Quantitative analysis of microbiota in patients with orthopedic structures on dental implants using the real-time pcr method

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Олимов A., Хайдаров A., & Ахмадалиев N. (2020). Quantitative analysis of microbiota in patients with orthopedic structures on dental implants using the real-time pcr method. in Library, 20(4), 83–87. извлечено от https://inlibrary.uz/index.php/archive/article/view/14292
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Аннотация

Microbiological research was carried out by PCR method in 42 persons aged 25-60 years (men - 24 and women - 18), installed and Osseo integrated DIO dental implants, which were prototyped by metal-ceramic crowns, with temporary fixation on Tempo Bond NE. After 20 days, a follow-up visit was scheduled. All the examined patients were taken biological substrate of gingival fluid from the para-implant furrow using paper endodontic pins (№ 25). Exposure of two pins was made within 30 seconds with the help of tweezers between the peri-implant cuff and the crown, biological substrate of gum liquid was taken from the parai-implant furrow. The biological substrate was transported and tested according to the manufacturer's recommendations.  When studying the content in patients with orthopedic constructions on dental implants by PCR method we observed high frequency of occurrence of Streptococcus sobrinus (69,3%), Streptococcus sanguis (48,6%) and Porphyromonas gingivalis (41,9).  Relatively similar data on the content of these microorganisms were obtained in the molecular genetic study of Streptococcus oralis samples (22.7%). Obviously, it is the combined effect of the most frequently diagnosed pathogens and peculiarities of interaction of anaerobic agents of parasitocenosis that can determine the nature of inflammatory and destructive process in the peri-implant zone.

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736

|

International

Journal of Pharmaceutical Research | Apr - Jun 2020 | Vol 12 | Issue 2

Research Article

Quantitative Analysis of Microbiota in Patients with

Orthopedic Structures on Dental Implants Using the Real-

Time PCR Method

AZIM OLIMOV, ARTUR KHAYDAROV, NUSRAT AKHMADALIEV
Tashkent State Dental Institute, Uzbekistan
E-mail:

Dr.khaydarovartur@mail.ru

Received: 12.10.19, Revised: 13.11.19, Accepted: 29.12.19

ABSTRACT

Microbiological research was carried out by PCR method in 42 persons aged 25-60 years (men - 24 and women
- 18), installed and Osseo integrated DIO dental implants, which were prototyped by metal-ceramic crowns,

with temporary fixation on Tempo Bond NE. After 20 days, a follow-up visit was scheduled.
All the examined patients were taken biological substrate of gingival fluid from the para-implant furrow using
paper endodontic pins (

25). Exposure of two pins was made within 30 seconds with the help of tweezers

between the peri-implant cuff and the crown, biological substrate of gum liquid was taken from the parai-implant
furrow.

The biological substrate was transported and tested according to the manufacturer's recommendations.
When studying the content in patients with orthopedic constructions on dental implants by PCR method we

observed high frequency of occurrence of Streptococcus sobrinus (69,3%), Streptococcus sanguis (48,6%) and
Porphyromonas gingivalis (41,9).
Relatively similar data on the content of these microorganisms were obtained in the molecular genetic study of

Streptococcus oralis samples (22.7%).
Obviously, it is the combined effect of the most frequently diagnosed pathogens and peculiarities of interaction
of anaerobic agents of parasitocenosis that can determine the nature of inflammatory and destructive process in

the peri-implant zone.

Keywords: PCR, pathogenic, opportunistic pathogens, internal interface, paraimplant area, implant, abutment.

INTRODUCTION

Dental implantation continues to be one of the
most important directions among the priority
problems of dentistry in the modern world.
Application of dental implants solves a
significant part of problems in case of partial
and complete absence of teeth, plays a decisive
role in restoring the function of chewing, helps
to correct and improve the aesthetics of the tooth

row, smile and face in general.
The experience of dental implants installation
continues to accumulate in all its aspects [2,3].
One of the conditions for the smooth
postoperative flow is the decrease of the
microorganisms’ level in the implant bed area
[1,4,9].
In spite of the fact that implantation in recent
years has a high level of success in the early

postoperative period, in the scientific literature
there is more and more information about the
risk of distant complications connected first of all
with the development of inflammation of tissues
surrounding the Osseo integrated implant
[4,7,9,12].
The most likely cause of development in the
tissues surrounding a functioning implant may
be penetration of the oral infection into the area
of implant-bone contact. The microbial

composition in peri-implant is now known and
represents a large variety of aerobic and
anaerobic microorganisms. The researchers
attach special importance to Prevotella
intermedia,

Porphyromonas

gigngivalis,

Actinobacillum actinomycetam commitans,
Bacteroides forsithus, Treponema denticola
[8,10,11]. The inflammatory process of tissues
in the peri-implant zone is the main reason of
bone tissue destruction and resorption in the

implant area.
The source of bacterial flora may be gaps and
hollow spaces in the inner surface (interface) of
the implant abutment, which will act as a
bacterial reservoir. Since usually used implants
consist of two parts - the implant and the
abutment - there is a connection place between
them, which is the inner surface (interface) of the
implant. The technologically acceptable gap is
from 2 to 5

μ

m, which is quite enough for

penetration of typical representatives of
pathogenic microflora of the oral cavity with
comparable sizes - from 0.5 to 2.0

μ

m.

OBJECTIVE OF THE STUDY

To detect pathogenic microorganisms in patients
with orthopedic constructions on dental implants
by PCR method.

ISSN 0975-2366
DOI:https://doi.org/10.31838/ijpr/2020.12.02.0110


background image

Azim Olimov et al / Quantitative Analysis of Microbiota in Patients with Orthopedic Structures on

Dental Implants using the Real – Time PCR Method

737

|

International

Journal of Pharmaceutical Research | Apr - Jun 2020 | Vol 12 | Issue 2

RESEARCH MATERIAL AND METHODS

In order to solve the stated goal we carried out
microbiological research in 42 people aged 25-
60 years (men - 24 and women - 18), who were
undergoing treatment at the Department of
Surgical Dentistry and dental implantology of
TGSI. This system has an implant-abutment
connection by the type of hexagonal connection
using a fixation screw. All patients had

orthopedic constructions (metal-ceramic crowns,
with temporary fixation on Tempo Bond NE)
installed. After 20 days, a follow-up visit was
scheduled. The oral consent of the patients was
obtained, to undergo the examination.
The fence of material for bacteriological
examination was carried out in the area of
implant gum furrow using paper endodontic
pins (

25). Exposure of two pins was made

within 30 seconds with the help of tweezers

between the peri-implant cuff and the crown,
biological substrate of gingival fluid was taken
from the parai-implant furrow. It was then
placed in a sterile Eppendorf plastic tube (1.5
ml) containing 1 ml of physiological solution.
The samples were stored and transported to the
laboratory at +4°C for 2 hours. Sample batches
were transported to the laboratory in thermal
containers with the refrigerant.

For DNA testing of the samples with the reagent
set "Ampley Prime® Florocenose-Aeroba" and
the reagent set for quantitative determination of
human

DNA

recommended

by

the

manufacturer were used, and the results were
calculated normalized values of microorganism
DNA concentration reflecting the number of
microorganism cells relative to human mucous
membrane cells.
At the stage of amplification with detection,

reagent tubes, DNA samples and control
samples were injected using disposable filter
tips. When preparing the amplification tubes the
total volume of reaction mixture was - 25 µl,
including the volume of DNA sample - 10 µl.
For one reaction 10 µl of PCR mixture of Aeroba
Florocoenosis and 5 µl of PCR buffer-H are
required. The mixture was prepared for the total
number of tested and control samples plus a

stock for several reactions.
The contents of the tubes were mixed with PCR-
mixture of Florocenosis-Aeroba, PCR-buffer-H,
deposited droplets on the vortex. In a separate
tube prepared a reaction mixture, mixed the
necessary

amount

of

PCR-mixture

of

Florocenose-Aeroba

and

PCR-buffer-H,

precipitated the drops on Vortex. The necessary
amount of tubes was taken for DNA
amplification of the tested and control samples.

Each tube was filled with 15 µl of the prepared
reaction mixture and 10 µl of DNA samples
obtained as a result of extraction from the
investigated samples.

Detection of the fluorescent signal is assigned
through the channels for fluorophores FAM,
JOE, ROX and Cy5. If other tests are performed
at the same time, detection is also assigned to
other used channels.
Tubes are placed in the cells of the reaction
module of the device and start the amplification
program with fluorescent signal detection.
On the basis of the obtained threshold cycle (ST)
values and on the basis of the given

concentration values for DNA calibrators K1 and
K2, the automatic construction of the calibration
straight line and calculation of the Genome
Equivalents (GE) values of streptocococcal DNA
in 1 ml of the investigated and control samples
are performed.
The obtained results of index evaluation were
processed in accordance with the principles of
medical statistics using the package of programs

"Excel-7", "Statistica 5.0" with the use of non-
parametric

methods

of

quantitative

characteristics analysis.

RESEARCH RESULTS AND DISCUSSION

The extraction of the internal space of a dental
implant using sterile paper endodontic pins (size
25) is the most optimal way to take the material
for molecular genetic research, due to the

excellent absorption capacity of the pins, the
possibility of taking the clinical material of a
certain volume and the elimination of trauma of
periimplant tissues.
Qualitative evaluation of the internal space
content of the dental implant by microorganisms
in clinical samples. While studying the content in
patients with orthopedic constructions on the
dental implants by PCR method we observed
high frequency of occurrence of Streptococcus

sobrinus (69,3%), Streptococcus sanguis
(48,6%) and Porphyromonas gingivalis (41,9%)
species (figure 1).
Relatively similar data on the content of these
microorganisms were obtained in molecular
genetic studies of Streptococcus oralis samples
(22.7%).
The above data indicate the predominance of
facultative anaerobic and bonded anaerobic

flora due to the lack of free access to oxygen in
the studied space.
The majority of species of the detected
microorganisms are conditionally pathogenic,
some - saprophytes. Such a high proportion of
anaerobic agents and their variety make it
difficult to identify a leading pathogen that could
be the "leader" of the infectious-inflammatory
process. Obviously, it is the combined effect of
the most frequently diagnosed pathogens and

the interaction of anaerobic agents of
parasitocinosis that can determine the nature of
the inflammatory and destructive process in the
peri-implant zone.


background image

Azim Olimov et al / Quantitative Analysis of Microbiota in Patients with Orthopedic Structures on

Dental Implants using the Real – Time PCR Method

738

|

International

Journal of Pharmaceutical Research | Apr - Jun 2020 | Vol 12 | Issue 2

Fig.1. Frequency of detecting pathogenic and

conditionally pathogenic bacteria of the

dental implant inner space by PCR method


If we take into account the fact that it is the foci
of the listed chronic infection that are often the
basis of periodontal disease, we can assume
that if bacteria can expand from the implant

interface into the oral cavity, inflammatory
processes can occur in the peri-implant tissues,
which subsequently lead to instability of the
implant.


CONCLUSION

.

Thus, we carried out work on standardization of
detection and quantitative determination of

pathogenic and conditionally pathogenic
bacteria of the internal space of dental implant
microorganisms in the clinical material by PCR
method in real time. The following conclusions
can be drawn:
1. According to the conducted research it is
possible to draw a conclusion that the presence
of the fixture and inner cavity superstructure in
the area of the dental implant articulation leads
to the fact that there is a migration of bacteria

and products of their vital functions from the
external and internal interface of the dental
implant, regardless of the linear sizes of these
spaces.
2. A considerable number of microorganisms
from Gram-positive coccuses to Gram-negative
sticks turned out to be able to penetrate through
the gap between the implant and the abutment.
3) It is possible to assume that the gap in the

area of the implant and abutment articulation
influences the processes of osseointegration and
inflammatory reaction in the peri-implant zone
tissues, but more detailed and profound
research is to be carried out on the interrelation
between the microleakage volume in the
articulation interface and bone tissue loss.


REFERENCES

1.

Bald L. Osteointegration: molecular, cellular
mechanisms // Clinical implantology and
dentistry. -1997. -

1. -pp.48–59.

2.

Paraskevich V.L. Dental implantology. Basics of
theory and practice: Scientific and practical

manual. -Minsk, 2002. -p.368.

3.

Nikolsky V.Yu. Dental implantation in severe
bone atrophy. -Samara, 2008. - – p.130.

4.

Sukhov V.D. Increase of efficiency of

prophylaxis of the early postoperative
complications at dental implantation: Cand. of
medical sciences: - Moscow, 2013. - p.22.

5.

Tamarova E.R., Masagutova N.R. Molecular
and genetic characteristics of the oral cavity
microflora at periodontitis // Vestnik of
Chelyabinsk State University. -2013. -

7. –

pp.70-71.

6.

Yakovlev, A.T.; Badrak, E.Yu. Investigation of
the microflora in the area of a dental implant
connection with an abutment (in Russian) //

Volgograd scientific-medical journal. -2015. -

1. -pp.46-49.

7.

Al Habashneh, R. Photodynamic therapy in
periodontal and peri-implant diseases // R.Al

Habashneh, F. A. R.Al Habashneh, F.A. Asa'ad,
Y. Khader, F. A. Asa'ad, Y. Khader //
Quintessence Int. - – 2015. - Vol. 46 (8). - –

pp. 677–690.

8.

Conrads G., de Soet J.J., Song L., Henne K.,
Sztajer H., Wagner-Dobler I. et al. Comparing

the cariogenic species Streptococcus sobrinus
and S. mutans on whole genome level (in
Russian) //J. Oral. Microbiol. -2014. -Vol.6. -

P.1-9.

9.

Esposito M. Treatment of periimplantitis: what

interventions are effective? A Cochrane
systematic review /M.Esposito, M.G. Grusovin,
H.V.Worthington //Oral Implantol. - – 2012. -

5. - pp. 21-41.

10.

Koyanagi T. Comprehensive microbiological in

periimplantitis and periodontitis /T. Koyanagi,
M. Sakamoto, Y. Takeuchi1, N. Maruyama //J.
Clinical Periodontology. - – 2013. - –

40. -

pp.218-226.

11.

Mesmer C. Clinical, microbiological and

immunological findings in peri-implantitis
patients with bar-retained lower removable
partial dentures, compared to a healthy

control group (12-month-follow-up) /C.
Mesmer, A. Forster, M. Antal, K. Nagy
//Fogorv. Sz. - – 2012. - –

105. - pp.59-64.

12.

Vered Y. Teeth and implant surroundings:
Clinical health indices and microbiologic

parameters

/Y.Vered,

A.Zini,

J.Mann

//J.Quintessence International. - – 2011. - –

42. pp.339-344.

13.

Amith

Kumar

B,

Prasanna

Habbu,

Thimmasetty, Lakshman, Prabha Hullatti, Ravi
Kumar S. "Phytosomes as Novel Drug
Delivery System for Herbal Medicine –A

Review." Systematic Reviews in Pharmacy 8.1
(2017), 5-7. Print. doi:10.5530/srp.2017.1.2

Streptoco

ccus

oralis,

22.7

Streptoco

ccus

sanguis,

48.6

Streptoco

ccus

sobrinus

, 69.3

Streptoco

ccus

haemolyt

icus,

16.4

Porphyro

monasgi
ngivalis,

41.9

Библиографические ссылки

Bald L. Osteointegration: molecular, cellular mechanisms // Clinical implantology and dentistry. -1997. -№ I. -pp.48-59.

Paraskevich V.L. Dental implantology. Basics of theory and practice: Scientific and practical manual. -Minsk, 2002. -p.368.

Nikolsky V.Yu. Dental implantation in severe bone atrophy. -Samara, 2008. - - p. 130.

Sukhov V.D. Increase of efficiency of prophylaxis of the early postoperative complications at dental implantation: Cand. of medical sciences: - Moscow, 2013. - p.22.

Tamarova E.R., Masagutova N.R. Molecular and genetic characteristics of the oral cavity microflora at periodontitis // Vestnik of Chelyabinsk State University. -2013. -№7. -pp.70-71.

Yakovlev, A.T.; Badrak, E.Yu. Investigation of the microflora in the area of a dental implant connection with an abutment (in Russian) // Volgograd scientific-medical journal. -2015. -№ I.-pp.46-49.

Al Habashneh, R. Photodynamic therapy in periodontal and peri-implant diseases // R.AI Habashneh, F. A. R.AI Habashneh, F.A. Asa'ad, Y. Khader, F. A. Asa'ad, Y. Khader // Quintessence Int. — 2015. - Vol. 46 (8). — pp. 677-690.

Conrads G., de Soet J.J., Song L., Henne K., Sztajer H., Wagner-Dobler I. et al. Comparing the cariogenic species Streptococcus sobrinus and S. mutans on whole genome level (in Russian) //J. Oral. Microbiol. -2014. -Vol.6. -P. 1-9.

Esposito M. Treatment of periimplantitis: what interventions are effective? A Cochrane systematic review /М.Esposito, M.G. Grusovin, H.V.Worthington //Oral Implantol. - - 2012. --№ 5.-pp. 21-41.

Koyanagi T. Comprehensive microbiological in periimplantitis and periodontitis /Т. Koyanagi, M. Sakamoto, Y. Takeuchi I, N. Maruyama //J. Clinical Periodontology. - - 2013. - - №40. -pp.218-226.

Mesmer C. Clinical, microbiological and immunological findings in peri-implantitis patients with bar-retained lower removable partial dentures, compared to a healthy control group (12-month-follow-up) /С. Mesmer, A. Forster, M. Antal, K. Nagy //Fogorv. Sz. - - 2012. - - № 105. - pp.59-64.

Vered Y. Teeth and implant surroundings: Clinical health indices and microbiologic parameters /Y.Vered, A.Zini, J.Mann //^Quintessence International. — 2011. — №42. pp.339-344.

Amith Kumar B, Prasanna Habbu, Thimmasetty, Lakshman, Prabha Hullatti, Ravi Kumar S. "Phytosomes as Novel Drug Delivery System for Herbal Medicine -A Review." Systematic Reviews in Pharmacy 8.1 (2017), 5-7. Print, doi: 10.5530/srp.2017.1.2

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