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Mechanism of Action of Calcium and Hydroxyl Ions of Calcium Hydroxide on Tissue and Bacteria
Carlos ESTRELA[1]
Gilson Blitzkow SYDNEY[2]
Lili Luescke BAMMANN[3]
Oswaldo FELIPPE JÚNIOR[4]
[1] Faculdade de Odontologia, Universidade Federal de Goiás,
Goiânia, GO, Brasil
[2] Faculdade de Odontologia, Universidade Federal do Paraná,
Curitiba, PR, Brasil
[3] Faculdade de Odontologia, Universidade Federal de Pelotas,
Pelotas, RS, Brasil
[4] Instituto de Química, Universidade de São
Paulo, São Paulo, SP, Brasil
Braz Dent J (1995) 6(2): 85-90 ISSN 0103-6440
| Introduction | Anti-bacterial
| Biological | Conclusions
| References |
The biological and bacteriological action of calcium hydroxide confer
to it its current success as an intracanal dressing. For this reason the
mechanism of action of calcium and hydroxyl ions on tissue and bacteria
deserves further study. The objective of the present paper is to analyze
and discuss the mechanism of action of calcium and hydroxyl ions on anaerobic
bacteria, starting from the isolated study of the influence of pH on these
bacteria, as well as the mechanism of action of calcium hydroxide on tissue.
Key words: calcium hydroxide, intracanal dressing, anaerobic
bacteria.
Introduction
Recent advances in cellular and molecular biology, biochemistry and microbiology
have brought about a better understanding and better definitions of certain
mysteries still present in endodontics. Modern thinking has been directed
towards the use of an intracanal dressing with a potentially effective
action against different types of respiratory bacteria (aerobic, anaerobic
and microaerophiles) which act by inhibiting the action of osteoclasts
present in the area of dental resorption and which favor the repair process
of altered periapical tissue.
The destruction of bacterial life is dependent on the conditions related
to their growth and multiplication, among which are physico-chemical factors
such as: temperature, pH, osmotic pressure, concentrations of oxygen, carbon
dioxide and substrate (Nolte, 1982). It has been noted that the response
of the periapical tissues to endotoxins produced by gram-negative bacteria,
which are predominant in infected radicular canals, assures an opportunity
for the repair of the destroyed tissue architeture. This fact can be observed
by means of reinsertion of the periodontal ligament and of the reintegration
of the alveolar bone, in conjunction with an osteocemental formation. For
this reason, the intracanal dressing must be effective against bacteria
which may have escaped and survived after root canal preparation, the control
of persistent exudate and the destructive action of the osteoclasts present
in external dental resorption.
The success of calcium hydroxide as an intracanal dressing is due to
its ionic effect observed by the chemical disassociation into calcium and
hydroxyl ions and its action on tissue and bacteria. Its capacity to stimulate
tissue repair through the induction of mineralization confirms the biological
action of calcium hydroxide (Holland, 1971; Binnie and Mitchell, 1973).
The superiority of its antibacterial action when compared to other substances
has also been shown (Bystron et al., 1985).
Estrela (1994) advocates that calcium hydroxide has resisted the test
of time due to two enzymatic properties: the property of inhibiting bacterial
enzymes by means of hydroxyl ions that act on the cytoplasmic membrane
of the bacteria (generating the antibacterial effect) and that of activating
tissue enzymes, such as alkaline phosphatase, which have an influence on
mineralization, leading to the mineralizing effect. The chemical and biological
dynamics which occur, respectively, in the ionic disassociation of calcium
hydroxide and its effect through tissue and bacterial cellular alterations
deserve careful discussion and investigation.
Anti-bacterial action of calcium hydroxide - mechanism of action of hydroxyl
ions on anaerobic bacteria
The greatest concern in the selection of any dressing is the knowledge
of its mechanism of action on the predominant bacterial flora. Antibiotics
provoke two types of effects on bacteria. They either inhibit growth or
reproduction or they lead to its death. These actions are exercised essentially
by interfering in the synthesis of the cell wall, altering the permeability
of the cytoplasmic membrane and interfering in protein synthesis.
From this line of reasoning, one can ask about the site of action of
calcium hydroxide. Could its mechanism of action be considered similar
to that of Penicillin or Cephalosporine, or identical to Nystatin or Polymyxin?
The answer given by the literature is that calcium hydroxide is an exceptional
antibacterial agent due to its elevated pH.
However, on adopting as a reference the effects of antibiotics against
bacteria, and more specifically the site of action, the phenomenon of the
action of calcium hydroxide as an antibacterial should be better elucidated.
For this reason it is important to analyze the isolated effect of pH on
bacterial growth and metabolism, and cellular division.
The essential enzymatic systems of bacteria have as their locale a cytoplasmic
membrane where they involve themselves in the last stages of the formation
of the cellular wall, participate in the bio-synthesis of lipids, and are
responsible for the conveyance of electrons, as enzymes involved in the
process of oxidative phosphorylation. Formed by a double phospholipoproteic
layer, it acts as an osmotic barrier to ionized substances and to large
molecules, being freely permeable to sodium ions and amino acids (selective
permeability). When necessary, they produce proteinase that hydrolyze proteins
and amino acids, since bacteria are generally incapable of utilizing macro-molecules.
The enzymes located in the cytoplasmic membrane relate themselves to
the conveyance of substances to the interior and to the exterior of the
cell, by the structuring of the cellular wall and respiratory activity.
Extra-cellular enzymes act on the nutrients, carbohydrates, proteins and
lipids which, by means of hydrolysis, favor digestion. To sum up, the enzymatic
systems of the cytoplasmic membrane take on primordial functions for the
bacteria, such as metabolism and cellular growth and division (Burnet and
Schuster, 1982).
On the other hand, the catalytic activity of the enzymes can be regulated
by variations of the pH of the medium. Each enzyme possesses an optimum
pH at which its velocity of reaction is maximum (Lehninger, 1986). However,
there is a difference between the internal pH of the bacteria and that
of the medium, possibly being responsible for the influence of pH in bacterial
cellular activity. As a matter of fact, the mechanism which maintains the
internal neutrality in unknown (Kodukula et al., 1988).
The enzymes present internally and externally in the cytoplasmic membrane
influence their complex metabolic reactions, the velocity of the chemical
reactions favored by those enzymes influenced by the substrate. It is believed
that the control of the flow of nutrients alters chemical conveyance through
the membrane which is essential to bacterial life (Nolte, 1982; Orten and
Neuhaus, 1982).
The energy necessary for the movement of organic nutrients and components
into the cell is obtained through a pH gradient present in the cytoplasmic
membrane which can be altered by a change of pH of the medium. The effect
of the pH on the chemical movement can be direct or indirect. It is direct
when the pH influences the specific activity of the proteins of the membrane,
with a combination with the specific chemical group. On the other hand,
the indirect effect can lead to alterations in the ionization states of
the organic components. The transfer through the membrane is facilitated
more to non-ionized components than to ionized ones. Depending on the pH
there will be an increase of nutrient availability, and an intense transfer
can induce inhibition and toxic effects on the cell. In this way, enzymatic
activity of bacteria is inhibited in conditions of elevated pH (high concentration
of hydroxyl ions) (Kodukula et al., 1988).
The influence of the pH on the transfer and permeability of the cytoplasmic
membrane probably explains the microbiological action of the hydroxyl ions
of calcium hydroxide in the control of bacterial enzymatic activity. The
conveyance of nutrients and the return of the catabolites through the cytoplasmic
membrane must be carried out naturally.
Another explanation about the behavior of hydroxyl ions on the cellular
membrane comes from the chemical mechanism which is related to lipidic
peroxidation. The loss of integrity of the membrane can be observed through
the destruction of unsaturated fatty acids or phospholipids. When the hydroxyl
ion removes atoms of hydrogen from the fatty acids, a free lipidic radical
is formed which, on reacting with the oxygen molecule, is transformed in
a lipidic peroxide radical. The peroxide thus formed can act as a new inductor,
drawing another hydrogen atom of a second unsaturated fatty acid, resulting
in another lipidic peroxide and another new free lipidic radical, transforming
itself into a chain reaction (Rubin and Farber, 1990).
For this reason, the elevated pH of calcium hydroxide, with values reaching
12.6, is due to the great liberation of hydroxyl ions which are capable
of altering the integrity of the bacterial cytoplasmic membrane through
the toxic effects generated during the transfer of nutrients or through
the destruction of the phospholipids of unsaturated fatty acids.
With respect to its antibacterial action, Estrela et al. (1994) raised
the hypothesis of the possibility of calcium hydroxide producing reversible
and irreversible bacterial enzymatic inactivation. The irreversible inactivation
can be observed in extreme conditions of pH over a long period of time
during which there is a total loss of biological activity of the cytoplasmic
membrane. The reversibility of enzymatic activity is encountered on the
return to the ideal pH. Lehninger (1986) relates that extreme values of
pH cause the uncoiling of many proteins with loss of their biological activities.
For many years the process of denaturation was thought to be irreversible.
However, if pH returns to its normal value, there is a return of native
structure of the lost biological activity, that is to say, there is renaturation.
Kodukula et al. (1988) also consider that a reactivation of catalytic activity
is possible when the enzyme resumes operating in an ideal pH.
It has also been observed that pH of the interior of the dental tubules
and of the external surface of the cement are not as high as the interior
of the canal which is in contact with calcium hydroxide paste. Estrela
et al. (1994), using a colorimetric method and universal indicating solution,
evaluated in vitro the diffusion of hydroxyl ions of calcium hydroxide
through the dentin in an inert atmosphere of nitrogen. They observed small
modifications of pH on the external surface of the apical cement as well
as in the interior of the radicular canal. In the group in which the vehicles
were anesthetic solution and saline solution, the pH of the apical cement
at 30 days was around 7 to 8, remaining unchanged at 60 days. Meanwhile,
in the group whose vehicle was polyethylene glycol, a pH of 7 to 8 in the
apical cement was only attained at 45 days, remaining the same at 60 days.
In the interior of the radicular canal all the calcium hydroxide pastes
maintained a pH of more than 12 during the 60 days of observation.
This alteration of pH on the surface of the apical cement and in the
interior of the radicular canal, when calcium hydroxide is used as an intracanal
dressing, is due to a greater or lesser dentinal permeability, to the velocity
of diffusion of hydroxyl ions, and to the degree of dentinal calcification
present.
Biological action of calcium hydroxide - mechanism of action on tissue
Calcium hydroxide, apart from its bacterial enzymatic inhibition which
represents an important antibacterial property, has the capability of activating
tissue enzymes which favor tissue restoration through mineralization.
The elevated pH of calcium hydroxide activates alkaline phosphotase
(Binnie and Mitchell, 1973; Tronstad et al., 1981), the best pH for the
activation of this enzyme varying with the type and concentration of substratum,
with the temperature and with the source of enzymes, the limits being from
8.6 to 10.3 (Thompson and Hunt, 1966).
Alkaline phosphatase is a hydrolytic enzyme that acts by means of the
liberation of inorganic phosphate from the esters of phosphate. It is believed
to be intimately related to the process of mineralization (Granstrom and
Linde, 1972). This enzyme can separate the phosphoric esters, freeing phosphate
ions which, once free, react with calcium ions from the blood stream to
form a precipitate, calcium phosphate, in the organic matrix. This precipate
is the molecular unit of hydroxyapatite (Seltzer and Bender, 1979).
Calcium hydroxide in direct contact with conjunctive tissue gives origin
to a zone of necrosis, altering the physico-chemical state of intercellular
substance which, through rupture of glycoproteins, seems to determine proteic
denaturation (Holland, 1971). The formation of mineralized tissue after
contact of calcium hydroxide with conjunctive tissue has been observed
from about the 7th to the 10th day (Holland, 1971; Binnie and Mitchell,
1973).
In this context, Holland (1971), while studying the repair process of
dental pulp after pulpotomy with calcium hydroxide, verified in the superficial
granulosis zone interposed between the zone of necrosis and the deep granulosis
zone, the existence of massive granulation. He goes on to report that these
structures are made up of calcium salts and calcium-protein complexes.
They show themselves to be birefringent to polarized light, reacting positively
to chloramilic acid and to Von Kossas's method, providing that part of
the calcium ions come from the protective material. Below the profound
granulation zone are the proliferation cellular zone and the normal pulp.
Similar results were obtained by Seux et al. (1991).
Other hydroxides, such as those of barium and strontium, by histochemical
methods have shown similar effects to those obtained by calcium hydroxide
(Holland et al., 1982), reaffirming the active participation of calcium
ions from the protective material in the areas of mineralization. Using
a different methodology, electronic sweep microscope and a micro-analyzer
of dispersion of X-ray (EDX), Wakabayashi et al. (1993) have obtained results
which agree with those of Holland et al. (1982).
However, one observes that the hydroxyl ion and the calcium ion of calcium
hydroxide act in a synergic way to mineralization. While the hydroxyl ions
activate the alkaline phosphatase favoring mineralization, the calcium
ions permit the reduction of permeability of new capillaries in granulated
tissue of depulped teeth, diminishing the quantity of intercellular liquid
and activating the acceleration of perophosphatase while at high concentration,
an important factor during mineralization (Heithersay, 1975), and they
act in the complement system activity of the immunological reaction (Tronstad
et al., 1981).
The mechanism of calcium hydroxide action can be altered in the presence
of carbon dioxide (weak oxidec acid) which, by means of chemical reaction,
transforms itself into calcium carbonate. The product thus formed is devoid
of biological and bacteriological properties due to pH exhaustion.
Estrela (1994) reports that, through chemical analysis, calcium hydroxide
pastes were examined for the calcium carbonate formation in conjunctive
tissue of dogs. In the paste containing polyethylene glycol 400 as a vehicle,
lower values of calcium carbonate mass were found. In periods of 45 to
60 days there was practically a stabilization. He goes on to point out
that after the initial reactions of calcium hydroxide with the tissue there
are strong motives to reduce the number of calcium hydroxide paste exchanges
during its use as an intracanal dressing, principally when the initial
inflamatory alteration is overcome.
Conclusions
It is the ionic disassociation of calcium hydroxide into calcium and hydroxyl
ions and their effect on bacteria and tissue which make their use so successful.
The mechanism of action of calcium hydroxide is directly influenced by
its high pH. This results in enzymatic tissue activation, parting from
alkaline phosphatase, and also in the inactivation of enzymes of the cytoplasmic
membrane of bacteria. These two enzymatic effects of mineralization, which
favors the process of tissue restoration, and bacteriological action, which
alters the integrity of the sites essential to bacterial metabolism, growth
and cellular division, are what confer to calcium hydroxide an important
place among intracanal dressings.
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Correspondence: Professor Carlos Estrela, Departamento de Cirurgia
e Medicina Oral, Faculdade de Odontologia, Universidade Federal de Goiás,
Praça Universitária s/n, Caixa Postal 35, 74001-970, Goiânia,
GO, Brasil
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