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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 12  |  Issue : 2  |  Page : 91-95

In Vitro Osteogenic Potential of Freeze-Dried Homologous Platelet-Rich Plasma


1 Department of Periodontology, Faculty of Dentistry, Universitas Gadjah Mada, Jl. Denta, Sleman, Yogyakarta, Indonesia
2 Clinical Dentistry Program, Department of Periodontology, Faculty of Dentistry, Universitas Gadjah Mada, Jl Denta, Sleman, Yogyakarta, Indonesia

Date of Submission16-Dec-2020
Date of Decision30-Mar-2021
Date of Acceptance27-Apr-2021
Date of Web Publication26-Jul-2021

Correspondence Address:
Kwartarini Murdiastuti
Lecturer, Department of Periodontology, Faculty of Dentistry, Universitas Gadjah Mada, Jl. Denta, Sleman, Yogyakarta, 55281
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/denthyp.denthyp_183_20

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  Abstract 


Introduction: Platelet-rich plasma (PRP) is paid attention for regenerative therapy because it clinically improves neoangiogenesis and periodontal regeneration. PRP can be made in form of homologous PRP (HPRP) from healthy, screened, and habitual blood donors and freeze-dried to increase stored time of HPRP and maintain growth factors. The purpose of this study is to evaluate freeze-dried homologous platelet-rich plasma (FD HPRP) on osteogenesis. Material and Methods: HPRP was taken from the blood bank and then a freeze-drying and γ-radiation process was carried out with doses of 20 and 25 KGy, respectively, for the sterilization. Blood was collected as much as 10 mL and then centrifuged at 700 rpm for 3 minutes to produce injectable platelet-rich fibrin (iPRF) as a positive control. Cell line MG63 were cultured to confluent, and then treated with FD HPRP 20 kGy, FD HPRP 25 kGy, iPRF, and one group non treated as a negative control. Osteocytes were determined based on morphology after hematoxylin staining to see the differentiation process. The data were analyzed using one-way analysis of variance (ANOVA). Results: There was a significant difference in the number of osteocytes between FD HPRP and negative control (P < 0.05). The number of osteocytes FD HPRP 25 was higher than FD HPRP 20. Conclusion: The present results indicate that FD HPRP could support the bone regeneration and used without any preparation like fresh PRP.

Keywords: freeze-dried platelet-rich plasma, injectable platelet-rich fibrin, osteogenesis, periodontal regeneration


How to cite this article:
Murdiastuti K, Olivia N, Kusumadewi Ww, Sumito N. In Vitro Osteogenic Potential of Freeze-Dried Homologous Platelet-Rich Plasma. Dent Hypotheses 2021;12:91-5

How to cite this URL:
Murdiastuti K, Olivia N, Kusumadewi Ww, Sumito N. In Vitro Osteogenic Potential of Freeze-Dried Homologous Platelet-Rich Plasma. Dent Hypotheses [serial online] 2021 [cited 2021 Sep 26];12:91-5. Available from: http://www.dentalhypotheses.com/text.asp?2021/12/2/91/322521




  Introduction Top


Periodontitis is an inflammatory disease of the periodontal tissues leading to destruction in periodontal tissues characterized by loss of support of the affected teeth, specifically periodontal ligament and the bone into which they are inserted.[1],[2] The ultimate goal of periodontal therapy is regeneration.[3] Platelet plays an essential role during wound-healing process in improving regeneration by providing growth factors, various cells, cytokines, and coagulation factors.[4] Growth factors are expressed during phases of healing and the key elements in promoting regeneration. These are considered as the most relevant factors in regenerative process.[5]

Platelet-rich plasma (PRP) is an autologous source of various growth factors obtained by isolating and concentrating fresh blood. Recently, PRP has been applied clinically to enhance bone and tissue healing. Several trials have shown that employing PRP when dental implant procedure osseointegration and bone regeneration.[6] PRP can be prepared from patient’s own blood (autologous PRP) and multiple donors (homologous PRP [HPRP]).[7] However, autologous PRP has a problem when patients with poor general health, multidrug therapy or hematologic disorders, and the volume blood absence required could often cause adverse effects, in particular in repeated treatments.[8] HPRP from healthy, screened, habitual blood donors possesses a number of advantages, such as easy preparation, ability to obtain more quantity at one time, high platelet counts rather than therapeutic ratios, and low cost.[9]

During clinical PRP application, one of the major unsupportive problems for long-term PRP storage is relatively short half-life of growth factors in PRP. Thus, patients have to donate large amounts of blood before surgery for preparation of a new PRP.[10] To overcome this limitation, PRP is made in the form of a dry powder called freeze-dried PRP (FD PRP). Freeze drying is a method to increase the storage time of HPRP and maintain the growth factors.[11] Nakatani et al.[12] explained that PRP can be preserved by freeze drying without loss of wound healing properties and contain various growth factors including platelet derived growth factor (PDGF), transforming growth factor beta (TGF-b), and vascular endothelial growth factor (VEGF), and these growth factors promote wound healing and bone regeneration. Another experiment comparing single or multiple injection applications in the wound area of diabetic mice with FD PRP containing trehalose, fresh PRP, multiple doses of recombinant VEGF, or no treatment demonstrated multiple doses of VEGF, and the use of FD PRP promoted a faster wound closure when compared with the nontreated group.[13]

Shiga et al.[11] revealed that FD PRP should be prepared in a clean room using good sterilization procedures to avoid infection. Sterilization can be achieved by gamma ray radiation without affecting the effectiveness of Freeze dried-platelet lysate (FD-PL) in chondrocytes, fibroblasts, osteoblasts, and BM-MSC cultures.[14] Sterilization is needed to enhance the bioactivity and storage of FD PRP for a long time. In the study of Muraglia et al.[15] FD PRP sterilized with gamma radiation at dose 25 kGy presented no colonies. Based on this result, this study employs gamma ray radiation of 20 and 25 kGy.

Besides PRP, there is another platelet concentrate introduced recently, which is injectable platelet-rich fibrin (iPRF).[16] iPRF development of PRF generation with main concept iPRF is lower centrifugation speeds in non-glass centrifugation tubes with fast time.[17] Some studies comparing iPRF with PRP against fibroblast,[18] osteoblast behavior,[9] cartilage regeneration,[19] antimicrobial efficacy,[20] and iPRF revealed better results. However, freeze-dried homologous PRP with iPRF is not yet compared. Therefore, in this study, iPRF is employed as positive control. The objective of this study is to evaluate osteogenic potential of freeze-dried homologous platelet-rich plasma (FD HPRP).


  Material and Methods Top


This experimental research is approved by the Ethics Commission, Faculty of Dentistry, Universitas Gadjah Mada, Indonesia with registration number No.00520/KKEP/FKG/-UGM/EC/2020. The research sample administering human osteoblast-like cells, MG63 cell line (ATCCR - CRL 1427TM) was divided into four groups (FD HPRP 20, FD HPRP 25, iPRF as positive control, and one nontreated group as negative control).

Freeze-dried homologous platelet-rich plasma

Donor blood bags were obtained from a blood bank in Indonesia with double blood bags. The blood bag and centrifugation bowl were balanced. The centrifugation bowl that had been balanced was placed into the centrifugation side facing away and the blood bag was aligned with the lobe of the cup. Rotate 32XG, temperature 4°C for 30 minutes. The centrifugation cup was removed slowly so that the blood did not mix. The blood was placed in the main bag of the extractor’s diploma slowly. Then, it was clamped and the connecting hose between the main and the satellite bags was opened. Plasma was flowed into the satellite bag and left in the main bag approximately 3 cm from the surface of the red blood cells concentrated. The connecting hose between the main and the satellite bags was then sealed employing an electric scaler, and the connecting hose was cut. Blood bags containing plasma from PMI were frozen at −40°C for 12 hours, then freeze-dried for 48 hours using a freeze-drier machine (Freeze Dryer Modulyo, Edwards), and finally the sterilization process with gamma radiation at doses 20 and 25 KGy was applied.

Injectable platelet-rich fibrin

Ten milliliters of blood was obtained and placed in a plastic tube (Gibco) without anticoagulant. Then it was centrifuged at 700 rpm for 3 minutes at room temperature. After centrifugation, iPRF was collected from the upper layer.[16]

Cell culture

Human osteoblast cell line MG-63 (ATCC Bethesda, USA) was cultured in a T75 Flask (TPP Switzerland) with Dulbecco’s Modified Eagle Medium (DMEM) (Gibco), FBS (Sigma), Penicillin Streptomycin (Gibco), Fungizone (Gibco) media incubated at 37°C and 5% CO2. When steoblast cells which were 80–90% confluent, the cell medium was removed. Then, DMEM (Gibco) was inserted into a flask containing osteoblasts. The cells were washed using trypsin-EDTA 0.25% (Gibco), so that the cells attached to the flask were released. The cells were placed in a 15 mL conical (Biologix) containing DMEM and then centrifuged at 1500 rpm for 5 minutes. The cells would settle at the conical base and DMEM was added and aspirated. The cells were seeded into a 96-well plate as much as 100 μL. The cells were incubated overnight and treated.

Morphology and differentiation

Identification of the number of cultured bone cells was performed by morphological observation after the cells were colored with hematoxylin-eosin staining. After the seventh day, the cell culture grown on top of the closing glass was washed with PBS (Gibco) and fixated in a 4% paraformaldehyde buffer solution (Sigma) for 24 hours. The cells were washed with aquades for 10 minutes with soaking three times, and then they were soaked in hematoxylin for 10 minutes and rinsed with aquades for 5 minutes. Then, it was soaked in eosin (Sigma) for 5 minutes, rinsed with aquades for 5 minutes, and dehydrated employing alcohol. After that, it is continued with immersion on xylol (Millipore) twice for 10 minutes. Then, the closing glass was affixed with a glass object and observed under a microscope with a magnification of 40 × 10 with 5 field of view.[21]

Statistical analysis

Data in this study are the number of osteocytes from FD HPRP 25, FD HPRP 20, iPRF and negative control was determined via the Shapiro–Wilk test to find out whether the data were normally distributed. The homogen variables were assessed by Levene test. Significant differences between the groups were examined using parametric methods. Intergroup differences were compared using analysis of variance (ANOVA) and post hoc comparison tests were performed for the demographic and clinical variables. Statistical analyses were conducted using statistical analysis software [Statistical Package for the Social Sciences (SPSS), Version 25.0, Indonesia]. P < 0.05 was considered statistically significant.


  Results Top


The data in this study are the number of osteocytes in the treatment of FD HPRP 20, FD HPRP 25, iPRF, and negative control [Figure 1]. The highest number of osteocytes in iPRF and the lowest in negative control [Figure 2]. A normality and homogenity test was performed using Levene test, which showed normal and homogen (P > 0.05). Then statistical analysis was continued using one-way ANOVA to find out whether there is a difference between four groups. One-way ANOVA test shows significant differences between FD HPRP 20, FD HPRP 25, iPRF, and negative control (P < 0.05) [Table 1]. Post Hoc LSD test shows that there was no significant difference (P > 0.05) between FD HPRP 20 and FD HPRP 25 or negative control, but there was significant difference between FD HPRP 20 and iPRF (P < 0.05) regarding the number of osteocytes. FD HPRP 25 was not significantly different from FD HPRP 20 (P > 0.05), but it was significantly different from iPRF and negative control (P < 0.05).
Figure 1 Hematoxilin-Eosin staining showed osteocytes in the treatment of (a) FD HPRP 20; (b) FD HPRP 25; (c) iPRF; (d) negative control

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Figure 2 Mean osteocytes number of each group. The highest number of osteocytes in iPRF and the lowest in negative control. The asterisk symbol in the figure denotes the significant difference between two groups, P < 0.05. iPRF, injectable platelet-rich fibrin

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Table 1 The mean and standard deviation number of osteocytes in HPRP 20, FD HPRP 25, iPRF, and negative control group

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  Discussion Top


Periodontitis is an inflammation of the tooth support tissue caused by microorganisms that can damage the periodontal ligaments and resorption of the alveolar bone, resulting in loss of attachment loss.[22] Goals of periodontal therapy are not only the arrest of periodontal disease progression, but also the regeneration of structures lost to disease where appropriate.[23] Periodontal regeneration is a complicated process involving many aspects, which are the control of infection and inflammation, recruitment of stem/progenitor cells, promotion of cell proliferation and differentiation, and new tissue formation.[22] Besides, periodontium composed of distinct layers of tissues and different biological cues should be provided to guide Periodontal Ligament (PDL), cementum, and alveolar bone formation.[24]

The outcome showed that FD HPRP could affect the osteogenesis process because the number of osteocytes was significantly higher than negative control. Osteogenesis is the process of bone formation by osteoblast cells. One of the stages in the osteogenesis process is a differentiation process (a transformation process or the change of cells into the form and functions of cells that change from osteoblasts to osteocytes).[21] The occurrence of differentiation process can be identified with morphological changes and the number of osteocytes in bone cell culture population. Many factors may affect proliferation cells and differentiation. The components can be either hormones or growth factors.[25] PRP containing growth factors such as PDGF-BB, TGF-1, Insulin-like growth factor (IGF-I), PDEGF, PDAF, and PF-4, released from platelets and involved in wound healing, are considered to be promoters of tissue regeneration.[26]

In present study, FD HPRP affected osteogenesis process signed with a number of osteocytes during the 7 days incubation. This result, according to Nakatani’s research,[12] is that FD PRP can increase in vivo bone regeneration similarly to fresh PRP. It indicates that biology activity of growth factors in FD PRP can be maintained. Pietramaggiori et al.[13] reported that growth factor bioactivity and its healing effect in FD PRP can be employed in the chronic wound healing. Horimizu et al.[27] asserted that the storage of FD PRP coated with a collagen sponge at 4°C did not cause a significant loss in bioactivity. According to the research performed by Kinoshita et al.[10] FD PRP can maintain PDGF pharmacological activity during 4 weeks of storage. PDGF induces osteogenic differentiation by enhancing collagen synthesis, bone cell proliferation, and tissue repair.[27]

The result of this study was that the number of osteocytes was higher in FD HPRP 25 than FD HPRP 20. It is due to the gamma ray dose applied in the sterilization process. Muraglia et al.[28] argued that FD PRP sterilized with gamma radiation at dose 25 KGy showed no colonies, thus improving the effectiveness of FD HPRP. Therefore, based on this theory and research, FD HPRP can be stored for a long time with the sterilization process in advance to maintain its content and avoid infection. FD HPRP has a benefit that it is easy to store, as its powder form enables storage in a refrigerator or even at room temperature (RT). It would be very convenient for mixing with bone grafts or other materials.[11]

Limitation of this research is the time, it is only done on the seventh day. Hence, for the next research, it can be a longer time to investigate more bone regeneration process. For future animal studies and clinical testing, it is necessary to validate this regenerative technology.


  Conclusion Top


FD PRP could affect osteogenic differentiation indicated by a higher number of osteocytes than negative control. FD HPRP 25 showed a higher number of osteocytes than FD HPRP 20. Based this study, it can be concluded that FD HPRP can be a regenerative treatment option in daily clinical practice because of easy preparation and storage for a long time.

Acknowledgment

This research was supported by public funds for the final project recognition program Gadjah Mada University Indonesia.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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