3-D21 1516 Tung Nguyen Thanh Vietnam-Rabit
3-D21 1516 Tung Nguyen Thanh Vietnam-Rabit
3-D21 1516 Tung Nguyen Thanh Vietnam-Rabit
Thuy-Duong Thi Nguyen1, My-Huong Le1, Tan-Tai Tran1, Tae-Sung Bae2, Tung Nguyen-Thanh3,4*
1. Odonto-stomatology Faculty, Hue University of Medicine and Pharmacy, Hue University, Hue 49000, Vietnam.
2. Department of Dental Biomaterials, School of Dentistry, Jeonbuk National University, Jeonju 561-756, South Korea.
3. Faculty of Basic Science, Hue University of Medicine and Pharmacy, Hue University, Hue 49000, Vietnam.
4. Institute of Biomedicine, Hue University of Medicine and Pharmacy, Hue University, Hue 49000, Vietnam.
Abstract
The target of this study was to establish an animal model for reconstructing critical-sized
alveolar defects, allow the experimental testing of advanced alveolar regeneration without causing
any damage to the health of the animal.
Alveolar bone defect operation was executed at the lateral side of the right central incisor to
create a bony deformity after tooth extraction in New Zealand White rabbit. They were sacrificed at
the fixed point of time to either harvest the skulls or perform Radiographic analysis and histological
survey with Hematoxylin and Eosin staining to evaluate the bone regeneration at operative site.
Clinical examination and imaging assessment was capable of presenting a critical-sized
alveolar bone defect. Soft tissue started to heal gradually, no signs of inflammation and swelling at
surgical site was reported and bone regeneration completed after 8 weeks. Radiographic survey
displayed the increasing density contrast after surgery, replaced the C-shaped radiolucent at the
surgical site. It also correspond the developement of bone in the harvested skulls, in which the
amount of bone started to rise from 0-8 weeks. Histological analysis showed the step-by-step
formation of different structures at surgical sites.. The bone and connective tissue completely filled
the defect after 8 weeks.
Our surgical procedure was capable of establishing an alveolar defect model in the central
incisor region of the rabbit maxilla. It demonstrates the capability of this model as a test bed for
assessing the healing capacity of soft tissues and alveolar bone regeneration.
Experimental article (J Int Dent Med Res 2021; 14(3): 887-895)
Keywords: Alveolar bone defect, animal model, rabbit maxilla, bone regeneration, tissue
engineering.
Received date: 25 May 2021. Accept date: 23 July 2021
have made an enormous progress in the field of mineral density, fracture firmness between
bone regeneration including stem cells-based rabbits and human being. Rabbits also
therapy, growth factors combined with demonstrate the remarkable bony metabolism
biomaterials. Many biomaterials have been and increased bone circulation rate, mostly
developed to overcome the shortcomings of cortical remodeling when compared to primates
autogenous bone and have achieved optimistic and other rodents. 14. Therefore, a rabbit model is
results using bone marrow, platelet, beta relevant to imitate an alveolar bone defect in
tricalcium phosphate (β-TCP), hydroxyapatite etc. human.
6 7 8
, , . Calcium phosphate - a key representative The aim of this study is to develop a
of biomaterials, has been proved of their model on New Zealand White rabbits
biocompability, bioactivity and osteo-conductivity (Oryctolagus cuniculus) to present a critical-sized
through many different researches 9. Meanwhile, alveolar defect and apply the materials right after
tissue engineering customarily uses one tooth extraction, which is similar to human
bioactive operator with key regenerative alveolar bone defects in case of immediate
functions, which are a mixture of activated implant after tooth extraction, periodontitis, etc.
molecules and growth factors to stimulate cells 10.
A promising approach has been tested recently is Materials and methods
the application of the osteo-inductive growth
factors such as bone morphogenetic proteins Animals and housing
(BMPs), vascular endothelial growth factor 30 New Zealand White Rabbits, at least
(VEGF), platelet-derived growth factor (PDGF) 6-8 weeks old and weighing 2 - 2.5 kg were
and transforming growth factor beta (TGF-β) operated following the standardized procedure
(protein therapy), which have been evaluated which was approved by animal ethical committee
and gained remarkable success in clinical of Hue University of Medicine and Pharmacy
practice and research 11. However, using only (Certificate No. H2019/069). All rabbits were
growth factors makes the stimulation become housed at the animal experimentation laboratory
oversimplified and insufficient, and probably turns which room temperature and humidity were
into the adverse effects 12. Within the past few maintained at about 25oC and 56%, respectively.
years, stem cell–based treatment has been A 12-hour light/dark schedule was used. Rabbits
broadly examined to prove the regenerative were given standard laboratory food pellets and
potential. Being known as playing a critical role in water ad libitum. The rabbits were divided into 0-,
cell proliferation and differentiate, many products 2-, 4-, 6-, 8-week postoperative sacrifice point of
of stem cells have been used: dental pulp stem time.
cells, bone marrow mononuclear cells, platelet Rabbit skull osteological survey
concentrates such as platelet-rich firin (PRF), Rabbits were sacrificed by lethal dose of
plate-rich plasma (PRP) 7, 8. These promise to ketamine injection (600 mg). Firstly, scalpel and
pave a new path for tissue engineering, blades, forceps, scissors were used to expose
especially in bone regeneration. the bones by cutting out the skin and muscle and
Many animal models have been removing all the adipose tissue and organs. The
developed to further discover the potential of bones of the rabbit were bubbled in 3% solution
biomaterials in bone regeneration and overcome of soda water for 1 hour to eliminate muscles and
the limitations of in vitro models in controlling the related structures effectively. Then it took 2 hours
complexity in the interactions between cells, for cooling and 3 hours for cleaning following the
growth factors, scaffold materials, etc. 13. The concept of Baker et al. (2003) and Van Cleave
goal of animal models is to test the capacity of (2010). After that, the remaining body tissue on
forming new bone of the material, avoid adverse the bones were rubbed by BP handle and blade
tissue response and minimize bone resorption. as much as possible. Lastly, they were soaked
Rabbits are the biggest animals in the small- into 10% bleaching water solution for 2 hours to
sized category. They are non-aggressive, easy to prevent further annihilation by microorganisms
house, handle, observe, reproducible and big and then dried in the sun for 10 hours.
enough to bear the trauma of the surgery and The rabbit skulls were then used for
anesthetization. Literature has reported some observation of the significant anatomical
points in common when compared the bone landmarks, especially the central incisor and the
alveolar bone at the distobuccal of the tooth. rabbits were intravenously injected with air.
Alveolar bone defect model creation Maxillae were dissected and stored in 10%
surgery paraformaldehyde. Then, samples were washed
30 New Zealand White Rabbits, at least with phosphate buffered saline (PBS) and
6-8 weeks old and weighing 2-2.5 kg were immersed in 10% Disodium Ethylene Diamine
operated following the standardized procedure Tetra Acetic acid (EDTA) for 8 weeks to
which was approved by animal ethical committee decalcification. Samples were sectioned and
of Hue University of Medicine and Pharmacy. 6 stained with Hematoxylin and eosin (H&E) stain
rabbits were used for histological survey and 6 to assess bone formation at the defect site.
were used to harvest the skulls. These two Measurement of bone formation
groups were further subdivided into 0-, 2-, 4-, 6-, The amount of bone formation was
8-weeks post-operatively, it was also the measured by calculating the difference between
sacrificing point of time, namely D0, 2W, 4W, 6W the initial size of bone defect from the size of the
and 8W, respectively. bone defect at 2 weeks, 4 weeks, 6 weeks and 8
30 minutes prior to surgery, each animal weeks after surgery. Both radiographic and
received an intramuscular injection of 1ml histological evaluation was done to estimate the
atropine sulfate which acts as preanesthetic newly formed bone.
agents to promote the effectiveness of Radiographic density were ranked
anesthesia and 0.5ml Tiletamine (Zoletil 50mg) following the scoring method of Miloro et al.;
15 minutes afterwards. Rabbits were put in a Score 1: no newly formed bone; Score 2:
quiet and dark place to get into sedation more contrast less than contiguous cancellous bone;
easily. The perimandibular and cervical areas Score 3: contrast is equal or higher than
were subsequently scrubbed with Povidine (2% contiguous cancellous bone, but less than
chlorhexidine gluconate). Three milliliters of cortical bone; Score 4: contrast is higher than
Xylocaine (Lidocaine HCl with 1:100,000 contiguous cortical bone 15. Bone regeneration
epinephrine) were injected subcutaneously along was compared according to experimental time (2,
the incision area to provide additionally local 4, 6 and 8 weeks).
anesthesia and reduce the volume of blood lost. Histological analysis was performed using
A linear incision around the marginal gingiva, the scoring method of Han et al., based on the
extending to the distal-buccal aspect of the percentage of newly formed bone and connective
central incisor, along the curvature of the tooth tissue in the defect area. The appearance of
was performed, soft tissues and periodontal bone at the defect as the replacement of
ligament were to expose the maxillary alveolus of connective tissues was the signal of bone
the central incisor. Subsequently, a carbide bur regeneration. Each sample was rated on a scale
with a low-speed hand piece was used to create of 0-10. Samples were read separately by 2
the osteotomy traversing through the lateral bony pathologists at 2 different point of times 16.
cortex, tooth roots, and trabecular bone along Statistical analysis
with saline irrigation. The amount of bone formation at 2 weeks,
Tooth root was gently luxated with a root 4 weeks, 6 weeks and 8 weeks after surgery
tip elevator and eventually removed with a dental were compared using a Mann-Whitney test. All
forcep. The oral mucosa was sutured with 5-0 data were expressed as Mean± SEM. A value of
absorbable Vicryl suture. The rabbits were fed p ≤ 0.05 was considered to be statistically
with soft diet and injected antibiotics in 3 days. significant.
The rabbits were cared of accordingly per Results
protocol and watched by veterinarian until the Characterization of alveolar defect
sacrifice point of time. model
Radiographic analysis The normal and the alveolar defect-
Rabbits were taken X-ray immediately created rabbit skull were visually inspected to
after surgery and postoperatively at 2, 4, 6, 8 uncover that the anterior maxilla held a pair of
weeks by X-ray. Images were analyzed using central incisors and a pair of accessory palatal
EZDent biomedical software. incisor. The central incisor appeared protrusive
Histological procedures while the accessory palatal incisor was far
At the end point (0, 2, 4, 6, 8 weeks) smaller and approximately half of it. The alveolar
socket of an extracted central incisor formed a blood vessels; CT, connective tissue; AD,
pocket-like cavity with a dimension of 7–8 mm adipose tissue; black arrow, osteoblast; blue
made it an ideal model for alveolar defect studies arrow, osteoclast; asterisk, osteocyte.
which is similar to the alveolar defect after
extraction in human. Frontal and lateral view of Histological analysis of a normal tooth
the skulls revealed the position and the depth of and surrounding tissues revealed various
the alveolar needed to be removed to extract the different structures such as alveolar bone,
central incisor. (Figure 1) periodontal ligament, blood vessels, connective
tissues, adipose tissues, etc. There were a plenty
of osteoblast, osteocyte at the alveolar bone site.
These structures played an important role in
nurturing the tooth and supporting soft and hard
tissue (Figure 2a-f). In fact, the
histomorphometric image of socket after
extraction showed the lack of blood vessels,
periodontal ligaments, the decreasing number of
connective tissues, osteoblasts and osteocytes,
which were the crucial part for bone formation
(Figure 2g-i).
2-week (p<0,05). Looking deeper into the data in blood vessels; CT, connective tissue; NB, new-
the bone score distribution chart, there was no formed bone. NS: not significant.
newly formed bone detected at 2-week. 33,3% of
rabbits scored 2, which were found to have new The comparison of histological images at
bone formation but the contrast was lower than the defect sites after 2,4,6,8 weeks showed bone
the adjacent contiguous cancellous bone at 4- healing pattern which correlated well with the
week. The rabbits scored 2 rose to 66,7% and radiographic findings. After 2 weeks, the blood
the rest scored 3, which had the contrast higher vessels started to penetrate inside the defects. 4
than adjacent contiguous cancellous but lower weeks after the surgery witnessed the
than the cortical bone at 6-week. At 8-week, appearance and proliferation of connective
66,7% of rabbits scored 3, exclusively there was tissues. The point of time after 6 weeks marks
the appearance of score 4 with 33,3%, which the presentation of new bone forming inside the
means 33,3% rabbits had the contrast higher connective tissues and the gradual decrease of
than cortical bone (Figure 3h). connective tissue. Finally, the bone formed were
mostly filled in the defects with the sink of
connective tissue after 8 weeks. (Figure 4a-f).
Newly formed bone and connective tissue
were scored based on the study of Han et al.
These scores were in correspondence with the
histological images, in which the new bone
formation had an upturn from 2-week to 8-week,
while the connective tissue rose from 2-week to
4-week then gradually dropped from 6-week. The
difference of the bone score was significant at
each point of time, except for the difference
between 4-week and 2-week (p<0,05). A
significant difference of connective tissue score
was found at each point of time (p<0,05). (Figure
4g, i). Looking closer to the distribution of bone
score which included 4 ranges of score: 0-2, 3-4,
5-6 and 7-8. All rabbits were scored 0-2 at 2-
week and 4-week, which means less than 20%
area of new bone formation were found. 6-week
marked the appearance of 20% rabbits with
score 3-4, which had 30-40% area of newly bone
formed. 8-week witnessed the presence of 10%
rabbits scored 5-6 and 10% of rabbits scored 7-8,
the rabbits scored 3-4 were doubled compared to
6-week and 20% left belonged to rabbits with the
score 0-2. The distribution of connective tissue
contained 5 ranges of score: 0-2, 3-4, 5-6, 7-8
Figure 4. Histological aspects of bone and and 9-10. 80% of rabbits scored 0-2 and the rest
connective tissue regeneration on alveolar defect scored 3-4 at 2-week. The majority of rabbits
model. scored 7-8 and the remaining scored 3-4 and 5-6
a. Normal alveolar bone. b. alveolar bone defect with the same percentage at 4-week. At 6-week
at day 0. c-f. Tissue healing after 2,4,6,8 weeks the score was distributed at 3-4 and 5-6 with the
of surgery g-h. Histograms showing mean and percentage of 60% and 40% respectively. 8-
the frequency distribution of newly formed week marked the return of the score 0-2 and 3-4
connective tisue scores after 2,4,6,8 weeks of with the rate of 40% and 60% respectively
surgery. i-j. Histograms showing mean and the (Figure 4h,j).
frequency distribution of newly formed bone
scores after 2,4,6,8 weeks of surgery AL,
alveolar bone; TR, tooth root; De, root dentin; BV,
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