Key words:
Leukocyte platelet rich fibrin - orthodontic tooth movement - plasma - platelet concentrate
- platelet-rich fibrin - platelet-rich plasma
INTRODUCTION
To date, several methods have been applied to enhance the postoperative healing process
and decrease inflammation.[1]
[2]
[3] Considering the local release of growth factors, platelets can be used as an adjunct
to stimulate the regenerative capacity of periosteum and enhance bone healing, especially
in postextraction cases.[4]
[5]
[6]
[7] Blood vessels provide the necessary growth factors and inhibitors to initiate the
osteogenic biomineralization cascade. Inevitable injury to blood vessels during oral
surgical procedures causes blood extravasation and subsequent platelet aggregation
and fibrin clot formation. Platelet activation results in formation of blood clots
and platelet plugs and subsequent secretion of bioactive proteins, necessary for tissue
regeneration and repair.[2]
[6]
[8]
Considering the bioactive properties of platelets in the healing process, they can
be used as a valuable therapeutic adjunct in medicine and dentistry.[9]
[10] Platelet rich plasma (PRP) and leukocyte platelet rich fibrin (LPRF) are the two
main autologous products derived from platelets.[11]
[12]
[13]
[14]
[15] Many studies have investigated the potential biological differences between these
two platelet concentrates.[15]
[16] Although they are both clinically effective in accelerating the healing process,
platelet rich fibrin (PRF) is low cost and easy to use and may be added to other bone
substitutes.[17] Compared to PRP, LPRF and PRF have demonstrated more sustained release of growth
factors from the fine and flexible fibrin matrix in their structure.[15]
[18] Furthermore, the presence of high concentrations of leukocytes in LPRF compared
to PRF plays a significant
role in enhanced release of some crucial growth factors such as transforming growth
factor beta 1.[19]
Resorptive remodeling of the alveolar ridge commonly occurs following tooth extraction.
This process may be beneficial in fixed orthodontic treatment of patients with severe
crowding.[20]
[22] Literature is rich with studies on the application of various bioactive grafts to
increase the bone maturation rate and enhance the rate of orthodontic tooth movement
(OTM).[23] Adequate volume of alveolar bone is a prerequisite for successful OTM during space
closure. However, application of different graft materials may enhance the bone filling
process and consequently shorten the “regional acceleratory phenomenon” cascade, which
includes the release of various growth factors after tooth extraction. LPRF contains
concentrated growth factors such as platelet derived growth factor, transforming growth
factor beta, and insulin like growth factor 1, which enriches the blood clot formed
in the extraction socket and subsequently enhances wound healing and bone regeneration
with no inhibitory effect on the natural healing process.[19]
[20]
[23]
Regarding the acceleration of tooth movement, limited publications are available on
the efficacy of submucosal injection of PRP to accelerate OTM and preserve the bone.[24]
[26] Considering the simultaneous positive effect of PRP/LPRF on bone healing, socket
preservation, and acceleration of tooth movement, the current study tested the application
of LPRF in tooth extraction sockets to evaluate its efficacy for acceleration of space
closure phase of orthodontic treatment. This study sought to evaluate whether LPRF
application can accelerate OTM. To the best of our knowledge, this is the first study
on the efficacy of LPRF for acceleration of OTM.
MATERIALS AND METHODS
Study design
This split mouth clinical trial evaluated the efficacy of application of LPRF in extraction
sockets for acceleration of OTM in fixed orthodontic patients. A split mouth design
was used to limit the effect of interpersonal variations on response to LPRF. The
study was conducted in the Department of Orthodontics and Dentofacial Orthopedics
of Shahid Beheshti University of Medical Sciences between March 2015 and March 2016.
This clinical trial was registered in www.irct.ir (IRCT2015100724405N1) and followed
the CONSORT statement as a guide for study design. Ethical approval was also obtained
from the Ethics Committee of Dental Research Center at Shahid Beheshti University
of Medical Sciences (protocol approval number: 9310).
Sample size calculation
Sample size was calculated to be thirty extraction sockets (n = 15 in each group) assuming the mean difference and standard deviation (SD) of 0.95,
type 1 error (α) of 0.05 and type 2 error (β) of 0.1 to achieve a statistical power
of 90% using N = (Z1 − α/2 + Z1 − β)2 × σ2/(△μ)2 with assumptions of α (significance level) = 0.05 ≥ Z1 − α/2 = 1.96, β = 0.1 (statistical
power = 0.9) ≥Z1 − β = 1.28, σ = 0.95, △μ = 0.95.
Participants and randomization
Participants were selected from patients referred to the Department of Orthodontics
and Dentofacial Orthopedics at Shahid Beheshti University of Medical Sciences. Male
and female orthodontic patients 12 years and older with extraction treatment plan
who met the following inclusion criteria were included presence of all maxillary and
mandibular permanent teeth except for third molars, comprehensive orthodontic treatment
plan of bilateral symmetric extraction of premolar teeth, no medication intake or
systemic disease, and full banding/bonding of teeth in both arches. The exclusion
criteria were pregnancy, history of previous orthodontic treatment, nonextraction
treatment plan, syndromic patients, systemic diseases, or medication intake such as
nonsteroidal anti inflammatory drugs, which would interfere with OTM and history of
previous orthodontic treatment. Each patient signed an informed consent form after
receiving a thorough explanation regarding the study. In each jaw, the LPRF application
quadrant was chosen by drawing lot, done by the corresponding author. In one quadrant
of each jaw, the extraction socket was preserved by immediate placement of LPRF as
the experimental group, while the other side served as the control group for secondary
healing.
Preparation of leukocyte platelet rich fibrin
The protocol for LPRF preparation was simple and included the collection of whole
venous blood from the brachial vein using a 10 mL syringe. The collected blood was
transferred into two sterile vacutainer tubes (9 mL) without anticoagulant and were
placed symmetrically into the centrifuge device. The intraspin tubes were immediately
centrifuged (PCO2 Process) at 2700 rpm for 12 min, after which, three layers were formed: red blood
cells at the bottom, upper straw colored cellular plasma, and the middle fraction
containing the fibrin clot and platelets. The upper straw colored layer was discarded,
and the middle fraction was collected, 2 mm below the lower dividing line, which was
LPRF. Fibrinogen which is initially concentrated in the upper part of the tube is
combined with the circulating thrombin following centrifugation to form fibrin. A
fibrin clot is then formed in the middle of the tube, right between the red corpuscles
at the bottom and acellular plasma at the top. Platelets are trapped massively in
the fibrin mesh [Figure 1a and b].
Figure 1: (a) Collection of venous blood from the brachial vein using a 10 mL syringe. (b)
Platelets are trapped massively in the fibrin mesh
The central part includes platelets trapped massively in the fibrin mesh. The success
of this technique entirely depends on the time interval between blood collection and
its centrifugation, which should be minimized. The blood sample without anticoagulant
starts to coagulate almost immediately upon contact with the glass and decreases the
required centrifugation time to concentrate fibrinogen. Adherence to the correct preparation
protocol and quick handling are critical to obtain clinically usable LPRF clot charged
with serum and platelets. Resistant autologous fibrin membranes may be available by
driving out the fluids trapped in the fibrin matrix.
Orthodontic tooth movement
Within the scope of orthodontic treatment with fixed appliances (ROTH American Orthodontics
Master/Mini Series 0.022 inch slot), patients requiring extraction of their first
premolars according to their orthodontic treatment plan after the leveling phase of
treatment underwent augmentation of extraction socket with LPRF unilaterally immediately
after extraction. Extraction of first premolars was performed a traumatically on both
sides. On one side of each jaw, the extraction socket was preserved by immediate placement
of LPRF in the extraction socket as the experimental group and the other sides served
as the control for secondary healing. The LPRF plugs were placed gently into the socket,
and the sockets were sutured using 4 0 Vicryl sutures (Ethicon). The teeth adjacent
to the defect were then pulled together by a NiTi closed coil spring (Ormco®, Orange,
California, USA) with constant level of force. A piece of 0.016 × 0.022 inch stainless
steel wire was used as the main archwire. The sites were examined weekly for any appliance
dislodgment. The treatment was considered finished when the amount of OTM was considered
clinically enough based on the clinical appraisal of individual pretreatment crowding
and proposed treatment plan.
The amount of OTM was measured by comparing the change in horizontal linear distance
between the mid marginal ridges of the adjacent teeth on a regular basis every 2 weeks
for 4 months (eight time points: before placement of LPRF [T0] and 2 weeks [T1], 4
weeks [T2], 6 weeks [T3], 8 weeks [T4], 10 weeks [T5], 12 weeks [T6], 14 weeks [T7],
and 16 weeks [T8]) after the study commencement until adequate canine retraction was
achieved which is different in each socket [Figure 2a and b]. If this distance decreases more in experimental side, it means that the teeth moved
faster than control side.
Figure 2: (a) The extraction socket was preserved by immediate placement of LPRF in the extraction
socket as the experimental group and the other side served as the control group for
secondary healing. (b) The exact amount of tooth movement were measured on the study
casts using a digital caliper
Statistical analysis
Statistical analysis was performed using SPSS 18 (SPSS Inc., PASW statistics for windows,Version
18.1. ChicGO). In this split mouth clinical trial, descriptive statistics, including
the mean and SD of OTM in each group, were measured at eight different time points.
The independent sample t test was used to evaluate the equality of means in both experimental and control
group. The random effect model was used for the comparison between the experimental
and control groups, and variables, which could be considered as personal differences,
were entered into the model using a split mouth design.
RESULTS
Thirty extraction sockets of eight patients (five males, three females, mean age of
17.37 years and SD of 12.48 years, and range 12–25 years) were assessed in this study.
All patients completed the follow up period. In seven patients, both maxillary and
mandibular arches and in one patient only the mandibular arch were assessed. [Table 1] shows the mean and SD of tooth movement in the control and experimental groups.
Table 1
Comparison of the distance between marginal ridges of the teeth adjacent to sockets
in millimeters between the experimental and control groups at each time point
|
Time
|
Point
|
Experimental
|
Control
|
95%
|
CI
|
n
|
Mean
|
SD
|
95%
|
CI
|
|
1
|
15
|
6.65
|
0.834
|
6.19
|
7.11
|
15
|
6.762
|
0.763
|
6.33
|
7.18
|
|
2
|
15
|
5.290
|
1.783
|
4.30
|
6.27
|
15
|
5.412
|
1.738
|
4.44
|
6.37
|
|
3
|
15
|
4.636
|
1.824
|
3.62
|
5.64
|
15
|
4.865
|
1.764
|
3.88
|
5.84
|
|
4
|
15
|
3.510
|
2.023
|
2.38
|
4.63
|
15
|
3.998
|
2.138
|
2.81
|
5.18
|
|
5
|
14
|
3.145
|
1.617
|
2.21
|
4.07
|
14
|
3.777
|
1.923
|
2.66
|
4.88
|
|
6
|
13
|
2.822
|
1.337
|
2.01
|
3.63
|
13
|
3.423
|
1.742
|
2.37
|
4.47
|
|
7
|
9
|
2.875
|
0.499
|
2.49
|
3.25
|
9
|
3.612
|
1.375
|
2.55
|
4.66
|
|
8
|
4
|
1.075
|
0.153
|
0.83
|
1.31
|
4
|
1.255
|
0.100
|
1.09
|
1.41
|
|
Total
|
100
|
4.123
|
2.081
|
−0.86
|
0.80
|
100
|
4.505
|
2.070
|
−0.71
|
0.94
|
In all eight time points, the mean linear measurements between mid marginal ridges
of teeth adjacent to extraction sites were less in experimental groups compared to
control. This distance decreased more in experimental side which means that the teeth
moved faster than control side.
According to the random effect model, the experimental group (LPRF placed in the extraction
socket) showed higher rate of OTM (P = 0.006). [Figure 3] shows the pattern of tooth movement in the two groups. The graph shows the higher
rate of OTM in the experimental group (P = 0.006). According to the random effect model, the teeth in the maxillary compared
to mandibular arches in both the experimental and control group did not show any significant
difference in the rate of OTM [P = 0.9 and P = 0.77, respectively, [Table 2].
Figure 3: Amount the distance between marginal ridges of the teeth adjacent to sockets in millimeters
at different time points. Data represent the mean values in the experimental and control
groups
Table 2:
Comparison of total tooth movement in the upper and lower arches between the experimental
and control groups
|
Group
|
Arch
|
Mean
|
SD
|
P
|
|
Experimental
|
Upper (7)
|
6.76
|
0.538
|
0.94
|
|
Lower (8)
|
6.56
|
1.058
|
|
Total (15)
|
6.65
|
0.834
|
|
Control
|
Upper (7)
|
6.96
|
0.695
|
0.77
|
|
Lower (8)
|
6.58
|
0.819
|
|
Total (15)
|
6.76
|
0.763
|
|
Total
|
Upper (14)
|
6.86
|
0.606
|
0.626
|
|
Lower (16)
|
6.57
|
0.914
|
[Table 2] shows the mean and SD of tooth movement (millimeters) in both the control and experimental
groups in the maxillary and mandibular arches.
DISCUSSION
The results of this study demonstrated that the distance between mid marginal ridge
points of crowns adjacent to extraction sites was less in experimental groups; therefore,
it showed the possible positive efficacy of LPRF application in the extraction socket
for acceleration of OTM including anchorage loss of posterior teeth. It means that
anterior and posterior teeth adjacent to extraction sites moved faster toward each
other in experimental groups. To the best of our knowledge, this is the first pilot
study aiming to evaluate the efficacy of LPRF for acceleration of OTM in humans. To
date, many surgically assisted approaches such as periodontally accelerated osteogenic
orthodontics, corticotomy, and micro osteoperforation with and without using PRP/LPRF
have been developed to decrease the treatment time of fixed orthodontic patients.
These approaches are all based on the regional acceleratory phenomenon.[27]
[28]
[29]
[30]
[31] Researchers tried to shift the invasive surgically assisted techniques to more conservative
approaches including micro osteoperforation,[31] piezopuncture,[32] and very recently mini implant facilitated micro osteoperforation,[33] which eliminate the need for flaps, bone grafting, and suturing. However, these
techniques may still traumatize the surrounding bone, undermine the periodontal support
of teeth, and cause patient discomfort.[33] In addition, the intensity and extensity of their acceleratory effect depend on
the intensity and extensity of the surgical insult, and therefore, conservative approaches
might not be able to trigger a distinctive acceleratory effect on OTM.[34] To simulate the effects of surgical insult without actual surgical trauma, local
injection of growth factors and cytokines, and recently, autologous
PRP were introduced to stimulate alveolar bone remodeling.[25]
[26]
[35] PRP was first introduced to dentistry in 1998 to be combined with autogenous bone
grafts to expedite bone maturation and result in higher bone density.[36] Hoaglin and Lines also conducted a study to evaluate the use of PRF for prevention
of localized osteitis following lower third molar extraction.[6] This retrospective review demonstrated that preventative treatment of localized
osteitis could be accomplished using a low cost, autogenous, soluble, biologic material,
PRF, and that PRF enhanced third molar socket healing/clot retention and greatly decreased
the clinical time required for postoperative management of localized osteitis.[6] Since then, many studies have evaluated the use of LPRF and PRF to facilitate implant
placement and periapical surgeries,[37]
[38] revascularization procedures,[39] perforation repair, and also bone regeneration in oral and maxillofacial region.[38]
Considering the efficiency of PRP for healing of the alveolar socket after tooth extraction,
there are controversies regarding its effect on postoperative pain, swelling, bleeding,
and postoperative discomfort.[13]
[40]
[41] Radiographic examinations revealed an early and significant increased radiographic
density at the PRP treated sockets in comparison to the ipsilateral not PRP treated
sites,[42]
[43] demonstrating the effect of PRP on early phase of bone healing. However, in a prospective
split mouth study conducted by Arenaz Bua et al., this acceleration in bone formation was not reported to last for more than 6 months.[41]
On the other hand, considering the inhibitory effect of high concentration of PRP
on bone cell division and bone density, a submucosal injection of PRP for acceleration
of OTM was reported by Liou in 2016.[24] This study reported dose dependent application of PRP for acceleration of tooth
movement and for alveolar bone loss at the pressure side of en masse anterior retraction.
However, submucosal injection of PRP is superior to other surgically assisted methods
since it is a noninvasive and clinically feasible method.[24] In this technique, PRP is injected through the attached gingiva into the oral mucosa.
The disadvantages of this technique include the necessity of administration of local
anesthesia before injection for pain control, probability of PRP leakage during injection,
and also the possibility of postinjection pain, discomfort, and mucosal swelling.
In this study, 15% of patients reported severe postinjection pain, which was correlated
with the PRP concentration level.[24] On the other hand, in orthodontic patients with extraction treatment plan, LPRF/PRP
can be placed in the extraction sockets and there would be no need for its submucosal
injection. However, controversies exist regarding the potential benefit of each procedure
and further studies are needed to fully clarify the advantages of each method of delivery
in extraction patients.
In vitro and in vivo studies have shown that the release of growth and healing factors peaks at around
7 days after buccal vestibular mucosal injection in rat models. However, this acceleratory
effect was transient and seemed to decrease over the next 2–3 weeks.[15]
[25] In a study by Güleç et al., on rat models, the difference in the amount of tooth movement between the experimental
(high concentration PRP) and control group was significant at 7, 14, and 21 days.
This result was in accordance with our study, in which the total amount of tooth movement
in the experimental group was significantly higher at T1 (2 weeks = 14 days) and T2
(4 weeks = 28 days). However, in the study by Güleç et al., the study duration was limited to 21 days after buccal vestibular injection, and
long term effects were not evaluated in their study.[25]
In a study by Liou on humans, it was stated that the clinical effect of a single submucosal
injection of PRP could last for 5–6 months clinically. The fastest rate of acceleration
was reported to be during the 2nd–4th month after the injection.[24] This result was in accordance with our study, in which the total amount of tooth
movement in the experimental group was significantly higher at T1 (2 weeks = 14 days)
and T2 (4 weeks = 28 days). In addition, based on this transient effect, they suggested
a multi PRP injection protocol based on the clinical objective including a single
injection of PRP at the beginning of treatment for the purpose of alignment and leveling
and another booster injection at 6 months after the first injection for the purpose
of anterior retraction or posterior protraction.[24]
One limitation of this study was lack of precise evaluation of whole blood and LPRF
content and concentration of growth factors in each patient. Liou assessed the platelet
count in 1 mL of the collected blood sample and final PRP sample.[24] In a recent study by Güleç et al., in 2017, the rate of OTM was evaluated in rats histomorphometrically after submucosal
injection of high and moderate concentrations of PRP.[25] They demonstrated that both high and moderate concentrations of PRP had a transient
acceleratory effect on OTM in rats. This result was in contrast to the previous studies
that reported
the inhibitory effect of PRP on bone metabolism only in high concentrations of PRP.[44] The authors discussed that the reason may be the presence of mechanical force (dynamic
loading) in contrast to static loading in the reported study.[25] Since PRP has a concentration dependent effect on bone turnover[44] and also OTM,[25] it may be stated that different LPRF concentrations may also have different effects
on bone turnover and OTM.[25] However, the split mouth design of this study aimed to compensate for these possible
interpersonal variations. Nevertheless, for future studies, it is highly recommended
to determine the concentrations of platelets and leucocytes in the whole blood and
LPRF samples by the use of ELISA before their application.
LPRF, similar to PRP products, is a mixture of various growth factors, cytokines,
and enzymes, which may have overlapping biological effects and the exact mechanism
of action of each ingredient is still unclear.[45] Many of these elements might demonstrate anti inflammatory effects, responsible
for the increased tissue healing capacity, and at the same time, many cytokines such
as tissue necrosis factors might aggravate the inflammatory response and lead to accelerated
tooth movement.[46] As stated in PRP studies, LPRF can also promote both inflammatory and anti inflammatory
responses and their exact effect could be mainly related to the timing of release,
concentration, and content of its growth factors.[47]
Considering the lack of available evidence on the effect of LPRF on the rate of tooth
movement in humans, our result is not comparable to the findings of other studies
in this field. The major limitation of this study was limited sample size and the
relatively negligible observed clinical effect on acceleration of OTM.
A major strength of this study was that it showed the possibility of application of
autologous blood derived LPRF in humans compared to allogenic PRP application in rat
models. Therefore, the observed result could not be attributed to immune reaction
to the allogenic PRP injection.[25]
[45] However, the inconvenience related to the need for venipuncture and blood drawing
procedure for preparation of LPRF should be considered before clinical application
of this method. Furthermore, more randomized clinical trials are recommended.
CONCLUSION
More decrease in horizontal linear measurement between the mid marginal ridges of
teeth in experimental groups than control groups means that application of LPRF may
accelerate OTM, particularly in cases with extraction treatment plan.
Financial support and sponsorship
Nil.
