Articolul-2
www.stomaeduj.com DENTAL MATERIALS
TESTING POSSIBILITIES OF MATERIALS USED IN
Original Articles
PERIODONTAL THERAPIES ON LABORATORY RATS
Ioana Mitruț1a , Horia Octavian Manolea1b* , Alex Ioan Sălan2c , Ioana Neagu3d , Andreea Stănuși4e ,
1e
Alexandra Drăghici
1
Department of Prosthesis Technology and Dental Materials, Faculty of Dentistry, University of Medicine and Pharmacy of Craiova, DJ-200349 Craiova, Dolj,
Romania
2
Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Medicine and Pharmacy of Craiova, DJ-200349 Craiova, Dolj, Romania
3
Department of Periodontology, Clinical Emergency County Hospital Craiova, DJ-200349 Craiova, Dolj, Romania
4
Department of Prosthodontics, Faculty of Dentistry, University of Medicine and Pharmacy of Craiova, DJ-200349 Craiova, Dolj, Romania
a
DDS, PhD student; e-mail: ioanamitrut@gmail.com; ORCIDiD: https://orcid.org/0000-0002-4843-1278
b
DDS, PhD, Professor, Head of Department; e-mail: horia.manolea@umfcv.ro; ORCIDiD: https://orcid.org/0000-0002-2710-5537
c
DDS,PhD, Teaching assistant; e-mail: alexsalan87@gmail.com; ORCIDiD: https://orcid.org/0000-0003-4806-0101
d
DDS, Periodontology resident; e-mail: oana_neagu15@yahoo.com; ORCIDiD: https://orcid.org/0000-0002-2179-5098
e
DDS, PhD Student; e-mail: andreeacazan22@yahoo.com; ORCIDiD: https://orcid.org/0000-0003-2520-7967
f
DDS, PhD Student; e-mail: ada.draghici21@gmail.com; ORCIDiD: https://orcid.org/0000-0003-1421-8425
ABSTRACT https://doi.org/10.25241/stomaeduj.2020.8(2).art.2
Introduction Periodontitis is an oral inflammatory disease of significant importance, that leads to gingival
inflammation, alveolar bone loss and has a high impact on the quality of life and general health. There
has been a continuous interest in the scientific world to test new therapies and therapeutic materials for
periodontal disease. One of the most critical tools to investigate mechanisms of periodontal pathogenesis
and test new therapeutic materials are animal models. In addition, there is a wide range of materials used
in periodontal therapy, especially in terms of bone augmentation, so choosing the ideal material is often
difficult to achieve.
Methodology In this article, we have evaluated two methods for testing artificially induced periodontal
defects - intra-orally and extra-orally - the biological adaptation of the materials used in periodontal and
bone regeneration techniques on animal models.
Results We have created two protocols for the extra-oral and intra-oral approaches. By following them we
have succesfully managed to create the periodontal defects and to apply the therapeutic materials. We have
also made a comparison between the two methods, and the possibilities of materials that can be used.
Conclusion Tests performed on animal models will remain an important asset for evaluating new approaches
for the improvement of tissue regeneration therapies. As there are continuous advances in the study of
dental materials, we also have to search for new, easy to perform, ethics-friendly methods to evaluate the
biological response of these materials.
KEYWORDS
Periodontal Disease; Animal Models; Alveolar Bone Loss; Bone Augmentation.
1. INTRODUCTION inflammatory disease. Therefore, the major goal of
periodontal therapy was to eliminate the pathological
Periodontal disease is known to have a a long history, organisms discovered in the dental plaque located
constantly accompanying the evolution of the on the surface of the tooth [3,4]. Periodontal therapy
human species. New data regarding its prevalence is complex and it includes: prevention strategies
confirm its high value up to 50% around the world, to control the inflammation level and regenerative
with the highest scores in the older population [1].
therapies of all supporting structures and tissues
It represents one of the major causes of tooth loss
which can compromise mastication, esthetics, self- [5]. Root planing leads to clinical improvement by
confidence, and quality of life [2]. disrupting the subgingival biofilm, which reduces
Microbial dental plaque has been accepted as the the amount of bacteria, resulting in a delay in the
primary etiological factor in the occurrence of repopulation of pathogenic microorganisms [6,7]
OPEN ACCESS This is an Open Access article under the CC BY-NC 4.0 license.
Peer-Reviewed Article
Citation: Mitruț I, Manolea HO, Sălan AI, Neagu I, Stănuși A, Drăghici A. Testing possibilities of materials used in periodontal therapies on laboratory
rats. Stoma Edu J. 2021;8(2):100-105
Received: April 06, 2020; Revised: April 27, 2021; Accepted: May 13, 2021; Published: May 17, 2021;
*Corresponding author: Prof. Horia Octavian Manolea, DDS, PhD; 2 Petru Rares Street, DJ-200349 Craiova, Dolj, Romania
Tel/Fax: +40766335216; e-mail: horia.manolea@umfcv.ro
Copyright: © 2021 the Editorial Council for the Stomatology Edu Journal.
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Today many biomaterials are available to use for 3.1. Study Population
Original Articles
periodontal regeneration. The vast majority of mate- We have performed the tests on male Wistar rats.
rials used in periodontal therapies are materials The animals were then kept in separate cages and
(such as chlorhedine, tetracycline, metronidazole) marked in batches. The cages were well ventilated,
used for therapeutic purposes in non-surgical with an alternation of 12 hours of light / dark, being
procedures. Lately there has been an increasingly maintained at a temperature of about 250C. The
frequent use of materials used in reconstructive animals were fed with standard fodder combined
surgical therapies. Synthetic bone grafts materials with granulate, and ad libitum water. Each animal
are available in particles with different diameters was registered and the data of the experiment were
and can offer the benefits of an unlimited amount, kept in a single register.
without the risk of transmitting the disease and
without the need to create an additional surgical 3.2. Working methodology
area. Polymers are widely used as a barrier material After studying the specialized literature, we selected
in guided tissue regeneration applications [8]. The two types of protocols that we adapted to be used in
interest of the researchers and the support from our project [10,11]. Thus, the testing methods of the
the companies allow for an enormous amount of materials used in this study were performed through
clinical and pre-clinical research to be carried out an intraoral and extraoral approach.
every day. Generally, the repopulation of cells on the The whole operation took place in the animal
root surface after periodontal surgery determines research facility of the university. The surgical area
the nature of the attachment that will form. After was equipped correspondingly and located in the
surgery and removal of biofilm, the root surface same living unit as the animals, so that the stress
of bone defects can be repopulated by epithelial could be limited as well as the potential danger
cells, gingival connective tissue cells, bone cells, or to the animals health. The surgery room was well
periodontal ligament cells [9]. disinfected before the operation with disinfectants
such as sodium hypochlorite, chlorine dioxide
2. AIM or glutaraldehyde solutions and the animals
and instruments were prepared so as to prevent
This study is part of a complex in vivo project that contamination and ensure the success of surgery.
aims to test new materials for gingival inflammation These interventions were performed under general
and alveolar bone loss on induced periodontal anesthesia with ketamine and narcoxide using
defects. The aim of the current study is to identify and Ketamidor 100mg / ml 20 IU (0.2 ml) and Xilazyn Bio
to compare different approaches in the induction of 2% 0.3 ml. The injection was made slightly to the
periodontal disease and bone defects. right of the white abdominal line.
We have studied, developed and compared two All experiments were conducted in accordance
different protocols for inducing periodontal with local guidelines on the welfare of experimental
defects on animal models. The two methods consist animals and with the approval of the Ethics Commi-
in two different approaches to the periodontium ttee of the Research Facility (No. 80/16.04.2019 and
and alveolar bone – through an intra-oral and extra- No. 101/23.09.2019).
oral approach, which will allow the further studies of
different materials used for periodontal disease. 4. RESULTS
This paper aimed to evaluate the different methods
of addressing artificially induced periodontal defects We have created both procedures for both intra-oral
through a study performed on laboratory animals. and extra-oral approaches. Both procedures have
The study is part of a multidisciplinary project, as it managed to induce periodontal defects, and they
integrates the clinical surgical field, and biomaterials have proved to be reproduced successfully.
science as well. By developing these protocols, we The procedure for the intraoral approach was
hope to bring our contribution to the field of dental performed on the lower incisors of the animal
materials and oral tissues in vivo studies. models.
As shown in Fig. 1, the depth of the gingival groove
3. MATERIALS AND METHODS measured with the help of a periodontal probe at the
level of each tooth in six points MV, V, DV, ML, L, DL.
The working methodology consisted in a series The procedure for the intraoral approach was
of steps starting from the creation of periodontal performed on the lower incisors of the animal
defects in the mandible of laboratory animals, models. As shown in Fig. 1, the depth of the gingival
clinical evaluation of the evolution of inflammation groove measured with the help of a periodontal
in the periodontium, application of materials studied probe at the level of each tooth in six points MV, V,
in defects. DV, ML, L, DL.
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Original Articles
Figure 1. Measuring the depth of the gingival groove using a Figure 2. Holding the retraction thread in place by a ligature
periodontal probe. with a 5/0 sterile non-absorbable silk thread.
Figure 3. Mechanical scaling and root planning with a Gracey Figure 4. Application of the regeneration material in the
periodontal curette. periodontal pocket.
After that, we have inserted a piece of a silk thread The method used in this study to create a peri-
used for gingival retraction, size 00, into the gingival odontal space in the vestibular area of the
sulcus, and performed sutures with a 5/0 suture of lower incisors and placement of a gingival
sterile non-absorbable silk to hold in place the gin- retraction thread, followed by the suture allowed us
gival chord. (Fig. 2) to obtain the periodontal defect after one week by
After 7 days, the rats in the study groups were maintaining the mechanical irritating factor at this
again anesthetized and the sutures and gingival level.
retraction threads were reoved. The periodontal For the extra-oral approach we chose as the initial
defect obtained in each animal was quantified by site the mandibular side and the bone defect to be
made on the mandibular molar level. After prepar-
noting the depth of the periodontal pocket measured
ing the animal for surgery, the correct identification
using a periodontal probe at the level of each tooth in
of the epithelium and hard tissue should guide the
six points (MV, V, DV, DL, L, ML), but also the
operator for the initial incision. A superficial incision
presence and location of inflammation areas.
is made for the first time to expose the masseter
After performing the mechanical scaling and root
muscle (Figs. 13, 14) and to have access to the ligament
planning, the materials studied were applied to
marks that extend in a posterior-anterior direction,
the corresponding study group by inserting them approaching the basilar edge of the mandible. An
directly into the periodontal defect from the depth incision can be made below the ligament line at the
of the periodontal pocket until it was completely masseter muscle to expose the mandible.
filled (Figs. 3,4). A distinct ligament usually covers the lateral area at
The intraoral approach in order to test the the level of the first molar; it should be dissected
materials used in periodontal therapies had the to ensure efficient flap take-off and proper surgical
advantage of an easier application for both access.
surgical and non-surgical materials, but is limited to After exposing the bone we identified the first
the incisor group, whose permanent eruption may molar and the opaquer area of bone in the form of a
affect the results obtained. "tear" that is a characteristic of the vestibular cortex.
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Original Articles
Figure 5. Appearance of the defect created in the mandibular bone. Figure 6. Appearance of the material shortly after its application in the
defect created in the mandibular bone.
Figure 7. Suture with resorbable threads of the masseter muscle. Figure 8. Suture of the skin with non-absorbable threads.
The area of creation of the defect was located at The gingival tissues become swollen, with pocket
the distal root of the first mandibular molar. Using a formation, accumulation of debris, and ulceration
surgical 4mm spherical drill, we created a bone at about 3 months of age. Alveolar bone resorption
defect measuring 8 mm length 4mm wide and 2mm underneath the gingiva causes the teeth to slide
depth. (Fig. 5) apart and eventually to exfoliate [12].
After the test materials were applied to the created Alveolar bone is constantly renewed by mode-lling
defect (Figs. 6,7), the muscle tissue was first reposi- and remodelling mechanisms in response to func-
tioned using absorbable sutures. tional demands, local and systemic factors. Nutri-
Once the proper muscle closure and the closure tional deficiencies in animals have been shown to
were ensured, the skin was repositioned with non- affect the periodontal tissues.
absorbable sutures. (Fig. 8) One study determined that dietary boron deprivation
alters periodontal alveolar bone modelling and
5. DISCUSSION remodelling by inhibiting bone formation [14].
Animal models have been frequently used in wound
This study aimed to evaluate the testing methods of healing studies primarily because of cost conside-
materials that can be used in periodontal therapies. rations.
The laboratory rats proved to have similar anatomy Wound contraction is considered to be the primary
of the periodontal tissues with those of humans [12]. healing method of rats as opposed to re-epitheliali-
Laboratory animals have a significant advantage be- sation seen in humans.
cause they can copy the cellular complexities that Since wound contraction is rapid the overall
occur in humans in vivo and are often more accurate healing time of rats is substantially reduced, unlike
than in vitro studies that takeplace on plastic surfa- re-epithelialisation which involves the creation of
ces with a limited number of cell types present [13]. new skin tissue.
Animal models are highly susceptible to periodontal The reduced healing time in rodent burn models
disease, as the gingival tissues go through different allows researchers to quickly study the mechanics of
stages once the plaque occurs. wound healing healing [15].
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Studies have proposed many protocols for in- advantage of easier access to the distal area of the
Original Articles ducing gingival inflammation leading to peri- arch, but but complex surgical procedures limit
odontal destruction, such as the placement of its use, excluding testing of materials used in non-
a retentive silk or ligature in the gingival sul- surgical therapies.
cus of the molars or incisors [10,12,16] or by the
injection of lipopolysaccharides or various periodon- 5. CONCLUSIONS
tal pathogens such as Prophyromonas gingivalis,
Aggregatibacter actinomycetemcomitans, [12,17, Tests performed on laboratory animals will remain
one important way to evaluate new approaches that
18]. improve modalities tissue regeneration currents. For
Both the method of inserting an interdental the materials used in periodontal therapies to be
floss and the repeated intra-gingival injection of optimally effective, doctors and researchers should
bacteria are suitable to analyse the pathogen- have sufficient knowledge of both the ways of
esis of periodontal disease and therapeutic applying the materials, as well as their properties
strategies to modulate the progression of the and also advantages and disadvantages.
disease. However, studies of reconstructive therapies In our study, the intraoral approach allowed us to
require surgically created periodontal defects [11]. easily obtain periodontal defects, but which can
The testing methods of the materials used in this be maintained only for a short period of time. The
study were performed through an intraoral and extra-oral approach allows access to the lateral area
extra-oral approach. The intraoral approach was to achieve periodontal defects for a longer time, but
achieved by initially creating the periodontal defect is more surgically aggressive.
by applying a retraction thread maintained in the
ACKNOWLEDGMENTS
gingival groove maintained by means of sutures,
None.
followed by the mechanical cleaning procedure and
the insertion of the tissue regeneration material. AUTHOR CONTRIBUTIONS
The extra-oral surgical approach was performed by IM: contributed to creating the concept and structure of the
creating cavities in the mandible of laboratory study, in establishing the experimental conditions for the intra-
animals, followed by the application of tissue rege- oral protocol, and performed the experimental surgery on
neration material. laboratory animals. HM: coordinated the experimental surgeries,
The intraoral approach has the advantage of an interpretation of the data, formulated the research conclusions
easier application for both materials in both surgi- and supervised the drafting of the article. AIS: participated in the
cal and non-surgical procedures, but is limited to drafting of the extra-oral protocol and performed the surgery on
the incisor group. The method used in this study laboratory animals with the extra-oral approach. IN: participated
to create a periodontal defect on the lower incisors in the writing and translation of the paper, contributed to
by placing a gingival retraction thread, helped us structuring the bibliographical references, and took the photos
obtain the periodontal defect after one week. during the surgeries. AS: participated in researching the bibliogra-
We found that the insertion in the gingival phical references, contributed to the introductory part and in
the drafting of the abstract. AD: participated in the surgical
sulcus of a small retraction chord combined with the
experiments, writing, editing and translation of the paper.
placement of a ligature acted as a continuous
All authors read and approved the final manuscript
source of irritation to the tissue and led to a more
aggressive result. FUNDING
Complex surgical procedures limit its use, excluding The authors declare that they have not received any funding.
testing of materials used in non-surgical therapies.
The extra-oral approach in order to test the COMPETING INTERESTS
materials used in periodontal surgery has the The authors declare that they have no competing interests.
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Ioana MITRUȚ
Department of Prosthesis Technology and Dental Materials
Faculty of Dentistry
University of Medicine and Pharmacy of Craiova
Craiova, Romania
CV
Dr. Mitruț Ioana has been a PhD student at the Department of Prosthesis Technology and Dental Materials of the University of
Medicine and Pharmacy of Craiova since 2018. She is also a Prosthodontics resident at the same university. Her PhD thesis is
entitled “Possibilities of using calcium fructoborate preparations in dentistry”. Her research interest are testing dental materials
and other biomaterials on biological tissues, the applications of boron based compounds in oral health. She has a lot of
experience in performing in vivo tests on laboratory animals.
Questions
1. The general goals of periodontal therapy are?
qa. Regenerative therapies;
qb. Multiple teeth extraction;
qc. Prosthetic therapies;
qd. Aesthetic therapies.
2. Laboatory rats can be used in periodontology research because:
qa. We can use as many animals as possible for tests;
qb. They have similar anatomy of the periodontal tissues with those of humans;
qc. They cannot copy the cellular complexities that occur in humans;
qd. Even if they are efficient they are less accurate than in vitro tests.
3. The maintenance of the animals consist of:
qa. Housing them in cages with continuous darkness;
qb. The surgery facility to be in another building to not disturb the animals in the cages;
qc. The cages should have an alternation of 12 hours of light / dark;
qd. The animals should be fed only once a day.
4. The possibilities for creating periodontal defects are?
qa. Injections of antibiotics;
qb. Placement of a retentive silk or ligature in the gingival sulcus;
qc. Extraction of healthy teeth;
qd. Food deprivation.
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