Section 12: Mineralized Tissues

1. Section 12: Mineralized Tissues 3. Ion exchangeMineralizationFluoride note: paper on mech of fluorosis: JDR 84, 832-6, 2005 3/3/06

2. Hydroxyapatite (HA): ion exchange • in biological HA, ions are continuously in motion, dissociating, associating, etc. (dynamic equilibrium) • even when there's no net precipitation or dissolution,exchange of ions is occurring • ion exchange rates depend on location: crystalinterior v slow slow surface v fast bulk solution hydration shell 1

3. HA: ions substituted • because bulk solutions in contact with HA vary in ion content, biological HA contains other ions: HA ions substitute ions Ca2+ Mg2+, Sr2+, Pb2+, Fe2+ PO43– CO32–HPO42– citrate3– OH–Cl–, F– , HCO3– 2

4. HA: ions substituted • because bulk solutions in contact with HA vary in ion content, biological HA contains other ions: HA ions substitute ions Ca2+ Mg2+, Sr2+, Pb2+, Fe2+ PO43– CO32–HPO42– citrate3– OH–Cl–, F– , HCO3– • most substitutions increase HA solubility • e.g., enamel solubility higher due to presence of 2–4% CO32– (1–2 CO32–/20 PO43– ) • the term carbonated apatite sometimes used • F– exception: substitution with F–lowers solubility 2

5. Ion exchange in HA: CO3= for PO43– trigonal tetrahedral 3

6. Mineralization • if a solution of calcium & Pi with ion product, e.g.,1.5 mM2 (supersaturated relative to HA) is prepared,no HA forms • if crystal of HA added,it will grow until solution's ion product = K'sp • these experiments illustrate: • Ca-Pi solutions tend to stay supersaturated • precipitation on existing crystals (seed or nucleator) is much faster • stoichiometry of HA formation: 10Ca2+ + 6HPO42– + 8OH–®Ca10(PO4)6(OH)2 + 6H2O 4

7. Mineralization in vitro • if above solution's ion product increased to >3 mM2, a different CaP salt forms: calcium hydrogen phosphate: CaHPO4.2H2OK'sp= 2.6mM2 aka brushite, amorphous calcium phosphate (ACP) this illustrates:though more soluble, other simpler salts form faster than HA • stoichiometry of formation & dissolution of ACP: Ca2+ + HPO42– + 2 H2O ® CaHPO4.2H2O • How do the first crystals form in mineralizing tissues? i.e., how is mineralization initiated? 5

8. Initiation (nucleation) Ca Pi _ _ _ _ OH + + _ – _ – + + + + + + + + + + + + + + + + heterogeneous nucleation (aka epitaxy):initiating template different from crystal components • first ions of crystal-to-be aligned by binding to surface of a protein Model of part ofinitiator protein:surface complementaryin shape & chargeto HA unit cell 6 after first few ions have bound

9. Initiation (nucleation) • this first layer of ions then binds additional complementary ions to form a second layer, etc. • eventually, growth produces the final crystal • initiator proteins:phosphoryns,bone sialoprotein II • have numerous phosphorylatedser side chains: binding sites for Ca2+ of crystal-to-be • cationic side chainssupply + charges: binding sites for PO43– & OH– _ _ + _ – + + + + + + + + 7 HA unit cell

10. Crystal Growth • matrix vesicles • phospholipid membrane-enclosed • contain bone sialoprotein II (osteoblasts) phosphoryns (odontoblasts) • produced by budding from osteoblasts/odontoblasts • contain transmembrane calcium pump (CaATPase) ATP hydrolysis drives Ca2+ transport ATP + H2O ®ADP + Pi matrix vesicle solid CaPi facil-itateddiffusion activetransport Ca2+ Pi Pi Ca2+ CaATPase 8

11. Crystal Growth matrix vesicle • growth of crystallites facilitated by concentrating calcium & Pi (maintains ion product > Ksp) • Pi also made by hydrolysis of • phosphoestersalkaline phosphatase ROPO3– + H2O ® ROH + Pi • pyrophosphate (PPi)pyrophosphatase PPi + H2O ® 2 Pi • membrane removed at some time after initiation of crystallization solid CaPi facil-itateddiffusion activetransport Ca2+ Pi Pi Ca2+ CaATPase 9

12. hole zone Mineralizationin bone &dentin tropocollagen ↑ • crystallitesinitially deposited inhole zones • eventually also deposited in spaces parallel to tropocollagen strands (pores) pore hst731du.gif crystallites pore hole zone hst731dl.gif adapted fromTen Cate, 5thed., Fig. 5-6 10 surface hole

13. Mineralization of enamel 2 2 ameloblastmovement 3 1 2 3 • matrix proteins • enamelins • nucleator ? • coat surface of mature HA crystallite • amelogenins • regulate HA crystallite growth • digested by proteases during maturation • stages of mineralization 1. nucleation (at DEJ)initiator may be dentin-based 2. 2-D growth (ribbons) 3. 3-D growth (maturation) dashed line indicates volume ofcrystal after maturation 11

14. Composition of mineralized tissues bone dentin enamelnew mature mineral, wt % 60 70 37 95 vol % 36 47 16 86 HA/total mineral 2/3 1 1 crystallite size, Ålength 200 1400width, thickness 50 800 pores, % of enamel surface area 1-2 organic, vol % 36 33 20 2 main protein collagen amelogenins enamelins H2O, vol % 28 21 64 12 12

15. Section 12: Mineralized TissuesFluoride 3/3/06

16. Fluoride metabolism • definitions • [F–]: 1 ppm = 1 mg/L (1 mg/kg in solids) = 50 µM • daily dose ≈ 1 mg from drinking ~ 1 L H2O with 1 ppm F – (dose = mg/L F–x volume ingested) • absorption • stomach & small intestine • via simple diffusion in stomach as HF (pKa = 3.5) • rate = k [F–] • t½ ≈ 30 min 13

17. Fate of absorbed fluoride • [F–] in plasma: ≈ 1µM for 1 mg daily dose • crosses placenta • present in breast milk • deposited in bone • via ion-exchange (non-enzymatic, at/near equilibrium) • adults: ~50% of dose • infants: 70-80% • rest excreted via kidneys • rate = k [F–] in plasma • t½ ≈ 4 hr (range: 2-9 hr) 14

18. Fluoride toxicity: lethal dentalfluorosis acutelethal chronicfluorosis • acute lethal:due to • F – inhibition of numerous enzymes • reaction of F – with Ca2+ in ECF (CaF2 precipitates) low [Ca2+] excites nerve & muscle cellshypocalcemic tetany laryngospasm 10 g 1 mg 10 1 g 100 mg daily fluoride dose (log scale) 15

19. Fluoride toxicity: fluorosis dentalfluorosis acutelethal chronicfluorosis • chronic fluorosis (aka skeletal fluorosis, osteofluorosis) • multi-year exposure • hypermineralization of bone resorption inhibited, formation increased* • ectopic mineralization of tendons, ligaments 10 g 1 mg 10 1 g 100 mg daily fluoride dose (log scale) *these effects are the basis for fluoride use in osteoporosis prevention/treatment 16

20. Fluoride toxicity: fluorosis dentalfluorosis acutelethal chronicfluorosis • dental fluorosis: enamel hypomineralization • fluorotic enamel contains more protein, less mineral • due to exposure to high [F–] during enamel formation • pore size >2% • probable contributory factors • impaired apoptosis of ameloblasts • inhibition of amelogenin-digesting proteases duringenamel maturation 10 g 1 mg 10 1 g 100 mg daily fluoride dose (log scale) 17

21. Effect of fluoride on caries & dental fluorosis 10 4 1. v mild: small chalky areas < 25% of surface 2. mild: white opacities < 50% of surface 3. moderate: brown staining; some on all surfaces 4. severe: extensive discoloration & hypoplasia 2 decayed, missing, filled 5 fluorosis index 0 10 1 0.1 ppm fluoride Fluorosis index (one version) 18

22. Anticaries effect of fluoride • presence of fluoride during enamel formation and/or remineralization results in partial replacement of OH– with F– in HA • molecular rationale for anticaries effect • F– ion smaller & more symmetrical than OH–, allowing better packing of ions • within unit cell • between unit cells (shared ions) • interionic attractions greater, so solid state stabilized, therefore solubility (K'sp) lower 19

23. Ion exchange: F– for OH– hydroxyapatite unit cell,with 1 OH– replaced by 1 F– for presenting 20

24. Anticaries effect of fluoride (cont’d) • HA crystals larger, have fewer defects (imperfections) • resulting enamel has less space between crystals (smaller pores) • stoichiometry of hydroxyapatite ↔ fluorapatite Ca10(PO4)6(OH)2 +2 F– ↔ Ca10(PO4)6F2 + 2 OH– (complete replacement of OH– by F– would yield 38,000 ppm F–) • maximum replacement <10% of this 21

25. Tooth surface proximity & F– concentration • HA nearest enamel surface has highest [F–] • surface HA has lower solubilitydue to higher F– content • surface accumulationprobably due to F–incorporation duringcrown maturation • additionally, F– is added by long-term ion-exchange & demineralization/remineralization ppm in H2O 1 2000 0.1 ppm fluoride 1000 25 50 22 microns from surface

26. Fluoride incorporation over time 4000 5 ppm in H2O • F – content of HA increases with age • dependent on [F–] in H2O • incorporation into HA by: remineralization ion exchange • F – presence in saliva inhibits net demineralization • topical F – application with high [F–] produces CaF2 deposit,providing slow-release of F– 2000 ppm F in surface enamel 1000 600 1 ppm in H2O 400 30 50 tooth age, years 23

27. Anticaries effect of fluoride:topical vs. systemic high systemic-F– thenlow topical-F– • people with enamel made in high-F– area who move to low-F– area have higher caries rate • people with enamel made in low-F– area who • move to high-F– area • use F– toothpastes have lower caries rate • people take on caries rate of region they move to • results suggest that F– presence during demineralization/ remineralization* is most important anticaries effect • i.e., topical exposure to F– more important than systemic exposure * acid challenges low systemic-F– thenhigh topical-F– (acid challenges) info from"Fluoride in Dentistry" chptr 19 24

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