Effect of carbonated hydroxyapatite incorporated advanced platelet rich fibrin

intrasulcular injection on the alkaline phosphatase level during orthodontic relapse

Ananto Ali Alhasyimi, Pinandi Sri Pudyani, Widya Asmara, and Ika Dewi Ana

Citation: AIP Conference Proceedings 1933, 030006 (2018);

View online: https://doi.org/10.1063/1.5023953
View Table of Contents: http://aip.scitation.org/toc/apc/1933/1
Published by the American Institute of Physics
 Effect of Carbonated Hydroxyapatite Incorporated
Advanced Platelet Rich Fibrin Intrasulcular Injection on the
Alkaline Phosphatase Level during Orthodontic Relapse
Ananto Ali Alhasyimi1,2, a), Pinandi Sri Pudyani2, b), Widya Asmara3, c), Ika Dewi Ana4, d)

1
Graduate School of Dental Sciences, Faculty of Dentistry, Universitas Gadjah Mada,
Yogyakarta 55281, Indonesia
2
Department of Orthodontics, Faculty of Dentistry, Universitas Gadjah Mada,
Yogyakarta 55281, Indonesia
3
Department of Microbiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada,
Yogyakarta 55281, Indonesia
4
Department of Dental Biomedical Sciences, Faculty of Dentistry, Universitas Gadjah Mada,
Yogyakarta 55281, Indonesia
a)
anantoali@ugm.ac.id
b)
pinandi@ugm.ac.id
c)
wied_as@ugm.ac.id
d)
Corresponding Author: ikadewiana@ugm.ac.id

Abstract. Nowadays, relapse in orthodontic treatment is considered very important because of high incidence of relapse
after the treatment. Alkaline phosphatase (ALP) as a biomarker of bone formation will decrease in compression sites
during relapse after orthodontic tooth movement. In this situation, manipulating alveolar bone remodeling to increase
ALP level is considered one of the new strategies to prevent relapse properly. In the field of tissue engineering, in this
study, carbonated hydroxyapatite (CHA) is expected to have the ability to incorporate advanced platelet rich fibrin
(aPRF). Next, CHA will retain the aPRF containing various growth factors (GF) until it reaches into a specific targeted
area, gradually degraded, and deliver the GF in a controlled manner to prevent relapse. Here, gingival crevicular fluid
(GCF) of 45 samples (n=45) were collected and levels of ALP were analyzed using UV-Vis 6300 Spectrophotometer at
405 nm wavelength. We found that there is a significant difference of ALP levels (p<0.05) in GCF between treatments
and control groups. ALP level was elevated significantly in CHA and CHA-aPRF groups at days 7 and 14 after
debonding compared with the control groups. The peak level of ALP was observed at days 14 after debonding in groups
C (0.789 ± 0.039 U/mg). Therefore, it can be concluded that the application of hydrogel CHA with controlled release
manner incorporated aPRF enhances bone regeneration by increasing ALP level.

Keywords: controlled release, hydrogel CHA, aPRF, alkaline phosphatase

INTRODUCTION

Relapse is a process in which the tooth arrangement changes after orthodontic treatment towards the initial
condition before treatment, and it is inevitable outcome from many factors including incomplete remodeling of
alveolar bone [1]. Previous studies have shown that the presence of alkaline phosphatase (ALP) activity in gingival
crevicular fluid (GCF) has been described as biological markers during bone remodeling for bone formation during
orthodontic tooth movement [2]. ALP activity is found higher at tension sites compared with compression sites
during orthodontic tooth movement [3]. The increasing osteoblast activities during bone formation will be
characterized by the increasing ALP expressions [4].

2nd Biomedical Engineering’s Recent Progress in Biomaterials, Drugs Development, and Medical Devices
AIP Conf. Proc. 1933, 030006-1–030006-6; https://doi.org/10.1063/1.5023953
Published by AIP Publishing. 978-0-7354-1625-3/$30.00

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 Stimulation of alveolar bone formation after orthodontic tooth movement is believed to effectively prevent the
relapse of moved teeth [5]. These findings suggest that manipulating alveolar bone remodeling after orthodontic
tooth movement is considered one of the main strategies to prevent relapse properly. Carbonated (hydroxy) apatite
(CHA) is thought to be one of an ideal candidate for enhancing alveolar bone remodeling since it has the same
interconnected porous structure with an inorganic component like cancellous bone [6]. Another advantage possessed
by hydrogel CHA lies in its ability to be drug delivery system in controlled release technology [7]. Platelet-rich
fibrin (PRF) is one of the latest generation of platelet concentrate which contains high levels GF that plays a central
role in tissue engineering applications to induce hemostasis and the bone healing process [8]. In vitro study proved
PRF may release autologous GF’s gradually and express stronger effect on proliferation and differentiation of
osteoblasts than platelet rich plasma [9]. Advanced PRF (aPRF) is a modification of PRF, by decreasing
centrifugation speed and an increasing centrifugation time, to allow higher quantities of platelet concentrations
compared to standard PRF [10] [11]. The objective of the present study iss to investigate the effect of hydrogel
CHA-aPRF intrasulcular injection on ALP levels containing in GCF’s rabbit after orthodontic tooth movement.

MATERIALS AND METHODS

Preparation of carbonate apatite incorporated advanced platelet rich fibrin

Hydrogel used in this study was prepared from β-type gelatin of bovine bone (Nitta Co., Japan) and
calcium hydroxyde (Ca(OH)2) in a gelatin-CHA compositions of 7:3 (weight %) then physically cross-linked. A
total of 0.37 gr sodium citrate (Sigma-Aldrich, Germany) was used as dispersant. The hydrogel was poured into 4×4
cm2 polyprophylene mold (BioBik, Japan) and frozen at -20˚C for 24 hours, then freeze dried for 24 hours in -40 ̊C
to obtain dried gelatin. Dehydrothermal treatment to crosslink gelatin-hydrogel was done at 140 ̊C for 72 hours.
PRF as an autologous GF was prepared from 10 ml of rabbit blood that was withdrawn from a peripheral vein of
the rabbit’s ear using needle 25 gauge (Terumo Company Ltd., Japan) then immediately centrifuged with a 1500
rpm for 14 minutes. At the end of the centrifugation process, 3 separate segments of blood components were
produced where the intermediate layer clot was isolated and then pressed with an aPRF processing box (Osung Ltd.,
USA) for 10 min (Fig. 1). The aPRF releasate has 4.78 times higher (2.017 × 103 platelets/mm3) than the whole
blood platelets. After preparation, it was loaded into the hydrogel CHA immediately. To electrically bind the PRF
into the microspheres, a total of 200 μL releasate PRF was dropped onto 10 mg of hydrogel-CHA microspheres and
incubated for 1 hour at 37°C
This research was approved by the Health and Medical Research Ethics Committee of Faculty of Dentistry,
Universitas Gadjah Mada (UGM) with number 00849/KKEP/FKG-UGM/EC/2016. A total of 45 specific pathogen-
free (SPF) New Zealand male rabbits (n=45), body weight 2500–3000 g, were obtained from the Animal
Experimental Unit of LPPT (Integrated Research and Testing Laboratory) of UGM (Yogyakarta, Indonesia). Rabbits
were anesthetized with an intramuscular injection of the mixture of 50 mg/kg body weight ketamine and 10 mg/kg
body weight xylazine. The lower incisors of all rabbits were subjected to orthodontic force and moved distally for a
period of one week using a Nickel-Titanium open coil spring 0.010” x 0.030” (DynaFlex, the Netherlands) that was
inserted between the straight wire bracket 0.022-inch slot (American Orthodontics®, USA) with a 0.016” stainless
steel wire. Activation force was approximately 50 cN, as measured by a dynamometer tension gauge (Medkraft,
USA). After one week, the springs were not reactivated and the distance was maintained during two weeks of
stabilization (retention) period. During stabilization period, injection of CHA-aPRF was given. The application of
the controlled release CHA-was grouped into Group B (CHA), Group C (CHA-aPRF), and Group A as a control
group without treatment with 15 Rabbits (n=15) in each group. A total of 50 μL hydrogel CHA-aPRF have gently
injected on the mesial side of both incisor incisor gingival sulcus at the sub-periosteum using 26 G x ½” disposable
syringe (OneMed, Indonesia) every 7 days. After 2 weeks stabilization period, the orthodontic appliances were
debonded, then the rabbits were sacrificed (0, 3, 7, 14, and 21 days after debonding) and the ALP levels were
measured.

Gingival crevicular fluid collection

Gingival Crevicular Fluid (GCF) of each sample in each group was taken on days 0, 3, 7, 14 and 21 respectively
after debonding and collected using sterile paper point size 15 (Paper Points ISO 0.2, Dentsply, Germany). Each
paper strip was inserted approximately 1 mm into the gingival sulcus of the mesial side of teeth (compression sites)

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and was left in situ for 30 seconds. A total of two dipped paper points was then placed into a 1.5 mL eppendorf tube
containing 350 μL of physiological saline solution. Afterwards, the tube was then centrifuged for 5 minutes at 2000
rpm in 4o C using a microcentrifuge refrigerator (Eppendorf 5424R, USA) to elute the GCF component from the
paper points completely. The paper points were then removed and the supernatants were kept frozen and stored at
−80°C until assayed.

Measurement of Alkaline phosphatase level

Alkaline phosphatase level was analyzed by UV-Vis 6300 Spectrophotometer (Jenway, UK). Approximately 50
mL of 40 mM Na2CO3 buffer (pH 9.8) mixed with 3 mM MgCl2 was put into Eppendorf tubes using pipettes. A total
50 μL of GCF samples and 50 μL of 3mM p-nitrophenyl phosphate were added to the same tubes. The tubes were
then incubated for 30 min at 37°C. The enzymatic reaction was terminated by addition of 50 mL of 0.6 M sodium
hydroxide (NaOH). Then, the p-nitrophenol produced was assayed by absorbance at 405 nm wavelength, referred to
a standard curve prepared from phosphatase substrates (Sigma 104, Sigma-Aldrich, St. Louis, USA). Enzyme
activity was presented in the form of enzyme unit (U) as μmol of p-nitrophenol released per minute at 37° C. The
ALP levels were then determined based on units (U) of activity versus the total protein content (mg) and were
expressed as U/mg. The data of the groups were subjected to a test of normality and homogeneity. With respect on
this, Analysis of Variance (ANOVA) was used to determine the significance between the groups. The significance
for all statistical tests was set at p<0.05, and least significant difference (LSD) post-hoc test was used to detect
pairwise disagreements between the groups.

(a) (b) (c) (d)

FIGURE 1. Images of the stages of obtaining aPRF releasate in rabbits (a) Blood samples after centrifugation (b) isolation of
middle layer using sterile scissor (c) pressing the PRF membrane to obtain the releasate
using PRF box (d) Appearance of the aPRF releasate.

RESULTS AND DISCUSSION

Mean and standard deviation of ALP levels between control and treatment groups is shown in Figure 2. The
result showed that ALP levels were found higher in the groups treated with CHA and CHA-aPRF group compared
to control groups. ALP has been reported as a biologic marker of osteoblastic activity during bone formation [12]
[13]. The previous study showed that there was a correlation between alveolar bone remodeling with changes in
ALP activities contained in GCF. It was suggesting that there were increased osteoblastic cells activity when ALP
levels found much higher and elevated [14] [4]. Osteoblast activity was elevated indicated that there is a new bone
formation. To prevent relapse, osteoblast is required to consistently stimulate bone formation [15]. Osteoblasts
consequently fill the trenches and tunnels of bone in the surfaces of the bone formed by osteoclasts, through
generating novel bone matrices within them and initiate bone apposition [16]. Osteoblasts also regulate

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osteoclastogenesis, a key factor for osteoclast differentiation, by expressing osteoprotegerin (OPG), which inhibits
RANKL (receptor activator of nuclear factor-κB ligand) binding RANK on osteoclast precursors [17].

FIGURE 2. Mean (standard deviation) of ALP levels (U/mg) from 3 groups tested

TABLE 1. One way Anova and LSD post-hoc test summary on ALP levels between 3 groups tested

Day p-value Groups CHA CHA-aPRF

Day 0 0,004* Control 0,003* 0,003*
CHA 0,876
CHA-aPRF
Day 3 0,000* Control 0,000* 0,000*
CHA 0,912
CHA-aPRF
Day 7 0,000* Control 0,000* 0,000*
CHA 0,024*
CHA-aPRF
Day 14 0,000* Control 0,000* 0,000*
CHA 0,000*
CHA-aPRF
Day 21 0,000* Control 0,000* 0,000*
CHA 0,072
CHA-aPRF
Values are presented as p-value by ANOVA,
*Significant differences between groups (p <0.05).

Table 1 showed that CHA significantly (p<0.05) increased ALP levels compared to control groups. Mechanism
of CHA for enhancing bone remodeling is known by raising the local concentration of calcium and phosphate ions
which are required for new bone formation [18]. CHA demonstrated high osteoconductivity and high expression of

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osteoblastic markers of differentiation, such as ALP, type I collagen, osteocalcin, and osteopontin [19]. Table 2
shows that there was a significant difference between group B and group C (p<0.05) on day 7 and 14 after
debonding. The result of this study is in accordance with previous in vivo studies proving that hydrogel CHA
incorporating GF was potential to promote angiogenic effects [7]. Angiogenesis may help oxygen and nutrients
delivery and serve as a route for bone precursor cells to reach the targeted site [20].
PRF contains high levels of GF’s that play a central role in hemostasis and the bone healing process, including
vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF),
epidermal growth factor (EGF) and transforming growth factor (TGF) [8]. These GF’s were gradually delivered in a
controlled manner and inducing osteogenesis through osteoconduction, osteoinduction and angiogenesis,
respectively. VEGF contains in PRF can induce the differentiation, migration and accumulation of osteocytes, and to
increase the activity of ALP [21], which is consistent with the results of this study. Previous in vitro studies have
reported that PRF could modulate osteoblast proliferation and upregulate ALP activity effectively [22] [23].
The peak level of ALP was observed in the group treated with CHA-aPRF at days 14 after debonding (0.79 ±
0.04 U/mg), which agrees with some previous studies [3] [12] [24]. It is supported by a prior study conducted by
Yokoya et al. [25] that the number of osteoclasts on the compression side decreased by day 14 when the highest
enzyme activities were expressed. A comparative in vitro study proved that exudates of PRF at day 14 expressed
maximum ALP activity [9]. On day 14 the enzyme activities were expected to show exactly at the final phase of
tooth movement when hyalinization occurs.

CONCLUSION

The results of this study indicate that intrasulcular injection of controlled release CHA incorporated aPRF has
potential effect to enhance alveolar bone remodeling and prevent orthodontic relapse as indicated by the significant
increases in ALP activity at day 7 and 14 after debonding. However, more studies are needed on a molecular and
cellular level, and parameters must be improved (such as RANKL, OPG, TGF, VEGF, type I collagen) to confirm
the efficacy and potency of CHA-aPRF to reduce the orthodontic relapse tendency in the future orthodontics clinic.

ACKNOWLEDGEMENTS

This research is supported by grant Bantuan Pendanaan Perguruan Tinggi Negeri Badan Hukum, Research
Directorate, Universitas Gadjah Mada, the Republic of Indonesia, in the fiscal year of 2017, under Contract No. 352/
DIT.LIT/ 2017.

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