Download Organization of Organic Matrix Components in Mineralized Tissues`

AMER. ZOOL., 24:945-951 (1984)
Organization of Organic Matrix Components in Mineralized Tissues'
STEPHEN WEINER
Incumbent of the Graham and Rhona Beck Career Development Chair,
Isotope Department, Weizmann Institute of Science,
Rehovot, Israel
SYNOPSIS. The organic matrix is thought to play an important role in controlling crystal
growth during the formation of skeletal hard parts. The structural organization of the
matrix macromolecular constituents can provide a key to understanding the nature of the
control processes. Although the data are limited, both vertebrate and invertebrate organic
matrices appear to be organized according to the same "basic motif," namely a core of
relatively hydrophobic structural macromolecules (usually proteins) and surface layers of
acidic proteins and polysaccharides. Analyses of the latter from different invertebrate
phyla using reversed phase high performance liquid chromatography, reveal that the same
two classes of macromolecules are present in each of the three cases studied, emphasizing
the fundamental importance of these components in crystal growth. Substantial information, at the molecular level, on the conformations and orientations of matrix constituents in relation to the mineral crystal lattice, is available only for mollusk shells, and to
some extent on vertebrate tooth enamel. In these cases the major matrix constituents are
aligned with one or more mineral crystallographic axes. These observations suggest that
the matrix performs active, specific roles in crystal growth. Although it is still premature
to assess the importance of various basic crystal growth mechanisms, the data available
do not preclude the possibility that epitaxial crystal growth is an important factor.
hydroxyapatite crystals in tooth dentin and
The growth of crystals within a pre- enamel [McConnell, 1962]). The crystal
formed organic structural framework (the faces that are expressed, as well as crystal
organic matrix) is a basic mode of skeletal size, are also usually characteristic of the
formation adopted by many different mineralized tissue. One particularly interorganisms (Lowenstam, 1981). The organic esting example is that of the foliated calcite
matrix is generally assumed to play an layers of some bivalve mollusks in which
important role in crystal growth and also the rare 1012 face is expressed (Runnecontributes to the biomechanical proper- gar, 1984). This face is a fast-growing one
ties of the formed mineralized tissue. The and would therefore not normally be found
ultrastructural and particularly the crys- in inorganically formed calcite. Many factallographic properties of the minerals tors can contribute to the control of crystal
formed by the "organic matrix mediated" growth, although the presence of the prebiomineralization process show that the formed organic matrix suggests that it perconditions under which crystals grow are forms some of the fundamental roles. It is
usually very well defined (Lowenstam, unlikely, however, that the matrix always
1981; Lowenstam and Weiner, 1983). functions in the same manner. Organic
Often only one particular form of a given matrices associated with amorphous minmineral will be found at a particular min- erals, for example, probably do not pereralization site (for example, calcite, ara- form the same functions as matrices assogonite, vaterite or monohydrocalcite for ciated with ordered crystals (Weiner et al.,
the carbonate minerals [Lowenstam, 1983a). Although some indications about
1980]). In some cases the degree of crys- the role of the organic matrix in crystal
tallinity is also specifically determined (for growth can be obtained from examining
example, the contrasting crystallinity of the properties of the mineral phase, clearly
the definitive evidence must come from the
matrices themselves, particularly with
respect to organization of the constituent
1
From the Symposium on Mechanisms of Calcification macromolecules, their molecular relations
in Biological Systems presented at the Annual Meeting to the mineral phase and the nature of their
of the American Society of Zoologists, 27-30 Decem- surface properties. This paper is a "status
ber 1983, at Philadelphia, Pennsylvania.
INTRODUCTION
945
946
STEPHEN WEINER
report" of the subject, relying to a large
extent upon information obtained from
invertebrate mineralization systems; a field
of study whose foundations were laid by a
few pioneers, prominent among whom is
Professor Karl Wilbur.
framework constituents varies substantially in different tissues, presumably
reflecting in part the various biomechanical requirements to which the skeletal hard
part is subjected. More recent comparative
information on the biochemistry of the
acidic matrix constituents from a protoBASIC MOTIF OF ORGANIC
zoan (the benthic foraminifer Heterostegina
MATRIX ORGANIZATION
depressa [Weiner and Erez, 1984]), an echiIn many mineralized tissues, the organic noderm (the sea urchin Paracentrotus livimatrix forms a two- or three-dimensional dans [Weiner and Erez, 1984]) and a molstructure onto which or into which the lusk (the cephalopod Nautilus repertus
crystals grow. Microscopic examination of [unpublished data]) show that in all three
thin sections of such matrix layers has, in shells the same two classes of soluble-acidic
the case of mollusk shells (Nakahara, 1979, macromolecules can be identified, primar1983) and vertebrate tooth enamel (Little, ily by their amino acid compositions (Table
1958; Yanagisawae/a/., 1981), revealed an 1). The one class is composed mainly of
internal organization of matrix constitu- proteins rich in aspartic acid, possibly with
ents (proteins and polysaccharides) which some covalently bound polysaccharides.
may be common to many other organic The other class contains proteins rich in
matrices. The more acidic hydrophilic con- serine with relatively small amounts of
stituents are closely associated with the aspartic acid. Infrared spectra of this class
mineral phase, whereas the more hydro- of matrix constituents show that relatively
phobic constituents are spatially removed large amounts of polysaccharide are presfrom the mineral. The two categories of ent (Worms and Weiner, unpublished). It
matrix components have been called is of interest to note that the latter class of
"framework" and "surface" constituents, macromolecules resembles some of the
for the more hydrophobic-insoluble mac- acidic "enamelin" proteins of vertebrate
romolecules and the more acidic soluble teeth, particularly with respect to amino
macromolecules, respectively (Weiner et al., acid composition.
1983a). (After dissolution of the mineral
Despite the paucity of information, a
phase by ethylenediaminetetraacetic acid more general view of organic matrix orga[EDTA] at neutral pH, followed by dialysis nization is emerging in which not only is
against water, some of the matrix constit- the basic motif of "framework" and "suruents remain insoluble and some dissolve. face" constituents applicable to matrices
The terms "soluble" and "insoluble" refer formed by widely divergent organisms, but
to these conditions.) Similar ideas were also within the assemblage of acidic matrix conproposed by Degens (1979) for mollusk stituents, the same types of macromoleshells and Glimcher (1981), Veis et al. cules are present. In order to assess whether
(1981), Termine et al. (1981) and others different matrices actually function
for vertebrate bones and teeth.
according to the same basic principles,
In a comparative review of the biochem- much more information is needed, priical properties of mineralized hard parts marily with respect to the conformations
from many different phyla, Weiner et al. and orientations of matrix constituents and
(1983a) concluded that the acidic matrix how these are related to the various minconstituents are present in all tissues for eral phases.
which data is available, whereas the
CONFORMATIONS AND ORIENTATIONS OF
"framework" constituents are not invariMATRIX MACROMOLECULES
ably present. For example, the matrix of
the large robust mollusk, Strombus gigas, is
In contrast to the large amount of biocomposed almost entirely of acidic "sur- chemical information available on matrix
face" constituents (Weiner, 1979). Fur- constituents (e.g., Crenshaw, 1982; Eastoe,
thermore, the biochemical nature of the 1979; Termine, 1980; Veis and Sabsay,
947
ORGANIZATION OF ORGANIC MATRIX
TABLE 2. Amino aad compositions of the Sep-Pak-A* and Sep-Pak-C'fractions obtained from the soluble organic matrix
fractions of the shells of a foraminifer, a sea urchin and a mollusk.
Phylum
Species
Foraminiferab
Echinodermata
Mollusca
Heterostrgina depressa
Paracentrotus Inidans
Xautilus Teprrlu?
Sep-Pak fractions
A
A
A
C
c
c
11.03
9.82
25.40
16.09
30.47
5.86
Thr
4.83
4.17
4.51
4.19
8.16
3.65
Ser
20.00
16.15
5.60
23.75
11.77
7.72
12.69
15.34
Glu + Gin
5.78
16.46
11.68
12.04
Pro
4.55
4.31
3.18
2.46
4.80
5.44
Gly
19.72
19.38
18.03
10.04
24.00
26.60
Ala
9.80
7.94
3.90
8.83
7.65
11.99
Val
4.69
3.07
2.43
4.98
3.06
0.99
—
Met
—
2.56
0.94
0.18
0.10
He
1.52
4.06
1.73
1.75
2.03
1.62
Leu
3.59
5.09
3.99
4.44
2.86
2.42
Tyr
1.66
1.06
2.15
2.15
4.32
1.26
Phe
2.34
2.11
3.60
2.83
1.26
1.38
His
2.07
0.80
0.78
1.75
3.02
3.20
Lys
7.12
1.52
3.36
1.68
3.77
3.87
Arg
—
0.38
0.96
0.20
0.27
1.32
* Sep-Pak-A fraction contains the soluble matrix constituents which are not retained by a Waters Associates
Sep-Pak C18 cartridge when dissolved in 0.05 M sodium acetate pH 6.5. The Sep-Pac-C fractions contain the
components released by a dimethyl sulfoxide flush after the cartridge was flushed previously with 50%
acetonitrile in 0.5 M sodium acetate pH 6.5. For details see Weiner and Erez (1984).
b
Previously reported in Weiner and Erez (1984).
Asp + Asnd
c
d
Also called N. belauensis
Amino acids listed as mole percent. Cysteine is absent or if present, in trace amounts.
1983; Krampitz, 1982; Krampitz et al.,
1983; Linde et al., 1980) relatively little is
known about the conformations and orientations of these macromolecules. Part of
the difficulty can be attributed to the fact
that for X-ray and electron diffraction
studies, the mineral needs to be partially
or completely removed. In the process,
some of the components, particularly the
more hydrophilic ones, rapidly dissolve. For
this reason, almost all the information
available is confined to the more hydrophobic "framework" constituents, and of
these substantial information is available
for only three different tissues; mollusk
shells, mammalian bone and teeth.
Mollusk shell nacreous layers have proven
to be very convenient for studying matrix
conformations and orientations, primarily
because of the regular layered arrangement of alternating sheets of mineral (aragonite) and matrix. Transmission electron
microscopy (TEM) of stained thin-sections
reveals that in individual matrix sheets as
many as five different layers can be observed. The two surface layers are com-
posed mainly of the soluble-acidic constituents (Nakahara et al., 1982; Weiner elai,
1983a). The core comprises a thin layer of
chitin (Nakahara, 1983) sandwiched
between two thicker layers of protein. Xray and electron diffraction studies of the
framework matrix constituent conformations show that the core chitin is in the /3form and the proteins primarily adopt the
antiparallel /3-sheet conformation. Similar
observations were made for prismatic and
foliated layers of mollusks, although the
chitin phase was not always detected using
X-ray diffraction (Weiner and Traub,
1980). The chitin polymers are oriented
approximately perpendicular to the protein polypeptide chains (Weiner and Traub,
1980, 1984; Weineretai, 19836). Thisplywood-like construction presumably contributes to the mechanical strength of the
matrix (Weiner and Traub, 1984).
Not all organic matrices have five layers.
The matrix of the gastropod, Strombus gigas
for example, has only electron-dense layers
when examined with the TEM (Bevelander
and Nakahara, 1980). Decalcification of the
948
STEPHEN WEINER
shell of Strombus with EDTA results in the
dissolution of more than 95% of the matrix.
Ion exchange chromatography of the soluble fraction shows that most of it is composed of aspartic acid-rich proteins (Weiner, 1979). Infrared studies of these
proteins together with their associated
acidic polysaccharides after extraction from
the shell show that both constituents
undergo conformational changes as a result
of calcium binding, with the proteins
adopting the /3-sheet conformation (Worms
and Weiner, unpublished).
T h e spatial relations between the
"framework" constituents and the mineral
phase in the nacreous layers of a cephalopod (Weiner and Traub, 1980), a bivalve
and a gastropod (Weiner et al., 19836) have
been determined by X-ray and electron diffraction respectively. In all three cases, the
same spatial relation between the organic
and mineral phase was observed, namely
the chitin fibrils are aligned with the a-axis
of aragonite and the protein polypeptide
chains with the 6-axis. The gastropod, Tectus, represents a particularly interesting
case as its matrix is composed of a mosaic
of relatively ordered areas and the aragonite crystal associated with each area is
aligned with the local matrix orientation
(Weiner et al., 19836). This type of matrixmineral arrangement is inconsistent with
theories of shell formation involving fields,
gradients or currents over large areas
(Weiner and Traub, 1983).
A number of alternative theories
explaining oriented crystal growth exist
(Mann, 1983) including the much discussed epitaxial growth theory (Irving,
1981). The observations summarized above
by no means prove that crystal growth in
mollusk shells occurs epitaxially upon a
matrix template. They do, however, show
that matrix macromolecules are ordered
and that a well-defined spatial relation exists
between the overgrowth phase (the mineral crystal) and the substrate (the matrix),
properties which are consistent with an
epitaxial model. To validate such a model,
much more information is required particularly with respect to the detailed molecular organization of the "surface" acidic
macromolecules (Weiner and Traub,
1983). For more detailed discussions of
alternative mechanisms of crystal growth
see Towe (1972), Crenshaw (1982), Krampitz (1982), and Mann (1983).
Collagen is the major "framework" constituent of vertebrate bone and dentin. Its con-
formation is undoubtedly the best understood of all the matrix components.
Collagen has a distinctive amino acid
sequence (Bornstein and Traub, 1979; Allman et al., 1979; Highberger et al, 1982),
which enables the three polypeptide chains
per molecule to twist into a rope-like triple
helical conformation (Fraser et al., 1979;
Bornstein and Traub, 1979; Traub et al.,
1969). These molecules, some 300 nm long
and 1.5 nm in diameter, self-assemble and
later crosslink in a staggered array to form
fibrils (Bornstein and Traub, 1979). The
fibrils contain gap regions about 35 nm
long between colinear molecules, the socalled "holes." It has been shown that the
first formed crystals of hydroxyapatite are
located in the gap regions and then spread
to the pore-like spaces within the fibril
(Berthet-Coliminas etal, 1979). The c-axes
of the hydroxyapatite crystals are more or
less aligned along the lengths of the collagen fibers (Glimcher, 1981). The precise
relations, at the molecular level, between
the mineral and organic phases, are not
known. The collagen of dentin and bone
is almost exclusively type I, which also predominates in skin, tendon and other nonmineralizing tissues. It thus appears that
collagen provides a structural framework
on and in which mineralization occurs (Katz
and Li, 1973), but that the direct control
of crystal growth is mediated by other components. Current models envisage one or
more of the acidic "non-collagenous" components binding to collagen and in turn
directing crystal formation (Glimcher,
1981; Veis and Sabsay, 1983; Termine et
al., 1981). There is, as yet, no direct evidence that acidic components are indeed
located between the collagen substrate and
the newly-formed crystal.
The enamel matrix constituents of vertebrate
teeth contain at least two different classes
of proteins. The amelogenins are rich in
proline, leucine and glutamine, and the
enamelins in glycine, aspartic acid, serine,
ORGANIZATION OF ORGANIC MATRIX
glutamic acid and alanine (Eastoe, 1963;
Eggert et al., 1973; Glimcher et al, 1977;
Termine^ a/., 1980; Fincham etal., 1981).
The enamelins are thought to be more intimately associated with the mineral phase
as they cannot be extracted from the tissue
without dissolving the mineral (Termine et
al, 1980). Conformational studies of demineralized enamel organic matrix using
X-ray diffraction show that some of the
proteins do adopt the /8-sheet conformation (Pautard, 1961), in some cases showing the cross-/3 pattern (Glimcher et al.,
1961; Bonar et al., 1965). Some investigators, however, report only diffuse X-ray
diffraction patterns (Pautard, 1963; Fearnhead, 1965). Part of the difficulty can be
attributed to the disordering effects of the
demineralization and drying processes, in
addition to the fact that enamel ultrastructure is very complex (Boyde, 1969; Skobe,
1976).
In a recent study of the enamel of partially demineralized and fixed rat incisors, Jodaikin et al. (1984) obtained X-ray
diffraction patterns in which the oriented
protein reflections (4.7A) and oriented
hydroxyapatite reflections could be related.
This was achieved by careful alignment of
the specimen such that the X-ray beam was
perpendicular to the two main sets of rods
(prisms). The results showed that the bulk
of the proteins, which adopt the /3-sheet
conformation, are aligned so that their
polypeptide chains are approximately perpendicular to the c-axes of the hydroxyapatite crystals in the same rod. The 4.7A
protein reflection is most intense in the
more mature zone of the tooth, where enamelin-like proteins are more prominent.
We conclude, therefore, that some of the
enamelin proteins do have a regular /3-sheet
conformation and are aligned in a specific
manner with respect to the hydroxyapatite
crystals with which they are associated,
properties commensurate with an important role for these components in crystal
formation. We do note that the repeat distance between equivalent calcium ions
along the c-axis of hydroxyapatite is 6.9A
and that the repeat distance between every
second residue along a protein polypeptide
chain is also about 6.9A. However, as the
949
protein polypeptide chains are aligned perpendicular to the hydroxyapatite c-axis, this
optimal lattice match does not occur, at
least for the bulk of the regularly ordered
protein. The significance of this observation is not understood as yet.
Besides the mineralized tissues mentioned above, very little is known about the
molecular conformations and orientations
of matrix constituents from other phylogenetic groups. Nothing is known about
the molecular arrangements of the acidic
matrix constituents on the matrix surfaces—the macromolecules which are in
direct contact with the mineral phase itself
and those which are most likely to be
involved in crystal nucleation, growth and
cessation of growth. Available information
on the possible functions of these acidic
macromolecules is usually of a speculative
nature based on some biochemical property of the molecules (e.g., Weiner and
Hood, 1975; Lee etal., 1977; Weiner, 1983)
or is surmised from the behavior of matrix
macromolecules in solution (e.g., Nawrot et
al., 1976; Wheeler^ al., 1981; Termine et
al., 1981; Blumenthal, 1981; Reynolds et
al., 1983; Stetler-Stevenson and Veis,
1983). Studies of the matrix organization
and structure of mollusk shells have also
lead to some speculative proposals in which
the nucleation site is thought to constitute
a small part of the matrix surface (Crenshaw and Ristedt, 1975; Weiner and Traub,
1984).
CONCLUDING REMARKS
Understanding the principles of crystal
growth in a preformed organic framework
is to a large extent a structural problem.
From the limited data available, few generalizations can be made and these may not
stand the test of time. The basic "motif
of matrix organization, viz., "framework"
macromolecules (when present) contributing primarily to mechanical properties
and "surface" macromolecules primarily
involved with crystal formation, can be recognized in widely divergent phyla which
employ the organic matrix-mediated process for forming their mineralized tissues.
In the few cases where information is available, the matrix macromolecules are
950
STEPHEN WEINER
proteins from oral tissues: II. The matrix proordered to an extent which is commensuteins in dentine and enamel from developing
rate with their performing "specific" funchuman deciduous teeth. Arch. Oral Biol. 8:633tions in crystal growth. It is premature to
652.
assess the importance of basic crystal Eastoe, J. E. 1979. Enamel protein chemistry—past,
growth mechanisms such as epitaxy,
present and future. J. Dent. Res. 58B:753-763.
although the data available to date do not Eggert, F. M., G. A. Allen, and R. C. Burgess. 1973.
Amelogenins—purification and partial characpreclude that this may indeed be a comterization of proteins from developing bovine
mon, basic mechanism by which crystal
dental enamel. Biochem.J. 131:471-484.
growth occurs.
Fearnhead, R. W. 1965. The insoluble organic comACKNOWLEDGMENTS
ponent of human enamel. In M. V. Stack and R.
W. Fearnhead (eds.), Tooth enamel, pp. 127-131.
John Wright and Sons, Bristol.
Fincham, A. G., A. B. Belcourt, and J. D. Termine.
1981. The molecular composition of bovine fetal
enamel matrix. In A. Veis (ed.), The chemistry and
I thank my colleagues Prof. Wolfie Traub
and Dr. Andrew Jodaikin for their most
helpful comments. This research was partly
biology of mineralized connective tissues, pp. 5 2 3 supported by a US-Israel Binational Sci529. Elsevier, New York and Amsterdam.
ence Foundation (BSF) grant.
Fraser, R. D. B., T. P. Macrae, and E. Suzuki. 1979.
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