|Year : 2019 | Volume
| Issue : 4 | Page : 322-328
Comparative evaluation of growth factors from platelet concentrates: An in vitro study
Anirban Chatterjee, Koel Debnath
Department of Periodontology, The Oxford Dental College, Bengaluru, Karnataka, India
|Date of Submission||10-Nov-2018|
|Date of Acceptance||13-Mar-2019|
|Date of Web Publication||1-Jul-2019|
Dr Koel Debnath
#106, Block-B, GK Jewel City Apartment, Kudlu Harlur Main Road, Kudlu, Bengaluru - 560 068, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim of the study was to compare and evaluate the various growth factors released for a period of 23 days from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM). Materials and Methods: A total of 15 systemically healthy controls were recruited and 10 ml of blood sample was withdrawn from the individual. Following the standard centrifugation protocol, PRF and PRFM were prepared. The platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), and insulin growth factor (IGF) were evaluated for 23 days. Results: The PDGF released from PRFM was statistically significant from PRF till the 15th day of release. The VEGF too had an increased release up till the 15th day from PRFM group as compared to PRF, but no statistically significant difference could be obtained. EGF from the 15th day to 23rd day had a greater release from PRFM group as compared to PRF group. FGF from 7th day to 23rd day had statistically significant difference in the PRFM group as compared to PRF group. TGF and IGF had statistically significant difference in PRFM group as compared to PRF group from 11th day to 23rd day and 1st to 17th day, respectively. Conclusion: The initial and robust release of GFs was seen in PRFM group at earlier days, whereas a steady and constant release of six GFs could be appreciated from PRF group upto 23rd day. Therefore, for a rapid and early healing and regeneration, both the platelet concentrates can be utilized in periodontal therapy.
Keywords: Growth factors, platelet-rich fibrin, platelet-rich fibrin matrix
|How to cite this article:|
Chatterjee A, Debnath K. Comparative evaluation of growth factors from platelet concentrates: An in vitro study. J Indian Soc Periodontol 2019;23:322-8
|How to cite this URL:|
Chatterjee A, Debnath K. Comparative evaluation of growth factors from platelet concentrates: An in vitro study. J Indian Soc Periodontol [serial online] 2019 [cited 2019 Nov 18];23:322-8. Available from: http://www.jisponline.com/text.asp?2019/23/4/322/261558
| Introduction|| |
Periodontal disease is characterized by gingival inflammation which results in the formation of periodontal pocket with the loss of the supporting alveolar bone and connective tissue around the teeth. The treatment plan plays a challenging role in eliminating the gingival inflammatory process and preventing the disease progression with the regeneration of tissues previously lost to the disease. It implies the formation of new bone and cementum and a functionally oriented periodontal ligament. This results with the formation of new cementum with the insertion of collagen fibers., In nature, the proteins responsible for coordinating these regenerative events are called growth factors. These naturally occurring growth factor molecules with matrix proteins are key regulators of various biological events. The growth factors are known to have pleiotropic effects on wound repair, and nearly all tissues including the periodontium.,
The use of recombinant growth factors has shown various issues, thus an alternating mode of the path with the combination of ease of preparation and physiologic delivery can be beneficial in the regeneration and successful treatment with the use of platelet concentrates. The various generations of platelets concentrates have been evaluated for the concentration and duration of release of growth factors.,
The platelet-rich fibrin (PRF), a second-generation platelet concentrate introduced by Choukroun et al. in 2001 has been widely used in the field of dentistry. It is known to provide immune-compatible growth factors at a very low cost and without the use of anticoagulants. PRF has shown a gradual and slow release of platelet-derived growth factor (PDGF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF). The slower release of growth factors over time is due to the ability of the fibrin matrix to store the proteins within its fibrin mesh as well as the cells capability to further release the growth factors into their surrounding microenvironment. The pleiotropic leukocytes have shown to be highly important immune cells which is capable of monitoring, assigning, and recruiting various cell types during the wound healing process.
The first-generation platelet concentrate, introduced by Marx et al. in 1998 is known to act on healing inducible cells to increase their numbers through mitogenesis and perform angiogenesis by stimulating vascular ingrowth. Its massive use in medicine and field of dentistry has been too evaluated. The major disadvantage in the utilization of the platelet concentrate is the tedious process of double centrifugation and also the addition of an anticoagulant. To simplify the process and utilize the regenerative potential of the platelet concentrates along with the growth factors led to the introduction of PRF matrix (PRFM). Various factors such as thrombin-fibrinogen concentration ratios and factors such as protein and ion concentrations including calcium are responsible for structural changes in the fibrin gel of the platelet concentrate. The PRFM preparation uses calcium gluconate along with a standard rotation per minute gravitational force and without the use of exogenous thrombin. The new protocol of preparation appears to be physically stronger than conventional PRP.
Thus, the present study aimed to compare and evaluate the release of growth factors for a period of 23 days.
| Materials and Methods|| |
The research was conducted in the Department of Periodontics, The Oxford Dental College and hospital. Ethical clearance was obtained from the institution. Fifteen healthy controls with an age range of 22–40 years were recruited for the study. The criteria implemented for participating in the nonrandomized prospective trial were as follows: systemically healthy, good compliance with oral hygiene instructions following initial therapy, participants requiring any form of periodontal surgeries such as depigmentation procedures and periodontal surgery; the presence of vital teeth, absence of mobile teeth, and did not undergo periodontal surgery for the past 6 months. The exclusion criteria included participants under antibiotic medication for the past 6 months, suffering from the chronic systemic condition, smoking individuals, and pregnant and lactating mothers. Informed consent was obtained from the participants fulfilling the criteria for enrolment in the trial. The clinical research trial was conducted from August 2016 to September 2016.
Blood sample collection
Ten milliliters of blood samples were collected from 15 controls (30 samples) by the principal investigator conducting the clinical trial. The samples were further processed for preparation of PRF and PRFM as per the conventional centrifugation protocol.
The 30 samples were further divided into Group A which included PRF membrane and Group B which included PRFM gel [Figure 1].
|Figure 1: Study flow chart. PRF – Platelet-rich fibrin; PRFM – Platelet-derived growth factor; N – Number of samples|
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Platelet concentrate preparation
A volume of 10 ml of blood sample was withdrawn from the antecubital vein from each individual. A volume of 5 ml of blood was transferred into glass vacutainer and 5 ml was transferred into Meresis PRFM kit (Meresis, Laboratory, Bengaluru, Karnataka, India).
Platelet-rich fibrin preparation
A volume of 5 ml of blood present in glass vacutainer was centrifuged in REMI 4 (R-4C, REMI Laboratory Instruments, Mumbai, Maharashtra, India) centrifugation machine for 2800 rpm for 12 min [Figure 2]. The gel obtained following the centrifugation had three layers. The top layer consisted of platelet poor plasma, the middle layer had the fibrin clot and the bottom layer consisted of concentrated red blood cells. The gel was then compressed in-between sterile gauze pieces and the supernatant was discarded. The membrane obtained was then transferred to the laboratory for the assessment of growth factors.
|Figure 2: Platelet rich fibrin preparation (a) Blood withdrawn from antecubital vein; (b) blood centrifuge at 2700 rpm for 12 minutes; (c) platelet rich fibrin gel obtained; (d) platelet rich fibrin membrane obtained|
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Platelet-rich fibrin matrix preparation
Five milliliters of blood sample was transferred to Merisis PRFM kit (Meresis, Laboratory, Bengaluru, Karnataka, India) using a single spin centrifugation method. It was centrifuged at rpm of 3000 for 10 min. The supernatant obtained at the top of gel was removed through syringe and activator containing 0.1% gluconate was added and mixed for 9–10 times. The gel obtained was again transferred to the laboratory for growth factor release estimation. The conventional method of PRFM gel requires the use of double spin for preparation which makes it a time-consuming method as depicted in [Figure 3].
|Figure 3: Platelet-rich fibrin matrix preparation (a) blood withdrawn from actecubital vein; (b) Meresis tube used for preparation of PRFM; (c) withdrawn blood placed in the Meresis tube; (d) centrifugation done at 3000 rpm; (e) supernatant fluid withdrawn; (f) activator added; (g) PRFM gel obtained|
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The growth factors released from both the platelet concentrates were evaluated with 4.5 ml of phosphate buffer saline and 15 of bovine serum albumin as a protease inhibitor.
The gels were transferred into 15 ml tube-containing the phosphate buffered saline and bovine serum albumin. Following the transfer of platelet concentrates, along with the buffer the mix was frozen at −80°C until the growth factor determination completed. Growth factors were extracted from patches by shaking (for 30 min) and three freeze-thaw cycles. Samples were centrifuged for 15 min at 3700 g and the supernatant were used for growth factor detection in ELISA kit as seen in [Figure 3]. The release of growth factors from both the platelet concentrates was measured with Picogram unit.
The release of six growth factors (PDGF, VEGF, TGF, IGF, and EGF) from both the platelet concentrates was statistically evaluated using SPSS software version 18 (SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago, IL, USA). A post hoc power analysis was designed to determine the power of the study.
The primary outcome using a level of 0.05 showed a sample size of 15 healthy controls indicating the power of the study to be 95% which stated that sample size was adequate. The statistical comparison of the growth factors release was evaluated using independent sample t-test. A value of P < 0.05 was considered as statistically significant.
| Results|| |
Platelet-derived growth factor release from platelet-rich fibrin and platelet-rich fibrin matrix over time
Analysis of growth factor release of PDGF at each time point as well as accumulated over time is displayed in [Figure 4]. It was found that after 1 day, significantly higher levels of PDGF was released from PRFM at a concentration of 173.24 when compared to PRF which was about 164.3. The growth factor release from PRFM group peaked at the highest concentration of 190.67 at 15 days, whereas PRF group showed a gradual increase from the 1st day to 15th day at a concentration of 180.5. From the 15th day to 23rd day PRF showed a release of 183.11. Statistically significant difference in the mean of growth factors was observed in PRFM and PRF groups where PRFM had higher mean values as compared to PRF.
|Figure 4: Platelet derived growth factors (PDGF) release from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM) for a period of 23 days|
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Vascular endothelial growth factor release from platelet-rich fibrin and platelet-rich fibrin matrix over time
The release of GFs quantified from both PRFM and PRF showed an increased concentration of PRFM at 256.48 and PRF 233.9 at 17 day. Thereafter, the growth factor release from both the platelet concentrates released was at constant rate till the 23rd day as shown in [Figure 5]. Statistically when evaluated the mean of both the platelet concentrates released from the 1st to 23rd day did not show statistically significant results.
|Figure 5: Vascular endothelial growth factor (VEGF) release from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM) for a period of 23 days|
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Release of endothelial growth factor from platelet-rich fibrin and platelet-rich fibrin matrix over time
The trend of release of EGF growth factor seen in PRF and PRFM groups showed an increase release of growth factor at the 3rd day from PRFM at 162.19 and PRF at a concentration of 154.92. The values of PRFM than gradually increased to 180.8 on the 17th day and then remained constant to 182.51 up to the 23rd day. With respect to PRF the growth factor released at the 17th day was 169.23 and remained constant up to 172.81 up to 30th day. Although there was no statistically significant results in both the platelet concentrates. The mean of growth factor release was higher in the PRFM group from the 3rd to 23rd day as depicted in [Figure 6].
|Figure 6: Epidermal growth factor (EGF) release from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM) for a period of 23 days|
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Fibroblast growth factor release from platelet-rich fibrin and platelet-rich fibrin matrix over time
The release of growth factors from PRF and PRFM has been quantified. The growth factor release from PRF increased from day 1 to day 19 from 120.32 to 140.06 and from PRFM the growth factor released was ranging from 127.33 to 151.44 as shown in [Figure 7]. There was no significant difference between the GF release from both platelet concentrates. However when statistically evaluated the GF release from the 7th to 23rd day has shown higher mean in the PRFM group as compared to the PRF group.
|Figure 7: Fibroblast growth factor (FGF) release from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM) for a period of 23 days|
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Transforming growth factor release from platelet-rich fibrin and platelet-rich fibrin matrix over time
Analysis of TGF growth factor release from the 1st to 23rd day was evaluated. It was observed that both the platelet concentrate till 9th day had constant release of GF of 119.24 and 126.19. However following days, PRFM had a gradual increase in GF and reached to peak on 19th day at 145.73 and PRF showed an increase of 133.67. Although statistically, both the platelet concentrate did not show a significant difference till the 9th day. However, from the 11th day to 23rd day, PRFM group had higher mean as compared to that of PRF group as observed in [Figure 8].
|Figure 8: Transforming growth factor (TGF) release from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM) for a period of 23 days|
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Release of insulin growth factor from platelet-rich fibrin and platelet-rich fibrin matrix over time
The trend in growth factor release seen from PRF and PRFM group revealed the GF peaked in PRFM group at 17th day at 94.36 and PRF was at 83.69 as depicted in [Figure 9]. However, following 17th day till 23rd days, there was a gradual and constant release of GF from both the platelet concentrate. Thus, when evaluated statistically PRFM group had shown higher mean and statistically significant as compared to PRF release of GF.
|Figure 9: Insulin growth factor (IGF) release from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM) for a period of 23 days|
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| Discussion|| |
The platelet concentrates ideal goal is to enhance periodontal regeneration. The magnificent role of these platelet concentrates significantly releases the most important growth factors which play a pivotal role in the wound healing process for a period of 1 month. The growth factors PDGF, TGF, and IGF as reported in various literature has strong mitogenic and anabolic activities further stimulating the proliferation of fibroblast and periodontal ligament cells, enhancing collagen synthesis., PDGF enhances osteoblastic cell proliferation leading to new bone formation. The dynamic VEGF is critical for neo angiogenesis during the wound healing and also facilitate in maintaining the integrity of endothelial cell lining of the blood vessel. The FGF and EGF are known to play an important role in the regulation of ectodermal and mesenchymal derived cell along being a potent chemotactic and mitogenic actions for the periodontal ligament fibroblast cells. Thus, the diverse action of the growth factors forms a key player in wound healing and regeneration.
The present study illustrates the quantitative release of growth factors from PRF and PRFM highlighting its immense regenerative potential. The results derived from the various growth factor release from both the platelet concentrates showed an increased blown out release of PDGF, VDGF, EGF, and FGF in first 1 week from PRFM gel as compared to PRF membrane which had a constant and gradual increase in release up till the 19th day and then consecutively delivered a slow and constant release till the 23rd day. Simultaneously, quantitatively, the highest increase in growth factors was also obtained from PRFM in the initial days as compared to PRF membrane.
The release of growth factors solely is dependent on the fibrin meshwork structure obtained after polymerization of the platelets and leukocyte concentration. The fibrin matrix of PRF is known to slowly release platelet cytokine accelerating the process of integration of fibrin network into regenerative site facilitating the endothelial cell migration helping in the process of neoangiogenesis, thus creating a perpetual process of healing.
The PRFM gel matrix contains high concentrations of nonactivated, intact, and functional platelets, within a fibrin matrix, that further release, a relatively increased concentration of growth factors over a period of 7 days. Although a drop in concentration of growth factor were observed soon after, it was presumed that initial high concentration release of growth factors would be sufficient to influence the healing of the wound with further maturation and remodeling. The pattern of growth factor release from PRFM could be attributed to the addition of calcium gluconate in the preparation of PRFM enabling a faster clot formation governing an immediately increased release of growth factor from the matrix gel., According to Schär et al. 2015, fibrin polymerization in PRFM led to an unstable matrix formation that disintegrated at a faster pace resulting in an increased release of the growth factors in the first few hours of preparation.,, An immediate release at an increased concentration seen for PDGF, VEGF, and EGF could be due to diffusion from plasma or an instant release from an activated platelets. The PRP gel preparation was evaluated from various commercially delivery system (Fibrinet, RegenPRP-Kit, Plateltex) where a different variation in the concentration of growth factors released were observed., The present study used the Meresis Kit (Meresis, Laboratory, Bengaluru, Karnataka, India) for PRFM preparation which was technically quite simple as compared to traditional mode of PRFM preparation.
The results derived in respect to PRF membrane were observed to be a constant and gradual increase in the release of growth factors. It could be attributed to a stronger fibrin architecture entrapping more number of leukocytes in the fibrin matrix. Thus, allowing an intense slow release of growth factor from the fibrin matrix.
The quantitative release of growth factors has been evaluated in various researches in the literature. The period of observing the growth factors release was maximum observed for 10 days. In a rare scenario, all six important growth factors have been evaluated together and compared with other platelet concentrates, whereas, the present study had observed the release of six important growth factors for a period of 23 days. The release of rapid and faster delivery of growth factors has been evaluated for 7 days from PRFM by Lucarelli et al. in 2010 where the concentration of PDGF-AA, PDGF-AB, VEGF, TGF1, EGF, and bFGF in the PRFM were greater at day 1 compared to following days. Su et al. in 2009 evaluated the PDGF-AB, TGFB1, VEGF, EGF, and IGF in PRF membrane but did not compare with any other platelet concentration. Gassling et al. too showed that osteoblasts and fibroblasts cultured with PRP or PRF demonstrated varying expression of various growth factors, but with those cultured with PRP favoring significantly higher levels of PDGF-AB and TGFB1 expression. Carroll et al. in 2009 evaluated the release of growth factors from PRFM matrix gel was a rapid release were observed with the maximum release from PDGF, EGF, and VEGF for a week. In a recent evaluation of PRF, A PRF, and PRP were the release of protein content and quantification of growth factors release had observed and recommended to use PRP in the areas of research where a faster delivery of growth factor is essential. However, the PRF group had shown a gradual and constant release of the growth factors. These results were consistent with the present finding where an abundant release of the growth factors within the first day were observed, and thereafter, a gradual decline in the release of the growth factors was seen in the PRFM as compared to PRF. The variation in the kinetics of growth factor was observed with the greatest release seen in PDGF, VEGF while FGF, IGF, and EGF had gradual. Miron et al. in 2017 evaluated the liquid formulation of PRF without the formulation of PRF termed injectable-PRF (i-PRF) without the use of anti-coagulants was investigated. PDGF-AA, PDGF-AB, EGF, IGF-1, TGF-β1, and VEGF were evaluated for a period of 10 days. I-PRF has shown a considerable increase for a long-term period in respect to PDGF-AA, PDGF-AB, EGF, IGF-1, and PRP has shown a higher level of TGF-β1 and VEGF for the same period. I-PRF had exhibited higher fibroblast proliferation and PRP had shown the highest cellular proliferation.
In the field of periodontal therapy, various functions of the regenerative potential of platelet concentrate have been advocated. The extensive uses of platelet concentrates have been evaluated in the intra bony defect where an excellent improvement in the clinical parameters has been observed., The promising results have also been established in class II furcation defects with the use of platelet concentrates as compared to open flap debridement. The fibrin matrix, the key player in growth factors release, can also show variability as per different age groups as observed in a study by Yajamanya et al. where histologically PRF membrane at third decade of life had shown loose fibrin matrix as compared to younger age groups. A study done by Agarwal et al. in 2016 stated that intra bony defect fill was statistically significant with the use of platelet-rich plasma as compared to demineralized freeze-dried bone graft.
Thus the present study is in accordance with the studies by Lucarelli et al., Gasssling et al. and Carroll et al. where an increased release of GFs from PRFM was observed at the initial period of time but eventually PRF had shown a constant and gradual release of GFs in 23-day analysis. Even though, both the platelet concentrates belong to the same family, their internal structure and biology are completely different. The variation in the impression of growth factors were observed, independently might play a positive impact during the healing.
The present study too had limitation as the protein release content from both the platelet concentrate was not evaluated as it is known to suggest the living progenitor cell ability to migrate the wound area to perform the process of regeneration. Furthermore, the fibrin matrix meshwork varies with an elderly age group individual which should also be evaluated in perspective of growth factor release. The recruitment of larger sample size in a wide range of population is warranted to establish the observation.
| Conclusion|| |
To the best of our knowledge, the present study is the first of its own kind where growth factors from the platelet concentrates for 23 days was evaluated. The key role of platelet concentrate is not the quantity of leukocytes, but its interlinkage to form the end results with fibrin, leukocytes, and growth factors. Thus, both the platelet concentrates can be considered as a biomaterial as it has an immense potential for the regenerative potential. For a better understanding of the platelet concentrates, future investigations studying the effects of each platelet formulation on cell behavior as well as in vivo study is required.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]