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Protein Synthesis in Wound alter Tooth Extraction in Pancreatectomized Diabetic Rats
Sylvestre Arnaldo GRANDINI1
Luiz Guilherme BRENTEGANI1
Arthur Belem NOVAES1
Renato Hélios MIGLIORINI2
1Faculdade de Odontologia de Ribeirão Preto,
Universidade de São Paulo Ribeirão Preto, SP, Brasil
2Faculdade de Medicina de Ribeirão Preto,
Universidade de São Paulo Ribeirão Preto, SP, Brasil
Braz Dent J (1990) 1(1): 25-30 ISSN 0103-6440
| Introduction | Material
and Methods | Results and Discussion | Conclusion
| References |
The incorporation of alanine C14 in protein synthesis was analyzed in
recently formed alveolar tissue after tooth extraction in partially-pancreatectomized
diabetic rats. The incorporation of alanine C'4 was higher in diabetic
animals than in treated diabetic and control groups. The results can be
explained by a .delay in bone tissue repair.
Key words: protein synthesis, wound healing, pancreatectomy,
diabetes, alanine C14.
Introduction
In wound healing, protein metabolism is fundamental for the repair of collagen,
a tissue which is dependent on the synthesis of a large quantity of a special
protein. Any factor that can affect either the intracellular mechanism
of tropocollagen synthesis or the extracellular mechanism of precipitation
and organization of collagen fibers will alter the evolution of the repair
process. The importance of carbohydrate and protein metabolism is emphasized
when we consider some systemic situations which affect the metabolism of
such substances. In this respect, diabetes deserves special attention because
of the generalized metabolic changes it causes. Insulin deficiency provokes
clear alterations of carbohydrate, protein and lipid metabolism. It has
been reported that insulin deficiency in diabetics can restrict physiological
stimuli to osteoblasts (Hernberg, .1952) and osteoclasts (Pinheiro, 1966)
with a consequent reduction in their number and activity. Grandini et al.
(1970) observed histologically that the process of alveolar repair in alloxan
diabetic rats is clearly retarded and that insulin administration to the
animals leads to a more favorable sequence of healing evolution. Wettenhall
et al. (1969) studied the action of insulin and growth hormone on collagen
synthesis in vitro and reported that connective tissue is sensitive to
insulin but not to growth hormone.
Since it has been well established that insulin acts favorably on the
processes of protein synthesis in several tissues (Krahl, 1%2), we undertook
the present study to determine whether the delay in alveolar healing reported
previously was simply dependent on the activity of protein synthesis. Thus,
the objective of the present investigation was to evaluate in vitro the
activity of protein synthesis using labelled amino acid alanine C14, in
the tissue formed following dental extraction in pancreatectomized diabetic
rats.
Material and Methods
Two hundred and forty male rats (Rattus norvegicus albinus, Wistar) weighing
140 to 220 grams were used. The animals were fed a balanced diet before
and after the experiment and were divided at random into three groups:
1) control, 2) pancreatectomized diabetic animals and 3) pancreatectomized
diabetic animals treated with insulin (NPH, Lilly).
Diabetes was induced by partial pancreatectomy. About 20'11v of the
animal's pancreas was maintained after performing the first step of the
Migliorini (1970) technique. To check the diabetic condition, the King
and Gardner (1947) method was used to determine blood glucose concentration
in three stages, I.e., 7 days after pancreatectomy, at extraction time,
and on the day of sacrifice. Only the animals which presented blood glucose
levels equal to or higher than 250 mg per 100 ml throughout the experiment
were utilized. The group of treated diabetic animals started to be treated
with 1 unit insulin two days after partial-pancreatectomy and the dose
was gradually increased up to 3 units per day and maintained at that level
until the day of sacrifice.
The extractions were performed at least 10 days after partial pancreatectomy.
Following general anesthesia by inhalation of sulfuric ether, the upper
right incisors were extracted using a specially adapted instrument, by
the method of Okamoto and Russo (1973). After surgery the animals were
given water ad libitum and only soft food in order to maintain the alveolus
free of food particles. As prevention against infection, the animals received
three daily intramuscular injections of 100 units of penicillin (Despacillin
R 400, Squibb).
Based on previous histological findings (Grandini, 1978), it was determined
that sacrifice time should be at the following periods after surgery for
the control group: 3, 4, 5, 7 and 10 days; and for the other 2 groups:
5, 7, 10, 12 and 14 days. Repair tissue was removed from the alveolus at
these times. The animals were sacrificed by decapitation and the right
maxillary alveolar processes were immediately removed and carefully dissected.
The distal bone wall of the alveolus was then removed for access to, and
collection of healing repair tissue. At sacrifice, material was also collected
from other animals kept at the same experimental conditions for histological
preparation in order to verify microscopically which kind of tissue had
been incubated.
Healing repair tissue fragments were incubated in buffered Krebs-Henseleit
(1932) medium which was balanced by a gaseous phase containing 95% 02 and
5'11v C02, with final pH between 7.3 and 7.5. To each incubation bottle
containing 2 ml medium 5 ml alanine labelled with C14 ( = 0.5 mc) was added.
The incubation bottles containing the material collected were exposed one
minute to 95% 02 and 5% CO2 and shaken for 2 hours at a constant temperature
of 37°c. After incubation, the material was pooled in 15-ml centrifugation
tubes. one drop of material and of saturated Na2SO4 was added and then
the material was mixed well. The quantity of c14 protein was determined
as reported by Migliorini and Manchester (1971).
Results and Discussion
The results obtained for each group during the postoperative period are
presented in Table 1 and Figure i. The activity of protein synthesis in
the recently formed alveolar tissue following dental extraction was calculated
as the average value obtained from all the incubation tubes, being the
estimation done for each 25 mg of tissue for 5W,000 ppm.
The new tissue formed and incubated presented good activity of protein
synthesis in the three groups of animals. This activity showed some changes
related to chronology, similar to the findings reported by Grandini et
al. (1970). In quantitative terms, however, alanine C14 incorporation into
protein was higher in the diabetic animals than in the treated diabetic
and control groups. Protein synthesis peaked on the fifth day in the control
group and on the tenth day in the diabetic group. In contrast, in the treated
diabetic group, protein synthesis peaked around the 5th, 7th and 10th postoperative
days, with slightly higher levels on the 7th day.
A hypothesis that could be considered to explain the high level of protein
synthesis in the diabetic animals is the lack of evolution from repair
tissue to bone tissue. Guimarães (1967) reported a longer healing
phase in diabetic animals, due to the fact that the fibroblastic phase
started later and progressed more slowly than in the control animals. In
the control group, starting on the 5th postoperative day, the alveolar
apical third was almost completely filled with recently formed osseous
trabeculae; the medial third presented mostly new connective tissue, and
the cervical part showed only granulation tissue. This picture was not
the same in the diabetic group in which the apical and part of the medial
third remained filled with newly formed repair tissue that only reached
some maturation and characteristics of young connective tissue by the 10th
postoperative day.
In the control group, tissue formation occurred throughout the alveolus,
whereas in the diabetic group it occurred mostly in the apical third and
in the treated diabetic group in the apical and medial thirds of the alveolus.
The animals of the treated diabetic group received insulin throughout
the experiment and showed marked improvement in alveolar repair, very close
to the healing rate observed in the control group.
The present results do not permit us to state with certainty that the
level of diabetes affects the healing process, because the sample studied
was obtained from animals with blood glucose levels of 250 to 618 mg/ml
(few of the animals used had blood glucose levels below 300 mg/ml). The
animals with higher blood glucose levels had frequent infections and showed
a quantitative reduction in repair tissue formation. This finding was also
reported by Otaka (1962) working with glycemia over 300 mg/ml.
The amino acid of choice for the present study was alanine C14 because
it is evenly incorporated into all proteins and therefore can be used to
estimate total protein synthesis.
Pancreatectomy was considered to be an excellent model of experimental
diabetes for the study of the metabolic effects of the acute absence of
insulin in rats and was helpful in the study of alveolar repair subsequent
to tooth extraction.
Conclusion
Diabetic subjects showed protein synthesis with more activity and a later
peak during healing when compared with those in the control and treated
diabetic groups. The timing of the activity of protein synthesis is altered
by insulin administration. Partial pancreatectomy proved to be an excellent
means of inducing diabetes in subjects for the study of wound healing in
dental sockets.
References
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healing subsequent to tooth extraction. Oral Surg Med Oral Path 45: 190-199,
1978
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de extração dental em rates cam diabete aloxanico (Estudo
Histológico). Bol Fac Farm Odont Ribeirão Preto 7: 47-71,
1970
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- Descrito, histométrico e histoquímico do tecido de granulação
cicatricial em rates diabéticos aloxanicos. Doctoral thesis, Faculdade
de Odontologia de Bauru 1967
Hemberg CA: The bone structure in alloxan induced diabetes mellitus
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Okamoto, T, Russo MC: Wound healing following tooth extraction. Histochemical
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Otaka Y: Histochemical studies on mucopolysaccharides in the field of
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Pinheiro CE: Estudo de alguns aspectos da ossificação
em rates diabéticos aloxânicos. Doctoral thesis, Faculdade
de Odontologia de Bauru 1966
Wettenhall REH, Schwartz PL, Bomstein J: Actions of insulin and growth
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Diabetes 18: 280-284, 1969
Correspondence: Dr. Sylvestre Arnaldo Grandini, Departamento
de Cirurgia, Faculdade de Odontologia de Ribeirão Preto, USP, 14050
Ribeirão Preto, SP, Brasil.
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