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98]

Original Article

Comparative evaluation of platelet

count and antimicrobial efficacy of
injectable platelet-rich fibrin with
other platelet concentrates: An in vitro
study
Prerna Ashok Karde, Kunal Sunder Sethi, Swapna Arunkumar Mahale,
Smita Uday Khedkar,1 Agraja Ganpat Patil, Chaitanya Pradeep Joshi

Departments of Abstract:
Periodontics and Background: Platelet concentrates are used in various medical procedures to promote soft- and hard-tissue
1
Microbiology, MGV’S regeneration. In recent times, their antimicrobial efficacy is also explored. However, various platelet concentrates
KBH Dental College have evolved which differ in the centrifugation protocols. One such recently introduced platelet concentrate is
and Hospital, Nashik, injectable platelet-rich fibrin (i-PRF) concentrate. Hence, the aim was to evaluate the antimicrobial property,
Maharashtra, India and platelet count of i-PRF in comparison to other platelet concentrates, i.e., PRF, platelet-rich plasma (PRP),
and control (whole blood). Materials and Methods: Blood samples were obtained from 10 chronic generalized
marginal gingivitis patients. Platelet concentrates were prepared using standardized centrifugation protocol.
Platelet count was evaluated by manual counting method using smear preparation of each sample. Subsequently,
antimicrobial activity against oral bacteria was examined on blood agar using disc diffusion method to quantify
the inhibitory effects. Results: Statistical significance was analyzed by one-way analysis of variance (ANOVA).
P <0.05 was considered statistically significant. Mean zone of inhibition around i-PRF (P < 0.01) and PRF (P < 0.05)
showed statistical significance. Although a distinct zone of inhibition was seen with PRP, it was not statistically
significant (P > 0.05). i-PRF showed statistically significant difference (P < 0.001) in platelet count when compared
Access this article online
to control. It was also significant when compared to PRP (P < 0.01), PRF (P < 0.001). Conclusion: i-PRF has
Website: maximum antimicrobial efficacy and higher platelet count in comparison to other platelet concentrates, thereby
www.jisponline.com indicating to have a better regenerative potential then others.
DOI: Key words:
10.4103/jisp.jisp_201_17
Antimicrobial, periodontitis, platelet counts, platelet-rich plasma
Quick Response Code:

INTRODUCTION In the past few years, various platelet concentrates

have evolved depending on the technique

P eriodontitis is an inflammatory and a

polymicrobial disease affecting, supporting
structures around the teeth. It results in
progressive attachment loss and bone loss.
Hence, the periodontal therapy is aimed at
regenerating the lost periodontal structures.
In recent times, platelet concentrates have
gained popularity in periodontal regenerative
therapy owing to its autologous nature. The
Address for rationale behind its regenerative potential
correspondence:
is the presence of various growth factors
Dr. Prerna Ashok Karde,
MGV’S KBH in the α–granules of platelets which are
Dental College and released at the local site on their activation. It
Hospital, Panchavati, thus promotes wound healing after surgical
Nashik - 422 003, periodontal therapy. Besides this, they also
Maharashtra, India. impart anti-inflammatory properties, thereby
E-mail: prerna.karde@
reducing postoperative pain and swelling. In
gmail.com
addition, few studies have also explored its
Submission: 27-08-2017 antibacterial potential although its mechanism
Accepted: 01-09-2017 is controversial.[1-3]
© 2017 Indian Society of Periodontology | Published by Wolters Kluwer - Medknow
employed which vary in their centrifugation
protocols. One such recently introduced
platelet concentrate by Joseph Choukron in
2014 is injectable platelet-rich fibrin (PRF) more
commonly referred as i-PRF™. It requires neither
any anticoagulant nor any additive. It is obtained
This is an open access article distributed under the terms of the
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License, which allows others to remix, tweak, and build upon
the work non-commercially, as long as the author is credited
and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com
How to cite this article: Karde PA, Sethi KS,
Mahale SA, Khedkar SU, Patil AG, Joshi CP.
Comparative evaluation of platelet count and
antimicrobial efficacy of injectable platelet-rich fibrin
with other platelet concentrates: An in vitro study.
J Indian Soc Periodontol 2017;21:97-101.
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Karde, et al.: i-PRF: A novel platelet concentrate
by centrifuging blood at low-speed 700 rpm 60 (G). This results
in PRF for use in liquid (injectable) form. It coagulates within few
minutes after the injection. It is believed to contain not only white
cells and platelets but also circulating stem cells and endothelial
cells. Hence, it is considered as a “blood concentrate” and not
just a platelet concentrate.[4,5]
At present, i-PRF is still in its infancy and needs to be explored
with regard to its properties. Hence, the present study was
designed to evaluate the platelet count, and antibacterial
efficacy of i-PRF in comparison with the other available platelet
concentrates, i.e., Platelet rich fibrin (PRF), Platelet rich plasma
(PRP), and control (whole blood).
MATERIALS AND METHODS
Participant selection
Blood samples were obtained from ten chronic generalized
marginal gingivitis patients who reported to the Department of
Periodontology at our college and volunteered to participate in
the study (mean age 32 ± 5.6 years). All were nonsmokers, with
no history of infection or any antibiotic use for at least 3 months
before the study. None of them gave history of any anticoagulant
or immunosuppressive therapy that might interfere with natural
coagulation process. None of them reported of any bleeding
or clotting disorders. All the patients gave written informed
consent before beginning the study. This research was approved
by the institutional ethical committee and strictly adhered to
the guidelines of the Declaration of Helsinki.
Sample preparation
A volume of 8 ml of blood was collected from each patient: 2 ml
was used for preparation of i-PRF, PRF, and PRP, remaining
2 ml with anticoagulant served as a control [Figure 1]. During
centrifugation, a tube containing 2 ml saline served as a counter
balance.
Injectable platelet-rich fibrin preparation
A volume of 2 ml of blood collected in noncoated vacutainer
without any additives and centrifuged at 700 rpm for 3–4 min.[5]
Platelet-rich fibrin preparation
A volume of 2 ml of blood collected in silicon-coated vacutainer
without any additives and centrifuged at 3000 rpm for 10 min.[6]
Platelet-rich plasma preparation
A volume of 2 ml of blood collected in 3.2% sodium citrate
anticoagulant-coated vacutainer. Initially, soft spin at 1000 rpm
for 10 min, followed by centrifuging top layer of plasma at
hard spin at 2000 rpm for 20 min. Finally, half of the superficial
plasma layer removed, and the platelet pellet suspended in the
remaining half of the plasma volume.[6]
Control
A volume of 2 ml blood collected in ethylenediaminetetraacetic
acid anticoagulant containing vacutainer which was not
subjected to any centrifugation.
Tests performed
Quantification of platelets
0.1 ml of i-PRF, PRP, and control were placed separately on glass
slide. Using the corner of a clean slide, held at 45°, the drop of the
98 Journal of Indian Society of Periodontology - Volume 21, Issue 2, March-April 2017
sample was spread on that slide. As PRF was obtained in clot form,
its platelet count was assessed indirectly by subtracting the platelet
count in the residual serum (remaining after PRF preparation) from
the platelet count of whole blood. All the slides were subjected
to Leishman staining [Figure 2]. Platelets were calculated by
traditional estimation method. In this method, 10 fields of the
smear were evaluated under oil immersion. The average was
then multiplied by 15,000. This gave the platelet count/cu mm.[7-9]
Evaluation of antimicrobial potential
Plaque sample collection
Supragingival plaque samples of subjects who volunteered
for blood sample collection were obtained using gracey
curette (Gracey curettte, Hu-Friedy Mfg. Co., LLC, Chicago,
USA). The sample was immediately transferred in tube
containing 5 ml saline and vortexed for 5 min to obtain uniform
suspension. Later, 0.1 ml of this suspension was then used to
plate blood agar (HiMedia Laboratories Pvt. Ltd., Mumbai,
India) using streak method.
Agar well-diffusion method
Wells were prepared in the inoculated agar plate. 0.1 ml of
i-PRF, PRP, and control were placed in those wells. A punched
piece of PRF membrane was placed on inoculated agar plate.
Inoculated blood agar plates were then incubated in an
incubator aerobically at 37°C for 48 h [Figure 3]. The same
procedure was repeated for all the samples.
RESULTS
Statistical analysis
For antimicrobial potential, the comparison between zones of
inhibition obtained around all the samples was analyzed by
one-way ANOVA. Comparative evaluation of the number of
platelets was also done using ANOVA. P <0.05 was considered
statistically significant. For statistical analysis, “SPSS-9”
software (IBM India Pvt Ltd, Bangalore, India) was used.
Quantification of platelets
Platelet count/mm3 obtained for i-PRF, PRP, PRF, and control
were 1,434,000 ± 75,233, 1,343,000 ± 81,486, 249,000 ± 61,319, and
291,000 ± 51,575, respectively. The platelet count of i-PRF was
statistically significant when compared to control (P < 0.001).
It was also significant when compared to PRP (P < 0.01) and
PRF (P < 0.001). This indicates that i-PRF has maximum number
of platelets in comparison to other concentrates.
Evaluation of antimicrobial potential
The antimicrobial efficacy was demonstrated by appearance
of a clear zone of inhibition around the samples. Mean zone of
inhibition (in cm) around i-PRF, PRF, and PRP were 1.42 ± 0.25,
1.3 ± 0.16, and 1.02 ± 0.12, respectively. Mean zone of inhibition
around i-PRF (P < 0.01) and PRF (P < 0.05) reached statistical
significance. Although a distinct zone of inhibition was seen with
PRP, it was not statistically significant (P > 0.05). These results
indicate that i-PRF has a significant inhibitory effect on growth
of oral bacteria in comparison to other platelet concentrates.
DISCUSSION
Periodontal regeneration involves various biologic events such
as cell adhesion, migration, proliferation, and differentiation
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Karde, et al.: i-PRF: A novel platelet concentrate
a b c d
Figure 1: Platelet concentrate samples obtained after centrifugation protocol.
(a) Injectable platelet-rich fibrin; (b) platelet-rich fibrin; (c) platelet-rich plasma; (d) control
a b
c d
Figure 2: Smear preparation of platelet concentrates observed under ×100 oil
immersion. Arrows indicate platelets. (a) Control; (b) injectable platelet-rich fibrin;
(c) platelet-rich plasma; (d) platelet-rich fibrin
Figure 3: Zone of inhibition using agar well diffusion method for injectable
platelet-rich fibrin, Platelet-rich plasma, platelet-rich fibrin, control
in an orchestrated sequence. Regenerative procedures with
the use of soft- and hard-tissue grafts are performed to attain
Journal of Indian Society of Periodontology - Volume 21, Issue 2, March-April 2017
periodontal restoration. Platelets play a vital role in wound
healing. They release growth factors such as platelet-derived
growth factor (PDGF), transforming growth factor (TGF-β),
and insulin-like growth factor once they are activated. Platelets
also secrete fibrin, fibronectin, and vitronectin, which act as a
matrix for the connective tissue and as adhesion molecules
for more efficient cell migration. Thus, they play a crucial
role in cell proliferation, collagen synthesis, and osteoid
formation.[6,10,13] This led to the development of numerous
techniques of autologous platelet concentrates over the years
such as PRP and PRF.
PRP although being an autologous preparation requires the
addition of thrombin and calcium for its activation. These
additives can result in the development of antibodies to the
clotting factors V, XI, and thrombin, thereby adversely affecting
the coagulation process and also can trigger an immune
reaction. PRF is the second generation platelet concentrate
introduced by Choukron (2001).It is easy to prepare and has
good handling characteristics. It does not involve any use of
bovine thrombin or anticoagulant which considerably reduces
the biochemical handling of blood as well as risks associated
with the use of any additives. PRF itself contains physiologically
available thrombin that is responsible for slow polymerization
of fibrinogen into fibrin resulting in a physiologic architecture
favorable to wound healing. This fibrin network protects the
growth factors from proteolysis. Besides, PRF also favors
the development of microvascularization leading to a more
efficient cell migration.[11]
I-PRF was introduced based on the similar concept as that
of PRF with added advantage of it being in injectable form.
This injectable form of PRF can be utilized alone or combined
easily with various biomaterials. Its protocol is based on
the concept that slower and shorter centrifugation spin
results in a higher presence of regenerative cells with higher
concentrations of growth factors.[12] In a recently conducted
study, it was observed that PRP slowly dissolved over a
period while i-PRF formed a small clot as a result of fibrin
components that acted as a dynamic gel with cells likely
contained within its hydrogel. It was therefore hypothesized
that an additional release of growth factors from i-PRF can
be excepted beyond 10 days although PRP was found to be
dissolved by that time.[5]
Evaluation of platelet counts can be done by various automated
devices. However, in a study done by Bajpai et al. to estimate the
platelet count by peripheral smear method and by automated
cell counter, it was observed that there was no significant
difference in the two methods. The authors concluded that the
method of platelet estimation by peripheral smear is useful as
a rapid, cheap method to assess platelet count.[8] In the present
study, all the samples except PRF were available in liquid form
which could be easily smeared on glass slide and stained.
As PRF was available in a clot form; its platelet count was
indirectly calculated by evaluating the platelets available in
residual serum. Similar evaluation was performed by Suchetha
et al. for estimation of platelet count in PRF clot.[14]
In the present study, it was observed that i-PRF had highest
number of platelet count and it was statistically significant.
This could be attributed to the low centrifugation speed
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Karde, et al.: i-PRF: A novel platelet concentrate
and time resulting in their higher number. Ghanaati et al.
introduced the “low-speed concept” for blood centrifugation
whereby lower centrifugation speeds were shown to contain
higher numbers of cells including leukocytes before the
formation of a fibrin clot.[12]
In the present study, it was observed that i-PRF and PRP
showed 503% and 464% rise in number of platelets. PRF clot
showed the concentration of about 87% of platelets when
compared to whole blood. Preparations with higher platelet
counts are known to release more growth factors. Miron
et al. demonstrated that in general PRP had higher early
release of growth factors whereas i-PRF showed significantly
higher levels of total long-term release of these factors. It was
concluded that i-PRF can release higher concentrations of
various growth factors and induce higher fibroblast migration
and expression of PDGF, TGF-β, and collagen1.[5]
Studies evaluating the antimicrobial efficacy of platelet
concentrates such as PRP and PRF have been previously carried
out. i-PRF being recently introduced is not much explored. In
the present study, it was observed that i-PRF showed maximum
zone of inhibition around oral microflora, i.e., 1.42 ± 0.25 (in cm).
The order of zone of inhibition from highest to lowest was
i-PRF > PRF > PRP. In one study, antimicrobial efficacy of
PRP and PRF was performed against Porphyromonas gingivalis
and Aggregatibacter actinomycetemcomitans. It was seen that P.
gingivalis and A. actinomycetemcomitans were inhibited by PRP
but not by PRF.[15] In another study, PRF demonstrated clear
zone of inhibition against subgingival plaque sample and
minimum amount of turbidity using PRF was confirmed by
colorimetric analysis.[16] In the present study, although a distinct
zone of inhibition was obtained with all test samples, it was
significant with only i-PRF and PRF. Various mechanisms have
been hypothesized regarding the mechanism of antibacterial
effect of platelet-derived preparations such as generation
of oxygen metabolites, including superoxide, hydrogen
peroxide, and hydroxyl free radicals; binding, aggregation,
and internalization of microorganisms, thereby enhancing
the clearance of pathogens from the bloodstream; release an
array of potent antimicrobial peptides.[16] Yeaman proposed
that direct interaction of platelets with microorganisms,
participation in antibody-dependent cell cytotoxicity and
engulfment by entrapped white blood cells within PRF
could result in direct bacterial killing. Besides this release of
myeloperoxidase, activation of the antioxidant responsive
elements and antigen-specific immune response have also
been suggested.[17]
Limitations
Comparative evaluation of release of growth factors could not
be performed due to lack of facility. In addition, identification
of bacteria inhibited by all the test samples was not performed.
Evaluation of antibacterial activity in dynamic environment
is required.
CONCLUSION
The use of platelet concentrates in periodontal regenerative
therapy is increasing nowadays. With i-PRF, its application
can be more feasible and minimally invasive without using
any additives for its preparation.
100 Journal of Indian Society of Periodontology - Volume 21, Issue 2, March-April 2017
Within the limitations of the study, i-PRF had the highest
number of platelets. Its antimicrobial potency was also highest
when compared to other platelet concentrates. Since there is
limited literature related to this novel platelet concentrate
further research is required to explore its properties. More
in vitro and animal studies are required to establish its healing
and regenerative efficiency.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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