a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9

Human dental plaque microcosm biofilms: Effect of nutrientvariation on calcium phosphate deposition and growth

Lisa Wong *, Chris H. Sissons

Dental Research Group, Department of Pathology and Molecular Medicine, Wellington School of Medicine and Health Sciences,

University of Otago, P.O. Box 7343, Wellington South, New Zealand

a r t i c l e i n f o

Article history:

Accepted 28 August 2006

Keywords:

Dental plaque

Plaque microcosm biofilm

Plaque mineralisation

Plaque growth

Biofilm

Abbreviations:

BMM, basal medium mucin

DMM, defined medium mucin

CPMU, calcium–phosphate–

monofluorophosphate–urea solution

a b s t r a c t

Plaque mineralisation is a multi-factorial process involving plaque pH, nucleation, inhibi-

tors and promotors. It is poorly understood because of its complexity.

Objective: To establish the effects of amino acids and peptones in the simulated oral fluid

BMM, a saliva analogue DMM and modifications of these on mineral deposition into dental

plaque biofilm microcosms.

Methods: Microcosms were cultured for up to 35 days in an Artificial Mouth pulsed with

sucrose, followed by 10 days periodic treatment with a pH 5.0 calcium–phosphate–mono-

fluorophosphate–urea solution (CPMU).

Results: Initial biofilm doubling times were 3–7 h, which then slowed and varied under the

different nutrient conditions although their pH behaviour was similar. In BMM, mineral

deposition was 20% that of DMM, but removal of BMM peptones increased deposition 12-

fold. Substitution of the amino acids in DMM by casein did not affect deposition levels, but

their removal leaving mucin the sole macronutrient, increased mineral deposition three-

fold, reaching 40 mmol Ca/g protein.

Conclusions: These substantial increases in mineral deposition when the macronutrient

concentration is reduced indicates probable changes in the nucleating, inhibitory and Ca-

binding properties of the simulated oral fluids themselves and/or changes in the plaque

microbiota and their crystal nucleators and inhibitors.

# 2006 Elsevier Ltd. All rights reserved.

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1. Introduction

Dental calculus is dental plaque heavily mineralised with

calcium (Ca) and phosphate (Pi) in large flat crystals,1,2 the

result of interactions between oral plaque microbiota, Ca and

Pi, and mineralisation nucleators and inhibitors in the oral

fluids. Calculus formation varies greatly among individuals.3

The relative importance of factors controlling the processes in

calculus formation is unclear.3,4 Complex multi-factorial

influences on the mineralisation of dental plaque include

the plaque fluid pH as a major driver4,5 which directly affects

calcium phosphate supersaturation with respect to hydro-

* Corresponding author. Tel.: +64 4 918 6311; fax: +64 4 389 5725.E-mail address: lisa.wong@otago.ac.nz (L. Wong).

0003–9969/$ – see front matter # 2006 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2006.08.006

xyapatite.6 We have shown that the generation of an alkaline

pH is an important factor modulating mineral deposition in

vitro.7 Other contributing factors include bacterial and

environmental8 nucleation9 and inhibitor/promotor mechan-

isms.10,11 Crystal formation is initiated by nucleation onto

matrix or exposed cell nucleators. For example, calcification of

the mineralising bacterium Corynebacterium matruchotii occurs

on an extracellular membrane proteolipid nucleation site12

which is a proton pump that removes protons produced

during hydroxyapatite crystallisation.13 Because of the inter-

ference of plaque removal procedures by calculus and the

involvement of calculus in periodontal disease, it is important

d.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9 281

to further understand the multi-factorial controls over plaque

mineralisation in order to develop procedures targeted at

minimising calculus formation.

A plaque mineralisation procedure designed to deposit

calcium phosphate minerals, including (fluor)hydroxyapa-

tites, into dental plaque in vivo14 was developed to provide a

calcium phosphate reservoir in plaque to protect tooth tissues

against a cariogenic acid attack and aid enamel remineralisa-

tion.15 It is based on a urea metabolism-induced pH-rise of

CPMU, a pH 5.0 solution of Ca, Pi, monofluorophosphate and

urea, which is slightly supersaturated with respect to

hydroxyapatite.14 Plaque calcium phosphate precipitation is

induced by plaque microbial urea metabolism which rapidly

raises the pH.16 Monofluorophosphate is hydrolysed to F and

Pi, by alkaline phosphatases,17 favouring the formation of

fluoridated hydroxyapatites. This procedure has been used to

mineralise microcosm plaque biofilms cultured in an ‘Artificial

Mouth’.7,18

The establishment of the ‘Multi-plaque Artificial Mouth’

(MAM) and microcosm dental plaques as realistic models of

the oral environment and natural plaques18–20 in which the pH

can be monitored21 and modified16 provides a laboratory

model which facilitates the study of plaque mineralisation.7 In

microcosm biofilms, the whole mixed oral flora is cultured in

vitro under standardised, controllable and manipulable

environmental and nutrient conditions simulating the human

Fig. 1 – Protocols for each experiment. I, Inoculation on days 1, 3

day 21 (Experiments 1 and 2). (B) Pre-mineralisation sampling on

day 17 (Experiment 3b); mineralisation for 10 days started on d

(Experiment 3a) and for 9 days on day 17 (Experiment 3b). (C) Mi

1), day 46 (Experiment 2), day 37 (Experiment 3a) and day 27 (E

oral cavity. Microcosm plaques have growth rates,22 composi-

tion,19 structure18 and pH behaviour23–25 similar to plaques in

vivo. An important feature of microcosm plaques is their likely

inclusion of the full range of plaque bacteria and metabolic

processes, including those which are unknown, making them

particularly suitable for studying complex processes such as

plaque mineralisation.

Application of CPMU to microcosm plaques for 6 min every

2 h causes plaque mineral levels to increase substantially and

is increased further by elevation of the plaque pH range.7 The

pH can be manipulated by altering the concentration of pH-

modifying substrates, such as urea, in the simulated oral

fluid16 and in CPMU.7 Addition of 20% human serum to the

simulated oral fluid supplied to plaque biofilms strongly

inhibited the deposition of calcium phosphate suggesting

direct inhibition of mineralisation by serum components and

hence gingival crevicular fluid.7 The aim of this study was to

extend this finding by establishing the effect of protein-related

components in simulated oral fluids on the levels of mineral

deposited from CPMU into plaque microcosm biofilms. The

simulated oral fluid, BMM,18 contains undefined components

(20 g/l, yeast extract, trypticase and proteose peptone),

basically modelling nutrients present in saliva. We have also

developed a chemically defined ‘artificial saliva’, DMM,26 for

large-scale culture of plaque microcosms. This enables

specific modification of its 47 components which include

and 5 for all experiments. (A) Insertion of pH electrodes on

day 34 (Experiments 1 and 2), day 27 (Experiment 3a) and

ay 35 (Experiment 1), day 36 (Experiment 2), day 27

neralisation stopped, plaques sampled day 45 (Experiment

xperiment 3b).

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9282

vitamins and growth factors, basal amino acids, salts and

amino acids at 5 g/l equivalent to the composition of casein as

the ‘protein equivalent’.

The hypothesis being tested was that peptides and yeast

extract in the simulated oral fluids inhibit CPMU-based

calcium phosphate deposition independent of modifications

to the pH. This was evaluated by comparing the effects of: (i)

the high peptone ‘simulated oral fluid’, BMM, (ii) the saliva

analogue, DMM and (iii) modifications to their macronutrient/

amino acid composition, on plaque calcium phosphate

deposition, plaque pH and pH responses to CPMU and growth.

2. Materials and methods

2.1. Experimental conditions for general plaque biofilmgrowth and mineralisation

Four experiments, summarised in Fig. 1, were carried out

using the MAM.18–20 Two experiments (Experiments 1 and 2)

examined the effect of the peptides and yeast extract in BMM

and two (Experiment 3a and b) investigated the effect of the

‘protein/peptide’ components of DMM on calcium phosphate

deposition from a standardised 10 day periodic treatment with

a mineralising procedure using CPMU and on plaque growth.

Dental plaque microcosm biofilms were initiated from

saliva that had been stimulated by chewing chicle gum and

was enriched with plaque bacteria by abstention from oral

hygiene for 24 h and collected immediately prior to inocula-

tion, a procedure approved by the Wellington Ethics Commit-

tee. The saliva was filtered through sterile glass wool to

Table 1 – Modifications to composition of simulated oral fluid

Experiment Abbreviationa Componentremoved

Equivalent‘protein’ (g/l)

BMM-based media

1, 2 BMM – 20

1 BMM/5 – 4

1 BMM[(TP,PP,YE)/2] – 10

1, 2 BMM[TP,PP] YE 15

1 BMM[TP,PP,GCHIb] YE 15

2 BMM[PP] TP,YE 5

2 BMM[mucin] TP,PP,YE 0

2 BMM[CA] TP,PP,YE 10

2 BMM[YE,CA] TP,PP 15

DMM-based media

3a, b DMM – 5

3a DMM[casein] – 5

3a DMM[mucin] Amino acids 0

a BMM, basal medium with mucin; TP, 5 g/l trypticase peptone; PP, 10 g/

DMM, defined medium with mucin.b Composition of GCHI enrichment (Remel Inc.). Concentration in 1 l of B

cocarboxylase, 0.13 mg/l p-aminobenzoic acid, 0.3 mg/l thiamine hydroch

hydrochloride, 259 mg/l L-cysteine hydrochloride, 10 mg/l adenine, 0.3 m

remove particulate material. For the culture of each plaque

microcosm, 1 ml of saliva was inoculated onto a 25 mm

diameter Thermanox1 coverslip (Nunc., Naperville, IL, USA) to

initiate biofilm growth. The coverslips were then placed into

the MAM plaque culture system. The microcosm plaques were

reinoculated on days 3 and 5 with 1 ml of saliva collected

immediately before reinoculation following the same protocol

as on day 0. They were grown unmineralised for 17–34 days

with BMM or DMM or their modifications (Table 1) at 35 8C in

an atmosphere of 5% CO2 in N2 supplied for 30-min every 2 h.

Other experiments (unpublished) suggest that plaque micro-

cosm composition is stabilised after about 7–9 days growth. In

the BMM-grown microcosms, measurement of growth was

stopped on day 21 and pH electrodes inserted. They were then

exposed for 9–10 days to CPMU with a standardised 12 h

regime consisting of four 6-min applications of 1.5 ml of CPMU

applied at t = 0, 2, 4 and 6 h and a 6 min application of 1.5 ml of

292 mmol/l (10%, w/v) sucrose at t = 8 h. CPMU contained

20 mmol/l CaCl2, 12 mmol/l NaH2PO4, 5 mmol/l Na2PO3F (MFP)

and 500 mmol/l urea at pH 5.0. The MFP was obtained from

Colgate–Palmolive New Zealand Ltd. and further purified.27

2.2. Experimental protocols for plaques cultured withmodified BMM

Standard BMM contained: 2.5 g/l partially purified pig gastric

mucin (Type III, Sigma Chemical Co., St Louis, MO, USA), 5 g/l

trypticasepeptone (TP) (BBL1, BectonDickinsonMicrobiological

Systems, MD, USA), 10 g/l proteose peptone (PP) (Oxoid, Unipath

Ltd., Basingstoke, UK), 5 g/l yeast extract (YE) (Difco Labora-

tories, Detroit, MI, USA), 33.5 mmol/l KCl, 2.5 mg/l haemin,

, BMM and saliva analogue, DMM

Modification to ‘protein/peptide’ components

Full composition, basal medium plus mucin (2.5 g/l), unmodified

(see text)

BMM itself diluted five-fold

Yeast extract, trypticase peptone and proteose peptone diluted

two-fold, mucin unmodified

Yeast extract removed, contains trypticase peptone, proteose

peptone and mucin

Yeast extract removed, contains trypticase peptone, proteose

peptone and mucin, also supplemented with GCHI enrichmentb

Yeast extract and trypticase peptone removed, contains proteose

peptone and mucin

Contains mucin only

Contains mucin and supplemented with casamino acids

Contains yeast extract, mucin and casamino acids

Full composition, defined medium plus mucin (2.5 g/l), unmodified,

contains ‘protein component’ amino acids (see text)

‘Protein component’ amino acids substituted with casein

Contains mucin only, no protein component

l proteose peptone; YE, 5 g/l yeast extract; CA, 10 g/l casamino acids;

MM: 1.0 g/l glucose, 2.5 mg/l diphosphopyridine nucleotide, 1.0 mg/l

loride, 0.1 mg/l Vitamin B12, 100 mg/l L-glutamine, 11 mg/l L-cystine

g/l guanine hydrochloride, 0.2 mg/l ferric nitrate.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9 283

adjusted to pH 7 and after autoclaving was supplemented with

5.8 mmol/l menadione, 1 mmol/l urea and 1 mmol/l arginine.18

The effect of nutrient variation was examined by modifying

the BMM peptide and yeast extract composition by supple-

mentation, dilution, or removal of components while the other

constituents (KCl, haemin, menadione, urea, arginine) and

mucin were kept constant (Table 1). In Experiment 1, standard

BMM was modified by: (i) diluting YE,TP and PP two-fold

(BMM[(TP,PP,YE)/2]); (ii) diluting these, plus mucin five-fold

(BMM/5); (iii) removing YE (BMM[TP,PP]); (iv) adding the

microbial growth factor supplement GCHI enrichment (Remel

Inc., Lenexa, KS, USA; composition; Table 1) to (iii)

(BMM[TP,PP,GCHI]). In Experiment 2, standard BMM was

modified by: (i) sequentially removing YE (BMM[TP,PP]), TP

(BMM[PP]) and PP from BMM until mucin remained the sole

macronutrient (BMM[mucin]), (ii) adding casamino acids (CA)

(Difco Laboratories, Detroit, MI, USA), at 10 g/l to the mucin-only

medium (BMM[CA]) and (iii) also returning YE (BMM[YE,CA]).

In Experiment 1, plaque microcosm biofilms were cultured

in a five-place MAM18,23 which, in Experiment 2, was extended

with a single-plaque chamber20 to grow a sixth biofilm. BMM,

or modifications of it (described in Table 1), were continuously

supplied at 3.3 ml/h/plaque. Periodic 8- or 12-hourly, 6 min

applications of 1.5 ml of 146 mmol/l (5% w/v) sucrose were

supplied to model meals (Fig. 1). In Experiment 1, sucrose was

supplied 8-hourly during plaque growth and pH measurement

and 12-hourly during treatment with CPMU. In Experiment 2,

sucrose was delivered 8-hourly during growth and during pH

measurement up to day 26, subsequent deliveries were 12-

hourly during pH measurement and CPMU treatment.

The plaque microcosm biofilms were first grown unminer-

alised for 21 days. Growth was measured daily as wet-weight

accumulation22 up to day 17, then micro-oesophageal pH and

reference electrodes inserted on day 21 and plaque pH

continuously measured.18,21,23 pH8h or pH12h values were

obtained during plaque growth and pH measurement con-

tinued during CPMU treatment. Mean pH8h or pH12h values

were derived from the pH reached immediately prior to an 8-

or 12-hourly sucrose application,7 and are a close approxima-

tion of the resting pH which is the steady-state biofilm pH not

influenced by sucrose metabolism and takes longer to

establish.16 On day 34, a pre-mineralisation subsample for

Ca, Pi, F and protein analysis was taken from each plaque

microcosm. The electrodes were removed for pre-mineralisa-

tion sampling and replaced, then the plaques treated with

CPMU for 10 days. At the end of CPMU treatment and 16 h after

the last CPMU application to allow free ions to be washed out,

up to 7 subsamples were taken from each plaque microcosm

and analysed for Ca, Pi, F and protein.

2.3. Experimental protocols for plaques cultured withmodified DMM

Standard DMM (pH 6.8) contained: pig gastric mucin (2.5 g/l),

urea (1.0 mmol/l), ions (in mmol/l: CaCl2, 1.0; MgCl2, 0.2;

KH2PO4, 3.5; K2HPO4, 1.5; NaCl, 10.0; KCl, 15.0; NH4Cl, 2.0), a

basal mixture of amino acids based on salivary levels of free

amino acids (n = 21), vitamins and growth factors (n = 17) and

‘protein/peptide’ equivalent amino acids to model the proteins

in saliva constructed to be equivalent to 5.0 g/l casein.26 The

concentrations of the ‘protein/peptide’ equivalent amino

acids were (in mmol/l): alanine, 1.95; arginine, 1.30; aspar-

agine, 1.73; aspartic acid, 1.52, cysteine, 0.05; glutamic acid,

5.41; glutamine, 3.03; glycine, 1.95; histidine, 1.08; isoleucine,

2.38; leucine, 3.68; lysine, 3.03; methionine, 1.08; phenylala-

nine, 1.73; proline, 3.68; serine, 3.46; threonine, 1.08; trypto-

phan, 0.43; tyrosine, 2.17; valine, 2.38.

In Experiment 3a, three plaque microcosms were grown

with DMM, or modifications of it (Table 1), supplied at 3.3 ml/h/

plaque with sucrose supplied 8-hourly during growth and 12-

hourly during mineralisation (Fig. 1). DMM was modified by: (i)

substituting the ‘protein/peptide’ equivalent amino acids with

casein (Sigma Chemical Co.) itself (5 g/l, DMM[casein]) and (ii)

removing the amino acids, leaving mucin only (DMM[mucin]).

The mucin, urea, salts, basal salivary amino acids and

vitamins and growth factors were kept constant. Microcosm

biofilms were grown for 27 days with growth measured to day

17. A pre-mineralisation subsample from each microcosm

biofilm was taken on day 27, 10 days of CPMU treatment

followed and then further plaque subsamples taken for post-

mineralisation Ca, Pi, F and protein analysis.

In Experiment 3b two biofilms were grown with unmodified

DMM for a shorter period before mineralisation. Biofilm

growth was measured to day 17, pre-mineralisation subsam-

ples were taken on day 17 and then the plaques treated with

CPMU for 9 days before subsamples were taken for Ca, Pi, F and

protein analysis.

2.4. Plaque growth and mineral analysis

Plaque growth was plotted on a linear wet-weight accumula-

tion scale to delineate growth patterns and on a logarithmic

scale to display growth rates. Doubling times (Dt) were

calculated as: Dt = (t2 � t1) 0.301/log (m2) � log (m1), where m1

and m2 are plaque biofilm biomass at plaque age t1 and t2.22

For mineral analyses, microcosm biofilm samples of 10–

190 mg wet-weight7 were dried over P2O5 to a constant dry-

weight, extracted with 1 ml of 0.5 mol/l perchloric acid,

centrifuged (16,250 � g, 10 min) and cell protein measured in

the pellet.28 In the perchloric acid supernatant, Ca was

measured by atomic absorption spectrophotometry,7 Pi by

molybdate reaction and spectrophotometry,29 and fluoride by

ion-specific electrode (Orion, Model 94–09, Cambridge, MA), to

give total mineral levels. For each microcosm biofilm, one pre-

mineralisation subsample was taken for Ca, Pi, F and protein

analysis.

2.5. Data and statistical analysis

Mean (�S.E.) pre-mineralisation levels of the single samples

for all BMM-grown microcosms and for all DMM-grown

microcosms were calculated, i.e. n equals the number of

plaques (Table 2). For each deposited ion, the pre-mineralisa-

tion level was subtracted from the post-mineralisation level

obtained for each mineralised microcosm plaque subsample,

then the mean (�S.E.) was calculated. The mean Ca:Pi molar

ratio (�S.E.) for each plaque was calculated as the mean of

individual Ca:Pi ratios of the subsamples of that plaque

microcosm. The theoretical Ca:Pi molar ratio for hydroxya-

patite (Ca10(PO4)6(OH)2) is 1.67. The mean F:Ca (�1000) molar

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9284

Table 2 – Effect of modifications to the composition of basal medium mucin (BMM) and defined medium with mucin (DMM)on calcium, phosphate and fluoride concentrations deposited in microcosm plaque biofilms mineralised with calcium–phosphate–monofluorophosphate–urea solution (mean W S.E.)

Experimental conditionsa nb Ca (mmol/gprotein)

Pi (mmol/gprotein)

F (mmol/gprotein)

Ca:Pi ratio ofdeposited mineral

F:Ca (�1000) ratio ofdeposited mineral

BMM and modifications

Pre-mineralisation: BMM 11 0.05 (0.02) 0.21 (0.02) 7.5 (7.2) 0.36 (0.13) 1005 (993)

Experiment 1(post-mineralisation)

BMM 5 2.70 (0.70) 1.76 (0.37) 125 (31) 1.81 (0.38) 51 (6)

BMM[TP,PP] 6 2.55 (0.60) 1.63 (0.34) 52 (9) 1.58 (0.08) 34 (13)

BMM[TP,PP,GCHI] 6 1.12 (0.32) 0.67 (0.10) 29 (7) 2.26 (0.94) 31 (5)

BMM[(TP,PP,YE)/2] 6 2.02 (0.59) 1.55 (0.29) 20 (9) 1.26 (0.26) 6 (3)

BMM/5 6 6.92 (0.91) 3.94 (0.47) 121 (11) 1.75 (0.04) 20 (5)

Experiment 2 (post-mineralisation)

BMM 5 1.85 (0.40) 1.63 (0.23) 92 (11) 1.24 (0.11) 57 (11)

BMM[TP,PP] 6 2.51 (0.69) 1.65 (0.37) 81 (35) 2.16 (0.13) 31 (6)

BMM[PP] 7 6.77 (0.68) 4.18 (0.35) 312 (80) 1.68 (0.02) 42 (9)

BMM[mucin] 7 31.14 (4.33) 19.60 (3.10) 384 (56) 1.65 (0.07) 13 (2)

BMM[CA] 6 15.86 (1.92) 8.56 (0.96) 602 (160) 1.89 (0.06) 37 (8)

BMM[YE,CA] 7 5.82 (1.00) 3.85 (0.49) 177 (70) 1.56 (0.09) 32 (9)

DMM and modifications

Pre-mineralisation: DMM 5 0.01 (0.001) 0.21 (0.14) 1.01 (0.51) 0.23 (0.14)c 150 (66)d

Experiment 3a (post-mineralisation)

DMM 6 15.3 (1.9) 9.4 (1.5) 190 (51) 1.67 (0.09) 12 (2)

DMM[casein] 6 15.7 (0.5) 10.0 (0.7) 355 (188) 1.59 (0.06) 22 (11)

DMM[mucin] 6 41.7 (11.6) 23.6 (6.8) 974 (148) 1.77 (0.04) 27 (4)

Experiment 3b (post-mineralisation)

DMM 4 11.3 (0.9) 7.27 (1.18) 481 (38) 1.60 (0.12) 43 (3)

DMM 4 13.5 (4.0) 7.61 (2.11) 371 (64) 1.76 (0.04) 32 (5)

a Abbreviations as in Table 1.b For pre-mineralisation values n = number of plaques, for post-mineralisation plaques n = number of subsamples analysed per plaque.c For one plaque the Pi concentration was below the level of detection. The limit of detection (0.01 mmol P/ml) was used in the calculation.d For three plaques the F concentration was below the level of detection. The limit of detection (0.001 nmol F/ml) was used in the calculation.

ratio (�S.E.) was calculated similarly from the F:Ca ratios of

subsamples for each plaque microcosm.

One-way analysis of variance on the log-transformed data

for each experiment, adjusting for multiple-comparison using

the Bonferroni inequalities was performed using SPSS (SPSS

Inc., Chicago, IL, USA).

3. Results

3.1. Effect of nutrient modification on the deposition of Ca,Pi and F in microcosm plaques

All microcosm plaques treated with CPMU mineralising

solution had increased Ca, Pi and F levels (p < 0.001;

Table 2). ANOVA analysis of the differences between miner-

alised plaque microcosms is shown in Table 3.

In Experiment 1, there was no significant difference in

mineral levels in BMM-grown and modified BMM micro-

cosms except for a three- to six-fold increase in Ca level

deposited in the plaque microcosm grown with BMM/5

compared to BMM[TP,PP,GCHI] and BMM[(TP,PP,YE)/2]

(Table 2; p < 0.05; Table 3). Due to the substantial spatial

differences in mineral deposition between different sites

within microcosm plaque biofilms7 yielding correspondingly

high variance of the mean, none of the other modifications

yielded statistically significant differences in Ca and Pi

deposition.

In Experiment 2, Ca and Pi levels were similar to the

equivalent microcosms in Experiment 1. Removal of YE from

BMM (BMM[TP,PP]) again had no significant effect. Removal

of TP from BMM[TP,PP] and then PP to give BMM[mucin],

yielded a stepwise increase in mineral deposition. With TP

removal (BMM[PP]), Ca and Pi levels increased above the level

in BMM 3.7- and 2.6-fold, respectively (Table 2; p < 0.001 and

p < 0.01, respectively; Table 3). Omitting PP also, leaving only

mucin (BMM[mucin]) resulted in Ca and Pi levels increasing

17- and 12-fold above the BMM-grown biofilm, to 31 and

20 mmol/g protein, respectively (Table 2; p < 0.001; Table 3).

The addition of casamino acids to BMM[mucin] decreased

CaPi deposition by 50% (Table 2; p < 0.001; Table 3). When YE

was re-added, Ca and Pi levels further decreased to levels

similar to the BMM[PP] biofilm of 6.8 and 4.2 mmol/g protein,

respectively (Table 2; p < 0.01 and p < 0.05, respectively;

Table 3).

In Experiments 3a and b in microcosms cultured in

standard DMM, CaPi deposition was similar, five- to eight-

fold greater than in BMM microcosms (p < 0.01; Table 3) and

corresponded to BMM containing 10 g/l casamino acids

(BMM[CA] in Experiment 2). In Experiment 3a, substitution

of the amino acids by casein did not affect Ca or Pi levels,

but their removal altogether leaving mucin as the only

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9 285

Table 3 – Significances from ANOVA analysis of log-transformed calcium, phosphate and fluoride levels in mineralisedmicrocosm plaque biofilms cultured in BMM and DMM and their modificationsa,b

a. Experiment 1

Experimental conditionsc nd BMM[TP,PP] BMM[TP,PP,GCHI] BMM[(TP,PP,YE)/2] BMM/5

BMM 5 y ��BMM[TP,PP] 6 � yBMM[TP,PP,GCHI] 6 ** yyyBMM[(TP,PP,YE)/2] 6 * y ��BMM/5 6

b. Experiment 2

Experimental conditionsc nd BMM[TP,PP] BMM[PP] BMM[mucin] BMM[CA] BMM[YE,CA]

BMM 5 *** yy *** yyy *** yyy � ** yBMM[TP,PP] 6 ** yy ***yyy�� *** yyy � * yyBMM[PP] 7 *** yyy * yBMM[mucin] 7 y *** yyyBMM[CA] 6 ** y �BMM[YE,CA] 7

c. Experiment 3a and b

Experimental conditionsc nd DMM[casein] DMM[mucin] DMM DMM

DMM (Experiment 3a) 6 * y �DMM[casein] (Experiment 3a) 6 * �DMM[mucin] (Experiment 3a) 6 ** y ** yyDMM (Experiment 3b) 4

DMM (Experiment 3b) 4

d. Experiment 1, 2, 3a and b

Experimental conditionsc nd BMM(Experiment 2)

DMM(Experiment 3a)

DMM(Experiment 3b)

DMM(Experiment 3b)

BMM (Experiment 1) 5 *** yyy ** y � ** yBMM (Experiment 2) 5 *** yyy ** yy �� ** yy ��DMM (Experiment 3a) 6

DMM (Experiment 3b) 4

DMM (Experiment 3b) 4

a Calcium (*), phosphate (y) and fluoride (�).b (*,y,�) significant at <0.05; (**,yy,��) significant at <0.01; (***,yyy,���) significant at <0.001.c Abbreviations as in Table 1.d n = number of subsamples analysed per plaque.

macronutrient present, yielded a three-fold increase to 42 and

24 mmol/g protein, respectively (p < 0.01; Table 3).

Mean Ca:Pi molar ratios of the deposited mineral for most

microcosm plaques ranged from 1.24 to 2.26 (Table 2) in all

experiments and were relatively similar to the range of

theoretical values for calcium phosphate phases (1.0–1.9230,31).

Where mineral deposition was substantial, Ca:Pi ratios tended

towards hydroxyapatite (1.67). The standard errors were large

as a result of a large variation in relative Ca and Pi levels in

subsamples obtained from each plaque biofilm. In all experi-

ments, F levels increased substantially following CPMU

treatment to yield variable F:Ca ratios in the range 6–57.

3.2. Plaque pH during microcosm plaque growth andmineralisation in BMM and its modifications

In Experiments 1 and 2, the plaque pH8h and pH12h values of 6.6

obtained during plaque growth, as approximations to the

resting pH, showed minor differences between plaque

biofilms. These differences were similar for corresponding

biofilms in both experiments. An exception was the 0.5 pH unit

lower resting pH of the microcosm cultured with GCHI

enrichment, which included 1 g of glucose (5.6 mmol)/l of

medium (Table 4).

The modifications to BMM did not greatly affect the plaque

pH response to CPMU and sucrose applications, with alkaline

pH fluctuations from 8.6 to 6.8 during CPMU applications and a

decrease to a minimum of pH 5.3 after a sucrose application,

illustrated by representative pH responses for microcosm

plaque biofilms cultured with BMM, BMM[TP,PP] and

BMM[mucin] in Experiment 2 (Fig. 2).

3.3. Microcosm plaque biofilm growth patterns

Plaques grown with reduced concentrations of ‘protein/

peptide’ generally grew slower than those cultured with full

composition BMM or DMM (Fig. 3a and b). In Experiment 1, the

removal of YE from BMM slowed growth after day 6 by

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9286

Table 4 – Plaque pH8h and pH12h values obtained after themeasurement of plaque growth in modified BMM andbefore exposure to CPMU (mean W S.E.) (Experiments 1and 2)

Experimental conditionsa nb pH

Experiment 1, pH8h

BMM 10 6.44 (0.05)

BMM[(TP,PP,YE)/2] 11 6.80 (0.17)

BMM/5 6 6.40 (0.07)

BMM[TP,PP] 10 6.61 (0.23)

BMM[TP,PP,GCHI]c 11 5.95 (0.03)

Experiment 2d, pH12h

BMM 12 6.47 (0.07)

BMM(TP,PP) 12 6.84 (0.05)

BMM(PP) 14 6.49 (0.03)

BMM(CA) 12 6.66 (0.03)

BMM(YE,CA) 15 6.73 (0.05)

a Abbreviations as in Table 1.b Number of pH8h (Experiment 1) or pH12h (Experiment 2)

measurements taken immediately before a sucrose-induced pH

decrease (Stephan curve).c contains 5.6 mmol/l glucose.d Number of pH12h values were obtained for BMM[mucin].

one-third. Addition of GCHI enrichment to BMM[TP,PP] slightly

prolonged rapid growth. Growth in the other modifications to

BMM was biphasic with rapid growth until day 7, slowed

growth until day 11, which then increased again. With

standard BMM, biphasic growth was less pronounced. Growth

of the BMM/5 plaque was also biphasic. Initially 80% of the

BMM plaque, it decreased to about two-thirds and then

increased to a similar rate after day 14 (Fig. 3b). Removal or

dilution of the nutrients yielded corresponding decreases in

biomass accumulation: BMM > BMM[(TP,PP,YE)/2] > BMM/5

(Fig. 3a). In Experiment 2, serial removal of peptides from

standard BMM also yielded decreases in wet-weight accumu-

lation (Fig. 3c and d). Plaques cultured with BMM[CA] or with

BMM[mucin] grew slowly with net accumulation only one-

third and one-tenth, respectively, of BMM but maintained a

constant growth rate during the later period of growth that

was similar to the other biofilms (Fig. 3d).

Fig. 2 – Plaque pH response to CPMU of three plaques in

Experiment 2 grown under different nutrient conditions

((—) BMM; ( - - - ) BMM[TP,PP]; (. . .) BMM[mucin]). pH data

collection was interrupted between t = 24.6 and 27 h.

In Experiments 3a and b, the DMM plaque biofilms grew

similarly to day 7 then growth differed slightly (Fig. 3e). For the

first 24 h, Dts ranged between 2.9 and 6.8 h. Substitution of the

protein-equivalent amino acids with casein itself resulted in a

period of slow then rapid growth after 5 days that was similar

to initial growth in DMM, subsequently falling to a growth rate

that was similar to the corresponding growth in DMM (Fig. 3f)

but at 50% biomass. Growth with mucin and salivary levels of

amino acids only reduced biomass accumulation to about 25%

compared to DMM and produced an apparent sequence of

reducing growth rates.

4. Discussion

Reductions in the concentrations of polypeptides in the

simulated oral fluid BMM and saliva analogue DMM, increased

calcium phosphate deposition substantially in microcosm

dental plaque biofilms following mineralisation with CPMU

under otherwise similar pH conditions and Ca and Pi supply.

This was a result of other changes to inhibitory and Ca-binding

properties of the simulated oral fluid itself and/or changes in

plaque composition or activities. These multi-factorial alter-

natives cannot be distinguished in these experiments but

nevertheless suggest that whatever the explanation, that in

addition to pH7 the presence of proteins and amino acids is

important in modulating plaque mineralisation processes and

hence probably affect calculus formation.

The levels of calcium phosphate deposited in BMM-grown

plaques were the same as those in similarly treated BMM

plaques previously reported, reaching 2 mmol/g protein.7

Mineral deposition in DMM-grown plaques was substantially

greater than in BMM microcosm plaques. In BMM, it was

reduced by high concentrations of the polypeptides present,

progressively less so as they were removed. Ca and Pi levels for

the BMM[mucin] biofilm reached levels 12–17-fold greater than

the full composition BMM biofilms. The ‘inhibitory effect’ of

the peptones and yeast extract was less potent than that of

20% serum7 which contains strong mineralisation inhibitors

such as high levels of albumin.32 DMM contains lower

concentrations of ‘protein/peptide’ equivalent amino acids

and does not contain the undefined yeast extract and peptone

components and under the standard application regime mean

Ca and Pi levels reached 13.4 and 8.1 mmol/g protein,

respectively. Ca:Pi ratios were generally similar to the values

for hydroxyapatite (1.67) and calculus (1.832). The decrease in

F:Ca molar ratios from the pre-mineralisation ratios indicated

F incorporation and the probable formation of fluoridated

mineral phases.

We have shown the plaque pH range to be a major factor in

regulating plaque mineralisation in microcosm dental plaques

under these conditions.7 Differences in pH did not explain the

present results. Although the modifications to BMM affected

plaque biofilm growth and subsequent calcium phosphate

deposition, the pH range during growth was similar for all

biofilms except for the biofilm grown with a low level of

continuously supplied glucose due to supplementation with

GCHI enrichment. Plaque pH range and response to CPMU

treatment also was virtually unaffected and was dominated by

pulsing with urea in CPMU.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9 287

Fig. 3 – Growth of microcosm dental plaque biofilms in unmodified and modified BMM and DMM (see text and Table 1) for up

to 17 days. (a) Linear plot to show the amount and pattern of plaque accumulation and (b) logarithmic plot to display growth

rates for Experiment 1 (—*—) BMM; (—!—) BMM[(TP,PP,YE)/2]; (—5—) BMM/5; (—*—) BMM[TP,PP]; (—&—)

BMM[TP,PP,GCHI]. (c) Linear and (d) logarithmic plots for Experiment 2 (—*—) BMM; (—*—) BMM[TP,PP]; (—!—) BMM[PP];

(—5—) BMM[mucin]; (—&—) BMM[CA]; (—&—) BMM[YE,CA]. (e) Linear and (f) logarithmic plots for Experiment 3a (—*—)

DMM; (—!—) DMM[casein]; (—5—) DMM[mucin]) and the mean of two plaques in Experiment 3b (—*—) DMM)).

In developing the chemically defined artificial saliva DMM,

an amino acid mixture was used because of the large amounts

needed.26 Therefore this bypasses the need for protein

degradation mechanisms required for plaque in vivo to

catabolise protein. The presence of valine and cystine

arylaminidases in BMM plaques and their absence in DMM26

suggests there may be selection of enzymes in plaque

microcosms necessary for the metabolism of the peptides in

BMM. Given the major effect of proteins and peptides on

mineral deposition, the presence of particular proteases and

peptidases in plaque and saliva may be important to the

removal of crystallisation inhibitors (and promotors) in vivo,

most likely by affecting crystal growth. Further simplification

in DMM compared to the in vivo oral environment included

the supply of CO2 to maintain the major plaque buffering

system, the CO2/HCO3� equilibrium.

Although proteins and possibly peptides, are clearly potent

modulators of plaque mineralisation, the sheer complexity

and multi-factorial nature of plaque mineralisation precludes

further definitive conclusions concerning mechanisms

involved. Nutrient acquisition is a major factor in plaque

ecosystem population dynamics and hence plaque composi-

tion and activity.33 Changes to plaque nutrient conditions also

alters the microbial composition of plaques (Sissons, unpub-

lished). The microbiota that was more prominent in the saliva

analogue DMM could have stronger nucleators for mineral

deposition. The mineralising ability of bacteria is strain-

specific.34–37 Likewise, the process of plaque mineralisation

resulting in the formation of calculus is thought to be

associated with strain-specific bacterial activities8 which

include alkali generation,38,39 Ca-binding,40 local Pi genera-

tion,41,42 nucleation by protelipid proton pumps,13 and

changes in protein expression with bacteria in biofilms.43,44

To distinguish between effects on cell-specific nucleators and

inhibitors and inhibitors present in the environmental oral

fluids more detailed study is required.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 8 0 – 2 8 9288

For plaque microcosm growth in these experiments, DMM

and BMM had initial doubling times similar to previous

BMM22 and DMM26 plaques. Subsequent Dts had a greater

range under the different nutrient conditions yielding

differences in plaque growth patterns and rates, reflecting

divergent growth. Growth rates decreased when the poly-

peptide concentrations were reduced, suggesting that the

acquisition of nutrients from macromolecules is a key

regulator of plaque growth. The abruptly varying growth

rates obtained in DMM when the amino acids were removed,

forcing nutrient acquisition from the macromolecules,

casein and mucin, suggests that intraplaque microbial

nutrient inter-relationships changed at these points during

microcosm maturation.

The study of plaque mineralisation has progressed little

over a number of years despite its importance in calculus

formation as well as caries, in particular, the deposition of

calcium phosphate mineral into plaque as a means of caries

protection and enamel remineralisaton.14,45 The models of the

oral environment, natural plaque and plaque mineralisation

procedure described here have shown the importance of

proteins/peptides as well as pH.7 They have considerable

potential for use in further studies into the multivariate

factors affecting plaque mineralisation to complement studies

with single species models.

Acknowledgements

This study was supported by the Health Research Council of

New Zealand, Wellington Medical Research Foundation and

the New Zealand Dental Association Research Foundation. We

thank Colgate–Palmolive New Zealand Limited for supplying

the monofluorophosphate and Gordon Purdie for statistical

advice.

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