doi:10.1111/iej.
12153
REVIEW
Osmolarity and root canal antiseptics
G. Rossi-Fedele1,2 & A. R. Guastalli3
1
Warwick Dentistry, The University of Warwick, Coventry, UK; 2Pontifical Catholic University of Rio Grande do Sul, Porto
Alegre, RS, Brazil; and 3Chemical Engineering, University of Barcelona, Barcelona, Spain
Abstract
Rossi-Fedele G, Guastalli AR. Osmolarity and root canal
antiseptics. International Endodontic Journal, 47, 314–320,
2014.
Antiseptics used in endodontics for disinfection pur-
poses include root canal dressings and irrigants.
Osmotic shock is known to cause the alteration of
microbial cell viability and might have a role in the
mechanism of action of root canal antiseptics. The
aim of this review was to determine the role of osmo-
larity on the performance of antiseptics in root canal
treatment. A literature search using the Medline elec-
tronic database was conducted up to 30 May 2013
using the following search terms and combinations:
‘osmolarity AND root canal or endodontic or antisep-
tic or irrigation or irrigant or medication or dressing
or biofilm; osmolality AND root canal or endodontic
or antiseptic or irrigation or irrigant or medication or
dressing or biofilm; osmotic AND root canal or end-
odontic or antiseptic or irrigation or irrigant or medi-
cation or dressing or biofilm; osmosis AND root canal
or endodontic or antiseptic or irrigation or irrigant or
medication or dressing or biofilm; sodium chloride
Introduction
Antiseptics are substances used to destroy harmful
micro-organisms or inhibit their activity in humans
and animals [http://www.nlm.nih.gov/mesh/MBrow
ser.html (accessed on 23 March 2013)]. Antiseptics
used in root canal treatment include irrigation solu-
Correspondence: Giampiero Rossi-Fedele, 10 Station Path,
Staines, Middlesex TW18 4LW, UK (Tel.: + 44 7841111387;
fax: +44 1784 888160; e-mail: grossifede@yahoo.com).
314 International Endodontic Journal, 47, 314–320, 2014
AND root canal or endodontic or antiseptic or
irrigation or irrigant or medication or dressing or bio-
film’. Publications were included if the effects of osmo-
larity on the clinical performance of antiseptics in
root canal treatment were stated, if preparations with
different osmolarities values were compared and if
they were published in English. A hand search of arti-
cles published online, ‘in press’ and ‘early view’, and
in the reference list of the included papers was carried
out following the same criteria. A total of 3274 publi-
cations were identified using the database, and three
were included in the review. The evidence available
in endodontics suggests a possible role for hyper-
osmotic root canal medicaments as disinfectants, and
that there is no influence of osmolarity on the tissue
dissolution capacity of sodium hypochlorite. There are
insufficient data to obtain a sound conclusion regard-
ing the role of hypo-osmosis in root canal disinfection,
or osmosis in any further desirable ability.
Keywords: antiseptics, osmolarity, osmotic shock,
root canal irrigant, root canal medicament, sodium
hypochlorite.
Received 23 April 2013; accepted 29 June 2013
tions and intracanal medicaments. Apart from their
antimicrobial capability, other advantageous proper-
ties suggested for irrigants include tissue-dissolving
attributes, debris and smear layer removal and lubri-
cation abilities. At the same time, medicaments
should ideally be able to create an environment for
the dental pulp to continue the lateral and apical for-
mation of hard tissue in incompletely formed roots
(Gutmann et al. 2006).
Colligative properties include vapour pressure, boil-
ing point, freezing point and osmotic pressure. These
are dependent on the concentration of solutes in the
© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

solution and are relevant to physiology, biochemistry
and clinical investigations. Some commonly used
expressions of concentration are osmolarity, osmolal-
ity, molarity and molality (Elmoazzen et al. 2009,


Sklubalovǎ & Zatloukal 2010). Although the termi-
nology associated with calculated and measured
osmotic activity is frequently muddled, there are fun-
damental differences between these expressions


(Erstad 2003, Sklubalovǎ & Zatloukal 2010).
Molarity (M) in chemistry is the number of moles
of solute per litre of solution, whilst molality (m) is
the number of moles of solute per kilogram of solvent.
Mole is the quantity of a substance that contains
6.022
1023 (so called ‘Avogadro’s number’) of parti-
cles (Pain 1977, Chang & College 2002). Osmolarity
(Mosm) and osmolality (mosm) express osmotic concen-
tration of solutes by means of the concentration of
osmotically active particles in the solution (Fazekas
et al. 2013). Osmolarity (number of osmol per litre of
solution; osmol L1) of a solution is directly propor-
tional to its molarity (number of moles per litre of
solution; mol L1); both are expressed in volume, and
consequently, they are temperature dependent. Osmo-
lality (number of osmol per kilogram of solvent;
osmol kg1) of a solution is directly proportional to
its molality (number of moles per kilogram of solvent;
mol kg1). For diluted solutions, it is accepted that
osmolarity and osmolality have the same value; if the
concentration is high, this assumption is incorrect. A
conversion factor is required to convert osmolality
into osmolarity, and the same can be applied to
molality and molarity. The relationship observed


results in the following equation (Eqn 1) (Sklubalovǎ
& Zatloukal 2009):
m M
¼ ð1Þ
mosm Mosm
Osmolarity and osmolality are units of concentra-
tion that take into account the dissociative effect,
although most ionic species do not completely dissoci-
ate, with less dissociation occurring in concentrated
solutions than in diluted solutions (Brady 1990). The
capacity of ionic solutes to dissociate in aqueous solu-
tions into charged particles can be expressed in terms
of osmotic pressure. This is defined as the tendency of
a solvent to pass freely, through a semipermeable
membrane, from a solution of lower concentration to
a solution of higher concentration in order to estab-
lish the equilibrium between solutions (osmosis pro-
cess) (Brady 1990); a different definition is the
external pressure in a system needed to prevent the
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Rossi-Fedele & Guastalli Osmolarity and root canal antiseptics
solvent from moving through a semipermeable mem-
brane (Scheie 1979).
Osmotic pressure for electrolytes may be calculated
from the van’t Hoff equation (Eqn 2), taking into
account the degree of dissociation (Brady 1990):
p ¼ iCRT ð2Þ
Where i is the van’t Hoff factor, C is the molarity of
the solution (mol L1), R is the ideal gas constant
(0.082 L atm K1 per mol or 8.314 J K1 per mol)
and T is the absolute temperature (K).
The osmoles of a solution are the standard units of
osmotic effect of a non-dissociating compound in a
solution. The mass of an osmol is the gram-molecular
mass of a solute osmotically equivalent to 1 mole of a
non-electrolyte dissolved in water (Waymouth 1970,


Pain 1977, Debnam 2005, Sklubalovǎ & Zatloukal
2009). For example, a solution with molarity of
0.1 mol L1 of sodium chloride has osmolarity of
0.2 Osm L1 as both Na+ and Cl affect indepen-
dently the osmotic pressure of an ideal solution.
The relation between osmolarity and osmotic pres-
sure is described in Eqn (3) (Wood 1999):
p
Mosm ¼ ð3Þ
RT
Table 1 Summarizes the concentration expressions
commonly used in physiology.
Models used to study osmotic pressure often look at
semipermeable membranes, pure solvents and mem-
branes permeable only to the solvent (Yokozeki 2006).
It is worth mentioning that highly rich and complex
behaviours occur because of the incomplete dissocia-
tion of even strong electrolytes in non-ideal solutions,
and the osmotic effect reduction due to complex inter-
actions between solvents, ions and macromolecules in
colloid and/or protein solutions (Yokozeki 2006). Glo-
bal concentration and osmotic pressure in a mixture
of solutes depend on the contribution of each solute or
sum of concentrations. The concentration of solutes
and its appropriate definition is essential in under-
standing the phenomenon (Yokozeki 2006).
Micro-organisms in nature rarely exist under ideal
conditions that will allow for their growth at maximal
rates. Instead, they are exposed to diverse kinds of
stress through suboptimal or hostile conditions (Matin
2009); osmotic pressure is one of the most significant
physical parameters with which bacteria must con-
tend (Scheie 1979). Osmotic stress can consist of
either ‘hyperosmotic shock’ due to an increase in
external osmolarity, or the opposite, ‘hypo-osmotic
shock’ (Csonka & Hanson 1991). Osmotic insult leads
International Endodontic Journal, 47, 314–320, 2014 315
 13652591, 2014, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.12153 by Universidad Rey Juan Carlos C/Tulipan S/N Edificio, Wiley Online Library on [17/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Osmolarity and root canal antiseptics Rossi-Fedele & Guastalli

Table 1 Concentration expressions commonly used in physiology

Concentration Standard
unit Symbol unit Formula Units References
1
Molarity M Mol Moles of solute/litres of solution mol L Pain (1977), Chang &
College (2002)
Molality m Moles of solute/kilograms of solvent mol kg1 Pain (1977), Chang
& College (2002)
Osmolarity Mosm Osmol /i
ni
Mi(a) Osm L1 Campbell (2004),
∑ci  pR1T1(b) or osmol L1 Wood (1999)
Osmolality mosm /i
ni
Mi(a) mi þ Bi m2i þ Ci m3i þ . . .(c) Osm kg1 Campbell (2004),
pR1T1(c) or osmol kg1 Elmoazzen et al. (2009),
Wood (1999)
Osmotic pressure p – p = RTC (b) atm(d) Campbell (2004),
p = iRTC (b) Brady (1990)
(a)
/i = osmotic coefficient of solute i, which express the degree of non-ideality of the solution. /i takes values between 0 and 1 for
the degree of molecular dissociation. ni = number of particles into which the solute i can be divided (for example sucrose: 1; NaCl:
2; Ca(OH)2: 3, etc.). Mi = molarity of solute i. mi = molality of solute i.
(b)
i is the van’t Hoff factor; C is the molarity of the solution (mol L1); R is the ideal gas constant (0.082 L atm K1 per mol or
8.314 J K1 per mol); T is the absolute temperature (K).
(c)
Polynomial in molality of the solute mi. The virial coefficients Bi and Ci can be determined experimentally.
(d)
When R is expressed as 0.082 L atm K1 per mol.

bacteria to react with mechanisms to escape, increase
robustness and shift from rapid growth to stasis, in
order to increase resistance (Novick 2003, Matin
2009). Stress response strategies are mediated by the
synthesis of different proteins aiming to neutralize and
escape the particular stress (specific stress response)
and to prevent and repair potential damage to vital
cell macromolecules such as proteins, DNA and the
cell envelope (general stress response) (Matin 2009).
The internal osmotic pressure in bacterial cells is
higher than that of the surrounding medium, result-
ing in pressure being exerted outwards on the cell
wall, a process known as turgor (Gutierrez et al.
1995). In case of hyperosmosis, bacteria are able to
produce and accumulate in the cytoplasm ‘compatible
solutes’ or ‘osmoprotectants’ (Str€ om et al. 1986,
Gutierrez et al. 1995) to match the concentration of
salts in the highly concentrated extracellular environ-
ment. The increase in the internal osmotic pressure
aims to stabilize proteins and the cell membrane and
prevents plasmolysis and bursting of the cell (Gutierrez
et al. 1995, Castenholz 2009, Morgan & Vesey 2009).
It is instructive to note that there is a limit in protec-
tion related to the ‘osmoprotectans’ accumulation that
depends on the severity and duration of the stress
(Csonka & Hanson 1991, Gutierrez et al. 1995). The
cytoplasmic membrane area, together with the cyto-
plasmic volume, could change significantly following a
rise in medium osmolality (Csonka & Hanson 1991).
Microbial membranes are permeable to water but
can regulate the movement of solutes and substances
entering or exiting their cells and catalyse exchange
reactions (Csonka & Hanson 1991, Denich et al.
2003). Membranes present with a certain degree of
fluidity to respond to perturbations, such as osmotic
pressure, in order to compensate for the suboptimal
conditions (Denich et al. 2003, Saier 2009). Water
loss can lead to membrane hardening and the poten-
tial loss of structural integrity (Castenholz 2009), and
to different states. In the initial state, the ‘solution
domain’ water is free flowing and metabolic reactions
almost certainly occur at reduced rates. Subsequently,
in a ‘gel domain’, water is no longer a continuous
solvent and metabolic reactions cease (Nienow 2009),
leading to the previously described stasis in order to
increase resistance (Matin 2009).
In the case of hypo-osmotic shock, an influx of
water into the cells occurs with a build-up of turgor
pressure. This is apparently not particularly deleteri-
ous, as bacteria are able to decrease the concentration
of internal ‘compatible solutes’ by catabolism, poly-
merization or excretion of these molecules, as well as
dilution by cellular growth or loss because of leaki-
ness of the membranes (Csonka & Hanson 1991).
The purpose of this article was to review the clini-
cal significance of osmolarity variations in root canal
antiseptics.
Literature search
A literature search, using the Medline electronic data-
base, was conducted for articles published up to the

316 International Endodontic Journal, 47, 314–320, 2014 © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

30 May 2013, using the following search terms and
combinations: ‘osmolarity AND root canal or
endodontic or antiseptic or irrigation or irrigant or
medication or dressing or biofilm; osmolality AND
root canal or endodontic or antiseptic or irrigation or
irrigant or medication or dressing or biofilm; osmotic
AND root canal or endodontic or antiseptic or irriga-
tion or irrigant or medication or dressing or biofilm;
osmosis AND root canal or endodontic or antiseptic
or irrigation or irrigant or medication or dressing or
biofilm; sodium chloride AND root canal or endodon-
tic or antiseptic or irrigation or irrigant or medication
or dressing or biofilm’.
Publications were included if the effects of osmolarity
on the clinical performance of antiseptics in root canal
treatment were stated, if preparations with different
osmolarities values were compared and if they were
published in English. The titles and abstracts of the
publications identified were initially screened by two
independent reviewers. Publications were included for
full-text evaluation by one reviewer (GRF). Full-text
assessment and data extraction were performed by one
reviewer (GRF) if the content of the abstracts met the
inclusion criteria. Furthermore, to include the most
recent publications, a hand search of articles published
online, ‘in press’ and ‘early view’ was performed. The
same search criteria were used as the electronic search
for the International Endodontic Journal, Journal of
Endodontics, Dental Traumatology, Australian End-
odontic Journal, European Journal of Oral Sciences and
Oral Surgery, Oral Medicine, Oral Pathology and Oral
Radiology. The reference lists of those articles included
were checked for additional articles of relevance. A
total of 3274 publications were identified using the
database, and three were included in the review.
Review
Antimicrobial effect
The role of hyperosmotic stress as antimicrobial agent
in endodontics has been studied in two investigations
from the same research group, with promising results
when used for a period of time comparable to intraca-
nal dressings (van der Waal et al. 2011, 2012). No
investigation has looked into the role of hypo-osmosis
in the endodontic milieu. Dual-species biofilms of
Enterococcus faecalis and Pseudomonas aeruginosa were
exposed to 6 mol L1 sodium chloride (NaCl) to raise
the osmotic value of the incubation broths and
assessed for biofilm growth viability and extracellular
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Rossi-Fedele & Guastalli Osmolarity and root canal antiseptics
matrix amount. Ninety-six hour biofilms were used
for viability assessment and incubated for periods of
time of between four and 168 h. When assessed by
conventional plate counting after 72 h, there was a
statistically significant reduction in colony-forming
units when compared with the negative controls.
Twenty-four-hour-old biofilm showed reduced mass
because of extracellular polysaccharide loss, as well as
alteration of cellular shape and size when exposed for
4 h to the stress (van der Waal et al. 2011). A second
publication looked into the possibility of organic salts
being used in addition to NaCl, to test whether two
different stresses will result in an enhanced effect or
‘double-hurdle’ strategy to eradicate E. faecalis in vitro.
In a preliminary screening, several organic acid salts
were tested for biofilm inhibitory ability, and it was
found that the dissolved salts of sodium sorbate (sor-
bic acid and sorbate ions) produced the greatest
reduction in metabolic activity. Subsequently, solu-
tions of potassium sorbate (KS), which dissociate in
the same active components as the latter, were used
together with NaCl in viability assays to test for syn-
ergistic effects. Forty-eight-hour biofilms were
immersed for 1 h in test solutions containing different
ratios of NaCl and KS, as well as the unmixed salts.
When looking at bacterial counts, 4 mol L1 NaCl
did not show statistically different colony-forming
units reduction. This suggests that hyperosmosis per
se has no antimicrobial effect in their experimental
conditions, whilst 1 mol L1 KS presented with a
‘non-clinically relevant’ effect. Conversely, when
mixed together, the salts showed an enhanced disin-
fection, with a mixture of 3 mol L1 NaCl and
1 mol L1 KS being extremely effective. These results
were explained by the authors as resulting from the
combination of hyperosmotic stress and intracellular
accumulation of substances potentially affecting cellu-
lar viability (van der Waal et al. 2012).
Tissue dissolution
Apart from the effect of osmolarity on the tissue disso-
lution ability of sodium hypochlorite, no other desir-
able property for antiseptics has been considered in
the literature included in this review.
The tissue dissolution ability of sodium hypochlorite
preparations appears to be unrelated to their osmolar-
ity values (Jungbluth et al. 2012). Although the
authors described osmolarity values as being ‘similar’,
without reporting any significance tests on this
characteristic, the values reported ranged from
International Endodontic Journal, 47, 314–320, 2014 317

Osmolarity and root canal antiseptics Rossi-Fedele & Guastalli
559.3  0.6 to 732.3  4.9 mOsm L1 (Jungbluth
et al. 2012). As the authors reported a summary data
set instead of the full underlying data, it is not possi-
ble for the reader to carry out robust statistical meth-
ods, although it can be inferred that there are
statistically significant differences between the osmo-
larity values of the solutions tested.
Discussion
The rationale for using a hyperosmotic medication is
to achieve an increase in osmotic values in the canal,
with subsequent ionic diffusion in those areas that are
inaccessible to chemo–mechanical preparation and
therefore antimicrobial action on those areas (van der
Waal et al. 2011, 2012); this is similar to the diffusion
of chlorine forms when sodium hypochlorite is used as
root canal irrigant (Jungbluth et al. 2012, Rossi-Fedele
et al. 2013). The role of root canal preparation to gain
‘radicular access’ for root canal antiseptics has been
previously suggested (Gulabivala et al. 2005).
When looking at the role of stress on bacteria, it is
important to take into account the fact that the vast
majority of bacteria grow in matrix-embedded biofilms
on a wide variety of surfaces, within which they are
protected from antibacterial actions of a different ori-
gin (Donlan & Costerton 2002). The presence of bio-
films in root canals of periapically affected teeth has
also been demonstrated using light and electronic
microscopy in a seminal study (Nair 1987). Biofilm-
embedded cells produce polymeric substances making
this matrix and, from the physiological standpoint,
have altered growth rates and are able to transcribe
specific genes that make the cells more resistant to
antimicrobial agents (Donlan & Costerton 2002). The
cells are protected by the matrix working as a diffu-
sional barrier, due to reduction in the external mole-
cule’s penetration into the interior of the structure
and because of the interactions between the matrix
material and the agent as such (Donlan & Costerton
2002). Interestingly, an in vitro investigation previ-
ously cited suggests biofilm showed reduced mass
because of extracellular polysaccharides loss through
hyperosmotic stress (van der Waal et al. 2011). There-
fore, this appears to be a potential mechanism by
which to overcome this particular survival strategy.
Based on this observation, the possibility of root canal
irrigation with a hyperosmotic solution, followed by
conventional antiseptics, appears worth investigating.
It has been purported that microbial cells have a
minimum cytoplasmic osmolality of approximately
318 International Endodontic Journal, 47, 314–320, 2014
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250 mosmol kg1, being equivalent to an osmotic of
pressure 0.6 MPa (Csonka & Hanson 1991). Mature
biofilms present with complex three-dimensional
structure, with microenvironments differing in
numerous characteristics, including osmolarity (Fux
et al. 2005). As antiseptics have diverse osmolarity
values, they may be expected to have osmotic stress
amongst their mechanisms of action; for example,
sodium hypochlorite preparations values reported in
the literature vary between 559.3  0.6 and
732.3  4.9 mOsm L1 (Jungbluth et al. 2012).
Thus, hyperosmotic shock can be expected to be one
of its antimicrobial action modalities. It should be
noted that the solute concentrations in clinical reality
are very likely to suffer from dilution because of the
leaching of interstitial fluids or blood, as well as in-
adequate removal of previously used irrigants prior to
the placement of intracanal antiseptics.
Hypo-osmotic preparations have been suggested to
cause leakiness of the membranes (Csonka & Hanson
1991). Therefore, theoretically this can cause easier
penetration of active components into the bacterial
cells. However, no investigation has looked at the
role of hypo-osmosis in root canal treatment. The
lack of investigation regarding hypo-osmotic antisep-
tics has implications for research. Traditionally, the
use of substances with low osmolarity as controls,
such as distilled water or tap water, having osmolar-
ity values near to zero (Lindskog & Blomlof 1982,
Hwang et al. 2011) has been considered question-
able, because of the subsequent risk of hypo-osmotic
shock (Casaroto et al. 2010), and the altering of the
viability of the micro-organisms of the control group.
However, there are no data in endodontology to
enable a sound conclusion to be drawn in this spe-
cific regard.
To put in a general context the role of sodium hypo-
chlorite’s chemical characteristics influence on its tis-
sue dissolution ability, it should be noted that pH and
concentration have been shown to play a role
(Rossi-Fedele et al. 2011, Jungbluth et al. 2012),
whilst surface tension, such as osmolarity, has no
effect (Jungbluth et al. 2012, Rossi-Fedele et al. 2013).
Conclusion
The in vitro evidence available suggests that hyperos-
motic preparations might have an antimicrobial role
as root canal medicaments, whilst the osmolarity of
sodium hypochlorite preparations has no effect on its
tissue dissolution ability.
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Rossi-Fedele & Guastalli Osmolarity and root canal antiseptics

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