aspects of biological apatite crystaljul 13, 2020 · denticle outer layer, eusthenoptern foodi...
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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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