|Ahead of print publication
Assessment of the low-speed centrifugation concept modified in the release of fibroblast growth factor-2 in Saudi healthy patient
Abdulrahman Alshehri1, Salah Alokaili2, Sami Shafik3, Nasser M Assery4, Naghshbandi Jafar5, Khalid Alhezaimi6
1 Aseer Dental Center, Saudi Ministry of Health, Aseer Region, Riyadh City, Saudi Arabia
2 Department Periodontal, Dental Center, Saudi Ministry of Health, King Saud Medical City, Riyadh City, Saudi Arabia
3 Department of Periodontal, Dental School, Riyadh Elm University, Riyadh City, Saudi Arabia
4 Eastman Institute for Oral Health, Rochester, New York, USA
5 School of Dentistry, Tishk International University, Erbil Kurdistan, Iraq
6 Department of Endodontics and Periodontics, Dental School, Riyadh Elm University, Riyadh City, Saudi Arabia
|Date of Submission||26-Dec-2019|
|Date of Decision||12-Mar-2020|
|Date of Acceptance||25-May-2020|
|Date of Web Publication||07-Aug-2020|
Ministry of Health, Aseer Region
Source of Support: None, Conflict of Interest: None
Introduction: Alteration of the centrifugation time and protocol may impact the release of platelet-rich fibrin (PRF) scaffolds. The current study purpose was to investigate the released levels of fibroblast growth factor-2 (FGF-2) in standard PRF (S-PRF) and low-speed centrifugation concept known as advanced PRF (A-PRF and A-PRF+). Measurements were done at five times interval over 42 days. The aim was to assess the FGF release and the relation between initial platelet counts and the concentrations of FGF-2 release using the following technique: (1) S-PRF, (2) A-PRF, and (3) A-PRF+.
Materials and Methods: Twenty-four blood samples were taken from eight random Saudi Arabian national healthy subjects enrolled in the investigation. Blood samples were processed using S-PRF, A-PRF, and A-PRF+ centrifugation protocols. Protein quantification was performed using enzyme-linked immunosorbent assay at 1, 7, 14, 28, and 42-day intervals.
Results and Discussion: A statistically significant difference in the mean of FGF-2 measurement between protocols at the 7th day where both S-PRF and A-PRF were significantly higher than A-PRF+ (P < 0.012). Initial platelets' significant count for S-PRF, A-PRF, and A-PRF+ was on day 1, day 7, and 7th day, respectively. Both protocols S-PRF and A-PRF yielded significantly higher release of the FGF-2 when compared to A-PRF+ in Saudi healthy subjects.
Keywords: Fibroblast growth factor-2, growth factor release, low-speed centrifugation, platelet, platelet-rich fibrin
|How to cite this URL:|
Alshehri A, Alokaili S, Shafik S, Assery NM, Jafar N, Alhezaimi K. Assessment of the low-speed centrifugation concept modified in the release of fibroblast growth factor-2 in Saudi healthy patient. Saudi J Oral Sci [Epub ahead of print] [cited 2020 Sep 23]. Available from: http://www.saudijos.org/preprintarticle.asp?id=291611
| Introduction|| |
Fibroblast growth factor-2 (FGF-2) corresponds to the basic FGF subtype of the FGF family, which is a member of the heparin-binding growth factor family. FGF-2 release positively enhances both bone regeneration and osteogenesis. Tan et al. investigated the influence of FGF-2 on bony defects in dogs. They found that periodontal defects could be regenerated by treatment with marrow stem cells that were transfected with FGF-2. Furthermore, in a primate model, FGF-2 provided statistically significant improvements in periodontal regeneration when it was applied topically to the created periodontal defects. Kitamura et al. investigated clinical activity of FGF-2 in stimulating periodontal regeneration of periodontal tissues lost by periodontitis. They suggested clinical efficacy expected from FGF-2 to increase rate alveolar bone height after 36 weeks compared to control group.
Ghanaati et al. were first to report the low-speed centrifugation concept (LSCC) which later had some modifications to the original preparation protocol. The main changes were the alteration of relative centrifugation force (RCF) through its reduction from RCF at 708 g to be 208 g, and this was called advanced platelet-rich fibrin (A-PRF) protocol. It was reported LSCC with RCF reduction may result in the improvement of the regeneration capacity of PRF matrices via higher concentration release of PRF. Centrifugation time was considered as one of the factors that impact the growth factor release and PRF stability which resulted in new protocol, namely A-PRF+.
A study investigated the distribution pattern of the platelets and growth factor release within three PRF matrices (PRF, A-PRF, and A-PRF+) prepared using different RCFs and centrifugation times. They concluded the LSCC may have helped to increased growth factor release in leukocytes and platelets within the solid PRF matrices. Furthermore, Fujioka-Kobayashi et al. reported the adjustments of centrifugation speed and time with the low-speed concept yielded an increase in growth factor release from PRF clots, which subsequently may directly influence tissue regeneration.
The current study aimed to investigate the following over a 42 days' time interval: (1) FGF-2 release levels in standard PRF (S-PRF) compared with the low-speed concept protocol (A-PRF and A-PRF+) and (2) the relation between initial platelet counts for each technique and FGF-2 concentrations. The hypothesis of this research was that both protocols have the same outcomes on the release of FGF.
| Materials and Methods|| |
Total of eight Saudi Arabia nationals and chosen randomly from the pool of subjects who met the inclusion criteria using (Microsoft Excel, Washington, United States). Exclusion criteria were bleeding disorders, diabetic, pregnant women, subjects who underwent chemotherapy and/or radiation therapy in the last 2 years, use of tobacco or other related products, and use any medication with known effect on blood volume in the last 3 years. A complete blood count was obtained for all enrolled individuals to record the platelet counts. All subjects voluntarily per the IRB guidelines have accepted and signed consent form.
Preparation of platelet-rich fibrin
Three vials of extracted blood were collected from each volunteered subject, age ranged between 25–45 years, and all subjects have signed the consent form. Blood extraction was performed using a 21-gauge × ¾” needle (PROCESS FOR PRF, Nice, France). Considering the distance of tube from rotor axis (rmax= 11) and a tube angle of 32°, 10 mL of whole blood without anticoagulant was centrifuged immediately at 2700 rpm (900 g) −12 min for S-PRF, 1300 rpm (210 g) −14 min for A-PRF, and 1300 rpm (210 g) −8 min for A-PRF+ for each subject using a centrifuge (HERMLE Z206A, manufactured by HERMLE Labortechnik GmbH, Wehingen-Germany). Fibrin clot of 4 ml of 10 ml was produced and collected after centrifugation. PRF clots were placed into prepared tubes with 5 ml of Dulbecco Modified Eagle Media (Abbexa Ltd., Innovation Centre, Cambridge Science Park, Cambridge, CB4 0EY, UK).
Protein quantification with enzyme-linked immunosorbent assay
To determine the amount of growth factors released from S-PRF, A-PRF, and A-PRF+ at 1, 7, 14, 28, and 42 days, samples were placed in shaking incubator (DAIHAN LABTECH CO., LTD, Namyangju City, Korea) and kept at 37°C. Protein quantification was performed using enzyme-linked immunosorbent assay (ELISA) (Mindray Bio-Medical Electronic Ltd., Nanshan, China). At each time intervals, 5 ml of the culture media was collected and frozen and 5 ml of culture media was added to replace the collected 5 ml. FGF-2 was quantified using an ELISA kit (Abbexa Ltd., Cambridge, United Kingdom) according to the manufacturer's protocol. Absorbance was measured per the manufacturer recommendations (450 nm) using a microplate reader. All samples had five independent testing were performed at the specified time to ensure calibration of testing device intervals.
Data analyses were performed using the Statistical Packages for the Social Sciences (SPSS) version 20 (Armonk, NY, USA: IBM Corporation) and GraphPad Prism 8 (GraphPad Software, San Diego, USA). FGF-2 experiments were measured using the mean ± standard deviation. Data normality, statistical interactions, and collinearity (i.e., variance inflation factor) were also assessed with the Kolmogorov–Smirnov and Shapiro–Wilk test.
Correlations were conducted using one-way ANOVA and Pearson correlation. We also performed a post hoc analysis of variance to measure the mean difference at each time interval and each preparation protocol using Tukey's honest significance difference. P ≤ 0.05 was considered statistically significant.
| Results|| |
Standard platelet-rich fibrin
S-PRF released FGF-2 mean was 2.6 pg/mL, 2.6 pg/mL, 2.7 pg/mL, 2.7 pg/mL, and 2.6 pg/mL in day 1, day 7, day 14, day 28, and day 42, respectively. The S-PRF had significant FGF release in the 7th and 14th days [Figure 1].
|Figure 1: Mean protein quantification of fibroblast growth factor-2 release at each time point *Significant at P≤ 0.05 level|
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Advanced platelet-rich fibrin
The mean of FGF-2 released by was 2.5 pg/mL, 2.6 pg/mL, 2.6 pg/mL, 2.8 pg/mL, and 2.4 pg/mL on day 1, day 7, day 14, day 28, and day 42, respectively. The significant release of FGF-2 was on 7 and 14 days [Figure 1].
Advanced platelet-rich fibrin+
FGF-2 release mean was 2.6 pg/mL, 2.2 pg/mL, 2.1 pg/mL, 2.4 pg/mL, and 2.3 pg/mL on their day 1, day 7, day 14, day 28, and day 42, respectively. The significant release of FGF-2 was reported on 7 and 14 days [Figure 1].
Intergroup comparisons showed a statistically significant difference in mean FGF-2 release (P = 0.019) between S-PRF and A-PRF+ on day 7 [Table 1]. Additionally, a statistically significant difference in mean of FGF-2 release (P < 0.033) between S-PRF and A-PRF+ was found also on day 14 [Table 1]. Mean initial platelet count was associated significantly to the mean of FGF-2 release on day 1 for S-PRF (P = 0.003) and day 7 for S-PRF (P = 0.011) and A-PRF (P = 0.041) [Table 2].
|Table 1: Multiple comparisons of the differences in mean fibroblast growth factor-2 release among standard platelet-rich fibrin, advanced platelet-rich fibrin, and advanced platelet-rich fibrin+|
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|Table 2: Correlation between fibroblast growth factor-2released (pg/mL) for each preparation protocol and the initial platelet count over time|
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The accumulated means of FGF-2 release showed statistically significant associations with mean initial platelet count for on day 1 for S-PRF (P ≤ 0.05) and on day 7 for S-PRF (P ≤ 0.05) and A-PRF (P ≤ 0.05) [Table 3] and [Figure 2].
|Table 3: Accumulated mean fibroblast growth factor- 2release based on the initial platelet count|
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|Figure 2: Total accumulated fibroblast growth factor-2 released over the 42-day period.†Marginally higher among the groups,†Significantly lower among the group|
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| Discussion|| |
In the present study, FGF-2 quantified on day 1 was generally similar among the three protocols (S-PRF, A-PRF, and A-PRF+). On day 7, both S-PRF and A-PRF maintained the same levels of FGF-2 release, whereas these levels start to decrease significantly with A-PRF+. These findings are not in agreement with El Bagdadi et al. report where they studied different growth factors, including vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and transforming growth factor (TGF)-beta1. Their findings reported that A-PRF+ exhibited the highest levels for VEGF over the study period. Regarding EGF and TGF-beta1, A-PRF, and A-PRF+ both were significantly higher than S-PRF. They also reported that growth factor concentrations increased over the study period with the maximal concentration recorded at day 7. This finding is similar to our finding where S-PRF and A-PRF showed significant growth factor release on the 7th day. Fujioka-Kobayashi et al. showed that A-PRF+ was the highest technique compared to others to release TGF-beta1, VEGF, EGF, and IGF-1 growth factors. This finding is different from our results where our findings revealed that S-PRF reported the highest concentrations in most study time intervals. The difference in our results with the previous works could be attributed to the difference in the chosen time intervals and the quantification methods.
The current study found positive correlations between mean FGF-2 release and initial platelet count were reported on day 1 for S-PRF and day 7 for both S-PRF and A-PRF. In the current study, the highest accumulated release of FGF-2 for S-PRF was in the 1st and 7th days, which is marginally higher when compared to A-PRF and A-PRF+. These findings are not in agreement with previous similar works.,, They reported that A-PRF+ released the highest amount of growth factors in individual and accumulated calculations.
To our knowledge, this is the first study to discuss the long-term release of FGF-2 from PRF matrix scaffolds extracted from healthy Saudi Arabian national using LSCC protocol. The limitation of the current study was the sample number and be equally distributed between gender for Saudi Arabia national.
We thank Nouf Alsaadon, laboratory specialist, for assistance with the laboratory procedures including ELISA.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]