PII: SO300-5712(96)00068-i Journal of Dentistry, Vol. 25, No. 6; pp.

475-484,1997
Copyright 0 1997ElsevierScienceLtd. All rights reserved
Printed in Great Britain
0300-5712/97$17.00+0.00
ELSEVIER

Artificial salivas for in vi&o studies of dental

materials*
V. W.-H. Leung and B. W. Darvell
Dental Materials Science Unit, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong

ABSTRACT
Objectives: An artificial saliva (AS) of defined composition is necessary for testing the performance of
materials that serve in the mouth as natural saliva is too variable. The chemistry involved is critically
important. Many AS recipes can be found in the literature, but the stability of tooth material, i.e.
hydroxyapatite (HAP), in most of these has not been addressed. In fact, few contain all major ionic
components with concentrations in the physiological range. The aim of the present study was (a) to
review reported AS formulae from their inception in 1931 to date, (b) to compare the stability of HAP in
various reported AS, and (c) to investigate the individual effects of ionic components present in the
reported formulae on the stability of HAP.
Methods: A computer algorithm, RAMESES, for solving multiple equilibrium equations, was employed
for all calculations.
Results: There was a marked difference between two groups, i.e. those with and without the presenceof
Ca; those with Ca were supersaturated with respect to HAP in the physiological pH range, the saturation
pH ranging from about 4.5 to 6.0. There was also an approximately 180-fold range in solubility at pH 7,
due to the individual effects of components such as phosphate, carbonate and citrate. Acetate, lactate and
sulphate showed smaller effects, others have no appreciable effect.
Conclusions: All components and equilibria of relevance to saliva must be included in the system for
detailed models. Continued systematic development of a standardized AS is essential. o 1997 Elsevier
Science Ltd. All rights reserved.

KEY WORDS: Artificial salivas, Tooth mineral, Ionic effects

J. Dent. 1997; 25: 475-484 (Received 3 June 1996; accepted 26 September 1996)

INTRODUCTION understood that duplicating exactly the properties of

human saliva is impossible due to the inconsistent and
An artificial saliva (AS) which reacts with the test unstable nature of natural saliva3. This instability also
material in a manner similar to that of natural saliva is makes natural saliva itself inappropriate for use in
a basic requirement of an artificial oral environment’, standardized in vitro studies. So the development of
as for example, for in vitro tests that require the an AS is essential for well-justified and controlled
chemical conditions pertaining in the mouth’. It is well experiments. There has indeed been a long history of
AS development, but one characterized by generally
*Part of the present findings has been presented at the 73rd haphazard and ad hoc formulation.
General Session of the International Association of Dental The application of an AS for studies of dental
Research conference (1995).
Correspondence should be addressed to: Dr V. W.-H. Leung,
materials can be found reported as early as 1931, when
Dental Materials Science, Prince Philip Dental Hospital, Souder and Sweeney4 studied mercury poisoning
34 Hospital Road, Hong Kong. Fax: (852) 2548-9464. E-mail: by dental amalgam restorations. Greenwood et al5
hrudlwh@hkusua.hku.hk. suggested another formula to study the failure of
476 J, Dent. 1997; 25: No. 6
stainless steel inlay retention posts. Worner6 modified
this slightly for a study of dental amalgam. That AS
was further modified for studies of the corrosion of
dental alloys7, and then employed for amalgam corro-
sion’. Muhler and Swenson’ suggestedan AS explicitly
based on the analysis of the saliva of 11 patients;
this was later modified for amalgam corrosion”.
Wasserman et al. ‘I in 1958 introduced an AS for a
study of dental calculus, which was later applied to
hydroxyapatite nucleation12. Meanwhile, Swartz et all3
studied the tarnishing of dental alloys and said that an
AS was employed to mimic oral conditions. However,
that recipe was not reported. In 1961, Vieira and
Marchi14 investigated the water absorption of polymers
in an AS which they introduced, and that was later
employed for a study of dental amalgam15.Fusayama
et aLI6 reported a recipe, claimed to be modified from
the (unreported) formulation of Swartz et aZ.13, for
corrosion of dental gold and amalgam. This
‘Fusayama’ formulaI was later employed for electro-
chemical and biological tests on dental materials17, for
nickel-based casting alloy corrosion’*, and fluoride
release from dental glass-ionomer”, and further modi-
fied for amalgam corrosion2’, galvanic currents between
gold and amalgam21and corrosion testing”. Hay and
Hartles23 introduced another recipe based on analysis
of human saliva24-27 to study properties of natural
saliva, but it was later employed to study the dissolution
of metallic mercury”. Carter et aZ.29 and Ross et aL3’
introduced yet another recipe of AS for dental amalgam
corrosion, according to a reported composition of
human saliva27.The Carter et a1.29 recipe was modified
for a study of the electrochemical behaviour of dental
gold alloys33,34,and further modified for corrosion
testing of dental amalgam35.Tani and Zucchi31 used a
different recipe for corrosion studies, based on un-
reported data of a human saliva analysis, which
was later employed for corrosion tests of copper-
manganese-nickel alloys32 and, modified, for amalgam
corrosion”. An AS was suggestedfor the treatment of
hypoptyalism36, while a bovine salivary gland extract
was suggested for the treatment of xerostomia. How-
ever, for the latter, quantitative values were reported for
only calcium and phosphate; other components were
not determined. Stookey and Stahlman’s AS to study
tooth enamel fluoride uptake was later employed for
studies of the caries process39and the leachability of
denture base acrylic4’. Darvell developed a formulation
for corrosion studies2,41,also based on reported human
saliva analysis data27,but whose components were, for
the first time, all explicitly tested for appreciable effects
on the corrosion process. That recipe, recognized to
be at an early stage of development, was later im-
proved42,43for studies of the calcium phosphate system
in the oral environment. In 1978, Shellis suggested a
recipe for cultural studies of dental plaque, based on the
reported data of several human saliva analyses45p53.
Demagistris54 suggested a formula of AS for physico-
mechanical characterization studies of polymers. An-
other recipe was introduced in 1985 by ten Cate et al.55
for a study of remineralization, modified from a re-
mineralizing solution used previously with artificial
enamel lesionss6; the nitrate in this is of dubious rel-
evance to the natural system, as is the inclusion of
cacodylate as a pH buffer. Sieck et al.57, in 1990,
developed another AS in a study of enamel fluoride
uptake; and which was claimed to be based upon the
formulation of the previous author56 but the concen-
trations of all components and the buffer solution
used were altered. This recipe57has also been utilized
recently in a study of dentinal tubule occlusion forma-
tie?*. Other new recipes continue to be reported,
for example in studies of Pd alloy corrosion59, cavity
varnish solubility6’, and the plaque-calcium-fluoride
system61.
Darve1141’62examined and reviewed most of the
reported AS recipes up to 1975. It was observed
that most formulations were the result of essentially
arbitrary compilations of substances present in human
saliva, but often with no regard for the solubilities of
components in the form specified2z41%62, e.g. solid sul-
phur and calcium carbonate’,“; and the unjustified
inclusion of some substances, e.g. sulphide and pyro-
phosphate. Indeed, the common failure to justify modi-
fications is noteworthy. Marek63 reported that Meyer
and Nally64 examined the behaviour of several dental
alloys in natural saliva65,Ringer solutions66’67,and five
different synthetic salivass,9316,29,31,
and indicated that,
among those tested, that proposed by Fusayama et aZ.16
produced results most closely approximating those
in natural saliva. Marek63 commented that synthetic
salivas of different formulations often differed substan-
tially in both acidity and buffering capacity. Leung42
extended the review by adding a number of formu-
1ae36,37,54 in addition to Darvell’s list41,62 and also
commented that many AS systemshad been used with
neither justification being offered nor reference to an
authority who does give that justification. The appro-
priate calcium content in particular had not been deter-
mined for any system at all, and that is the constituent
most essential for tests made in contact with tooth
materials. Therefore, Leung42,based on the preliminary
formula of Darvel12,investigated the calcium phosphate
system in saliva-like media. Solubility isotherms for
hydroxyapatite, with and without CO,, through a
large part of the physiological pH range, were estab-
lished42,43.Holland22 did a comparative corrosion study
using two of the AS recipes, i.e. those of Darvell’ and
Fusayama et a1.16, and various sodium chloride solu-
tions with different pH, and commented that the SO~U-
tion from Darvel12had a composition similar to natural
saliva with respect to ions and low-molecular-weight
components. This made it at least potentially useful
also for casting alloys, besides the original purpose of
amalgam corrosion testing. Holland’s results showed
that Fusayama’s solution l6 was more corrosive to the
 Leung and Darvell: Artificial salivas for materials testing
casting alloys. Ranking by the corrosion sensitivity of
amalgam, according to Holland22, the results using
Fusayama’s solution l6 were consistent with the clinical
experience of the materials. However, this cannot be a
true criterion for choosing the most appropriate AS
formula as Fusayama’s AS was clearly shown to deviate
from natural saliva22,i.e.
. in the presenceof sulphur and
higher concentration of urea (reactive components for
the corrosion process).
It is argued that the most fundamental stage in the
development of an AS is to clarify the importance of
each component in the saliva system with respect to
identified ‘sensitive receivers’. One of these would be
tooth tissue, since this ought to be stable with respect to
the test medium43. This testing can only be done
piecemeal by studying individual effects and in the
context of the whole system. Accordingly, the present
investigation was to compare the stabilities of the
principal tooth mineral, i.e. HAP, in terms of its
solubility isotherm in different AS recipes and to study
the effects of individual components on the stability of
HAP. This was done numerically.
NUMERICAL ANALYSIS
A computer program, RAMESES VIII ~1.97s in Basic
(Basic PDS, v 7.1, Microsoft, Redmond, WA, USA),
using the published algorithm68p72,was employed for
solving the multiple equilibrium equations to calculate
the solubility isotherms. The concentrations of the ionic
components of the various AS systems are shown in
Table I. The standardized formation constants p for all
the solution species and solid HAP were recalculated
from the component species from the reported values
(Table II). Since the various sources presented their
data in a variety of styles, the recalculation provides
uniformity and conciseness.The selection of constants
was based upon the reported conditions for their deter-
mination, as done previously by other authors78-81.In
fact, the ionic strength effect on the present system has
been shown to be negligible42. The inapplicability and
inappropriateness of the available activity coefficient
equations as well as, in particular, the use of ionic
strength have been discusseda2.We favour the view
that hydration of solution species, as advocated by
Heyrovskas3, is the underlying physico-chemical expla-
nation of the non-ideality of solution properties,
although the implications for speciation are not yet
fully worked out.
The apparent stabilities of HAP in the various AS
formulae were compared according to their reported
compositions. However, it is apparent that [Cal, when
included, would obscure the underlying behaviour with
respect to HAP. So a second series of calculations was
performed omitting the stated [Cal. A third series
omitted [P] as well, better to understand the role of
other components. The effect of dissolved CO, was then
477
tested in similar fashion. In addition, a number of
simple systemswere tested to determine the incremental
effect on the HAP isotherm of various components.
The basis of the calculations has been set out
in detai169-73,and only a summary of the problem
is outlined here. The equilibrium equations are
represented in matrix algebra notation thus:
CP=L, (1)
where C(r x s) is the coefficient matrix for the Y
equilibrium equations for s species; P(s x 1) is the
vector of logarithmic concentrations, i.e. Pi=lOg(Xi),
where (XJ=(species i); and L(s x 1) is the vector of
constants, where Lj=log(~j), Kjzstability constant of
the jth equilibrium equation. There must be charge
balance:
XTQ=O, (2)
where X(s x 1) is the vector of species’ concentrations;
and Q(s x 1) is the vector of their charges; as well as
mass balance:
T - MTX=O, (3)
where M(s x t) is the matrix of component multi-
plicities; and T(t x 1) is the analytical totals vector for
those t components. RAMESES efficiently solves this
system of equations 1, 2 and 3 which, being non-linear,
has no explicit solution. The degree of saturation is
obtained from the vector W(u x 1):
W=H- NTP,
(4)
where N(s x u) is the multiplicity matrix for the u
solids; H(u x 1) is the vector of the logarithmic solubil-
ity products. A point on the solubility isotherm corre-
sponds to W,=O, and therefore linear interpolation for
zero-crossing was used to determine this71.
In the present system, there were 83 solution
equilibria and 17 components (see Table II).
The calculations were performed on a grid covering
the ranges log,,[HAP]= -7 to - 1 (step 0.25) mol/l
(as HAP=(Ca2+),(P0,3-)3(OH-)) and pH=3-8 (step
0.125 unit). In each, charge balance at the required pH
was obtained by adjusting [Na+], [Kf] or [Cl-]; the
concentrations of these speciestherefore do not match
the published recipe but at one point. It is easiest to
visualize this as a titration with HAP at a given pH until
saturation is attained.
Some modification of the systems studied were
inevitable. Thus the solid S in AS(4,l l), Br and I
in AS(S), as well as cacodylate and HEPES (N-2-
hydroxyethylpiperazine-N’-2’-ethane sulphonic acid)
buffers in AS(20) and AS(21), respectively, were not
included (cf. Table I>. The solubility of solid sulphur
(which is irrelevant anyway) is too low41, and
equilibrium constants for cacodylate, HEPES (both
Tab/e 1. Artificial saliva recipes (concentrations of components are in mmol/l)

AS Authors Na K Ca MQ NH, CI PO, so, co3 SCN Lac tit Urate Urea CO,(g) Other PH
- .~~ 5
0-l
1 Souder and Sweeney (1931)' 0.62 1.03 0.25 1.84 0.31
2 Greenwood et al. (1 937)5 14.64 58.60 5.80 32.19 16.79 5.16 *
6.7
3 Worner et al. (1 937)6 14.64 42.52 5.80 32.19 8.75 5.17
4 Schoonover and Souder (1941)' 14.64 58.60 5.80 32.19 16.79 5.16
5 Muhler and Swenson (1947)9 11.89 5.37 5.99 0.02 12.21 5.06 5.99 16.65 S(s) 0.05 6.9-7.1
6 Wasserman et al. (1958)" 84.16 4.69 1.02 76.72 3.87 6.43
7 Vieira and Marchi (1961)14 1.85 4.54 13.69 3.62 2.27 1.19 8.32
8 Fusayama et al. (1963)'" 15.33 5.37 5.40 0.02 23.02 4.23 16.65 Na,S 15.34; P207 0.01
9 Hay & Hartles (1966)'3 13.04 18.18 2.14 0.42 5.61 15.43 5.45 10.12 2.23 Br 0.07; I 0.01
10 Carter et al. and Ross et al. (1 967)29 29.83 21.79 2.90 28.07 3.08 17.86 3.40 17.86
11 Tani and Zucchi (1 967)3' 16.21 25.05 19.73 1.20 14.91 5.32 *
6.7
12 Guthrow ef a/. (1967)" 11.85 6.71 5.99 0.02 13.55 5.99 16.65 S(s) 0.05; P,O, 0.01 7.1-7.2
13 Brugirard et a/. (1971, 1 973)33.34 33.50 21.79 28.07 4.13 17.86 3.40 2.16
14 Melter and Ledion (1 972)35 33.50 21.79 28.08 2.98 17.86 3.40 21.64
15 Matzker and Schreiber (1 973)36 14.43 21.05 1.27 0.48 34.03 1.96 1.03 7.2
16 Stookey and Stahlman (1 976)38 1.94 0.72 0.29 1.41 0.81 0.29
17 Darvell (1978)' 19.31 4.11 8.01 4.67 7.14 2.38 0.78 0.07 0.09 3.33
18 Shellis (1978)@ 11.55 20.50 1.43 0.21 4.35 23.22 5.10 6.45 2.30 0.05 0.06 2.86
19 deMagistris (1981)54 4.35 20.97 18.90 2.74 2.07
20 ten Cate et al. (1985)55 1.50 150.90 150.00 0.90 NO, 3; Cacodylate 20 7
21 Sieck et al. (1 990)57 30.68 1.09 32.18 0.68 F 0.0026; HEPES 50 7
22 Leung (1989)“' and 28.16 25.74 4.10 29.84 4.67 2.24 0.78 0.95 0.11 3.30 0.04
Leung and Darvell (1991)43
23 Goehlich and Marek (1 990)s9 21.48 25.27 20.12 3.62 17.86 5.14 9.99 0.10
24 Papadogiannis et al. (1991)"' 51.14 1 .oo 37.08 1 .oo Acet 15.06
25 Blake-Haskins et al. (1992)6' 13.00 141.00 1.50 144.80 4.00 8.20
26 Olsson ef a/. (1994)*' 11.90 5.37 7.16 26.50 5.00 67.00 H,S 0.02
27 Olsson et al. (1994)" 16.20 25.02 19.70 1.36 5.32 10.00 0.10

* Data not reported; pH as in original report.

 Leung and Darvell: Artificial salivas for materials testing 479

Table 2. Data used in the calculation of the HAP solubility isotherms. The standardized formation constants (p)
were recalculated from their sources using as components: H,O, CO*(g), H’, Na’, K’, NH,‘, Ca*‘, Mg’-, Cl-,
POd3-, SCN-, lactate-, citrate3-, urate-, urea, P*O, 4- , S*-. Only references not given previously43 are shown

Ref, Species P Ref.

CaOH+ 2.540x1 O-l3 H,(citrate) 1 .2o2x1o’3

Ca(O% 6.015~10-*~ H*(citrate)- 1.585x1 0”
CaHPO, 5.159x10’4 H(citrate)*- 6.918x1 O5
CaH,PO,+ 2.747~10’~ Ca(citrate)- 1.738x1 O3
CaPO,- 2.900x1 o6 Citrate(-H)4- 1.000x1 0-l’
OH- 1.012x10-‘4 CaHJcitrate)’ 1.995x1 0”
&PO, 4.719x1 02’ CaH(citrate) 8.509x1 0’
H*PO,- 3.350x1 o19 Ca(Hcitrate)*n- 2.951xl0’8
HPOpm 2.114x10’* Ca(citrate)+ 1.047x1 06
CO&q) 1.862x1 0 Ca(urea)*+ 5.250x10-’
HP& 2.290x10-* Ca(urea)*n+ 1.290x1 o-1
HCO,- 3.630~10-~ HSCN 7.940x1 om3
CO+ 2.689x1O-‘6 H(urate) 7.692x103
CaCO, 8.119x10-‘* H*(urate)’ 1.932x1 o9
CaHCO,+ 6.453x1 O-5 Urea(-H)- 5.013xl0’3
NaCO,- 7.582x1 O-l7 H(urea)’ 1.289
NaHCO, 5245x1 O-6 NH,(w) 5.435x10-‘0
77
H(lactate) 4.266x1 O3 MgOH’ 1.995x1 o-‘*
77
Ca(lactate)+ 1.844 MgSCN’ 1.000x 1o-’
Ca(lactate), 1.738x1 0 MgCO, 3.544x10-19 77
73 77
H(acetate) 5.721~10~ MgHPO, 1.334xl0’4
73
Ca(acetate)+ 1.514x10 MgH,PO,+ 1.334x1 020 77

73
Na(acetate) 6.607x10-’ Nd-WO& 4.470x1 oz9 77
74 77
H,F’O& 6.011~10~~ MgPO,- 2.512~10~
74 77
V%k 5.894x103’ MgSO, 1.995x1 0
74
HJPO&- 1.700x1 03* MgCI’ 1.000x1 o-’ 77
75
CaH,PO,),- 1.452x1 O= MgWCO,), 7.569x1 O-29 77
75
Ca2H2W4)2 1.331 xl 033 Mg(citrate)- 8.318~10 73
43
CaH,PO,H,CO,+ 7.674x1 0” Mg(citrate)+ 7.079x1 o3 73
43
CaH*PO,HCO, 2.432x1 0” Mg(urea)*+ 4.898x1 0-l 73
76
Ca*HPO,CO, 1.535x1 O8 Mg(urea),z+ 1.202x1 o-’ 73
76
Ca*PO,CO,- 7.674x1 0-’ HP*O,s- 3.890x10’ 7,
77
KSO,- 6.920 H*P,O+ 2.455x1 016 77
77
NaSO,- 4.900 H,P,O,- 3.802x1 0” 77
77
HSO,- 3.160x1 0 VA 1.905x1 0’9 77
7,
CaSO, 2.692x1 0’ MgP*O,z- 2.344x1 O5 77
77
HNO, 3.981x105 MgHP*O,- 5.888x1 O’* 7,

NaNO, 77
1.410x1 0-l Ca2P207 6.310x10* 77
77
KNO, 6.030x10-’ HS- 1 .ooox1o’3 77

CaNO,+ 77
1.150 W 6.918xlO’9 77
77
WNOJ, 5.010x1 o-’
KCI 77
7.943x1 o-’ HAP 1.375x1 045
NaCl 9.120x10-’

also irrelevant), Br and I with other components in the
AS systems were absent from the literature. Two AS
recipes, AS(2,l l), could not be explicitly tested because
the actual amounts of CO, present were not specified
and this would have a significant effect on the stability
of HAP43 (see below). However, CO, at 0.035 bar
was included for the calculation for those two ASS on
the basis that this partial pressure lies within the
physiological range.
RESULTS AND DISCUSSION
Figure 1 shows the results for the reported AS recipes.
Three clear groups of isotherms, i.e. (a), (b) and (c),
were found. The vertical part of e;bcl_h
of the solubility
isotherms in group (a) indicates a maximum pH (or so
called critical pH) at which the recipe would give
complete solution of the ingredients. These calculated
solubility limits range from pH 4.47 to 6.01. The Ca and
PO, contents in those AS recipes range from 0.25 to
7.16 and 0.31 to 5.1 mmol/l, respectively. This demon-
strates that the definition of a fixed ‘critical’ pHs4 to
determine de- and remineralization potential is mean-
ingless. The separation of the three groups was due to
the presence of the two most important components:
i.e. calcium for group (a) and carbonate (or CO,) for
(c), producing the deviations from condition (b).
When [Cal was omitted, groups (a) and (b) in Fig. I
were merged (Fig. 2). The spread of behaviour between
the CO,-free and CO,-containing groups is noteworthy.
480 J. Dent. 1997; 25: No. 6
(4
-I 5 6 7 8
PH
Fig. 7. Solubility isotherms, log(concentration of HAP) against pH;
AS systems with the presence of all reported components. It is
observed that isotherms fall into three groups: (a) excess Ca, from
right to left at lower margin, AS 1, 16, 21/24, 15, 6, 25, 18, g/IO, 8,
5, 26; (b) from top to bottom at right margin, AS 11, 14, 13, 17,
20/7/4/3/2, 19, 12; and (c) with CO,, as (b), AS 27, 23, 11, 22, 2.
-3
-6
-7
3 4 5 6 7 8
PH
Fig. 2. Solubility isotherms, log(concentration of HAP) against pH;
AS systems excluding calcium. Isotherms fall into two groups: (a)
the list consists of those for (a) and (b) in Fig. 7 and (b) with CO,
(list as in Fig. I).
Further omitting [P] (Fig. 3) shows that most of the
variation in the CO,-free group was due to variation in
the amount of [PI; however, there remain appreciable
differences above pH 6.5.
Figures 2 and 3 show two distinct sets of isotherms,
which are distinguished by the absence (a) or presence
(b) of carbonate (or CO,). Figure 4 demonstrates the
effect of 0.035 bar COz on all ASS by the wider spread
of the isotherms labelled as group (b) in which the
amounts of Ca present were not high enough to attain
saturation with respect to HAP (group (a)). The posi-
tion of the full set of curves in Fig, 5 similarly shows the
effect of CO, having omitted Ca; this can be compared
with Fig. 2. The spread of the curves in Fig. 5, again,
r -4
y -5
-6
3 4 5 6 7 8
PH
Fig. 3. Solubility isotherms, log(concentration of HAP) against pH;
AS systems excluding calcium and phosphate. Isotherms fall into
two groups: (a) all except list (b); and (b) with CO,, AS 23127,
22/2/I 1.
-6
3 4 5 6 7 8
PH
Fig. 4. Solubility isotherms, log(concentration of HAP) against pH;
AS systems with 0.035 bar CO,. Isotherms fall into two groups: (a)
excess Ca, from right to left, AS 21, 24, 6, 15, 25, 18, 9, IO, 3/8, 2,
4/5, 26; and (b) from top, AS 22/l/20, 11/27/16, 19/14, 7/23/13/17,
12.
demonstrates effects due to variation in the ionic back-
ground, The effect of carbonate has previously been
demonstrated both experimentally and numerically42a43;
the numerical modelling required the postulation of two
novel Ca-PO,-CO, complexes to account for the exper-
imental data. These complexes are included in the
calculation now.
Starting with a ‘plain’ solution of Na, Cl (Fig. 6,
curve (a)), the effects of the addition of P04, CO,, and
citrate individually are illustrated. The concentrations
of these components were as in AS(22) (Table I). The
effects on the solubility were due to the formation of
complexes or ion pairs between those components and
the calcium or phosphate ions from the dissolved HAP.
 Leung and Darvell: Artificial salivas for materials testing 481

-2

-6

-7
3 4 5 6 7 8 4 5 7 8

PH
Fig. 5. Solubility isotherms, log(concentration of HAP) against pH;
AS systems with 0.035 bar CO, excluding calcium.
Fig, 7. Solubility isotherms, log(concentration of HAP) against pH;
addition of the same components in Fig. 4, incrementally, i.e. (a)
plain system=Na, Cl; (b) (a)+Cit+PO,; (c) AS(22) without CO,; and
(d) AS(22)=(c)+CO,.

h
-4.6

-4.8
r
P
- -5.0
-IE
-5.2

-5.4

4 5 6 7 8
PH 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8
Fig. 6. Solubility isotherms, log(concentration of HAP) against pH; PH
addition of individual components; (a) phosphate (PO,), (c) citrate Fig. 8. Solubility isotherms, log(concentration of HAP) against pH;
(Cit), and (d) CO,, individually to the plain system (b), i.e. system addition of components, i.e. (b) lactate (Lac), (c) sulphate (SO,),
with only water, Na and Cl. and (d) acetate (Acet) individually to the plain system (a).

Citrate, which forms complexes with Ca, pushes the
isotherm markedly upwards (curve (c)). An even larger
effect is due to the formation of Ca-P04-CO, com-
plexes (curve (d)). However, as expected, adding PO,
depresses the isotherm (b).
Figure 7 shows the successive contributions of the
components with the major effects, i.e. Cit, PO,, CO,.
The difference between curves (b) and (c) is due to the
remaining components of AS(22) (except CO,) taken
together. The effects of acetate (as in AS(24)), lactate
(AS(22)) and sulphate (AS( 16)), were small (Fig. S), and
no appreciable effects were observed from remaining
components, individually, in all mentioned AS recipes.
However, this is not meant to imply that the omission
of those components is appropriate because, as the
undulation of curve (c) in Fig. 7 shows, the net effect of
all such components may be important. At least, these
components should not be ignored for a detailed model,
bearing in mind that stability with respect to HAP is
only one criterion of many for the appropriateness of
an AS. Curve (b), in Fig. 7, with the more obvious
undulation for the plain system with added PO, and
Cit, demonstrates how PO, and citrate compete with
each other across the range of pH. In comparison with
curve (c), AS(22) without carbonate, the damping effect
due to the presence of the remaining components in
AS(22), other than CO,, is shown.
The large variation in solubilities of HAP between
AS formulae emphasizes that (1) most compositions
were chosen arbitrarily without taking into account the
stability of tooth mineral, i.e. HAP, and (2) some
formulae could not represent the aqueous situation of
482 J. Dent. 1997; 25: No. 6
saliva reacting with the tooth mineral. All reported ASS
containing Ca were supersaturated with respect to
HAP over the normal physiological pH range (Fig. I).
Clearly, for these, the Ca content is too high. This could
be due to analytical data for human saliva being used
uncritically. A large portion of Ca is normally bound by
proteins in human saliva85, so the analytical total Ca
cannot reflect the situation in the absence of protein.
In fact, proteins were not present in any formulae
that were supersaturated with respect to Ca, and no
explanations have been offered as to what the formulae
were based upon.
Each component of an AS has some role in the
process being studied, whether it be corrosion, pH
buffering, water sorption or, fundamentally, the
stability of tooth material. However, most often, no
attempts have been made to clarify the effects of their
components on the materials to be tested or even to
justify the existence of the individual components.
Sixteen out of the 27 AS recipes (Table l) had no
justification for their formulation whatsoever. It follows
that test results in these cases cannot themselves
be justified, especially as no corroborative data were
offered.
It is self-evident that an AS should be similar to
human saliva for oral conditions to be approached in
in vitro simulation studies. Major deviations, unless
explicitly shown to be irrelevant to the system in
hand, disqualify. Likewise, ad Izoc modifications with-
out sound reasoning are unacceptable in principle.
Factors which will, in due course, need to be considered
include tonicity, buffer capacity, minor inorganic com-
ponents and other organic substances.Calcium content
clearly needs to be adjusted to suit the pH chosen as
well as total composition, depending on purpose and
the need for tooth tissue to be stable in contact with the
solution. Only by a systematic approach, as has been
argued already2,42,43,will the essential medium for
many kinds of test be rationalized and standardized.
This approach entails the retention in all contexts of
any component once it has been shown to play a role in
any context. The much larger questions of the concen-
tration sensitivity of any process for any component, as
for Ca regarding HAP, remains.
Finally, a numerical model can only be as good as the
equilibria included, and many that might be expected
have not yet been reported. A comprehensive collection
of formation constants is essential if a fuller under-
standing is to be obtained. Nevertheless, improvement
of the model will not affect the nature of the broad
differences between recipes, and the refining of a formu-
lation will only improve the generality of application.
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