Int. J. Morphol.

,
33(3):1146-1150, 2015.

Electrical Stimulation in the Bone Repair

of Defects Created in Rabbit Skulls

Estimulación Eléctrica en la Reparación Ósea de Defectos Creados en Cráneos de Conejos

C. Silva*; S. Olate**; L. Pozzer*; M. Muñoz***,****; M. Cantín*****; F. Uribe**,****** & J. R. de Albergaría-Barbosa*

SILVA, C.; OLATE, S.; POZZER, L.; MUÑOZ, M.; CANTÍN, M.; URIBE, F. & DE ALBERGARIA-BARBOSA, J. R. Electrical
stimulation in the bone repair of defects created in rabbit skulls. Int. J. Morphol., 33(3):1146-1150, 2015.

SUMMARY: Electrical stimulation has been used in different conditions for tissue regeneration. The aim of this study was to
analyze the tissue response of defects created in rabbit skulls to electrical stimulation. Two groups were formed, each with 9 New Zealand
rabbits; two 5 mm defects were made, one in each parietal, with one being randomly filled with autogenous bone extracted as particles and
the other maintained only with blood clotting. The rabbits were euthanized at 8 weeks and 15 weeks to then study the samples collected
histologically. In the 8-week analysis bone formation was observed in the defects in the test and control filled with bone graft, whereas the
defects with clotting presented a very early stage of bone formation with abundant connective tissue. At 15 weeks an advanced stage of bone
regeneration was identified in the defects with bone graft, whereas no significant differences were found in the electrically stimulated defects.
In conclusion, electrical stimulus does not alter the sequence of bone formation; new studies could help establish patterns and influences of
the stimulus on bone regeneration.

KEY WORDS: Electrical stimulus; Bone regeneration; Bone graft.

INTRODUCTION

Bone grafts are widely used for the reconstruction of Being aware of the biological processes present in
atrophic alveolar ridges. Autogenous bone and replacement bone regeneration, the use of different technologies could
bones have been analyzed in different scenarios, yielding improve conditions for integration. The application of
results that vary based on different research models and electrical stimuli has been used frequently in neurosurgery,
characteristics of the bone defects. obtaining satisfactory results in terms of bone regeneration
(Oishi & Onesti, 2000). In oral and maxillofacial surgery,
Thus, it has been determined that particle size is a however, there is limited information; recent studies by our
factor in bone regeneration capacity (Klüppel et al., 2013) group (Buzzá et al., 2014) showed that there were no
and that the vascularization properties of the recipient bed significant differences between the groups with application
might also be relevant in bone integration (de Oliveira et al., of electrical therapy and the groups without electrical therapy
2013). The types of materials used as well as their agents of in implant-associated defects, whereas Kaynak et al. (2005)
application are also factors that may permit enhanced reported a significant relation in periodontal regeneration
adaptation and bone integration of the defect (Duque Netto related to electric therapy.
et al., 2013). Within this wide network of factors that might
influence tissue regeneration capacity, the continuous The aim of this study was to identify the bone repair
autogenous graft is the gold standard in bone integration model in defects created in rabbit skulls using electrical
(Olate et al., 2007), such that its use as a control in different stimulation.
studies is an important element.

*
Division of Oral and Maxillofacial Surgery, State University of Campinas, Campinas, Brazil.
**
Division of Oral and Maxillofacial Surgery, Universidad de La Frontera, Temuco, Chile.
***
Center for Biomedical Research, Universidad Autónoma de Chile, Temuco, Chile.
****
Universidad Científica del Sur, Lima, Perú.
*****
PhD Program in Morphology, Universidad de La Frontera, Temuco, Chile.
******
PhD Program in Medical Sciences, Universidad de La Frontera, Temuco, Chile.

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 SILVA, C.; OLATE, S.; POZZER, L.; MUÑOZ, M.; CANTÍN, M.; URIBE, F. & DE ALBERGARIA-BARBOSA, J. R. Electrical stimulation in the bone repair of defects created in rabbit skulls.
Int. J. Morphol., 33(3):1146-1150, 2015.

MATERIAL AND METHOD The animals were euthanized at 8 and 15 weeks (4

and 5 animals at each stage) from the initial surgery, for
which an overdose of anesthetic was applied.
Eighteen male albino New Zealand rabbits were used,
with an average weight of 3.0 kg and aged between 3 and 6 Histology. Samples were obtained using sections of the skull
months. All the animals were kept on a diet of solid food made with a saw and drill-bits, staying at least 1 cm from
and water ad libitum. the edge of the defect created. The samples were kept in
buffered formalin for 24 h to be decalcified later with 0.5%
Surgery. The anesthesia used was 2 (2,6-xylidine) - 5,6- nitric acid for 20 days. Next, the pieces were subjected to
dihydro-4H-1,3 thiazine hydrochloride in a dose of 2 mg/kg diaphanization and slides were prepared with routine
of body weight combined with ketamine hydrochloride in a techniques using 6 mm serial sections and H&E staining for
dose of 10 mg/kg; atropine sulfate (0.05 mg/kg) was also study under an optical microscope.
used to reduce intraoperative respiratory risks.

After carrying out a trichotomy on the calvarium of RESULTS

the animal and using aseptic and antiseptic techniques with
chlorhexidine 0.5%, the surgical area was isolated with sterile
fields typical of these procedures. The study was without complications and free of any
other alterations in the operating protocol.
A mid-line incision was made in the calvarium,
extending between the frontal and occipital bone; the 8-week period
periosteum of the zone was completely separated. The
osteotomies were performed using 5 mm trephines, making Control group. Non-grafted defect: The hole within the
two holes in each side of the skull, taking care not to perforate defined bone and the presence of connective tissue were
the dura mater. The osteotomies were done with a low-speed identified; islands of bone tissue were observed in the cen-
motor and constant irrigation with saline. tral portion at the initial stages of formation.

One side was randomly selected to be maintained Grafted defect: The graft component had vascularization
without any type of filling; the other side was filled with the and the presence of medullary bone tissue. The process was
bone extracted from both sectors and used in particle form. at an advanced stage, although some necrotic elements were
Plane sutures were used as per normal protocols. Under these observed. There was a large amount of connective tissue.
conditions four groups were created: Group 1 was the con-
trol and Group 2 the experimental. Intraoperative and Experimental group. Non-grafted defect: Considerable
postoperative antibiotic and analgesic therapy was areas of connective tissue were found in the hole; in the center
administered regularly to all the animals. of the defect there were centers of calcification surrounded
by fibrous connective tissue and with limited vascularity.
Electrical stimulation. Forty-eight hours after the surgery,
electrical stimulation was applied to the experimental group, Grafted defect: The border between the pre-existing bone
for which a box was designed where the body was kept and the graft had a high level of vascularization and an
(horizontally) to ensure the fixed position of the animal, advanced stage of organization; the graft appeared to be
keeping the head exposed outside the box. A device fixed incorporated with the formation of two corticals. In some
with elastics was installed on the animal’s skull, which sections, small areas of necrotic bone were observed, with a
contained the electrodes that came in contact with the skull, good expression of the reparation tissue predominating.
with the orbital and maxillomandibular area free of any
fixation. 15-week period

The Healtec Celular system (Healtec Electromedicina Control group. Non-grafted defect: Defined limits were
Ltda.) was used with frequencies from 20 to 350 MgHz, observed between the pre-existing bone and the defect
spaces of 1 MgHz, impulses of 85 microseconds and created with a large amount of fibrous connective tissue;
adjustable pauses in 80, 160, 320 and 640 cycles; the greater vascularization was observed than at the previous
monitored outputs had a depth of action of 25 cm and power stage, showing a defined medullary tissue together with
of 1 to 4.5 W. The animals received 160 cycles for 1 hour a adipose tissue.
day for 21 days (3 weeks). Grafted defect: The edge of the defect was difficult to defi-
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 SILVA, C.; OLATE, S.; POZZER, L.; MUÑOZ, M.; CANTÍN, M.; URIBE, F. & DE ALBERGARIA-BARBOSA, J. R. Electrical stimulation in the bone repair of defects created in rabbit skulls.
Int. J. Morphol., 33(3):1146-1150, 2015.

ne due to the presence of newly formed bone tissue with an between the newly formed bone in the defect and the pre-
adequate level of maturation. There was still fibrous existing bone; two well-defined corticals were observed in
connective tissue in a smaller amount; the newly formed the upper and lower area that integrated with the bone
bone tissue was of good quality and with an adequate number surrounding the bone defect.
of cells involved.

Experimental group. Non-grafted defect (Fig. 1): A DISCUSSION

significant amount of newly formed bone tissue was observed
with two well-defined corticals and fibrous tissue remnants;
there was adequate vascularization between the edge of the Bone reconstruction with the aim of achieving
defect and the newly formed tissue. functional loading through dental implants has been analyzed
in multiple studies. A range of non-autogenous materials have
Grafted defect (Fig. 2): No differences were observed been used with varying success (de Oliveira et al., 2014;
Duque Netto et al.), providing important information in terms
of tissue regeneration.

The biological and mechanical conditions observed

in alveolar defects are varied, so that the analysis performed
on an animal model can give valuable information for these
analyses. Al-Nawas & Schiegnitz (2014) conducted a meta-
analysis, comparing several bone substitutes with autogenous
grafts, and reported that there were no differences in the
implant survival comparing the two materials; nevertheless,
a limitation in the clinical application of these results is
indicated in the size of the defect and the volume and real
regeneration capacity from the materials used. This limitation
makes sense, where some studies have reported that the
biological capacity of the recipient, vascularization of the
zone and bone quality of the recipient bed would have an
important role in bone regeneration (de Oliveira et al., 2013).
Fig. 1. Histological image of bone repair in the graft with filling
after 15 weeks in the control group (NB= new bone, PB= previously Although some bone substitutes have demonstrated
present bone, CT= connective tissue, BV= blood vessel). adequate versatility, the continuous autogenous bone graft
was the only one able to reach regeneration based on the
three principles of osteogenesis, osteoinduction and
osteoconduction (Olate et al.; Netto et al., 2013). Thus, when
new elements are applied to the regenerative process the
gold standard is needed to then analyze other types of
materials.

In neurosurgery, the electrical application for

osteogenesis has a well-documented history (Oishi &
Onesti); however, it has not had a significant impact on oral
and maxillofacial surgery. The model applied in this study
has been published previously with the use of dental implants
(Buzzá et al.), concluding the absence of significant
differences in bone regeneration with or without the
application of electrical stimuli. Kaynak et al. studied the
electrical effect on periodontal regeneration in a canine
Fig. 2. Histological image (10X) of the bone graft defect from the model, obtaining favorable and significant results at the sites
test group (with application of electrical stimulus) (NB= new bone, where electrical potential was applied as therapy. In a
PB= previously present bone, CT= connective tissue, BV= blood systematic review on bone regeneration for spinal fusion
vessel). they showed that any of the protocols validated on electrical
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 SILVA, C.; OLATE, S.; POZZER, L.; MUÑOZ, M.; CANTÍN, M.; URIBE, F. & DE ALBERGARIA-BARBOSA, J. R. Electrical stimulation in the bone repair of defects created in rabbit skulls.
Int. J. Morphol., 33(3):1146-1150, 2015.

stimulation resulted in arthrodesis formation, although al., 1995). Similar conclusions were published by Yoshimura
limited relevant studies had been conducted to examine this et al. (1993), who indicated greater bone formation in
condition (Tian et al., 2013). periodontal defects treated with guided bone regeneration
and biodegradable membranes. It is possible that the
The number of samples in the study limited the difficulty in adapting clinical application systems decreases
possibility of statistical development between the groups; the study into alveolar bone regeneration through the
however, the bone formation model was similar in the two application of electrical stimuli; Kaynak et al., also indicated
groups. The autogenous bone graft group with electrical a greatest bone formation at sites treated with electrical
stimulation presented a more advanced stage of bone stimuli. Our descriptive results indicate that electrical
regeneration, indicating a possible positive response. stimulus can be a positive influence on bone regeneration,
Nevertheless, considering the analysis times for euthanasia, although the model for delivering electricity in this study
the differences were mainly in the 15-week group, coinciding may alter the final results of this experiment.
in that the control group also presented an advanced stage
of bone regeneration. It can be concluded that electrical stimulus does not
alter the sequence of bone formation in defects generated in
The most accepted model of action for electrical rabbit skulls. New studies could reveal the real influence of
stimulation is that it would induce osteogenesis (Kubota et this type of stimuli on bone regeneration.

SILVA, C.; OLATE, S.; POZZER, L.; MUÑOZ, M.; CANTÍN, M.; URIBE, F. & DE ALBERGARIA-BARBOSA, J. R. Estimulación
eléctrica en la reparación ósea de defectos creados en cráneos de conejos. Int. J. Morphol., 33(3):1146-1150, 2015.

RESUMEN: La estimulación eléctrica ha sido empleada en diferentes condiciones para la regeneración de tejidos. El objetivo
de esta investigación es analizar la respuesta tisular de defectos creados en cráneo de conejos a la estimulación eléctrica. Se formaron 2
grupos con 9 conejos de raza New Zealand cada uno; en ellos se realizaron dos defectos de 5 mm, uno en cada parietal, siendo aleatoriamente
uno rellenado con el propio hueso autógeno extraído en forma de partículas y el otro mantenido solo con coagulo sanguíneo; se realizó
la eutanasia a las 8 semanas y a las 15 semanas para luego estudiar histológicamente las muestras recolectadas. En el análisis de 8
semanas se observó formación ósea en los defectos test y control rellenados con injerto óseo mientras que los defectos con coagulo
presentaron un estado muy precoz de formación ósea, observándose abundante tejido conectivo. A las 15 semanas se identificó un estado
avanzado de regeneración ósea en los defectos con injerto óseo, donde no se apreció diferencias importantes en los defectos estimulados
eléctricamente. Se concluye que el estimulo eléctrico no altera la secuencia de formación ósea; nuevos estudios podrían contribuir a
establecer patrones e influencias del estimulo eléctrico en la regeneración ósea.

PALABRAS CLAVE: Estimulo eléctrico; Regeneración ósea; Injerto óseo.

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