© 2019 Polish Dental Association
O R I G I N A L PA P E R
EVALUATION OF KERATINISED GINGIVAL FIBROBLAST
PROLIFERATION AFTER PROTEFIX® GEL APPLICATION,
IN THE ASPECT OF WOUND HEALING IN ELDERLY PATIENTS
Barbara Sterczała1, Julita Kulbacka2, Jolanta Saczko2, Marzena Dominiak1
1
Department of Dental Surgery, Wrocław Medical University, Poland
Department of Molecular and Cell Biology, Wrocław Medical University, Poland
2
ABSTRAC T
Introduction: Atrophic mucous membranes in elderly patients become more susceptible to all kinds of injuries: chemical, biological, and mechanical, including iatrogenic. Thus, the healing processes are difficult and
challenging. Therefore, it is important to implement preparations to support tissue regeneration in postoperative
recommendations. Acceleration of the recovery process would significantly improve the quality of life of elderly
patients.
Aim: The aim of the pilot study was the evaluation of the keratinised gingival fibroblast proliferation process after
the use of Protefix® gel in vitro.
Material and methods: An in vitro pilot study of human fibroblasts obtained from healthy donors and isolated
from keratinised gingival tissues. The primary cell line of human fibroblasts was stimulated by 5%, 50%, and 100%
Protefix® gel for 20 min and 24 h.
Results: In this in vitro pilot study, it was shown that Protefix® gel significantly stimulated the proliferation
of human fibroblasts from primary culture.
Conclusions: Summarising, the conducted pilot study showed significant stimulation of proliferation of human
fibroblasts after exposure to Protefix® gel at a concentration of 5%. The obtained results indicate that the tested
product may promote the healing of surgical wounds, which is especially important in elderly patients in whom
the healing process is more complicated.
Key words: gerodontology, cell culture, Protefix®.
J Stoma 2019; 72, 1: 9–12
DOI: https://doi.org/10.5114/jos.2019.86486
INTRODUCTION
The quality of life and the activity of geriatric patients
depends to a large extent on the condition of the oral
cavity. Xerostomia, burning mouth syndrome (BMS),
oral candidiasis, prosthetic stomatitis, and mucous injury due to: poor denture adhesion, mucosal part rough-
ness, and thermal or allergic reaction (caused by the release of free monomer) are some of the more common
reasons for elderly patients to report to the dental office [1, 11, 18]. As well as the pain, discomfort of elderly
patients is caused by the lack of teeth; therefore, because
of rehabilitation of the stomatognathic system, they are
often referred to the dental surgeon. Pre-prosthetic pro-
Address for correspondence: Barbara Sterczała, Department of Dental
Surgery, Wrocław Medical University, 26 Krakowska St., 50-425 Wrocław,
Poland, e-mail: Barbara.sterczala@umed.wroc.pl
O F F I C I A L J O U R N A L O F T H E P O L I S H D E N TA L A S S O C I AT I O N
O R G A N P O L S K I E G O TO WA R Z Y S T WA S TO M ATO L O G I C Z N E G O
Received: 26.04.2019 • Accepted: 14.05.2019 • Published: 28.06.2019
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Małgorzata Dudek, Iwona Soika, Weronika Jończyk, Anna Turska-Szybka, Dariusz Gozdowski, Dorota Olczak-Kowalczyk
Barbara Sterczała, Julita Kulbacka, Jolanta Saczko, Marzena Dominiak
cedures preparing for treatment include procedures in
the field of muco-gingival surgery, including: fraenoplasty of lip or cheek, and vestibuloplasty [4, 6]. Patients
also report for the removal of papillary hypertrophy
of the palatal mucosa and fibromuscular hypertrophy
of the mucous membrane, and gingiva in the form of fissile granulomas, which are the result of unfavourably
distributed chewing forces of the lower teeth directed to
the upper total denture causing so-called combination
syndrome [7, 10].
Geriatric patients are a group requiring a special dental approach, considering the anatomical and physiological changes resulting from the aging process, accompanying psychosomatic diseases, and taken medications.
This is often interdisciplinary care. Diabetes, hypertension, hormonal changes in the menopausal period, and
deficient diseases such as anaemia or Sjögren syndrome
are the cause of symptoms such as dry mouth and burning mucous. These symptoms are persistent in everyday
life. In addition, renewal processes are slower. Therefore,
it is important to implement in the post-treatment recommendations preparations that will support tissue regeneration.
Currently, there are numerous products available
that locally relieve the pain caused by damage to the mucous and have a protective effect against the action
of mechanical, biological, or chemical stimuli. Depending on the application and the time of their use, we can
distinguish different forms of preparations, e.g.:
• pastes administrated immediately after the treatment,
having a fibrous, self-hardening, or elastic consistency: Peripac®, Coe Paki® and Coe Pak®, Septo-pack®;
they are usually administered for 8-10 days provided
that the patient respects the oral hygiene regimen;
• rinses containing chlorhexidine, e.g. Eludril®, Corsodyl®, Curasept®, KIN®;
• gels: Elugel®, Perio-KIN®, to provide proper oral hygiene.
We can also distinguish preparations according their
origin:
• animal like: Solkoseryl Dental Adhesive Paste® containing a composition of protein-free dialysate calf
blood or Collgel® derived from the skin of silver
carp;
• plant like: Baikadent®, Protefix®.
Protefix® is an easy-to-apply preparation due to its
gel form. It contains in its composition clove oil, which
has an antiseptic effect, which is of colossal importance
in elderly people susceptibility to bacterial, fungal, or
viral infections; it acts also as an analgesic. Due to its
glyceroloxidtriester content, the preparation has an extended period of penetration of active substances into
the damaged tissue [12, 14].
The aim of our pilot study was to evaluate the keratinised gingival fibroblast proliferation after the use
of Protefix® gel in vitro.
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MATERIAL AND METHODS
CELL CULTURE
In this in vitro pilot study, the human fibroblasts obtained from healthy donors and isolated from keratinised
gingival tissues were used according to the procedures
described in Patent No. P 3812045 (Saczko, 2008) [16].
The cells were grown in Dulbecco Modified Medium
(DMEM, Sigma-Aldrich) containing 10% foetal bovine
serum (FBS) and enriched in a 5% antibiotic/antifungal
solution (Sigma). Serum is crucial for cell cultivation because it contains valuable growth factors, mineral salts,
lipids, hormones, and adhesion factors. It stimulates cell
growth, has a positive effect on the buffering properties
of the medium, and protects the cells against mechanical
damage. The culture medium was changed twice a week.
Cell culture and all procedures were performed under
sterile conditions. The cells were maintained in a humidified atmosphere at 37°C and 5% CO2. For further
experiments, fibroblast cells were subjected to trypsinisation (0.25% trypsin-EDTA, Sigma-Aldrich) and seeded into 96-well plates for further testing.
MITOCHONDRIAL ACTIVITY OF FIBROBLASTS IN
THE MTT TEST
The primary cell line of human fibroblasts was resuspended into transparent 96-well plates (Nunc). After
24 hours, Protefix® gel was added at the following dilutions: 5%, 50%, and 100%. Then the cells were incubated
for 20 minutes or 24 hours. After incubation, an MTT assay (Sigma-Aldrich) was performed to evaluate the cell
viability. The method is based on the detection of mitochondrial enzyme activity - succinate dehydrogenase.
It converts the orange water-soluble tetrazolium salt
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) to formazan. This product has the form of
dark purple, insoluble crystals. Due to their dissolution
with isopropanol (they are not soluble in water), a coloured solution is formed. The intensity of the coloured
reaction is verified spectrophotometrically at 570 nm.
The amount of the resulting coloured product indicates
the oxidative activity of the mitochondria in the cells,
and thus the number of viable cells in the entire population (the more reduced the MTT, the greater the number
of metabolically active cells.) The measurements were
performed on a MultiskanTM FC microplate photometer
(Thermo Scientific).
RESULTS
In this in vitro pilot study it was shown that Protefix®
gel significantly stimulated the proliferation of human
fibroblasts derived from primary culture. After exposiJournal of Stomatology * http://www.jstoma.com
Evaluation of fibroblast proliferation after Protefix® gel application
200
180
Viability [% of control cells]
tion to the Protefix® gel at a concentration of 5%, fibroblasts revealed a significant increase in the colour reaction of formazan crystals, in particularly after 24 hours
of incubation. The cell viability increased by as much
as 50% in comparison to untreated control cells. With
the increasing concentration of the preparation, i.e.:
50% and 100%, a proportional decrease in the proliferation of the examined cells was observed. The results
of cell viability are shown in Figure 1.
160
140
120
100
80
60
40
DISCUSSION
20
0
The aging process, systemic diseases, or medicines
that are taken cause the mucous membrane of elderly patients to become dry and smooth. Cytologically, an increased number of eosinophils is observed.
The processes of apoptosis dominate. In the connective
tissue the number of cells decreases, including fibroblasts. This leads to destruction of the capillaries. Vascular walls are damaged, which causes the weakening
of ion transport, and this determines the reduction
of alkaline and acid phosphatase [5, 8]. Atrophic mucous in elderly patients becomes more susceptible to all
kinds of injuries: chemical, biological, and mechanical,
including iatrogenic.
Mediators of inflammatory and immunological reactions are responsible for the wound healing process,
including regulating molecules, i.e. cytokines, which
are produced by cells of the immune system but also
by fibroblasts. Fibroblasts release growth factors that
affect various biochemical processes such as mitogenesis, chemotaxis, angiogenesis, and morphogenesis,
which are involved in the tissue regeneration process.
At the wound site, fibroblasts that are unable to perform
their functions are also damaged. In the healthy and efficient body, fibroblasts migrate from neighbouring areas.
However, systemic diseases, bacterial, viral, or fungal infections limit cell migration. Another factor necessary
for proper wound healing is angiogenesis from the vascular bed, allowing nutrition and constant delivery
of systemic growth and cellular factors [3, 9, 13].
It is therefore important to implement a post-treatment preparation, especially for geriatric patients, which
will: be easy to use, be biocompatible, show adhesion to
tissues, and be anallergic. In addition to the soothing effect, it will also help to regenerate damaged tissue, thus
minimising the risk of parathypical processes. Protefix®
from the preparations available on the market, thanks
to the glyceroloxydtriester in the composition, creates
a lipid film on the gingivae, without causing excessive
pressure of the mucosa, thus protecting the affected oral
mucosa against abrasion. At the same time, it does not
cause the anaemisation of the damaged site and creates
space for cell proliferation. In addition, the included
clove oil has antifungal and antiviral activity. After application, it soothes irritations resulting from the use
J Stoma 2019, 72, 1
100
50
5
Dilution [%]
20 min
24 h
FIGURE 1. The effect of 5%, 50%, and 100% Protefix
gel on human gingival fibroblasts after 20 minutes and
24 hours of the exposure. The data represent the mean
values of the minimum repetition from three independent repetitions
of newly made prosthetic restorations and relieves
pain [12, 14]. It shows antibacterial activity, especially
for Escherichia coli, Mycobacterium phlei, Bacillus subtilis, Streptococcus aureus, which certainly prevents infection of the wound, especially in people with dry oral mucosa, thus supporting the migration of cells from areas
adjacent to the wound [15, 17]. Clove oil also has a local
effect to improve blood circulation through antioxidant
activity and improves cell nutrition.
Our pilot study confirms the stimulation of fibroblast
proliferation by Protefix® gel, and therefore the appropriateness of using the product immediately after damage
to the mucosa. Proliferation of fibroblasts stimulated
with the preparation supports the release of fibroblast
growth factor (FGF), which induces collagen synthesis.
This connection was confirmed in the histological studies by Żurek et al. [20]. Also, the correlation between
the amount of FGF and the proliferation of periodontal regeneration cells was confirmed in the research by
Yu et al. [19]. The extracellular matrix (EMC) modulated in this way may affect periodontal regeneration.
However, Dominiak et al. showed that the numerous
growth factors that last up to four days affect the reduction of pain and swelling after surgical procedures [4].
CONCLUSIONS
Summarising, the conducted pilot study showed
a significant stimulating effect on human fibroblast proliferation after exposure to Protefix® gel at a concentration of 5%. The obtained results indicate that the tested
product may considerably enhance the healing of surgical wounds, which is especially important in elderly patients in whom the healing process is more problematic.
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Barbara Sterczała, Julita Kulbacka, Jolanta Saczko, Marzena Dominiak
ACKNOWLEDGMENTS
The research was partially supported by the Polish
Society of Dentistry and Statutory Funds of the Department of Department of Dental Surgery, Wrocław Medical University, Poland.
18. Villa A, Connell CL, Abati S. Diagnosis and management of xerostomia and hyposalivation. Ther Clin Risk Manag 2014; 11: 45-51.
19. Yu HCh, Huang FM, Lee SS, Yu ChCh, Chang YCh, et al. Effects
of fibroblast growth factor-2 on cell proliferation of cementoblasts. J Dent Sci 2016; 11: 463-467.
20. Żurek J, Dominiak M, Botzenhart U, Bednarz W. The use of biostatic fascia lata high allograft as a scaffold for autologous culture
of fibroblast – an in vitro study. Ann Anatomy 2014; 199: 104-108.
CONFLICT OF INTEREST
The authors declare no potential conflict of interests
with respect to the authorship and/or publication of this
article.
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Journal of Stomatology * http://www.jstoma.com