineralised matrix No apatite crystal formation Initiation of nucleation by hydroxyapatite crystals of dentin Initial mineral- OCTACALCIUM PHOSPHATE

72. Maturation Gradual completion of mineralisation Starts from height of crown progresses cervically, from dentinal end of the rods Integration of 2 processes- 1. Each rod matures from depth to surface 2. Sequencing of maturing rods from cusp towards cervical line. Begins before matrix has reached full thickness Matrix deposited on inner surface, mineralisation on outer surface of recently deposited matrix Incisal and occlusal region mature ahead of cervical region

73. ULTRASTRUCTURE: Growth of crystals Increase in size from about 1.5-25 μ m during maturation phase Tuftelin- nucleation of enamel crystals Other proteins- regulate mineralisation, binding to specific surface of crystal , further deposition RATE OF FORMATION =4 μ m/day 1mm thickness= 40days Loss of organic matrix is caused by withdrawal of protein and water

74. WHY IS AMELOGENESIS UNIQUE? FEATURES AMELOGENESIS DEVELOPMENT OF OTHER MINERALISED STRUCTURE IN TOOTH SECRETORY CELLS Epithelial Ectomesenchymal MINERALISATION By non collagenous protein Collagen has main role MATRIX Lacks collagen 90% absorbed by ameloblast Collagen is the main protein No absorption MINERALISATION OF MATRIX DURING FORMATION Partial No ORGANIC PHASE Absent Present (Osteoid, Predentin, Cementoid ) REGENERATION No- ameloblast undergo apoptosis Regenerate throughout life

75. Clinical consideration ENAMEL HYPOPLASIA ENAMEL HYPOCALCIFICATION FLUOROSIS

76. Enamel regeneration Enamel cannot regenerate or remodel on its own Various methods are employed to attempt regeneration of enamel Synthetic enamel fabrication , tissue engineering etc Can done by Hydrothermal method- Controlled release of Ca from Ca-EDTA Hydrothermal transformation- Octacalcium phosphate rod to Hydroxyapatite nanorods and using hydrogen peroxide containing pastes PAMAM-COOH solution- organic template on demineralised enamel produce hydroxyapatite crystals

77. Alternative cell sources for enamel formation are- [ Jayasudha et al: Enamel regeneration- Current progress and challenges, September 2014]

78. enamel biomimetics Biomimetic methods Enamel acts like a single crystal Replication of Enamel Translucency- should regenerate the highly organised structure of enamel Fundamental difficulty in clinical application- low solubility of calcium phosphate - difficult to remineralise deep lesion Crystal precipitate randomly - will not rebuild enamel structure- develop stabilising agents

79. Example of some enamel biomimetic materials [ Rodrigo.S.Lacruz et al: Dental enamel formation and implication for oral health and disease, May 2017]

80. references Rodrigo.S.Lacruz et al: Dental enamel formation and implication for oral health and disease, May 2017 Jayasudha et al: Enamel regeneration- Current progress and challenges, September 2014 Michel Goldberg el al : Dentin: structure, composition and mineralisation, January 2011 Yue Sa et al : Compositional, structural and mechanical comparisons of normal enamel and hypomaturation enamel, August 2014 Rick J. Rauth et al: Dental Enamel: Genes Define Biomechanics, December 2009

81. J.P. Simmer et al: Molecular mechanisms of dental enamel formation, 1995 Jansen et al: Permeability of normal enamel, 1951 Orban’s Oral Histology and Embryology(13 th Edition): G.S.Kumar Tencate’s Oral Histology, Development, Structure and Function(8 th Editon ): Anonio Nanci Textbook of Oral Anatomy, Histology, Physiology and Tooth Morphology (1 st Edition) : Dr K. Rajkumar , Dr. R. Ramya