AMER. ZOOL., 24:965-976 (1984)
Organic Matrices and Mineral Crystallites in
Vertebrate Scales, Teeth and Skeletons1
NORMAN E. KEMP
Division of Biological Sciences, The University of Michigan,
Ann Arbor, Michigan 48109
SYNOPSIS. The earliest fossil vertebrates, the ostracoderms, more than 500,000,000 years
ago had skeletons which included the hard tissues calcified cartilage, bone, dentine and
enamel. It seems plausible that the modes of mineralization pioneered in these primitive
tissues have persisted throughout vertebrate radiation without major changes. All four
types utilize the calcium phosphate mineral apatite to form inorganic crystallites which
develop in close association with matrix proteins. In the collagen-associated tissues—
calcified cartilage, bone and dentine—the short, needle- or lath-like crystallites develop
along collagen fibrils. Non-collagenous components of the matrix in these tissues include
proteoglycans, glycoproteins and matrix vesicles. Special calcium-binding or phosphatebinding constituents may promote crystal nucleation and growth. In enamel (enameloid)
large hexagonal crystallites develop in association with the glycoproteins amelogenin and
enamelin secreted by cells of the inner dental epithelium (ameloblasts). Enamel (enameloid)
of the shark tooth resembles mammalian enamel in size and shape of crystallites. Biochemical and immunohistochemical techniques are being used in several laboratories to
determine how these proteins control the mineralization of enamel, and whether they are
homologous in all vertebrate classes.
a new type of head with a highly differentiated
brain and special sense organs, gills
Evidence from comparative anatomy,
embryology, histology and paleontology around the pharyngeal slits, a hypomeric
dates the beginning of vertebrate phylog- muscular investment around the digestive
eny more than 500 million years ago in the tube, and a mineralized integumental exoCambrian Period of the Paleozoic Era. skeleton as well as a mineralized axial endoBeing a vertebrate means having a back- skeleton. It seems likely that ectomesenbone or vertebral column consisting of car- chyme of neural crest origin, probably
tilage or bone. Yet the transition from the derived phylogenetically from the nerve
invertebrates to vertebrates brought net of protochordates, played a major role
changes in all organ systems and probably in organization of the new vertebrate head
resulted from the gradual accumulation of and also pioneered in the differentiation
many mutations over millions of years. of vertebrate mineralized tissue (Schaeffer,
Deuterostome protochordates laid the 1977; Hall, 1981; Gans and Northcutt,
groundwork for evolution of vertebrates 1983).
Selection of apatite as the mineral for
through acquisition of a hollow dorsal nerve
cord, a supportive notochord, segmental strengthening organic matrices secreted by
trunk muscles and pharyngeal slits (Romer, epidermal and mesenchymal cells was a key
1945; Gregory, 1951; Berrill, 1955; Gans choice for the future of vertebrate radiaand Northcutt, 1983; Northcutt and Gans, tion. Minerals of this group are among the
first to crystallize out of molten magma as
1983).
To become vertebrates the protochor- it erupts from an active volcano. Apatite
dates had to change their life-style from minerals, composed of calcium phosphate
passive filter feeding to active, predatory and associated ions which commonly
suction feeding. Along the way they evolved include hydroxyl, fluoride and carbonate,
are abundant in the earth's crust as major
constituents of the commercially valuable
sedimentary rock phosphorite and as
1
From the Symposium on Mechanisms ofCalafication accessory inclusions in most igneous rocks
in Biological Systems presented at the Annual Meeting (McConnell, 1973). Apatites are harder and
of the American Society of Zoologists, 27-30 Decemmore durable than the calcium carbonate
ber 1983, at Philadelphia, Pennsylvania.
INTRODUCTION
965
966
NORMAN E. KEMP
minerals (vaterite, aragonite, calcite) of
most invertebrate skeletons. Providing the
building material for a superior type of
supportive skeletal system, however, may
not have been the primary advantage of
apatitic crystallization. Dissolved phosphate concentrations are low both in sea
water and fresh water; hence the calcium
phosphate minerals may have been selected
by vertebrate progenitors primarily as a
means for concentrating phosphorus, an
indispensable element for cellular structure and function (McLean and Urist,
1968). Apatite crystallites deposited in the
integument may have functioned in waterproofing the body surface, and also may
have served as a storage dump for excess
phosphate which could not be readily
excreted through the kidneys (Smith, 1932;
Berrill, 1955).
Although the vertebrates early adopted
the phosphatic mode of mineralization and
have exploited it, they were not the first
to discover it. Even among the most primitive animals, the Protozoa, there are some
species which store calcium phosphate
granules and crystallites intracellularly.
Pautard (1976) has described intracellular
clusters of hydroxyapatite crystallites in the
ciliate Spirostomum ambiguum. Inarticulate
brachiopods, e.g., Lingula, have constructed their shells from hydroxyapatite
since their fossil record began in the Cambrian period. Fossil conodonts, which are
tooth-like remains of an unknown type of
invertebrate or early vertebrate, are apatitic (Rhodes, 1954). Some species of corals, annelids, arthropods and molluscs utilize calcium phosphate in their skeletons
(Pautard, 1961).
Until recently the earliest known fossil
remains of vertebrates were fragments of
the skeletons of pteraspid ostracoderms
from Middle Ordovician strata (Romer,
1945; Moy-Thomas and Miles, 1971) about
450 million years old. Fragments of the
dermal skeleton of Astraspis desiderata Walcott, Eriptychius americana Walcott and
Eriptychius orvigi proved that a mineralized
dermal armor had already evolved; furthermore, some head fragments of Eriptychius possessed prisms of calcified cartilage in the endoskeleton of the skull
underlying dermal scales (Denison, 1967;
Kemp and Westrin, 1979). The age horizon of the ostracoderms has how been
pushed back to the upper Cambrian beyond
500 million years ago by the discovery of
dermal skeletal fragments in the Deadwood Formation of northeastern Wyoming. These fossils have rounded dermal
scales similar to those of Anatolepis heintzi
previously described from the Lower
Ordovician level at Spitzbergen, Norway
(Repetski, 1978). Ostracoderms encased in
bony plates and scales flourished for over
a million years until Upper Devonian time,
when their fossil record terminated. Their
vertebrae lacked centra but probably consisted of paired cartilaginous neural arches
as in modern lampreys.
The principal mineralized tissues of vertebrates are calcified cartilage, bone, dentine and enamel (enameloid), all of which
were already present in the agnathan ostracoderms (Moss, 1964, 1968, 1977; 0rvig,
1967, 1977; Halstead, 1969, 1973, 1974;
Hall, 1975; Patterson, 1977; Schaeffer,
1977; Kemp and Westrin, 1979). The dermal plates, scutes and scales of the ostracoderms consisted of a lower layer of compact, lamellar bone, a middle layer of
spongy bone, and a superficial adornment
of rounded protuberances called odontodes (0rvig, 1977). These "dermal teeth"
or "denticles" contained a dental papilla
and mineralized layers of dentine and
enamel (enameloid). The term aspidin has
been used to distinguish the acellular bone
of the Heterostraci from the cellular bone
of the Osteostraci (Moss, 1964).
Cosmoid scales of the lobe-finned fishes
(Crossopterygii) and fossil lung fishes (Dipnoi) had a base of compact bone called isopedine, a layer of spongy bone, a dentinelike layer called cosmine, and a thin cap of
enamel (enameloid). Ganoid scales of many
extinct ray-finned fishes (Actinopterygii)
and a few modern survivors, e.g., the bichir
Polypterus, were similar to cosmoid scales
except that both the bony basal layer of
isopedine and the enamel (enameloid) layer
were greatly thickened. This primitive type
of ganoid scale, termed palaeoniscoid, has
been modified to the lepidosteoid type having only the basal bony layer and the enamel
MINERAL CRYSTALLITES IN VERTEBRATES
967
(enameloid) layer of ganoin, as in the gar- how mammals calcify cartilage in the
fish Lepisosteus. Scales of teleost fish, known epiphyseal plates of their growing long
as bony-ridge scales (Lagler et al., 1962), bones, for example. If the principle of hard
are of two types: cycloid scales in soft-rayed tissue homology holds, we may extend our
teleosts, e.g., the carp, and ctenoid scales insight backwards and surmise that Eripin spiny-rayed teleosts, e.g., the perch. Both tychius at the dawn of vertebrate evolution
types consist of an inner fibrillary layer of could calcify its neurocranial cartilage the
collagenous connective tissue, and an outer same way the shark Squalus does today
calcified layer of bony ridges (circuli) (Kemp and Westrin, 1979). Knowledge
arranged concentrically around a central about normal calcification of hard tissues
focus. The exposed posterior part of the provides the basis for understanding
scale is ridged externally in the cycloid type pathological calcification or decalcificabut bears tooth-like cteni in the ctenoid tion, as in soft tissue calcinosis, osteopotype. Kresja (1979) considers the homol- rosis, osteomalacia, osteogenesis imperogy of the layers in cycloid and ctenoid fecta and dentinogenesis imperfecta.
scales as enigmatic but proposes that the
fibrillary layer may correspond to the basal
HARD TISSUE MINERALIZATION
bony layer of earlier types and that the
Apatite minerals include the following
bony layer may be the homologue of a den- (McConnell, 1973): menetite, CaHPO4;
tine layer. He also hypothesizes that the brushite, CaHPO 4 -2H 2 O, whitlockite,
exposed ridges or cteni on the posterior Ca3(PO4)2; octacalcium phosphate, Ca8H2part of these scales, which are covered by (PO4)6-5H2O; hydroxyapatite, Ca10(PO4)6epidermis, may be homologous to the (OH)2; fluorapatite, Ca10(PO4)6F2; chloraenamel layer of earlier types. In some fishes, patite, Ca10(PO4)6Cl2; dahllite, a carbonate
e.g., Carassius auratus, the fibrillary layer of hydroxyapatite; and francolite, a carbonthe scale becomes calcified with hydroxy- ate fluorapatite. Apatites may incorporate
apatite crystals, but the extent of calcifi- alkali metals, Mg, Sr, Ra, trace elements,
cation of this layer varies in different species and citrate (Vaughan, 1981). The biologic
(Onozato and Watabe, 1979).
apatite of vertebrate hard tissues is comVertebrae with centra first evolved in monly designated hydroxyapatite, although
primitive gnathostome fishes, the placo- McConnell (1973) and Featherstone et al.
derms, which persisted in the fossil record (1983) advise that it is probably usually a
until the Permian period. Modern cyclo- carbonate hydroxyapatite and therefore
stomes (lampreys and hagfishes) have lost dahllite.
the ability to mineralize their cartilaginous
It has been proposed that calcification in
skeletons or teeth (hagfish), but all other the hard tissues begins with the deposition
living vertebrates—cartilaginous fish of amorphous calcium phosphate,
(Chondrichthyes), bony fish (Osteich- Ca9(PO4)6 and may include phases of depothyes), amphibians, reptiles, birds and sition of whitlockite, brushite and octacalmammals—mineralize their hard tissues in cium phosphate. Eventually, however, submuch the same way as did the ostraco- stituted hydroxyapatite is the only mineral
derms. Tissue types have remained form detectable (Hohling et al., 1981; Palaremarkably conservative despite the mul- mara et al., 1981; Landis and Navarro,
tifarious expressions of form in the evo- 1983). Fluoride substitution promotes
lution of scales, teeth and skeletons. Schaef- growth of crystal size in the enameloids of
fer (1977) has proposed "that enameloid, teeth and scales of sharks and teleost fish
enamel, dentine and dermal bone are (LeGeros and Suga, 1980).
homologous as specific types of calcified
Mineralogically apatite crystals exhibit
dermal tissue in all groups of vertebrates the hexagonal class of symmetry, characin which they occur." I believe that the terized by three equal horizontal axes of
same principle applies for endoskeletal cal- symmetry (a,, a , a ) and a vertical axis (c)
2
s
cified cartilage and bone. How a shark cal- of different parameter
perpendicular to the
cifies its cartilaginous skeleton is probably horizontal axes (Wade and Mattox, 1960).
968
NORMAN E. KEMP
In order for crystallization to occur from
solution the product of the concentrations
(or activities) must exceed the solubility
product constant (S.P.C. or K sp ) for precipitation of the phosphate salt. This value
is defined as the ion product (I.P.) of the
dissolved ions at concentrations (activities)
when rates of solution and precipitation
are equal, i.e., at equilibrium. Thus
[Ca 2+ ] s x [PO43"]2 = Ksp. Ca,(PO4)2, and
- l o g Ksp. = pKsp Cas(PO4)2 (McLean and
Urist, 1968). Orthophosphoric acid,
H 3 PO 4 , is a weak tribasic acid which dissociates in blood plasma to produce primary, secondary or tertiary orthophosphate ions, H 2 PO 4 '-, HPO 4 2 -, and PO43~.
At pH 7.4 human blood plasma has 85%
of its total inorganic phosphate (P,) as
HPO 4 2 -, 15% as H 2 PO 4 '-, and 0.0035% as
PO43~. Pyrophosphate (H4P2O7) is present
in small amounts in such compounds as
thiamin pyrophosphate. It has been determined that at low pH values (<4.0) the
solubility of calcium phosphate is governed
by the Ks p of primary calcium phosphate,
Ca(H 2 PO 4 ) 2 H 2 O, whereas at higher pH
values up to 6.0 it is governed by the Ksp
of secondary calcium phosphate (brushite),
CaHPO 4 -2H 2 O. At still higher pH values
the latter compound is unstable and undergoes spontaneous hydrolysis leading to an
equilibrium state in which hydroxyapatite
crystals are formed. These solubility characteristics account for the deposition of
hydroxyapatite at physiological pH (Neuman and Neuman, 1958).
Simkiss (1975) has discussed the relationship between blood plasma concentrations of P ; and mineralization of bone.
Mammalian blood has a [Ca2+] x [P;] ion
product of 1.67 (mmole/liter) 2 . Bone mineral will dissolve in artificial blood plasma
to yield a S.P.C. of about 0.83 (mmole/
liter)2; hence blood appears to be supersaturated with respect to ion concentrations necessary for precipitation of mineral. In fact, however, bone mineral will
not precipitate from solution until a S.P.C.
of 4.3 (mmole/liter) 2 is attained. Paradoxically plasma is undersaturated for spontaneous precipitation. Neuman and Neuman (1958) have reasoned that blood and
extracellular fluid must be nearly equivalent in composition but that the "interstitial fluid" of bone must have a different
ionic composition. They postulate that
there must be "some diffusion barrier, some
ion gradient or ion pump" between bone
fluid and extracellular fluid, and that the
explanation lies in the properties of the
osteoid matrix.
Neuman and Neuman (1958) have suggested that evolution might have selected
either of two mechanisms to induce new
crystal formation: (1) a booster mechanism
for enriching concentrations of calcium and
phosphate ions; or (2) a catalytic method
for promoting crystal seeding by epitaxy.
It is well known that hydroxyapatite and
isolated bone mineral can induce growth
of crystals by epitaxy, but in the matrices
of cartilage or osteoid there are no crystals
to induce initial seeding. Neuman and
Neuman concluded that a possible candidate for initial seeding might be an organic
crystalline substance and suggested collagen as the only such material available in
cartilage and bone. Fleisch and Neuman
(1961) have proposed that pyrophosphate
may inhibit calcification of bone and must
be removed by pyrophosphatase before
mineralization can proceed. Pyrophosphate thus may function as a crystal poison
(Simkiss, 1964,1975). Both pyrophosphate
with its P-C-P linkage and diphosphonates
with P-O-P linkage are potent inhibitors of
bone mineral deposition (Jung et ah, 1973).
According to a theory proposed by
Glimcher and Krane (1968), the "hole
zones" between the tropocollagen molecules packed within collagen fibrils are the
sites of initial hydroxyapatite nucleation.
The hole zones measure 400 A x 15 A and
would provide enough space for about 50%
of the mineral in bone. They believe that
these sites may raise the local concentrations of calcium and phosphate ions by first
binding phosphate, but that the relationship to collagen is not necessarily epitactic
(review by Hancox, 1972). Growth of crystals would be restricted initially by the space
within collagen fibrils and later by the
interfibrillar space as crystals elongate in
parallel with the fibrils (Glimcher, 1981).
MINERAL CRYSTALLITES IN VERTEBRATES
969
the earliest step in mineralization may be
the accumulation of amorphous calcium
Collagen is the principal protein of the phosphate within matrix vesicles, followed
matrix of bone, dentine and calcified car- by its conversion to hydroxyapatite crystilage; hence their mineral crystallites may tallites. Upon rupture of the vesicles, crysbe classified as collagen-associated. The tallites are freed to become aligned with
same is true of tooth cementum, which may collagen fibrils secondarily (Sayegh et al.,
be considered a type of bone. Both bone 1974). Membrane vesicles develop by
and dentine contain Type 1 collagen like abscission from scleroblasts and thus posthat in non-mineralized connective tissue, sess the phospholipids and hydrolytic
whereas cartilage contains Type 2 collagen enzymes associated with the cell mem(Linsenmayer, 1981). The fibrils of carti- brane, including alkaline phosphatase, pylage are less than 500 A in diameter, but rophosphatase, ATPase, and 5'-AMPase
those of bone may be more than 1,000 A (Anderson, 1973; Peress^a/., 1974). Lanthick. Despite these differences, the crys- dis and Glimcher (1982) have observed
tallites of calcified cartilage, bone and den- paniculate mineral deposits in the matrix
tine are similar, being relatively short and of growth plate cartilage but not within the
needle- or lath-shaped, in contrast to the matrix vesicles; hence they express doubt
much larger hexagonal crystals of enamel. about the presumed role of the vesicles in
Non-collagenous constituents of the the genesis of mineralization.
ground substance undoubtedly influence
That the initial deposits of bone mineral
the pattern of mineralization in the colla- may be amorphous rather than crystalline
gen-associated hard tissues. Proteoglycans, is a concept stemming from the demonconsisting of a backbone of protein with stration by Eanes and Posner (1965) that
attached glycosaminoglycans, are more amorphous calcium phosphate converts to
abundant in cartilage than in bone. Gly- hydroxyapatite crystals in vitro. Later it was
coproteins with shorter carbohydrate shown (Eanes et al., 1973) that the amorchains, on the other hand, are more prev- phous phase consists of particles 200-2,000
alent in bone. The glycoproteins include A in diameter, which adhere in branching
sialoprotein and osteocalcin, a calcium- chains. Eanes and co-workers hypothesize
binding protein which may bind to hy- that amorphous calcium phosphate may be
droxyapatite crystals (review in Vaughan, the precursor for the patchy nodular
1981). Degradation of proteoglycans may deposits of hydroxyapatite crystals without
be a prelude to mineralization of collage- any visible relationship to collagen fibrils,
nous matrices by rendering collagen fibrils as observed in the early stages of bone minmore accessible to mineralizing ions (Dzie- eralization. In support of this view, Gay
wiatkowski et al, 1968; Kobayashi, 1971; (1977) has shown that amorphous bone
Woessner, 1971; Kemp and Westrin, 1979). mineral is associated with randomly oriImmunofluorescent studies have shown, ented hydroxyapatite crystals in newly ossihowever, that proteoglycans are not lost in fying regions of quail and chick limb bones,
calcified cartilage matrix (Poole et al., but that in highly calcified regions the crys1982).
tals are oriented along collagen fibrils.
An alternative to the "collagen theory" Quantitatively amorphous calcium phosof crystallite nucleation in collagen-asso- phate accounts for 35% of bone mineral
ciated hard tissues is the "matrix vesicle" (Termine and Posner, 1967). Deposition
theory of Anderson (1969) and Bonucci of amorphous calcium phosphate granules
(1970). These investigators and others sub- may begin within the mitochondria of calsequently have shown that membrane- cifying cartilage cells or of developing bone
bound vesicles about 1,000 A in diameter osteoblasts (Mathews, 1972;Landis, 1981).
are abundant in the matrices of calcifying Somehow this intracellular mineral store
cartilage, bone and dentine (Anderson, may be mobilized and delivered to the
1973; Ornoy et al., 1983). It is believed that extracellular matrix as an important source
COLLAGEN-ASSOCIATED MINERALIZATION
970
NORMAN E. KEMP
of the mineral which deposits there
(Simkiss, 1976).
Several specific types of metabolites
secreted into the matrix of collagen-associated hard tissues have been targeted as
agents to facilitate mineralization. The glycoprotein osteocalcin containing 7-carboxyglutamic acid becomes helical after
binding calcium and subsequently adheres
strongly to hydroxyapatite (Hauschka and
Carr, 1982). The protein osteonectin binds
selectively both to collagen and hydroxyapatite. Osteonectin-collagen complexes
bind free calcium ions and also synthetic
apatite crystals in vitro (Termine et al,
1981). Calcification-initiator protein (CIP)
is a disulfide-bonded glycoprotein with
marked calcium-binding capacity (Urist et
al, 1977). A class of phosphate carrier proteins called phosphophoryns (Veis and
Sabry, 1983) have been extracted from
dentine matrix. They are rich in aspartic
and serine residues and most of the serine
residues are phosphorylated. Both the carboxyl and phosphate groups of these compounds bind calcium with high affinity. It
is theorized that they may assist both in
crystal nucleation and in regulation of crystal growth. Phospholipids have also been
implicated in the mineralization process.
Calcium-phospholipid-phosphate complexes (Ca-PL-PO 4 ) readily induce hydroxyapatite crystallization in vitro and may
behave similarly in vivo (Boskey and Posner, 1982). Phosphatidyl serine in the
membranes of matrix vesicles may form
complexes with calcium and inorganic
phosphate (PS-Ca-P,) and thus assist in the
delivery of mineralizing ions to nucleation
sites (Yaari et al., 1982).
MINERALIZATION OF ENAMEL
The matrix of enamel in mammalian
teeth is secreted by ameloblasts of the inner
dental epithelium. The hexagonal crystallites of human enamel are about 40 nm in
diameter (Arends et al, 1983) and attain
average dimensions of 26.3 nm thick x
68.3 nm wide (Kerebel et al, 1979). In
comparison with the crystallites of dentine,
they are much larger in diameter and much
longer. Length estimates vary, but they may
be several microns long. Stages in secretion
of ameloblastic vesicles from Tomes' processes and changes in shape of ameloblasts
after secretion have been described by
Reith (1970) for the rat. Ameloblasts that
are ruffle-ended appear to be more active
than smooth-ended ones during the mineralizing phase (Takano et al., 1982).
Although it is generally accepted that the
enamel layer is homologous in amphibians,
reptiles and mammals, the homology of the
comparable layer in fish teeth has long been
controversial (0rvig, 1967; Poole, 1967;
Moss, 1968, 1977; Kemp and Park, 1974).
Poole (1967) has proposed the term enameloid for this layer in fishes pending clarification of the cells secreting its matrix.
One group of investigators have maintained that the enamel (enameloid) layer
in shark teeth is not true enamel but instead
a modified type of dentine secreted by
odontoblasts. Another group have held that
this tooth cap layer is true enamel, resulting from the secretory activity of ameloblasts (Moss, 1977). My own observations
(Kemp, 1974; Kemp and Park, 1974) and
those of Kerebel et al., (1977) support the
latter view by demonstrating that shark
tooth ameloblasts are secretory and that
the crystallites which develop in the
enameline matrix are large and hexagonal
like those of mammalian teeth.
Scanning electron micrographs illustrating the appearance of the enamel layer and
basal bony plate of sharks' teeth are shown
in Figures 1-4. Tooth specimens from
three different shark species, Negaprion
brevirostris, Carcharhinus menisorrah and
Triaenodon obesus, were fixed in 95% alcohol, immersed in Clorox to remove basal
connective tissue and dental papilla, dried
in air, sputter-coated with gold, and
observed with a JEOL (JSM-U3) scanning
electron microscope. Figure 1 shows a row
of calcified teeth in Negaprion. Figure 2 is
an enlargement of the boundary between
the enamel cap layer and basal plate. Crystallites of bone in the basal plate are needlelike. Figure 3 at high magnification shows
the narrow, elongate crystallites on the
outer surface of the enamel layer. Here on
the surface adjacent to ameloblasts during
MINERAL CRYSTALLITES IN VERTEBRATES
development the crystallites appear to be
coated with an assembly of beads, which
may be helically wound matrix proteins not
removed by Clorox treatment. Figure 4
shows the much larger, more mature crystallites on the inner surface of the enamel
layer bordering the dental papilla. Here
the crystallites are hexagonal and have lost
the beaded coating of immature crystallites.
It appears from morphological evidence
that the enamels of shark teeth and mammalian teeth are homologous. There is,
however, one prominent distinction
between the enamel layers in these two
groups. Odontoblastic processes extend
only through the dentine layer of mammalian teeth, whereas in shark teeth they
extend beyond the dentino-enamel junction well into the enamel layer. The term
enameloid is useful to the extent that it
connotes this histological difference in the
shark tooth enamel layer, but the term may
be misleading if used to mean that the mode
of mineralization is different in sharks and
mammals.
Shellis and Miles (1974) have interpreted the "collar enameloid" covering the
teeth of some teleosts as containing both
collagen and amelogenin-like protein of
epithelial cell origin. Herold (1974), however, concluded that the cap layer of pike
teeth is true ectodermal enamel like that
of mammals. Smith (1978, 1979) interprets
the surface sculpturing of fossil crossopterygian fish teeth and also of teeth from
the extant coelacanth Latimeria chalumnae
as enamel-like, deposited under the control of the inner dental epithelium. Meinke
(1982) likewise has described ectodermally
derived enamel on the dermal denticles of
the fossil coelacanth Spermatodus pustulosus.
Mammalian enamel proteins are glycoproteins of two types, called amelogenins
and enamelins (Eastoe, 1974; Fincham et
ai, 1982a; Slavkin et ai, 1983). Amelogenins are hydrophobic proteins with high
levels of proline, glutamic acid, histidine
and leucine (Herold et ai, 1980; Termine
et ai, 1980). They constitute 80% of the
total enamel proteins and account for most
971
of the matrix during the secretory phase
of enamel development (Belcourt et al.,
1983; Slavkin et al., 1983). Amelogenins
have molecular weights up to 40,000 daltons and are subdivided into a C-group with
molecular weights from 15,000 to
> 30,000, and a J-group with molecular
weights of about 5,000 (Fincham et al.,
1982a). Proportions of the proteins of
higher molecular weight decrease, while
proportions of smaller polypeptides
(J-group) increase during tooth maturation (Fincham et al., 19826).
Enamelins are acidic glycoproteins rich
in aspartic acid, serine, glutamic acid and
glycine (Termine et al., 1980). They have
molecular weights ranging from 8,000 to
72,000 daltons, tending toward the lower
end of the range in mature enamel (Fincham et al., 1982a). These proteins adsorb
strongly to hydroxyapatite and are conserved with advancing tooth maturation
while amelogenins are diminishing (Termine et ai, 1980).
Utilization of antibodies to amelogenins
and enamelins has opened an exciting new
chapter in the comparative biochemistry
of amelogenesis. Graver et al. (1978) have
shown that antiserum to bovine amelogenins reacts with protein in pre-ameloblasts
and also in secretory ameloblasts. Newly
deposited enamel matrix reacts lightly,
whereas mature enamel does not react
except for thin layers along the dentinoenamel junction and adjacent to ameloblasts. Herold et al. (1980) have demonstrated that fluorescent antibodies to bovine
amelogenin cross react with the inner dental epithelium of the mouse, pig, tokay,
gecko, the salamander Notophthalmus viridescens, and the shark Squalus acanthias. The
enameloid matrix of Squalus dermal denticles fluoresced even more brightly than
did the enameloid of Squalus teeth when
exposed to the antibody. From these results
they concluded that "enameloid matrix
contains ectodermally derived amelogenins as well as mesodermally derived proteins."
Slavkin and associates (Slavkin et al.,
1982) have shown that rabbit anti-mouse
enamel protein antisera react specifically
972
NORMAN E. KEMP
FIG. 1. Scanning electron micrograph of file of calcified teeth from a 40-cm lemon shark, Negaprion brevirosiris,
oriented with anteriormost tooth facing upward at bottom of micrograph. Enamel layer (E) covers cusp above
bony basal plate (B). Scale bar = 1 mm. x 30.
FIG. 2. Enlarged view of border between enamel layer (E) and basal plate (B) in calcifying tooth of a 60-cm
gray reef shark, Carcharhinus menisorrah. Crystallites in basal bone are relatively short and needle-like. Scale
bar = 1 urn. x 5,000.
MINERAL CRYSTALLITES IN VERTEBRATES
with ameloblasts growing either in vivo or
in vitro. Such antisera also cross react with
the matrix around epithelial cells in the
horny cap of the unmineralized teeth of
the hagfish Eptatretus stoutii (Slavkin et ai,
1983). Electrophoretic evidence indicates
that the hagfish tooth cap matrix proteins
have molecular weights of 52-55 kilodaltons and therefore may be in the class of
enamelins. According to E. E. Graham
(personal communication), the current
immunofluorescent techniques do not distinguish between amelogenins and enamelins. Mammalian-type amelogenins have
not yet been positively identified in the
cyclostomes and chondrichthyan fish. Kollar (1982) has suggested using enamel protein antisera to test the homology of the
proteins secreted by chick ameloblasts
experimentally induced to differentiate by
combining mouse dental papilla with chick
pharyngeal epithelium grafted into the eyes
of nude mice hosts.
How the two types of enamel proteins,
amelogenins and enamelins, control crystallite nucleation and growth in the matrix
of tooth enamel, and whether the mechanism of mineralization of enamel is homologous from sharks to man are two intriguing puzzles which may soon be solved.
ACKNOWLEDGMENTS
I wish to thank Dr. Alex S. Tompa for
assistance in the literature search for this
contribution in honor of Professor Karl M.
Wilbur, whose research on biomineralization has been an inspiration to both of us.
I am also grateful to Professor Dominic D.
Dziewiatkowski for information about
chemical aspects of mineralization, and
Professor Gerald R. Smith for information
about fossil ostracoderms. My investigations on skeletal calcification in vertebrates
973
have been supported by NSF Grant GB
4317 and USPHS Grant AM 13745.
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