archives of oral biology 59 (2014) 241–250
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Histometric evaluation of dental alveolar repair in malnourished rats in the intrauterine or postnatal phase Fa´bio A.S. Sartorato *, Cla´udia A.M. Mura, Sandra R.R. Lucas Laboratory of Developmental Biology, Department of Morphology and Genetics, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, SP, Brazil
article info
abstract
Article history:
Objective: Nutritional aggravations during pregnancy or during the early stages of postnatal
Accepted 25 November 2013
development can impair bone development; thus, we aimed to assess the effects of food
Keywords:
Design: Thirty-six Wistar rats were divided into three groups: (C) 12 pups were obtained
restriction on the dental alveolar bone repair process using histometric analysis. Food restriction
from control mothers with food intake at ease; (GR) 12 pups from mothers subjected to 70%
Gestational malnutrition
food restriction during pregnancy; (PNR) 50% of maternal food restriction during lactation
Postnatal development
and 50% of restriction for the 12 pups after weaning. At three months of age, the upper right
Dental alveolar repair
incisor was extracted from the pups. After 14 or 28 days, the pups were sacrificed for evaluation of newly formed bone area (NB) and total bone area (TA) in the medial and apical thirds of the alveolus. Results: In the apical third of the alveolus, the ratio of NB/TA was greater at 28 days for all groups and there was no damage to any of the groups. In the medial third, the ratio was higher at 28 days for the C and GR groups. The PNR group did not show an evolution of alveolar dental repair. Compared between the thirds, all groups exhibited a higher percentage of newly formed bone in the medial third area, at any time point after surgery. Conclusions: The percentage of the total alveolar area covered by newly formed bone (NB/TA) revealed a late preference in the process of alveolar repair in the medial third, although only in the PNR group. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Annually, approximately 30 million children are born with low weight, representing approximately 24% of all births during this period. These children often face serious shortand long-term health problems because low birth weight is the greatest determinant of mortality or morbidity and disability in childhood with repercussions extending into adult life.1
Foetal programming is the phenomenon by which changes in foetal growth and development in response to the intrauterine environment generate permanent effects.2 Thus, several clinical studies have shown that certain parameters, such as bone structure, physiology and metabolism,3,4 bone mass,5,6 its growth7 and its mineral content,8 can be programmed by environmental influences during intrauterine life. Experimental models used for the study of foetal programming theory have demonstrated that nutrition is an important modifiable factor because it determines significant
* Corresponding author at: Departamento de Biologia do Desenvolvimento, Universidade Federal de Sa˜o Paulo, Rua Botucatu, 740, Edifı´cio Leita˜o da Cunha, 28 andar, Sa˜o Paulo, SP, CEP: 04023-900, Brazil. Tel.: +55 11 55764262; fax: +55 11 55764848. E-mail address: [email protected] (Fa´bio A.S. Sartorato). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.11.014
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archives of oral biology 59 (2014) 241–250
Gestation
Lactation
Extraction
21-23 days
28 days
3 months age
Mothers
Euthanasia 14 d
28 d
Pups
C-M
ad libitum
ad libitum
C
ad libitum
GR-M
70% restriction
ad libitum
GR
ad libitum
PNR-M
ad libitum
50% restricition
PNR
50% restriction
Fig. 1 – Nutritional scheme imposed to the pregnant rats and offspring.
changes in bone parameters. Nutritional aggravations arising from protein, energetic or protein-caloric restrictions imposed on animals during pregnancy or during the early stages of their postnatal development can impair bone development.9–14 In addition, specific phenomena of the alveolar bone repair process after tooth extraction have been elucidated using experimental models.15–17 Thus, by imposing a food restriction at different time periods of offspring development, we aimed at evaluate distinctly the potential repercussions of this nutritional aggravation on bone formation and development, which can compromise the dental alveolar repair process of the adult rat using histometric analyses.
2.
Materials and methods
All procedures were approved by the institutional animal research committee. Twelve-week-old Wistar male and female rats were used for breeding. Pregnant animals were transferred into individual plastic cages, enabling the manipulation on their diets during the different developmental phases of their offspring. First, the control group consisting of mothers (C-M, control mother) was established. Control mothers received commercial feed ad libitum throughout gestation and lactation. Once the average daily dietary intake of these animals was determined, some of the animals were subjected to a dietary restriction according to their experimental group. In the group of rats subjected to constraint only during pregnancy (GR-M, gestational restriction mother), the diet was reduced by 70% compared to the normal average daily ingestion of the C-M group throughout the time points. On the day of birth, these mothers received feed ad libitum during lactation. In addition to this restricted group, a group of mothers with postnatal restriction (PNR-M, postnatal restriction mother) was established, in which the rats were deprived of 50% of the average normal diet ingested by C-M animals, but only for 28 days of lactation. At birth, the groups of pups were divided according to the group of pregnant mother rats: Group C, puppies of control mothers, which continued to receive feed ad libitum after weaning; GR group, pups from mothers who have a 70% food restriction only during gestation and after weaning received feed ad libitum; Group PNR; pups of mothers that were subjected to 50% food restriction during the entire lactation period, and continued to be subjected to this restriction until the time of euthanasia.
To avoid differences in the nutritional status of the offspring resulting from differences in the number of animals per litter, only six young males pups were left with each mother. If the litter generated was less than 6 males, then the females of this litter were used to obtain the minimum number of animals until the end of lactation, thus ensuring the same amount of breast milk for all animals. After weaning, only males were used in this experiment. This nutritional scheme was maintained until the animals were sacrificed at either 14 or 28 days after tooth extraction, which was performed when the animals reached adulthood (Fig. 1). All animals used during the experiment were maintained under controlled conditions of temperature (23 1 8C) and relative humidity (55 5%), with artificial lighting via a fluorescent lamp with a photo-period of 12 h of light and 12 h of dark. The animals were fed commercial Labina–Purina chow, which consisted of ingredients listed in Table 1.
2.1.
Surgical procedure
After general anaesthesia with ketamine (10% Dopalen, Vetbrands, Brazil) and xylazine (Anasedan 2%, Vetbrands, Brazil), the male rats had their upper right incisor extracted, with respective to the dental germ. The tooth avulsion was achieved with the aid of a chisel and forceps adapted for such purpose. At the end of the procedure, after applying simple gingival using a mononylon suture wire 5–0, all of the animals received a single dose of antibiotic (Veterinarian Pentabiotic Small Animals, Fort Dodge Animal Health Ltd.).
2.2.
Histological procedures
After 14 or 28 days of post-operative care, six animals from each group (C, GR and PNR) were sacrificed using excessive anaesthesia. After soft tissue dissection, the maxilla was separated from the skull via a crosscut surfacing the distal face of the last upper molar. The samples were fixed in 10% formalin for 48 h and decalcified in a solution of formic acid and sodium citrate.18 During decalcification, the samples were
Table 1 – Commercial chow composition. Moisture not more than Crude protein not less than Crude fat not less than Crude fibre not more than Minerals not more than Calcium not more than Phosphorus not more than
13% 23% 4% 5% 10% 1.3% 0.85%
archives of oral biology 59 (2014) 241–250
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Fig. 2 – A. Suture between the maxillary and incisive bone corresponding to the region of the first cut and the dotted line indicates the 2nd cut; 2B. Apical-, medial- and cervical-thirds.
Fig. 3 – Cross-sections of the apical- and medial-thirds with histological structures. Anatomical details used for characterization of the histological sections: (A) dental alveolus, (B) lower board of maxillary bone, (C) vomeronasal organ, (D) nasolacrimal duct, (E) opposite incisor, (F) lower board of incisive bone.
sectioned transversely into thirds: apical-third, medial-third and cervical-third. To achieve this, a cut was made at the suture between the incisive bone and maxillary bone and another cut was made approximately 2 mm prior to this suture (Fig. 2A). Each third was placed separately in paraffin (Fig. 2B). Semiseriate cross sections were obtained of the alveolar medial- and apical-thirds, with a thickness of 6 mm using a precision microtome (Leica, model RM 2265, United Kingdom). There was a range of 48 mm between the sections (Fig. 3). Of these sections, four sections were stained with Harris-Haematoxylin and Eosin (HE) and selected for histometric analysis to estimate the total bone area and newly formed bone area (Fig. 4).
2.3.
Histometric measures
Images of the selected sections were acquired using a light microscope with an attached camera (Olympus BX50) using the lens of 2 times. The images were projected onto a computer screen and measured using an image analysis programme (Quantimet 500IW-Leica Qwin V3, Cambridge, England). For quantification of the total bone area (TA), we used the manual design feature of the alveolar contour to calculate the defined area. For the newly formed bone area
(NB), we used the detection feature using similar colours after previous marking of such areas.
2.4.
Statistical analyses
All results were expressed as the mean standard error. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 15.0 for Windows, Chicago, IL, USA. For the analysis of the number of pups, the pups’ weight at birth and histometric parameters of the test of analysis of variance (ANOVA) were used. For the analysis of body weight and food intake on the pups, Analysis of variance with repeated measures was used. When necessary, multiple comparisons were analyzed using the Bonferroni test. Differences were considered significant when p < 0.05.
3.
Results
3.1.
Number of offspring and pup weight at birth
The food restriction imposed on the mothers during pregnancy did not affect the number of pups at birth (Table 2).
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archives of oral biology 59 (2014) 241–250
Table 2 – Mean and standard error of the number of pups per litter at birth. The number in parenthesis represents the number of litters. C
GR
PNR
11.42 0.36 (7)
10.71 0.74 (7)
11.00 0.93 (11)
* p < 0.05.
Fig. 4 – Image of the alveolus medial-third, highlighting the total area (TA), connective tissue (CT) and newly formed bone (NB).
situation remained. In addition, there was a significant weight gain in all groups; however, PNR animals exhibited lower values of weight gain compared to the other groups. When we evaluating the evolution of body weight of six males pups in groups C, GR and PNR from the time of tooth extraction until 14 days after euthanasia in Fig. 6B or 28 days in Fig. 6C, the animals from group C and GR did not present significant differences during this entire experiment compared to body weight. In addition, the animals in groups C and GR, in the three periods observed, showed higher values than the animals in group PNR, which had reduced food intake.
3.3.
Fig. 5 – Postnatal animal weight at birth. Results were expressed as the mean W SE. *p < 0.05 vs. (C), ^p < 0.05 vs. (PNR).
However, the effect of this diet during the intrauterine development was reflected in the lower pup weight in the GR group at birth (Fig. 5).
3.2.
Evolution of body weight
The offspring body weight evolution of the three study groups may be accompanied from the time of weaning until euthanasia, as shown in Fig. 6. To facilitate the assessment, the days were grouped into periods and weight comparisons between groups were made in the initial, intermediate and final period. In Fig. 6A, one can follow the weight gain of the offspring from weaning until the day of surgery for extraction of the upper right central incisor. The pups in the PNR had significantly lower weights compared to the other groups during the first period. Throughout this stage of postnatal development, the puppies in the C group demonstrated a greater body weight from the period 5 when compared with the animals from the GR and PNR groups and these animal weight differences were only accentuated in the PNR group during this period. In the final phase, that is, in period 10, this
Evolution of dietary intake
Because the male pups showed increased differences in body weight evolution after weaning, we assess their food intake pattern (g/100 g body weight). To facilitate the assessment of the relative dietary intake on offspring from the time of weaning until dental extraction surgery, the days were grouped in periods and the food intake comparisons between groups were made in the initial, intermediate and final periods. As shown in Fig. 7, with advancing age, independent of the group, the animals significantly decrease their intake of food. A comparison between groups revealed that the PNR group exhibited a lower intake compared to the C and GR groups, independent of time.
3.4.
Histometric measures
3.4.1.
Total bone area (TA)
The alveolar apical third total area decreased significantly over time due to bone resorption in the three groups of animals examined. The PNR animals remained with a smaller alveolar area compared to the other groups, in the two-thirds of the animals studied, and at any time during the post-operative assessment (Table 3).
3.4.2.
Newly formed bone (NB)
The dynamics of new bone formation as a function of time after surgery was shown in Table 4. The results obtained in the apical third of the alveolus showed that all animal groups subjected to euthanasia 28 days after teeth extraction presented a greater area of newly formed bone compared to animals evaluated at 14 days. In the medial-third, only animals of the C group presented the same dynamics of bone formation displayed in the apical-third over time. The other groups showed no significant increases in the amount of bone tissue. In addition, the PNR animals presented a smaller extent of newly formed bone area in the apical-third compared to the C and GR groups at any evaluation time. In the medial-third, this difference was significant only at 28 days.
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archives of oral biology 59 (2014) 241–250
Fig. 6 – Evolution of the body weight of male pups. Results were expressed as the mean W SE. (A) From weaning to extraction, *p < 0.01 vs. (C), ~p < 0.01 vs. (GR); (B) from extraction to euthanasia at 14 days, *p < 0.001 vs. (C), ~p < 0.001 vs. (GR) and (C) from extraction to euthanasia at 28 days, * p < 0.001 vs. (C), ~p < 0.001 vs. (GR). (A) Periods 1 (29th – first day after weaning), 5 (53th until 60th), 10 (92nd – day before extraction); (B) Periods 1 (extraction day), 3 (6th until 10th), 5 (14th – euthanasia day); (C) Periods 1 (extraction day), 4 (11th until 15th), 8 (28th – euthanasia day).
3.4.3.
Relationship NB/TA
Considering that the NB/TA relationship represents the percentage of the total area of the alveolus third covered by newly formed bone, we examined if the NB/TA relationship of the apical third of the alveolus was significantly
greater at 28 days compared to 14 days, independent of the group studied. In the alveolus medial-third, this evaluation revealed that the PNR animals did not show the same dynamic of bone repair compared to the other groups over time after surgery, (Table 5).
Table 3 – Total alveolus bone area (TA) in square micrometres (mm2) of the three groups studied: control (C), gestation restriction (GR) and postnatal restriction (PNR). Values represent mean W standard error. TA Apical Medial ^ *
14 28 14 28
d d d d
p < 0.01 (14 days) vs. (28 days). p < 0.001 (PNR) vs. (C and GR).
C
GR
PNR
5,619,404.9 327,235.4 5,038,747.2 244,282.9^ 5,011,560.3 150,004.5 4,889,556.4 176,664.8
5,247,593.2 129,832.6 4,979,919.6 212,502.6^ 5,519,200.7 152,539.4 5,223,592.3 189,424.0
4,400,310.3 305,355.5* 3,474,474.6 185 448.2^,* 4,118,819.3 097,164.4* 4,011,957.3 311,961.6*
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Table 4 – Newly formed bone area of the alveolus (NB) in square micrometres (mm2) of the three groups studied: control (C), gestation restriction (GR) and postnatal restriction (PNR). Values represent the mean W standard error. NB Apical Medial ^ *
14 28 14 28
d d d d
C
GR
PNR
1,965,259.3 216,144.8 2,169,483.9 195,891.4^ 2,564,101.8 106,782.9 3,008,592.5 138,066.7^
1,934,043.6 104,086.6 2,034,286.8 246,848.5^ 2,966,603.2 243,853.2 3,326,738.0 187,050.1
1,481,865.0 242,310.0* 1,342,738.2 151,041.3^,* 2,389,398.0 266,267.3 2,243,881.0 281,236.0*
p < 0.05 (14 days) vs. (28 days). p < 0.01 (PNR) vs. (C and GR).
Table 5 – Newly formed bone area and total area ratio (NB/TA) of the three groups studied: control (C), gestation restriction (GR) and postnatal restriction (PNR). Values represent the mean W standard error. NB/TA
C
GR
PNR
Apical
14 d 28 d
0.347 0.022 0.434 0.038^
0.369 0.018 0.403 0.031^
0.330 0.036 0.387 0.039^
Medial
14 d 28 d
0.512 0.019 0.616 0.017^
0.535 0.032 0.637 0.026^
0.583 0.065 0.558 0.051
^
4.
p < 0.001 (14 days) vs. (28 days).
Discussion
Bone growth may be programmed using genetic and/or environmental influences during intra-uterine life, which indicates that, in addition to the hereditary basis, environmental factors such as nutrition might interfere with bone development in rats10 as well as in humans.19 Several experimental studies9–14 have shown that malnutrition imposed in the early stages of development may have a negative effect on bone structure in adults. In this study, food restriction was imposed at different stages of animal development, enabling the comparison of these aggravations specifically on the alveolar bone repair process. Although some studies have previously demonstrated13,20 that the reduction in osteoblastic activity resulting from malnutrition determines the loss in bone growth and development, there is still a lack of studies evaluating the injury potential in the alveolar bone repair process. Most food restriction methods used in experimental studies have included limiting the amount of food ingested,11,12,21–23 reduction or withdrawal of dietary proteins9,10,13,14,20,24–29 and reducing uterine circulation via the ligature of one of uterine artery branches.30 All of these studies differ not only on the type of diet manipulated
but also the time and duration of food restriction, which makes it difficult to compare between the results obtained. Importantly, the gestational restriction imposed on female rats did not result in a decrease in the number of pups per litter, as previously observed by Woodall et al.31 who also subjected pregnant rats to global nutrient restriction. This result appears consistent even when mothers were subjected to protein restriction.10,20,27 However, using this last restriction mode, the litters consisted of fewer pups with greater weight, as an alternative to increasing the survival rate of newborn animals.13 The importance of an adequate intake of nutrients by the mother during pregnancy, to ensure normal foetal growth20,22,27,30 could be observed in our experimental model as we compared the weight of newborn puppies since the gestational malnutrition imposed resulted in a remarkable reduction in birth weight. In this study, we considered the lactation period to be 28 days after birth.20 The average body weight of the pups at weaning showed that the animals that had suffered from food restriction during gestation (GR) did not differ from those of the control group. This finding suggested that if there was a compromise in milk offering,32 or33 in the protein content and other components, it must not have been emphasised because it secured the proper growth of the pups. However, we cannot rule out the possibility that
Fig. 7 – Relative food intake of male pups from weaning to extraction. Results were expressed as the mean W SE. *p < 0.001 vs. (C) ~p < 0.01 vs. (GR). Periods 2 (30th until 36th), 6 (61st until 68th), 10 (92nd – day before extraction).
archives of oral biology 59 (2014) 241–250
these animals presented a better efficiency in the use of milk due to reduced metabolic activity. However, the food restriction imposed during intrauterine development was significantly reflected in the weight of these animals after weaning, once they presented smaller body weights until they reached 90 days of life, even with a relative dietary intake similar to the control group. However, it was only at more advanced ages, that these animals exhibited a recovery in body weight. Similar results were found in rats at 160 days of age, whose mothers were exposed to 50% of food restriction during pregnancy.34 Next to weaning, rats subjected to postnatal restriction (PNR) showed a body weight that was 50% lower than the animals from the other two groups, which indicated severe malnutrition.35 A sharp reduction also occurred in the weight of the pups from mothers exposed to 50% of food restriction during lactation.34 Remmers et al.23 found a preferences of approximately 60% in weight of the pups restricted during lactation and suggested that the enforced malnutrition during this initial period of development determined the low weight of these animals even in adult life. The low weight at weaning can be due to damage in the quantity and/or quality of milk offered when the animals were young. Casein serves as a source of nitrogen for amino acids and nucleic acids that ensure growth during the lactation period,36 thus, a lower amount of protein in milk due to the enforcement of protein malnutrition determined the lower weight of pups at weaning. Although pups of the PNR group showed weight gain until the surgery, this increase was always less than that obtained by the other two groups. Similar damage in the growth of offspring occurred when the animals were subjected to 65% food restriction since the last gestational week until they reached 70 days of age.22 Specifically, the smaller supply of nutrients was decisive to the growth pattern exhibited by these animals. We selected to perform the histometric evaluations on transverse sections of the alveolar socket.37 Consistent with previous studies, we also had great difficulty in obtaining longitudinal cuts parallel to the long axis of the tooth, which would be important for the simultaneous observation of all three thirds representing the entire alveolar socket. In addition, the sidewalls of the alveolar socket would be poorly represented in these cuts. We assessed the bone repair only in the apicaland medial-thirds of the alveolar socket because we judged it to be inappropriate to use the same approach for the evaluation of the cervical-third because it presents a greater potential of interference in the repair process, resulting from the extraction technique and their anatomical fragility, which may make it more difficult to compare the results between the groups. In addition, this region arguably presents greater physiological reabsorption following the dental extraction of the molars.38,39 The main proposal of histometric analysis was to assess the percentage of the total area of the alveolar socket covered by newly formed bone (NB/TA) to obtain a quantitative result to enable the comparison of bone repair between the groups analyzed. However, the assessment of the total area that represents the size of the alveolus in the histological section (TA) and the newly formed bone (NB) separately enabled a better understanding of the participation of each of these parameters in this process. These evaluations have become important for the food restriction model during critical phases of prenatal and
247
postnatal development because there was a possibility that injury may have definite repercussions on the size achieved by the face bones of the animal, similar to what has been observed in the jaws.9 Even when this dietary restriction was imposed just after weaning, this deleterious effect could be observed in adult rats subjected to protein restriction in this period.40 In experimental conditions, when protein–calorie malnutrition was imposed throughout the intrauterine and postnatal development, its deleterious effect on the splanchnocranium structures was evident with a reduction in the height and length of the incisive bone.41 This may be explain in part by the results of TA in animals belonging to the PNR group. In addition, the TA evaluation in two postoperative times facilitated the understanding of the effect of time on the alveolar bone loss process in these animals due to dental extraction. Interestingly, the phenomenon of bone reabsorption of alveolar socket in rat incisors has not been largely explored in the literature. However, some experimental studies performed on molars also have been performed with temporal assessments. Thus, the early onset of this process at 7 days post-operative,16 the compromise of the vestibular wall at 14 and 28 days after extraction42 and vestibular and palatal walls using computerised tomography after 14, 28 and 42 days postoperative, with an emphasis at 28 days,38 had been previously established. Interestingly, our results showed a greater tendency of alveolar reabsorption with a consequent decrease in TA in the apical-third at 28 days postoperative in all groups studied. Although this parameter had been significantly lower in animals from the PNR group because of the effect of food restriction in the dimension affected by the splanchnocranium of these animals. The medial-third of these sockets did not present the same temporal pattern of reabsorption; however, the nutritional aggravations imposed on to PNR animals could also be evidenced in this third. The NB of the apical third over time was higher at 28 days of sacrifice compared to 14 days in groups C and GR. In this third, in addition to the lower alveolar area, the PNR group showed lower amounts of newly formed bone. In the medial third, the difference in function in the repair speed between the thirds may have been influenced by food restriction in the process of newly bone formation, which can be observed even in animals of the GR group. Evaluation of the NB/TA relationship in each third was important because the alveolar reabsorption process occurs simultaneously with the newly bone formation, as previously described by several studies.43–45 However, the values obtained in the PNR group were assessed with more accuracy, avoiding potential misinterpretations of the results as a function of the decrease in craniofacial size. Thus, the alveolar reabsorption begins at 7 postoperative days and reaches its maximum value at 30 days.43 Throughout the trial period of our study, the animals of different groups were suffering from bone reabsorption, which was associated with new formation. However, the animals of the PNR group did not show an increase in relationship to the NB/TA in the medial-third over time, indicating the localised negative influence of the nutritional aggravation imposed on the bone repair process. Despite the difficulty of comparing the results as a function of the different techniques used, evaluation periods and age of
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the animal, some studies have highlight some important aspects of the alveolar repair process. Among these studies, the histometric study of alveolar repair of rat incisors in longitudinal cuts estimated that at 14 days, approximately 40% of the alveolus was filled by newly formed bone in the apicaland medial-thirds.44 However, as the days passed, the reparation of the medial-third supplanted the apical-third. Our results indicated a predominance of the percentage of alveolar repair in the medial-third at 14 and 28 days after surgery. Another result obtained from the longitudinal sections of the alveoli incisors of adult female rats indicated that the advantage of the medial-third in relationship to the apical-third was at 14 days after surgery.46 Moreover, the histological cutting position of the alveolus determined only a slight difference in the percentage of newly formed bone in the medial-third, when the process was rated at the same time postoperatively.47 When the repair was evaluated in longitudinal cutting and calculated as the total amount of this process, the results showed that only 32% of the alveoli of control rat incisors was repaired after two weeks of extraction.48 However, our results revealed important percentage differences between the two thirds studied, for this group of animals. Depending on the tooth studied, different evolutions of the alveolar repair process were established. For example, while molar alveoli showed a 30% increase in the percentage of newly formed bone between 14 and 21 days of postoperative39,49, in the incisors, the difference of this NB/TA relationship did not show such an extensive increase between the evaluation periods. The histometric results also showed that the alveolar bone repair continued until 28 days after surgery. This was consistent with the results of other studies,37,50 independent of the tooth extracted. While some studies have claimed that over time, the amount of connective tissue decreased was replaced by trabeculae bone that was newly formed and the total bone healing process goes beyond 3 weeks,51 other studies have stated that the process of alveolar bone repair in the apical-third continues until 45 days17 and others have indicated that it reaches 60 days postoperatively.43,45 Radiographical evaluations also indicate a longer period of bone repair mainly in the region of the bone crest.52 Using histometric analysis, we concluded that the total area percentage of the alveolus covered by newly formed bone (NB/TA) revealed a late preference in the process of alveolar repair to the medial-third in animals that were severely malnourished (PNR); however, these animals presented a greater impairment of the TA in the apicalthird of the alveolus at the time of evaluation. The animals in the GR group did not demonstrate the same pattern of late new bone formation (NB) compared to the controls in the medial-third.
Funding None.
Competing interest We declare that we have no conflict of interest.
Ethical approval We declare that all the handling and care of the animals was performed according to the ‘‘European Convention for Protection of Vertebrate Animals for Experimental and other Scientific Purposes.’’ This project was approved by the Research Ethics Committee of the Federal University of Sa˜o Paulo (UNIFESP) and approved under n8 0577/07.
Acknowledgment This study was supported by CAPES (Coordenaca˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.archoralbio.2013.11.014.
references
1. World Health Organization. Feto-maternal nutrition and low birth weight. 2013http://www.who.int/nutrition/topics/ feto_maternal/en/.html. 2. Sayer AA, Cooper C. Fetal programming of body composition and musculoskeletal development. Early Hum Dev 2005;81:735–44. 3. Cooper C, Javaid MK, Taylor P, Walker-Bone K, Dennison E, Arden N. The fetal origins of osteoporotic fracture. Calcif Tissue Int 2002;70:391–4. 4. Devlin MJ, Bouxsein ML. Influence of pre- and peri-natal nutrition on skeletal acquisition and maintenance. Bone 2012;50:444–51. 5. Schlu¨ssel MM, Castro JAS, Kac G, Silva AAM, Cardoso VC, Bettiol H, et al. Birth weight and bone mass in young adults from Brazil. Bone 2010;46:957–63. 6. Mølgaard C, Larnkjær A, Mark AB, Michaelsen K. Are early growth and nutrition related to bone health in adolescence? The Copenhagen cohort study of infant nutrition and growth. Am J Clin Nutr 2011;94(suppl):1865S–9S. 7. Javaid MK, Cooper C. Prenatal and childhood influences on osteoporosis. Best Pract Res Clin Endocrinol Metab 2002;16:349–67. 8. Dennison EM, Cooper C, Cole AZ. Early development and osteoporosis and bone health. J Dev Orig Health Dis 2010;1:142–9. 9. Alippi RM, Meta MD, Olivera MI, Bozzini C, Schneider P, Meta IF, et al. Effect of protein-energy malnutrition in early life on the dimensions and bone quality of the adult rat mandible. Arch Oral Biol 2002;47:47–53. 10. Mehta G, Roach HI, Langley-Evans S, Taylor P, Reading I, Oreffo ROC, et al. Intrauterine exposure to a maternal low protein diet reduces adult bone mass and alters growth plate morphology in rats. Calcif Tissue Int 2002;71:493–8. 11. Fetoui H, Mahjoubi-Samet A, Jammousi K, Ellouze F, Guermazic F, Zeghala N. Energy restriction in pregnant and lactating rats lowers bone mass of their progeny. Nutr Res 2006;26:421–6.
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12. Cesani MF, Orden AB, Oyhenart EE, Zucchi M, Mun˜e MC, Pucciarelli HM. Growth of functional cranial components in rats submitted to intergenerational undernutrition. J Anat 2006;209:137–47. 13. Lanham SA, Roberts C, Cooper C, Oreffo ROC. Intrauterine programming of bone. Part 1: alteration of the osteogenic environment. Osteoporos Int 2008;19:147–56. 14. Lanham SA, Roberts C, Perry MJ, Cooper C, Oreffo ROC. Intrauterine programming of bone. Part 2: alteration of skeletal structure. Osteoporos Int 2008;19:157–67. 15. Kanyama M, Kuboki T, Akiyama K, Nawachi K, Miyauchi FM, Yatani H, et al. Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction. Arch Oral Biol 2003;48:723–30. 16. Hikita H, Miyazawa K, Tabuchi M, Kimura M, Goto S. Bisphosphonate administration prior to tooth extraction delays initial healing of the extraction socket in rats. J Bone Miner Metab 2009;27:663–72. 17. Prado RF, Silveira VAS, Rocha RF, Vasconcellos LMR, Carvalho YR. Effects of experimental osteoporosis and low calcium intake on postextraction sockets of rats. Int J Exp Pathol 2012;93:139–47. 18. Morse A. Formic acid-sodium citrate decaldification an butyl alcohol dehydration of teeth and bones for sectioning in paraffin. J Dent Res 1945;24:143–53. 19. Gale CR, Martyn CN, Kellingray S, Eastell R, Cooper C. Intrauterine programming of adult body composition. J Clin Endocrinol Metab 2001;86:267–72. 20. Oreffo ROC, Lashbrooke B, Roach HI, Clarke NMP, Cooper C. Maternal protein deficiency affects mesenchymal stem cell activity in the developing offspring. Bone 2003;33:100–7. 21. Lucas SRR, Miraglia SM, Gil FZ, Coimbra TM. Intrauterine food restriction as a determinant of nephrosclerosis. Am J Kidney Dis 2001;37:467–76. 22. Martı´n MA, Ferna´ndez E, Pascual-Leone AM, Escriva´ F, Alvarez C. Protein calorie restriction has opposite effects on glucose metabolism and insulin gene expression in fetal and adult rat endocrine pancreas. Am J Physiol Endocrinol Metab 2004;286:E542–50. 23. Remmers F, Schreuder MF, Gemke RJBJ, Delemarre-van de Waal HA. Energy intake and resting energy expenditure in adult male rats after early postnatal food restriction. Br J Nutr 2008;99:1149–56. 24. Alippi RM, Meta MD, Boyer PM, Bozzini CE. Catch-up in mandibular growth after short-term dietary protein restriction in rat during the post-weaning period. Eur J Oral Sci 1999;107:260–4. 25. Reichling TD, German RZ. Bones, muscles and visceral organs of protein-malnourished rats (Rattus norvegicus) grow more slowly but for longer durations to reach normal final size. J Nutr 2000;130:2326–32. 26. Fagundes ATS, Moura EG, Passos MCF, Oliveira E, Toste FP, Bonomo IT, et al. Maternal low-protein diet during lactation programmes body composition and glucose homeostasis in the adult rat offspring. Br J Nutr 2007;98:922–8. 27. Gosby AK, Stanton LML, Maloney CA, Thompson M, Briody J, Baxter RC, et al. Postnatal nutrition alters body composition in adult offspring exposed to maternal protein restriction. Br J Nutr 2009;101:1878–84. 28. Jones DC, Bernstein M, German RZ. Catch-up and targeted growth following variable duration protein restriction: effects on bone and body mass. J Morphol 2011;272:485–96. 29. Shen Q, Xu H, Wei L, Chen J, Liu H. Intrauterine growth restriction and postnatal high-protein diet affect the kidneys in adult rats. Nutrition 2011;27:364–71. 30. Engelbregt MJT, van Weissenbruch MM, Lips P, van Lingen A, Roos JC, Delemarre-van de Waal HA. Body composition and bone measurements in intra-uterine growth retarded and early postnatally undernourished male and female rats
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
249
at the age of 6 months: comparison with puberty. Bone 2004;34:180–6. Woodall SM, Breier BH, Johnston BM, Gluckman PD. A model of intrauterine growth retardation caused by chronic maternal undernutrition in the rat: effects on the somatotrophic axis and postnatal growth. J Endocrinol 1996;150:231–42. O’Dowd R, Kent JC, Moseley JM, Wlodek ME. Effects of uteroplacental insufficiency and reducing litter size on maternal mammary function and postnatal offspring growth. Am J Physiol Regul Integr Comp Physiol 2008;294: R539–48. Derrickson ME, Lowas SR. The effects of dietary protein levels on milk protein levels and postnatal growth in laboratory mice (Mus musculus). J Mammal 2007;88:1475–81. Howie GJ, Sloboda DM, Vickers MH. Maternal undernutrition during critical windows of development results in differential and sex specific effects on postnatal adiposity and related metabolic profiles in adult rat offspring. Br J Nutr 2012;108:298–307. Corte´s E, Gonza´lez C, Betancourt M, Ortiz R. Assessment of DNA damage in spleen, bone marrow, and peripheral blood from malnourished rats by single cell gel electrophoresis assay. Teratog Carcinog Mutagen 2001;21:231–47. Blackburn DG, Hayssen V, Murphy CJ. The origins of lactation and the evolution of milk: a review with new hypotheses. Mamm Rev 1989;19:1–26. Okamoto T, Fialho AC. Estudo histolo´gico comparativo entre dois me´todos de obtenc¸a˜o de cortes de alve´olos de ratos. Rev Odontol da UNESP 1990;19:63–74. Jee J, Lee W, Lee BD. The influence of alendronate on the healing of extraction sockets of ovariectomized rats assessed by in vivo micro-computed tomography. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:e47–53. Aguirre JI, Altman MK, Vanegas SM, Franz SE, Bassit ACF, Wronski TJ. Effects of alendronate on bone healing after tooth extraction in rats. Oral Dis 2010;16:674–85. Miller JP, German RZ. Protein malnutrition affects the growth trajectories of the craniofacial skeleton in rats. J Nutr 1999;129:2061–9. Herrmann AK. Efeitos da desnutric¸a˜o prote´ico-calo´rica intrauterina e po´s-natal sobre as estruturas craniofaciais. Estudo experimental em ratos. Universidade Federal de Sa˜o Paulo; 2001. [Tese de mestrado]. ¨ .. The effect of alendronate on Altundal H, Gu¨vener O.O resorption of the alveolar bone following tooth extraction. Int J Oral Maxillofac Surg 2004;33:286–93. Shimizu M, Sasaki T, Ishihara A, Furuya R, Kawawa T. Bone wound healing after maxillary molar extraction in ovariectomized aged rats. J Electron Microsc 1998;47:517–26. Teo´filo JM, Brentegani LG, Lamano-Carvalho TL. A histometric study in rats of the effect of the calcium antagonist amlodipine on bone healing after tooth extraction. Arch Oral Biol 2001;46:375–9. Teo´filo JM, Brentegani LG, Lamano-Carvalho TL. Bone healing in osteoporotic female rats following intra-alveolar grafting of bioactive glass. Arch Oral Biol 2004;49:755–62. Machado GJR, Dias SMD, Bosco AF, Okamoto T, Castro JCB, Dornelles RCM. Action of nicotine and ovariectomy on bone regeneration after tooth extraction in rats. J Oral Maxillofac Surg 2010;68:2675–81. Luvizuto ER, Queiroz TP, Dias SMD, Okamoto T, Dornelles RCM, Garcia Jr IR et al. Histomorphometric analysis and immunolocalization of RANKL and OPG during the alveolar healing process in female ovariectomized rats treated with oestrogen or raloxifene. Arch Oral Biol 2010;55:52–9. Fracon RN, Teo´filo JM, Moris IC, Lamano-Carvalho TL. Treatment with paracetamol, ketorolac or etoricoxib did not
250
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hinder alveolar bone healing: a histometric study in rats. J Appl Oral Sci 2010;18:630–4. 49. Reis-Filho CR, Silva ER, Martins AB, Pessoa FF, Gomes PVN, Arau´jo MSC, et al. Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats. Arch Oral Biol 2012;57:469–76. 50. Pereira MC, Zecchin KG, Campagnoli EB, Jorge J. Ovariectomy delays alveolar wound healing after molar
extractions in rats. J Oral Maxillofac Surg 2007;65: 2248–53. 51. Lamano-Carvalho TL, Bombonato KF, Brentegani LG. Histometric analysis of rat alveolar wound healing. Braz Dent J 1997;8:9–12. 52. Bodner L, Kafe I, Littner MM, Cohen J. Extraction site healing in rats. A radiologic densitometric study. Oral Surg Oral Med Oral Pathol 1993;75:367–72.
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Histometric evaluation of dental alveolar repair in malnourished rats in the intrauterine or postnatal phase Fa´bio A.S. Sartorato *, Cla´udia A.M. Mura, Sandra R.R. Lucas Laboratory of Developmental Biology, Department of Morphology and Genetics, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, SP, Brazil
article info
abstract
Article history:
Objective: Nutritional aggravations during pregnancy or during the early stages of postnatal
Accepted 25 November 2013
development can impair bone development; thus, we aimed to assess the effects of food
Keywords:
Design: Thirty-six Wistar rats were divided into three groups: (C) 12 pups were obtained
restriction on the dental alveolar bone repair process using histometric analysis. Food restriction
from control mothers with food intake at ease; (GR) 12 pups from mothers subjected to 70%
Gestational malnutrition
food restriction during pregnancy; (PNR) 50% of maternal food restriction during lactation
Postnatal development
and 50% of restriction for the 12 pups after weaning. At three months of age, the upper right
Dental alveolar repair
incisor was extracted from the pups. After 14 or 28 days, the pups were sacrificed for evaluation of newly formed bone area (NB) and total bone area (TA) in the medial and apical thirds of the alveolus. Results: In the apical third of the alveolus, the ratio of NB/TA was greater at 28 days for all groups and there was no damage to any of the groups. In the medial third, the ratio was higher at 28 days for the C and GR groups. The PNR group did not show an evolution of alveolar dental repair. Compared between the thirds, all groups exhibited a higher percentage of newly formed bone in the medial third area, at any time point after surgery. Conclusions: The percentage of the total alveolar area covered by newly formed bone (NB/TA) revealed a late preference in the process of alveolar repair in the medial third, although only in the PNR group. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Annually, approximately 30 million children are born with low weight, representing approximately 24% of all births during this period. These children often face serious shortand long-term health problems because low birth weight is the greatest determinant of mortality or morbidity and disability in childhood with repercussions extending into adult life.1
Foetal programming is the phenomenon by which changes in foetal growth and development in response to the intrauterine environment generate permanent effects.2 Thus, several clinical studies have shown that certain parameters, such as bone structure, physiology and metabolism,3,4 bone mass,5,6 its growth7 and its mineral content,8 can be programmed by environmental influences during intrauterine life. Experimental models used for the study of foetal programming theory have demonstrated that nutrition is an important modifiable factor because it determines significant
* Corresponding author at: Departamento de Biologia do Desenvolvimento, Universidade Federal de Sa˜o Paulo, Rua Botucatu, 740, Edifı´cio Leita˜o da Cunha, 28 andar, Sa˜o Paulo, SP, CEP: 04023-900, Brazil. Tel.: +55 11 55764262; fax: +55 11 55764848. E-mail address: [email protected] (Fa´bio A.S. Sartorato). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.11.014
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Gestation
Lactation
Extraction
21-23 days
28 days
3 months age
Mothers
Euthanasia 14 d
28 d
Pups
C-M
ad libitum
ad libitum
C
ad libitum
GR-M
70% restriction
ad libitum
GR
ad libitum
PNR-M
ad libitum
50% restricition
PNR
50% restriction
Fig. 1 – Nutritional scheme imposed to the pregnant rats and offspring.
changes in bone parameters. Nutritional aggravations arising from protein, energetic or protein-caloric restrictions imposed on animals during pregnancy or during the early stages of their postnatal development can impair bone development.9–14 In addition, specific phenomena of the alveolar bone repair process after tooth extraction have been elucidated using experimental models.15–17 Thus, by imposing a food restriction at different time periods of offspring development, we aimed at evaluate distinctly the potential repercussions of this nutritional aggravation on bone formation and development, which can compromise the dental alveolar repair process of the adult rat using histometric analyses.
2.
Materials and methods
All procedures were approved by the institutional animal research committee. Twelve-week-old Wistar male and female rats were used for breeding. Pregnant animals were transferred into individual plastic cages, enabling the manipulation on their diets during the different developmental phases of their offspring. First, the control group consisting of mothers (C-M, control mother) was established. Control mothers received commercial feed ad libitum throughout gestation and lactation. Once the average daily dietary intake of these animals was determined, some of the animals were subjected to a dietary restriction according to their experimental group. In the group of rats subjected to constraint only during pregnancy (GR-M, gestational restriction mother), the diet was reduced by 70% compared to the normal average daily ingestion of the C-M group throughout the time points. On the day of birth, these mothers received feed ad libitum during lactation. In addition to this restricted group, a group of mothers with postnatal restriction (PNR-M, postnatal restriction mother) was established, in which the rats were deprived of 50% of the average normal diet ingested by C-M animals, but only for 28 days of lactation. At birth, the groups of pups were divided according to the group of pregnant mother rats: Group C, puppies of control mothers, which continued to receive feed ad libitum after weaning; GR group, pups from mothers who have a 70% food restriction only during gestation and after weaning received feed ad libitum; Group PNR; pups of mothers that were subjected to 50% food restriction during the entire lactation period, and continued to be subjected to this restriction until the time of euthanasia.
To avoid differences in the nutritional status of the offspring resulting from differences in the number of animals per litter, only six young males pups were left with each mother. If the litter generated was less than 6 males, then the females of this litter were used to obtain the minimum number of animals until the end of lactation, thus ensuring the same amount of breast milk for all animals. After weaning, only males were used in this experiment. This nutritional scheme was maintained until the animals were sacrificed at either 14 or 28 days after tooth extraction, which was performed when the animals reached adulthood (Fig. 1). All animals used during the experiment were maintained under controlled conditions of temperature (23 1 8C) and relative humidity (55 5%), with artificial lighting via a fluorescent lamp with a photo-period of 12 h of light and 12 h of dark. The animals were fed commercial Labina–Purina chow, which consisted of ingredients listed in Table 1.
2.1.
Surgical procedure
After general anaesthesia with ketamine (10% Dopalen, Vetbrands, Brazil) and xylazine (Anasedan 2%, Vetbrands, Brazil), the male rats had their upper right incisor extracted, with respective to the dental germ. The tooth avulsion was achieved with the aid of a chisel and forceps adapted for such purpose. At the end of the procedure, after applying simple gingival using a mononylon suture wire 5–0, all of the animals received a single dose of antibiotic (Veterinarian Pentabiotic Small Animals, Fort Dodge Animal Health Ltd.).
2.2.
Histological procedures
After 14 or 28 days of post-operative care, six animals from each group (C, GR and PNR) were sacrificed using excessive anaesthesia. After soft tissue dissection, the maxilla was separated from the skull via a crosscut surfacing the distal face of the last upper molar. The samples were fixed in 10% formalin for 48 h and decalcified in a solution of formic acid and sodium citrate.18 During decalcification, the samples were
Table 1 – Commercial chow composition. Moisture not more than Crude protein not less than Crude fat not less than Crude fibre not more than Minerals not more than Calcium not more than Phosphorus not more than
13% 23% 4% 5% 10% 1.3% 0.85%
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243
Fig. 2 – A. Suture between the maxillary and incisive bone corresponding to the region of the first cut and the dotted line indicates the 2nd cut; 2B. Apical-, medial- and cervical-thirds.
Fig. 3 – Cross-sections of the apical- and medial-thirds with histological structures. Anatomical details used for characterization of the histological sections: (A) dental alveolus, (B) lower board of maxillary bone, (C) vomeronasal organ, (D) nasolacrimal duct, (E) opposite incisor, (F) lower board of incisive bone.
sectioned transversely into thirds: apical-third, medial-third and cervical-third. To achieve this, a cut was made at the suture between the incisive bone and maxillary bone and another cut was made approximately 2 mm prior to this suture (Fig. 2A). Each third was placed separately in paraffin (Fig. 2B). Semiseriate cross sections were obtained of the alveolar medial- and apical-thirds, with a thickness of 6 mm using a precision microtome (Leica, model RM 2265, United Kingdom). There was a range of 48 mm between the sections (Fig. 3). Of these sections, four sections were stained with Harris-Haematoxylin and Eosin (HE) and selected for histometric analysis to estimate the total bone area and newly formed bone area (Fig. 4).
2.3.
Histometric measures
Images of the selected sections were acquired using a light microscope with an attached camera (Olympus BX50) using the lens of 2 times. The images were projected onto a computer screen and measured using an image analysis programme (Quantimet 500IW-Leica Qwin V3, Cambridge, England). For quantification of the total bone area (TA), we used the manual design feature of the alveolar contour to calculate the defined area. For the newly formed bone area
(NB), we used the detection feature using similar colours after previous marking of such areas.
2.4.
Statistical analyses
All results were expressed as the mean standard error. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 15.0 for Windows, Chicago, IL, USA. For the analysis of the number of pups, the pups’ weight at birth and histometric parameters of the test of analysis of variance (ANOVA) were used. For the analysis of body weight and food intake on the pups, Analysis of variance with repeated measures was used. When necessary, multiple comparisons were analyzed using the Bonferroni test. Differences were considered significant when p < 0.05.
3.
Results
3.1.
Number of offspring and pup weight at birth
The food restriction imposed on the mothers during pregnancy did not affect the number of pups at birth (Table 2).
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Table 2 – Mean and standard error of the number of pups per litter at birth. The number in parenthesis represents the number of litters. C
GR
PNR
11.42 0.36 (7)
10.71 0.74 (7)
11.00 0.93 (11)
* p < 0.05.
Fig. 4 – Image of the alveolus medial-third, highlighting the total area (TA), connective tissue (CT) and newly formed bone (NB).
situation remained. In addition, there was a significant weight gain in all groups; however, PNR animals exhibited lower values of weight gain compared to the other groups. When we evaluating the evolution of body weight of six males pups in groups C, GR and PNR from the time of tooth extraction until 14 days after euthanasia in Fig. 6B or 28 days in Fig. 6C, the animals from group C and GR did not present significant differences during this entire experiment compared to body weight. In addition, the animals in groups C and GR, in the three periods observed, showed higher values than the animals in group PNR, which had reduced food intake.
3.3.
Fig. 5 – Postnatal animal weight at birth. Results were expressed as the mean W SE. *p < 0.05 vs. (C), ^p < 0.05 vs. (PNR).
However, the effect of this diet during the intrauterine development was reflected in the lower pup weight in the GR group at birth (Fig. 5).
3.2.
Evolution of body weight
The offspring body weight evolution of the three study groups may be accompanied from the time of weaning until euthanasia, as shown in Fig. 6. To facilitate the assessment, the days were grouped into periods and weight comparisons between groups were made in the initial, intermediate and final period. In Fig. 6A, one can follow the weight gain of the offspring from weaning until the day of surgery for extraction of the upper right central incisor. The pups in the PNR had significantly lower weights compared to the other groups during the first period. Throughout this stage of postnatal development, the puppies in the C group demonstrated a greater body weight from the period 5 when compared with the animals from the GR and PNR groups and these animal weight differences were only accentuated in the PNR group during this period. In the final phase, that is, in period 10, this
Evolution of dietary intake
Because the male pups showed increased differences in body weight evolution after weaning, we assess their food intake pattern (g/100 g body weight). To facilitate the assessment of the relative dietary intake on offspring from the time of weaning until dental extraction surgery, the days were grouped in periods and the food intake comparisons between groups were made in the initial, intermediate and final periods. As shown in Fig. 7, with advancing age, independent of the group, the animals significantly decrease their intake of food. A comparison between groups revealed that the PNR group exhibited a lower intake compared to the C and GR groups, independent of time.
3.4.
Histometric measures
3.4.1.
Total bone area (TA)
The alveolar apical third total area decreased significantly over time due to bone resorption in the three groups of animals examined. The PNR animals remained with a smaller alveolar area compared to the other groups, in the two-thirds of the animals studied, and at any time during the post-operative assessment (Table 3).
3.4.2.
Newly formed bone (NB)
The dynamics of new bone formation as a function of time after surgery was shown in Table 4. The results obtained in the apical third of the alveolus showed that all animal groups subjected to euthanasia 28 days after teeth extraction presented a greater area of newly formed bone compared to animals evaluated at 14 days. In the medial-third, only animals of the C group presented the same dynamics of bone formation displayed in the apical-third over time. The other groups showed no significant increases in the amount of bone tissue. In addition, the PNR animals presented a smaller extent of newly formed bone area in the apical-third compared to the C and GR groups at any evaluation time. In the medial-third, this difference was significant only at 28 days.
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Fig. 6 – Evolution of the body weight of male pups. Results were expressed as the mean W SE. (A) From weaning to extraction, *p < 0.01 vs. (C), ~p < 0.01 vs. (GR); (B) from extraction to euthanasia at 14 days, *p < 0.001 vs. (C), ~p < 0.001 vs. (GR) and (C) from extraction to euthanasia at 28 days, * p < 0.001 vs. (C), ~p < 0.001 vs. (GR). (A) Periods 1 (29th – first day after weaning), 5 (53th until 60th), 10 (92nd – day before extraction); (B) Periods 1 (extraction day), 3 (6th until 10th), 5 (14th – euthanasia day); (C) Periods 1 (extraction day), 4 (11th until 15th), 8 (28th – euthanasia day).
3.4.3.
Relationship NB/TA
Considering that the NB/TA relationship represents the percentage of the total area of the alveolus third covered by newly formed bone, we examined if the NB/TA relationship of the apical third of the alveolus was significantly
greater at 28 days compared to 14 days, independent of the group studied. In the alveolus medial-third, this evaluation revealed that the PNR animals did not show the same dynamic of bone repair compared to the other groups over time after surgery, (Table 5).
Table 3 – Total alveolus bone area (TA) in square micrometres (mm2) of the three groups studied: control (C), gestation restriction (GR) and postnatal restriction (PNR). Values represent mean W standard error. TA Apical Medial ^ *
14 28 14 28
d d d d
p < 0.01 (14 days) vs. (28 days). p < 0.001 (PNR) vs. (C and GR).
C
GR
PNR
5,619,404.9 327,235.4 5,038,747.2 244,282.9^ 5,011,560.3 150,004.5 4,889,556.4 176,664.8
5,247,593.2 129,832.6 4,979,919.6 212,502.6^ 5,519,200.7 152,539.4 5,223,592.3 189,424.0
4,400,310.3 305,355.5* 3,474,474.6 185 448.2^,* 4,118,819.3 097,164.4* 4,011,957.3 311,961.6*
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Table 4 – Newly formed bone area of the alveolus (NB) in square micrometres (mm2) of the three groups studied: control (C), gestation restriction (GR) and postnatal restriction (PNR). Values represent the mean W standard error. NB Apical Medial ^ *
14 28 14 28
d d d d
C
GR
PNR
1,965,259.3 216,144.8 2,169,483.9 195,891.4^ 2,564,101.8 106,782.9 3,008,592.5 138,066.7^
1,934,043.6 104,086.6 2,034,286.8 246,848.5^ 2,966,603.2 243,853.2 3,326,738.0 187,050.1
1,481,865.0 242,310.0* 1,342,738.2 151,041.3^,* 2,389,398.0 266,267.3 2,243,881.0 281,236.0*
p < 0.05 (14 days) vs. (28 days). p < 0.01 (PNR) vs. (C and GR).
Table 5 – Newly formed bone area and total area ratio (NB/TA) of the three groups studied: control (C), gestation restriction (GR) and postnatal restriction (PNR). Values represent the mean W standard error. NB/TA
C
GR
PNR
Apical
14 d 28 d
0.347 0.022 0.434 0.038^
0.369 0.018 0.403 0.031^
0.330 0.036 0.387 0.039^
Medial
14 d 28 d
0.512 0.019 0.616 0.017^
0.535 0.032 0.637 0.026^
0.583 0.065 0.558 0.051
^
4.
p < 0.001 (14 days) vs. (28 days).
Discussion
Bone growth may be programmed using genetic and/or environmental influences during intra-uterine life, which indicates that, in addition to the hereditary basis, environmental factors such as nutrition might interfere with bone development in rats10 as well as in humans.19 Several experimental studies9–14 have shown that malnutrition imposed in the early stages of development may have a negative effect on bone structure in adults. In this study, food restriction was imposed at different stages of animal development, enabling the comparison of these aggravations specifically on the alveolar bone repair process. Although some studies have previously demonstrated13,20 that the reduction in osteoblastic activity resulting from malnutrition determines the loss in bone growth and development, there is still a lack of studies evaluating the injury potential in the alveolar bone repair process. Most food restriction methods used in experimental studies have included limiting the amount of food ingested,11,12,21–23 reduction or withdrawal of dietary proteins9,10,13,14,20,24–29 and reducing uterine circulation via the ligature of one of uterine artery branches.30 All of these studies differ not only on the type of diet manipulated
but also the time and duration of food restriction, which makes it difficult to compare between the results obtained. Importantly, the gestational restriction imposed on female rats did not result in a decrease in the number of pups per litter, as previously observed by Woodall et al.31 who also subjected pregnant rats to global nutrient restriction. This result appears consistent even when mothers were subjected to protein restriction.10,20,27 However, using this last restriction mode, the litters consisted of fewer pups with greater weight, as an alternative to increasing the survival rate of newborn animals.13 The importance of an adequate intake of nutrients by the mother during pregnancy, to ensure normal foetal growth20,22,27,30 could be observed in our experimental model as we compared the weight of newborn puppies since the gestational malnutrition imposed resulted in a remarkable reduction in birth weight. In this study, we considered the lactation period to be 28 days after birth.20 The average body weight of the pups at weaning showed that the animals that had suffered from food restriction during gestation (GR) did not differ from those of the control group. This finding suggested that if there was a compromise in milk offering,32 or33 in the protein content and other components, it must not have been emphasised because it secured the proper growth of the pups. However, we cannot rule out the possibility that
Fig. 7 – Relative food intake of male pups from weaning to extraction. Results were expressed as the mean W SE. *p < 0.001 vs. (C) ~p < 0.01 vs. (GR). Periods 2 (30th until 36th), 6 (61st until 68th), 10 (92nd – day before extraction).
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these animals presented a better efficiency in the use of milk due to reduced metabolic activity. However, the food restriction imposed during intrauterine development was significantly reflected in the weight of these animals after weaning, once they presented smaller body weights until they reached 90 days of life, even with a relative dietary intake similar to the control group. However, it was only at more advanced ages, that these animals exhibited a recovery in body weight. Similar results were found in rats at 160 days of age, whose mothers were exposed to 50% of food restriction during pregnancy.34 Next to weaning, rats subjected to postnatal restriction (PNR) showed a body weight that was 50% lower than the animals from the other two groups, which indicated severe malnutrition.35 A sharp reduction also occurred in the weight of the pups from mothers exposed to 50% of food restriction during lactation.34 Remmers et al.23 found a preferences of approximately 60% in weight of the pups restricted during lactation and suggested that the enforced malnutrition during this initial period of development determined the low weight of these animals even in adult life. The low weight at weaning can be due to damage in the quantity and/or quality of milk offered when the animals were young. Casein serves as a source of nitrogen for amino acids and nucleic acids that ensure growth during the lactation period,36 thus, a lower amount of protein in milk due to the enforcement of protein malnutrition determined the lower weight of pups at weaning. Although pups of the PNR group showed weight gain until the surgery, this increase was always less than that obtained by the other two groups. Similar damage in the growth of offspring occurred when the animals were subjected to 65% food restriction since the last gestational week until they reached 70 days of age.22 Specifically, the smaller supply of nutrients was decisive to the growth pattern exhibited by these animals. We selected to perform the histometric evaluations on transverse sections of the alveolar socket.37 Consistent with previous studies, we also had great difficulty in obtaining longitudinal cuts parallel to the long axis of the tooth, which would be important for the simultaneous observation of all three thirds representing the entire alveolar socket. In addition, the sidewalls of the alveolar socket would be poorly represented in these cuts. We assessed the bone repair only in the apicaland medial-thirds of the alveolar socket because we judged it to be inappropriate to use the same approach for the evaluation of the cervical-third because it presents a greater potential of interference in the repair process, resulting from the extraction technique and their anatomical fragility, which may make it more difficult to compare the results between the groups. In addition, this region arguably presents greater physiological reabsorption following the dental extraction of the molars.38,39 The main proposal of histometric analysis was to assess the percentage of the total area of the alveolar socket covered by newly formed bone (NB/TA) to obtain a quantitative result to enable the comparison of bone repair between the groups analyzed. However, the assessment of the total area that represents the size of the alveolus in the histological section (TA) and the newly formed bone (NB) separately enabled a better understanding of the participation of each of these parameters in this process. These evaluations have become important for the food restriction model during critical phases of prenatal and
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postnatal development because there was a possibility that injury may have definite repercussions on the size achieved by the face bones of the animal, similar to what has been observed in the jaws.9 Even when this dietary restriction was imposed just after weaning, this deleterious effect could be observed in adult rats subjected to protein restriction in this period.40 In experimental conditions, when protein–calorie malnutrition was imposed throughout the intrauterine and postnatal development, its deleterious effect on the splanchnocranium structures was evident with a reduction in the height and length of the incisive bone.41 This may be explain in part by the results of TA in animals belonging to the PNR group. In addition, the TA evaluation in two postoperative times facilitated the understanding of the effect of time on the alveolar bone loss process in these animals due to dental extraction. Interestingly, the phenomenon of bone reabsorption of alveolar socket in rat incisors has not been largely explored in the literature. However, some experimental studies performed on molars also have been performed with temporal assessments. Thus, the early onset of this process at 7 days post-operative,16 the compromise of the vestibular wall at 14 and 28 days after extraction42 and vestibular and palatal walls using computerised tomography after 14, 28 and 42 days postoperative, with an emphasis at 28 days,38 had been previously established. Interestingly, our results showed a greater tendency of alveolar reabsorption with a consequent decrease in TA in the apical-third at 28 days postoperative in all groups studied. Although this parameter had been significantly lower in animals from the PNR group because of the effect of food restriction in the dimension affected by the splanchnocranium of these animals. The medial-third of these sockets did not present the same temporal pattern of reabsorption; however, the nutritional aggravations imposed on to PNR animals could also be evidenced in this third. The NB of the apical third over time was higher at 28 days of sacrifice compared to 14 days in groups C and GR. In this third, in addition to the lower alveolar area, the PNR group showed lower amounts of newly formed bone. In the medial third, the difference in function in the repair speed between the thirds may have been influenced by food restriction in the process of newly bone formation, which can be observed even in animals of the GR group. Evaluation of the NB/TA relationship in each third was important because the alveolar reabsorption process occurs simultaneously with the newly bone formation, as previously described by several studies.43–45 However, the values obtained in the PNR group were assessed with more accuracy, avoiding potential misinterpretations of the results as a function of the decrease in craniofacial size. Thus, the alveolar reabsorption begins at 7 postoperative days and reaches its maximum value at 30 days.43 Throughout the trial period of our study, the animals of different groups were suffering from bone reabsorption, which was associated with new formation. However, the animals of the PNR group did not show an increase in relationship to the NB/TA in the medial-third over time, indicating the localised negative influence of the nutritional aggravation imposed on the bone repair process. Despite the difficulty of comparing the results as a function of the different techniques used, evaluation periods and age of
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the animal, some studies have highlight some important aspects of the alveolar repair process. Among these studies, the histometric study of alveolar repair of rat incisors in longitudinal cuts estimated that at 14 days, approximately 40% of the alveolus was filled by newly formed bone in the apicaland medial-thirds.44 However, as the days passed, the reparation of the medial-third supplanted the apical-third. Our results indicated a predominance of the percentage of alveolar repair in the medial-third at 14 and 28 days after surgery. Another result obtained from the longitudinal sections of the alveoli incisors of adult female rats indicated that the advantage of the medial-third in relationship to the apical-third was at 14 days after surgery.46 Moreover, the histological cutting position of the alveolus determined only a slight difference in the percentage of newly formed bone in the medial-third, when the process was rated at the same time postoperatively.47 When the repair was evaluated in longitudinal cutting and calculated as the total amount of this process, the results showed that only 32% of the alveoli of control rat incisors was repaired after two weeks of extraction.48 However, our results revealed important percentage differences between the two thirds studied, for this group of animals. Depending on the tooth studied, different evolutions of the alveolar repair process were established. For example, while molar alveoli showed a 30% increase in the percentage of newly formed bone between 14 and 21 days of postoperative39,49, in the incisors, the difference of this NB/TA relationship did not show such an extensive increase between the evaluation periods. The histometric results also showed that the alveolar bone repair continued until 28 days after surgery. This was consistent with the results of other studies,37,50 independent of the tooth extracted. While some studies have claimed that over time, the amount of connective tissue decreased was replaced by trabeculae bone that was newly formed and the total bone healing process goes beyond 3 weeks,51 other studies have stated that the process of alveolar bone repair in the apical-third continues until 45 days17 and others have indicated that it reaches 60 days postoperatively.43,45 Radiographical evaluations also indicate a longer period of bone repair mainly in the region of the bone crest.52 Using histometric analysis, we concluded that the total area percentage of the alveolus covered by newly formed bone (NB/TA) revealed a late preference in the process of alveolar repair to the medial-third in animals that were severely malnourished (PNR); however, these animals presented a greater impairment of the TA in the apicalthird of the alveolus at the time of evaluation. The animals in the GR group did not demonstrate the same pattern of late new bone formation (NB) compared to the controls in the medial-third.
Funding None.
Competing interest We declare that we have no conflict of interest.
Ethical approval We declare that all the handling and care of the animals was performed according to the ‘‘European Convention for Protection of Vertebrate Animals for Experimental and other Scientific Purposes.’’ This project was approved by the Research Ethics Committee of the Federal University of Sa˜o Paulo (UNIFESP) and approved under n8 0577/07.
Acknowledgment This study was supported by CAPES (Coordenaca˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.archoralbio.2013.11.014.
references
1. World Health Organization. Feto-maternal nutrition and low birth weight. 2013http://www.who.int/nutrition/topics/ feto_maternal/en/.html. 2. Sayer AA, Cooper C. Fetal programming of body composition and musculoskeletal development. Early Hum Dev 2005;81:735–44. 3. Cooper C, Javaid MK, Taylor P, Walker-Bone K, Dennison E, Arden N. The fetal origins of osteoporotic fracture. Calcif Tissue Int 2002;70:391–4. 4. Devlin MJ, Bouxsein ML. Influence of pre- and peri-natal nutrition on skeletal acquisition and maintenance. Bone 2012;50:444–51. 5. Schlu¨ssel MM, Castro JAS, Kac G, Silva AAM, Cardoso VC, Bettiol H, et al. Birth weight and bone mass in young adults from Brazil. Bone 2010;46:957–63. 6. Mølgaard C, Larnkjær A, Mark AB, Michaelsen K. Are early growth and nutrition related to bone health in adolescence? The Copenhagen cohort study of infant nutrition and growth. Am J Clin Nutr 2011;94(suppl):1865S–9S. 7. Javaid MK, Cooper C. Prenatal and childhood influences on osteoporosis. Best Pract Res Clin Endocrinol Metab 2002;16:349–67. 8. Dennison EM, Cooper C, Cole AZ. Early development and osteoporosis and bone health. J Dev Orig Health Dis 2010;1:142–9. 9. Alippi RM, Meta MD, Olivera MI, Bozzini C, Schneider P, Meta IF, et al. Effect of protein-energy malnutrition in early life on the dimensions and bone quality of the adult rat mandible. Arch Oral Biol 2002;47:47–53. 10. Mehta G, Roach HI, Langley-Evans S, Taylor P, Reading I, Oreffo ROC, et al. Intrauterine exposure to a maternal low protein diet reduces adult bone mass and alters growth plate morphology in rats. Calcif Tissue Int 2002;71:493–8. 11. Fetoui H, Mahjoubi-Samet A, Jammousi K, Ellouze F, Guermazic F, Zeghala N. Energy restriction in pregnant and lactating rats lowers bone mass of their progeny. Nutr Res 2006;26:421–6.
archives of oral biology 59 (2014) 241–250
12. Cesani MF, Orden AB, Oyhenart EE, Zucchi M, Mun˜e MC, Pucciarelli HM. Growth of functional cranial components in rats submitted to intergenerational undernutrition. J Anat 2006;209:137–47. 13. Lanham SA, Roberts C, Cooper C, Oreffo ROC. Intrauterine programming of bone. Part 1: alteration of the osteogenic environment. Osteoporos Int 2008;19:147–56. 14. Lanham SA, Roberts C, Perry MJ, Cooper C, Oreffo ROC. Intrauterine programming of bone. Part 2: alteration of skeletal structure. Osteoporos Int 2008;19:157–67. 15. Kanyama M, Kuboki T, Akiyama K, Nawachi K, Miyauchi FM, Yatani H, et al. Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction. Arch Oral Biol 2003;48:723–30. 16. Hikita H, Miyazawa K, Tabuchi M, Kimura M, Goto S. Bisphosphonate administration prior to tooth extraction delays initial healing of the extraction socket in rats. J Bone Miner Metab 2009;27:663–72. 17. Prado RF, Silveira VAS, Rocha RF, Vasconcellos LMR, Carvalho YR. Effects of experimental osteoporosis and low calcium intake on postextraction sockets of rats. Int J Exp Pathol 2012;93:139–47. 18. Morse A. Formic acid-sodium citrate decaldification an butyl alcohol dehydration of teeth and bones for sectioning in paraffin. J Dent Res 1945;24:143–53. 19. Gale CR, Martyn CN, Kellingray S, Eastell R, Cooper C. Intrauterine programming of adult body composition. J Clin Endocrinol Metab 2001;86:267–72. 20. Oreffo ROC, Lashbrooke B, Roach HI, Clarke NMP, Cooper C. Maternal protein deficiency affects mesenchymal stem cell activity in the developing offspring. Bone 2003;33:100–7. 21. Lucas SRR, Miraglia SM, Gil FZ, Coimbra TM. Intrauterine food restriction as a determinant of nephrosclerosis. Am J Kidney Dis 2001;37:467–76. 22. Martı´n MA, Ferna´ndez E, Pascual-Leone AM, Escriva´ F, Alvarez C. Protein calorie restriction has opposite effects on glucose metabolism and insulin gene expression in fetal and adult rat endocrine pancreas. Am J Physiol Endocrinol Metab 2004;286:E542–50. 23. Remmers F, Schreuder MF, Gemke RJBJ, Delemarre-van de Waal HA. Energy intake and resting energy expenditure in adult male rats after early postnatal food restriction. Br J Nutr 2008;99:1149–56. 24. Alippi RM, Meta MD, Boyer PM, Bozzini CE. Catch-up in mandibular growth after short-term dietary protein restriction in rat during the post-weaning period. Eur J Oral Sci 1999;107:260–4. 25. Reichling TD, German RZ. Bones, muscles and visceral organs of protein-malnourished rats (Rattus norvegicus) grow more slowly but for longer durations to reach normal final size. J Nutr 2000;130:2326–32. 26. Fagundes ATS, Moura EG, Passos MCF, Oliveira E, Toste FP, Bonomo IT, et al. Maternal low-protein diet during lactation programmes body composition and glucose homeostasis in the adult rat offspring. Br J Nutr 2007;98:922–8. 27. Gosby AK, Stanton LML, Maloney CA, Thompson M, Briody J, Baxter RC, et al. Postnatal nutrition alters body composition in adult offspring exposed to maternal protein restriction. Br J Nutr 2009;101:1878–84. 28. Jones DC, Bernstein M, German RZ. Catch-up and targeted growth following variable duration protein restriction: effects on bone and body mass. J Morphol 2011;272:485–96. 29. Shen Q, Xu H, Wei L, Chen J, Liu H. Intrauterine growth restriction and postnatal high-protein diet affect the kidneys in adult rats. Nutrition 2011;27:364–71. 30. Engelbregt MJT, van Weissenbruch MM, Lips P, van Lingen A, Roos JC, Delemarre-van de Waal HA. Body composition and bone measurements in intra-uterine growth retarded and early postnatally undernourished male and female rats
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
249
at the age of 6 months: comparison with puberty. Bone 2004;34:180–6. Woodall SM, Breier BH, Johnston BM, Gluckman PD. A model of intrauterine growth retardation caused by chronic maternal undernutrition in the rat: effects on the somatotrophic axis and postnatal growth. J Endocrinol 1996;150:231–42. O’Dowd R, Kent JC, Moseley JM, Wlodek ME. Effects of uteroplacental insufficiency and reducing litter size on maternal mammary function and postnatal offspring growth. Am J Physiol Regul Integr Comp Physiol 2008;294: R539–48. Derrickson ME, Lowas SR. The effects of dietary protein levels on milk protein levels and postnatal growth in laboratory mice (Mus musculus). J Mammal 2007;88:1475–81. Howie GJ, Sloboda DM, Vickers MH. Maternal undernutrition during critical windows of development results in differential and sex specific effects on postnatal adiposity and related metabolic profiles in adult rat offspring. Br J Nutr 2012;108:298–307. Corte´s E, Gonza´lez C, Betancourt M, Ortiz R. Assessment of DNA damage in spleen, bone marrow, and peripheral blood from malnourished rats by single cell gel electrophoresis assay. Teratog Carcinog Mutagen 2001;21:231–47. Blackburn DG, Hayssen V, Murphy CJ. The origins of lactation and the evolution of milk: a review with new hypotheses. Mamm Rev 1989;19:1–26. Okamoto T, Fialho AC. Estudo histolo´gico comparativo entre dois me´todos de obtenc¸a˜o de cortes de alve´olos de ratos. Rev Odontol da UNESP 1990;19:63–74. Jee J, Lee W, Lee BD. The influence of alendronate on the healing of extraction sockets of ovariectomized rats assessed by in vivo micro-computed tomography. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:e47–53. Aguirre JI, Altman MK, Vanegas SM, Franz SE, Bassit ACF, Wronski TJ. Effects of alendronate on bone healing after tooth extraction in rats. Oral Dis 2010;16:674–85. Miller JP, German RZ. Protein malnutrition affects the growth trajectories of the craniofacial skeleton in rats. J Nutr 1999;129:2061–9. Herrmann AK. Efeitos da desnutric¸a˜o prote´ico-calo´rica intrauterina e po´s-natal sobre as estruturas craniofaciais. Estudo experimental em ratos. Universidade Federal de Sa˜o Paulo; 2001. [Tese de mestrado]. ¨ .. The effect of alendronate on Altundal H, Gu¨vener O.O resorption of the alveolar bone following tooth extraction. Int J Oral Maxillofac Surg 2004;33:286–93. Shimizu M, Sasaki T, Ishihara A, Furuya R, Kawawa T. Bone wound healing after maxillary molar extraction in ovariectomized aged rats. J Electron Microsc 1998;47:517–26. Teo´filo JM, Brentegani LG, Lamano-Carvalho TL. A histometric study in rats of the effect of the calcium antagonist amlodipine on bone healing after tooth extraction. Arch Oral Biol 2001;46:375–9. Teo´filo JM, Brentegani LG, Lamano-Carvalho TL. Bone healing in osteoporotic female rats following intra-alveolar grafting of bioactive glass. Arch Oral Biol 2004;49:755–62. Machado GJR, Dias SMD, Bosco AF, Okamoto T, Castro JCB, Dornelles RCM. Action of nicotine and ovariectomy on bone regeneration after tooth extraction in rats. J Oral Maxillofac Surg 2010;68:2675–81. Luvizuto ER, Queiroz TP, Dias SMD, Okamoto T, Dornelles RCM, Garcia Jr IR et al. Histomorphometric analysis and immunolocalization of RANKL and OPG during the alveolar healing process in female ovariectomized rats treated with oestrogen or raloxifene. Arch Oral Biol 2010;55:52–9. Fracon RN, Teo´filo JM, Moris IC, Lamano-Carvalho TL. Treatment with paracetamol, ketorolac or etoricoxib did not
250
archives of oral biology 59 (2014) 241–250
hinder alveolar bone healing: a histometric study in rats. J Appl Oral Sci 2010;18:630–4. 49. Reis-Filho CR, Silva ER, Martins AB, Pessoa FF, Gomes PVN, Arau´jo MSC, et al. Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats. Arch Oral Biol 2012;57:469–76. 50. Pereira MC, Zecchin KG, Campagnoli EB, Jorge J. Ovariectomy delays alveolar wound healing after molar
extractions in rats. J Oral Maxillofac Surg 2007;65: 2248–53. 51. Lamano-Carvalho TL, Bombonato KF, Brentegani LG. Histometric analysis of rat alveolar wound healing. Braz Dent J 1997;8:9–12. 52. Bodner L, Kafe I, Littner MM, Cohen J. Extraction site healing in rats. A radiologic densitometric study. Oral Surg Oral Med Oral Pathol 1993;75:367–72.
