|Year : 2019 | Volume
| Issue : 1 | Page : 23-27
|Comparative evaluation of angiogenesis using a novel platelet-rich product: An in vitro study
Treesa William Gomez1, Rajesh V Gopal1, Faisal M.A Gaffoor1, Santhosh T.R Kumar2, Girish C Sabari1, R Prakash2
1 Department of Conservative and Endodontics, Noorul Islam College of Dental Science, Kerala University of Health Sciences, Thiruvananthapuram, Kerala, India
2 Department of Biotechnology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India
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|Date of Submission||28-May-2018|
|Date of Decision||20-Jun-2018|
|Date of Acceptance||12-Dec-2018|
|Date of Web Publication||14-Feb-2019|
| Abstract|| |
Background: In vivo angiogenesis is normal and vital process in growth and development, wound healing, and formation of granulation tissue wherein new blood vessels form from preexisting vessels as part of revascularization. Platelet-rich products promote wound healing associated with angiogenesis. Biomaterials such as titanium were found to be angiogenic. Unlike in vivo situations, in vitro angiogenesis, study cells, within a controlled environment.
Aims: The aim of this study is to evaluate the angiogenic potential of a novel platelet-rich product.
Materials and Methods: Blood was drawn from volunteers with informed consent. Blood samples were centrifuged to obtain platelet-rich products. Platelet concentrates prepared were platelet-rich plasma (PRP), platelet-rich fibrin, and a novel platelet-rich product which is titanium-prepared PRP (TPRP), obtained using titanium. The study which compared platelet concentrate was divided into four groups subjected to tissue culture. Phase-contrast microscope was used to determine the rate of growth by cell counting.
Statistical Analysis: ANOVA was used for comparison within groups and post hoc for multiple comparisons.
Results: TPRP group showed granular ground substance. Group with platelet-rich fibrin (PRF) shows a high rate of growth whereas those with TPRP showed better growth rate when compared to its counterpart, PRP.
Conclusions: This is the first study which introduces TPRP. Previous studies have proved that titanium-prepared PRF has better structural quality than its counterpart platelet-rich fibrin. This study concludes that TPRP has better angiogenic potential than its counterpart PRP. Further in vivo studies are needed to promote TPRP as a new generation of platelet products.
Keywords: Angiogenesis; platelet-rich products; titanium
|How to cite this article:|
Gomez TW, Gopal RV, Gaffoor FM, Kumar ST, Sabari GC, Prakash R. Comparative evaluation of angiogenesis using a novel platelet-rich product: An in vitro study. J Conserv Dent 2019;22:23-7
|How to cite this URL:|
Gomez TW, Gopal RV, Gaffoor FM, Kumar ST, Sabari GC, Prakash R. Comparative evaluation of angiogenesis using a novel platelet-rich product: An in vitro study. J Conserv Dent [serial online] 2019 [cited 2020 Nov 26];22:23-7. Available from: https://www.jcd.org.in/text.asp?2019/22/1/23/252244
| Introduction|| |
Platelet-rich plasma (PRP) seems to enhance neovascularization which may accelerate healing process and promote scar tissue of better histological quality. PRP is a plasma fraction in which several growth factors are concentrated at high levels. In recent years, the biological effects on various cells of the active soluble releasate that is isolated following platelet activation of PRP have been reported. PRP induces the proliferation, migration, and tube formation of vascular endothelial cells which are major processes in angiogenesis. PRP promotes angiogenesis in vitro and in vivo.
In Dentistry, restoring vitality to immature tooth, using these platelet concentrates, is in progress. PRP is potentially an ideal scaffold in revascularization treatment. PRP is the first generation of platelet-rich products. Joseph Choukroun et al. considered platelet-rich fibrin (PRF) as the second generation. This fibrin biomaterial consisted of a fibrin matrix polymerized in a tetramolecular structure, the incorporation of platelets, leukocyte and cytokines, and the presence of circulating stem cells. PRF is a living biomaterial. Dohan Ehrenfest quantified a significant release of growth factors during at least 7 days. Lee et al. evaluated long-term treatment with 1% PRP-enhanced proliferation of the human dental stem cells by 120 h and showed the most significant enhancement at 96 h.
Thor et al. studied that whole blood in contact with titanium alloy resulted in binding of platelets to the material surface and in the generation of thrombin–antithrombin complexes. PRF may offer ease of use, simple handling, and enhanced delivery of growth factors during bone augmentation procedures. Ozdemir et al. observed that when used in conjunction with titanium barriers, PRF can increase quality of newly formed bone and enhance rate of bone formation due to concentration of growth factors. Shivashankar et al. stated that the use of PRP as a potentially ideal scaffold for regenerative endodontic therapy has been documented in literature.
Due to its noncorrosive properties, titanium has excellent biocompatibility. Yeli et al. observed that silver and titanium particles were introduced into dental composites, respectively, to introduce antimicrobial properties and enhance biocompatibility of composites.
PRF was prepared using the most biocompatible titanium, a novel platelet-rich product. Platelet activation by titanium seemed to offer some high characteristics of the novel titanium-prepared PRF (TPRF). TPRF method is based on the hypothesis that it may be potentially beneficial if a titanium tube is used instead of a glass tube in the classical leukocyte-rich PRF method. The fibrin of TPRF seemed more tightly woven and thicker than that of the classic leukocyte-rich PRF.
In this study, titanium tubes are used to prepare PRP to introduce titanium-prepared PRP (TPRP) and study its angiogenic potential.
Several of the existing angiogenesis assays incorporate a means for investigating endothelial mechanisms of cell proliferation and migration. Despite these developments, there is no gold standard assay. Irvin et al. in their review stated that proliferation assays measured by cell counting are used to study in vitro angiogenesis.
| Materials and Methods|| |
Preparation of blood products
PRP, platelet-rich fibrin, the novel TPRP, and PRP activated with calcium chloride to prepare blood samples were collected from three healthy adult volunteers, who were free from systemic diseases and not under any medication. The volunteers gave their informed consent, and the study was conducted as per the revised Helsinki Declaration. The study protocol was approved by the Institutional Committee of Ethics.
Platelet-rich plasma preparation
It includes three formulations; PRP, novel TPRP, and PRP activated by calcium chloride which were prepared initially. Six milliliters of blood was drawn from antecubital vein of each volunteer using syringe. Blood samples were transferred to nine (2ml) vaccum tubes containing sodium citrate as anticoagulant. Tubes kept on electric roller for mixing the anticoagulant and labeled. Contents of three tubes were transferred to three titanium tubes. Titanium tubes were manufactured at Jayon Implants, Palakkad. Titanium alloy Ti6Al4V was used to construct the tubes and were made rough. All tubes were subject to centrifugation (5810R) at a speed of 1600 rpm for 7 min. Eppendorf pipette was used to pipette the first supernatant and transfer to six centrifuge tubes and three titanium tubes. All nine tubes were again subject to centrifugation to obtain the second supernatant. Thus, PRP and TPRP were obtained. To three tubes containing PRP, 50 μl of 10% calcium chloride was added and left for homogenization to obtain the third formulation, activated PRP.
Platelet-rich fibrin preparation
Speed of blood collection and transfer to centrifuge was done immediately since anticoagulants are not used, and coagulation begins immediately after contact with tube. Five milliliters of blood was drawn and centrifuged at a speed of 2000 rpm for 7 min. Fibrins were separated from their red blood cell. The same was repeated to obtain three fibrin preparations.
The study groups were divided into four groups to compare the angiogenic potential of platelet concentrates on endothelial cells. Tissue culture was done using endothelial cells and growth medium was provided in the laboratory by Lonza. Twelve preparations from four formulations such as PRP, TPRP, activated PRP, and PRF were subject to tissue culture with endothelial cells. Cells were revived and incubated at 37°C. They were thawed and centrifuged at 3000 rpm for 3 min. Cell pellets were transferred to T25 flasks with EGM-2 medium and maintained in the incubator and monitored for attachment. After they were confluent, for cell seeding, trypsinization was done and transferred into twelve well plates. EGM-2 medium was added and maintained in the incubator and monitored. Prepared platelet extracts such as PRP, activated PRP, TPRP, and PRF were added to three wells each along with EGM-2 medium. To wells containing experimental controls for cells, growth medium was added along with fetal bovine serum as positive control and no growth medium for negative controls. Preparations and controls were subject to tissue culture. Culture wells were incubated for 21–24 h at 37°C and observed from time to time for attachment and change of medium. Cultures were monitored to 72 h. Once they were confluent, wells were viewed under phase-contrast microscopy and photographs were taken [Figure 1].
|Figure 1: Cells viewed under phase-contrast microscope. Group A: PRP, Group B: PRP and CaCl2, Group C: TPRP, Group D: PRF. Number of cells × 103 in 0.75 cm2/well. Scale bar is 100 μm. PRP: Platelet-rich plasma, TPRP: Titanium-prepared platelet-rich plasma, PRF: Platelet-rich fibrin|
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Growth was detected by cell counting (number of cells × 103 in growth area of 0.75 cm2/well). Group A was PRP and cells. Group B was activated PRP and cells. Group C was TPRP and cells. Group D was PRF and cells. PRF was the standard to which comparisons were made.
Cell counting was done using software with grid system and data were entered in Excel and analyzed using software SPSS 16.0 version (SPSS Inc., Chicago, IL, USA). Normality was evaluated using Kolmogorov–Smirnov test. ANOVA was done for comparison of quantitative variables and data were analyzed with Friedman's test for ranking. Post hoc test was done for multiple and pairwise comparison between and within groups. P < 0.05 was considered statistically significant.
| Results|| |
Results were expressed from cell counting of culture wells viewed under phase-contrast microscope. Obtained values were used to study the rate of cell growth. Comparisons using ANOVA and post hoc showed PRF and PRP (P = 0.006), PRF and PRP: CaCl2 (P = 0.001), and PRF and TPRP (P = 0.039). Friedman's test, ranking was highest for Group D which were cultures with PRF, ranked as 4.00 showing the highest growth rate. Group B: Cultures with PRP activated with calcium chloride were ranked least with least amount of cell growth. Group C: Cultures with TPRP showed a good amount of growth of cells but were lesser than Group D and ranked 3.00. Group A: Cultures with PRP showed a comparatively lesser amount of cell growth than Groups D and C and were ranked 1.83. The study is considered statistically significant. Group C alone shows the presence of a granular network/ground substance.
| Discussion|| |
Platelets can be activated by addition of thrombin/calcium chloride which induces release of growth factors. During inflammation, platelets are activated naturally by thrombin. Activation of platelets by addition of calcium chloride only mimics inflammatory process in-vitro, but their effects on growth of endothelial cells were found to be negative. The fact that extracellular matrix present in tissue cultures activates platelets explains that an additional activation with CaCl2 may be unnecessary. The presence of granular network formed in culture with TPRP is unique and is absent in all other three groups [Figure 1] and [Figure 2]. The study supports that PRF promotes neovascularization faster compared to other platelet products. It also supports that TPRP is superior to its counterpart PRP which may be due to increased availability of growth factors in the presence of hemocompatible titanium.
|Figure 2: Comparison of ground substance of PRF samples and TPRP samples as viewed under phase-contrast microscope. Cells embedded in a granular ground substance in TPRP samples, whereas no such granular ground substance was seen in PRF samples. Scale bar is 100 μm. PRP: Platelet-rich plasma, TPRP: Titanium-prepared platelet-rich plasma, PRF: Platelet-rich fibrin|
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Activated platelets release a range of growth factors (e.g., transforming growth factor-beta, platelet-derived growth factor, and epidermal growth factor EGF), cytokines (e. g., interleukin [IL-1β] and IL-8), and angiogenic factors (e.g., vascular endothelial growth factor and angiopoietin-1). Kakudo et al. studied the capacity of PRP releasate to stimulate tissue regeneration which is thus believed to be due to the stimulatory activities of the growth factors released from activated platelets on proliferation of progenitor cells with vascularization at local sites.
Titanium passivates itself in vivo by forming an adhesive oxide layer. Hemocompatibility is a key property for biomaterials that come into contact with blood. Gorbet and Sefton studied that thrombus formation and inflammation involve activation of both coagulation and complement system, initiation of these systems causes plasma protein adsorption on the surface followed by activation and adhesion of platelets and leukocytes.
The use of titanium as well as tantalum and indium in an in vitro study by Hong et al. displayed pronounced thrombogenic properties. Thrombogenic activity of the material could be useful for onset of a rapid start of the healing process in healing of bone. Marino et al. and Marx et al. proposed the use of PRP as a viable technique to obtain a high concentration of growth factors. The rationale behind the use of PRP is the assumption that it may improve wound healing by increasing levels of growth factors in the wound site after degranulation of platelets. Raines et al. studied that the microstructure and high energy surfaces of titanium induce its ability to produce angiogenesis during osseointegration. Titanium substrate features control osseointegration by enhancing angiogenesis at the material–tissue interface. Titanium surface microtopography and energy interact through α2β1 signaling to regulate expression of angiogenic growth factors. Tunalı et al. modified the initial L-PRF method by changing the structure of the centrifuge tubes and used a more biocompatible material, titanium. They hypothesized that TPRF may last a bit longer in the tissue.
Angiogenesis in vitro can draw a pathway to revascularization of the pulp clinically. Influences on rates of angiogenesis by these different blood products reflect rate of revascularization of the pulp. Revitalizing the adult pulp is a great challenge due to microvasculature within a nearly closed chamber. From the perspective of regenerative endodontics, in root canals of molar teeth, PRP is a good adjuvant, since it is a fluid when compared to PRF which is in gel form.
Hypothesis from the essence of this study is that hemocompatible biomaterials such as titanium are adjutants in repair and revascularization, and TPRP is a better adjuvant than PRP and therefore may be used in posterior teeth instead of PRP. We can apply this also to different branches of medicine wherein revascularization is critical, for example, orthopedics and cardiovascular surgery.
| Conclusions|| |
This study concludes that cultures with PRF show the highest rate of cell growth. Cultures with TPRP show more cell growth than its counterpart PRP but lesser than those with PRF. Cultures with activated PRP show the least amount of cell growth [Graph 1].
Since cultures with TPRP show better angiogenesis than with its counterpart PRP, comparisons with other blood products as per Dohan Ehrenfest's classification need to be studied. Although PRF is quickly prepared chair side and is considered a gold standard in healing among other platelet-rich products, we need to conduct further experiments on novel titanium-prepared blood products mainly the physical form of titanium to be incorporated for preparations such as “TPRF” which is hypothetically necessary while attempting to promote angiogenesis due to its quick preparation. Further in vivo studies are needed to promote TPRP as a new generation of platelet products.
We would like to thank the team of scientist in the cancer research unit at Rajiv Gandhi Centre for Biotechnology, Trivandrum, Kerala, India, for their technical support and also Dr. Ravisankar M.S., Dr. Rethi Gopakumar, and Dr. Deepak Chandran.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Treesa William Gomez
Thaivillakam House, Moongode P. O, Thiruvananthapuram - 695 144, Kerala
Source of Support: None, Conflict of Interest: None
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