Aspects of Biological Apatite Crystal

(4-1)Two pathways for apatite crystal formation(OCP and CDL pathways)

(4-2) Crystal damage related to fluoride, cadmium exposure

and estrogen deficiency:

based on CDL pathway(Crystal defects and osteoporosis)

(4-3) The Event of Pathological Calcification(Vascular calcification)

(4-4) Bone crystal resorption by osteoclasts(Event of endocytosis)

<M. KAKEI, M. YOSHIKAWA, T. SAKAE, H. MISHIMA>

(4-1)Two pathways for apatite crystal formation

1: Octacalcium phosphate (OCP) pathway :

The OCP pathway, mediated by mineral precursor OCP, first appeared around the Cambrian period.

In the absence of F ions, the crystal formation gradually progresses to form hydroxyapatite.

With the presence of F ions, the process of crystal formation will facilitates to form fluoroapatite.

Localization: the shell of Lingula unguis, conodont, chiton radular teeth , shark tooth enameloid and placoid

denticle outer layer, Eusthenoptern foodi tooth enameloid and dermal denticle outer layer, etc.•

2: Central dark line (CDL) pathway:

The CDL pathway without mediating OCP precursor evolved around the Silurian period.

The CDL pathway needs the supply of carbonate ions to neutralize the inhibitory effect of Mg ions by forming

huntite minerals. Therefor, carbonic anhydrase plays a key role in initiating the crystal nucleation.

F, Cd ions or Es deficiency inhibit the synthesis or activity of carbonic anhydrase, causing the failure of crystal

nucleation.

Localization: Tooth enamel, dentin, bone, goldfish scales, shark tooth dentin, shark placoid denticle inner layer,

Eusthenoptern foodi tooth dentin and dermal denticle inner layer, baleen of baleen whales, etc.

Two different crystal characters: one is CDL-free type

and the other is CDL-bearing type.

b

a: enameloid crystals of Scapanorthynchus texanus, b: enamel crystals of

Palaeoloxodon naumanni, c: dentin crystal of Palaeoloxodon naumanni,

d: bone crystal of Eusthenopteron foodi. Arrows: CDLs

Bars = 10 nm J. Fossil Res. 48(2), 53-65. (2016)

With permission

** Figs reflect two different mechanisms of crystal formation.

J. Fossil Res. 48(2), 53-65. (2016)

** OCP pathway first appeared, followed by CDL pathway.

Apatite crystals observed in conodont tooth apparatus (dentin: a),

shark placoid scales (outer layer: b) and chiton radular teeth (c)

(CDL-free)

a = b = c (bar = 10 nm)

cba

a

b c

d

e

OCP(b), apatite(c) lattice lines and the conversion of OCP into

apatite form observed in the shell of Lingula unguis

(b = c): bar = 10 nm, ( d = e): bar = 10 nm, (a-e): no stain

J. Fossil Res. 48(2), 53-65. (2016)

* One of OCP lattice line (d, arrow) produced two lattice lines of

apatite (e, small arrows). Same event to see: J. Electron microscopy 58(6), 393-398. (2009)

The conversion of OCP into HA form

The lattice fringes of both OCP and HA

OCP HA

OCP HA

Gar pike

Before conversion After conversion

a

b c

TEM observations of the matured crystals of enamel,

dentin and bone

(CDL-bearing)

a: enamel, b: dentin, c: bone crystals No stain

Arrows: CDLs J. Fossil Res. 48(2), 53-65. (2016)

a b

No stain Bar = 10 nm

CDL lattice images of before (a) and after (b) the electron

beam damage

** CDL does not produce two lattice lines, showing

the different character between OCP and CDL.

Nakahara H. and Kakei M. Josai Shika Daigaku kiyo 12, 1–7. (1983)

e

fd

c

b

a g

TEM observations of the early stage of crystal developments in

tooth enamel, dentin and bone

(a, d and f): bar = 500 nm, (b, c, e and g): bars = 10 nm, (a-c, e and g): double stained sections,

(d and f): no stain. J. Fossil Res. 48(2), 53-65. (2016)

Ribbon-shaped structures in enamel. (a: cross section, b, c: longitudinal

section; Needle-shaped minerals in dentin and bone (d, f, e, g)

* No lattice fringe is observed within the envelope structure at

the early stage.

a

b c

* Crystal nucleation and growth take place within

an organic envelope.

Crystal development of the rat tooth enamel

Arrow heads: organic envelope; arrows: CDLs. (a = b = c): bar = 10 nm,

double staining . J. Fossil Res. 48(2), 53-65. (2016) (modified)

a b c

Arrow heads: organic envelope; arrows: CDLs.

(a and b): double staining; (c): no stain. (a = b = c, bar

= 10 nm)

Kakei, M., Sakae, T. and Mishima, H（2007）In : Biomineralization: from

paleontology to materials Science (eds. Arias, J.L. and Fernandez, M.S.).

Editorial Universitaria, Santiago, Chile, pp.107–115.

TEM observations of an organic envelope and apatite crystals in

the inner layer of shark placoid scales and goldfish scales

a b c

Arrow heads: organic envelope; arrows: CDLs. (a):

double staining; (b and c): no stain. (a = b = c, bar =

10 nm)

J. Fossil Res. 48(2), 53-65. (2016) (modified)

(shark) (goldfish)

Apatite crystals in the baleen whale

a b c d

(a): cross-section of a baleen, bar=150 μm, (b): bar= 100 nm,

(c and d): 10 nm, (b-d): no stain Arrows: CDLs

Human sound enamel Caries

The central area of an enamel crystal is abundant

in carbonate

Carbonate and magnesium contents of minerals at various

developmental stages of the rat calvaria

* Only trace amount of carbonate in the mineral phase at the earliest

(amorphous) stage of calcification. More information: Ann. Anat. 179, 311-316. (1997)

Newborn: the amorphous rich stage, 6-days: the nucleus rich stage, 12-W: the matured stage

** Huntite minerals (1123 cm-1) develop prior to the first

apatite lattice (960 cm-1)

Raman analysis of the nucleation site of apatite crystal

D: 960 +1123 cm-1 :

Crystal nucleation

(central dark line

formation)

A: 1123 cm-1 : Huntite

development

Casciani F S, Etz E S, Newbury D E, and Doty

S B Scan. Electron Microsc. 2, 383–91. (1979)

With permission

1123 cm-1 960 cm-1

960 cm-1 : apatite PO4 peak1123 cm-1 : huntite CO3 peak

The thermal decompositions of OCP and huntite minerals

* The decomposition of huntite minerals begins from around

500 C, while OCP decomposed at a lower temperature of 150 C.

DTA

Wei

ght/

%

He

at F

low

/μV

-80.00

-60.00

-100.00

-40.00

-20.00

0.00

-100.0

-20.0

0.0

20.0

40.0

200.00.0 800.0 900.0400.0 600.0

Temperature/℃

-80.0

-60.0

-40.0

TG

Bigi et al. J. Inorg. Biochem. 40, 293-299. (1990) With permission

Huntite

OCP

J Fossil Res. 38, 43–48. (2005) (modified)

The CDL is still existed after the heat treatment, differentiating CDL from

OCP. The disappearance of CDLs begins from around 500 C.

TEM observation of CDL after heat treatment

Enamel (a-c) and bone crystals (d-f) after heat treatment.

(400-600 C) Arrows = CDLs

Compositional and structural changes of bone crystals by heating

* Creating larger crystals by heating at above 700 ℃ may contribute to the increase

of crystallinity. (2019 BIOM XV, modified) Related article: Yanagi S. Bulletin of TIRI 3, 96-97. (2008), Greiner et al. In:

Biomoneralization: from molecular and nano-structural analyses to environmental science. (eds. K. Endo et al.) Tsukuba, Japan, pp. 21-29.

Bone crystals were heated at 600 (a), 700 (b) and 1,000 ℃ (c).

(a); Each crystal appears to be obscure and

some crystals amalgamate with each other

(bar = 25 nm ). A few crystals preserve CDL.

(inset, arrows, bar = 10 nm)

(b) and (c); Various sizes of large crystals were formed. No CDLs were observed

anymore. (b); heated at 700 ℃. (c); Heated at 1,000 ℃. (bars = 120 nm ).

The scheme of apatite formation process via CDL pathway

1: Amorphous mineral, consisting Ca, PO4 and Mg ions within an organic envelope. Mg ions: an inhibitory effect on

the mineralization process (LeGeros R Z. (1981) Prog. Crystal Growth Charact. 4, 1–45).

2: The carbonate ions supplied by CA generate huntite minerals (small dots), eliminating the inhibitory effect of Mg

ions.

3: Creation of first apatite lattice line.

4: The first lattice line together with huntite minerals creates the CDL.

5 and 6: The crystal growth.

J. Fossil Res. 48(2), 53-65. (2016)

(4-2) Crystal damage related to fluoride, cadmium exposure and estrogen

deficiency: based on CDL pathway

(Crystal defects and osteoporosis)

The exposure to fluoride (F) chemicals suppresses the synthesis of carbonic

anhydrase, regardless of F content.

The cadmium (Cd) exposure reduces the carbonic anhydrase activity by

replacing Zn with Cd, regardless of its content.

Estrogen (Es) deficiency reduces the synthesis of carbonic anhydrase.

During the crystal nucleation, the failure of carbonate supply results in an

irreversible damage to tooth and bone crystals. Crystal perforation in enamel

and increase of amorphous minerals in bone. In particular, combination of F

(or Cd) chemical and estrogen deficiency accelerates the development of

osteoporosis.

フッ素症 正常

白濁して透明性がない

Arrow indicates lack of

transparency

F-affected deciduous tooth Normal permanent tooth

Raman analysis

a ：Tooth enamel

b : F 30min treatment

c : F 5hrs treatment

d : Synthetic apatite

e : F 24hrs treatment of

synthetic apatite

f : Fluorapatite (shark)

** Conversion of hydroxyapatite to fluoroapatite

never occurred after fluoride treatment Similar findings reported in: Tsuda H, Ruben J, and Arends J. (1996) Eur. J. Oral Sci. 104, 123–131.

J. Hard Tissue Biology 21, 257-265. (2012) With permission

Electron microscopy of tooth enamel

** The central area of an enamel crystal showed less acid resistance

than the peripheral area due to the richness of carbonate.

This may give misconceptions about F scheme for caries prevention.

Low mag.

High mag.

sound (a, d) caries (b, e) F exposure (c, f)

LM C COVX OVXLM C CCd FCdF

31K31K

A B

aa b

b

Immunoblot analysis of CA (carbonic anhydrase) in

immature enamel matrix proteins

** A: F exposure suppresses the synthesis of CA, while Cd

exposure decreases the enzymatic activity.

** B: Es deficiency also weakens the synthesis of CA.

a: amid black stain, b: immunoblot

Proc. Jpn. Acad., Ser. B 85, 500-507. (2009) Proc. Jpn. Acad., Ser. B 89, 340-347. (2013)

A B1 32

1

3

2

C

sound F exposure Es deficiency Cd exposure

Schematic explanation of the mechanisms of crystal structure defects

caused by F ,Cd ions, and Es deficiency (A 1-3), and crystal defects

in rat tooth enamel (B and C).

J. Fossil Res. 48(2), 53-65. (2016)

** Supply of carbonate ions is necessary for the crystal nucleation.

a: Control

b: Cd 100 ppm exposure

c: Es deficiency (ovary-ectomy)

d: Combination of Es deficiency

and Cd 100 ppm exposure

Micro-CT analysis of tibia

** Trabecular architecture is remarkably loosened by the

combined effects of Es deficiency and Cd exposure.

Proc. Jpn. Acad., Ser. B 89, 340-347. (2013)

The effects of cadmium exposure and Es deficiency

on the calvaria (Soft X-ray radiography)

Proc. Jpn. Acad., Ser. B 89,

340-347. (2013)

a: Control

b: Cd 100 ppm

c: OVX (ovary-ectomy)

d: OVX-Cd 100 ppm

** Combination of Es deficiency and Cd exposure (d) showed

the increase of the radiolucent area, resulting in a labyrinthine

pattern (d).

a b c

d e f

Electron micrographs of minerals observed in both

radiolucent and radiopaque areas of calvaria

(Cd, F exposure)

** Cd exposure with Es deficiency increases amorphous minerals in

bone (d).

** Similarly, F exposure with Es deficiency increases amorphous

minerals in bone. To see: Adv Tech Biol Med 2016, 4:1 http://dx.doi.org/10.4172/2379-1764.1000170

(a and d ): radiolucent area, (b and e): radiopaque area, (c and f):

control, a-c: Low mag, d-f: High mag. Arrows: CDLs Proc. Jpn. Acad., Ser. B. 89, 340–347. (2013)

Comparative study of CA activity in immature

enamel tissue affected by F or Cd exposure

Enzymatic activity was measured by a differential gas pressure method.

Kodama E. Master thesis, Tokyo Gakugei University (2007) (in Japanese)

Cont: Control, Cd20: Cd 20 ppm, Cd40: Cd 40 ppm, Cd100: Cd 100 ppm, F2: F 2 ppm

J. Hard Tissue Biology 21, 257-265. (2012) (modified)

** F shows a 20-fold detrimental effect more than that of Cd.

The effects of harmful chemicals and Es deficiency on the

calvaria (Soft X-ray radiography)

* * The harmfulness of F exposure exceeds that of Cd exposure.

Light microscopic study of the trabecular architecture

beneath the metaphyseal plate of rat tibia

** Combined effects of Es deficiency and F exposure accelerate the

bone fragility, showing more severe than the case of Cd. Adv Tech Biol Med

2016, 4:1 http://dx.doi.org/10.4172/2379-1764.1000170 (modified)

a: Control

b: OVX (ovary-ectomy)

c: F 1.0 ppm

d: OVX-F 1.0 ppm

e: OVX-Cd 2.0 ppm

a b c

d e

In vascular lesion, both CDL-free and –bearing crystals are observed.

(4-3) The Event of Pathological Calcification

(Vascular calcification)

J. Fossil Res. 48(2); 53-65. (2016)

* Crystals formed under pathological conditions are quite

different from those formed in normal bone.

(4-4) Bone crystal resorption by osteoclasts (OS)

(Event of endocytosis)

* TEM shows that bone crystals were resorbed by endocytosis.Related reports: Hancox N.M., Boothroyd M.D. and Boothroyd B. (1961) J. Biophysic. Biochem. Cytol. 11, 651-661.

Boothroyd B. (1964) J. Cell Biology 20, 165-173.

OS

OS

Conclusions

(4-1) Two pathways for apatite formation

OCP pathway first appeared at the Cambrian. OCP pathway can employ F ions to facilitate the conversion process,

resulting in fluorapatite.

CDL pathway might have appeared around the Silurian. CDL pathway never creates fluorapatite. Carbonic anhydrase

initiates the crystal nucleation in this system . CDL is not identical to OCP.

(4-2) Crystal damage related to fluoride, cadmium exposure

and estrogen deficiency: based on CDL pathway

(crystal defects and osteoporosis)

Chemicals (F, Cd) and Es deficiency cause the crystal structure defects in calcified hard tissues, such as perforated

crystals in enamel and amorphous minerals in bone.

Combined effects of harmful chemicals (F, Cd) and Es deficiency accelerate the development of postmenopausal

osteoporosis.

The declining bone formation may be the primary cause of osteoporosis.

(4-3) The Event of Pathological Calcification

(Vascular calcification)

The process of crystal formation under pathological conditions is different from that of bone.

No soft tissue cells can differentiate into hard tissue-forming cells under the pathological events .

(4-4) Bone resorption by osteoclasts

(Event of endocytosis)

TEM observation shows that bone crystals seemed to be resorbed by endocytosis.

Supplementary

The enzyme of enolase is highly essential for the glycolysis pathway, which is the first step of energy (ATP) production

system, in the living world. Fluoride is said to kill oral bacteria by inhibiting the synthesize of this enzyme. (Warburg

O. and Christian W. (1942) Biochem. Z. 310, 384-421)

There is a report saying that fluoride could cause hypochromic microcytic anemia according to an animal study. (Vijaya

Bhaskara Rao and Vidyunmala S. (2009) American-Eurasian J. Toxicological Science 1, 81-83.)

Similarly, it has been reported that cadmium intake caused anemia based on animal study. (Horiguchi H., Oguma E. and

Kayama F. (2011) Toxicological Science 122, 198-210.)

Crystals formed under vascular calcification event were quite different from those observed in bone, contradicting the

phenotypic change theory that soft tissue cells convert into bone-forming cells. : On the basis of little information,

crystals formed under pathological condition are expected to be different from those formed in normal bone. (Faure G,

Daculsi G, Netter P, Gaucher A. and Kerebel B. Scan. Electron Microsc. (1982) (Pt 4), 1629-34.)

FOP known as an intractable disease, is worth considering why immunosuppression could suppress the heterotopic

ossification of soft tissues. (Kaplan et al. (2007) J. Bone Joint Surg. Am.89, 347-357) Muscular tissue of FOP patients

is said to be vulnerable to an external stimulus. Structure of apatite crystals is not fully elucidated.

Regarding the remineralization effect of F. leaving it up to you after reading these reports. (J. Electron Microsc （2003）,

2, 471-76.; J. Hard Tissue Biology(2012) 21(3), 257-266.)

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